587166 玖、發明説明： 【發明所屬之技術領域】 本發明爲延續美國專利申請號10/035〇14，2001年12月24日。 【先前技術】 多樣分析步驟及裝置爲常見使用溢流道檢驗來偵測存在於測試樣品 分析物的存在及/或濃度。舉例，免疫系統的免疫分析利用機器，其中抗體 產生對存在抗原的反應’其對生物體爲爲致病物或外來物。這些抗體及抗 原，如免疫反應能夠結合另一個，因此產生高度特殊反應機器能夠使用來 偵測在生物體樣品中特殊抗原的存在及濃度。 這些已知技術的免疫方法其使用與可偵測複合物的免疫反應標籤，因 此分析物可以被分解分析。舉例，“夾層形式“檢驗一般包含混合有抗體 的測試樣品及分析物。這些抗體爲移動及連結在一標籤或探子，如染色 蠟，膠狀金屬溶液，或放射性同位素。此混合物接著接觸包含一對分析物 固定不動的抗體帶或區的色層分析材料。色層分析材料通常形成類似量尺 的支板。當分析物與標的抗體混合到達在色層分析材料上固定不動的抗 粗’發生結合且結合標的抗體於區域中的小範圍。其指出分析物的存在。 此技術可被使用來包含定量及半定量結果。此種夾層形式檢驗的例子揭示 於美國專利編號4168146，Grubb等人及436624丨，T〇m等人。 一種替換的技術爲“競爭形式“檢驗。“競爭形式“檢驗，標籤一般 標定分析物或分析物-類似物，其競爭結合與與任何未標的分析物的抗體存 在於樣per中。:f兄爭檢驗一般使用來偵測分析物如半抗原，每一個半抗原爲 一價僅能夠結合一個抗體細胞。競爭免疫分析裝置的例子描述於美國專利 編號 423560卜 Deutsch 等人，4442204，Liotta，及 5208535，Buechler 等 許多這些檢驗依靠校正來提供有根據且有意義的結果，特别是半定量 及定量偵測。特别的，外部及内部分析系統一般被利用。在一個外部校正 系統中，一個標準曲線一般包含一連續已知分析物數量的標準品，且包含 E:\PATENT\PK.00108\pk-001-0827\pk-00l-0827.doc2003/6/t2 587166 樣品的結果與鮮轉峨_取錄品中分析物的絲及/或數量 校正万油當輯簡單且簡單操作。然而，其—般與環境及—批—批 程度干擾，且不可靠。 便利的内部校正系統，換言之，一般利用一種有校正區及檢波區的薄 膜在分析物捕麵上朗定的。林的，常見_输正系統爲使用生物 捕捉劑（如抗體）於校正區。此生物捕獲劑貴，易損壞，且在整體作用物 準的數量難以控制。另夕卜，生物捕捉劑亦傾向於降低耗時。 士種$要乂即存在的準確溫泥道校正彳、統爲可被控制及不昂貴， 且不顯著降低耗時。 、 【發明内容】 提及本發明的具體實施例，溢流道檢驗（如央層，競爭，等）被揭示 ^偵測測試樣品中分析物的存在及數量。此檢驗包含_個多孔膜，其在液 體流動間與包含特殊、结合物及可侧探子的探子配對。舉例，在一些 具恤貝施例中，可偵測的探子從發光體，催化劑，螢光物質，化學發 光物^，放射性標籤，可見標籤，微脂粒，及結合物。在一種特殊^ 具體貫施例中，可偵测的探子包含乳膠微粒。 人多孔膜亦定義爲一包含能夠結合分析物或配對探子的捕捉劑之 =波區。在一些具體實施例中，舉例，捕捉劑可由抗原，半抗原，抗 也’及衩合物中選擇。檢波區能夠產生檢波信號以指出分析物的存在。 另外，幫助存在於樣品中分析物數量的偵測，多孔膜亦定義一個 才又正區，其中聚電解質爲非分散固定於多孔膜。校正區包還一個或多 個包έ 解質的校正區（如線，點，等）。聚電解質能與探子結合 物結合。使用於校正區的聚電解質一般有任何需要的電荷。雖然不需 2 ’聚電解質的電荷可選擇與探子電荷相反的電荷，因此，幫助相對 電荷分子間離子結合形成。舉例，在一具體實施例中，聚電解質爲負 電荷。在此例子，聚電解質，在一些具體實施例中，從包含聚離胺酸， 聚乙醇胺，環氧氣丙烷官能聚胺及/或聚氨基胺，聚二丙烯二甲基-氣化 鋁，陽離子纖維素，及相似物中選擇。 E:\PATENT\PK-001 08\pk-001-0827\pk-001-0827.doc2003/6/p 6 聚電％質固定於校正區中一般可以多種方式達成。舉例，在一具體實 施例中，具電荷之聚電解質分子可以與某些存在於多孔膜的官能基形成離 子結合。同樣的，爲了形成更固定不變，聚電解質有時與存在於多孔膜上 的έ此基共價鍵結。舉例，在一具體實施例中，可交聯的聚電解質，如環 氧氣丙垸官能聚胺及/或聚氨基胺可與多孔膜交聯。 一個校正區產生的信號，其接著可與檢波信號比較來偵測在測試樣品 中分析物存在或崎數量。舉例，在—些具體實施例中，校正信號可見且 可與檢波信航較。然而，校正信號亦可與通職器使㈣檢波信號比 較。如螢光讀取機，顏色強度讀取機，及相似。若需要，一種校正曲線可 由校正信號與已知量的分析物之縣賊立。—個生成，麟接著使用於 偵測在測減樣品中未知量的分析物。 ，及本發明另-個具體實施例，揭示_個侧測試樣品t分析物存在 及數量的溢跋檢驗。溢流道檢驗包好孔膜林液體流動間與包含特 ，結合物及可制探子的探子配對。當此接觸時，配娜子夠行與測 4^中的分析物結合，因此配聰子/讀物複合物及未結合配對 探子形成。進一步，多孔膜定義爲檢波區。捕捉劑非遍佈性固定於檢 波區内的乡。触織姆合配聰子/分析物複合物以產生檢 波信號。缝的聚電解質非散佈映於校正區中多孔膜上。校正區能 產，校正信號_檢波信號比較，其中在測試樣樣品中_對分析物 含量由比較檢波信號欲校正信號來偵測。 本發明提及P嬉體實摘，揭示試樣品中分析物 、、如、’、抗原）存在及數量的溢流道檢驗。溢流道檢驗包含多孔膜其在 设動間與包的.殊結合物及可彳貞測探子的探子配對。舉例，在一 ，具體實摘巾，爲對分析物翻的結合膜。多孔膜定義—個檢波 ，，其中固定數量的捕捉劑非遍佈性固定於檢波區内的多孔膜。捕捉 j (如，抗體）可結合分析物（如，抗原），因此測試樣品的分析物 2對探付固定數量的捕捉劑競爭。檢波區能夠產生檢波信號。定 =的聚電解質非散佈固定於校正區中多孔膜上。校正1能產生校正信 W與檢波信號比較，其中在測褪樣品中_對分析物含量由比較 E:\PATENT\PK-001 08\pk-001 -0827\pk-001-0827.doc2003/6/12 587166 fe波彳5 ϊ虎欲校正信號來偵測。 仍提及本發明另一個具體實施例，揭示一個偵測測試樣品中分析 物（如，抗原）存在及數量的溢流道檢驗。檢驗包含多孔膜其在液體 泥動間與包含特殊結合物（如抗體）及可偵测探子的探子配對。當此 接觸時，配對探子夠行與测試樣品中的分析物結合，因此配對探子/ 刀析物la物及未結合配對探子形成。多孔膜定義一個檢波區，其中 固足數1的捕捉劑非遍佈性固定於檢波區内的多孔膜。捕捉劑（如， 抗，）可與未結合配對探子結合物，其中檢波區能夠產生檢波信號。 ^的聚電解質非散佈眺於校正區中多孔膜上。校正區能I生校正 信號以與檢波信號比較，其中在測試樣樣品中的相對分析物含量由比 較檢波信號欲校正信號來偵測。 本發明其他特色及觀點更詳細揭示於下。 【實施方式】 定義 秦 如在此使用‘分析物“一般提及爲被偵測的物質。舉例，分析物可包 含抗原物質，半抗原，抗體，及結合物。分析物包含，但不限制，毒素， 有械物貝，蛋白貝，胜肽，微生物體，胺基酸，核酸，荷爾蒙，類固醇， 維他命，禁物（包含提供治療目的如提供非法目的一樣），、細菌，病毒微 粒及代谢物或任何上面物質的抗體。一些分析物特别的例子包含鐵蛋白， 肌酸肝酶MIB (CK-ΜΒ);高地辛；二苯妥因；苯基巴比妥；卡巴氮平； Λ克Μ素，紫菌素；茶鹼；丙戊酸；奎尼丁；黄體素（LH);濾泡刺激素 (FSH );***；黄自同素；IgE抗體；維生素B2微球蛋白；糖化血色素 (Gly.Hb)’皮質醇；洋地黄毒；N-乙醯普魯卡因胺（NApA);普魯卡因 胺；德國痳疹病毒，如德國痳疹IgG及德國痳疹IgM :弓蟲抗體，如弓蟲 IgG (Toxo-IgG)及弓蟲lgM (ToxCK[gM);睾固酮；水楊酸；乙酿基胺基 苯；B型肝炎表面抗原（HbsAg); B型肝炎中心抗原抗體，如抗B型肝炎 中心抗原IgG及IgM( Anti-HBC );人體免疫缺乏病毒i及％ mvi及2 ); 人類Τ細胞白血病病毒！及2 ( HTLV ) ; β型肝炎e抗原（HbeAg ) ; β型587166 发明 Description of the invention: [Technical field to which the invention belongs] The present invention is a continuation of US Patent Application No. 10/035001, December 24, 2001. [Prior art] Various analysis steps and devices are commonly used to detect the presence and / or concentration of analytes in a test sample using overflow channel testing. For example, an immune analysis of the immune system uses a machine in which an antibody produces a response to the presence of an antigen ' which is pathogenic or foreign to an organism. These antibodies and antigens, such as an immune response, can bind to another, so a highly specialized response machine can be used to detect the presence and concentration of specific antigens in a biological sample. These known techniques of immunization use immunoreactive tags with detectable complexes so that the analyte can be broken down for analysis. For example, a "sandwich" test typically includes a test sample and analyte mixed with antibodies. These antibodies are mobile and attached to a tag or probe, such as stained wax, colloidal metal solutions, or radioisotopes. This mixture is then contacted with a chromatographic analysis material comprising a pair of analyte-immobilized antibody bands or regions. Chromatographic analysis materials often form support plates similar to a ruler. When the analyte and the target antibody are mixed to reach the anti-rough ' immobilized on the chromatographic analysis material, binding occurs and the target antibody binds to a small area in the region. It indicates the presence of the analyte. This technique can be used to include quantitative and semi-quantitative results. Examples of such sandwich-form inspections are disclosed in U.S. Patent Nos. 4,168,146, Grubb et al. And 436624, Tom et al. An alternative technique is a "competitive form" test. A "competitive format" test, which typically labels an analyte or analyte-analog, competes for binding with antibodies to any unlabeled analyte in a sample. The: f test is generally used to detect analytes such as haptens. Each hapten is a cell that can bind to only one antibody at a single price. Examples of competitive immunoassay devices are described in U.S. Patent No. 423560, Deutsch et al., 