TWI235159B - Anti-human hemoglobin monoclonal antibodies for detecting fecal occult blood, producing hybridomas and uses thereof - Google Patents
Anti-human hemoglobin monoclonal antibodies for detecting fecal occult blood, producing hybridomas and uses thereof Download PDFInfo
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1235159 A71235159 A7
本發明係有關對人類血紅素且右 、、 系/、有專一性又單株抗體, 製孩抗體之融合瘤及其於檢測糞便潛血之用途。 胃腸癌及消化道之病變經常引起腸胃道之出血,而 液往往會隨糞便排出體外,因此檢驗糞便中是否各有:二 血(稱糞便潛血),可用來早期診斷消化道之病變,尤其: 大腸/直腸癌之敏感性及專一性最高。 /、、 目前國内常用之糞便潛血檢測法為化學 予去,由於血紅素 中心血基質具有假-過氧化酶活性,叾能使某些無色化合 物經氧化反應而呈現顏色。其中最常 口 q化學法係為癒瘡 木月旨法(guaiac method): 2H2〇 + 〇2 血紅素+顯色劑 Hb + H2〇2 癒瘡木脂之氧化 〇2 +癒瘡木壙—經氧化之癒瘡木脂 (無色) (藍色)The present invention relates to a fusion antibody specific to a human heme and a right, a line, a single antibody, a child antibody, and its use for detecting fecal occult blood. Gastrointestinal cancer and gastrointestinal lesions often cause gastrointestinal bleeding, and the fluid is often excreted with the feces. Therefore, it is checked whether the feces each have two blood (called fecal occult blood), which can be used for early diagnosis of gastrointestinal diseases, especially: Colorectal / rectal cancer has the highest sensitivity and specificity. / 、 At present, the faecal occult blood detection method commonly used in China is chemical removal. Because the central blood matrix of heme has pseudo-peroxidase activity, it can cause some colorless compounds to show color through oxidation reaction. Among them, the most commonly used chemical method is the guaiac method: 2H2〇 + 〇2 hemoglobin + chromogen Hb + H2〇2 oxidation of guaiac oleum + guaiac wood— Oxidized Acne Lignans (Colorless) (Blue)
經濟部智慧財產局員工消費合作社印製 C請先閱讀背面之注音?事項再填寫本頁) 酚 奎寧酮 惟化學法之專一性差,容易被受測者所 可所攝取之食物或藥 物所影響而出現偽陽性。如動物血紅紊;5如& 、久新鮮蔬果中都可 能含有造成偽陽性之物質,故受測者於二 、一天前便不可食用 58055.DOC\en - 4 - . 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐) 1235159 A7 ----_ B7___ ^ 五、發明說明(2、) 紅肉及多種蔬果類食品,因此造成許多不便。 k體抗原特異結合之免疫分析技術已發年 / 丁 並已配 (請先閱讀背面之注意事項再填寫本頁} 合色層分析法發展出簡便、快速之方法,即使是任何無鲈 驗之個人,亦可以肉眼判讀結果,如簡易層析式檢驗法。 簡易層析式檢驗法一般以二種原理來施行,—為競爭刑 免疫反應,一為三明治免疫反應。競爭型反應乃樣品接觸 區位於檢驗片之一端,與待測樣品相接觸,所吸附之樣品 以毛細現象移向另一端移動,以致濕透整個檢驗片。指示 劑區接近樣品接觸區,該區以乾燥型式聚集有指示劑-抗 體結合體。測試區位于指示劑區之上方,該區固定有顯色 反應物’如待測物-载體抗原結合體。它可和抗體指示劑 結合體發生反應而顯現出一肉眼可判讀的訊號。該載體抗 原可為如白蛋白、球蛋白、血藍質、鐵蛋白質等。 經濟部智慧財產局員工消費合作社印製 反應測試時,將檢驗片之樣品接觸區端與待測樣品相接 觸。樣品液依毛細現象向上浸溼,並回溶抗體-指示劑結 合物。若樣品中含有待測物,則會與測試區上的測試物-載體抗原結合韓相互競爭有限的抗體-指示劑結合劑。故 樣品中測試物含量愈高,測試區陽性訊號愈弱。由測試區 訊號反應之有無及強弱,可判定樣品中是否含有測試物及 其濃度。 三明治型反應,則是將對抗待測抗原的二個抗體(單株 抗體或多株抗體,一個抗體固定在測試區,另一個抗體和 指示劑相連接。若樣品中含待測抗原,則待測抗原會先和 抗體-指示劑結合體先反應,而後移動至測試區和被固定 58055. D〇C\en " 5 - 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐) l235l59 A7 B7 五、發明說明(3_ ) ^ 的另一個抗體作用,而被捉取固定於測試區,而出現肉眼 可見的訊號。待測抗原濃度愈高,陽性訊號愈強。 蓉於免疫分析法於專一性及方便性上之優點,亟需發展 本國之免疫檢驗材料與方法。 發明概述 本發明之目的為提♦供對人類血紅素具有專一性之單株抗 體。 本發明之另一目的為提供產製該抗體之融合瘤。 本發明之另一目的為進一步提供檢測糞便潛血之方法, 其檢測試劑與套組。 圖式簡要說明 圖1 ·免疫二明治層析式檢驗試劑。其中(1)為樣品接 觸,其利用毛細現象使滴於玻璃纖維膜下端之樣品往硝化 纖維膜處移動;(2)為抗體指示劑區,於硝化纖維膜下方之 玻璃纖維膜上吸附有膠體金_單株抗體46.24結合體;為 反應測試區,硝化纖維膜上所喷之c線為難抗老鼠免疫球 蛋白(對照線)V T線為單株抗體37·24 (受試線);及⑷為液 體吸附區。測試時,試劑上若只顯現一條線(對照線)則為 陰性反應;若顯現兩條線(對照線及受試線)則為陽性反 應。 I明之詳述 本發明首先提供兩種對人類血紅素具有專一性之單株抗 體,以及分別產製該單株抗體之融合瘤37 46及46 24。