TWI306152B - - Google Patents

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13 I _會月~~口篆丘.替換貝 發咽騰44— 發明領域 本發明係關於糖鏈[如彼等連接於蛋白質（蛋白多糖、 糖蛋白）或脂質而存在者]或自由糖類之結構分析的領域。 引言 由單糖(糖）單元組成之寡糖及多糖係藉由糖苷鍵彼此 連結。該糖鏈如同DNA或蛋白質鏈般，具有兩個不同末端 。於糖鏈之情形下，彼等末端爲還原端(相當於直鏈糖分子 之醛基)及非還原端。然而，不同於蛋白質及DNA者，糖 類亦可爲分支狀，且基本上於糖類中之各糖單元係供作可 選用之分支點。 有許多種蛋白質鍵結於糖類。此等蛋白質大多數專一 地鍵結於特定的短鏈寡糖序列。外源凝集素(lectin)爲自植 物分離出之與糖類鍵結的蛋白質。爲達本申請案目的，”外 源凝集素”一詞亦包括得自動物之糖鍵結性蛋白質(如”哺乳 類外源凝集素”）。抗體爲專一地辨識特定分子結構之蛋白 質。抗體如同外源凝集素般，亦可辨識糖結構。糖苷酶爲 切割糖鏈內之糖苷鍵的酵素。糖苷酶亦可專一地辨識特定 的寡糖序列。糖基轉移酶爲切割糖鏈並進一步將糖單元轉 移至新生成端之一的酵素。 多糖類結構之測定技術並未發展得如蛋白質分析'及 DNA分析技術般快速。此乃由於隨著DNA相關硏究領域 之基礎發現的事實，使人們意識到DNA的重要性曾被大大 地低估。這導致數十年來對於DNA分析方法及對於〇Na 本身之致力硏究。再者，隨著改良及簡化之DNA分析方法 4 本紙張尺度適用中國國家^準(CNS)A4規格（210 X 297公釐) -- -^----------—^^4 j 丨— ί— 訂---------. (請先閱讀背面之注意事項再填寫本頁)13 I _ 月月~~口篆丘.Replacing Beifa pharynx 44 - FIELD OF THE INVENTION The present invention relates to sugar chains [as they are attached to proteins (proteoglycans, glycoproteins) or lipids] or free sugars) The field of structural analysis. Introduction Oligosaccharides and polysaccharides composed of monosaccharide (sugar) units are linked to each other by glycosidic bonds. This sugar chain has two different ends like a DNA or protein chain. In the case of a sugar chain, these ends are a reducing end (corresponding to an aldehyde group of a linear sugar molecule) and a non-reducing end. However, unlike proteins and DNA, the saccharide may also be branched and essentially serve as an optional branch point for each saccharide unit in the saccharide. There are many kinds of proteins that bind to sugars. Most of these proteins are specifically bonded to specific short-chain oligosaccharide sequences. Lectin is a sugar-bonded protein isolated from plants. For the purposes of this application, the term "exogenous lectin" also includes carbohydrate-binding proteins (such as "mammalian lectins"). An antibody is a protein that specifically recognizes a specific molecular structure. Antibodies, like lectins, also recognize sugar structures. A glycosidase is an enzyme that cleaves a glycosidic bond in a sugar chain. Glycosidases can also specifically identify specific oligosaccharide sequences. A glycosyltransferase is an enzyme that cleaves a sugar chain and further transfers the sugar unit to one of the newly formed ends. The technique for the determination of polysaccharide structures has not evolved as fast as protein analysis and DNA analysis techniques. This is due to the facts found in the field of DNA-related research that have made people realize that the importance of DNA has been greatly underestimated. This has led to decades of research on DNA analysis methods and on 〇Na itself. Furthermore, with the improved and simplified DNA analysis method 4 This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) ---^-----------^^ 4 j 丨— — — order ---------. (Please read the notes on the back and fill out this page)

1306152 五、發明說明（ 的問世，蛋白質結構及功能分析之技術開始使用越來越多 由DNA技術衍生之工具。例如蛋白質結構之測定經常藉由 反轉基因技術進行，例如獲得一小段蛋白質序列，並由其 對應之mRNA序列[該mRNA序列爲現今於大多數情形下易 於獲得，正如同可自資料庫獲得之多數物種(包含人類）的 大部分mRNA序列]推論出其餘的胺基酸序列。 相較於DNA及蛋白質分析技術之進步，大多數哺乳類 蛋白質之極重要部分(亦即，其連接於糖類及聚糖之部分） 的分析呈普遍落後。 糖分Ϊ (gly comolecules)之重要性因青黴素之發現而顯 著，青黴素爲一種細菌細胞壁之糖分子合成抑制劑，其可 能爲目前所發現成效最大的抗生素。 另一實例爲肝素之醫療用途，肝素爲一種抑制凝血的 聚糖，現今被廣用於醫藥。具醫學重要性之糖分子的進一 步實例包含：葡糖胺聚糖(GAGs)、硫酸乙醯肝素、細胞素（ 如IL-8、TNF)、化學增活素（chemokines)(如酸性纖維母細 胞生長因子）及各種生長因子。前述細胞素、化學增活素及 生長因子亦可鍵結於GAGs及其他多糖類，因此考量以彼 等作爲外源凝集素。 多糖類結構之測定爲糖生物學(glycobiology)發展的重 要基礎。糖生物學之硏究涉及如上述之細菌細胞壁、血液 聚糖等各種主題，亦涉及與病毒性疾病(如HIV感染）、自 體免疫性疾病（如胰島素依賴型糖尿病及類風濕性關節炎） 、及不正常細胞生長（如於癌症中發生者）有關之生長因子 5 — -- -------- -- , --— ,———裝--------訂--------- (請先閱讀背面之注意事項再填寫本頁) 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 1306152 A7 A7 B7 五、發明説明（3) 及細胞表面受體結構。 (請先閲讀背面之注意事項再填寫本頁) 於醫學之其他領域，如隱形眼鏡、人造皮之供給、補 缺學之發展上’多糖類爲良好的候選材料。再者，多糖類 使用於多種非醫學領域如紙業。此外’食品及藥品業當然 使用大量的各種多糖類及寡糖類。 上述所有領域中，需要改良糖類分析技術，以達品質 控制、硏究時之結構測定之目的’以及供進行結構功能分 析。 蛋白質及DNA定序領域已於多年前引進先進的分析方 法。組成DNA及蛋白質之成分僅藉由一種連結方式彼此連 結(於DNA爲5’至3’磷酸橋，於蛋白質爲胜肽鍵）。DNA僅 含有4種不同成分(核酸），而蛋白質含有約20種不同成分( 胺基酸）。雖然存有改質之胺基酸，但必須先使用DNA模 版，依遺傳密碼合成蛋白質。因此，存於新合成之蛋白質 中的胺基酸數量及種類僅限於在遺傳密碼中所示之有限的 胺基酸類型。該密碼爲共通者，在所有生命體中僅有些微 差異。 經濟部智慧財產局員工消費合作社印製 基於上述結構理由，蛋白質及DNA之結構分析在今曰 爲一種不需要高度熟練人員之簡單、快速、且相對地便宜 的程序。 相反地，有多種糖類結構之分析方法已被發展出來， 其各自具有缺點。迄今無法不使用複雜的方法而藉由使用 單一技術測定多糖，甚至於寡糖之全部序列。關於此難題 有許多理由。第一，糖類不依模版合成。由於缺乏結構訊 _；_ 6 本紙張尺度適用中國國家標準（CNS)八4規格（21GX297公廣） 1306152 A7 B7 五、發明説明（φ) (請先閲讀背面之注意事項再填寫本頁) 息，硏究者必須假設其構築單元係選自迄今已知之任何糖 單元。此外，此等單元可能己遭改質，例如於合成期間添 加硫酸根基而改質。 第二，糖單元間之連結處多。若糖單元連結於己糖’ 則該糖可連結於Cl、C2、C3、C4或C6原子中之任一者。 此外，於C1原子之連結可爲α或/3構型。 第三，糖類可爲分支狀，而進一步使其結構以及具有 相同數目及種類之糖單元的可能結構的數量複雜化。 第四個難題在於許多糖間之結構差異小之事實，因爲 糖單元間之差別可能僅在於羥基之位置(差向異構體）。 先前技藝 已開發出許多種糖類結構之分析方法。 W〇93/24503揭示一種依序去除寡糖之還原端的單糖 單元的方法，該方法係將位於還原端之單糖轉換成其酮或 醛型，接著使用肼切割位於該單糖及該寡糖鏈中之下一個 單糖間之糖苷鍵。使該自由單糖自該寡糖鏈單離’再藉層 析法鑑定。重複該方法，直至所有單糖被分離。 經濟部智慧財產局員工消費合作社印製 WO 93/22678揭示一種定序未知寡糖之方法，該方法 係依其基本結構作假設，接著自多種定序工具（如糖苷酶) 中選出預期可以得到最高量之結構訊息的一種工具。此方 法需要某些與寡糖結構有關之基本訊息(通常爲單糖之組成 )。該方法亦說明以定序劑進行之反應既貴又浪費時間的事 實，因此需求一種減少此等支出的方法。 W〇93/22678揭示一種藉由探測固定於VLSI晶片上之 7 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1306152 A7 B7 五、發明説明（s) (請先閲讀背面之注意事項再填寫本頁) 探針單一陣列如寡去氧核苷酸，而檢測分子之方法。此文 獻教示大量探針可黏結於固定表面上，且可使用於VLSI晶 片上之邏輯電路，藉由各種方法檢測該等探針與待分析物 (analyte)間的反應。 EP 421，972揭示一種定序寡糖的方法，該方法係藉由 在寡糖的一端作標記，將所標記的寡糖區分成等分試樣， 以不同的試劑混合物（如糖苷酶)處理各等分試樣，匯集不 同的反應混合物，接著使用層析法分析反應產物。此方法 僅對N-連結型聚糖有效，因該類聚糖於糖鏈與蛋白質連結 之點具有共通結構。0-連結型聚糖間之變異較多，此方法 未適用於此等在基本結構上具有較大變異的寡糖。 因此，本發明目的在於提供一種克服前述先前技藝方 法上的所有問題之糖類結構分析的方法。 因此，本發明提供一種藉由單一技術，而不需要組合 藉由不同技術獲得之結果即可達最終結果之糖類結構分析 〇 本發明方法適用於寡糖以及多糖之結構分析。 經濟部智慧財產局員工消費合作社印製 本發明方法更適用於自動化，因此提供一種可以提供 基本上足以獨特地鑑定指定之寡糖或多糖的訊息之簡單而 快速的分析。 本發明進一步提供鑑定所得之寡糖或多糖序列的方法 〇 本發明之進一步目的及優點將由下文之說明而變得明 顯。 __ 8 本紙法尺度適用中國國^^CNS ) A4規格（210X297公釐） 1306152 Η ί2· I4修正 年月日、 補无 ____B7 I --------- - 五、發明說明（j) 發明大綱 本發明主要係關於糖類之結構分析方法，該方法包括 Σ〇於一表面上放置多數個基本上爲序列專一性及/或位 點專一性之鍵結劑； b) 使該表面與欲分析之糖類接觸，或與包括該糖類之 多數個片段的混合物接觸； c) 洗滌或者去除未鍵結之糖類或糖類片段； d) 將基本上爲序列專一性及/或位點專一性之標記，或 基本上爲序列專一性及/或位點專一性之標記的混合物添加 至步驟c)製得之表面； e) 獲得鍵結於該表面之標記的一個或多個影像；及 f) 自該影像推演出所分析之該種糖類的關於本身 (identity)的訊息。 於本發明方法之較佳具體例中，該基本上爲序列專一 性及/或位點專一性之標記係產色鍵結劑（chromogemc binding agents)。本文所用之”產色鍵結劑”一詞包含所有鍵 結於糖類且具有不同顏色或可檢測之標記的所有藥劑，而 在鍵結於糖類之後，使該糖類獲得該顏色或其他標記。除 了具有在可見光範圍內之固有的、立即可觀察到的顏色之 化學結構以外，使用之其他標記包含螢光基、生物素標籤 、酵素（可用於促使形成呈色產物的反應者）、磁性標記及 同位素標記等。前述列舉之可檢測之標記僅係用以達到說 明之目的，而無限定或完全揭露之用意。有時，本文所用 9 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公f ) -----------裝-------- - 訂------ (請先閱讀背面之注意事項再填寫本頁) 1306152 經濟部智慧財產局員工消費合作社印製 Α7 Β7 五、發明説明（"I ) 之”顏色”一詞（例如於上述方法的步驟(e)所述者)亦包含任 何可檢測之標記。 於本發明方法之較佳具體例中，係藉由簡單的目視檢 測該表面，再與標準物比較而獲得結構訊息。或者，於另 一較佳具體例中，步驟(f)包括光學濾器之使用。於再一較 佳具體例中，於該表面上顯現之顏色影像係藉由照相捕攫 ，接著以數字予以顯示。 雖然本發明方法可使用任何適當的基本上爲序列專一 性之鍵結劑，本發明特定而言，係關於使用外源凝集素作 爲基本上之序列專一性及/或位點專一性鍵結劑。於本發明 另一較佳具體例中，該適當的基本上爲序列專一性及/或位 點專一性之鍵結劑係抗體。 以基本上爲序列專一性及/或位點專一性之方式鍵結於 糖類的任何適當的呈色(或可檢測之)物質皆可使用作爲基 本上之序列專一性及/或位點專一性產色鍵結劑。不過，一 般而言，該基本上爲序列專一性及/或位點專一性之產色鍵 結劑係產色外源凝集素或產色抗體。於本發明之一較佳具 體例中，該產色鍵結劑係呈色外源凝集素。又較佳之具體 例中，需要使用螢光劑或經生物素標示之外源凝集素或抗 體。於更佳之具體例中，該基本上爲序列專一性之產色鍵 結劑係經酵素標記之抗體。 於另一方向，本發明方法進一步包括以在鍵結於糖類 之後可以切割糖鏈之基本上爲序列專一性的藥劑處理該糖 。該處理可於該糖類與該表面接觸以前進行。或者，該處 10 I紙張尺度適用中國國家標準（CNS ) A4規格（210X297公ϋ ----- ； -------、玎------0 (請先閲讀背面之注意事項再填寫本頁) U 12 1 4 年月曰1306152 V. The invention, the technology of protein structure and functional analysis began to use more and more tools derived from DNA technology. For example, the determination of protein structure is often carried out by reverse gene technology, such as obtaining a small protein sequence, and The rest of the amino acid sequence is deduced from its corresponding mRNA sequence [this mRNA sequence is readily available in most cases today, just as most mRNA sequences of most species (including humans) available from the database]. Compared to advances in DNA and protein analysis techniques, the analysis of the most important parts of most mammalian proteins (ie, their attachment to sugars and glycans) is generally lagging behind. The importance of glycy comolecules is due to penicillin It is found that penicillin is a sugar molecule synthesis inhibitor of bacterial cell wall, which may be the most effective antibiotic found so far. Another example is the medical use of heparin, a glycan that inhibits blood coagulation, which is widely used today. Further examples of medically important sugar molecules include: glycosaminoglycans (G AGs), heparin sulfate, cytokines (such as IL-8, TNF), chemokines (such as acidic fibroblast growth factor) and various growth factors. The aforementioned cytokines, chemical stimulating hormones and growth Factors can also be linked to GAGs and other polysaccharides, so consider them as exogenous lectins. The determination of polysaccharide structure is an important basis for the development of glycobiology. The study of glycobiology involves the above Various themes such as bacterial cell walls and hemoglobin are also involved in viral diseases (such as HIV infection), autoimmune diseases (such as insulin-dependent diabetes and rheumatoid arthritis), and abnormal cell growth (such as cancer). Among the occurrences of growth factors 5 -- -- -- -- -- -- -- -- , -- -- -- -- -- -- -- -- -- ( Please read the notes on the back and fill out this page.) This paper size applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1306152 A7 A7 B7 V. Description of invention (3) and cell surface receptor structure. Please read the notes on the back and fill out this page.) In other areas of medicine For example, the supply of contact lenses, artificial leather, and the development of supplements is a good candidate for polysaccharides. Furthermore, polysaccharides are used in a variety of non-medical fields such as paper. In addition, the food and pharmaceutical industry certainly uses a large variety of Sugars and oligosaccharides. In all of the above fields, improved saccharide analysis techniques are needed to achieve quality control, structural determination for research purposes, and for structural functional analysis. The field of protein and DNA sequencing has been introduced many years ago. Analytical method: The components constituting DNA and protein are linked to each other only by a linkage method (the DNA is a 5' to 3' phosphate bridge, and the protein is a peptide bond). DNA contains only four different components (nucleic acids), while proteins contain about 20 different components (amino acids). Although there is a modified amino acid, it is necessary to first synthesize the protein according to the genetic code using a DNA template. Therefore, the amount and type of amino acid present in the newly synthesized protein is limited to the limited type of amino acid shown in the genetic code. This password is a common one and only slightly different in all living organisms. Printed by the Ministry of Economic Affairs, the Intellectual Property Office, and the Consumer Cooperatives. Based on the above structural reasons, structural analysis of proteins and DNA is now a simple, fast, and relatively inexpensive procedure that does not require highly skilled personnel. Conversely, analytical methods with a variety of carbohydrate structures have been developed, each with disadvantages. It has hitherto been impossible to determine the polysaccharide, even the entire sequence of oligosaccharides, by using a single technique without using complicated methods. There are many reasons for this problem. First, the sugar is not synthesized according to the template. Due to the lack of structure information _; _ 6 This paper scale applies to China National Standard (CNS) VIII 4 specifications (21GX297 public) 1306152 A7 B7 V. Invention description (φ) (Please read the back of the note before filling this page) The researcher must assume that the building unit is selected from any sugar unit known to date. In addition, such units may have been modified, for example by adding sulfate groups during synthesis. Second, there are many connections between sugar units. If the saccharide unit is attached to a hexose', the sugar can be attached to any of the Cl, C2, C3, C4 or C6 atoms. Further, the linkage to the C1 atom may be in the alpha or /3 configuration. Third, the saccharide may be branched, further complicating its structure and the number of possible structures having the same number and type of saccharide units. The fourth difficulty lies in the fact that many structural differences between sugars are small, since the difference between sugar units may be only at the position of the hydroxyl group (epimer). Previous Techniques A number of analytical methods for the construction of carbohydrates have been developed. W〇93/24503 discloses a method for sequentially removing a monosaccharide unit at the reducing end of an oligosaccharide, which converts a monosaccharide at a reducing end into its ketone or aldehyde form, followed by cleavage at the monosaccharide and the oligosaccharide A glycosidic bond between the monosaccharides in the sugar chain. The free monosaccharide was identified by the single detachment from the oligosaccharide chain. This method is repeated until all the monosaccharides are separated. WO 93/22678, published by the Intellectual Property Office of the Intellectual Property Office of the Ministry of Economic Affairs, discloses a method for sequencing unknown oligosaccharides, which is based on its basic structure and then selected from various sequencing tools (such as glycosidases). A tool for the highest amount of structural information. This method requires some basic information about the structure of the oligosaccharide (usually the composition of the monosaccharide). This method also illustrates the fact that the reaction with the sequencer is both expensive and time consuming, and therefore requires a method of reducing such expenditure. W〇93/22678 discloses a method for detecting 7 paper sizes fixed on a VLSI wafer. The Chinese National Standard (CNS) A4 specification (210X297 mm) 1306152 A7 B7 5. Inventive Note (s) (Please read the back Precautions Fill in this page) A single array of probes such as oligodeoxynucleotides, and methods for detecting molecules. This document teaches that a large number of probes can be bonded to a fixed surface and that the logic used on the VLSI wafer can be used to detect the reaction between the probes and the analytes by various methods. EP 421,972 discloses a method for sequencing oligosaccharides by separating the labeled oligosaccharides into aliquots by treatment at different ends of the oligosaccharide, and treating them with different reagent mixtures (eg glycosidases) Aliquots were pooled with different reaction mixtures, followed by chromatographic analysis of the reaction products. This method is effective only for N-linked glycans, since the glycans have a common structure at the point where the sugar chain is linked to the protein. There is a large variation between 0-linked glycans, and this method is not applicable to such oligosaccharides having a large variation in basic structure. Accordingly, it is an object of the present invention to provide a method of saccharide structure analysis that overcomes all of the aforementioned prior art methods. Accordingly, the present invention provides a saccharide structure analysis that achieves the final result by a single technique without the need to combine the results obtained by different techniques. 〇 The method of the present invention is applicable to structural analysis of oligosaccharides and polysaccharides. Printed by the Ministry of Economic Affairs, Intellectual Property Office, Staff Consumer Cooperatives The method of the present invention is more suitable for automation, thus providing a simple and quick analysis that provides information that is substantially sufficient to uniquely identify a given oligosaccharide or polysaccharide. The invention further provides a method of identifying the resulting oligosaccharide or polysaccharide sequence. Further objects and advantages of the present invention will become apparent from the following description. __ 8 This paper method scale applies to China's country ^^CNS) A4 specification (210X297 mm) 1306152 Η ί2· I4 revision date, no ____B7 I --------- - V. Invention description (j SUMMARY OF THE INVENTION The present invention is primarily directed to a method of structural analysis of a saccharide comprising placing a plurality of substantially serial-specific and/or site-specific bonding agents on a surface; b) rendering the surface Contacting the saccharide to be analyzed, or contacting a mixture comprising a plurality of fragments of the saccharide; c) washing or removing unbound sugar or carbohydrate fragments; d) being substantially sequence specific and/or site specific a label, or a mixture of substantially sequence specificity and/or site specificity, added to the surface prepared in step c); e) obtaining one or more images of the label bound to the surface; and f) The information about the identity of the sugar analyzed by the image is derived from the image. In a preferred embodiment of the method of the invention, the substantially sequence specificity and/or site specificity is a chromogemc binding agent. As used herein, the term "chromogenic bonding agent" encompasses all agents that are bound to a saccharide and have a different color or detectable label, and after binding to the saccharide, the saccharide is given the color or other indicia. In addition to the chemical structure with an intrinsic, observable color in the visible range, other labels used include fluorophores, biotin labels, enzymes (which can be used to promote the formation of color-developing products), magnetic labels And isotope labeling. The above-identified detectable labels are used for the purpose of illustration only and are not intended to be limiting or fully disclosed. Sometimes, the 9 paper sizes used in this article apply to the Chinese National Standard (CNS) A4 specification (210 X 297 public f) -----------装-------- - order--- --- (Please read the note on the back and fill out this page) 1306152 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed Β7 Β7 5, invention description ("I) the word "color" (for example, in the above method The step (e) also includes any detectable mark. In a preferred embodiment of the method of the invention, the surface is visually detected by simple visual inspection and compared to a standard to obtain a structural message. Alternatively, in another preferred embodiment, step (f) includes the use of an optical filter. In still another preferred embodiment, the color image appearing on the surface is captured by photography and then displayed numerically. Although any suitable substantially sequence specific binding agent can be used in the methods of the present invention, the present invention specifically relates to the use of exogenous lectins as substantially sequence specificity and/or site specific binding agents. . In another preferred embodiment of the invention, the appropriate binding agent-specific antibody is substantially sequence specific and/or site specific. Any suitable color (or detectable) substance that binds to the saccharide in a manner that is substantially sequence specific and/or site specific can be used as substantially sequence specificity and/or site specificity. Color bonding agent. However, in general, the chromogenic bonder which is substantially sequence specific and/or site specific is a chromogenic lectin or chromogenic antibody. In a preferred embodiment of the invention, the chromogenic bonding agent is a color lectin. In a further preferred embodiment, it is desirable to use a fluorescent agent or biotin to label a lectin or antibody. In a more preferred embodiment, the substantially sequence-specific color-forming bonding agent is an enzyme-labeled antibody. In another aspect, the method of the invention further comprises treating the sugar with a substantially sequence specific agent that cleaves the sugar chain after binding to the sugar. This treatment can be carried out before the sugar contacts the surface. Or, the 10 I paper scale is applicable to the Chinese National Standard (CNS) A4 specification (210X297 public ϋ ----- ; -------, 玎 ------ 0 (please read the back note first) Please fill out this page again) U 12 1 4 months