4442204, Liotta, and 5208535, Buechler, etc. Many of these tests rely on calibration to provide evidence-based and meaningful results, especially semi-quantitative and quantitative detection. In particular, external and internal analysis systems are generally used. In an external calibration system, a standard curve generally contains a standard with a known number of analytes, and contains E: \ PATENT \ PK.00108 \ pk-001-0827 \ pk-00l-0827.doc2003 / 6 / t2 587166 The result of the sample and the freshly transformed E_The silk and / or quantity correction of the analyte in the enrolled product is easy and simple. However, its level of interference with general and environmental and batch-by-batch levels is unreliable. Convenient internal calibration system. In other words, a thin film with a calibration zone and a detection zone is generally used to determine the analyte capture surface. Lin's, a common system is the use of biological capture agents (such as antibodies) in the calibration area. This bio-capture agent is expensive, easily damaged, and the quantity of the whole acting standard is difficult to control. In addition, biocapture agents also tend to reduce time consumption. There is an accurate warm mud track correction, which can be controlled and inexpensive, and does not significantly reduce time consumption. [Summary of the Invention] Referring to the specific embodiment of the present invention, overflow channel inspection (such as central layer, competition, etc.) is revealed. ^ Detect the presence and quantity of analytes in the test sample. This test consists of a porous membrane that is paired with a probe containing special, bound, and lateral probes between liquid flows. For example, in some shirted embodiments, detectable probes are from illuminants, catalysts, fluorescent substances, chemiluminescent substances ^, radioactive tags, visible tags, microlipids, and conjugates. In a particular embodiment, the detectable probe comprises latex particles. A human porous membrane is also defined as a wave region containing a capture agent capable of binding to an analyte or a paired probe. In some embodiments, for example, the capture agent may be selected from an antigen, a hapten, an antibody, and a conjugate. The detection zone can generate a detection signal to indicate the presence of the analyte. In addition, to help detect the amount of analytes present in the sample, the porous membrane also defines a positive region, where the polyelectrolyte is non-dispersively fixed to the porous membrane. The calibration area package also contains one or more degraded calibration areas (such as lines, points, etc.). Polyelectrolytes can bind to probe conjugates. Polyelectrolytes used in the calibration zone generally have any required charge. Although the charge of the 2 ' polyelectrolyte is not required, the charge opposite to the probe charge can be selected, and therefore, it assists the formation of intermolecular ions of the relative charge. For example, in a specific embodiment, the polyelectrolyte is negatively charged. In this example, the polyelectrolyte, in some specific embodiments, comprises polyionine, polyethanolamine, epoxy propane-functional polyamines and / or polyaminoamines, polydipropylenedimethyl-aluminum vaporized, and cationic fibers. Choice of similar ingredients. E: \ PATENT \ PK-001 08 \ pk-001-0827 \ pk-001-0827.doc2003 / 6 / p 6 Fixing the charged mass in the calibration area can be achieved in many ways. For example, in a specific embodiment, a charged polyelectrolyte molecule can be ionically bound to certain functional groups present in a porous membrane. Similarly, in order to form a more fixed, polyelectrolyte is sometimes covalently bonded to a thiol group present on a porous membrane. For example, in a specific embodiment, a crosslinkable polyelectrolyte, such as an epoxy propionate-functional polyamine and / or a polyaminoamine, can be crosslinked with a porous membrane. A signal generated by a calibration zone can then be compared to the detection signal to detect the presence or amount of analyte in the test sample. For example, in some embodiments, the correction signal is visible and comparable to the detection signal. However, the calibration signal can also be compared with the detection signal of the universal instrument. Such as fluorescent readers, color intensity readers, and similar. If desired, a calibration curve can be established by the calibration signal and a known amount of analyte. One generation was then used to detect an unknown amount of analyte in the subtracted sample. , And another specific embodiment of the present invention, reveals the overflow test of the presence and quantity of the analyte in each side test sample t. The overflow channel test covers the liquid flow cell of the porous membrane forest and is paired with a probe containing a special compound, a conjugate, and a detectable probe. When this contact occurs, the gamete is able to bind to the analyte in the test, so the gamete / reader complex and unbound paired probes are formed. Further, a porous membrane is defined as a detection region. The capture agent is non-persistently fixed in the township within the detection area. The zombie is mated to the satoshi / analyte complex to generate a detection signal. The slitted polyelectrolyte is non-dispersed on the porous membrane in the calibration zone. In the calibration zone, the calibration signal is compared with the detection signal. In the test sample, the analyte content is detected by comparing the detection signal with the signal to be corrected. The present invention refers to the actual extraction of the P body, and reveals the overflow