几 此二株融合瘤已於民國八十八年六月二十五日寄存於食 58055. D〇C\en 〇 _ -D - 、 * 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公爱) (請先閱讀背面之注咅?事項再填寫本頁)Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs. C Please read the note on the back? Please fill in this page again for details.) Phenol Quinone The chemical specificity is poor, and it is easy to be affected by foods or medicines that can be ingested by the test subject, resulting in false positives. Such as animal hemoglobin; 5 such as & and long fresh vegetables and fruits may contain substances that cause false positives, so the testees should not eat them two or one days ago. 58055.DOC \ en-4-. This paper standard applies to China Standard (CNS) A4 specification (210 X 297 mm) 1235159 A7 ----_ B7___ ^ V. Description of the invention (2) Red meat and a variety of fruits and vegetables, causing a lot of inconvenience. The k-antigen specific binding immunoassay technology has been available for many years / Ding has been equipped (please read the precautions on the back before filling out this page) The combined color layer analysis method has developed a simple and fast method, even if any Individuals can also interpret the results with the naked eye, such as the simple tomographic test. The simple tomographic test is generally implemented by two principles: the competitive immune response and the sandwich immune response. The competitive response is the sample contact area. It is located at one end of the test piece, which is in contact with the sample to be measured. The adsorbed sample moves to the other end by capillary phenomenon, so as to wet the entire test piece. The indicator area is close to the sample contact area, and the indicator is collected in a dry form in this area. -Antibody binding body. The test area is located above the indicator area, and the area is fixed with a color reaction reagent such as the test substance-carrier antigen binding body. It can react with the antibody indicator binding body to show a naked eye. Interpreted signal. The carrier antigen can be, for example, albumin, globulin, hemocyanin, ferritin, etc. Printed response from the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs During the test, the end of the sample contact area of the test piece is in contact with the sample to be tested. The sample solution is wetted up by the capillary phenomenon, and the antibody-indicator conjugate is dissolved back. If the sample contains the test object, it will contact the test area. The test substance-carrier antigen binding on the antibody-indicator binding agent has limited competition with each other. Therefore, the higher the content of the test substance in the sample, the weaker the positive signal in the test area. From the presence or absence of the signal reaction in the test area, the strength of the signal can be determined in the sample. Whether it contains the test substance and its concentration. In the sandwich type reaction, two antibodies (single or multiple antibodies against the test antigen) are fixed, one antibody is fixed in the test area, and the other