五、發明說明（ 1306152 理可於去除未鍵結之糖類以後，但於添加基本上爲序列專 一;性之產色鍵結劑以前進行。 於本發明方法之特佳具體例中，該表面爲濾紙，而該 基本上爲序列專一性的藥劑係以預先限定(pre_defined)之順 序排列於該爐紙上。 於另一方面，本發明提供一種糖分子鑑別(GMID)卡, 列出依據本發明方法之快速地前述較佳具體例獲得之糖類 結構分析的資料。於一較佳具體例中，所用之基本上爲序 列專一性的試劑係以代號示於GMID卡上。於GMID卡之 另一較佳具體例中’在分析時使用之基本上爲序列專一性 的藥劑之組合係以獨特的代號表示。 本發明亦關於一種固體支承體，其包括代表糖類或糖 類序列或片段之預先限定順序的多數個可目視或可檢測之 標記。 另一方面’本發明亦包括選擇一組可供上述方法使用 之基本上爲序列專一性的產色鍵結劑的方法，該方法包括 下列步驟： a) 獲得欲分析之糖類的全部或部分單糖組成(MC) ; b) 選擇一組含有η個可以鍵結於該糖類中所含之單糖 類的基本上爲序列專一性及/或位點專一性之標記； c) 修正於步驟b)獲得之該組基本上爲序列專一性及/或 位點專一性之標記，以確保該組中沒有兩個標記具有相同 的顏色或是可檢測之標記； d) 修正於步驟c)選擇之該組基本上爲序列專一性及/或 11 本紙張尺度適用中國國家標準（CNS)A4規袼（210 X 297公釐） (請先閱讀背面之注意事項再填寫本頁) ▼裝--------訂·-----丨丨 1306152 \7 B7 95. 11 IB j'E 年月E-丨二t 補无 五、發明說明（1 ) 位點專一性之標記，以減少該組標記與該等基本上爲序列 專一性及/或位點專一性之鍵結劑或與其他基本上爲序列專 一性及/或位點專一性之標記間的交互反應。 於本方法之一較佳具例中，欲分析之糖類的MC係由 相關之糖類的MC推測。於另一較佳具體例中，欲分析之 糖類的MC係藉由對該糖類進行完整之MC分析而獲得。 於本方法之另一較佳具體例中，η値介於1與4之間。 本發明亦關於用以選擇於上述糖類結構分析方法中使 用的一組基本上爲序列專一性的標記之軟體，該軟體包括 a) 輸入，用以提供單糖組成(MC): b) 配對次程式，用以配對可以鍵結於該糖類中所含之 單糖的η個基本上爲序列專一性或位點專一性之標記配合 i c) 修正次程式，用以修正由次程式b)配對之一組基本 上爲序列專一性或位點專一性之標記，該次程式能夠以減 少該標記與該等基本上爲序列專一性或位點專一性之鍵結 劑或與其他基本上爲序列專一性或位點專一性之標記間的 交互反應爲基礎而選擇該標記； d) 再次修正次程式，能夠確保該組中沒有兩個標記真 有相同的顏色或是可檢測之特徵。 本發明亦關於一種獲得糖類之結構相關資料的方法， 包括下列步驟： a)使該糖類與能夠切割該糖鏈之基本上爲序列專一性 12 本紙張尺度適用中國國家標準（CNS)A4規格（210 χ 297公爱） ----------- (請先閱讀背面之注音？事項再填寫本頁) I — — — — — — — — 1306152 Α7 Β7 五、發明説明（ίΰ) 的藥劑反應； (請先閲讀背面之注意事項再填寫本頁) b)使該糖類與能夠鍵結於該糖類之基本上爲序列專一 性的藥劑反應； 0直接或間接地將標籤引入該糖類中，包含使用能夠 鍵結於該糖類的經標示之基本上爲序列專一性的藥劑，及 d)檢測含有切割劑之反應中、及不含切割劑之反應中 之標籤所在。 本發明又關於一種糖類之結構分析的方法，包括任何 順序之下列步驟： a) 提供一種糖類； b) 使該糖類與第一基本上爲序列專一性的藥劑反應； c) 使該糖類或其片段與第二基本上爲序列專一性的藥 劑反應； d) 使該糖類或其片段與第三基本上爲序列專一性的藥 劑反應； e) 視需要使用至少一種不同的第二或第三基本上爲序 列專一性的藥劑重複步驟c至d ; 經濟部智慧財產局員工消費合作社印製 其中，可以在並行而獨立之反應中使用相同的糖類’ 並以至少一種不同的第一、第二、或第三基本上爲序列專 一性的藥劑進行步驟a)至e)，但其條件爲該糖類係經標示 者及/或一種或多種該第一、第二、或第三基本上爲序列專 一性的藥劑係經標示者，或者將一種標籤導入該糖類中’ 且在導入該標籤的步驟之後的一個或多個步驟檢測出該標 籤，且其另一條件爲至少一個第一、第二及/或第三基本上 _；_ 13 本纸張尺度適用中國國家g ( CNS ) A4規格ϋΐ〇Χ297公釐） 1306152 I 95.11 1 β /Λ 年月補无 Α7 Β7 五、發明說明（ 爲序列專一性的藥劑爲切割劑。該標籤較佳爲螢光性標籤 本發明亦關於其中該第一、第二、或第三基本上爲序 列專一性的藥劑中之至少一者被固定的方法，其中該固定 化之藥劑不是作爲該切割劑之基本上爲序列專一性的藥劑 進一步包括於本發明範疇中的方法爲其中該第一基本 上爲序列專一性的藥劑係切割劑。 本發明範疇亦包括其中該第二基本上爲序列專一性的 藥劑被固定的方法。於一較佳具體例中，該切割劑可爲糖 苷酶或糖基轉移酶，該第二基本上爲序列專一性的藥劑係 外源凝集素，而該第三基本上爲序列專一性的藥劑係抗體 於一較佳具體例中，該第三基本上爲序列專一性的藥 劑係外源凝集素。進一步包括於本發明範疇中的方法爲其 中該第三基本上爲序列專一性的藥劑係切割劑的方法。 本發明亦關於上述方法，其中進一步包括推衍出糖類 序列之步驟。 本發明進一步關於其中該第三基本上爲序列專一性的 藥劑係糖苷酶或糖基轉移酶的方法。 於本發明上述方法之較佳具體例中，若干個該第一基 本上爲序列專一性的藥劑係固定於相同的基材上。較佳， 所有的第一基本上爲序列專一性的藥劑皆固定於單一基材 上。 14 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） •-------—--------訂--------- (請先閱讀背面之法意事項再填寫本頁) 1306152 A7 B7 95. ii 16 條正 年月曰n } m .tl 五、發明說明（丨λ) 於本發明方法中使用之第一基本上爲序列專一性的藥 劑較佳選自外源凝集素及抗體。該第二或第三基本上爲序 列專一性的藥劑較佳爲將經標示的單糖單元引導至該糖類 上之糖基轉移酶。 於該方法之另一較佳具體例中係使用一個以上之標籤 ，且使用之各標籤可被獨立地檢測。 本發明亦關於上述方法，其中該第二及第三基本上爲 序列專一性的藥劑同時存於反應中，但其一者活化之後， 藉由改變緩衝液條件才使另一者活化，因此該等基本上爲 序列專一性的藥劑中之一者因該改變而不活化，同時使另 一個基本上爲序列專一性的藥劑被活化。 又較佳爲，可同時添加數個第三基本上爲序列專一性 的藥劑’但其一者活化之後，藉由改變緩衝液條件才使另 一者活化’因此該等基本上爲序列專一性的藥劑中之一者 或更多者因該改變而不活化，同時使另一個基本上爲序列 專一性的藥劑被活化。較佳一個或更多個該等第三基本上 爲序列專一性的藥劑係糖苷酶或糖基轉移酶。 本發明亦關於上述方法中每個該第一或第二基本上爲 序列專一性的藥劑係固定於僞陣列(virtual array)之獨立單 元上的方法。該陣列較佳爲MASDA陣列。 此外’本發明係關於一種藉由使用糖苷酶或其等效物 依序分解而分析糖類結構的方法。於一較佳具體例中，該 藉由依序分解而分析糖類結構的方法包括下列步驟： a)遮蔽該糖類之還原端； 15 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） (請先閱讀背面之注意事項再填寫本頁)5. Description of the Invention (1306152) may be carried out after removal of unbound sugars, but prior to the addition of a substantially sequence-specific coloring bonding agent. In a particularly preferred embodiment of the method of the invention, the surface is Filter paper, and the substantially sequence specific drug is arranged on the paper in a pre-defined order. In another aspect, the invention provides a sugar molecular identification (GMID) card, listing the method according to the invention The data of the saccharide structure analysis obtained by the above preferred embodiment is rapidly described. In a preferred embodiment, the substantially sequence-specific reagents are indicated by the code on the GMID card. In a preferred embodiment, the combination of agents that are substantially sequence specific for use in the analysis is represented by a unique designation. The invention also relates to a solid support comprising a predetermined sequence representing a sequence or fragment of a carbohydrate or carbohydrate. A plurality of visually or detectable labels. On the other hand, the invention also includes selecting a set of substantially color-specific bond-forming bonds that can be used in the above methods. The method comprises the steps of: a) obtaining all or part of the monosaccharide composition (MC) of the saccharide to be analyzed; b) selecting a group comprising η substantially monosaccharides which can be bonded to the saccharide contained in the saccharide Marking for sequence specificity and/or site specificity; c) correcting the set of markers obtained in step b) to be essentially sequence specificity and/or site specificity to ensure that there are no two markers in the group Have the same color or detectable mark; d) Correct the group selected in step c) is basically sequence specific and / or 11 paper scale applicable to China National Standard (CNS) A4 regulations (210 X 297 PCT) (Please read the notes on the back and fill out this page) ▼装--------订·----丨丨1306152 \7 B7 95. 11 IB j'E Year E-丨Two t complements five, invention descriptions (1) site specificity markers to reduce the set of markers and these substantially sequence specificity and / or site specificity of the bonding agent or other substantially sequence Interaction between specificity and/or site specificity. In a preferred embodiment of the method, the MC of the saccharide to be analyzed is presumed by the MC of the relevant saccharide. In another preferred embodiment, the MC of the saccharide to be analyzed is obtained by performing a complete MC analysis of the saccharide. In another preferred embodiment of the method, η値 is between 1 and 4. The invention also relates to a set of substantially sequence-specifically labeled software for use in the above-described saccharide structure analysis method, the software comprising a) input for providing a monosaccharide composition (MC): b) pairing times a program for matching n substantially serial-specific or site-specific markers that can be bound to the monosaccharide contained in the carbohydrate with an ic) correction subroutine for correcting the subprogram b) A set of markers that are substantially sequence specific or site specific, and which is capable of reducing the labeling of the labeling with the substantially sequence specificity or site specificity or other substantially sequence specificity Selecting the marker based on the interaction between the markers of sex or site specificity; d) revising the secondary program to ensure that no two markers in the group have the same color or detectable features. The invention also relates to a method for obtaining structurally related information of a saccharide comprising the steps of: a) applying the saccharide to a substantially sequence specificity capable of cleavage of the oligosaccharide; 12 paper scales applicable to the Chinese National Standard (CNS) A4 specification ( 210 χ 297 公公) ----------- (Please read the phonetic transcription on the back? Then fill out this page) I — — — — — — — — 1306152 Α7 Β7 V. Invention description (ΰ) Pharmacy reaction; (please read the note on the back and then fill out this page) b) react the saccharide with a substantially sequence-specific agent capable of binding to the saccharide; 0 introduce the label directly or indirectly into the saccharide The inclusion of a labeled substantially sequence specific agent capable of binding to the saccharide, and d) detecting the label in the reaction containing the cleavage agent and in the reaction containing no cleavage agent. The invention further relates to a method of structural analysis of a saccharide comprising the steps of: a) providing a saccharide; b) reacting the saccharide with a first substantially sequence specific agent; c) rendering the saccharide or the saccharide thereof The fragment is reacted with a second substantially sequence-specific agent; d) reacting the saccharide or fragment thereof with a third substantially sequence-specific agent; e) using at least one different second or third base as needed Repeat steps c to d for the sequence specificity agent; the Ministry of Economic Affairs Intellectual Property Office employee consumption cooperative prints it, which can use the same sugar in parallel and independent reactions' and at least one different first, second, Or the third substantially sequence-specific agent is subjected to steps a) to e), provided that the saccharide is labeled and/or one or more of the first, second, or third substantially sequence specific The medicinal agent is labeled, or a label is introduced into the saccharide' and the label is detected at one or more steps after the step of introducing the label, and the other condition is A first, second and / or third basically _; _ 13 paper size for the Chinese national g (CNS) A4 size ϋΐ〇Χ 297 mm) 1306152 I 95.11 1 β / Λ Years of the month Α 7 Β 7 5 Description of the Invention (The sequence specificity agent is a cleavage agent. The label is preferably a fluorescent label. The invention also relates to at least one of the first, second, or third substantially sequence specific agents. A method of immobilization wherein the immobilized agent is not substantially sequence specific as the cleavage agent. Further included in the scope of the present invention is the first substantially sequence specific drug system cutting The invention also includes a method wherein the second substantially sequence specific agent is immobilized. In a preferred embodiment, the cleavage agent can be a glycosidase or a glycosyltransferase, the second substantially a sequence-specific drug-specific lectin, and the third substantially sequence-specific antibody antibody is in a preferred embodiment, the third substantially sequence-specific drug-specific lectin A method further included in the scope of the present invention is a method of the third substantially sequence-specific drug-based cleavage agent. The present invention also relates to the above method, further comprising the step of deriving a saccharide sequence. The method of the third substantially sequence-specific drug glycosidase or glycosyltransferase. In a preferred embodiment of the above method of the present invention, a plurality of the first substantially sequence-specific drug system are immobilized Preferably, all of the first substantially sequence-specific agents are immobilized on a single substrate. 14 This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm). •-------—--------Book--------- (Please read the back of the law and then fill out this page) 1306152 A7 B7 95. ii 16 positive Year 曰n } m.tl V. Description of the Invention (丨λ) The first substantially sequence-specific agent used in the method of the present invention is preferably selected from the group consisting of a lectin and an antibody. Preferably, the second or third substantially sequence specific agent is a glycosyltransferase that directs the indicated monosaccharide unit to the saccharide. In another preferred embodiment of the method, more than one label is used and each label used can be independently detected. The invention also relates to the above method, wherein the second and third substantially sequence-specific agents are simultaneously present in the reaction, but after activation, the other is activated by changing the buffer conditions, thus One of the agents that are substantially sequence specific is not activated by the change, while another agent that is substantially sequence specific is activated. Further preferably, a plurality of third substantially sequence-specific agents can be added simultaneously. 'But after one of them is activated, the other is activated by changing the buffer conditions. Thus, these are substantially sequence specific. One or more of the agents are not activated by the change, while another agent that is substantially sequence specific is activated. Preferably, one or more of said third substantially sequence-specific drug glycosidases or glycosyltransferases. The invention also relates to a method of immobilizing each of the first or second substantially sequence specific agents in a separate array of virtual arrays in the above method. The array is preferably a MASDA array. Further, the present invention relates to a method for analyzing a saccharide structure by sequentially decomposing using a glycosidase or its equivalent. In a preferred embodiment, the method for analyzing the structure of a saccharide by sequential decomposition comprises the steps of: a) masking the reducing end of the saccharide; 15 the paper size is applicable to the Chinese National Standard (CNS) A4 specification (210 X 297 gong) PCT) (Please read the notes on the back and fill out this page)

1306152 A7 B7 五、發明說明（0) b) 藉由以糖苷酶或其等效物與糖反應，而曝露出另一 個還原端； c) 標不該另一個速原端； d) 視需要使用不同的糖苷酶或其等效物重複步驟a至c 〇 再者，本發明係關於一種如更前文所述之分析糖類結 構的方法，其中係依上述用以獲得糖類之結構相關資料的 方法蒐集資料，再將該等資料整合，由而衍生出該糖類之 結構訊息。 本發明亦關於使用依上文用以獲得糖類之結構相關資 料的方法獲得之資料構築糖類之序列圖譜的方法，該方法 包括下列步驟： a) 使用上述方法蒐集辨識序列三元組(triplet)， b) 鑑定第一型三元組，該三元組爲序列(第一辨識位點 )'(糖苷酶)-(第二辨識位點)之三元組， c) 鑑定第二型三元組，該三元組爲序列(糖苷酶)-(第一 辨識位點)-(第二辨識位點)之三元組， d) 依類似處對該等三元組加以分類， e) 以不同的糖苷酶辨識位點比較三元組， f) 依在糖上之發生順序排列該等三元組， g) 排列該等糖苷酶辨識位點， h) 檢查該等三元組之相容性， i) 以單一縱列順序排列糖苷酶以及第一及第二基本上 舄序列專一性的藥劑之辨識序列，及 16 ---I----I I ill —---訂---1----- (請先閱讀背面之注意事項再填寫本頁) 尺度適用中國國家標準（CNS)A4規格（210 X 297公f ) 1306152 A7 B7 五、發明説明（1气） j) 將該等辨識序列(位點)轉譯成多糖序列。 此外，本發明關於一種構築糖類之序列圖譜的方法， 該方法進一步包括下列步驟： k) 修正重疊之問題， l) 輸出序列， m) 檢查所有可得之資料， 由而構築實際的糖類序列模型。 本發明亦關於上述構築糖類之序列圖譜的方法’其中 步驟m)包括檢查依上述方法獲得之其他資料，以獲得糖類 之結構相關性資料，由而進一步構築糖類之序列圖譜。 本發明亦關於糖類結構之分析設備，該設備提供呈平 面結構之第一基本上爲序列專一性的藥劑之陣列，使得各 個第一基本上爲序列專一性的藥劑座落於該平面結構之特 定區域，並進一步提供使待分析物與該陣列反應之裝置、 洗滌裝置、使一個或多個第二及第三基本上爲序列專一性 的藥劑與該陣列反應之裝置、及檢測與該糖類或該第二或 第三基本上爲序列專一性的藥劑連結之標籤的檢測裝置。 另一方面，本發明提供一種糖類結構的分析設備，該 設備具有許多等分量的珠粒(aliquots of beads)，每等分量皆 帶有不同的第一基本上爲序列專一性的藥劑，進一步設置 使待分析物與該等等分量的珠粒分別反應之裝置、洗滌裝 置、使一個或多個第二及第三基本上爲序列專一性的藥劑 與該等等分量的珠粒反應之裝置、及檢測與該糖類或該第 二或第三基本上爲序列專一性的藥劑連結之標籤的檢測裝 本紙張尺度通用中國國家標準（CNS ) A4規格（210X297公釐） ---------Ipt.! f請先閲讀背面之注意事項再填{馬本頁) 、1T_1306152 A7 B7 V. INSTRUCTIONS (0) b) Exposing another reducing end by reacting a glycosidase or its equivalent with a sugar; c) marking the other end of the velocity; d) using as needed Repeating steps a to c for different glycosidases or their equivalents, the present invention relates to a method for analyzing the structure of a saccharide as described above, wherein the method is as follows for obtaining structural data relating to saccharides The information is then integrated into the structure to derive the structural information of the sugar. The present invention also relates to a method of constructing a sequence map of a saccharide using data obtained by the method for obtaining structural data relating to saccharides, the method comprising the steps of: a) collecting an identification sequence triplet using the above method, b) identifying a first type of triplet, which is a triplet of the sequence (first recognition site) '(glycosidase)-(second recognition site), c) identifying a second type triplet , the triad is a triplet of a sequence (glycosidase)-(first recognition site)-(second recognition site), d) is classified according to the similarity of the triplet, e) is different The glycosidase recognition sites are compared to the triads, f) the triads are arranged in the order in which they occur on the sugar, g) the glycosidase recognition sites are arranged, h) the compatibility of the triads is checked i) identifying the sequence of the glycosidase and the first and second substantially 舄 sequence specificity in a single column, and 16 ---I----II ill —------1 ----- (Please read the note on the back and then fill out this page) Scale applies to China National Standard (CNS) A4 specification (210 X 297 public f) 1306152 A7 B7 Five DESCRIPTION invention (gas 1) J) Identification of the other sequence (site) sequences translated polysaccharides. Furthermore, the present invention relates to a method of constructing a sequence map of a saccharide, the method further comprising the steps of: k) correcting the problem of overlap, l) outputting the sequence, m) examining all available data, thereby constructing a practical saccharide sequence model . The present invention also relates to the above method for constructing a sequence map of a saccharide, wherein step m) comprises examining other materials obtained by the above method to obtain structural correlation data of the saccharide, thereby further constructing a sequence map of the saccharide. The invention also relates to an analysis device for a saccharide structure, the device providing an array of first substantially sequence-specific agents in a planar configuration such that each of the first substantially sequence-specific agents is localized to the planar structure a region, and further providing means for reacting the analyte with the array, a washing device, means for reacting one or more second and third substantially sequence specific agents with the array, and detecting the sugar or The second or third substantially substantially sequence-specific drug-linked label detecting device. In another aspect, the present invention provides an analysis apparatus for a saccharide structure having a plurality of aliquots of beads, each having a different first substantially sequence-specific agent, further set a device for reacting an analyte with the equal amount of beads, a washing device, a device for reacting one or more second and third substantially sequence-specific agents with the equal amount of beads, And detecting the label of the label linked to the saccharide or the second or third substantially sequence-specific agent. The paper size is generally Chinese National Standard (CNS) A4 specification (210X297 mm) ------- --Ipt.! f Please read the notes on the back and fill in {{this page), 1T_

La 經濟部智慧財產局員工消費合作社印製·La Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing ·

1306152 五、發明說明（丨7) 置。 本發明方法可用於硏究寡糖或多糖結構。彼等方法於 當此等寡糖或多糖與其他分子如胜肽、蛋白質或脂質偶合 時亦可適用。明確言之，本發明方法可用於葡糖胺聚糖 (GAGs)包含肝素、硫酸乙醯肝素、硫酸軟骨素、硫酸皮膚 素等之結構硏究。 本發明之所有上述及其他特徵及優點，將由其較佳具 體例之下述詳細且未予限制之實例進一步說明。 圖式簡沭 由較佳具體例之詳細說明及所附圖式將更清楚瞭解本 發明，其中： 圖1說明由適用於低溫滅菌羊乳(A及B)、未經滅菌之 羊乳(C及D)以及牛乳(E)的糖分子鑑別(GMID)卡。 圖2爲適用於各種脂多糖樣本之GMID卡複製品。卡 A至E分別對應於LPS#1、7、10、15及16。 圖3爲高級邏輯流程圖，其說明選擇一組呈色外源凝. 集素之演算法。 發明之詳細說明 下文說明本文所用之某些詞彙，以達闡明之目的。 “基本上爲序列專一性的藥劑”意指可鍵結於糖類之藥 劑，且該鍵結通常爲序列專一性，亦即，該藥劑將僅鍵結 於單糖單元之特定序列。不過，此序列專一性並非絕對， 因該藥劑可鍵結於其他相關序列(如其中一個或更多個糖缺 失、被置換或***之單糖序列）。該藥劑亦可鍵結於指定之 18 (請先閱讀背面之注意事項再填寫本頁) 訂--- s'. 本纸張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 1306152 ，气Η,.修正A7 牛月__ 五、發明說明（丨上） 單糖序列以外之一個或更多個不相關之序列或單糖。該基 本上爲序列專一性的藥劑通常爲蛋白質’如外源凝集素、 糖專一性抗體或糖苷酶或糖基轉移酶。外源凝集素之實例 包含自下列植物分離之外源凝集素：1306152 V. Description of invention (丨7). The method of the invention can be used to investigate oligosaccharide or polysaccharide structures. These methods are also applicable when these oligosaccharides or polysaccharides are coupled with other molecules such as peptides, proteins or lipids. Specifically, the method of the present invention can be applied to structural studies of glycosaminoglycans (GAGs) including heparin, heparin sulfate, chondroitin sulfate, dermatan sulfate, and the like. All of the above and other features and advantages of the present invention will be further clarified by the following detailed and non-limiting examples of preferred embodiments thereof. BRIEF DESCRIPTION OF THE DRAWINGS The invention will be more clearly understood from the detailed description of the preferred embodiments and the accompanying drawings, wherein: Figure 1 illustrates the un-sterilized goat milk (C and C) suitable for low temperature sterilization of goat milk (A and B) And D) and the milk molecular identification (GMID) card for milk (E). Figure 2 shows a GMID card replica suitable for use in various lipopolysaccharide samples. Cards A to E correspond to LPS #1, 7, 10, 15, and 16, respectively. Figure 3 is a high level logic flow diagram illustrating the algorithm for selecting a set of colored exogenous coagulation elements. DETAILED DESCRIPTION OF THE INVENTION Certain words used herein are set forth below for the purpose of illustration. "Substantially sequence-specific agent" means a drug that can be bonded to a saccharide, and the linkage is typically sequence specific, i.e., the agent will only bind to a particular sequence of monosaccharide units. However, this sequence specificity is not absolute, as the agent may be bonded to other related sequences (e.g., one or more sugar sequences that are missing, replaced or inserted). The medicine can also be keyed to the designated 18 (please read the note on the back and fill out this page). Order--- s'. This paper size applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm). 1306152, gas Η,. Amendment A7 牛月__ V. Description of the invention (丨) One or more unrelated sequences or monosaccharides other than the monosaccharide sequence. The agent which is substantially sequence specific is usually a protein such as a lectin, a glycospecific antibody or a glycosidase or a glycosyltransferase. Examples of exogenous lectins include lectins from the following plants:

Cona valia ensiformis Anguilla anguilla ，\、麥（Tiidcum vulgaris) M ^ M {Datura stramoniuim)Cona valia ensiformis Anguilla anguilla , \, Mai (Tiidcum vulgaris) M ^ M {Datura stramoniuim)

Gal an th us ni valis) 高麗槐(Maacir/a 落花生XArachis hypogaea) 西洋接骨木(Α/ώ加CM 侮紅豆（Erythrina cristagalli) β： M. (Lens culinaris) S (Glycine max) 菜豆[Phaseoius vulgaris)Gal an th us ni valis) Maacir/a Xarachis hypogaea Western elders (Erythrina cristagalli) β: M. (Lens culinaris) S (Glycine max) Bean [Phaseoius vulgaris)

Allomyrina dichotoma Dolichos biflorus 百脈根屬之從·ασσ/σ厶即 Ulex europaeus M.M{Ricinus communis) 除了外源凝集素之前述實例以外，其他生物活性化合 物如細胞素、化學增活素及生長因子亦具有黏結於GAGs 及多糖類之活性，因此考慮以外源凝集素達到本發明目的 19 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐) 一' -- I 慧 (請先閱讀背面之注意事項再填寫本頁) • at ί I n n-f n « — —— — — I — 4 1306152 qs. ϋ. 16年月El補充 A7 B7 五 發明說明（/；? 糖苷酶之實例包含半乳糖苷酶、半乳糖苷酶、 Ν-乙醯基胺基己糖苷酶、甘露糖苷酶、甘露糖苷酶 、α-墨角藻聚糖苦酶(Fucosidase)等。此等酵素中’有某些 酵素可能依其分離來源而具有不同的專一性。 上述酵素爲商業上可獲得者’如購自奧斯佛糖系統 (Oxford Glycosystems)股份有限公司[艾明頓（Abingdon) ’ 〇X14 1RG，英國]、西格瑪（处脱)化學公司[聖路易斯’ Mo.，美國]、或是派爾斯（Pierce)[POB· 117 ’洛克弗 (Rockford)，61105 美國]。 “切割劑”爲一種於其辨識序列處切割糖鏈之基本上爲 序列專一性的藥劑。典型之切割劑爲糖苷酶’包含外糖苷 酶及內糖苷酶，及糖基轉移酶。不過’可以切割糖苷鍵之 化學試劑亦可作爲切割劑’只要其基本上爲序列專一性者 即可。本說明書全文中之”切割劑(cleaving agent或cleavage agent)”一詞與”能進行切割之基本上爲序列專一性的藥劑” 具有相同意義。 “辨識序列”爲被基本上爲序列專一性的藥劑辨識之單 糖序列。辨識序列通常包括2-4個單糖單元。辨識序列之 一實例爲Gal/3 1-3 GalNac ’其被自純化得 之外源凝集素所辨識。當單糖被基本上爲序列專一性的藥 劑辨識時，爲達本文目的’該等單糖可以定義爲辨識序列 ‘糖”爲任一種直鏈或支鏈之寡糖或多糖。用於本文中 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） ------------------—-訂--------- (請先閱讀背面之注意事項再填寫本頁) 1306152 A7 B7 五、發明説明) 之此一詞彙有別於此技藝中之大體意義，於技藝中’該詞 亦包括多糖類及聚糖之糖結構等。 “繪製圖譜(mapping)”係指定義多糖鏈上特定之經預先 限定的圖樣之順序’爲一種導致最終獲得糖類序列的方法 ，亦即，完成糖之所有構成要素之決定。 “序列圖譜，，係辨識位點於糖類上出現之先後順序。 “單糖”爲一種單一糖單元，例如己糖、丁糖或戊糖。 單糖之明確實例包含半乳糖（Gal)、N-乙醯基-半乳糖胺 (GalNAc)、甘露糖(Man)、葡萄糖(Glc)等。 於本發明較佳具體例中，前文所述以及下文說明之方 法可用於藉由測定治療活性藥劑或其活性片段之結構’而 篩選治療劑。 於本發明之更佳具體例中，本發明分析及繪製圖譜之 方法對於使治療活性藥劑發揮其最大效用上更爲有效’因 其可以評估各種治療活性藥劑之糖苷化程度，並對彼等藥 劑作比較。例如，技藝中已知，於聚糖鏈非還原端處之半 乳糖可能與來自循環系統之糖蛋白鏈之完整部分的快速去 除有關。當使用糖蛋白作爲治療活性藥劑時，該屬性可以 戲劇性地影響與此等糖蛋白有關之藥物動力學參數。本發 明因此亦有關於上述方法在治療活性藥劑之發展及使其最 佳化上之用途。 於另一較佳具體例中，本發明係關於本文所述方法於 疾病診斷上之用途。本發明分析方法之診斷用途之一實例 爲比較及/或鑑定自細菌分離之脂多糖(LPSs)，以確定微生 21 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） t---------»^— 〈請先閲讀背面之注意事項再填寫本頁)Allomyrina dichotoma Dolichos biflorus from the genus ασσ/σ厶, ie Ulex europaeus MM{Ricinus communis) In addition to the aforementioned examples of lectins, other biologically active compounds such as cytokines, chemolysins and growth factors also have Adhesive to the activity of GAGs and polysaccharides, so consider exogenous lectin to achieve the purpose of the invention. 19 This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) A ' -- I Hui (please read the back first) Note: Please fill in this page) • at ί I n nf n « — —— — — I — 4 1306152 qs. 16. 16 years El Supplement A7 B7 Five Inventions (/;? Examples of glycosidases contain galactose Glycosidases, galactosidase, Ν-ethionyl hexosaminidase, mannosidase, mannosidase, α-fucosidase, etc. There are certain enzymes in these enzymes. It may have different specificities depending on its source of separation. The above enzymes are commercially available 'as purchased from Oxford Glycosystems, Inc. [Abingdon' 〇X14 1RG, UK] , Sigma (Exit) Chemical Company [St. Louis 'Mo., USA], or Pierce [POB· 117 'Rockford, 61105 US]. "Cleavage" is a recognition sequence The cleavage of a sugar chain is essentially a sequence-specific agent. A typical cleavage agent is a glycosidase comprising an exoglucosidase and an endoglycosidase, and a glycosyltransferase. However, a chemical that can cleave a glycosidic bond can also be used as a cleavage. The agent 'as long as it is substantially sequence specific. The term "cleaving agent or cleavage agent" throughout the specification has the same meaning as "the agent which is capable of undergoing cleavage and is substantially sequence specific" An "identification sequence" is a sequence of monosaccharides recognized by a substantially sequence-specific agent. The recognition sequence typically includes 2-4 monosaccharide units. An example of an identification sequence is Gal/3 1-3 GalNac 'which is Purified by a foreign lectin. When a monosaccharide is identified by a substantially sequence-specific agent, for the purposes of this document, 'the monosaccharide can be defined as the recognition sequence 'sugar' as either linear or Chain of oligosaccharides or polysaccharides. For the purposes of this paper, the Chinese National Standard (CNS) A4 specification (210 X 297 mm) is used. ------------------ Order --------- (Please read the note on the back and then fill out this page) 1306152 A7 B7 V. Invention Description) This vocabulary is different from the general meaning of this technique. The term also includes the sugar structure of polysaccharides and glycans. "Mapping" refers to the order in which a particular pre-defined pattern on a polysaccharide chain is defined' as a means of ultimately obtaining a carbohydrate sequence, i.e., the determination of all constituent elements of the sugar. "Sequence map, the order in which the recognition sites appear on sugars. "Single sugar" is a single sugar unit, such as hexose, butyrate or pentose. A clear example of a monosaccharide contains galactose (Gal), N -Ethyl galactosamine (GalNAc), mannose (Man), glucose (Glc), etc. In a preferred embodiment of the invention, the methods described above and described below can be used to determine therapeutically active agents or The therapeutic agent is screened for the structure of the active fragment. In a more preferred embodiment of the invention, the method of analyzing and mapping the present invention is more effective for maximizing the utility of the therapeutically active agent 'because it can evaluate various therapeutic activities The degree of glycosidation of the agents is compared to their agents. For example, it is known in the art that galactose at the non-reducing end of the glycan chain may be associated with rapid removal of the intact portion of the glycoprotein chain from the circulatory system. When glycoprotein is used as a therapeutically active agent, this property can dramatically affect the pharmacokinetic parameters associated with such glycoproteins. The present invention therefore also relates to the above methods in the treatment Development of a medicinal agent and its use for optimization. In another preferred embodiment, the invention relates to the use of the method described herein for the diagnosis of a disease. An example of the diagnostic use of the analytical method of the invention is a comparison. And/or identification of lipopolysaccharides (LPSs) isolated from bacteria to determine the microbiological 21 paper size applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) t---------»^- Please read the notes on the back and fill out this page.)