channel inspection of the presence and quantity of the analyte (such as, ', antigen) in the test sample. The overflow channel test consists of a porous membrane that is paired with a special conjugate and a probe that can detect the probe between the set. For example, in one example, a concrete towel is a binding membrane that turns over the analyte. Porous membrane definition—a detection wave, in which a fixed amount of capture agent is non-pervasively fixed to a porous film in the detection zone. Capture j (e.g., an antibody) can bind to an analyte (e.g., an antigen), so analyte 2 of the test sample competes for a fixed number of capture agents. The detection area can generate a detection signal. The fixed polyelectrolyte is non-dispersed and fixed on the porous membrane in the calibration zone. Calibration 1 can generate a calibration letter W compared with the detection signal, where in the measured sample _ the analyte content is compared by E: \ PATENT \ PK-001 08 \ pk-001 -0827 \ pk-001-0827.doc2003 / 6 / 12 587166 fe wave 彳 5 ϊ Tiger wants to correct the signal to detect. Still referring to another embodiment of the present invention, an overflow channel test is disclosed that detects the presence and amount of an analyte (e.g., an antigen) in a test sample. The test consists of a porous membrane that is paired with liquid probes that contain special binders (such as antibodies) and detectable probes. When this contact occurs, the paired probe is sufficient to bind to the analyte in the test sample, so the paired probe / knife precipitate and the unbound paired probe are formed. The porous membrane defines a detection region in which the capturing agent having a fixed number of 1 is non-pervasively fixed to the porous membrane in the detection region. The capture agent (eg, anti-) can be combined with an unbound paired probe, where the detection zone is capable of generating a detection signal. The polyelectrolyte is non-spread on the porous membrane in the calibration zone. The calibration area can generate a calibration signal for comparison with the detection signal, wherein the relative analyte content in the test sample is detected by comparing the detection signal with the signal to be corrected. Other features and perspectives of the present invention are disclosed in more detail below. [Embodiment] Definition Qin Ru uses the term “analyte” to be generally referred to as the detected substance. For example, the analyte may include antigenic substances, haptens, antibodies, and conjugates. The analytes include, but are not limited to, Toxins, organic shellfish, protein shellfish, peptides, microorganisms, amino acids, nucleic acids, hormones, steroids, vitamins, contrabands (including for the purpose of providing treatment, such as illegal purposes), bacteria, viral particles and metabolites or Antibodies to any of the above. Specific examples of some analytes include ferritin, creatine liver enzyme MIB (CK-MB); digoxin; diphenytoin; phenylbarbital; carbazapine; Λgram M, Puromycin; theophylline; valproic acid; quinidine; lutein (LH); follicle stimulating hormone (FSH); estradiol; aflatoxin; IgE antibody; vitamin B2 microglobulin; glycated hemoglobin (Gly.Hb ) 'Cortisol; Digitalis Poison; N-Ethylprocainamide (NApA); Procainamide; German rash virus, such as German rash IgG and German rash IgM: Toxoplasma antibodies, such as bow IgG (Toxo-IgG) and Toxoplasma lgM (ToxCK [gM); testosterone; Salicylic acid; Ethylaminobenzene; Hepatitis B surface antigen (HbsAg); Hepatitis B center antigen antibodies, such as anti-Hepatitis B center antigens IgG and IgM (Anti-HBC); Human immunodeficiency virus i and% mvi and 2); human T-cell leukemia virus! and 2 (HTLV); beta hepatitis e antigen (HbeAg); beta type
Ε \ΡΛΤΕΝΤ\Ρκ.〇〇l 〇8\pk-〇〇 | -0827\pk-001 -0827.doc2003/6/12 Q 肝炎e抗原抗體Unti-HBe);促甲狀腺素（TSH );甲狀腺素 她亞 甲狀腺素（TcM T3 );自由甲狀腺素（Free T3 );癌胚抗體（叫；癌 胎蛋白（婚）。»顧及_物質包含，但躺娜制，铸 基安非它命’巴比妥，如異戊巴比妥，西可巴比妥，戊巴比妥，苯基巴比 文，及巴比妥；安恩香重氮類，如利眠寧，樂安定；***驗，如印度*** 及***於；古柯鹼；坦尼·，麥角二乙胺；甲酉同；鴨片，如***，嗎啡， 可待因，二氫嗎啡酮，二氫可待因，美_，綠可待醜，超二氯嗎钟 及馨粟：翊|j定；及普祕芬。其他可能的分析物揭示於美國專利編號 4366241，等人。 如在此使用“測試樣品“―般提及爲疑似包含分析物的物質。測試樣 品可直接使用如包倾來源或聽縣特性。職樣品可從任何 生物體來源取得，如生職，包含血液，唾液，眼淚，腦脊髓液，汗液， 尿液，乳汁’腹水，沙聲，關節液，《液，羊水，或類似。測試樣品可 在使用時先叙’如從血鱗備錢，稀液，及她。處理方法可包 含過渡，蒸館’濃縮’與干擾物反應，及添加試劑。除了生理液外，其他 液體樣品可以使用如水’食物產品，及像環境及食品檢驗性能。另外，疑 似包含分析物的固體材料可如峨樣品般使用。在—些例子中，其有利於 修飾一固體測試樣品形成液態或釋放分析物。 詳細描述 現在詳細提及本發明多樣具體實施例，_❹個樣品呈現於後。每一 個例子由本發明的方法提供，但不局限於本發明。事實上，其將呈現這些 技術技能’多樣修飾及變化可由本發_成但獨開本發明翻^範園。 舉例，特徵説明或描述如部份具體實施例。因此，其傾向於本發明覆蓋這 些修飾及變化如在其申請專利範園内。 一般，本發明指出一個溢流道;^驗的内部校正系統。特别的，本發明 利用一個或多個在校正區内的聚電解質其由檢驗的多孔膜定義之。聚電罅 質被構成以結合探子及/或流過檢驗的探子結合物，因此產生一個能與檢波 信號比較的校正信號。其發現，不近内部校正系統提严偵測在鄉試樣品中 分析物存在的準確方法，使用於系統中的材料亦爲可被控制，不貴，且不 ΕΛΡΑΤΕΝΤΛΡΚ-001 08\pk-001 -0827\pk-001 -0827.doc2003/6/12 9 傾向於降低耗時。 提及第-至三圖，舉例，在—個可以由本發明提及形成的夹層式溢流 (2〇) 〇 ^ (20) 由堅硬材料（未顯示）支撐的多孔薄膜⑻。_般，多孔薄膜⑵）可 由任何多種賴樣品可穿過的材料形成。舉例，使用來形成多孔薄膜（a) 的材料包含，但不限制，天然，合成，或自然發生合成修飾材料，如多醋 類（纖維素材料如紙及纖維素衍生物，如醋酸纖維素，及氮纖維素）；矽 膠；無機材料，如銘衍生物，石夕薄土，硫化鍰，或其他無機細緻分散材料 -至分散於多孔聚合基礎，與聚合物如氣化乙埽，氯化乙稀-丙歸共聚物， 及氣化乙埽-乙烯醋酸共聚物；衣物，天然發生（如棉）及合成（如财龍或 尼龍）；多孔膠體，如矽膠，洋菜膠，葡萄聚醣，及明膠；聚合薄膜，如 聚丙烯胺；及相似。在一個特别的具體實施例中，多孔薄膜（23)由硝酸 纖維素及/或聚醚材料。其必須知道，專有名詞“硝酸纖維素“‘及爲纖維 素硝酸酯，其可以僅爲硝酸纖維素，或混合硝酸酯及其他酸，如有丨至7 個蚊基的脂肪族叛酸。 最初偵測在测試樣品内的分析物（40)，一個使用者可直接塗抹測試 樣品穿過一部份的多孔薄膜（23)，其接著可以移動到達一個或多個偵測 及校正區（揭示於下）。可替換的，測試樣品首先塗抹至抽樣墊上，其與 多孔薄膜（23)流動聯繫。舉例，如第一至三圖所示，側向流動檢驗（2〇) 可包^般構成來接收樣品的抽樣塾（21 )。一些適合可被使用形成抽产 墊（21 )的材料包含，但不限制，硝酸纖維素，纖維素，多孔聚乙埽塾二 及玻瑞纖維滤紙。若需要，樣品垫（21 )亦可包含一個或多個檢驗前處理 試劑，散佈或非散佈黏附。 在説明的具體實施例中，測試樣品從抽樣墊（21 )移動至結合塾（22 ) (由第一圖箭頭（29)所示）其位在連接抽樣墊（2〗）末端的位置。結合 墊（22)由測試樣品可以穿過的材料製成。舉例，在一具體實施例，結人 墊（22)由玻璃纖維形成。 除了簡單允許測試樣品穿過，結合墊（22)亦完成一般功能。舉例， 在一些具體實施例中，多樣探子（41 )(見第二圖）可釋放塗抹至結合塾 E:\PATENT\PK-OOI 08\pk-001 -0827\pk-001 -0827.doc2003/6/l 2 ^ (22 )。