antibody is connected to the indicator. If the sample Containing the test antigen, the test antigen will first react with the antibody-indicator conjugate, then move to the test area and be fixed 58055. D〇C \ en " 5-This paper size applies Chinese national standards (CNS ) A4 size (210 X 297 mm) l235l59 A7 B7 V. Another antibody function of the invention description (3_) ^ was captured and fixed in the test area, and a signal visible to the naked eye appeared. The higher the concentration, the stronger the positive signal. Due to the specificity and convenience of immunoassay methods, there is an urgent need to develop domestic immunoassay materials and methods. SUMMARY OF THE INVENTION The purpose of the present invention is to provide human hemoglobin with Specific monoclonal antibody. Another object of the present invention is to provide a fusion tumor producing the antibody. Another object of the present invention is to further provide a method for detecting fecal occult blood, a detection reagent and a kit thereof. BRIEF DESCRIPTION OF THE DRAWINGS 1 · Immunochromatographic chromatographic test reagents, where (1) is the sample contact, which uses capillary phenomenon to move the sample dropped on the lower end of the glass fiber membrane to the nitrocellulose membrane; (2) the antibody indicator area, which is used for nitration Colloidal gold_single antibody 46.24 conjugate is adsorbed on the glass fiber membrane under the fiber membrane; for the reaction test area, the c-line sprayed on the nitrocellulose membrane is difficult to resist mouse immunoglobulin (control line) and the VT line is a single antibody 37 · 24 (test line); and ⑷ is the liquid adsorption area. During the test, if only one line (control line) appears on the reagent, it is a negative reaction; if two lines (control line and test line) are displayed, it is a positive reaction. Detailed description of the invention The present invention first provides two single-body antibodies specific for human heme, and fusion tumors 37 46 and 46 24 which produce the single-body antibodies, respectively. Several of these two fusion tumors have been deposited in Food 58055 on June 25, 1998. D〇C \ en 〇_ -D-、 * This paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 Public Love) (Please read the note on the back? Matters before filling out this page)
--------訂·--------. 經濟部智慧財產局員工消費合作社印製 1235159 A7-------- Order · --------. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 1235159 A7
五、發明說明(4 ) 口口工業發展研究所之菌種保存及研究中心,其寄存編號分 別為 CCRC 960096及 CCRC 960097。 (請先閱讀背面之注意事項再填寫本頁) 另本發明亦提供一種利用該單株抗體37 46及/或46 24檢 驗糞便潛血之方法。本方法可用於診斷消化道之病變,例 如胃潰瘍、胃癌、食道潰瘍、食道癌、十二指腸潰瘍、大 腸/直腸癌及其息肉(p〇lyp 。其中較佳為診斷大腸/直腸 癌。 所有習知之抗體/抗原結合偵測方法皆可適用於實施本發 明之檢驗方法,例如酵素連結免疫吸附分析法(EUSA)、三 明治分析法、競爭性分析法、乳膠凝集反應、層析免疫分 析法等。 另本發明亦提供一種利用該單株抗體37 46及46 24檢驗糞 便潛血之試劑,其可包含⑴樣品接觸區,抗體指示劑 區’(3)反應測試區,(4)液體吸附區,及(5)載體。其中該 載體係為一或多種可提供毛細現象並吸附液體之多孔性物 -質所構成,例如濾紙、硝化纖維膜、耐龍膜、玻璃纖維膜 等。#日示劑可辱例如有色乳膠顆粒(c〇l〇re(j latex particies)、 至屬膠負溶液(metallic colloidal solution),如碎膠溶液 經濟部智慧財產局員工消費合作社印製 (colloidal silica particles)及染料膠質溶液(dye solution)等皆為 商品化之產品。 本發明之另一目的係提供一種利用該單株抗體37 46及或 46.24檢驗糞便潛血之套組。