*1T 經濟部智慧財產局員工消費合作社印製 1306152 A7 B7 五、發明説明（j) 物病原體之一致性。下文實例6說明使用本發明方法對不 同微生物之LPS樣本進行之比較。 於一更佳具體例中，本發明係關於本發明方法於食品 及/或飮料分析上之用途。此分析可包含使用GMID方法， 使食品或飮料樣本與已知之標準物作比較，以確定其物種 來源。例如，下文實例5說明不同來源之乳品樣本的 GIMD分析。另一實例係檢測及鑑定食品及飮料製品中之 細菌污染物，如於下文實例6中說明之LPS分析。 於一更佳具體例中，本發明提供上述方法於經遺傳改 質(GM)之農作物及由其衍生之產物的分析上之用途。GM 作物之實例包含彼等產生人體化抗體(humanized antibody 該抗體爲糖蛋白）的作物，及產生改質之澱粉或其他多糖類 之作物。 本發明提供一種分析糖鏈的方法。本發明使用生物反 應機制，例如彼等涉及能夠辨識短鏈寡糖序列之藥劑及酵 素的機制。相對於先前技藝之方法，本發明使用多數個反 應（亦即，訊息充裕之分析）。這使得本發明方法可以完全 避免使用先前技藝中所用之昂貴的技術，如以源後衰變陣 列輔助之鐳射解吸附/離子化質譜儀（post source decay matrix-assisted laser desorbtion/ionization mass spectrometry ； PSD MALDI-MS)、HPLC-MS、或結合快速原子撞擊之質譜 儀。 依據本發明之較佳具體例，在糖類(於還原端經標示者 )與第一基本上爲序列專一性的藥劑反應後，進行檢測步驟 22 (請先閲讀背面之注意事項再填寫本頁) >裝· 訂 經濟部智慧財產局員工消費合作社印製 $紙張尺度it财關家辟（CNS ) A4驗(110X297公釐) — 1306152 A7 B7 五、發明説明（>G) 。標籤之存在表示在該糖類中存有該第一基本上爲序列專 一性的藥劑之辨識位點<2。極應注意的是此步驟提供使用 者有關於該糖類中存有何種外源凝集素鍵結位點之相關訊 息。待與可切割糖類之辨識序列纟的第二基本上爲序列專 一性的藥劑反應後’重複該檢測步驟。無標籤則表示該第 一及第二辨識位點之順序爲心心還原端。 爲了進一步說明本發明方法，吾等兹假設該第一基本 上爲序列專一性的藥劑爲一種帶有辨識位點a之凝集素。 該欲分析之糖類係未經標示者。於第二步驟，添加可辨識 特定糖類序列纟之經標籤的抗體。接著進行檢測步驟，以 顯示是否該抗體辨識該糖類。於此情形下，撇開所用之外 源凝集素不談，所有反應皆爲正反應。待洗滌去除未鍵結 之抗體後，添加糖苷酶。該糖苷酶具有辨識位點C。進行 第二檢測步驟。在所有反應中之訊號皆消失時，該等辨識 位點之順序必爲或是3^4。另一方面，當訊號存在 時，辨識位點之順序可能是或03。 於本發明上述具體例中，彼等位點與還原端間之相關 性並未確立。不過，由其中該糖類經標示之上述第一個具 體例與方才所述之其中該第二基本上爲序列專一性的藥劑 經標示的具體例之組合，將容易地提供訊息。因此，於本 發明之進一步具體例中，標示該糖類之還原端。接著於第 一步驟，使之與各種外源凝集素鍵結。第二步驟，鍵結業 經標示之抗體。該等外源凝集素及抗體則以可以獨立地被 檢測出之不同標籤予以標示。因此’該二種標籤茲可於第 23 **~ _________. -· ~ - 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閲讀背面之注意事項再填寫本頁) ▼裝· 如 經濟部智慧財產局員工消費合作社印製 1306152 Α7 Β7 五、發明説明（yl ) 一及第二檢測步驟各自獨立地被檢測出來。第三步驟，添 加糖苦酶。接著進行第三及第四檢測步驟，以證實該二種 標籤存於各反應中。若該二種標籤於添加糖苷酶之前即存 在，則有若干可能性。第一，若該二種標籤於添加糖苷酶 之後仍然存在，則該辨識位點之順序爲^3-纟-還原端。第 二，若該二種標籤於添加糖苷酶之後皆喪失，該辨識位點 之順序必爲還原端。第三，若該糖標籤存在，而該 抗體標籤喪失，則該辨識位點之順序必爲還原端。 第四，若該抗體標籤仍存在，而該糖標籤喪失，則該辨識 位點之順序可能爲士纟-C-還原端或還原端。 可進行一組類似的反應，其中，先以切割劑分解糖類 ，再於其後續步驟與鍵結劑反應。 例如，於一較佳具體例中，使還原端經標示之糖類與 第一基本上爲序列專一性的藥劑(其可爲帶有辨識序列<3之 糖苷酶)反應。於對照組之反應，不對該經標示之糖進行處 理。接著使彼等反應進一步獨立地與固定化之第二基本上 爲序列專一性的藥劑(其可爲帶有辨識序列纟之外源凝集素 )反應。待洗滌未鍵結之糖類後，進行檢測步驟。標籤之存 在表示於該糖類中存有位點纟。藉由使其中存有該第一基 本上爲序列專一性之藥劑的反應與其中不含第一基本上爲 序列專一性之藥劑的獨立對照組之反應進行比較，可確定 糖苷酶對於標籤之存在的影響。例如，若與帶有辨識序列 6之外源凝集素鍵結以後在對照組反應測得標籤，而於其 中該第一基本上爲序列專一性的藥劑係帶有辨識序列β之 24 本紙張尺度適用中國國家標準（CNS > Α4規格（210X297公釐） (請先閲讀背面之注意事項再填寫本頁) 、1Τ L# 經濟部智慧財產局員工消費合作社印製 1306152 A7 經濟部智慧財產局員工消費合作杜印製 五、發明説明（sx) 糖苷酶的反應測不到該標籤時，該辨識位點之順序爲办-心 還原端。另一方面，若於對照組及糖苷酶之反應皆含有標 籤，表示該辨識位點之順序爲心纟-還原端。該辨識位點s 可能不會座落於該糖類內部，亦即，可能不會存於該糖類 序列中。 本發明上述具體例可與多種第一基本上爲序列專一性 的藥劑倂用。彼等藥劑通常係於各自獨立的反應中使用’ 但彼等反應共同具有一個對照組反應。亦可於一個反應中 使用一個以上之第一基本上爲序列專一性的藥劑。由此等 多樣化反應獲得大量的結構相關性訊息。 於本發明之另一具體例使用未經標示的糖。在以帶有 辨識序列2之糖苷酶分解後，使該糖類與固定化之外源凝 集素反應。洗滌去除未鍵結之糖類，接著以帶有辨識位點 c之經標示的抗體與該已鍵結之糖類片段反應。洗滌去除 未鍵結之抗體之後檢出該標籤，表示c係位於與該外源凝 集素鍵結之糖類片段上。該鍵結位點之序列可能是e-纟^或 /^74。該還原端與該辨識位點序列之相關位置不能由此反 應測出。若於對照組（不含糖苷酶)之反應檢測出該標籤， 而於含有糖苷酶之反應則否時，表示該辨識位點之序列爲 办-CM。於此具體例中，若亦進一步單獨使用不同的糖苷酶 、及/或與各種外源凝集素、及/或不同的抗體組合，將增 加所得訊息的量。 顯然，更前文所述之本發明具體例(其中該第三基本上 爲序列專一性的藥劑提供切割步驟)實質上相當於恰於前段 25 ^^尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閲讀背面之注意事項再填寫本頁) 琴 、1T- 加 1306152 \7 37 五、發明說明（Θ) 說明之具體例(其中該第一基本上爲序列專一性的藥劑提供 切割步驟）。本發明該二具體例間之差異在於：在更前文所 述之具體例中，可藉由在與該第三基本上爲序列專一性的 藥劑反應之前檢測標籤之存在觀察切割劑的影響，而於恰 於前段說明之具體例中，必需使用不含切割劑之對照組反 應，以測定該切割試劑之影響。雖然如此，於不同具體例 獲得之訊息範圍實質上相當，同時，用以處理該訊息及得 自該訊息之辨識位點序列(即，三元組)的方法亦相當。 上述實例係以該糖類呈線性，且該糖苷酶在該糖序列 中具有辨識位點的假設爲基礎。不過，糖類中之糖苷酶辨 識位點之存在通常將由分析與其他外源凝集素間之反應而 迅速證實。若任一反應中帶有外源凝集素的兩個標籤中的 任一個於添加糖苷酶之後喪失，則該糖苷酶必須在該糖類 中具有辨識位點。 可使用糖基轉移酶作爲基本上爲序列專一性的藥劑。 糖基轉移酶將依特定的序列圖樣(辨識序列），在糖類序列 中之特定點添加糖單元。因此，若使用於反應中之單糖業 經標示，可將指示糖基轉移酶辨識位點之存在的新標籤導 入該糖鏈中。當然，由糖基轉移酶導入之標籤應可與所用 之其他標籤區分。 當如上文般以經標示之抗體進行一組反應時，該抗體 可在多數反應中鍵結，但有極少數例外。此乃由於外源凝 集素與抗體間之糖類辨識序列部分重疊之故。於此情形下 ，與某些外源凝集素間之反應將呈負反應。因此，應使用 26 ------- ί ---------* 丨訂--------. (請先閱讀背面之注意事項再填寫本頁) 本紙張尺度適用中國國家標準（CNS)A4規格（210 * 297公釐） 1306152 A7 B7 五、發明説明（>Mr) 此訊息推衍3-7個糖單元的長度，因爲外源凝集素與抗體 之辨識序列各爲2-4個糖單元。 若上述第一及第二檢測步驟間無牴觸，則可同時進行 該二檢測步驟。 至於進一步實例，若在第一反應順序中，與外源凝集 素之第一反應係發生於末端經標示的多糖鏈上之位點a， 且與糖苷酶之第二反應係發生於多糖鏈上之位點則於 位點c發生之與第三基本上爲序列專一性的藥劑間之反應 導入可與該第一標籤區分的第二標籤。因此，由該二標籤 的存在將顯示該三個位點之順序爲還原端或he-心還 原端。 另一方面，若僅測得第二標籤，則存在兩種可能性： (1)該多糖中缺乏該第三基本上爲序列專一性的藥劑之辨識 位點，或(2)含有辨識位點之該多糖的一部分被該第二基本 上爲序列專一性的藥劑切割。 上述情形可由其中該第二基本上爲序列專一性的藥劑 消失、或該藥劑係在無法進行切割的緩衝條件下使用的第 二反應順序證實。若在第二反應順序中，在與該第三基本 上爲序列專一性的藥劑反應後無法測得該第二標籤，表示 該第三基本上爲序列專一性的藥劑在該糖類上缺乏鍵結位 點。另一方面，第二標籤之存在表示該切割位點位於該第 一與第三基本上爲序列專一性的藥劑的鍵結位點之間，亦 即，該糖類上之位點順序爲還原端。 於本發明方法中，不需進行彼等一個接著一個、後者 27 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閲讀背面之注意事項再填寫本頁) ▼裝· 經濟部智慧財產局員工消費合作社印製 1306152 A7 B7 五、發明説明（V厂） 依存於前者之結果的反應。相反地，本發明提供一種可進 行多種反應的方法，而使得可由單一組反應進行上述推論 。例如，於一組反應中，可使不同種的第一基本上爲序列 專一性的藥劑與在各反應中皆相同之第二及第三基本上爲 序列專一性的藥劑一起使用。第二組反應可同時進行，由 而使該反應中除了該第二基本上爲序列專一性的藥劑消失 或不活化以外，皆與該第一組反應一致。 然而，於中間檢測步驟獲得之訊息可有利地用於在以 下步驟中排除對於基本上爲序列專一性的藥劑的某些選擇 。已知，當使用於待分析之糖類上不具辨識位點的基本上 爲序列專一性的藥劑作爲第二或第三基本上爲序列專一性 的藥劑時，無法提供訊息。因此，於本發明之糖類經標籤 化的具體例中，在該糖鍵結於該第一基本上爲序列專一性 的藥劑之後進行檢測的結果，可用於由已鍵結糖類之第一 基本上爲序列專一性的藥劑之數量上選擇該第二基本上爲 序列專一性的藥劑。 於本發明之另一具體例中，在檢測步驟已完成，且如 上文般獲得訊息後，以不同的藥劑重複該第三基本上爲序 列專一性的藥劑之添加。對於在第一次添加該第三劑後仍 存有該二種標籤的反應而言，應用相同的考量，以解釋上 述結果。不過於僅存留一個標籤的情形下，可以推知該切 割劑是否在該第一基本上爲序列專一性的藥劑之辨識位點 與帶有該標籤之基本上爲序列專一性的藥劑之辨識位點(或 還原端)之間進行切割。在彼種情形下，該剩餘的標籤將在 28 本紙張尺度適用中國國家標準（CNS ) A4規格（2丨〇乂297公釐） (請先閲讀背面之注意事項再填寫本頁) >裝- -訂 經濟部智慧財產局員工消費合作社印製 1306152 A7 ______ B7_____ 五、發明説明（y(j 與不同種第三基本上爲序列專一性的藥劑反應後消失。 於本發明之又一具體例中，可添加不同的第二基本上 (請先聞讀背面之注意事項再填寫本頁) 爲序列專一性的藥劑，該種藥劑可帶有與在第一組反應中 添加之第二基本上爲序列專一性的藥劑不同或相同的標籤 。藉由另一種可切割糖鏈的第三基本上爲序列專一性的藥 劑之添加，將提供進一步的訊息。 於本發明之再一具體例中，添加各自帶有其標籤的二 種或更多種不同之第二基本上爲序列專一性的藥劑，彼等 標籤可獨立於其他使用之任一種標籤地被檢出。於添加切 割劑後喪失之類的標籤，如同上述對於僅使用一種第二基 本上爲序列專一性的藥劑進行實驗的考量般，將提供有關 該第二基本上爲序列專一性的藥劑之鍵結位點位置之訊息 〇 經濟部智慧財產局員工消費合作社印製 熟習技藝之人士應顯見：藉由如上述般進行充足之大 量反應，可獲得專一對應於特定糖類之反應的指紋 (fingeirpdnt)。此外，可能將如上文略述般獲得之部分序歹[J 訊息”摺疊(collapse)”成該糖類之完整序列。由於反應數目 可能非常龐大（如下文詳述），本發明方法不似先前技藝方 法侷限於對寡糖的分析。本方法亦可用於測定多糖(亦即具 有許多糖單元之大型糖類)之結構。 於本發明之一具體例中，該第一基本上爲序列專一性 的藥劑爲外源凝集素。該第一基本上爲序列專一性的藥劑 亦可爲抗體或其他序列專一性的藥劑。 本發明之另一具體例提供多種具有不同序列專一性的 29 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1306152 A7 B7 經濟部智慧財產局員工消費合作社印製 五、發明說明（>/ ) 外源凝集素或糖類專一性抗體，彼等外源凝集素或抗體以 某種陣列固定於基材上，例如類似現今用於形成寡糖陣列 的晶片之超大型積體(VLSI)電路晶片。用以製造此等晶片 及鍵結於該等晶片之試劑的方法說明於例如W0 93/22678 〇 於另一具體例中’本發明提供一種固定化之第一基本 上爲序列專一性的藥劑之僞陣列。此種僞陣列之一實例使 用MASDA粒子，該實例由本文發明者說明於PCT申請案 (IL-97/00105)，該案全文包含於本文作爲參考文獻。該第一 基本上爲序列專一性的藥劑在各自獨立的反應中(如，在25 個不同的反應中)係被固定於MASDA粒子上。接著可使僞 陣列的各部分皆與相同的第二基本上爲序列專一性的藥劑 反應，該反應之較佳進行方式爲自各MASDA反應取出一 份試樣，混合該等試樣，並使該混合物與該第二基本上爲 序列專一性的藥劑反應。必要時，使部分僞陣列與不同種 第二基本上爲序列專一性的藥劑反應，然後可以分別留置 所有的MASDA反應；或者可將一部分僞陣列合倂成混合 物，以與單一種第二基本上爲序列專一性的藥劑反應，同 時分別留置該僞陣列的其他部分，以與不同的第二基本上 爲序列專一性的藥劑反應。同樣地，亦可合倂或分別留置 僞陣列的各部分，以與該第三基本上爲序列專一性的藥劑 反應。 亦可使用珠粒作爲固定劑。技藝中已說明多種用於達 到鍵結胜肽及蛋白質之目的的不同珠粒，參見例如派爾斯 30 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） (請先閱讀背面之注意事項再填寫本頁)*1T Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing 1306152 A7 B7 V. Description of invention (j) Consistency of pathogens. Example 6 below illustrates the comparison of LPS samples of different microorganisms using the method of the present invention. In a more preferred embodiment, the invention relates to the use of the method of the invention for the analysis of food and/or dips. This analysis may involve using a GMID method to compare a food or dip sample to a known standard to determine its source of species. For example, Example 5 below illustrates GIMD analysis of dairy samples from different sources. Another example is the detection and identification of bacterial contaminants in food and tanning products, as described in Example 6 below for LPS analysis. In a more preferred embodiment, the invention provides the use of the above method for the analysis of genetically modified (GM) crops and products derived therefrom. Examples of GM crops include crops that produce humanized antibodies (the glycoproteins), and crops that produce modified starch or other polysaccharides. The present invention provides a method of analyzing a sugar chain. The present invention employs biological reaction mechanisms such as those involved in the identification of agents and enzymes capable of recognizing short-chain oligosaccharide sequences. The present invention uses a majority of responses (i.e., analysis of sufficient information) relative to prior art methods. This allows the method of the present invention to completely avoid the use of expensive techniques used in the prior art, such as post source decay matrix-assisted laser desorbtion/ionization mass spectrometry; PSD MALDI. -MS), HPLC-MS, or mass spectrometer combined with fast atomic impact. According to a preferred embodiment of the present invention, after the saccharide (labeled at the reducing end) is reacted with the first substantially sequence-specific agent, the detecting step 22 is performed (please read the back note and then fill in the page) > Installed by the Ministry of Economic Affairs, Intellectual Property Bureau, Staff Consumer Cooperatives, printed on the paper scale, it is the wealth of the family (CNS) A4 test (110X297 mm) — 1306152 A7 B7 V. Invention description (>G). The presence of the label indicates that the identification site <2 of the first substantially sequence-specific agent is present in the saccharide. It should be noted that this step provides the user with information about which lectin-binding sites are present in the saccharide. The detection step is repeated after reacting with a second substantially sequence-specific agent of the cleavable saccharide recognition sequence 纟. The absence of a label indicates that the order of the first and second identification sites is the cardiac reduction end. To further illustrate the method of the invention, we assume that the first substantially sequence specific agent is a lectin with recognition site a. The sugar to be analyzed is not labeled. In a second step, a tagged antibody that recognizes a particular carbohydrate sequence is added. A detection step is then performed to show if the antibody recognizes the carbohydrate. In this case, regardless of the use of the lectin, all reactions are positive. After washing to remove unbound antibody, a glycosidase is added. This glycosidase has a recognition site C. Perform the second detection step. When the signals in all reactions disappear, the order of the identification sites must be either 3^4. On the other hand, when the signal is present, the order of identifying the sites may be or 03. In the above specific examples of the present invention, the correlation between the sites and the reducing sites was not established. However, it will be readily provided by the combination of the first specific embodiment in which the saccharide is labeled and the specific embodiment of the second essentially sequence specific agent described herein. Thus, in a further embodiment of the invention, the reducing end of the saccharide is indicated. Next, in the first step, it is bonded to various exogenous lectins. In the second step, the labeled antibody is labeled. These lectins and antibodies are labeled with different labels that can be detected independently. Therefore, the two labels can be used in the 23rd **~ _________. -· ~ - This paper size applies to the Chinese National Standard (CNS) A4 specification (210X297 mm) (please read the notes on the back and fill out this page) ▼装· As for the Ministry of Economic Affairs, Intellectual Property Office, Staff Consumer Cooperatives, Printing 1306152 Α7 Β7 V. Invention Description (yl) The first and second detection steps are independently detected. In the third step, a sugar enzyme is added. The third and fourth detection steps are then performed to confirm that the two tags are present in each reaction. If the two tags are present before the addition of the glycosidase, there are several possibilities. First, if the two tags are still present after the addition of the glycosidase, the order of the recognition sites is the ^3-纟-reducing end. Second, if the two tags are lost after the addition of the glycosidase, the order of the recognition sites must be the reducing end. Third, if the sugar tag is present and the antibody tag is lost, the order of the recognition sites must be the reducing end. Fourth, if the antibody tag is still present and the sugar tag is lost, the order of the recognition sites may be the gill-C-reducing or reducing end. A similar set of reactions can be carried out in which the saccharide is first decomposed with a cleavage agent and then reacted with a bonding agent in a subsequent step. For example, in a preferred embodiment, the reducing end labeled saccharide is reacted with a first substantially sequence specific agent (which may be a glycosidase having the recognition sequence <3). The labeled sugar was not treated in the reaction of the control group. The reactions are then further independently reacted with an immobilized second substantially sequence-specific agent (which may be a lectin with a recognition sequence). After the unbonded sugars are to be washed, a detection step is performed. The presence of the label indicates that there is a site in the sugar. The presence of a glycosidase for the tag can be determined by comparing the reaction in which the first substantially sequence specific agent is present and the independent control in which the first substantially sequence specific agent is absent Impact. For example, if a lectin binding with a recognition sequence 6 is followed by a reaction in the control group, the label is determined, and wherein the first substantially sequence-specific drug system carries the identification sequence β of 24 paper scales. Applicable to Chinese national standards (CNS > Α4 specifications (210X297 mm) (please read the notes on the back and fill out this page), 1Τ L# Ministry of Economic Affairs Intellectual Property Bureau employees consumption cooperatives print 1306152 A7 Ministry of Economic Affairs Intellectual Property Bureau employees Consumption cooperation du printing system 5, invention instructions (sx) glycosidase reaction can not detect the label, the order of the identification site is the heart-heart reduction end. On the other hand, if the control and glycosidase reaction The label is included, indicating that the sequence of the recognition site is a palpebral-reducing end. The recognition site s may not be located inside the saccharide, that is, may not exist in the saccharide sequence. It can be used with a variety of first, substantially sequence-specific agents. These agents are usually used in separate reactions. 'But they have a control reaction together. More than one first substantially sequence-specific agent is used. A large number of structural correlation messages are obtained by such diverse reactions. Another embodiment of the invention uses unlabeled sugars. After the glycosidase of sequence 2 is decomposed, the saccharide is reacted with the immobilized lectin. The unbound carbohydrate is washed and removed, followed by the labeled antibody with recognition site c and the bonded carbohydrate fragment. The label is detected after washing to remove the unbound antibody, indicating that the c line is located on the carbohydrate fragment bound to the foreign lectin. The sequence of the binding site may be e-纟^ or /74. The position of the reducing end and the sequence of the recognition site cannot be determined by the reaction. If the label is detected in the reaction of the control group (without glycosidase), and the reaction is detected in the case of the reaction containing glycosidase, the identification is indicated. The sequence of sites is CM-CM. In this particular example, if a different glycosidase is used alone, and/or combined with various lectins, and/or different antibodies, the amount of information obtained will be increased. Obviously, more The specific example of the present invention (wherein the third substantially sequence-specific agent provides a cutting step) is substantially equivalent to the Chinese National Standard (CNS) A4 specification (210×297 mm) which is exactly 25 cm in the previous paragraph ( Please read the notes on the back and fill out this page.) Piano, 1T-plus 1306152 \7 37 V. Description of the invention (Θ) A specific example of the description (where the first is basically a sequence-specific agent providing a cutting step). The difference between the two specific examples of the present invention is that, in the specific example described above, the effect of the cutting agent can be observed by detecting the presence of the label before reacting with the third substantially sequence-specific agent. In the specific example just described in the previous paragraph, it is necessary to use a control reaction containing no cleavage agent to determine the influence of the cleavage reagent. Nonetheless, the range of messages obtained in different specific examples is substantially equivalent, and the method for processing the message and the sequence of identification sites (i.e., triples) from the message is comparable. The above examples are based on the assumption that the saccharide is linear and the glycosidase has a recognition site in the sugar sequence. However, the presence of a glycosidase recognition site in carbohydrates will usually be rapidly confirmed by the reaction between the assay and other lectins. If either of the two tags carrying the lectin in either reaction is lost after the addition of the glycosidase, the glycosidase must have a recognition site in the saccharide. Glycosyltransferases can be used as agents that are substantially sequence specific. Glycosyltransferases will add sugar units at specific points in the carbohydrate sequence, depending on the particular sequence pattern (identification sequence). Thus, if the monosaccharide used in the reaction is indicated, a new label indicating the presence of a glycosyltransferase recognition site can be introduced into the sugar chain. Of course, the label introduced by the glycosyltransferase should be distinguishable from the other labels used. When a set of reactions is carried out with the indicated antibodies as above, the antibodies can bind in most reactions with a few exceptions. This is due to the partial overlap of the carbohydrate recognition sequence between the lectin and the antibody. In this case, the reaction with certain lectins will be negative. Therefore, you should use 26 ------- ί ---------* ---------. (Please read the notes on the back and then fill out this page) Applicable to China National Standard (CNS) A4 Specification (210 * 297 mm) 1306152 A7 B7 V. Invention Description (>Mr) This message derives the length of 3-7 sugar units because of the identification of lectins and antibodies. The sequences are each 2-4 sugar units. If there is no contact between the first and second detection steps, the two detection steps can be performed simultaneously. As a further example, if in the first reaction sequence, the first reaction with the lectin occurs at position a on the terminally indicated polysaccharide chain, and the second reaction with the glycosidase occurs on the polysaccharide chain. The site then introduces a second label distinguishable from the first label from the reaction between the site c and the third substantially sequence specific agent. Therefore, the order of the three sites will be shown as the reducing end or the he-heart reverting end by the presence of the two tags. On the other hand, if only the second tag is measured, there are two possibilities: (1) the recognition site of the third substantially sequence-specific agent is absent in the polysaccharide, or (2) contains a recognition site. A portion of the polysaccharide is cleaved by the second substantially sequence specific agent. The above situation can be confirmed by the second reaction sequence in which the second substantially sequence-specific agent disappears or the agent is used under buffer conditions in which cleavage is not possible. In the second reaction sequence, the second label cannot be detected after reacting with the third substantially sequence-specific agent, indicating that the third substantially sequence-specific agent lacks a bond on the sugar. Site. In another aspect, the presence of the second tag indicates that the cleavage site is between the bonding sites of the first and third substantially sequence-specific agents, that is, the sites on the saccharide are in the reducing end. . In the method of the present invention, there is no need to carry out one after another, the latter 27 paper size is applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) (please read the back note first and then fill in this page) ▼装· Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed 1306152 A7 B7 V. Invention Description (V Factory) The reaction depending on the result of the former. In contrast, the present invention provides a method in which a plurality of reactions can be carried out such that the above inference can be made from a single set of reactions. For example, in a set of reactions, a first substantially sequence specific agent of a different species can be used with the second and third substantially sequence specific agents that are identical in each reaction. The second set of reactions can be carried out simultaneously such that the first set of reactions are identical except for the disappearance or inactivation of the second substantially sequence specific agent. However, the information obtained in the intermediate detection step can advantageously be used to exclude certain selections of agents that are substantially sequence specific in the following steps. It is known that when a substantially sequence-specific agent having no recognition site on a saccharide to be analyzed is used as the second or third substantially sequence-specific agent, no information can be provided. Therefore, in the specific example of the labeling of the saccharide of the present invention, the result of the detection after the sugar is bonded to the first substantially sequence-specific agent can be used for the first basic by the bonded saccharide. The second substantially sequence specific agent is selected quantitatively for the sequence specificity agent. In another embodiment of the invention, after the detecting step has been completed and the message is obtained as above, the addition of the third substantially sequence specific agent is repeated with different agents. For the reaction in which the two labels were still present after the first addition of the third agent, the same considerations were applied to explain the above results. However, in the case where only one label remains, it can be inferred whether the cleavage agent is at the recognition site of the first substantially sequence-specific drug and the identification site of the agent having substantially sequence specificity of the tag. Cut between (or the reducing end). In this case, the remaining label will be applicable to the Chinese National Standard (CNS) A4 specification (2丨〇乂297 mm) on 28 paper scales (please read the notes on the back and fill out this page) > - - Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed 1306152 A7 ______ B7_____ V. Description of the invention (y(j) disappears after reacting with a different type of third essentially sequence-specific agent. Another specific example of the present invention In the middle, you can add a different second basic (please read the back note first and then fill out this page). For the sequence-specific drug, the agent can carry the second basic added to the first group reaction. A different or identical label for a sequence-specific agent. A further message will be provided by the addition of a third substantially sequence-specific agent that cleaves the sugar chain. In yet another embodiment of the invention, Two or more different second substantially sequence specific agents each with their label are added, and the labels can be detected independently of any of the other labels used. After the addition of the cutting agent The label of the loss, as described above for the experiment using only a second substantially sequence specific agent, will provide information on the location of the bonding site of the second substantially sequence specific agent.人士The Ministry of Economic Affairs' Intellectual Property Office staff consumption cooperatives should read the familiar skills: by performing a sufficient amount of reactions as described above, a fingerprint (fingeirpdnt) specific to the reaction of a particular saccharide can be obtained. In addition, it may be as above Summarize the partially obtained sequence [J message "collapse" into the complete sequence of the saccharide. Since the number of reactions may be very large (as detailed below), the method of the invention is not limited to oligosaccharides as compared to prior art methods. The method can also be used to determine the structure of a polysaccharide (i.e., a large saccharide having a plurality of saccharide units). In one embodiment of the invention, the first substantially sequence specific agent is a lectin. The first substantially sequence specific agent may also be an antibody or other sequence specific agent. Another embodiment of the present invention provides multiple 29 paper grades with different sequence specificity apply to China National Standard (CNS) A4 specification (210X297 mm) 1306152 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed 5, Invention Description (>/ ) External agglutination Or saccharide-specific antibodies, such as lectins or antibodies, are immobilized on a substrate in an array, such as a very large integrated (VLSI) circuit wafer similar to the wafers used today to form oligosaccharide arrays. The methods of such wafers and reagents bonded to the wafers are described, for example, in WO 93/22678. In another embodiment, the invention provides a pseudo-array of immobilized first substantially sequence-specific agents. One example of such a pseudo-array uses MASDA particles, which is illustrated by the inventors herein in the PCT application (IL-97/00105), which is incorporated herein in its entirety by reference. The first substantially sequence-specific agent is immobilized on the MASDA particles in separate reactions (e.g., in 25 different reactions). Each portion of the pseudo-array can then be reacted with the same second substantially sequence-specific agent, preferably by taking a sample from each MASDA reaction, mixing the samples, and The mixture is reacted with the second substantially sequence specific agent. If necessary, a portion of the pseudo-array is reacted with a different second substantially sequence-specific agent, and then all of the MASDA reactions may be left separately; or a portion of the pseudo-array may be combined into a mixture, with a single second For sequence-specific drug reactions, the other portions of the pseudo-array are separately retained to react with different second substantially sequence-specific agents. Similarly, portions of the pseudo array can also be combined or separately retained to react with the third substantially sequence specific agent. Beads can also be used as a fixing agent. A variety of different beads have been described in the art for the purpose of bonding peptides and proteins. See, for example, the Palis 30 paper scale for the Chinese National Standard (CNS) A4 specification (210 X 297 mm) (please read first) Note on the back and fill out this page)