當包含於結合塾（22 )時，這此谈斗「 μ (40 ) ( 4〇 ) ( 2i } f ( ^ 賺物^ (跡—）存在檢驗 钟可以產生信號的物質爲視覺可_或由儀器設備制的计被使 二H (41 )。多樣合適的探子可包含發光體；放射體標籤；肉眼可見 ^減’包含膠狀金屬及非金屬微粒（如黄金），純微粒，酵素絲 =機聚合乳膠微粒；微齡或其他包含信號產生基㈣小囊;'及相似。 舉例’-些適合如探子般使用的酵素揭示於美國專利編號彻⑷仙臟 :其全結合於此./顧探子系統_子鱗紐鱗酸酶及基質 I監四吐_544|3_引朵嶙酸，或衍生物或相似物，或基質4·甲基伞 型花酸顧。在-替換的探子系統，探子可微螢光物質，沒有酵素操作器， 需要屋生檢波錢。航分子，域光m素，玫瑰紅及其衍生物或 相似物’爲適合在此反應巾佩探子。此魏材料前可購得的例子包含 ^ Mdc— Prcbes，Inc•販售的商標名稱“⑽触⑽“（紅5嶋^ TransfluoSphere ‘‘（ 543/620)的營光羧化微球體，如“丁_㈣“及$ 及6-羧基四甲基若丹明—樣，其亦由副⑽以加⑹⑻販售。 »可制，純微粒（麵提及爲“珠粒％v‘微雜“）亦可如 探子般使用，S此提供樣品直接讀取分析物存在或濃度耗讀出而不需要 進-步信號產生劑。在-些例子中，使用於量檢驗的微粒亦可以提供信號 (如光線吸收）其產生區域，其中有不同信號的微粒停留在薄膜⑵）的 位置。 、 做探子（41 )使用的微粒形式亦多樣化。舉例，天然產生微粒，如細 胞核’類菌質體，質體，細胞質體，哺乳動物細胞（如紅血球宿主），單 細胞微生物（如細菌），多醣體（如洋菜膠）及相似物，可被使用。進一 步，合成微粒亦可以被使用。舉例，在一具體實施例中：可被染劑染色的 合成乳膠微粒可如探子（41 )般利用雖然任何可吸收或共價結合至結合 樣式的乳膠微粒可在本發明中使用，乳膠微粒一般由聚苯乙烯，丁二烯苯 乙烯，苯乙烯丙埽酸—乙埽三聚物，聚甲基甲基丙烯酸，聚乙歸甲基丙缔酸， E:\PATENT\PK-001 08\pk-〇〇 1 -〇827\pk-001 -0827.doc2003/6/1 11Ε \ ΡΛΤΕΝΤ \ Ρκ.〇〇l 〇8 \ pk-〇〇 | -0827 \ pk-001 -0827.doc2003 / 6/12 Q Hepatitis e antigen antibody (Unti-HBe); thyrotropin (TSH); thyroxine Her subthyroxine (TcM T3); free thyroxine (Free T3); carcinoembryonic antibody (called; oncofetoprotein (marriage). »Taking into account _ substances contained, but made by Lianna, cast amphetamine 'barbitur , Such as isoprene barbiturate, sicoba barbiturate, pentobarbital, phenobarbital, and barbiturate; diazepam, such as rimienine, leazepam; cannabis test, such as India Cannabis and cannabis; ***e; tannol, ergot diethylamine; metformin; duck flakes, such as heroin, morphine, codeine, dihydromorphone, dihydrocodeine, beauty_, green Can be ugly, super dichloromethane and Xinsu: 翊 | jding; and Promifen. Other possible analytes are disclosed in U.S. Patent No. 4,346,241, et al. If the "test sample" is used here, it is suspected to be mentioned Substances that contain analytes. Test samples can be used directly, such as pour source or Tingxian characteristics. Professional samples can be obtained from any biological source, such as health, including blood , Saliva, tears, cerebrospinal fluid, sweat, urine, breast milk 'ascites, sand, joint fluid, fluid, amniotic fluid, or similar. Test samples can be described before use', such as preparing money from blood scales, dilute fluid , And her. Processing methods can include transitions, steaming halls 'concentrated' react with interfering substances, and adding reagents. In addition to physiological fluids, other liquid samples can use food products such as water, and environmental and food inspection performance. In addition, suspected Analytical-containing solid materials can be used like E-samples. In some examples, it is useful to modify a solid test sample to form a liquid or release an analyte. Detailed Description Now reference is made in detail to various specific embodiments of the present invention, a sample Presented later. Each example is provided by the method of the present invention, but is not limited to the present invention. In fact, it will present these technical skills' various modifications and changes can be made by the present invention, but the invention of the invention can be turned into a model garden. Examples The feature description or description is like some specific embodiments. Therefore, it is intended that the present invention cover these modifications and changes as in its patent application park. Generally, the present invention indicates an overflow channel; an internal calibration system for inspection. In particular, the present invention utilizes one or more polyelectrolytes in the calibration zone, which are defined by the inspection porous membrane. The polyelectrolyte is constructed to Combining probes and / or probe combinations that pass through the test, thereby generating a calibration signal that can be compared with the detection signal. It was found that the internal calibration system has been used to accurately detect the presence of analytes in rural test samples, and is used in The materials in the system are also controllable, not expensive, and not ΕΛΡΑΤΕΝΤΛΡΚ-001 08 \ pk-001 -0827 \ pk-001 -0827.doc2003 / 6/12 9 tend to reduce time consumption. Figure, for example, in a sandwich-type overflow (20) (20) porous membrane supported by a hard material (not shown) that can be formed by the present invention. In general, porous membranes ⑵) can be formed from any of a variety of materials that a sample can pass through. For example, the materials used to form the porous film (a) include, but are not limited to, natural, synthetic, or naturally occurring synthetic modified materials such as polyacetates (cellulosic materials such as paper and cellulose derivatives such as cellulose acetate, And nitrocellulose); Silicone; Inorganic materials, such as Ming derivatives, Shixi thin soil, sulfide, or other inorganic finely dispersed materials-to disperse on the basis of porous polymerization, and polymers such as gaseous ethyl acetate, ethyl chloride Dilute-acrylic acid copolymers, and vaporized acetamidine-vinyl acetate copolymers; clothing, naturally occurring (such as cotton) and synthetic (such as Cailong or nylon); porous colloids, such as silicone, agar gum, glucosan, And gelatin; polymeric films such as polyacrylamine; and similar. In a particular embodiment, the porous film (23) is made of nitrocellulose and / or polyether material. It must be known that the proper term “nitrocellulose” is cellulose nitrate, which can be only nitrocellulose, or a mixture of nitrate and other acids, such as 7 to 4 mosquito-based aliphatic rebel acids. The analyte (40) is initially detected in the test sample. A user can directly apply the test sample through a portion of the porous membrane (23), which can then be moved to one or more detection and calibration zones ( Revealed