且該套組可進一步包含可用於 檢測糞便潛血之習知化學試劑,例如癒瘡木脂化學法, 化學法,氨基比林化學法,聯苯胺化學法,或鄰甲苯胺化 58055.DOC\en „ 7 - ' , 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐) 1235159 A7 B7 經濟部智慧財產局員工消費合作社印製 五、發明說明(5:) , 學法之試劑。 茲以下述實例進一步說明本發明,但該等實例不應視為 本發明之限制條件。 實例1鼠源融合瘤37.46及46.24之製備 純化之人類血紅素(Sigma,cat. H-7379,lot. 32h9329 ) 與弗氏佐劑(Frened’s adjuvant,Gibco)調配成 1 : 1 (v/v) 之乳化液,其中除第一次免疫注射時使用完全佐劑 外,其後均使用不完全佐劑,將此乳化液以皮下注射 之方式免疫6至8週之BALB/C小白鼠(每隻注射100微 克之人類血紅素),一週注射一次,連續注射四週, 爾後每隔四週補強一劑(每隻注射40微克之人類血紅 素),共進行四次,於進行細胞融合前三天,再以靜 脈注射(不含佐劑)追加一劑。 三天後犧牲小白鼠並取出脾臟,將脾臟細胞與鼠源骨 髓瘤F 0細胞株(ATCC CRL1646)進行融合,利用50%之 聚乙二醇400(PEG400)以1 ·· 3 (脾臟細胞:鼠源骨髓瘤 F〇細胞株),之比例融合後,將細胞懸浮於RPMI培養基 (HAT,20%FCS) ’並將其稀釋成每毫升105個細胞後,種 入9 6穴培養盤中(每穴0.2毫升)培養。7日後,以吸附 人類血紅素之ELISA盤測試細胞培養上清液,選取與人 類血紅素具有專一性之融合瘤,再以限制稀釋法予以 單株化。 經此方法分離出單株細胞之鼠源融合瘤細胞株37.46 及46.24。其所分泌之單株抗體皆為IgG, kappa輕鏈。其 58055. D〇C\en - 8 - ^ ^ (請先閱讀背面之注意事項再填寫本頁)V. Description of the invention (4) The storage numbers of the strain preservation and research center of the Koukou Industrial Development Institute are CCRC 960096 and CCRC 960097, respectively. (Please read the precautions on the back before filling out this page) In addition, the present invention also provides a method for testing fecal occult blood using the single antibody 37 46 and / or 46 24. This method can be used to diagnose gastrointestinal lesions, such as gastric ulcer, gastric cancer, esophageal ulcer, esophageal cancer, duodenal ulcer, colorectal / rectal cancer and polyps (polip. Among them, the diagnosis of colorectal / rectal cancer is preferred. All known antibodies / Antigen-binding detection methods can be applied to implement the detection methods of the present invention, such as enzyme-linked immunosorbent assay (EUSA), sandwich analysis, competitive analysis, latex agglutination, chromatographic immunoassay, etc. The invention also provides a reagent for testing fecal occult blood using the single antibody 37 46 and 46 24, which may include a tritium sample contact area, an antibody indicator area '(3) a reaction test area, (4) a liquid adsorption area, and (5 ) Carrier, where the carrier is composed of one or more porous materials that can provide capillary phenomenon and absorb liquids, such as filter paper, nitrocellulose membrane, nylon film, glass fiber membrane, etc. # 日 示 剂 can shame such as Colored latex particles (c latex particies), metal colloidal solution (eg colloidal solution) Colloidal silica particles and dye solution are all commercial products. Another object of the present invention is to provide a kit for testing fecal occult blood using the single antibody 37 46 and 46.24. And the kit can further include conventional chemical reagents that can be used to detect fecal occult blood, such as guaiac chemistry, chemistry, aminopyrine chemistry, benzidine chemistry, or o-toluidine 58055.DOC \ en „7-', this paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1235159 A7 B7 Printed by the Consumers’ Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 5. Description of the invention (5 :) The following examples further illustrate the present invention, but these examples should not