A 裝 訂·- 1306152 A7 A7 B7 五、發明説明（θ) (請先閲讀背面之注意事項再填寫本頁) 公司(Pierce)之商品目錄，第0-222至0-231及T-155至T-200頁。該第一基本上爲序列專一性的藥劑(較佳爲外源凝 集素)可與珠粒鍵結。此方法之優點爲：該等珠粒接著可被 分成等分試樣，並與不同的第二及/或第三基本上爲序列專 一性的藥劑反應，由而進一步增加由本發明方法提供之訊 息量。 經濟部智慧財產局員工消費合作社印製 各種基本上爲序列專一性的藥劑之反應條件爲技藝中 已知者。此外，熟習技藝之人士可以各種基本上爲序列專 一性的藥劑進行一連串試驗，計算其於各種反應條件下之 鍵結活性。知悉於何種反應條件下將使特定之基本上爲序 列專一性的藥劑反應，以及於何種反應條件下該藥劑仍不 反應，可有利地用於控制其中含有若干基本上爲序列專一 性的藥劑之反應。例如，第二及第三基本上爲序列專一性 的藥劑可以同時添加至反應中，但藉由改變反應條件，可 以僅使該第二基本上爲序列專一性的藥劑被活化。接著可 以選擇進一步改變反應條件，以使該第二基本上爲序列專 一性的藥劑不活化，同時活化該第三基本上爲序列專一性 的藥劑。下表1列出某些反應條件之實例。除了表1所列 之pH及溫度數據以外，可硏究其他因子，如所含之金屬 如鋅，或陽離子(如錳、鈣、鈉）之鹽如氯化鈉鹽，以發現 最佳反應條件，或於何種條件下將使特定之基本上爲序列 專一性的藥劑活化，而其他藥劑不活化。 31 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1306152 A7 B7 五、發明說明（1) 表1 : 某些基本上爲序列專一;丨生的藥劑之反應條件 條件組密碼 條件序號 PH 溫度 CC) 酵素 1 3.5 30 刀豆召_半嘲漉有:酿 2 5.0 37 內a-N-乙醯基半乳糖穿酶 a-U-墨角藻聚糖芽酶 召-1，2-半乳糖棚 3 5.0 25 牛腎臓之α -墨角藻聚糖并贿 4 7.2 25 咖啡豆α-半乳糖芽酶 5 5.8 55 B. Fragilis之內-半乳糖穿酶 6 6.2 25 雜纖茼酺 7 4.3 37 牛睪九之Θ 1-3,4,6-半乳糖苷 酶 購自生物群相公司 (Biodiversa) 2-9.5 50 Gly 001-02 購自生物群相公司 3.0- 8.0 50 Gly 001-04 購自生物群相公司 2-11 50 Gly 001-06 •符號表示可藉外在條件分離之酵素組群。 •群相(Diversa)公司製造之嗜熱性內/外糖芽酶在不同 --— —— — — —--· I I (請先閱讀背面之注意事項再填寫本頁) ¼. 經濟部智慧財產局員工消費合作社印製 的pH及溫度時具有極大的活性變異。 •可能的條件亦可爲金屬等Zn、Mn、Ca、NaCl。 可利用蛋白質的官能基如胺基、羧基、羥基或硫醇基 進行該第一基本上爲序列專一性的藥劑之固定化作用。例 如，可如上提之PCT公開案所述般，藉由與環氧基矽甲烷 反應，使玻璃支承體藉環氧基予以官能化。環氧基與胺基 32 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 1306152 A7 B7 五、發明說明（尸） 如離胺酸殘基之自由ε-胺基反應。另一種以電冶材料(如 金)轉化表面的機制，亦說明於該pct公開案。由於此等材 料與硫醇基形成穩定共軛，因此蛋白質可藉由半胱胺酸殘 基之自由硫醇基直接連接於此等物質。此外，硫醇基可藉 由習知化學方法，或藉由與含有一個或多個硫醇基及與月女 基反應之基的分子(如半胱胺酸之Ν-羥基丁二醯亞胺酯)反 應而導入該蛋白質中。可切割硫醇基之交聯劑如二硫代雙( 丁二醯亞胺基丙酸酯)亦可與蛋白質的胺基反應。以锍劑還 原後，將使交聯劑曝露出其自由硫醇基。 可藉由習知方法將標籤導入基本上爲序列專一性的藥 劑或糖類中。標籤包含連接於該糖類或基本上爲序列專一 性的藥劑，且不會干擾該糖類或藥劑之功能的任何可檢測 之基。標籤可爲酵素，如過氧化酶及磷酸酯酶。原則上， 亦可使用酵素如葡萄糖氧化酶及yS-半乳糖苷酶。須注意， 若該糖含有與此酵素反應的單糖單元，則該糖類可能被改 質。其他可以使用之標籤包含螢光性標籤，如螢光黃 (Fluorescein)、德州紅、螢光素黃（Lucifer Yellow)、薔薇紅 、尼爾(Nile)紅、四甲基-薔薇紅-5-異硫代氰酸酯、1，6-二苯 基-1,3,5-己三嫌、順式-帕拉里油酸(Parinaric acid)、藻紅素 、別藻藍蛋白（八11〇口1^(^311丨11)、4’,6-二昧基-2-苯基 (DAPI)、豪艾克(Hoechst)33258、2-胺基苯甲醯胺等。其餘 之標籤包含電子密集之金屬如金、配位子、半抗原如生物 素、放射活性之標籤等。 標示糖類之實例包含： 33 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） ---— — — — — —----- (請先閱讀背面之注意事項再填寫本頁) Ή 經濟部智慧財產局員工消費合作社印製 1306152 A7 B7 五、發明説明（今（） 1. 使用還原端之顏色標籤(如，香豆素-120) 2. 使用經14C-放射活性標示之糖+糖苷酶(序列專一性糖 之合成） 3. 使用經3H-放射活性標示之糖+糖苷酶(序列專一性糖 之合成） 4. 使用經螢光基標示之糖+糖苷酶(序列專一性糖之合 成） 5. 使用經螢光基標示之外源凝集素或mAbs 6. 使用經螢光基標示之呈色或經酵素連結之mAbs 7. 使用生物素末端進行標示 8. 構築特殊之糖序列，並使用專一辨識該序列之抗體 或外源凝集素。 酵素性標籤之檢測係以ELISA領域中周知及於酵素檢 測上慣用之其他技術進行。該等酵素爲商業行爲可得者， 如自公司(如派爾斯)獲得。 螢光性標籤需要於特定波長激發，並於不同波長檢測 。螢光之檢測方法爲技藝中周知者，且已發表於諸多文章 及教科書中。關於此議題之文獻選擇可見於派爾斯公司 1994年商品目錄之0-124至0-126頁。螢光性標籤可經商 業行爲自公司如西格瑪或上述之派爾斯公司購得。 使標籤與蛋白質及糖偶合爲技藝中已知之技術。例如 ，以螢光性或放射活性標籤標示糖類所用之市售套組係購 自奧斯佛糖系統（Oxford Glycosystems)公司，艾明頓 (Abingdon)，英國。標示蛋白質用之試劑及其用法說明書可 34 (請先閲讀背面之注意事項再填寫本I) 、?τ 經濟部智慧財產局員工消費合作社印製 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1306152 A7 經濟部智慧財產局員工消費合作社印製 五、發明説明（ji) 自上述之派爾斯公司購得。 偶合通常係藉由使用官能基如羥基、醛基、酮基、胺 基、锍基、羧酸等基予以進行。可經商業行爲購得若干與 此等基反應之標籤如螢光性標籤。此外，可使用其一端與 標籤反應，而另一端與蛋白質或糖類反應之雙官能基交聯 劑。交聯劑的使用可能有利於防止蛋白質或糖類功能的喪 失。不過，其他任何可以保持蛋白質或糖類之原始功能之 適當偶合技術均可使用。 不過，熟習技藝之人士應顯見，可使用大量之公知方 法使蛋白質偶合於指定之支承體，或使標籤偶合於蛋白質 或糖類。 可使用適當方法如技藝中已知之方法進行標籤之檢測 。某些檢測方法說明於上述之WO 93/22678中，該案全文 之揭露皆倂於本文。特別適合本發明之方法係該公開案中 說明之CCD檢測器之方法。此方法可與能吸收特定頻率之 光的標籤倂用，以遮斷通過VLSI表面之試驗光源，因此， 該CCD感應器於經該標示劑鍵結之區域測到較少光量。該 方法亦可利用此等標籤吸收激發頻率之光的事實，而用於 螢光性標籤。此外，該CCD感測器亦可用於檢測該螢光性 標籤在激發後之發射。來自激發光之發射訊號的分離，可 藉由使用對不同波長具有不同敏感度之感應器、或藉由瞬 間解析、或二者倂用而達成。 茲將藉由下述實例進一步說明本發明。 實例1 _____ 35 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閲讀背面之注意事A Binding·- 1306152 A7 A7 B7 V. Description of Invention (θ) (Please read the note on the back and fill out this page) Catalogue of the company (Pierce), 0-222 to 0-231 and T-155 to T -200 pages. The first substantially sequence specific agent (preferably a lectin) can be bonded to the beads. An advantage of this method is that the beads can then be divided into aliquots and reacted with different second and/or third substantially sequence specific agents to further increase the information provided by the method of the invention. the amount. Printed by the Ministry of Economic Affairs, the Intellectual Property Office, and the Consumer Cooperatives. The reaction conditions for the various sequence-specific drugs are known in the art. In addition, those skilled in the art can perform a series of tests on a variety of substantially sequence specific agents to calculate their binding activity under various reaction conditions. Knowing under which reaction conditions a particular essentially sequence specific agent will react, and under which reaction conditions the agent will not react, may advantageously be used to control a number of substantially sequence specificities therein. The reaction of the agent. For example, the second and third substantially sequence specific agents can be added to the reaction simultaneously, but by altering the reaction conditions, only the second substantially sequence specific agent can be activated. The reaction conditions can then be selected to be further altered such that the second substantially sequence specific agent is not activated while activating the third substantially sequence specific agent. Table 1 below lists examples of certain reaction conditions. In addition to the pH and temperature data listed in Table 1, other factors such as metals such as zinc or salts of cations such as manganese, calcium and sodium such as sodium chloride can be investigated to find the optimum reaction conditions. Under what conditions, a particular agent that is substantially sequence specific will be activated, while other agents are not activated. 31 The paper size applies to the Chinese National Standard (CNS) A4 specification (210X297 mm) 1306152 A7 B7 V. INSTRUCTIONS (1) Table 1: Some basically sequence specific; reaction conditions of the medicinal reagents No. PH Temperature CC) Enzyme 1 3.5 30 Beans Beans _ Semi-Mocking 漉: Stuffed 2 5.0 37 Within aN-Ethyl galactose permease aU-Mocus geranase buds-1,2-galactose shed 3 5.0 25 Aureus sputum α-Mocus sulphate and bribe 4 7.2 25 Coffee bean α-galactosylase 5 5.8 55 B. Within Fragilis - galactose permease 6 6.2 25 茼酺 茼酺 7 4.3 37 牛睪9之Θ 1-3,4,6-galactosidase was purchased from Biodiversa 2-9.5 50 Gly 001-02 from Biocommunity Inc. 3.0- 8.0 50 Gly 001-04 from bio Group Phase 2-11 50 Gly 001-06 • Symbols indicate enzyme groups that can be separated by external conditions. • The thermophilic endo/exo-glycanase produced by Diversa is different ----------------------------------------------------------------------------------------------------------------- The pH and temperature printed by the employee consumption cooperatives have great activity variations. • Possible conditions may also be Zn, Mn, Ca, and NaCl such as metals. The immobilization of the first substantially sequence-specific agent can be carried out using a functional group of the protein such as an amine group, a carboxyl group, a hydroxyl group or a thiol group. For example, the glass support can be functionalized by an epoxy group by reaction with epoxy methane as described in the PCT publication above. Epoxy and Amine 32 This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1306152 A7 B7 V. Description of the invention (cadaver) Reacts from the free ε-amine group of the amine acid residue. Another mechanism for converting surfaces by electro-chemical materials such as gold is also described in the pct publication. Since these materials form a stable conjugation with the thiol group, the protein can be directly attached to the substance by the free thiol group of the cysteine residue. In addition, the thiol group can be obtained by a conventional chemical method or by a molecule which reacts with one or more thiol groups and a group which reacts with a ruthenium group (for example, cysteine-hydroxybutyric imine) The ester) is introduced into the protein by reaction. Crosslinkers which can cleave thiol groups such as dithiobis(butanediamine propionate) can also react with the amine groups of the protein. Upon reduction with the tanning agent, the crosslinker will be exposed to its free thiol group. The label can be introduced into a substantially sequence specific drug or saccharide by conventional methods. The tag comprises an agent attached to the saccharide or substantially sequence specific and does not interfere with any detectable group of functions of the saccharide or agent. Labels can be enzymes such as peroxidase and phosphatase. In principle, enzymes such as glucose oxidase and yS-galactosidase can also be used. It should be noted that if the sugar contains a monosaccharide unit that reacts with the enzyme, the sugar may be modified. Other labels that can be used include fluorescent labels such as Fluorescein, Texas Red, Lucifer Yellow, Rose Red, Nile Red, Tetramethyl-Rose Red-5- Isothiocyanate, 1,6-diphenyl-1,3,5-hexatriene, cis-palatinic acid (Parinaric acid), phycoerythrin, allophycocyanin (eight 11 〇) Mouth 1^(^311丨11), 4',6-diamidino-2-phenyl (DAPI), Hoechst 33258, 2-aminobenzamide, etc. The remaining labels contain electrons. Intensive metals such as gold, ligands, haptens such as biotin, radioactive labels, etc. Examples of labeled sugars include: 33 This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) -- -————————————----- (Please read the notes on the back and fill out this page) Ή Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed 1306152 A7 B7 V. Invention Description (Today (1) Use Color label of the reducing end (eg, coumarin-120) 2. Use 14C-radioactive labeled sugar + glycosidase (synthesis of sequence specific sugar) 3. Use 3H-radioactive label Sugar + glycosidase (synthesis of sequence-specific sugars) 4. Use of fluorescent-labeled sugar + glycosidase (synthesis of sequence-specific sugars) 5. Use fluorescent-based labeling of lectins or mAbs 6 Use fluorescent-labeled color-coded or enzyme-linked mAbs 7. Label with biotin terminus 8. Construct a specific sugar sequence and use an antibody or lectin that specifically recognizes the sequence. The assays are performed by other techniques well known in the ELISA art and used in enzyme assays. These enzymes are commercially available, such as from companies such as Pyle. Fluorescent labels need to be excited at specific wavelengths, and Detected at different wavelengths. Fluorescence detection methods are well known in the art and have been published in many articles and textbooks. The literature selection for this topic can be found in the Perth company's 1994 catalogue from 0-124 to 0-126. Fluorescent labels are commercially available from companies such as Sigma or the above-mentioned Pears Corporation. Coupling labels with proteins and sugars is known in the art. For example, fluorescent or radioactive The commercially available kits for labeling sugars are purchased from Oxford Glycosystems, Abingdon, UK, reagents for labeling proteins and instructions for their use. 34 (Please read the notes on the back first) Fill in this I), ?τ Ministry of Economic Affairs Intellectual Property Bureau employees consumption cooperatives printed paper scale applicable to China National Standard (CNS) A4 specifications (210X297 mm) 1306152 A7 Ministry of Economic Affairs Intellectual Property Bureau employees consumption cooperatives printed five, invention Description (ji) Purchased from the above-mentioned Pears Company. Coupling is usually carried out by using a functional group such as a hydroxyl group, an aldehyde group, a ketone group, an amine group, a thiol group, a carboxylic acid or the like. A number of labels, such as fluorescent labels, that are reactive with such groups are commercially available. Further, a difunctional crosslinking agent which reacts with a label at one end and a protein or a sugar at the other end can be used. The use of crosslinkers may be beneficial to prevent loss of protein or carbohydrate function. However, any other suitable coupling technique that preserves the original function of the protein or sugar can be used. However, it will be apparent to those skilled in the art that a large number of well known methods can be used to couple a protein to a designated support or to couple a tag to a protein or carbohydrate. Labeling can be performed using suitable methods, as is known in the art. Some of the detection methods are described in the above-mentioned WO 93/22678, the entire disclosure of which is incorporated herein by reference. A method which is particularly suitable for the present invention is the method of the CCD detector described in the publication. This method can be used with a label that absorbs light of a specific frequency to block the test light source passing through the surface of the VLSI, and therefore, the CCD sensor detects less light in the area bound by the labeling agent. The method can also be used for fluorescent labels by utilizing the fact that these labels absorb light of the excitation frequency. In addition, the CCD sensor can also be used to detect the emission of the fluorescent label after excitation. The separation of the transmitted signals from the excitation light can be achieved by using sensors having different sensitivities to different wavelengths, or by instantaneous resolution, or both. The invention will be further illustrated by the following examples. Example 1 _____ 35 This paper scale applies to the Chinese National Standard (CNS) A4 specification (210X297 mm) (please read the notes on the back first)

1· 項再填I :寫本頁) 裝- 訂 1306152 A7 B7 修正h複充 五、發明說明g ) 使用抗體作爲第一及第二序列專一件的苹部丨夕 糖分子分析. 此實例進一步說明依據發明分析糖分子之技術。使用 抗體作爲第一及第二序列專一性的藥劑。下表列出兩種不 同的糖類之反應結果，爲達說明目的，該二種糖類以HS 及NS表示。 該等糖之結構如下： MFLNH-II(HS):1· Item Refill I: Write this page) Pack - Book 1306152 A7 B7 Amendment h Refill 5, Invention Description g) Use the antibody as the first and second sequence of the individual part of the 丨 糖 糖 molecular analysis. This example further A technique for analyzing sugar molecules according to the invention. Antibodies are used as first and second sequence specific agents. The table below shows the results of the reaction of two different sugars, which are indicated by HS and NS for illustrative purposes. The structure of these sugars is as follows: MFLNH-II (HS):

Lex SO GicNAcP(“)Lex SO GicNAcP(")

Gaip(l-3)GJcNAcp(l-3；Gaip(l-3)GJcNAcp(l-3;

Ga]P(卜4) Glc <請先閱讀背面之注意事項再填寫本頁) T antigen NS：Ga]P (Bu 4) Glc <Please read the notes on the back and fill out this page) T antigen NS:

Le-XLe-X

GalSli)^ GlcNAcp(l-6)GalSli)^ GlcNAcp(l-6)

· I I — I ί I 訂----— I — I I· I I — I ί I order----- I — I I

Gaip(1-4) Glc —~Gaip(1-4) Glc —~

Leb Fuca(1-2)Galp(1-3) 表2列出糖類與該第一及第二序列專一性的藥劑[爲抗 丁-抗原、Lewlsx(Lex)或Lewisl/C原（Leb)之抗體]間之反應結 果。該第一基本上爲序列專一性的藥劑係固定於基體(較佳 係固相微粒子)上。該第二基本上爲序列專一性的藥劑係以 螢光劑，亦即，尼爾紅或綠色予以標示。此外，該糖類之 還原端係以作爲該第二基本上爲序列專一性的藥劑之標記 36 不紙張尺度刺巾関r.#i7cNS)A4~iii"(210,： 297公爱） 1306152 Α7 Β7 五、發明説明 ’且可明顯地與尼爾紅或綠色標籤區別之標籤予以標示。 表2列出糖類HS之反應，而表3列出糖類NS之反應。 -- &2 於某體卜 鍵結之糖類 抗T-杭原 抗Lex 杭Leb HS HS 第二 mAb 尼爾紅杭Lex 訊號 尼爾紅，還原端 還原端 firT- 表3 於某體卜 抗T-杭原 抗Lex 杭Leb 鍵結之糖類 NS NS 第二 mAb 綠色抗Leb _紅抗Lex 訊號 綠色’還原端 尼爾紅，還原端 (請先閱讀背面之注意事項再填寫本頁) _裝· 槪括而言，若使用所示抗體作爲第一基本上爲序列專 一性的藥劑(欄），則可於糖類HS或NS(列)之反應中測得下 述訊號： 表4 於某體h 抗T-杭原 杭Lex 抗Leb HS 尼爾紅，還原端 還原端 NS 綠色，還原端 尼爾紅，還原端 NS 綠色，還原端 尼爾紅，還原端 在標籤已被測得，且己記錄各反應之結果以後，添加 第三基本上爲序列專一性的藥劑。此實例以一種第三基本 上爲序列專一性的藥劑進行兩個獨立的反應。然後，該帶 37 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 訂1 經濟部智慧財產局員工消費合作社印製Leb Fuca (1-2) Galp (1-3) Table 2 lists the drugs specific for the first and second sequences of the sugar [for anti-butyl-antigen, Lewlsx (Lex) or Lewis/C original (Leb) The result of the reaction between the antibodies]. The first substantially sequence-specific drug system is immobilized on a substrate (preferably solid phase microparticles). The second substantially sequence specific drug is indicated by a fluorescent agent, i.e., neil red or green. In addition, the reducing end of the saccharide is labeled as a second substantially sequence-specific agent. 36 No paper-scale thorns. r.#i7cNS) A4~iii" (210,: 297 public) 1306152 Α7 Β7 5. The description of the invention 'and the label that can be clearly distinguished from the Neil red or green label is marked. Table 2 lists the reactions of the sugars HS, while Table 3 lists the reactions of the sugars NS. -- & 2 in a body bond bond sugar anti-T- Hangyuan anti-Lex Hang Leb HS HS second mAb Neil Red Hang Lex signal Neil red, reducing end reducing end firT- Table 3 in a certain body anti- T-Hangyuan anti-Lex Hang Leb bond sugar NS NS second mAb green anti-Leb _ red anti-Lex signal green 'reducing end Neil red, restore end (please read the back note before filling this page) _ loading · In other words, if the indicated antibody is used as the first substantially sequence-specific agent (column), the following signals can be measured in the reaction of saccharide HS or NS (column): Table 4 h anti-T-Hangyuan Hang Lex anti-Leb HS Neil red, reducing end NS green, reducing end Neil red, reducing end NS green, reducing end Neil red, the reducing end has been measured, and After recording the results of each reaction, a third substantially sequence specific agent is added. This example performs two independent reactions with a third essentially sequence specific agent. Then, the tape is 37. The paper size is applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm). 1 Printed by the Intellectual Property Office of the Ministry of Economic Affairs

1306152 五、發明說明（>夕 (請先閱讀背面之注意事項再填寫本頁) 有糖分子之固體相可有利地區分成等分試樣，以與α 1_2墨 角藻聚糖苷酶或外半乳糖苷酶(第三基本上爲序列專一 性的藥劑）。此外，可以第一及第二基本上爲序列專一性的 藥劑進行三組反應。 表5 :1306152 V. INSTRUCTIONS (> eve (please read the note on the back and fill out this page). The solid phase with sugar molecules can be advantageously divided into aliquots to match α 1 2 fucosidase or outer half. Lactosidase (the third essentially sequence-specific agent). In addition, three sets of reactions can be performed for the first and second substantially sequence-specific agents.