below). Alternatively, the test sample is first applied to a sampling pad, which is in flow connection with the porous film (23). For example, as shown in Figures 1 to 3, the lateral flow test (20) can be constructed to receive a sample of samples (21). Some suitable materials that can be used to form the production pad (21) include, but are not limited to, nitrocellulose, cellulose, porous polyethylene and fiberglass filter paper. If desired, the sample pad (21) may also contain one or more pre-test processing reagents, scattered or non-dispersed. In the illustrated specific embodiment, the test sample is moved from the sampling pad (21) to the bonding pad (22) (shown by the arrow (29) in the first figure) at a position connected to the end of the sampling pad (2). The bonding pad (22) is made of a material through which the test sample can pass. For example, in a specific embodiment, the knotting mat (22) is formed of glass fiber. In addition to simply allowing the test sample to pass through, the bonding pad (22) also performs general functions. For example, in some embodiments, the multiple probes (41) (see the second figure) can be released and applied to the bond 塾 E: \ PATENT \ PK-OOI 08 \ pk-001 -0827 \ pk-001 -0827.doc2003 / 6 / l 2 ^ (22). When included in conjunction with 塾 (22), this argument "μ (40) (4〇) (2i) f (^ earning ^ (trace-) the existence of the test clock can produce a signal is visually _ or by The instrument is made of H 2 (41). A variety of suitable probes can include light emitters; radiator tags; visible to the naked eye ^ minus' including colloidal metal and non-metal particles (such as gold), pure particles, enzyme silk = Organic polymer latex particles; young age or other vesicles containing signal-generating bases; 'and similar. Examples'-some enzymes suitable for use as probes are disclosed in U.S. Patent No. Tossin's Dirty: It's all incorporated here./Gu Probing System _Zygoscale Neuraminidase and Matrix I Supervisory Surgery_544 | 3_ Inosine acid, or derivative or analogue, or matrix 4 · methylumbelliferous acid Gu. In-replacement probe system The probe can be a micro-fluorescent substance, without an enzyme manipulator, and it requires room detection money. Aeronautical molecules, domain light m, rose red and its derivatives or similar are 'suitable for wearing the probe in this reaction. Before this Wei material Commercially available examples include ^ Mdc—Prcbes, Inc 5 嶋 ^ TransfluoSphere ”(543/620) of Yingguang carboxylated microspheres, such as“ Ding_㈣ ”and $ and 6-carboxytetramethylrhodamine, are also sold by the subsidiary »Can be produced, pure particles (referred to as" bead% v 'micro impurities ") can also be used as a probe, this provides samples to directly read the presence of analytes or concentration consumption read without further steps Signal generating agent. In some examples, the particles used for quantity inspection can also provide a signal (such as light absorption) in the area where the signal is generated, in which particles with different signals stay in the film ⑵). Use as a probe (41) The microparticle form is also diversified. For example, naturally occurring microparticles such as nucleus-like plastids, plastids, cytoplasmic bodies, mammalian cells (such as red blood cell hosts), unicellular microorganisms (such as bacteria), and polysaccharides (such as agar Glue) and the like can be used. Further, synthetic particles can also be used. For example, in a specific embodiment: synthetic latex particles that can be dyed by a dye can be used like a probe (41) although any absorbable or Covalent binding to binding The latex microparticles can be used in the present invention. The latex microparticles are generally composed of polystyrene, butadiene styrene, styrene propionic acid-acetamer terpolymer, polymethacrylic acid, and poly (ethylidene methyl methacrylate). Associative acid, E: \ PATENT \ PK-001 08 \ pk-〇〇1 -〇827 \ pk-001 -0827.doc2003 / 6/1 11
苯乙婦-順丁烯二酐共聚物，聚乙烯醋酸，聚乙埽利定，I一个7來J 烯對苯二甲酸，丙烯亞硝酸，氣乙埽-丙烯酸，及相似，或乙醛，羧基，胺， 氫氧基’或連氨衍生物。其他適合的微粒揭示於美國專利編號5670381，Acetophenone-maleic anhydride copolymer, polyethylene acetic acid, polyethylidene, 1 to 7 ene terephthalic acid, propylene nitrite, acetonitrile-acrylic acid, and the like, or acetaldehyde, Carboxyl, amine, hydroxyl 'or hydrazine derivatives. Other suitable microparticles are disclosed in U.S. Patent No. 5708381,
Joe等人及5252459，Tarcha等人，其結合於此。商業可購得的適合的染色， 乳膠微粒包含Bang’s Laboratory，Inc.販售的羧化乳膠珠粒。 當利用時，微粒探子（41 )主要直徑一般多樣化如需要取決因素如選 擇的械粒形式，薄膜孔大小，及薄膜複合物。舉例，在_些具體實施例中， 微粒探子（41 )主要直徑範圍爲〇 〇1微米至1〇〇微米，且在一些具體實施 例中，從0.1械米至75微米。在特殊的具體實施例中，微粒探子（41 )有 0.3微米的直徑。在一些例子中，薄膜（23 )的的孔大小爲〇1至〇 3微米。 當分配於結合塾（22 )上，探子（4丨）,能夠與分析物（奶）直接結合 (共饧或非共彳貝）。然而，其需要以一些方法修飾探子（41 )，因此其更準 確與分析物⑽）結合。在一些例子中，探子（Μ)亦可與某些特殊結合 物件（90)修飾，其爲非共價（如吸收）及/或共價接觸以形成探子结合物 (42)〇 特殊的結合薄膜-般提及爲有特殊結合對的薄M，如兩種不同的分子 ，中-個分子化學及/或物理結合至第二個分子。舉例，免疫活性特殊結合 薄膜包含抗原’半抗原，及複合物，包含由職重組方法或胜肽合成形 成。柷體可爲單株或多株抗體，一種重組蛋白或混合物或碎片，和抗體及 其他特殊結合物質混合-樣。這些抗體詳細的置備及其適合使用如特殊結 合物質一樣爲已知的技術技能。 'α 其他常見的特殊結合對包含但不限制，生物素及㈣素，碳水化合物 =蛋白，核酸片段（包含探子及_職雜交試驗_捉核酸片 又以偵測標的核酸片段），包含由重組方式形成的互補胜肽片段，分子受 器及動器，荷爾蒙及荷爾蒙結合蛋白，酵素輔因子及酵素，酵物制劑及 =，及相似。進—步，特殊結合對包含的分子相似於原始特殊結合物質。 例，分析物碎片衍生物，如分析物類似物可被使用如至少一個常見於八 析物的抗原蛋白。 、刀 41) 這些特殊的結合物件（9G )-般可使祕够樣敝術來與探子（ e 她_0010物01_082物咖^^^ 12 如探子(41)，共卿 化乙酿，，硫醇，環氧化物及其他反應或 鍵、·，α &此基，和殘留的自由基和陽離子 . 、 于自由基一樣，完成穿過蛋白質戀鎖 反【種表面&祕^；可如魏基解 包含-相對高表面濃度的極性基群。另卩^了 口因爲餘姊表面可 3= i 子中’如聚麵盼，微粒能夠直接與半白質共價結合 而不需要進一步的修飾。 可及第―及二圖’ ~種包含分析物MO的測試樣品最初 可利用抽樣塾（21 )。從抽雜，測試樣品可移動至結合签（22)，立中分 析物（則結合至探子結合物（42 )的特殊結合物件⑼）以形成探子結 合物/分析物複合物（49 )。然而，因爲結合塾（22 )與多孔膜⑻流動 =合’探子集合/分析物複合物（49)可從結合塾（22)遷移到存在於多孔 薄膜（23 )上的檢波區（η )。 檢波區（31)亦可包含固定捕捉劑（45)。雖然不需要，其亦可需要 其捕捉劑（45 )可從多樣典型或材料類型(如抗體）如特殊結合物件（9〇 ) -樣被使贿形絲子結合物（42 )。這雜鋪（45 )供應如探子結合 物/分析物複合物（49 )固定的結合位置。在_些例子中，分析物（4〇 )， 如杬體，抗原，等有兩個結合位置。