be considered as limiting conditions of the present invention. Example 1 Preparation of Purified Human Heme (Sigma, cat. H-7379, Murine Fusion Tumors 37.46 and 46.24) lot. 32h9329) and Freund's adjuvant (Gibco) to prepare a 1: 1 (v / v) emulsion, with the exception of using the complete adjuvant for the first immunization injection and subsequent use of Full adjuvant, this emulsion was injected subcutaneously into BALB / C mice (each injected with 100 micrograms of human hemoglobin) for 6 to 8 weeks, injected once a week for four consecutive weeks, and then strengthened every four weeks Agents (40 micrograms of human hemoglobin per injection) were administered four times, and three days before cell fusion, an additional dose was administered intravenously (without adjuvant). Three days later, the mice were sacrificed and the spleen was removed. Spleen cells were fused with murine myeloma F 0 cell line (ATCC CRL1646), and 50% polyethylene glycol 400 (PEG400) was used to 1. · 3 (spleen cells: murine myeloma F 0 cell line), After the ratios are fused, the cells are suspended in RPMI medium (HAT, 20% FCS) and diluted to 105 cells per ml, and then seeded into 96-well culture plates (0.2 ml per well) and cultured. After 7 days, the cell culture supernatant was tested with an ELISA plate that adsorbed human heme, and fusion tumors specific to human heme were selected, and then singulated by the limiting dilution method. The mouse-derived fusion cell lines 37.46 and 46.24 of a single cell were isolated by this method. The monoclonal antibodies secreted by them are IgG, kappa light chain. Its 58055. D〇C \ en-8-^ ^ (Please read the notes on the back before filling this page)
裝---- 訂--------- Φ. 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐) 1235159 A7 B7 五、發明說明(8 ) 磷酸鹽缓衝液,ρΗ=7·〇),於室溫下阻斷3〇分鐘,取出 硝化纖維膜置入恆溫(20°c )恆濕(24%濕度)之乾燥室中 乾燥。取長寬為7公分X 30公分之背板,在由上方算起 4.2公分及4.9公分以裁刀各劃一刀,撕開4 2公分上方 紙片,將NC模沿撕開部分下緣往上黏貼。再取2 4公分 X 30公分的濾紙(Whatman,3 MM Chr)沿撕開部分之背板 上緣往下黏貼。撕去背板4·9公分下方紙片,將含膠體 金-單株抗體46.24結合體混合物之玻璃纖維膜條片,置 於背板中央紙片未撕層之中間位置。將對折好之4.2公 分X 30公分之玻璃纖維膜沿背板下緣往上黏貼。最後 取30公分的3Μ膠帶由纖維膜條片上方約3亳米處往下黏 貼。將黏貼組合的試劑片切成7公分χ 〇 8公分的條片 (如圖1所示)置於試劑盒下蓋,再蓋上 开|上上盍,恆溫恆濕 乾燥18小時後,待測活性。 測試時,於試劑盒上的樣品孔放入約2〇〇微升樣品, 樣品為不同濃度的人類血紅素,稀釋液為〇1 %檸檬酸 鹽緩衝劑ρΗ,8.0+0.05% triton。其結果如下表2所示 表2 (請先閱讀背面之注意事項再填寫本頁)Packing ---- Order --------- Φ. This paper size is applicable to China National Standard (CNS) A4 (210 X 297 mm) 1235159 A7 B7 V. Description of the invention (8) Phosphate buffer solution , ΡΗ = 7.0), block at room temperature for 30 minutes, remove the nitrocellulose membrane and place it in a drying room with constant temperature (20 ° c) and constant humidity (24% humidity) for drying. Take a backboard with a length of 7 cm x 30 cm, and draw 4.2 cm and 4.9 cm from the top with a cutter, tear off the 4 2 cm piece of paper, and stick the NC die along the lower edge of the torn part. . Then take 2 4 cm X 30 cm filter paper (Whatman, 3 MM Chr) and stick it along the upper edge of the back plate of the torn part. The paper sheet below the back panel 4 · 9 cm was torn off, and a glass fiber membrane strip containing a colloidal