於添加 α卜3,4墨角藻聚糖苷酶之後的反應： 於某體上 抗Τ-抗原 抗Lex 杭Leb HS 澴原端 NS 表6 :添加得自肺炎雙球菌之外点·半乳糖苷酶[EC3.2.1.23 商品目錄編號1088718，購自柏林格曼罕明(Boehringer Mannheim)，68298，曼罕明，德國]之後的反應 . 於基體上 抗T-抗原 抗Lex 杭Leb _ HS 尼爾紅 NS 綠色 尼爾紅 表7 :於添加α 1-2墨角藻聚糖苷酶之後的反應: 於基體上 抗T-抗原 杭Lex 杭Leb HS 辰爾紅，澴原端 澴原端 NS 澴原端 茲可由上文蒐集之資料構築糖分子鑑別(GMID)卡。有 關糖類HS之此等訊息的實例列於表8，關於糖類NS者列 於表9。 表8 _ 於基體上 抗T-抗原 抗Lex 抗Leb _ 0 m爾紅，澴原端 澴原端 1 還原端 — . 2 尼爾紅， 38 本紙張尺·度適用中國國家標準（CNS)A4規格（210 χ 297公釐） 經濟部智慧財產局員工消費合作社印製 1306152 A7 B7 3 尼爾紅’還原端 還原端 表9 於基體上 抗T-抗原 抗Lex 抗LeB 0 綠色，還原端 尼爾紅，還原端 1 - - 2 綠色 尼爾紅， 3 還原端 該第二及第三基本上爲序列專一性的藥劑之一致性不 須於此資料表中揭露。永遠辨認試劑之特定組合的特定代 碼(上表中之1、2或3)已足夠達到比較之目的。 實例2 侬序標元:澴原端之計畫. 誠如上述說明及實例所示’本發明方法有利地用於標 示所欲硏究之糖類的還原端。不過，由於此標不技術可以 擴展至糖類內之位點’因此可藉由提供更多訊息而促成本 發明方法。由於事實上可藉由使用內糖芽酶切割後標示該 還原端，而標示糖鏈內部’因此可以獲得在糖鏈內之經標 示的還原端。由於相較於最初之還原端’該糖鏈內經標示 的還原端必須離該第一、第二及第三基本上爲序列專一性 的藥劑之鍵結位點更近’因此’內部構築之經標不的還原 端的使用提供了額外的訊息。再者’可能藉由如下文說明 之方法依序標示還原端，而依在所欲硏究之糖鏈上的順序 鑑定不同糖苷酶的位點。 兹於下述步驟中更詳細地說明依序標示還原端的方法 39 本紙張尺度適用中國國家標準（CNS)A4规格（210x297公幻 IT (請先閲讀背面之注意事項再填寫本頁) 1306152 A7 B7 五、發明說明（γ ) 1. 遮蔽: (請先閱讀背面之注意事項再填寫本頁) 使具有還原端之多糖於含有NaBIWNaOH，pH 11.5的 溶液中保溫。 此處理遮蔽還原端，因此該多糖現在缺乏還原端(RE) 〇 2. 曝露： 以內糖苷酶處理步驟1之多糖。若該多糖內部存有該 內糖苷酶之辨識位點，則將因切割該多糖而產生新的還原 端。該溶液現在含有兩種糖類：於內糖苷酶位點具有新曝 露的RE之片段，以及RE被遮蔽之第二片段。 1.標示還原端： 此反應可藉由使用例如2-胺基苯甲醯胺(奧斯佛糖系統 公司出售之用以標示糖類的套組，示於1994年商品目錄， 第62頁)予以進行。待於高氫濃度及高溫度(H+/T)條件下， 接著還原的反應完成後，混合物中含有兩種片段，其一是 其還原端經標示者，另一者因其還原端被遮蔽之事實而仍 未被標示。 經濟部智慧財產局員工消費合作社印製 標示還原端之另一途徑爲還原胺化反應。使含有芳胺 基之螢光性化合物與還原端之醛官能基反應。接著使所得 CH=N雙鍵藉由例如使用硼氫化鈉還原成CH2-N單鍵。此 技術係FACE(螢光團輔助之糖電泳)套組之一部分，該套組 可購自美國，加州，諾法特(Novato)之Glyko公司，該技術 詳細說明於例如Glyko公司之商品目錄第8-13頁(已倂於本 40 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 1306152 A7 五 經濟部智慧財產局員工消費合作社印製. 、發明説明（认） 文作爲參考文獻）。 iA第二內糖并醃反應： 接著可使第二內糖苷酶與該糖混合物反應。此新的反 應混合物茲具有三種片段，一種帶有完整的還原端，第二 種帶有由2-胺基苯甲醯亞胺標示之還原端，第三種片段帶 有遮蔽之還原端。 實例3 息與基本上爲序列專一件的藥劑之—荔列砭齊 ίΐ衍出結構訊息 此實例進一步說明本發明方法，亦即，藉由使用如更 即文所述之一組反應，產生與糖類結構有關之資料。此實 例進一步證明由該組反應可推衍出序列訊息。 於某些情形下，所用之試劑可能不會如公知之資料(如 由商品目錄獲得之資料)所預期般完全反應。例如，如更下 文所述之外源凝集素曼陀羅凝集素係於西格瑪之商品目錄 中所列出可以鍵結GlcNac者。然而，於更下文詳述之反應 中，顯示DSA鍵結於香豆素120衍生之Glc(Glc-AMC)。顯 見Glc-AMC之作用實質上類似GicNac，此乃因彼等化合物 間之結構類似。再者，由下述結果顯見所用之內半乳糖苷 酶不僅切割半乳糖殘基，亦切割用以連接Glc-AMC基與剩 餘之糖的鍵結。 顯然，本發明實務中所用之基本上爲序列專一性的藥 劑，於某些情形下，可能具有與公開之資料(如商品目錄) 所示藥劑的專一性不同的良好專一性。此等反應可藉由使 41 、紙張尺度適用中國國家標準（CNS ) A4規格（2i〇X297公釐） ；---------— (請先閱讀背面之注意事項再填寫本頁) 訂 b. 1306152 A7 B7 五、發明説明 用本發明方法與已知結構之糖類快速地鑑定。接著可使所 得結果與預期結果比較，其間之差異將可用於辨認所用之 基本上爲序列專一性的藥劑間之不同專一性。接著可以貯 存此種在基本上爲序列專一性的藥劑之良好專一性與公開 之資料間的差異性，以使用此等藥劑進行未知糖類結構之 特性分析。 下文中，使用末端經標示之戊糖及各種外源凝集素及 糖 說明本發明。該戊糖之結構爲GaH(l，4)[Fuc-a (UW-GlcNAc-^djyGal/Sd^yGlc。該戊糖於 GlcNAc 位 置處分支，且該GlcNAc之位置3及4分別鍵結墨角藻聚糖 及半乳糖。該戊糖之還原端(Glc)係以香豆素-120(7-胺基-4-甲基香豆素，可購自例如西格瑪，商品目錄編號A 9891)予 以標示。可如上文般，藉由使用芳族胺官能基標示還原端 ，而進行偶合反應。當香豆素-120於312 nm激發時，發出 藍色螢光。使用內-/5-半乳糖苷酶(EG，柏林格曼罕明公司) 及外-1,3-墨角藻聚糖苷酶[FD，新英格蘭生物實驗室(New England Biolabs)]作爲第一及第二基本上爲序列專一性的藥 劑。該二試劑的反應條件係如NEB商品目錄所述有關外_ 1,3-墨角藻聚糖苷酶的反應條件。 進行三組反應。第一組包含墨角藻聚糖苷酶(FD)及內-半乳糖苷酶(EG)，第二組僅包含FD，第三組僅包含eg。 第四組反應不含酵素，作爲對照組。 爲了確定酵素已經分解糖類，使用薄層層析法(TLC)， 對各反應進行尺寸區分(size-separated)。 42 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） ---------φ^-- (請先閲讀背面之注意事項再填寫本頁)Reaction after addition of α3,4-fucosylglycosidase: Anti-Τ-antigen against Lex HS 澴 NS at the end of the body NS Table 6: Adding point galactosides from Pneumococci The reaction of the enzyme [EC3.2.1.23 catalog number 1088718, purchased from Boehringer Mannheim, 68298, Manhaming, Germany]. Anti-T-antigen against Lex hang Leb _ HS on the substrate Red NS Green Neil Red Table 7: Reaction after addition of α 1-2 fucoidase: Anti-T-antigen on the substrate Lex Hang Leb HS Chen Erhong, 澴原澴澴原 NS 澴原The Molecular Identification (GMID) card can be constructed from the information collected above. Examples of such messages for sugar HSs are listed in Table 8, and those with sugar NS are listed in Table 9. Table 8 _ Anti-T-antigen on the substrate anti-Lex anti-Leb _ 0 m red, 澴 澴 澴 澴 1 1 — — — — — — — — 尼尔 尼尔 尼尔 尼尔 尼尔 尼尔 尼尔 尼尔 尼尔 尼尔 尼尔 尼尔 尼尔 尼尔 尼尔 尼尔 尼尔 尼尔 尼尔 尼尔 本 本 本 本 本 本 本 本Specifications (210 χ 297 mm) Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed 1306152 A7 B7 3 Neil Red 'Reducing End Reducing End Table 9 Anti-T-antigen against Lex Anti-LeB 0 Green on Green Substrate Red, reducing end 1 - - 2 green Neil red, 3 reducing end The consistency of the second and third essentially sequence specific agents is not disclosed in this data sheet. It is sufficient to always identify the specific code of a particular combination of reagents (1, 2 or 3 in the above table) for comparison purposes. EXAMPLE 2 标 标 澴 澴 澴 澴 澴 澴 之 . . . ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ However, since this standard technique can be extended to sites within the saccharide, it is possible to cost the inventive method by providing more information. Since the inside of the sugar chain can be labeled by labeling the reducing end after cleavage with an endoglycosidase, a labeled reducing end within the sugar chain can thus be obtained. Since the indicated reducing ends in the sugar chain must be closer to the bonding sites of the first, second and third substantially sequence-specific agents than the original reducing end, the internal structure is constructed. The use of the unsuccessful restore side provides additional information. Further, it is possible to sequentially identify the reducing ends by the method described below, and identify the sites of the different glycosidases according to the order of the sugar chains to be studied. In the following steps, the method of sequentially indicating the reducing end is described in more detail. 39 This paper scale applies the Chinese National Standard (CNS) A4 specification (210x297 phantom IT (please read the back note first and then fill this page) 1306152 A7 B7 V. INSTRUCTIONS (γ) 1. Masking: (Please read the notes on the back and then fill out this page) Keep the polysaccharide with reducing end in a solution containing NaBIW NaOH, pH 11.5. This treatment masks the reducing end, so the polysaccharide Now lacking the reducing end (RE) 〇 2. Exposure: The polysaccharide of step 1 is treated with endoglycosidase. If the endogenous glycosidase recognition site is present in the polysaccharide, a new reducing end will be produced by cutting the polysaccharide. The solution now contains two sugars: a fragment of the newly exposed RE at the endoglycosidase site, and a second fragment that is masked by the RE. 1. Labeling the reducing end: This reaction can be performed by using, for example, 2-aminobenzamide Amine (a set sold by Osborne Systems, Inc. to label sugars, shown in the 1994 Catalogue, page 62) was carried out. Under high hydrogen concentration and high temperature (H+/T) conditions, then reduction Reaction After the formation, the mixture contains two kinds of fragments, one of which is the labeling end of the reducing end, and the other is still not marked because of the fact that the reducing end is obscured. The Department of Economic Intelligence, the Intellectual Property Bureau, the employee consumption cooperative, printed the labeling reduction end. Another route is a reductive amination reaction. The arylamine-containing fluorescent compound is reacted with the aldehyde functional group at the reducing end. The resulting CH=N double bond is then reduced to CH2-N by, for example, using sodium borohydride. This technology is part of the FACE (Fluorescence-Assisted Glucose Electrophoresis) kit, which is available from Glyko Corporation of Novato, California, USA, and is described in detail in products such as Glyko. Tables 8-13 (Applicable to the 40th paper scale applicable to China National Standard (CNS) A4 specification (210 X 297 mm) 1306152 A7 Five Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing. ) as a reference) iA second inner sugar and pickling reaction: The second endoglycosidase can then be reacted with the sugar mixture. The new reaction mixture has three fragments, one with a complete reducing end, and the second The reducing end is indicated by 2-aminobenzimidazole, and the third fragment has a reducing end of the mask. Example 3 is a drug that is essentially a sequence-specific one. Structural Messages This example further illustrates the method of the present invention, i.e., by generating a data relating to the structure of the saccharide by using a group reaction as described in more detail. This example further demonstrates that sequence messages can be derived from the set of reactions. In some cases, the reagents used may not be as fully reactive as would be expected from known information (such as those obtained from the catalog). For example, as described below, the source lectin mandala lectin is attached to Sigma. The GlcNac can be linked to the list listed in the catalog. However, in the reaction detailed below, DSA is shown to be bonded to coumarin 120-derived Glc (Glc-AMC). It is apparent that the effect of Glc-AMC is substantially similar to that of GicNac due to the similar structure between the compounds. Further, it is apparent from the following results that the endo-galactosidase used not only cleaves the galactose residue, but also cleaves the bond for linking the Glc-AMC group to the remaining sugar. It will be apparent that the substantially sequence specific agents used in the practice of the present invention, in some instances, may have good specificity that differs from the specificity of the agents shown in the published materials (e.g., catalogues). These reactions can be made by applying the Chinese National Standard (CNS) A4 specification (2i〇X297 mm) to 41 and paper scales; --------------- (Please read the notes on the back and fill out this page) B. 1306152 A7 B7 V. DESCRIPTION OF THE INVENTION The method of the present invention is rapidly identified with sugars of known structure. The resulting results can then be compared to the expected results, and the differences therebetween can be used to identify the different specificities between the substantially sequence specific agents used. The difference between the good specificity of the substantially sequence specific agent and the published data can then be stored to analyze the identity of the unknown carbohydrate structure using such agents. Hereinafter, the present invention will be described using terminally labeled pentoses and various lectins and sugars. The structure of the pentose is GaH(l,4)[Fuc-a (UW-GlcNAc-^djyGal/Sd^yGlc. The pentose is branched at the position of GlcNAc, and the positions 3 and 4 of the GlcNAc are respectively bonded to the ink horn Algal and galactose. The reducing end of the pentose (Glc) is given by coumarin-120 (7-amino-4-methylcoumarin, available from, for example, Sigma, catalog number A 9891) Labeling. The coupling reaction can be carried out by labeling the reducing end with an aromatic amine functional group as above. When coumarin-120 is excited at 312 nm, it emits blue fluorescence. The inner-/5-half emulsion is used. Glycosidase (EG, Berlin Geman & Hamming) and exo-1,3-fucosidase [FD, New England Biolabs] as the first and second essentially sequence specific The reaction conditions of the two reagents are as described in the NEB catalogue for the reaction conditions of the exo-1,3-fucosidase. Three sets of reactions are carried out. The first group contains fucosidase ( FD) and endo-galactosidase (EG), the second group contains only FD, the third group contains only eg. The fourth group of reactions does not contain enzymes, as a control group. For sugars, use thin-layer chromatography (TLC) to size-separate each reaction. 42 This paper scale applies to Chinese National Standard (CNS) A4 specification (210X297 mm) -------- -φ^-- (Please read the notes on the back and fill out this page)

、tT, tT

LP 經濟部智慧財產局員工消費合作社印製 1306152 B7 五、發明説明（([〇) 區分後，可藉由將TLC板曝露於紫外光而檢測該板上 之糖類。結果說明於下文。LP Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed 1306152 B7 V. INSTRUCTIONS (([〇) After separation, the TLC board can be exposed to ultraviolet light to detect the sugar on the board. The results are explained below.

反應4中未添加糖苷酶，因此該糖類係完整的，且在 板上僅移動一小段距離。反應2之片段在分子量上排名第 二’而反應1及3之片段顯示相同的分子量。由此等資料 可以推知，該糖類上之糖苷酶位點之序列爲FD--EG--還原 端(香豆素標籤）。 再藉由更前文所述之一組反應試驗上述戊糖。使用外 源凝集素作爲第一及第二基本上爲序列專一性的藥劑。此 等外源凝集素[Anguilla Anguilla凝集素(AAA)，商品目錄編 號L4141 ;落花生凝集素(PNA)，商品目錄編號L0881 ;篦 麻凝集素(RCA I) ’商品目錄編號L9138 ;兵丑凝集素(LCA) ’商品目錄編號L9267 ; Arbus Precatorius凝集素(APA)，商 品目錄編號L9758]可購自西格瑪。外源凝集素亦可購自其 他公司。例如，RCA I可購自派爾斯，商品目錄編號39913 。外源凝集素係藉由印跡法固定於硝基纖維素濾膜上。 反應緩衝液係帶有1 mM CaCl及1 mM MgCl之磷酸 鹽緩衝鹽液(PBS)。待與凝集素鍵結後，以含於反應緩衝液 中之1% BSA遮蔽瀘膜。使用不含外源凝集素及以1〇微克 BSA作爲固定之蛋白質的反應作爲對照組。 ' 43 本紙張从適财關家標準（CNS ) A4規格（210X297公釐) ^ (請先閱讀背面之注意事項再填寫本頁) 訂 經濟部智慧財產局員χ消費合作社印製 1306152 A7 B7 五、發明説明Of丨） 反應結果示於表9。加號表示存有312 nm螢光，其代 表經香豆素標示之還原端的存在。表中之編號1-4表示上 文定義之反應。 表10 AAA PNA LCA DSA RCA I 1 ++ 2 ++ -h-h ++ 3 -Η- 4 ++ ++ -Η- ++ 由表9(反應4-對照組)所示結果證明外源凝集素AAA 、PNA、DSA及RCA I與糖類鍵結。因此，墨角藻聚糖、 Gal(l-3)GlcNAc、GlcNAc及半乳糖/GalNAc必定存於該糖類 中，此係因彼等糖類分別爲AAA、PNA、DSA及RCA-Ι所 辨識之糖類結構。其進一步證明上述半乳糖苷酶、墨角藻 聚糖苷酶及內-万-半乳糖苷酶辨識該糖類中之***序列。 此等序列分別爲 Fuc-(l-3/l-4)GlcNAc 及 GlcNAc/5(l-3)Gal冷 (l-3/4)Glc/GlcNAc。 由於當使用AAA(鍵結於墨角藻聚糖)作爲固定化之外 源凝集素時，該還原端可被二種糖苷酶之任一者切割，因 此可進一步地推論二糖苷酶位點係座落於墨角藻聚糖與還 原端之間。另一方面，對於含有DSA的反應而言，當使用 DSA作爲固定化藥劑時，由於任一種糖苷酶皆無法切除還 原端’可以推知GlcNAc單糖係位於糖苷酶位點與還原端 之間，或者，該Glc直接鍵結於香豆素。 再者，以PNA作爲固定化藥劑之反應顯示：該還原端 44 ^紙張尺度適用中國國家標準（ CNS了A4規格（210X297公釐） -- (請先閲讀背面之注意事項再填寫本頁) 琴 訂 經濟部智慧財產局員工消費合作社印製 1306152 經濟部智慧財產局員工消費合作杜印製 A7 B7 五、發明説明（fL) 僅有在使用內-/3-半乳糖苷酶(反應1及3)時方能切除。這 表示內-石-半乳糖苷酶位點係位於PNA位點與還原端之間 。另一方面，墨角藻聚糖苷酶位點必定位於PNA位點與糖 類之另一端之間。 當以上述資料作爲考量時，可能提出如下所示之糖類 序列：No glycosidase was added to reaction 4, so the saccharide was intact and moved only a short distance on the plate. The fragment of reaction 2 ranked second in molecular weight' and the fragments of reactions 1 and 3 showed the same molecular weight. From this data, it can be inferred that the sequence of the glycosidase site on the saccharide is FD--EG--reducing end (coumarin label). The above pentose sugar was tested by a group reaction as described above. Exogenous lectins are used as the first and second substantially sequence specific agents. Such lectins [Anguilla Anguilla lectin (AAA), catalog number L4141; groundnut agglutinin (PNA), catalog number L0881; castor lectin (RCA I) 'catalog number L9138; arsenal agglutinin (LCA) 'Product catalog number L9267; Arbus Precatorius lectin (APA), catalog number L9758) is available from Sigma. Exogenous lectins are also available from other companies. For example, RCA I is available from Pyle, catalog number 39913. The lectin is immobilized on a nitrocellulose filter by blotting. The reaction buffer was loaded with 1 mM CaCl and 1 mM MgCl phosphate buffered saline (PBS). After binding to the lectin, the ruthenium membrane was blocked with 1% BSA contained in the reaction buffer. A reaction containing no lectin and 1 μg of BSA as a fixed protein was used as a control group. ' 43 This paper is from the Financial Standards (CNS) A4 specification (210X297 mm) ^ (Please read the note on the back and fill out this page). The Ministry of Economic Affairs, Intellectual Property Bureau, Consumer Cooperatives, Print 1306152 A7 B7 V. DESCRIPTION OF THE INVENTION The results of the reaction are shown in Table 9. The plus sign indicates the presence of 312 nm fluorescence, which represents the presence of the reducing end indicated by the coumarin. Numbers 1-4 in the table indicate the reactions defined above. Table 10 AAA PNA LCA DSA RCA I 1 ++ 2 ++ -hh ++ 3 -Η- 4 ++ ++ -Η- ++ Proved lectin by the results shown in Table 9 (Reaction 4-control) AAA, PNA, DSA, and RCA I bind to sugars. Therefore, fucoidan, Gal(l-3)GlcNAc, GlcNAc, and galactose/GalNAc must be present in the saccharide because they are AAA, PNA, DSA, and RCA-Ι identified sugars. structure. It further demonstrates that the above galactosidase, fucosidase and endo-gal-galactosidase recognize the cleavage sequence in the saccharide. These sequences are Fuc-(l-3/l-4)GlcNAc and GlcNAc/5(l-3)Gal cold (l-3/4)Glc/GlcNAc, respectively. Since the reducing end can be cleaved by either of the two glycosidase when AAA (bonded to fucoidan) is used as the immobilized lectin, the disaccharidase site can be further inferred. It is located between the fucoidan and the reducing end. On the other hand, in the case of DSA-containing reaction, when DSA is used as the immobilization agent, since either glycosidase cannot cleave the reducing end, it can be inferred that the GlcNAc monosaccharide is located between the glycosidase site and the reducing end, or The Glc is directly bonded to the coumarin. Furthermore, the reaction of PNA as an immobilization agent shows that the reduction end 44 ^ paper scale is applicable to the Chinese national standard ( CNS A4 specification (210X297 mm) -- (please read the back note before filling this page) Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed 1306152 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperation Du printing A7 B7 V. Invention Description (fL) Only in use -/3-galactosidase (Reactions 1 and 3 It can be excised. This means that the endo-calcium galactosidase site is located between the PNA site and the reducing end. On the other hand, the fucosidase site must be localized to the PNA site and the sugar. Between one end. When considering the above information, the following sugar sequence may be proposed:

Fuc a (l-3,l-4)GlcNAc(l-3)Gal(l-4)Glc/GlcNAc----還原 端 上述具體例證明本發明方法可以依據相對地少的反應 產生多種資料，包含序列訊息。此序列訊息中的某些細節 可能尙未完成，例如，上述糖類中之墨角藻聚糖與GlcNAc 間之連結爲（1-3)或（1-4)連結。當戊糖之單糖組成已知時， 上述分析才能產生該戊糖的所有詳細資料。不過，相較於 習知技藝方法，即使缺乏單糖組成資料，所得訊息仍然非 常精確。 實例4 衍-生部分或完整的序列訊皁 本發明方法適用於自動化系統。因此，上述步驟，例 如實例1至3，可以使用供混合、等分試樣、反應及檢測 之自動化系統予以進行。然後可以進一步處理藉由此種自 動化方法獲得之資料，以便摺疊出繪製部分或完整序列訊 息之圖譜訊息。下文進一步詳細說明處理此等資料的方法 〇 在冤集所有資料以後，對添加糖苷酶以前之反應獲得 一 .____ 45 尺度適财0®家轉（CNS ) A4規格（21GX297公釐) —- (請先閱讀背面之注意事項再填寫本頁) >裝.Fuc a (l-3, l-4) GlcNAc(l-3)Gal(l-4)Glc/GlcNAc----Reducing end The above specific examples show that the method of the present invention can generate a variety of materials based on relatively few reactions, Contains sequence messages. Some details in this sequence of messages may not be completed. For example, the link between the fucoidan and GlcNAc in the above saccharide is (1-3) or (1-4) linkage. The above analysis produces all the details of the pentose sugar when the monosaccharide composition of the pentose sugar is known. However, the resulting message is very accurate even in the absence of monosaccharide composition data compared to conventional techniques. EXAMPLE 4 Deriving a Partial or Complete Sequence Soap The method of the invention is applicable to automated systems. Thus, the above steps, such as Examples 1 through 3, can be carried out using automated systems for mixing, aliquoting, reaction and detection. The data obtained by such an automated method can then be further processed to fold out the map information for drawing partial or complete sequence information. The method for processing such data is described in further detail below. After collecting all the data, the previous reaction to the addition of glycosidase yielded a .____ 45 scale suitable fortune 0® conversion (CNS) A4 specification (21GX297 mm) --- ( Please read the notes on the back and fill out this page. > Install.