到達檢波區（31 )，這些結合位置之 一由探子結合物/分析物複合物（49)的特殊結合物件（9〇)佔用。然而， 分析物（40)的自由結合位置可結合至固定捕捉劑（45)，一種新形成的 二聚物（50 )探子結合物（42 )顯示分析物（40 )的存在。因此，偵測特 殊分析物（40)是否存在於测試樣品中，一個使用者可簡單分析檢波區 (31)〇 然而，雖然一個檢波區可指出分析物存在，其一般難以僅使用檢波區 來偵測在測試樣品中的分析物相對濃度。因此提及本發明，檢驗亦包含校 正區其可以與檢波區結合來偵測在测試樣品的特殊分析物。舉例，再次提 及第一至三圖，説明一種包含校正區（32 )的溢流道檢驗（2〇 )的具體實 施例。在此具體實施例中，校正區（32)在多孔膜上形成，且位在檢波區 (31 )下行的位置。控制區（32 )提供結合劑（47 )，其能夠結合任何殘 E:\PATENT\PK-001 08\pk-001 -0827\pk-001 -0827.doc20〇3/6/12 13 587166 留的探子（41 )及/或穿過薄膜（23)長度的探子結合物（42)。特别的， 開始接觸測試樣品，任何探子（41 )及/或探子結和（42 )沒有結合分析物 (40 )與複合物（49 )移動穿過檢波區（31 )。在檢波區（31 )，如:述， 複合物（49)結合捕捉劑（45)及固定留下。然而，未結合探子（41^及 /或探子結合物（42 )持續移動穿過檢波區（31 )，且進入多孔膜（23 )的 校正區（32 )。在校正區（32 )，這些未結合探子（41 )及/或探子結合物（幻） 接著與結合劑（47 )結合。當固定於結合區（％ )，探子（Μ )刃或探子 結合物（42 )可看見，由視覺或其他方法，因此使用者可比較在檢波區（ 中的信號強度與在校正區（32)中的信號強度。 提及本發明具體實施例，結合劑（47)可包含聚電解質，其可結合掠 子（41 )及/或探子結合物（42)。聚電解質可包含正或負價網狀物，:一 般自然形成的的電價網狀物—樣。舉例，—些有正電價電㈣合適的例子 包含’但不限制，聚離胺酸（由Sigma_Aldrieh ―丨c。，⑹S山此 聚乙胺；環氧氣丙院官能聚胺及/或聚氨基胺，如聚二乙醇胺: 醜；聚二科二甲基舰㉝；陽離子纖維素衍生物，如纖維 素a物或纖維素與四級水溶性辟騎生物；及相似。在—特殊具 ί- I SC'23〇M ^ H'1〇° ( ^ Nati〇nal StarCh & Ch--1, Inc. 此^’.二中纖維素触物包含啸水溶性銘單體，可被使用。然而，- 二/、毛㈣電解質合適的例子’如聚（乙婦-共-甲基丙歸酸，納鹽）及 目^其亦必須知道兩性聚電解質（如有極性及非極性）。舉例，在—些 電解質包含’但不限制，聚苯基协甲基2·乙婦基利定引朵〕 # 雖二鉍’其由 Ρ〇1_Γ S_e，InC. 〇f D〇rVa1，Canada 購得。 決於天朗，糊嫩雜麵可多樣取 電價允斗m子—物，夕孔膜，及相似。特别的，聚電解質的散机 佳的電價。因此，舉例，也正電價的聚電解質-般有較 此，在-些例子Ϊ 及/或探子結合物（42 )，其—般爲負電荷。因 (32)中柯中，這些分子間的離子相互作用允許需要的結合在校正區 間需要的、纟過/雜，離子交互關餘要利職到在校正區（32) ”喊讀電㈣亦可與有綱電荷的探子（41)及/或探 E:\PATENT\PK-001 〇8'pk-001-〇827\pk-001 -0827.doc2003/6/12 14 子結合物（42)。 因爲聚電解質被設計來結合探子（41)及/或探子結合物（42)乙提件 校正信號，其-般需要躲電解f爲絲布，關定於从膜（23)表面。 相反的’心子（41)及/或探子結合物（42)能由使用者探求至校正檢驗來 立即偵測。因此，聚電解質可以—些方法使用至多孔膜（23)此聚電解質 不能散布於多孔膜（23)基礎。_的，聚電解質—般形成離子及/或與存 在於多孔膜（23)表面的官能基共辑結，因此其保留賴定。雖俠不需 要’在聚電解質與多孔膜間形成共價鍵結被需要永久狀電解質。. 舉例’在—具體實施财，被_來形絲電解質醉财先在溶液 中形成，且接著直接使肚多孔膜⑻。多樣溶液（如有機溶液，水， 寺）可被利用來形成溶液。被利用，單體聚合作用最初使用加孰，電子束 輕射’自由絲合作用，及相似。在—些例子中，如單體聚合作用，其與 某些多孔膜（23)官能基形成共價鍵結，因此固定產生的聚電㈣。舉例， f-具體實施例中’乙埽胺單體可與存在s—些多孔膜（如硝酸纖維 表面的衮甚。 力-個具體貫施例，聚電解質可形成直到使用至多孔膜（23)。若需 t =質首先使时機溶劑，水相_麵軸。之後，聚_質溶液 二夕孔膜（23 )’且接著乾燥。乾燥後，聚電解質可，如上所述， 鳩上的㈣_子結合。舉例，在-具體實施例中，正 醇胺可與存在於—些多孔膜（如硝酸纖維素）表面的負 形成離子鍵結。 另外，聚電解質亦可使用多種已知技術連結至多孔膜（23 )。舉例， =些^實施财，環魏能絲驗/紐驗可被如可連結， 電解f ^樣使用。這些材料關子揭示料國專利編號 Η ’㈣及3772076 ’ Keim，彻⑹，其全結合於此，且由 及 π/’ ^ 〜1 商標設計 KymeneTM 出售。Kymene™450 ，：多======= W孔職合师架。在-麵體魏财，交_溫度細爲抑至 E:\PATENT\PK-001 08\pk-001 -〇82Tvpk-0〇l -〇827.doc2003/6/l 2 15 587166 12〇C，且父聯時間範圍爲1〇至6〇〇秒。 必项Γ:首'1，在多孔膜（23)上之非分散固定聚電解質技術揭示於上，其 上^:何其他非翻賴定聚電解«合物可被使用於本發明。事實 且，賴祕示範'_，其可被制於本發明。糊，在-此 啡rt^32)—般可提供任何數量的不同校正區，因此使用者可在測| ^ 了中更子偵嚼殊分析物的濃度。在更多的具體實施例中，舉例 ，區（32)包含兩個或多個不同校正的校正區（如線，點，等）。人 :=體：：’二f:正區(25 ) ’ ( 26 )，及(27 )形成- «:條線= 位在 方:心一可位在，一條線的位置上，其平行測試樣品穿過檢驗流動的 :Τ個具體貫施例中，如第四Β圖所示，三個校正區(25a }，( 26a )， 或(27a)可位在形成點的位置上，其平行測試樣品穿過檢驗流動的方 二二Γ仔中，使用者可在較少時間中比較校正信號與檢波信號，因 爲母-個校正區大致上產生_個校正信號。 在多ϊΐ ) ’（26)及/或（27)可與不隨量的結合劑（47)先安裝 夕膜（23)上，因此每一個校正區（25)，（26)及（27)在探 =或探子結合物（42)可纽不同的信號強度。在每—個校正區中的結合 =47)全邵數量可使用不同大小的校正區及/或在每-個校正區有多樣濃 =合劑(47)體積而多樣化。一般來説，在校正區中的結合劑 浪度範圍從0.01%至25%的溶液重。 若需要，過多的探子分子可使用於檢驗（2〇)中，因此每一個校正區 125 )’（26 )及（27 )全部達到且先預測潛在的信號強度。因此，在校正 ，（25)’（26)及（27)上的探子（4"數量可先預定，因爲使用於校正 :(25 ) ’（ 26 )及（27 )的結合劑（47 )數量爲先預定且已知的濃度。比 幸又可在校正區（25),(26)及（27)強度及檢量線（Μ)上形成以計算分 E:\PATE^PK.〇0|〇8\pk-〇〇,.〇827\pk-〇01.〇827d〇c2〇〇3/6/12 16 析物（4〇)存在於測試樣品中的量。比較步驟發生，力口上讀取設備幫助或 使用其他技術。 校正及測試樣品可以在同時指出相同的情況，因此提供準確的定量結 杲’與增加的敏感度。檢驗（20)亦可使用於半定量偵測。特别的，當多 重校正區（25)，（26)及（27)提供信號強度範圍，檢波區⑶）的信號 強度可與校正區（25)，（26)及（27)的信號強皮比較。以強度範圍爲基 礎，其中檢波區（31 )下降，分析物（4〇 ) 一般的濃度範圍可被偵測。若 需要，檢波區（31 )及校正區（25)，（26)及（27)間的信號比例可與已. 知分析物濃度範_分析物濃度緣製成平面_產生校正曲線，如第五圖 所π。偵》則未知測試樣品的量，信號比可接著轉換成鎖提及之校正曲線之 为析物歌度。然而，當使用螢光劑偵測在測試樣品中分析物94〇的量，接 收器，接收裝置可使用來測量在檢波 (31)及校正區⑶）中營光劑產 生的量，且因此作恰當的比較以偵測測試樣品中分析物的數量。 除了上述情況的複合物，溢流道檢驗（2〇)亦可包含額外的複合物。 舉例，提及第-至三圖，檢驗（2〇)亦可包含燈芯墊（28)。燈芯藝（28) 接收移動穿過全部多孔膜（23 )的液體。如已知的技術技能，燈芯塾（28 ) 可幫助促進毛細作用及液體流過薄膜（23)。 雖然多種檢驗雜的具體實施例描述於上，其必須知道，本發明的檢 驗-般有任何需要的結構，且不包含所有上述的複合物。進—步，其他檢 L已知的的;^合物不在此制提及，亦可伽於本發明。舉例，多樣檢驗 …構描述於美國專利編號5395754，Lamb〇tte等人；56期81，j〇u等人， 及6194220，Malick等人，其全結合於此。另外，其亦知道，本發明提及 的說爭檢驗亦軸。競爭檢驗的技術及結構脱知的驗技能。 舉例，在-具體實施例中，溢流道檢驗（2〇)揭示於上，説明於第一 至三圖可簡單修飾形成利用探子結合物（42)的競爭檢驗，其包含特殊的 結合物件（9〇)同樣對分析物（4〇)如結果，分析物（々ο)及探子結合物 (、42)將競爭在檢波區（31)中已定量的捕捉劑（45)。一般而言，因爲 刀析物（4〇)未結合’其將快速穿過多孔膜，且佔據在檢波區⑶）内較 大的結合位置。任何未結合的探子結合物（42 )將軸至校正區（32)其 EAPATENT\PK-〇〇| 〇8\pk-〇〇|.〇827\pk-001-0827.doc2003/6/12 γη t其可與結合劑（47)結合。產生於校正區（32)的信號可魅生於檢波 區（3!)的信號比較，其中在測試樣品令分析物的相對含量爲檢波信號強 度相反的比例及校正信號強度直接比例。 相同的’在另-個具體實施例中，一種競爭檢驗可利用捕捉劑（Μ) 形成，其同樣針對分析物（4〇)。因此，因此，在此具體實施例中，探子 結合物（42)最初結合至分析物以形成三複合物Μ)。未結合的探子社 合物（42 )與三複合物（奶）接著移動至檢波區（31 )。其中未結合探子 結合物（42 )與觀足劑（45 )結合。任何殘餘未結合探子結合物⑹與 三複合物（49)將移動至校正區（32)，其中其競爭已定量的結合劑（4外 因此在校正區（U )巾產生·號可與在檢波區（31 )中產生聽號比較，> 其中測試樣品中的分析物相對含量爲檢波信號強度相反的比例及校正信 號強度直接比例。 本發明由下列的例子更加了解。 例子一 在半4紙夾層檢驗内校正區有效利用多樣聚電解質的能力被證明。最 初二由硝酉父纖維素製成的廳p〇re sx多孔膜樣品層壓於3〇公分長度的一 支接板夕樣聚電解質溶液接著溶解於膜樣品上。聚電解質溶使用塑膠 移液滴管頭，或使雜解機如打溶解於膜樣品。在使錄乙醇胺後， 膜在37°C乾燥1小時。 測試的聚電解質溶汸: 聚電解質 淨電荷 供應商 商標 測試濃度範圍 (重％) _ 聚離胺酸 正 Sigma-Aldrich Sigma 2-25 _ 致乙醇胺 正 Sigma-Aldrich 〇 Aldrich 2-25 環氧氣丙烷 正 Sigma-Aldrich Aldrich 2-25 二甲基-氯化鋁 正 Sigma-Aldrich Aldrich 2-25 聚異戊二烯七甲基2乙 基利定引朵 兩性 Polymer Source 無 2-25 E:\PATENT\PK-OOt 08\pk-001 -0827\pk-001 -0827.doc2003/6/12 18 聚乙烯甲基丙烯酸鈉 負 Sigma-Aldrich Aldrich 2-25 層壓膜接著剪成小段半試紙狀。一種纖維素纖維燈芯墊（MilliP〇re Co·)黏附於半試紙的一個末端。膜的其他末端嵌入多個探子及探子結合物 懸浮物。特别的，下列探子被測試： 探子 顏色 微粒大小 (微米） 淨電何 供應商 杂色羧化乳膠 珠粒 藍色 0.3 正 Bang’s Laboratory，Inc. 螢光羧化乳膠 珠粒 紅色 0.2 正 Molecular Probes, Inc. 綠色 0.5 黄色 1.0 酸性紅37 紅色 無 、 正 Sigma-Aldrich 半試紙亦嵌入探子結合物懸浮物中。特别的，上述所提的探子與抗-心 反應蛋白單株抗體（anti-CRP Mab )，抗-黄體素單株抗體（anti-LH Mab ) 及使用已知技術的抗白蛋白原多株抗體（anti_Pab )結合。舉例，1〇〇毫升 〇·5微米勞光羧化微粒（由Molecular pr〇bes，inc·購得）蕞初乙緩衝鹽 溶液（PBS )、凊洗2次，接著二次懸浮於2〇〇亳升pBS中。懸浮物，添加 5毫克碳二醚且混合物混合丨小時。微球體以硼酸緩衝液清洗2次，且再 清洗。微球粒二次懸浮於185毫升的石朋酸緩衝液中。15毫升的a_LH單株 抗體（9·7 *克/¾升）接著添加致懸浮物，且允許混合反應3小時。之後， 200毫升1M的乙醇胺水樣溶液添加至反應混合2〇分鐘。微球體接著此用 PBS清洗2次，且儲存於pbs中。 探子及探子結合物包含水及a%單雜酸氧乙埽三_聚合(一種 由Sigma Aldnch Tween2〇講得的非離子表面活性劑）。產生的探子濃度 範園從麵至5毫克/毫升且探子結合物的濃度範圍微〇 2至ι〇毫克/毫 升。