gold-single antibody 46.24 conjugate mixture was placed in the middle of the unteared layer of the paper sheet in the center of the back sheet. Paste the folded 4.2 cm x 30 cm glass fiber membrane along the lower edge of the back plate. Finally, take 30 cm of 3M tape and stick it down about 3mm above the fiber membrane strip. Cut the combined reagent piece into 7 cm x 08 cm strips (as shown in Figure 1) and place it on the lower cover of the kit, and then cover the open | upper top, and dry at constant temperature and humidity for 18 hours. active. During the test, about 200 microliters of sample was placed in the sample well on the kit. The samples were human heme at different concentrations, and the dilution was 0.1% citrate buffer ρΗ, 8.0 + 0.05% triton. The results are shown in Table 2 below. Table 2 (Please read the precautions on the back before filling out this page)
裝--------訂---------^^^1 經 濟 部 智 慧 財 產 局 員 工 消 費 合 作 社 印 製Packing -------- Order --------- ^^^ 1 Printed by the Consumer Affairs Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs
a :人類血紅素濃度單位為微克/亳升 b : +++表清楚強線; 表清楚強線; 表清楚線; 58055. D0C\en -11 - 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐) 1235159 A7 B7 五、發明說明( 士表弱線; -表無線條顯現。 由表2之結果可知 度為0.15微克/毫升。 本發明可偵測人類血紅素之最低濃 (請先閱讀背面之注意事項再填寫本頁)a: The unit of human hemoglobin concentration is micrograms / liter. b: +++ indicates a clear line; indicates a clear line; indicates a clear line; 58055. D0C \ en -11-This paper scale applies Chinese National Standard (CNS) A4 Specifications (210 X 297 mm) 1235159 A7 B7 V. Explanation of the invention (weak lines on the wristwatch;-wireless bars on the watch. From the results in Table 2, it can be known that the degree of hemoglobin is 0.15 μg / ml. Rich (Please read the precautions on the back before filling this page)
裝---- 訂..--------#. 經濟部智慧財產局員工消費合作社印製 58055. DOC\en 12- 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐)Packing ---- Order ..-------- #. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 58055. DOC \ en 12- This paper size is applicable to China National Standard (CNS) A4 (210 X 297 mm)
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US11175292B2 (en) | 2016-05-30 | 2021-11-16 | Eiken Kagaku Kabushiki Kaisha | Anti-human hemoglobin monoclonal antibody or antibody kit, insoluble carrier particle to which anti-human hemoglobin monoclonal antibody is immobilized, and measurement reagent and measurement method using same |
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US11175292B2 (en) | 2016-05-30 | 2021-11-16 | Eiken Kagaku Kabushiki Kaisha | Anti-human hemoglobin monoclonal antibody or antibody kit, insoluble carrier particle to which anti-human hemoglobin monoclonal antibody is immobilized, and measurement reagent and measurement method using same |
TWI800483B (en) * | 2016-05-30 | 2023-05-01 | 日商榮研化學股份有限公司 | Anti-human hemoglobin monoclonal antibody or antibody set, anti-human hemoglobin monoclonal antibody immobilized insoluble carrier particle, and measurement reagent or measurement method using the same |
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