、tT 經濟部智慧財產局員工消費合作社印製 1306152 A7 ___B7____ 五、發明説明（1^丨） 的檢測訊號以及添加糖苷酶（並與之反應）以後之反應獲得 的檢測訊號進行比較。對與糖苷酶反應後消失的彼等訊號 作標記。上述標記動作較佳係藉由製備此等訊號之說明表 (list)而達成，該表於後文稱爲第一說明表。接著可以針對 所輸入之各資料確立該多糖類上兩個位點之身分。於該(視 需要之僞)陣列處之位置表示該第一基本上爲序列專一性的 藥劑。若與糖苷酶反應前已測得訊號，則該藥劑之辨識位 點必定存於該多糖類中。訊號(例如與該第二基本上爲序列 專一性的藥劑有關之訊號)消失，表示該糖苷酶在該第一及 第二基本上爲序列專一性的藥劑之辨識位點間作切割。因 此，辨識位點之順序爲(第一基本上爲序列專一性的藥劑)-( 糖苷酶)-(第二基本上爲序列專一性的藥劑）。若以糖苷酶分 解後，該還原端的訊號仍然存在，則可確立該辨識序列對 應於該還原端的相對順序；否則，必須將兩種可能性(a-b-c 及c-b-a)皆列入考量。爲達說明目的，”第一基本上爲序列 專一性的藥劑之辨識位點”一詞，於下文將記爲”第一辨識 位點”，”第二基本上爲序列專一性的藥劑之辨識位點”一詞 ，將記爲”第二辨識位點”，而”糖苷酶之辨識位點”將記爲” 糖苷酶”。 現在可以建立上述型式之辨識位點三元組(第一型三元 組)的第二說明表： (第一辨識位點Μ糖苷酶)-(第二辨識位點）。 此外，現在可以建立關於在添加糖苷酶之後所有訊號 皆存留(第二型三元組)之(視需要之僞)陣列的位置： 一 46 適用^國國家標準（CNS ) Α4規格（210X297公釐） (請先閲讀背面之注意事項再填寫本頁) &gt;裝. 、1Τ 1306152 A7 B7 五 y4.12.14 ^ re 年月曰 補充 、發明說明（4f) ； · (糖苷酶)-(第一辨識位點)-(第二辨識位點）。 顯然’足量之三元組以分子序列的方式定義分子，亦 即’將僅有一個糖類序列可以含有所有已發現之三元組。 當可以獲得分子長度的訊息時，可能需求較少量的三元組 。若分子中之總糖含量已知，則所需之三元組數目亦可較 少。糖分子量及總單糖含量二者皆可由熟習技藝之人士已 知之先前技藝方法推衍。 獲得序列訊息(亦即，將三元組摺疊成辨識位點之圖譜 )的方法說明如下。 將三元組辨識位點之第二及第三說明圖評估爲一致(三 個辨識位點中有三個相同）、高度類似(三個辨識位點中有 兩個相同）及些微類似（三個辨識位點中有一個相同）。基於 說明之目的，茲假設該多糖爲線性多糖，例如聚糖肝素之 糖類部分。 然後，藉由使用上述第二及第三說明圖製備一組三元 組之說明圖’其中，.於該說明圖組中之各說明圖含有共用. 一種相同糖苷酶辨識序列的三元組。藉由使所有含有特定 糖苷酶辨識序列的三元組與所有含有第二種糖 辨識序 列的三元組進行比較，可以將該多糖序列區分成4個區域 ，其範圍係由分子之第一端至糖穿酶1(片段a)、由糖苦酶 1至糖苷酶2(片段b)、及由糖苷酶2至分子之第二端(片段 c) 1 〈第一端&gt;&lt;糖苷酶1&gt;&lt;糖苷酶2&gt;&lt;第二端&gt; 在第二型三元組中之具有不同糖苷酶位點的相同辨識 47 本纸張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） -----------ΦΜ--------訂---------# (請先閱讀背面之注意事項再填寫本頁)tT Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed 1306152 A7 ___B7____ V. The invention description (1^丨) detection signal and the detection signal obtained by adding glycosidase (and reacting with it) after the reaction. These signals are marked for disappearance after reaction with glycosidase. Preferably, the above marking action is achieved by preparing a list of such signals, which is hereinafter referred to as a first explanatory table. The identity of the two sites on the polysaccharide can then be established for each data entered. The first substantially sequence specific agent is represented by the position at the (as needed pseudo) array. If a signal has been detected before the reaction with the glycosidase, the recognition site of the agent must be present in the polysaccharide. The signal (e.g., the signal associated with the second substantially sequence specific agent) disappears, indicating that the glycosidase cleaves between the recognition sites of the first and second substantially sequence specific agents. Thus, the order of recognition sites is (first essentially sequence specific agent) - (glycosidase) - (second essentially sequence specific agent). If the signal at the reducing end is still present after the glycosidase is decomposed, the relative order of the recognition sequence corresponding to the reducing end can be established; otherwise, both possibilities (a-b-c and c-b-a) must be considered. For illustrative purposes, the term "first identification site for a sequence-specific drug" is hereinafter referred to as "first recognition site", "the identification of a second substantially sequence-specific agent" The term "site" will be referred to as "second recognition site" and "tagsidase recognition site" will be referred to as "glycosidase". A second explanatory table of the above-described type of recognition site triplet (type 1 triple) can now be established: (first recognition site glucosidase) - (second recognition site). In addition, it is now possible to establish the position of the (optional pseudo-) array of all signals remaining after the addition of the glycosidase (type II triad): a 46 applicable national standard (CNS) Α 4 specification (210X297 mm) (Please read the precautions on the back and fill out this page) &gt;装., 1Τ 1306152 A7 B7 5 y4.12.14 ^ re 曰月曰 Supplement, invention description (4f) ; · (glycosidase)-(first identification Site) - (second recognition site). Obviously a 'sufficient triad defines the molecule in a molecular sequence, ie, there will be only one carbohydrate sequence that can contain all the triples found. When a message of molecular length is available, a smaller number of triples may be required. If the total sugar content in the molecule is known, the number of triads required may also be less. Both the molecular weight of the sugar and the total monosaccharide content can be deduced from prior art methods known to those skilled in the art. A method of obtaining a sequence message (i.e., folding a triplet into a map of recognition sites) is described below. The second and third explanatory maps of the triad identification sites were evaluated as identical (three of the three identification sites were identical), the height was similar (two of the three identification sites were the same), and slightly similar (three One of the identification sites is the same). For the purposes of this description, it is assumed that the polysaccharide is a linear polysaccharide, such as a sugar moiety of a glycoheptin. Then, an explanatory diagram of a set of triplets is prepared by using the second and third explanatory diagrams described above, wherein each of the explanatory diagrams in the explanatory diagram group contains a triple of the same glycosidase recognition sequence. By comparing all the triads containing the specific glycosidase recognition sequence to all the triples containing the second sugar recognition sequence, the polysaccharide sequence can be divided into four regions, the first of which is the first end of the molecule. To sugar permease 1 (fragment a), from glycoprotein 1 to glycosidase 2 (fragment b), and from glycosidase 2 to the second end of the molecule (fragment c) 1 <first end> &lt;glycosidase 1&gt;&lt;Glycosidase 2&gt;&lt;2nd&gt; The same identification with different glycosidase sites in the second type of triplet 47 This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) -----------ΦΜ--------Book---------# (Please read the notes on the back and fill out this page)

1306152 五、發明說明（f) 位點(其中該等辨識位點係位於相對於各糖苷酶位點之相同 方向者)依其所在位置，係區域a或c中之候選位置。在第 二型三元組中具有不同糖苷酶位點的相同辨識位點之其中 該等辨識位點係位於不同方向者(例如’其中一種二兀組係 於還原端之方向’另一種三元組係於非還原端之方向），係 區域b(亦即，位於兩種糖脊酶位點之間）中之候選位置。 在第一型三元組中之具有不同糖苷酶位點的相同辨識 位點，係區域a(或c)中之第一或第二辨識位點中之一者的 候選位置’而該第一或第二辨識位點中之另一者係位於區 域c(或a)。亦即’若該第一或第二辨識位點中之一者係位 於區域a ’則該第一或第二辨識位點中之另一者必定位於 區域c，反之亦然。該等第一或第二辨識位點不可能位於 區域b。 在第一型三元組中具有不同糖苷酶位點的相同辨識位 點(其中一指定之辨識位點係以還原端之方向存於該等三元 組中之一者’而以非還原端之方向存於另一三元組之中）， 係區域b中之該辨識位點的候選位置° 當該等三元組中之若干辨識位點的上述位置相關性已 確定後，可使用邏輯推理，對該所有的辨識序列作一特定 方向的排序。此階段稱爲序列圖譜°若排序足量之辨識序 歹,則可由而推衍出該糖類之全部序列。由於該方法無法 確定糖分子量，故鏈長未知。因此，若各辨識位點間之重 疊度不足’則於該序列之某些區域中可能有其他糖單元存 在。若此等糖單元未落於所用之任一種基本上爲序列專一 48 (請先閱讀背面之注意事項再填寫本頁) ----- -丨丨訂- ------- 1306152 A7 _____B7 _ 五、發明説明（年(） (請先閲讀背面之注意事項再填寫本頁) 性之藥劑的辨識位點內，則無法被檢測出。不過，在此情 形下，亦可藉由先獲得代表糖鏈長的分子量，再獲得其總 單糖含量，而得到完整的序列訊息。 另一種縮小序列圖譜中之間隙的可能性係實例2之方 法，該方法係使用糖苷酶依序分解，而推衍出序列訊息。 經濟部智慧財產局員工消費合作社印製 存於糖類中之分支點可能如上文之引言般使本方法複 雜化。關於此點之補救方法係使用糖苷酶製備分子斷片， 並分析此等部分結構。於此等部分結構中之分支含量明顯 低於整個分子中之含量。此外，可以使用專一地辨識分支 點的試劑。此等試劑之實例係例如上文實例1中所用之抗 體。此等抗體中之每一者鍵結於含有至少一個分支點的一 種糖類序列。再者，可以獲得辨識分支之糖類結構的特定 酵素及外源凝集素。例如酵素支鏈澱粉酶(EC 3.2.L41)辨識 分支結構。此外，可藉由使用分支之糖結構作爲抗原，生 成抗體。再者，可以產生能鍵結特定糖類結構(包含分支之 糖類結構）的胜肽（參照，Deng SJ、MacKenzie CR、 Sadowska J ' Michniewicz J ' Young NM ' Bundle DR ' Narang ; Selection of antibody single-chain variable fragments with improved carbohydrate binding by phage display. J. Biol. Chem· 269 ， 9533-38 ， 1994) 〇 此外，現存之糖類結構的知識將於許多情形下精確地 預測分支點的存在。例如，N-連結之聚糖具有有限數量的 結構，如奧斯佛糖系統公司之商品目錄第6頁所示者。此 等結構之範圍係單分支(monoantennary)至五分支。較複雜 49 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1306152 94 1 年月日1,t止 _二補充_ 五、發明說明（q) 之結構類似於較簡單之結構，但具有其他外加之糖類殘基 。因此，若鑑定爲單分支之結構，則可藉由鑑定該等外加 之殘基，並使此等資料與N-連結之聚糖結構資料庫比較， 而簡單地預測更複雜之結構中的分支點。 尤其甚者，藉由分析依本發明方法蒐集之資料，經常 可以邏輯性地推衍分支點之存在及位置。例如，若兩個辨 識位點(記爲5及约係位於不同分支上，以位點位於還原端 與分支點之間的糖苷酶分解後，將導致還原端標記喪失。 然而，兩個辨識位點a及6之標記仍將存留。若使用位於 分支點與辨識位點a之間的糖苷酶，則辨識位點6與還原 端標記將被切除。不考慮分支點之可能性時，此將表示辨 識位點6係位於辨識位點a與還原端之間。然而，若使用 位於辨識位點6與分支點與之間的糖苷酶，則還原端標記 與辨識位點s將被切除。同樣地，不考慮分支之可能性時 ，此將表示辨識位點2係位於還原端與辨識位點6之間。 此等推論顯然彼此不互容，因此僅於假設辨識位點a與 係位於兩個不同的分支上時，方可被用於解釋。該分支點 係位於辨識位點^與6及上述之第一個糖苷酶之間。上文 所用之其他糖苷酶係各位於介於分支點與各辨識位點〇或 W之間的分支上。 因此，當使用本發明方法中之辨識分支結構的藥劑作 爲基本上爲序列專一性的藥劑時，可能推衍出糖分子中之 分支點的存在及位置的相關訊息。接著可使用此訊息構築 該結構之各分支的序列圖譜，而產生完整之分支結構的序 50 Ξ dj 阳阳它神;隹 /Γ'ΚΚ：、Λ is / 01 η V 907 Λν ^ \ -----------裝--------訂-------- (請先閱讀背面之注意事項再填寫本頁) 1306152 A7 B7 五、發明説明（4分） (請先閲讀背面之注意事項再填寫本頁) 列圖譜。於此結構中之間隙因而可如未分支之糖類的情形 般，依本發明，亦即，藉由使用其他反應、藉由以糖苷酶 分解而縮小，由而使得間隙存在之分子區域被專一地分離 ，以依本發明方法，並藉由如更前文所述之依序以糖苷酶 分解，進一步分析。 總括言之，如本發明之測定糖類序列及/或繪製該糖類 結構之圖譜的方法包括下列步驟= 1. 蒐集第一及第二型三元組 2. 依相似性，對該等三元組進行分類 3. 以不同的糖苷酶辨識位點，對該等三元組進行比較 4. 依於該糖類上之發生順序排序該等三元組 5. 排序該等糖苷酶辨識位點 6. 檢查該等三元組之相容性 7. 以單一順序方向排序糖苷酶與第一及第二基本上爲 序列專一性的藥劑之辨識序列 8. 將該等辨識序列(位點)轉譯成多糖序列 9. 校正”重疊”之問題 10. 輸出序列 經濟部智慧財產局員工消費合作社印製 11. 再次檢查所有可得之資料 在進行上述之步驟5之後，建立糖苷酶位點之初步順 序。接著於步驟6檢查所預測之各三元組是否與上述順序 一致。然後依據資料之矛盾處，生成與該三元組資料相符 之新模型。再以所有三元組之資料測試該模型。接著進行 更多反應，以便取得更多涉及該三元組之相關辨識位點的 51 本紙張尺度適用中國國家標準（CNS ) A4规格（210X297公釐） 1306152 A7 B7 五、發明說明（”） 矢量訊息。 待進行上述步驟8(其中，該依序排列之辨識位點係被 轉譯成實際之單糖單元)之後，可以提出糖類序列之模型。 爲了測試該模型，必須回答若干問題。第一個問題爲：何 者將是尙具有此相同之序列圖譜的最短序列？於此階段， 不須考量分子量及單糖組成之訊息(若可獲得）。此途徑僅 供建立結合了所有可獲得之具有儘可能地少的矛盾之資料 的序列。關於此點，第二個必須回答的問題係：是否此最 短序列仍與在該觀點上所有可得之資料相符(排除視需要之 分子量及單糖組成資料）？第三個必須回答的問題係：是否 有適合所建立之序列圖譜的其他序列存在？若答案是肯定 的，則接著可以使用下述問題測驗彼等外加序列：各序列 模型與三元組訊息，以及與其他視需要之資料如於分子量 、單糖組成、及生物學已知之典型糖類結構上之訊息的相 符程度如何？ 最後，將以所有三元組、單糖組成、在糖分子量及結. 構組成上之先前技藝、及由生物學上存在之類似結構所爲 之預測，對依上述步驟1-10發現之最佳的序列模型進一步 測試。藉由此重複試驗，檢視出所得資料與序列模型間之 矛盾處，若可能，改良該序列模型，使之更能代表該等資 料。 實例5 乳品樣本之.糖分子鑑別(GMID)分析 此實例之目的在於證明GMID技術可用於分析及比較 52 --------裝！丨 — 丨—訂--------- (請先閱讀背面之注意事項再填寫本頁) 大秘桀尺疳谪用由圃®犮棵進規格（210 X 297公穿） 1306152 A7 B7 五、發明説明（P) 乳品樣本。 A.膜及第一層外源凝集素： 下文說明之實驗中所用的支承面(supporting surface)係 硝基纖維素膜。該膜之製法如下： 1. 切取硝基纖維素膜，將其上表面劃分成9 X 6個方塊 (每個方塊爲3平方毫米)的陣列。將該等膜置於吸收用紙 上，並以筆標示每張膜左上方的方塊。 2. 將凍乾的外源凝集素再懸浮於水中，使其終濃度爲1 毫克/毫升。該再懸浮之外源凝集素（及對照用溶液：5%牛 血淸白蛋白）經予振盪混合，各溶液取1微升加至印跡膜 (blot)上之28個方塊(藉由在下文舉例說明之基本印跡膜畫 (請先閱讀背面之注意事項再填寫本頁) 訂 上陰影而表示)之一。1306152 V. DESCRIPTION OF THE INVENTION (f) Sites (wherein the recognition sites are in the same orientation relative to each glycosidase site) are the candidate positions in region a or c depending on their location. In the second type of triplet, the same recognition sites with different glycosidase sites, wherein the recognition sites are located in different directions (for example, 'one of the two groups is in the direction of the reducing end' and another ternary The group is in the direction of the non-reducing end) and is the candidate position in the region b (i.e., between the two sugar spine enzyme sites). The same recognition site having a different glycosidase site in the first type of triplet, the candidate position of one of the first or second recognition sites in the region a (or c) and the first Or the other of the second recognition sites is located in region c (or a). That is, if one of the first or second recognition sites is located in the region a' then the other of the first or second recognition sites is located in the region c, and vice versa. The first or second recognition sites are unlikely to be located in region b. The same recognition site having different glycosidase sites in the first type of triplet (one of the designated recognition sites is present in the direction of the reducing end in one of the triads) and the non-reducing end The direction is stored in another triad), the candidate position of the identification site in region b. When the above positional correlation of several recognition sites in the triples has been determined, logic can be used. Reasoning, sorting all the identification sequences in a particular direction. This phase is called the sequence map. If a sufficient number of identification sequences are sorted, the entire sequence of the sugar can be derived. Since the method cannot determine the molecular weight of the sugar, the chain length is unknown. Therefore, if the overlap between the recognition sites is insufficient, then there may be other sugar units in some regions of the sequence. If these sugar units do not fall in any of the sequences used, they are basically sequence specific 48 (please read the notes on the back and fill out this page) ----- -丨丨定- ------- 1306152 A7 _____B7 _ V. Invention description (Year () (Please read the note on the back and fill in this page) The identification of the drug can not be detected. However, in this case, you can also use The molecular weight representing the length of the sugar chain is obtained, and the total monosaccharide content is obtained to obtain a complete sequence message. Another possibility of narrowing the gap in the sequence map is the method of Example 2, which is sequentially decomposed using glycosidase. The sequence message is derived. The branch of the Ministry of Economic Affairs' Intellectual Property Office employee consumption cooperative printed in the sugar may complicate the method as described above. The remedy for this is to use glycosidase to prepare molecular fragments. And analyze the partial structures. The branch content in these partial structures is significantly lower than the content in the entire molecule. In addition, reagents that uniquely identify the branch points can be used. Examples of such reagents are, for example, The antibody used in Example 1. Each of these antibodies is bonded to a saccharide sequence containing at least one branch point. Further, specific enzymes and lectins which recognize the saccharide structure of the branch can be obtained, for example, an enzyme branch. The amylase (EC 3.2.L41) recognizes the branched structure. In addition, the antibody can be produced by using a branched sugar structure as an antigen. Further, a peptide capable of binding a specific saccharide structure (including a branched saccharide structure) can be produced. (Reference, Deng SJ, MacKenzie CR, Sadowska J 'Michniewicz J 'Young NM 'Bundle DR ' Narang ; Selection of antibody single-chain variable fragments with improved carbohydrate binding by phage display. J. Biol. Chem· 269 , 9533-38 , 1994) In addition, the knowledge of existing carbohydrate structures will accurately predict the existence of branching points in many cases. For example, N-linked glycans have a limited number of structures, such as the catalogue of Osborne Systems. The pages shown on page 6. The range of these structures is monoantennary to five branches. More complex 49 paper scales for Chinese countries Quasi (CNS) A4 specification (210X297 mm) 1306152 94 1st month, day 1, t _ _ _ _ 5, invention description (q) The structure is similar to the simpler structure, but with other added sugar residues. Thus, if identified as a single-branched structure, the branches in a more complex structure can be simply predicted by identifying the additional residues and comparing the data to the N-linked glycan structural database. point. In particular, by analyzing the data collected by the method of the present invention, it is often possible to logically derive the existence and location of the branch points. For example, if two recognition sites (denoted as 5 and the system are located on different branches, the glycosidase located between the reduction end and the branch point is decomposed, resulting in loss of the reducing end label. However, two recognition bits The markers at points a and 6 will remain. If the glycosidase between the branch point and the recognition site a is used, the recognition site 6 and the reducing end marker will be deleted. When the possibility of the branch point is not considered, this will It is indicated that the recognition site 6 is located between the recognition site a and the reduction site. However, if a glycosidase located between the recognition site 6 and the branch point is used, the reduction end label and the recognition site s will be cleaved. Ground, regardless of the possibility of branching, this would indicate that the recognition site 2 is located between the reducing end and the recognition site 6. These inferences are clearly incompatible with each other, so only the hypothetical identification site a and the system are located at two It can be used for interpretation on different branches. The branch point is located between the recognition sites ^ and 6 and the first glycosidase described above. The other glycosidase systems used above are located at the branch point. On the branch between each recognition site 〇 or W Therefore, when the agent for identifying the branched structure in the method of the present invention is used as a substantially sequence-specific agent, it is possible to derive a message related to the existence and position of the branch point in the sugar molecule. This message can then be used to construct the The sequence map of each branch of the structure, and the order of the complete branch structure is generated 50 Ξ dj Yang Yang Ito; 隹 / Γ 'ΚΚ:, Λ is / 01 η V 907 Λν ^ \ --------- --Install -------- Order --------- (Please read the back note and then fill out this page) 1306152 A7 B7 V. Invention description (4 points) (Please read the back Precautions. Fill in this page. Columns. The gaps in this structure can thus be as in the case of unbranched sugars, in accordance with the invention, that is, by using other reactions, by being reduced by glycosidase, by The molecular regions in which the gaps are present are specifically separated for further analysis according to the method of the present invention and by sequential glycosidase decomposition as described above. In summary, the sugar sequence is determined according to the present invention and/or Or the method of drawing the map of the saccharide structure includes the following steps: 1. Collecting the first and second types of triplets 2. Sorting the triplets according to similarity 3. Identifying the triplets with different glycosidase recognition sites 4. Depending on the sugars Sorting the triplets in order of occurrence 5. Sorting the glycosidase recognition sites 6. Checking the compatibility of the triplets 7. Sorting the glycosidase in a single sequential direction with the first and second substantially sequences Specific identification sequence of the drug 8. Translating the identification sequence (site) into a polysaccharide sequence 9. Correcting the problem of "overlap" 10. Output Sequence Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed 11. Recheck all The resulting data is subjected to step 5 above to establish a preliminary sequence of glycosidase sites. Next, in step 6, it is checked whether the predicted triplets are consistent with the above order. Then, based on the contradiction of the data, a new model corresponding to the data of the triad is generated. The model was tested with data from all triples. Then more reactions are carried out to obtain more of the relevant identification sites involving the triad. The paper size is applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) 1306152 A7 B7 V. Invention Description (") Vector After the above step 8 (where the sequentially identified recognition sites are translated into actual monosaccharide units), a model of the carbohydrate sequence can be proposed. In order to test the model, several questions must be answered. The question is: Which one will be the shortest sequence with this same sequence map? At this stage, there is no need to consider the molecular weight and monosaccharide composition information (if available). This approach is only for the purpose of establishing a combination of all available A sequence of potentially contradictory data. The second question that must be answered is whether this shortest sequence is still consistent with all available data at that point of view (excluding the molecular weight and monosaccharide composition as needed) The third question that must be answered is: Is there any other sequence that fits the established sequence map? If the answer is yes, then The additional sequences can be tested using the following questions: how well each sequence model and triplet message, and how well it meets other desirable data such as molecular weight, monosaccharide composition, and information on typical sugar structures known in biology. Finally, it will be predicted by all the triads, monosaccharide composition, previous techniques of sugar molecular weight and structure, and similar structures that exist biologically. The best sequence model is further tested. By repeating the test, the contradiction between the obtained data and the sequence model is examined, and if possible, the sequence model is modified to make it more representative of the data. Example 5 Dairy sample. Sugar Molecular Identification (GMID) Analysis The purpose of this example is to demonstrate that GMID technology can be used to analyze and compare 52 -------- loaded! 丨 丨 订 - order--------- (please read the back Precautions and then fill out this page.) 桀 圃 疳谪 ( ( ( ( 210 210 210 210 210 210 210 210 210 210 210 210 210 210 210 210 210 210 210 210 210 210 210 306 306 306 306 306 306 306 306 306 306 306 306 306 306 306 306 306 306 Lectin: The following description The supporting surface used in the process is a nitrocellulose membrane. The membrane is prepared as follows: 1. The nitrocellulose membrane is cut and the upper surface is divided into 9 X 6 squares (each square is 3 square millimeters). Array of the membranes, place the membranes on the absorbent paper, and mark the squares at the top left of each membrane with a pen. 2. Resuspend the lyophilized lectin in water to a final concentration of 1 mg/ml. The resuspended exogenous lectin (and the control solution: 5% bovine blood albumin) was mixed by shaking, and 1 μL of each solution was added to 28 blocks on the blot (by One of the basic imprinted film paintings (please read the note on the back and then fill out this page).