在时鐘之後，每個樣品之分開的校正線接著注意來偵測若探子/探 子結合物爲視覺可偵測。 E:\PATENT\PK-00I 08\pk-00I-0827\pk-00|-0827.doc2003/6/I2 J9 聚離胺酸，聚乙醇胺，聚二甲基胺-環氧氣丙垸及聚_ 丙埽二甲基-氣 化，示幾乎完全捕捉探子，且當捕捉劑量大於探子及探子結“時土，·其 膜上結合。在上述聚電解質之巾，轉胺酸錢乙醇胺在捕捉效^ C»最佳（S探子及探子結合物數量少於驗j量時，少數探子或探子辞人 物蔓延過校正線）；線的品質（線的形狀及清楚的邊緣）；及散撥ϋ 分鐘後殘留的線形狀）。 另外，兩性之聚異戊工烯-b-N-甲基2-乙埽基利定引来由直接捕捉電荷 及探子複合物。同樣的，負電荷之聚乙婦甲基丙_鈉直接捕捉正電荷乳 綠，如Bang，s Labomtory，Inc•的染色的羧化胺基終止乳膠粒，及其結合 ，。其亦發獅成好的控制/校正線。有趣的，一些聚電解質亦捕捉相同電 何的探子雜子結合物。糊，聚6醇麵_化及絲終止乳膠粒且其 ^體結合於膜上以形成控制/校正線。然❼，捕捉微粒與相同電荷如聚電解 質傾向展财過_較她微粒與㈣電荷聚電㈣大的散撥。 例子二 本發明控制聚乙醇胺在内校正區校正一種半試紙夾層檢驗捕捉的能 力證明。最初，由硝酸纖維素製成的讀ip〇re sx多孔膜樣品層壓於3〇 :分長度的一支撐板。聚乙醇胺溶液水樣溶液接著以塑膠移液滴管頭以^ 6 % ’ 2% ’及7.4%溶解於膜上。在塗抹聚乙醇胺後，膜在3r>c乾燥i小時。 層壓膜接著切成小半試紙狀。一種纖維素纖維燈芯墊（Mmip⑽c〇 ) im於半試紙的-個末端。膜的其他末端嵌入多種如例子—描述的探子及 ，子結合鮮物。錢材塗抹娜子及/悲子結合騎物1G分鐘後， 每個樣品的分解校正線接著被觀察制，絲子/探子結合物爲視覺可偵 測。 觀察，其被偵測當過多藍色乳膠粒（03μη1，Bang，SLaboratory，Inc) 時’由7.4%聚乙醇胺溶液形成較由聚乙醇胺形成校正線呈現較大強度 的校正線被使㈣。另外，其亦制當紅色螢綠粒触〇【_LH祕結合時， 由7.4%聚乙醇胺溶液形成較由16%聚乙醇胺形成校正線呈現較大強度的 校正線被使用。 例子三 E:\PATENT\PK-00I 08\pk-001-0827\pk-00I-0827.doc2003/6/12 2〇 本發明聚乙醇胺在内校正區校正一種半試紙夾層檢驗使用的能力被 證明。最初，由硝酸纖維素製成的Milipore SX多孔膜樣品層壓於3〇公分 長度的一支撐板。 一種7.4%聚乙醇胺溶液水樣溶液溶解於Mmp〇re级膜上以形成單一 校正線，且抗-c-反應蛋白（anti-CPR)單株抗體（Mab A58〇4，丨毫克/亳 升，由BiosPaciflc，Inc·購得）溶解於膜上形成檢量線。膜在37亡乾燥丨小 時。層壓膜接著切成小半試紙狀。一種纖維素纖維燈芯墊（Mmip〇rcC〇.) 黏附於膜的一個末端。. ' -個半試紙塗抹至控制井，其包含Tween 2〇,抗-c反應蛋白（anti_CRp ) Mab結合至藍色乳膠粒（anti-CPR Mab粒）及水，同時其他半試紙塗抹至 測試井包含C-反應蛋白（CRP )，Tween 20，anti-CRP Mab結合至藍色乳 膠粒（anti-CPR Mab粒），及水。每個井中的混合物沿半試紙移動至檢量 線，校正線及試紙的燈芯墊。 試紙與混合物塗抹制試井上，CRp分析物硫_CRp Mab粒在檢量 線上捕捉，.同時任域留未結合抗_CRP Mab粒由聚乙雜溶液在校正線 上捕捉。因此，10分鐘後，藍色線在檢量線及校正線上出現。同樣的，試. 紙與混合物塗抹於控制井上，全部抗_CRp Mab粒在校正線上捕捉。如結 果，一條藍色線僅在校正線上出現。 例子四 本發明聚乙醇胺在内校正區校正一種全試紙夾層檢驗使用的能力被 过明。取初’由硝奴纖維素製成的兩個Miiip〇re sx多孔膜樣品層壓於3〇 公分長度的一支撐板。一種纖維素纖維燈芯墊（Millipore C〇·)黏附於膜的 一個末，同時纖維素纖維樣品墊（Miiiip〇re c〇·)黏附於膜的其他端。一 種玻璃纖維結合墊（MilliporeCo.)亦位在緊臨樣品墊的薄膜位置。 一種7.4%聚乙醇胺溶液水樣溶液溶解於MiUp〇re sx膜上以形成單一 才父正線。結合墊装載抗-C-反應蛋白（anti_cPR)單株抗體（Mab A5804，1 耄克/毫升’由BiosPacific，Inc·購得）粒以形成檢量線。膜在37¾乾燥1 小時。 試紙的樣品墊接著塗抹至控制井，其僅包含磷酸緩衝鹽溶液（PBS)， E:\PATENT\PK-001 08\pk-001 -0827\pk-001 -0827.doc2003/6/12 21 587166 同時其他試紙的的樣品錄抹酬試井，其包含c反應蛋白（CRp)，^ 6 %Tween 20，及水。每一個井的混合物沿試紙移動至檢量線，校正線，及 試紙的燈芯墊。 試紙與混合物塗抹於測試井上’ CRP分析物由抗_CRp獅粒在检量 線上捕捉，_任何_未結合抗—CRP Mab粒錄乙_溶液在校必泉 上捕捉。因此，10分鐘後，藍色線在檢量線及校正線上出現。同樣的，試 紙與混合物塗抹於㈣井上，全雜_CRP Mab粒在校錢上捕捉。如結 果，一條藍色線僅在校正線上出現。 ° 例子五 本發明聚乙醇胺在内校正區校正一種半試紙夾層檢驗使用的能力被 證明。最初，由硝酸纖維素製成的Milipore SX多孔膜樣品層壓於3〇公分 長度的一支撐板。 一種7.4%聚乙醇胺溶液水槔溶液溶解於Milipore SX膜上以形成單一 校正線，且抗β-黄體素(anti^-LH )單株抗體(Mab，1毫克/毫升，由Fitzgerald Industrues Int’I，Inc.購得）溶解於膜上以形成檢量線。膜在ye乾燥1小 時。層壓膜接著切成小半試紙狀。一種纖維素纖維燈芯墊（Mmip〇reC〇.) 黏附於半試紙的一個末端。 一個半試紙塗抹至控制井，其包含Tween 20，抗-α黄體素（anti-a-LH ) Mab結合至藍色乳膠粒（anti-a-LH Mab珠粒）及水，同時其他半試紙塗抹 至測試井包含β-黄體素（LH )，Tween 20，anti-α-黄體素（anti-a-LH ) Mab 結合至藍色乳膠珠粒（anti-a-LH Mab粒），及水。每個井中的混合物沿半 試紙移動至檢量線，校正線及試紙的燈芯塾。 試紙與混合物塗抹於測試井上，LH分析物結合抗-α-LH Mab粒在檢 量線上以抗β-LH Mab粒捕捉，同時任何殘留未結合抗_a_LH Mab粒由聚 乙醇胺溶液在校正線上捕捉。因此，10分鐘後，藍色線在檢量線及校正線 上出現。同樣的，試紙與混合物塗抹於控制井上，全部抗_a_LH Mab粒在 校正線上捕捉。如結果，一條藍色線僅在校正線上出現。 例子六 本發明聚乙醇胺在内校正區校正一種全試紙夾層檢驗使用的能力被 E:\PATENT\PK-001 08\pk-001 -0827\pk-001 -0827.doc2003/6/12 22 587166 =:酸纖維素製成的-細一一- -種7·4%聚乙醇胺溶液水樣溶液溶解於膜上以形成單一校Joe et al. And 5252459, Tarcha et al., Incorporated herein. Commercially available suitable dyed, latex microparticles include carboxylated latex beads sold by Bang's Laboratory, Inc .. When used, the main diameter of the particle probe (41) is generally diversified, depending on factors such as the selected mechanical particle form, film pore size, and film composite. For example, in some specific embodiments, the particle probe (41) has a main diameter ranging from 0.001 micrometers to 100 micrometers, and in some specific embodiments, from 0.1 micrometers to 75 micrometers. In a particular embodiment, the particle probe (41) has a diameter of 0.3 microns. In some examples, the pore size of the film (23) is from 0.01 to 0.3 micron. When distributed on the bound osmium (22), the probe (4 丨) can directly bind to the analyte (milk) (co- or non-co-shell). However, it needs to modify the probe (41) in some way, so it binds more precisely to the analyte ⑽). In some examples, the probe (M) can also be modified with some special binding objects (90), which are non-covalent (such as absorption) and / or covalently contact to form the probe binding (42). Special binding film -Generally referred to as thin M with special binding pairs, such as two different molecules, one of which is chemically and / or physically bound to a second molecule. For example, the immunocompatibility-specific binding membrane includes an antigen 'hapten, and a complex including a recombinant method or peptide synthesis. The carcass can be a single or multiple antibodies, a recombinant protein or a mixture or fragment, mixed with antibodies and other special binding substances. The detailed preparation of these antibodies and their suitability for use are as well-known technical skills as special binding substances. 'α Other common special binding pairs include, but are not limited to, biotin and lutein, carbohydrates = proteins, nucleic acid fragments (including probes and _ hybridization tests _ catch nucleic acid fragments and detect target nucleic acid fragments), including by recombinant Complementary peptide fragments, molecular receptors and actuators, hormones and hormone-binding proteins, enzyme cofactors and enzymes, enzyme preparations, and similar. Further, the special binding pair contains molecules similar to the original special binding substance. For example, analyte fragment derivatives, such as analyte analogs, can be used, such as at least one antigen protein commonly found in octaanalytes. (41, knife 41) These special combination objects (9G)-generally can make the secret enough to stabbing with the spies (e she_0010 物 01_082 物 咖啡 ^^^ 12 As the spies (41), Gongqinghua Brewery, Thiols, epoxides, and