此實驗中所用之外源凝集素列於表11。 經濟部智慧財產局員工消費合作社印製 表11 外源凝集素 製造商 商品目錄編號 WGA 載體(Vector) MK2000 SBA 載體 MK2000 PNA 載體 MK2000 DBA 載體 MK2000 UEAI 載體 MK2000 CON A 載體 MK2000 RCA I 載體 MK2000 BSLI 載體 MK2OO0 一 SJA 載體 MK3000 LCA 載體 MK3000 53 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1306152 五、發明说明（) A7 ______B7The exogenous lectins used in this experiment are listed in Table 11. Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperatives Printed Table 11 Exogenous Lectin Manufacturers Catalog Number WGA Vector (Vector) MK2000 SBA Carrier MK2000 PNA Carrier MK2000 DBA Carrier MK2000 UEAI Carrier MK2000 CON A Carrier MK2000 RCA I Carrier MK2000 BSLI Carrier MK2OO0 A SJA carrier MK3000 LCA carrier MK3000 53 This paper scale applies to China National Standard (CNS) A4 specification (210X297 mm) 1306152 V. Invention description () A7 ______B7

Swga 載體 MK3000 PHA-L 載體 MK3000 ' PSA 載體 MK3000 AAA - PHA-E 載體 MK3000 PNA 魯賓(Leuven) LE-408 LCA 西格瑪 L9267 DSA 西格瑪 L2766 APA - WGA 魯賓 LE-429 傑卡林(Jacalin) __mm_ LE-435 5% BSA 沙比昂(Savyon) M121-033 3.將所製之印跡膜置於90 mm培養皿中。 4. 藉由在各培養皿中添加10毫升任一種熟習技藝之人 士周知之適當的遮蔽用溶液(例如，5%牛血淸白蛋白）遮蔽 該等印跡膜。 5. 將含有存於遮蔽用溶液中之印跡膜的培養皿置於旋 轉上，在室溫下藉由旋轉(50 rpm)，緩緩搖振2 '小時(或 於4°C放置隔夜，不旋轉）。 經濟部智慧財產局員工消費合作社印製 (請先閱讀背面之注意事項再填寫本頁) k. 6. 接著在各培養皿中添加10毫升洗液，以洗滌該等印 跡膜。任一種常備之緩衝溶液(如磷酸鹽緩衝溶液)可用於 進行該洗滌步驟。該等培養皿藉由緩緩旋轉(50 rpm)5分鐘 予以洗滌。此步驟一共進行3次，每次皆傾出舊洗液，並 置換以新鮮溶液。 B.添加乳品樣本： 所用之乳品樣本如下： 1. 牛之超高溫滅菌保久乳(3%)，購自以色列之Ramat haGolan乳品公司（批號522104); 2. 低溫滅菌羊乳’購自以色列之Mechek乳品公司（批 54 本紙張又度適用中國國家標準（CNS ) A4規格（2丨〇&gt;&lt;297公釐） 1306152 A7 ___B7___ 五、發明説明（P_) 號1及2); (請先閲讀背面之注意事項再填寫本頁) 3.未經滅菌之羊乳，購買所同2(批號3及4)。 乳品樣本稀釋至10%體積/體積，於每一印跡膜添加 5毫升之各樣本。 製備前述各乳品樣本之複製印跡膜。此外’製備另一 對不添加糖類之印跡膜(負對照組）。 接著使該等印跡膜於室溫搖振下保溫1小時。 C.呈色外源凝集素： 由先前對於受試乳品之單糖組成的了解’以及藉由依 下文實例7所述之演算法爲基礎的電腦程式之應用’選擇 下列呈色外源凝集素：Con A ’ VVA。 於洗液中製備該二種外源凝集素之混合物，使得彼等 呈色外源凝集素之濃度爲2毫克/毫升。 .1# 使500微升各外源凝集素混合物於如上文製備之印跡 膜上保溫。藉由測定螢光黃於520 nm處之螢光，以及(於 生物素化之外源凝集素的情形下)測定生物素與HRP-鏈黴 凡淀(streptavidin)溶液反應後產生之TMB藍色之訊號，而 讀取各印跡膜。 經濟部智慧財產局員工消費合作社印製 由經FITC標示及經生物素標示之外源凝集素獲得之 結果，分別示於表12及表13。此等表中所示之結果以〇 至3級計量，其中，〇表示在雜訊層級以下之訊號，而1-3 之結果表示扣除不含糖類之對照組所得結果後之正訊號(雜 訊以上）。 由此等有關低溫滅菌羊乳(批號1及2)、未經滅菌之羊 55 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐）Swga carrier MK3000 PHA-L carrier MK3000 ' PSA carrier MK3000 AAA - PHA-E carrier MK3000 PNA Rubin LE-408 LCA Sigma L9267 DSA Sigma L2766 APA - WGA Rubin LE-429 Jacallin __mm_ LE -435 5% BSA Savyon M121-033 3. Place the blotted membrane in a 90 mm Petri dish. 4. The blotting membranes are masked by adding 10 ml of any appropriate masking solution (e.g., 5% bovine blood albumin) known to those skilled in the art. 5. Place the petri dish containing the blotting membrane stored in the masking solution on the rotation and slowly shake it for 2 'h at room temperature by rotating (50 rpm) (or overnight at 4 °C, no Rotate). Printed by the Intellectual Property Office of the Ministry of Economic Affairs and the Consumer Cooperatives (please read the notes on the back and fill out this page) k. 6. Next, add 10 ml of the washing solution to each dish to wash the printed films. Any of the standing buffer solutions (e.g., phosphate buffer solutions) can be used to carry out the washing step. The dishes were washed by gentle rotation (50 rpm) for 5 minutes. This step was performed a total of 3 times, each time the old lotion was poured out and replaced with a fresh solution. B. Adding dairy samples: The dairy samples used are as follows: 1. Cattle UHT long-lasting milk (3%) purchased from Ramat haGolan Dairy Company of Israel (batch number 522104); 2. Low temperature sterilized goat milk 'purchased from Israel Mechek Dairy Company (Batch 54 papers are again applicable to China National Standard (CNS) A4 specifications (2丨〇&gt;&lt;297 mm) 1306152 A7 ___B7___ V. Invention Description (P_) Nos. 1 and 2); (Please first Read the precautions on the back and fill out this page. 3. 3. Unsterilized goat's milk, purchase the same 2 (lots 3 and 4). The dairy sample was diluted to 10% by volume/volume, and 5 ml of each sample was added to each blotting membrane. A replicated blotting membrane of each of the aforementioned dairy samples was prepared. In addition, another pair of imprinted membranes without a saccharide (negative control group) was prepared. The blotting membranes were then incubated for 1 hour at room temperature with shaking. C. Color-presenting lectin: The following color-extracting lectins were selected from the previous understanding of the monosaccharide composition of the tested dairy products and the application of computer programs based on the algorithm described in Example 7 below: Con A 'VVA. The mixture of the two exogenous lectins was prepared in a wash so that the concentration of the color lectin was 2 mg/ml. .1# 500 μl of each lectin mixture was incubated on the blotting membrane prepared as above. By measuring the fluorescence of fluorescein at 520 nm and (in the case of biotinylated lectins) the TMB blue produced by the reaction of biotin with HRP-streptavidin solution The signal is read, and each blot film is read. Printed by the Intellectual Property Office of the Ministry of Economic Affairs, the Consumers' Cooperatives. The results obtained by FITC and biotin-labeled lectins are shown in Tables 12 and 13, respectively. The results shown in these tables are measured on a scale of 33, where 〇 represents the signal below the level of the noise level, and the result of 1-3 represents the positive signal after the result of subtracting the control from the sugar-free control group (noise) the above). This is related to low-temperature sterilization of goat milk (lots 1 and 2), unsterilized sheep 55 paper size applicable to China National Standard (CNS) A4 specification (210X297 mm)

1306152 五 、發明說明（$&gt; 乳(批號3及4)及牛乳之結果獲得之糖分子鑑別(GMID)卡示 於圖1(分別爲A至E)。外源凝集素1至24之位置於每張 卡1頂端由左向右成列表示。 D.結果說明： 產生GMID之乳品樣本表示，於此等樣本中之多糖含 有對於專一辨識下列單糖之外源凝集素產生正結果之糖類 a. 葡萄糖/甘露糖(Con A、PSA及LCA); b. GlcNac(WGA 及 DSA)。 低溫滅菌羊乳樣本對於下列單糖產生正結果： a. 葡萄糖/甘露糖(Con A、PSA及LCA); b. GlcNac(DSA)。 未觀察到此等受試批號間之外源凝集素反應性的差異 未經滅菌之羊乳樣本對於下列單糖產生正結果： a. 葡萄糖/甘露糖(Con A、PSA及LCA); b. GlcNac(DSA)。 總括而言，牛乳樣本與羊乳樣本間之差異僅在於前者 與WGA反應。經低溫滅菌與未經滅菌之羊乳樣本之間’ 除了在低溫滅菌羊乳之訊號強度明顯較低以外，基本上# 無差異。 56 (請先閱讀背面之注意事項再填寫本頁) 裝--------訂-----I--. 1306152 A7 B7 經濟部智慧財產局員工消費合作社印製 五、發明説明（5^f) 表12 — 一 — — 一 _ — — 一 —· — m ίΝ Ο ο — — o — Ο 〇 〇 〇 o 〇 (Ν CM &lt; : - = : = - = : = . _ CM &lt; - - - . - :- - = = - : 另 &lt; 2 - - - . : = = = = = _ CTv C . .: _ _ - - : - - _ : CO 一 一 m m (N CN CN CN CN (N CN 卜 ο 〇 — 〇 o Ο 〇 一 — —· — VO ο 〇 〇 〇 o o Ο 〇 〇 〇 〇 〇 &lt; : - - - = - = = - - 守 ο 〇 o o o o ο 〇 〇 o o 〇 cn CN CM fN (N CN CN CN &lt;N &lt;N CN fN &lt;N CN CN fN CN fN (N CN CN (N CN CN CN CN — — CN CN CN (N CN CN CN CN CN CN CN CN 〇 Ο Ο — — CN CN cv Ο ο o 〇 〇 § Ο Ο _ _ 一 一 OQ m m m cn rn rn m cn rn rn r*· rn m m rn rn cn m ΓΛ m rn m ό Ο Ο 一 — o 〇 O Ο O O o Ο CN cs CN CN cs CN C^J CN CN CN CN CN m m m cn cn m fn r〇 m m cn m rn ο ο o o o o o Ο o o O ο CN rn m rn cn rn m m rn m — Ο Ο — 一 — 一 一 - &lt;N CN 04 &lt;N 账 * 槩 枭 φ 枭 φ cn •3画 S麗 济 彐麵 —璲 =tt 於 彐画 裂籐 3翻 &lt;N域 肿圃 3蘧 S ^ 埤画 彐媛 iu =tt πς 件麵 茗1 埤画 彐垮 寸 4^i =tt tf\ 57 (請先閲讀背面之注意事項再填寫本頁) 攀裝·1306152 V. Description of Invention ($&gt; Milk (Batch No. 3 and 4) and Milk Molecular Identification (GMID) card obtained as shown in Figure 1 (A to E, respectively). Location of lectin 1 to 24 The top of each card 1 is shown from left to right. D. Results Description: The GMID-derived dairy samples indicate that the polysaccharides in these samples contain sugars that produce positive results for lectins that specifically recognize the following monosaccharides. a. Glucose/mannose (Con A, PSA and LCA); b. GlcNac (WGA and DSA). Low temperature sterilized goat milk samples produce positive results for the following monosaccharides: a. Glucose/mannose (Con A, PSA and LCA) b. GlcNac (DSA). Differences in lectin reactivity between these test lots were not observed. Unsterilized goat milk samples produced positive results for the following monosaccharides: a. Glucose/mannose (Con A, PSA and LCA); b. GlcNac (DSA). In summary, the difference between the milk sample and the goat milk sample is only the former reaction with WGA. Between the low temperature sterilization and the unsterilized goat milk sample The signal intensity of low temperature sterilized goat milk is significantly lower, basically # no difference. 56 (Read first Note: Please fill out this page) Pack--------Book-----I--. 1306152 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing 5, invention description (5^f) Table 12 — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — - :- - = = - : Another &lt; 2 - - - . : = = = = = _ CTv C . .: _ _ - - : - - _ : CO 一一mm (N CN CN CN CN (N CN 卜ο〇— 〇o Ο 〇 — — — — — VO ο 〇〇〇oo Ο 〇〇〇〇〇&lt; : - - - = - = = - - 守ο 〇oooo ο 〇〇oo 〇cn CN CM fN (N CN CN CN &lt;N &lt;N CN fN &lt;N CN CN fN CN fN (N CN CN (N CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN — — CN CN cv Ο ο o 〇〇§ Ο Ο _ _ 一一OQ mmm cn rn rn m cn rn rn r*· rn mm rn rn cn m ΓΛ m rn m ό Ο Ο 一 — o 〇O Ο OO o Ο CN cs CN CN cs CN C^J CN CN CN CN CNm cn cn m fn r〇mm cn m rn ο ο ooooo Ο oo O ο CN rn m rn cn rn mm rn m — Ο Ο — 一—一一一- &lt;N CN 04 &lt;N Account* 槩枭φ 枭φ cn •3画S丽济彐面—璲=tt 彐 彐 彐藤3翻&lt;N domain swollen 3蘧S ^ 埤画彐媛iu =tt πς 茗面茗1 埤画彐垮 inch 4^i =tt tf\ 57 (Please read the back note first and then fill out this page ) Climbing ·

、1T I ^fluB λΓιιν m 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1306152 A7 __^_B7 五、發明説明（ 13 ml 經濟部智慧財產局員工消費合作社印製 〇 〇 CM CN CN CN CN fN (N CN (N (Ν (*Λ CN 〇 〇 οι CN — 一 一 一 〇 〇 Ο Ο CN CN &lt; CN &lt; &lt; σ\ &lt; - OQ 〇 〇 CN CN CN CN &lt;N CN 0-1 CN CN Oi 卜 〇 〇 CN 〇 〇 Ο 〇 d：:- *〇 o Ο Ο Ό 〇 〇 〇 o o Ο o O o o Ο Ο &lt; 'g* 〇 〇 —· 一 一 一 — o o o ο ο m g 〇 &lt;N 一 一 一 二 〇 〇 〇 cs CN CN as 〇 〇 一 一 *— — oa 一 fN &lt;N (N CN CN iN — 一 一 一 r** —· 一 CN CN CN CN CN CM (N CN CN CN Ό 〇 〇 &lt;N CN CN CN CS — - — — —1 ^r — — CN (N (N CN CN (N CN CN CN CN 〇 〇 〇 〇 〇 〇 〇 〇 〇 〇 Ο Ο CN 〇 〇 CN fN fN (M rs CN CN CN (N CN — 〇 〇 fN CS CN CS CN &lt;N tN fN CN CN 账 . 滅 戰 凝 铟 m m 4. tr m 3麵 -铤 济 4iL Ί1Τ 3® ttf 3S 街_ 3籐 m IJZ τΚ 济画 通 杜麵 3蘧 呀JJZ =tt τκ Hf画 3孃 n 衣 、?τ (請先閲讀背面之注意事項再填寫本頁) 本紙張尺度適用中國國家榇準（CNS ) A4規格（2〗〇X297公釐） 1306152, 1T I ^fluB λΓιιν m This paper scale applies Chinese National Standard (CNS) A4 specification (210X297 mm) 1306152 A7 __^_B7 V. Invention description ( 13 ml Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing 〇〇CM CN CN CN CN nN (N (Ν (*Λ CN 〇〇οι CN - 一一一〇〇Ο Ο CN CN &lt; CN &lt;&lt; σ\ &lt; - OQ 〇〇CN CN CN CN &lt; N CN 0-1 CN CN Oi 〇〇 〇〇 〇〇Ο : d::- *〇o Ο Ο 〇〇〇 〇〇〇oo Ο o O oo Ο Ο &lt; 'g* 〇〇—· 十一—ooo ο ο mg 〇&lt;N 一一二〇〇〇cs CN CN as 〇〇一一* — oa a fN &lt;N (N CN CN iN — 十一一 r** —·一CN CN CN CN CN CM (N CN CN CN Ό 〇〇&lt;N CN CN CN — — — — 1 ^r — — CN (N (N CN CN (N CN CN CN CN 〇〇〇〇〇〇〇〇〇〇 Ο Ο CN 〇〇CN fN fN (M rs CN CN CN (N CN — 〇 〇fN CS CN CS CN &lt;N tN fN CN CN 帐. 战战 indium mm 4. tr m 3 face - 4 4iL Ί1Τ 3® ttf 3S street _ 3 vine m IJZ τΚ 济画通杜面3蘧JJZ =tt τκ Hf draw 3 Niang n clothing, ?τ (please read the back note first and then fill out this page) This paper scale applies to China National Standard (CNS) A4 specification (2〗 〇 X297 mm) 1306152

五、發明說明（g) 實例6 脂多糖類之糖分子鑑別CQMID)分析 大體上使用實例5所述之方法，對購自西格瑪化學公 司（美國，密蘇里州，聖路易斯市)之5種不同脂多糖(LPS#1 、7、10、15及16)進行GMID分析。所用之呈色外源凝集 素爲 ECL、WGA、VVA 及 SBA。 由LPS#1、7、10、15及16獲得之GMID卡示於圖2( 分別爲A至E)。由此圖可以看出彼等GMID卡對於各種不 同的脂多糖提供獨特的”指紋”，且可以用於鑑定在含有細 菌或其產物之混合物的樣本中存在之此等化合物。 實例7 選擇呈色外源凝集素之方法 在選擇實例5及6中舉例說明之多糖分析方法中所用 之呈色外源凝集素時，有許多因子必須列入考量。在此等 考量中’各所選擇之外源凝集素必須具有可區別之顏色或 其他可檢測之標記，.亦必須減少外源凝集素間之交互作用 。舉例說明在呈色標記之選擇上所用之演算法的流程圖示 於圖3。圖3所示之演算法開始於η個呈色外源凝集素(或 其他可檢測之標記)之選擇作用101，該起始之選擇作用則 係依所欲分析的糖類之部分或全部單糖組成獲得之相關訊 息予以進仃。在下一步驟102中’檢查所選擇之外源凝集 素的顏色’以便檢視所選擇之顏色的一致性/不一致性。若 所選擇組群之顏色相同，使程序進行至步驟1 〇3，否則以 步驟104繼續該流程。於步驟1〇3，以另一種同屬相同鍵 (請先閱讀背面之注意事項再填寫本頁) - — II I I I I · I--I I I I I - 59 1306152 Α7 Β7 五、發明説明（1) 結類型的外源凝集素(亦即，具有相同的單糖鍵結專一性的 外源凝集素)取代已發現之具有非特異顏色的外源凝集素之 一；再使流程進行至步驟102。於步驟104，測試n個所選 擇之外源凝集素，以檢測外源凝集素彼此間或與前文實例 5所述方法之第一階段所用之非呈色型外源凝集素間之任 何交互反應。若發現交互反應，則以步驟105繼續該程序 ，否則，使流程進行至步驟106，結束該演算法。於步驟 105，已證實會與另一個外源凝集素起交互反應之外源凝集 素之一，係以不會產生交互反應之外源凝集素予以取代； 接著使流程進行至步驟102。以步驟106結束該演算法。 在此欲強調的是，當η値小，且當糖類具有簡單的單 糖組成時’上述演算法可藉由操作者本身徒手操作選擇程 序之各步驟而施用。或者(特別是當η爲較大之數目，或單 糖組成更複雜時），可藉由被設計用於完成上述演算程序之 電腦程式進行該程序。 上述實例已證實本文所述方法之有效性。然而彼等實 例僅係用以輔助達成舉例說明之目的。熟習技藝之人士應 明瞭，在所用之基本上爲序列專一性的藥劑、彼等藥劑之 反應條件、固定化技術、以及在標示、反應及檢測步驟之 順序上可以有許多變化產生，彼等變化皆不超脫本發明範 疇。 ______60 本紙張尺度適用中國國家樣準（CNS ) Α4規格（210X29&lt;7公釐） (請先閲讀背面之注意事項再填寫本頁) *1Τ —0. 經濟部智慧財產局員工消費合作社印製V. INSTRUCTIONS (g) Example 6 Lipid Polysaccharide Sugar Molecular Identification CQMID) Analysis In general, five different lipopolysaccharides purchased from Sigma Chemical Company (St. Louis, Missouri, USA) were used as described in Example 5. (LPS #1, 7, 10, 15, and 16) perform GMID analysis. The colored exogenous lectins used were ECL, WGA, VVA and SBA. The GMID cards obtained by LPS #1, 7, 10, 15, and 16 are shown in Figure 2 (A to E, respectively). It can be seen from this figure that their GMID cards provide a unique "fingerprint" for a variety of different lipopolysaccharides and can be used to identify such compounds present in a sample containing a mixture of bacteria or products thereof. Example 7 Method for Selecting Color Exogenous Lectin When selecting the colorectal lectin used in the polysaccharide analysis method exemplified in Examples 5 and 6, many factors must be considered. In these considerations, each selected lectin must have a distinguishable color or other detectable label. It is also necessary to reduce the interaction between lectins. A flow chart illustrating an algorithm used in the selection of color-coded markers is shown in FIG. The algorithm shown in Figure 3 begins with the selection action 101 of η colored exogenous lectins (or other detectable markers), which are selected as part or all of the monosaccharides of the sugar to be analyzed. The relevant information for the composition is obtained. In the next step 102, 'check the color of the selected foreign lectin' to view the consistency/inconsistency of the selected color. If the colors of the selected groups are the same, the program proceeds to step 1 〇 3, otherwise the process continues with step 104. In step 1〇3, the same kind of the same key (please read the back of the page and then fill in the page) - II IIII · I--IIIII - 59 1306152 Α7 Β7 V. Description of invention (1) Type of knot The lectin (i.e., the lectin having the same monosaccharide-binding specificity) replaces one of the lectins having a non-specific color that has been found; and the flow proceeds to step 102. At step 104, n selected lectins are tested to detect any interaction between the lectins or non-chromophoric lectins used in the first phase of the method described in Example 5 above. If an interaction is found, the process continues with step 105; otherwise, the process proceeds to step 106 to end the algorithm. At step 105, it has been confirmed that one of the exogenous lectins will interact with another lectin, which is replaced by a lectin that does not cause an interaction; and then the flow proceeds to step 102. The algorithm ends with step 106. It is emphasized here that when η is small and when the saccharide has a simple monosaccharide composition, the above algorithm can be applied by the operator's own steps of the selection process by hand. Or (especially when η is a larger number, or the composition of the monosaccharide is more complicated), the program can be performed by a computer program designed to perform the above calculation procedure. The above examples have demonstrated the effectiveness of the methods described herein. However, their examples are only used to assist in the purpose of illustration. Those skilled in the art should be aware that there may be many variations in the sequence specificity of the agents used, the reaction conditions of the agents, the immobilization techniques, and the sequence of labeling, reaction, and detection steps, and their variations. None of the scope of the invention is exceeded. ______60 This paper size is applicable to China National Standard (CNS) Α4 specifications (210X29&lt;7 mm) (please read the notes on the back and fill out this page) *1Τ —0. Printed by the Ministry of Economic Affairs, Intellectual Property Bureau, Staff Consumer Cooperative

Claims (1)

1306152 )&amp;年1306152 )&amp;year ASB8C8D8ASB8C8D8 六、申請專利範圍 1_ 一種糖類結構之分析方法，包括： a) 於一基材的表面上提供多數個具有不同鍵結專一性 之不同的基本上爲序列專一性或位點專一性之鍵結劑，其 鍵結該糖類的糖類辨識序列，其中一些該多數個不同.的基 本上爲序列專一性或位點專一性之鍵結劑係固定在該基材 的相同表面上； b) 使該表面與欲分析之糖類接觸，或與包括該糖類之 多數個片段的混合物接觸； .c)洗滌或去除未鍵結之糖類或糖類片段； d) 將基本上爲序列專一性或位點專一性之標記，或基 本上爲序列專一性或位點專一性之標記的混合物添加至步 驟c)製得之表面； e) 獲得鍵結於該表面之標記的一個或多個影像；及 f) 自該影像推衍出所分析之該種糖類之關於其本身 (identity)的結構訊息。 2. 如申請專利範圍第1項之方法，其中該標記爲產色 鍵結劑(chromogenic binding agent)，而其中該標記之影像爲 在該表面上顯現之顏色。 3. 如申請專利範圍第1項之方法，其中步驟f)包括目 視檢測該表面及與標準物比較。 4. 如申請專利範圍第1項之方法，其中步驟f)包括光 學濾器之使用。 5. 如申請專利範圍第1項之方法，其中步驟e)包括對 該影像予以攝影或數位化該影像。 1 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐） (請先閱讀背面之注意事項再填寫本頁) 訂： 1306152 B8 C8 D8 六、申請專利範圍 6. 如申請專利範圍第1項之方法，其中該基本上爲序 列專一性或位點專一性之鍵結劑爲外源凝集素。 7. 如申請專利範圍第1項之方法，其中該基本上爲序 列專一性或位點專一性之鍵結劑爲抗體。 8. 如申請專利範圍第2項之方法，其中該基本上爲序 列專一性或位點專一性之產色鍵結劑爲呈色外源凝集素。 9. 如申請專利範圍第2項之方法，其中該基本上爲序 列專一性或位點專一性之產色鍵結劑爲營光性外源凝集素 〇 10. 如申請專利範圍第2項之方法，其中該基本上爲 序列專一性或位點專一性之產色鍵結劑爲經生物素標示之 外源凝集素。 11. 如申請專利範圍第2項之方法，其中該基本上爲 序列專一性或位點專一性之產色鍵結劑爲螢光性抗體。 12. 如申請專利範圍第2項之方法，其中該基本上爲 序列專一性或位點專一性之產色鍵結劑爲經生物素標示之 抗體。 13. 如申請專利範圍第2項之方法，其中該基本上爲 序列專一性或位點專一性之產色鍵結劑爲經酵素標示之抗 體。 14. 如申請專利範圍第1項之方法，其進一步包括以 能夠切割糖鏈之基本上爲序列專一性的藥劑處理該糖類。 15. 如申請專利範圍第14項之方法，其中使該糖類於 與該表面接觸以前先經處理。 2 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐) ................... ! (請先閱讀背面之注意事項再塡寫本頁) 、-° A8册C8D8 1306152 六、申請專利範圍 16. 如申請專利範圍第Η項之方法，其中該糖類之處 理係於去除未鍵結之糖類以後’但於添加該基本上爲序列 專一性之產色鍵結劑以前進行。 17. 如申請專利範圍第1至16項中任一項之方法，其 中該表面爲濾紙，且其中該基本上爲序列專一性的藥劑係 以預先限定的順序排列於該濾紙上。 18. 如申請專利範圍第1至16項中任一項之方法，其 係用於發展治療活性之藥劑。 19. 如申請專利範圍第1至16項中任一項之方法，其 係用於篩選治療活性之藥劑。 20. 如申目靑專利軺圍弟1至16項中任一項之方法，1¾ 係用於食物或飲料分析。 21. 如申請專利範圍第1至16項中任一項之方法，其 係用於分析遺傳改質(GM)之農作物或由彼等農作物衍生;^ 產物。 22. —種固體支承體，其包括預先限定順序的代表糖 類或糖類序列或片段之多數個可目視或可檢測之標記，其 中該固體支承體係可獲得自： a) 於一基材的表面上提供多數個具有不同鍵結專〜性 之不同的基本上爲序列專一性或位點專一性之鍵結劑，其 中一些該多數個不同的基本上爲序列專一，性或位點專〜性 之鍵結劑係固定在該基材的相同表面上； b) 使該表面與欲分析之糖類接觸，或與包括該糖類之 多數個片段的混合物接觸； 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐） 請 閲 讀 背 Φ 意 事 1 負 Η 頁 訂 1306152 B8 C8 D8 . - · 六、申請專利範圍 C)洗滌或去除未鍵結之糖類或糖類片段；以及 d)將基本上爲序列專一性或位點專一性之標記，或基 本上爲序列專一性或位點專一性之標記的混合物添加至步 驟C)獲得之表面。 23. —種選擇一組可供如申請專利範圍第1項之方法 使用之基本上爲序列及/或位點專一性的標記之方法，該方 法包括下列步驟： a)獲得欲分析之糖類的全部或部分單糖組成(MC); .b)選擇一組含有η個可以鍵結於該糖類中所含之單糖 的具有不同鍵結專一性之基本上爲序列專一性或位點專一 性之標記； ' c) 修正於步驟b)獲得之該組基本上爲序列專一性或位 點專一性之標記，以確保該組中沒有兩個標記具有相同的 顏色或是可檢測之特徵； d) 修正於步驟c)選擇之該組基本上爲序列專一性或位 點專一性之標記，以減少該組標記與該等基本上爲序列專 一性或位點專一性之鍵結劑或與其他基本上爲序列專一性 或位點專一性之標記間的交互反應。 24_如申請專利範圍第23項之方法，其中該所欲分析 之糖類的MC係由相關之糖類的MC推測。 25.如申請專利範圍第23項之方法，其中該所欲分析 之糖類的MC係藉由對該糖類進行完整之MC分析而獲得 〇 26·如申請專利範圍第23項之方法，其中η値介於1 4 ‘丨丨丨 _____________________ 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐） ---------------------------------曼！！ {請先閱讀背面之注意事項再填寫本頁) 、-tf 1306152 B8 C8 D8 六、申請專利範圍 '與4之間。 27. —種用以選擇可供如申請專利範圍第1項之方法 中使用的一組基本上爲序列專一性及/或位點專一性的標記 之軟體，該軟體包括： a) 輸入，用以提供單糖組成(MC); b) 配對次程式，用以配對可以鍵結該糖類中所含之單 糖的η個具有不同結合專一性之基本上爲序列專一性或位 點專一性之標記； .c)修正次程式，用以修正由與次程式b)配對之一組基 本上爲序列專一性及/或位點專一性之標記，該次程式能夠 以減少該標記與該等基本上爲序列專一性及/或位點專一性 之鍵結劑或與其他基本上爲序列專一性及/或位點專一性之 標記間的交互反應爲基礎而選擇該標記； d)再次修正次程式，能夠確保該組中沒有兩個標記具 有相同的顏色或是可檢測之特徵。 28. —種糖類之結構分析的方法，包括下列步驟： a) 提供一種糖類； b) 使該糖類與一些第一基本上爲序列專一性的藥劑反 應，其中該第一基本上序列專一性的藥劑係固定在同一基 材上； c) 使該糖類或其片段與第二基本上爲序列專一性的藥 劑反應； d) 使該糖類或其片段與第三基本上爲序列專一性的藥 劑反應； 5 本紙張尺度適用中國國家標準(CNS)A4規格(210 x297公釐） ......................…… (請先閲讀背面之注意事項再填寫本頁) 訂： # 1306152 截 __ 六、申請專利範圍 e)視需要使用至少一種不同的第二或第三基本上爲序 列專一性的藥劑重複步驟c至d ; (請先閱讀背面之注意事項再填寫本頁) 其中，可以在並行而獨立之反應中使用相同的糖類， 並以至少一種不同的第一、第二、或第三基本上爲序列專 一性的藥劑進行步驟a)至e)，其中該糖類係經標示者及/或 一種或多種該第一、第二或第三基本上爲序列專一性的藥 劑係經標不者或者將一種標籤導入該糖類中，且在導入該 標籤的步驟之後的一個或多個步驟檢測出該標籤，且其另 一修件爲該第一基本上爲序列專一性的藥劑係鍵結劑且至 少一個第二或第三基本上爲序列專一性的藥劑爲切割劑， 其中該第一、第二、或第三基本上爲序列專一性的藥劑之 每一者具有不同的結合及/或切割專一'性。 29. 如申請專利範圍第28項之方法，其中該第一、第 二、或第三基本上爲序列專一性的藥劑中之至少一者被固 定，且其中該固定化之藥劑不是作爲該切割劑之基本上爲 序列專一性的藥劑。 30. —種糖類之結構分析的方法，包括下列步驟： a) 提供一種糖類； b) 使該糖類與一些第一基本上爲序列專一性的藥劑反 應； c) 使該糖類或其片段與第二基本上爲序列專一性的藥 劑反應； d) 使該糖類或其片段與第三基本上爲序列專一性的藥 劑反應； 6 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐） A8i 1306152 六、申請專利範圍 e)視需要使用至少一種不同的第二或第三基本上爲序 列專一性的藥劑重複步驟c至d ; 其中，可以在並行而獨立之反應中使用相同的糖類， 並以至少一種不同的第一、第二、或第三基本上爲序列專 一性的藥劑進行步驟a)至e)，其中該糖類係經標示者及/或 一種或多種該第一、第二或第三基本上爲序列專一性的藥 劑係經標示者或者將一種標籤導入該糖類中，且在導入該 標籤的步驟之後的一個或多個步驟檢測出該標籤，且其另 一條件爲該第一基本上爲序列專一性的藥劑係非固定化切 割劑且至少一個第二或第三基本上爲序列專一性的藥劑係 固定在相同的基材上，其中該第一、第二、或第三基本上 爲序列專一性的藥劑之每一者具有不同的結合及/或切割專 一性。 31. 如申請專利範圍第30項之方法，其中該第二基本 上爲序列專一性的藥劑係被固定。 32. 如申請專利範圍第30項之方法，其中該切割劑係 糖苷酶或糖基轉移酶，該第二基本上爲序列專一性的藥劑 係外源凝集素，而該第三基本上爲序列專一性的藥劑係抗 體。 33. 如申請專利範圍第28項之方法，其中該第三基本 上爲序列專一性的藥劑係切割劑或外源凝集素。 34. 如申請專利範圍第28項之方法，其進一步包括推 衍出糖類序列之步驟。 35. 如申請專利範圍第28項之方法，其中該第三基本 7 本紙張尺度適用中國國家標準(CNS)A4規格(210 x 297公釐) ........-.............…… (請先閱讀背面之注意事項再塡寫本頁) 訂： A8B8C8D8 1306152 六、申請專利範圍 上爲序列專一性的藥劑係糖苷酶或糖基轉移酶。 36. 如申請專利範圍第28項之方法，其中所有的第一 序列專一性的藥劑皆固定於單—基材上。 37. 如申請專利範圍第28項之方法，其中該第一基本 上爲序列專一性的藥劑係選自外源凝集素及抗體。 38. 如申請專利範圍第28項之方法，其中該第二或第 三基本上爲序列專一性的藥劑係將經標示的單糖單元引導 至該糖類上之糖基轉移酶。 39. 如申請專利範圍第28項之方法，其中使用一個以 上之標籤，且所用之各標籤可獨立地被檢測出。 40. 如申請專利範圍第28項之方法，其中該第二及第 三基本上爲序列專一性的藥劑同時存於反應中，但其一者 活化之後，藉由改變緩衝液條件才使另一者活化，因此該 等基本上爲序列專一性的藥劑中之一者因該改變而不活化 ，同時使另一個基本上爲序列專一性的藥劑被活化。 41. 如申請專利範圍第28項之方法，其中同時添加數 個第三基本上爲序列專一性的藥劑，但其一者活化之後， 藉由改變緩衝液條件才使另一者活化，因此該等基本上爲 序列專一性的藥劑中之一者或更多者因該改變而不活化， 同時使另一個基本上爲序列專一性的藥劑被活化。 42. 如申請專利範圍第40項之方法，其中一個或更多 個該等第三基本上爲序列專一性的藥劑係糖苷酶或糖基轉 移酶。 43. 如申請專利範圍第28項之方法，其中每個該第一 8 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐） ......................-!- ί請先閲讀背面之注意事項再塡寫本頁) 訂 A8B8C8D8 1306152 六、申請專利範圍 或第二基本上爲序列專一性的藥劑係固定於僞陣列(virtual array)之獨立單元上。 44. 如申請專利範圍第43項之方法，其中該陣列爲多 抗原血清診斷陣列(MASDA)。 45. 如申請專利範圍第28項之方法，其進一步包括藉 由使用糖苷酶或其等效物依序分解而分析糖類結構以作爲 在申請專利範圍第28項步驟d)後進行之方法，該方法包括 下列步驟： .a)遮蔽該糖類之還原端； b)藉由以糖苷酶或其等效物與該糖類反應，而曝露出 另一個還原端； C)標7K該另一個還原端； d)視需要使用不同的糖苷酶或其等效物重複步驟&amp;至c 〇 46. 如申請專利範圍第28項之分析糖類結構的方法， 其中係依如申請專利範圍第28及45項之方法蒐集資料， 再將該等資料整合，以由其中衍生出該糖類之結構訊息。 47. 如申請專利範圍第28項之方法，其中該一個或多 個標籤係螢光性標籤。 48. —種使用如申請專利範圍第28項所得之資料構築 糖類之序列圖譜的方法，該方法包括下列步驟： a) 使用如申請專利範圍第28項之方法蒐集辨識序列三 元組(triplet)， b) 鑑定第一型三元組，該三元組爲序列(第一辨識位點 9 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐) 4 (請先閲讀背面之注意事項再填寫本頁) 訂 1306152 韻 ____SI 六、申請專利範圍 Μ糖穿酶)·(第二辨識位點)之三元組， c) 鑑定第二型三元組，該三元組爲序列(糖苷酶)_(第一 辨識位點)-(第二辨識位點)之三元組， d) 依類似處對該等三元組加以分類， e) 以不同的糖苷酶辨識位點比較三元組， f) 依在糖類上之發生順序排列該等三元組， S)排列該等糖苷酶辨識位點， h)檢查該等三元組之相容性， .i)以單一縱列順序排列糖苷酶以及第一及第二基本上 爲序列專一性的藥劑之辨識序列，及 j) 將該等辨識序列(位點)轉譯成多糖序列。 49. 如申請專利範圍第48項之構築糖類之序列圖譜的 方法，進一步包括下列步驟： k) 修正重疊之問題， l) 輸出序列， m) 檢查所有可得之資料，由而構築實際的糖類序列模 型。 50. 如申請專利範圍第49項之方法，其中步驟⑹包 括使用如申請專利範圍第28項獲得之資料，如申請專利範 圍第46項檢查進一步構築糖類之序列圖譜的其他資料。 51. 一種糖類結構之分析方法，包括： a)於一表面上提供多數個不同的基本上爲序列專一性 或位點專一性之鍵結劑’其中各試劑係對一類多數糖類具 有專一性，因此該多數試劑不具有對特定糖類序列的絕對 10 ^纸張尺度適用中國國家標準(CNS)A4規格(210 * 297公釐) (請先閲讀背面之注意事項再填寫本頁) 訂 A8B8C8D8 1306152 六、申請專利範圍 序列專一性； b) 使該表面與欲分析之糖類接觸，或與包括該糖類之 多數個片段的混合物接觸； c) 洗滌或去除未鍵結之糖類或糖類片段； d) 將基本上爲序列專一性或位點專一性之標記，或基 本上爲序列專一性或位點專一性之標記的混合物添加至步 驟c)製得之表面； e) 獲得鍵結於該表面之標記的一個或多個影像；及 .f)自該影像推衍出所分析之該種糖類之關於其本身的 訊息。 52. —種用於至少部分決定糖類序列的方法，其包括 決定多數固定化試劑與該糖類或該糖類片段的結合模 式以及多數標記與該糖類或糖類片段的結合模式，其中該 模式係根據申請專利範圍第1至16項中任一項的方法所決 定；以及 藉由組合該結合模式形成至少部分序列資訊。 53. —種糖分子鑑別(GMID)卡，其包括該固定化試劑 與該糖類或該糖類片段結合的模式以及該標記與該糖類或 糖類片段結合的模式，其中該模式是該糖類或糖類片段的 鑑認特徵且係根據申請專利範圍第1至16項中任一項的方 法所決定。 54. 根據申請專利範圍第1或28項的方法，其中該 基材係珠粒。 11 本纸張尺度適用中國國家標準（CNS)A4規格(210 X 297公釐) ...................— (請先間讀背面之注意事項再塡寫本頁) #: 1306152 A8 B8 C8 D8 六、申請專利範圍 55·根據申請專利範圍第22項的固體支承體，其中該 固體支承體係珠粒。 56. 根據申請專利範圍第 51項的方法，其中該表面 係珠粒。 57. 根據申請專利範圍第1或28項的方法，其中該 基材係陣列。 58. 根據申請專利範圍第22項的固體支承體，其中該 固體支承體係陣列。 59. 根據申請專利範圍第 51項的方法，其中該表面 係陣列。 ......................續-—— (請先閲讀背面之注意事項再填寫本頁) 訂： 12 1紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐〉VI. Patent Application 1_ A method for analyzing a sugar structure, comprising: a) providing a plurality of substantially sequence-specific or site-specific bonds having different bond specificities on the surface of a substrate. a saccharide recognition sequence that binds to the saccharide, wherein some of the plurality of substantially different sequence-specific or site-specific bonding agents are immobilized on the same surface of the substrate; b) The surface is contacted with a sugar to be analyzed, or with a mixture comprising a plurality of fragments of the sugar; c) washing or removing unbound sugar or carbohydrate fragments; d) being substantially sequence specific or site specific a label, or a mixture of substantially sequence specificity or site specificity, added to the surface prepared in step c); e) obtaining one or more images of the label bound to the surface; and f) The image derives the structural information about the identity of the sugar being analyzed. 2. The method of claim 1, wherein the label is a chromogenic binding agent, and wherein the image of the label is a color that appears on the surface. 3. The method of claim 1, wherein step f) comprises visually detecting the surface and comparing it to a standard. 4. The method of claim 1, wherein step f) comprises the use of an optical filter. 5. The method of claim 1, wherein step e) comprises photographing or digitizing the image. 1 This paper size is applicable to China National Standard (CNS) A4 specification (210 X 297 mm) (please read the note on the back and fill out this page) Order: 1306152 B8 C8 D8 VI. Patent application scope 6. If the patent application scope The method of item 1, wherein the substantially sequence-specific or site-specific binding agent is a lectin. 7. The method of claim 1, wherein the substantially specific sequence or site specificity binding agent is an antibody. 8. The method of claim 2, wherein the chromogenic bonding agent which is substantially sequence specific or site specific is a coloring lectin. 9. The method of claim 2, wherein the chromogenic bond agent which is substantially sequence specific or site specific is a camping lectin 〇 10. as claimed in claim 2 The method wherein the chromogenic bond agent which is substantially sequence specific or site specific is a biotin-labeled lectin. 11. The method of claim 2, wherein the chromogenic binding agent which is substantially sequence specific or site specific is a fluorescent antibody. 12. The method of claim 2, wherein the chromogenic bond agent which is substantially sequence specific or site specific is a biotinylated antibody. 13. The method of claim 2, wherein the chromogenic bond agent which is substantially sequence specific or site specific is an enzyme labeled with an enzyme. 14. The method of claim 1, further comprising treating the saccharide with a substantially sequence specific agent capable of cleaving the sugar chain. 15. The method of claim 14, wherein the saccharide is treated prior to contact with the surface. 2 This paper size applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) ................... ! (Please read the notes on the back and then 塡。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 Sequence specificity of the color-bonding agent was previously performed. 17. The method of any one of claims 1 to 16, wherein the surface is a filter paper, and wherein the substantially sequence specific agent is arranged on the filter paper in a predefined order. 18. The method of any one of claims 1 to 16 for use in the development of a therapeutically active agent. 19. The method of any one of claims 1 to 16, which is for screening a therapeutically active agent. 20. For the method of any of the patents, 1 to 16 of the patent, 13⁄4 is used for food or beverage analysis. 21. The method of any one of claims 1 to 16, which is for the analysis of genetically modified (GM) crops or derived from their crops; 22. A solid support comprising a plurality of visually or detectable labels representing a sequence or segment of a saccharide or saccharide in a predefined sequence, wherein the solid support system is obtainable from: a) on a surface of a substrate Providing a plurality of keying agents having substantially unique sequence specificity or site specificity with different bond specificities, some of which are substantially different in sequence specificity, sex or site specificity The bonding agent is immobilized on the same surface of the substrate; b) contacting the surface with the sugar to be analyzed or with a mixture comprising a plurality of fragments of the sugar; the paper scale is applicable to the Chinese National Standard (CNS) A4 Specifications (210 X 297 mm) Please read the back Φ Meaning 1 Negative Η Page 1306152 B8 C8 D8 . - · VI. Patent scope C) Wash or remove unbonded sugar or sugar fragments; and d) Basic A label for sequence specificity or site specificity, or a mixture of substantially sequence specificity or site specificity is added to the surface obtained in step C). 23. A method of selecting a set of markers substantially sequence and/or site specific for use in the method of claim 1 for the method of claim 1, the method comprising the steps of: a) obtaining a saccharide to be analyzed All or part of the monosaccharide composition (MC); .b) selecting a set of n-specific single-specific or site-specific specificity with different bond specificities that can be bonded to the monosaccharide contained in the saccharide Marking; 'c) correcting the set of markers obtained in step b) to be substantially sequence specific or site specific, to ensure that no two markers in the group have the same color or detectable features; Correcting the set of markers selected in step c) to be substantially sequence specific or site specific, to reduce the set of markers and the substantially sequence specificity or site specificity of the bonding agent or other Basically the interaction between markers of sequence specificity or site specificity. The method of claim 23, wherein the MC of the saccharide to be analyzed is estimated by the MC of the relevant saccharide. 25. The method of claim 23, wherein the MC of the saccharide to be analyzed is obtained by performing a complete MC analysis of the saccharide, and the method of claim 23, wherein η値Between 1 4 '丨丨丨_____________________ This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) --------------------- ------------Man! ! {Please read the notes on the back and then fill out this page), -tf 1306152 B8 C8 D8 VI. Apply for a patent range between 'and 4. 27. A software for selecting a set of substantially sequence specificity and/or site specificity markers for use in the method of claim 1 of the patent application, the software comprising: a) input, To provide a monosaccharide composition (MC); b) a pairing subroutine for pairing n of the monosaccharides contained in the saccharide with substantially different specificity for sequence specificity or site specificity Mark; .c) a modified subroutine for correcting a tag that is substantially unique to sequence specificity and/or site specificity by pairing with subprogram b), the program being able to reduce the mark and the basics Selecting the marker based on the interaction between the sequence specificity and/or site specificity binding agent or other markers that are essentially sequence specificity and/or site specificity; d) The program ensures that no two markers in the group have the same color or detectable features. 28. A method of structural analysis of a saccharide comprising the steps of: a) providing a saccharide; b) reacting the saccharide with some first substantially sequence specific agent, wherein the first substantially sequence specificity The agent is immobilized on the same substrate; c) reacting the saccharide or fragment thereof with a second substantially sequence specific agent; d) reacting the saccharide or fragment thereof with a third substantially sequence specific agent 5 The paper size applies to the Chinese National Standard (CNS) A4 specification (210 x 297 mm) ............................ (Please read the back Precautions Please fill out this page) Order: # 1306152 截__ VI. Patent application scope e) Repeat steps c to d as needed using at least one different second or third essentially sequence specific agent; Read the notes on the back and fill out this page. Wherein, the same saccharide can be used in parallel and independent reactions, and the steps are performed with at least one different first, second, or third substantially sequence specific agent. a) to e), wherein the sugar is labeled And/or one or more of the first, second or third substantially sequence specific drugs are labeled or introduced into the saccharide and one or more after the step of introducing the label The step of detecting the label, and the other modification is the first substantially sequence-specific drug-based bonding agent and the at least one second or third substantially sequence-specific agent is a cleavage agent, wherein Each of the first, second, or third substantially sequence specific agents has a different binding and/or cleavage specificity. 29. The method of claim 28, wherein at least one of the first, second, or third substantially sequence specific agents is immobilized, and wherein the immobilized agent is not the cut The agent is essentially a sequence specific agent. 30. A method of structural analysis of a saccharide comprising the steps of: a) providing a saccharide; b) reacting the saccharide with some first substantially sequence specific agent; c) causing the saccharide or fragment thereof 2. A substantially sequence-specific drug reaction; d) reacting the saccharide or fragment thereof with a third substantially sequence-specific agent; 6 This paper size applies to the Chinese National Standard (CNS) A4 specification (210 X 297 metric) PCT) A8i 1306152 VI. Scope of Application e) Repeat steps c to d as needed using at least one different second or third substantially sequence specific agent; wherein the same can be used in parallel and independent reactions a saccharide, and performing steps a) through e) with at least one different first, second, or third substantially sequence specific agent, wherein the saccharide is labeled and/or one or more of the first, The second or third substantially sequence specific drug is labeled by the labeler or introduced into the saccharide, and the label is detected at one or more steps subsequent to the step of introducing the label And another condition is that the first substantially sequence-specific drug-based non-immobilized cleavage agent and at least one second or third substantially sequence-specific drug system are immobilized on the same substrate, wherein Each of the first, second, or third substantially sequence specific agents has a different binding and/or cleavage specificity. 31. The method of claim 30, wherein the second substantially serial-specific pharmacy is immobilized. 32. The method of claim 30, wherein the cleavage agent is a glycosidase or a glycosyltransferase, the second substantially sequence-specific agent is a lectin, and the third is substantially a sequence Specific drug antibody. 33. The method of claim 28, wherein the third is substantially a sequence specific drug cleavage agent or a lectin. 34. The method of claim 28, further comprising the step of deriving a carbohydrate sequence. 35. The method of claim 28, wherein the third basic 7 paper size is applicable to the Chinese National Standard (CNS) A4 specification (210 x 297 mm) ........-.... ............... (Please read the notes on the back and write this page first) Order: A8B8C8D8 1306152 VI. The patent system is a sequence-specific glycosidase or glycosyltransferase. 36. The method of claim 28, wherein all of the first sequence specific agents are immobilized on a single substrate. 37. The method of claim 28, wherein the first substantially sequence-specific agent is selected from the group consisting of a lectin and an antibody. 38. The method of claim 28, wherein the second or third substantially sequence specific agent directs the labeled monosaccharide unit to a glycosyltransferase on the saccharide. 39. The method of claim 28, wherein more than one label is used and the labels used are independently detectable. 40. The method of claim 28, wherein the second and third substantially sequence specific agents are simultaneously present in the reaction, but after one of the activations, the buffer condition is changed to cause the other The agent is activated such that one of the substantially sequence-specific agents is not activated by the change, while another substantially sequence-specific agent is activated. 41. The method of claim 28, wherein a plurality of third substantially sequence-specific agents are simultaneously added, but after activation, the other is activated by changing the buffer conditions, thus One or more of the agents that are substantially sequence specific are not activated by the change, while another substantially sequence specific agent is activated. 42. The method of claim 40, wherein the one or more of the third substantially sequence specific antibodies are glycosidase or glycosyltransferase. 43. For the method of claim 28, each of the first 8 paper sizes is subject to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) ............ ..........-!- ίPlease read the notes on the back and write this page again.) A8B8C8D8 1306152 VI. The scope of patent application or the second basic sequence specificity is fixed in pseudo On a separate unit of the array (virtual array). 44. The method of claim 43, wherein the array is a multi-antigen serum diagnostic array (MASDA). 45. The method of claim 28, further comprising analyzing the saccharide structure by sequential decomposition using a glycosidase or its equivalent as a method performed after step d) of claim 28, The method comprises the steps of: .a) masking the reducing end of the saccharide; b) exposing the other reducing end by reacting with the saccharide by a glycosidase or its equivalent; C) the other reducing end of the label 7K; d) repeating the steps &amp; to c 〇 46 using different glycosidases or their equivalents as needed. For example, the method for analyzing the saccharide structure of claim 28, wherein the application is in accordance with the scope of claims 28 and 45 The method collects data and integrates the data to derive a structural message of the sugar therefrom. 47. The method of claim 28, wherein the one or more labels are fluorescent labels. 48. A method of constructing a sequence map of a saccharide using the data obtained in claim 28 of the patent application, the method comprising the steps of: a) collecting an identification sequence triplet using the method of claim 28; , b) Identification of the first type of triplet, the triplet is the sequence (first identification site 9 This paper size applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 4 (please read the back Note: Please fill out this page) Book 1306152 Rhyme ____SI 6. Apply for the patent range Μ Sugar permease) · (Second identification site) triple, c) Identify the second type triad, the triad is a triplet of the sequence (glycosidase) _ (first recognition site) - (second recognition site), d) classifying the triplets according to similarities, e) identifying sites with different glycosidase Comparing the triads, f) arranging the triads in the order in which they occur on the sugar, S) arranging the glycosidase recognition sites, h) checking the compatibility of the triads, .i) Sequence alignment of glycosidase and identification of first and second essentially sequence specific agents Columns, and j) translate the recognition sequences (sites) into a polysaccharide sequence. 49. The method of constructing a sequence map of carbohydrates as set forth in claim 48 of the patent scope further comprises the steps of: k) correcting the problem of overlap, l) outputting the sequence, m) examining all available data, thereby constructing the actual sugar Sequence model. 50. If the method of claim 49 is applied, the step (6) includes the use of information obtained in accordance with item 28 of the scope of the patent application, such as the application of the 46th item in the scope of the patent application for further construction of a sequence map of the sugar. 51. A method of analyzing a saccharide structure, comprising: a) providing a plurality of different bonding agents that are substantially sequence specific or site specific on a surface, wherein each reagent is specific to a majority of the saccharides, Therefore, most of the reagents do not have an absolute 10^ paper scale for a specific carbohydrate sequence. The Chinese National Standard (CNS) A4 specification (210 * 297 mm) is applicable (please read the back note before filling this page). A8B8C8D8 1306152 , applying for patent range sequence specificity; b) contacting the surface with the sugar to be analyzed, or with a mixture comprising a plurality of fragments of the sugar; c) washing or removing unbound sugar or sugar fragments; d) a label substantially of sequence specificity or site specificity, or a mixture of substantially sequence specificity or site specificity is added to the surface prepared in step c); e) obtaining a label bonded to the surface One or more images; and .f) derived from the image the information about the sugar being analyzed about itself. 52. A method for at least partially determining a saccharide sequence comprising determining a mode of binding of a plurality of immobilizing agents to the saccharide or the saccharide fragment and a mode of binding of a plurality of labels to the saccharide or saccharide fragment, wherein the pattern is based on the application Determined by the method of any one of claims 1 to 16, and at least part of the sequence information is formed by combining the combination modes. 53. A saccharide molecular identification (GMID) card comprising a pattern of binding of the immobilizing agent to the saccharide or the saccharide fragment and a pattern of binding of the label to the saccharide or saccharide fragment, wherein the pattern is the saccharide or saccharide fragment The authentication feature is determined according to the method of any one of claims 1 to 16. 54. The method of claim 1 or 28, wherein the substrate is a bead. 11 This paper size applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) ................... — (Please read the back of the note first) Further, please write this page) #: 1306152 A8 B8 C8 D8 VI. Patent Application No. 55. The solid support according to claim 22, wherein the solid support system beads. 56. The method of claim 51, wherein the surface is a bead. The method of claim 1 or 28, wherein the substrate is an array. 58. The solid support of claim 22, wherein the solid support system is arrayed. 59. The method of claim 51, wherein the surface is an array. ......................Continued-- (Please read the notes on the back and fill out this page) Order: 12 1 Paper scale applies to Chinese national standards ( CNS) A4 specification (210 X 297 mm)