other reactions or bonds, ·, α & this group, and the remaining free radicals and cations. Like free radicals, completes the protein love through anti-species surface & secret ^; 可For example, the Weiji solution contains-a relatively high surface concentration of polar groups. In addition, because the surface of the Yu sister can be 3 = i in the ions, such as polyamines, particles can directly covalently bind to semi-white matter without the need for further Modifications can be obtained as shown in Figures ― and ② ~ The test samples containing the analyte MO can be initially sampled (21). From miscellaneous, the test sample can be moved to the binding sign (22), and the analyte (the binding To the special binding object 探 of the probe conjugate (42)) to form a probe conjugate / analyte complex (49). However, because the binding 塾 (22) flows with the porous membrane = = the probe set / analyte complex (49) Migration from bonded osmium (22) to the detection region existing on the porous film (23) η). The detection area (31) may also contain a fixed capture agent (45). Although not required, it may also require that its capture agent (45) be selected from a variety of typical or material types (such as antibodies) such as special binding objects (90). ) -Like brittle filament conjugate (42). This hybrid (45) provides a fixed binding site such as a probe conjugate / analyte complex (49). In some examples, the analyte (40) ), Such as corpus callosum, antigen, etc., have two binding sites. Arriving at the detection zone (31), one of these binding sites is occupied by a special binding object (90) of the probe conjugate / analyte complex (49). However, The free binding position of the analyte (40) can be bound to the fixed capture agent (45), a newly formed dimer (50) probe conjugate (42) shows the presence of the analyte (40). Therefore, the detection of special Whether the analyte (40) is present in the test sample, a user can simply analyze the detection area (31). However, although a detection area can indicate the presence of the analyte, it is generally difficult to use only the detection area to detect Relative concentration of analytes in the compounds. The invention is therefore referred to The test also includes a calibration zone that can be combined with the detection zone to detect special analytes in the test sample. For example, the first to third figures are mentioned again to illustrate a spillway test (2) that includes a calibration zone (32) 〇) specific embodiment. In this specific embodiment, the correction zone (32) is formed on the porous membrane and is located at a position downward from the detection zone (31). The control zone (32) provides a binding agent (47), which Capable of combining any residual E: \ PATENT \ PK-001 08 \ pk-001 -0827 \ pk-001 -0827.doc20〇3 / 6/12 13 587166 Remaining probes (41) and / or through the film (23) Length probe combination (42). In particular, starting to contact the test sample, any of the probes (41) and / or probe knots (42) do not bind the analyte (40) and the complex (49) and move through the detection zone (31). In the detection area (31), as described above, the complex (49) is bound to the capture agent (45) and fixed. However, the unbound probes (41 ^ and / or probe conjugates (42)) continue to move through the detection zone (31) and enter the calibration zone (32) of the porous membrane (23). In the calibration zone (32), these unbound probes The binding probe (41) and / or probe combination (magic) are then combined with the binding agent (47). When fixed to the binding area (%), the probe (M) blade or probe combination (42) can be seen by visual or Other methods, so the user can compare the signal strength in the detection area (and the signal strength in the correction area (32). Referring to the specific embodiment of the present invention, the binding agent (47) may include a polyelectrolyte, which can (41) and / or probe combination (42). The polyelectrolyte may include positive or negative valence networks, such as generally formed electricity price networks, such as, for example, those with positive electricity prices. Examples include, but are not limited to, polyamino acids (from Sigma_Aldrieh ― 丨 c., ⑹S mountain this polyethylamine; epoxy polyacrylamide functional polyamines and / or polyaminoamines, such as polydiethanolamine: Ugly; Polydiamine Dimethyl warfare; cationic cellulose derivatives, such as cellulose a or fibers It is similar to the fourth-class water-soluble bizarre creature; and similar. In—Special with ί- I SC'23〇M ^ H'1〇 ° (^ Nati〇nal StarCh & Ch--1, Inc. This ^ '. 二The cellulose substrate contains water-soluble monomers, which can be used. However,-2 /, suitable examples of hairy electrolytes such as poly (ethyl-co-methylpropionate, sodium salt) and mesh ^ It must also be aware of amphoteric polyelectrolytes (if polar and non-polar). For example, some electrolytes include 'but not limited to, polyphenyl co-methyl 2. ethynylidine] [though dibismuth' its It was purchased from Po1_ΓS_e, InC. 〇f D〇rVa1, Canada. Depending on the Tianlang, the mixed surface of the paste can be obtained from a variety of electricity prices, such as pores, pore membranes, and the like. In particular, polyelectrolytes Therefore, for example, polyelectrolytes that also have positive electricity prices are generally better than this, in some examples and / or probe conjugates (42), which are generally negatively charged. Because (32) In Ke, the ionic interactions between these molecules allow the required combination of the transition / heterogeneity required in the calibration interval. Ion interactions should be beneficial to the calibration area (32). ㈣It can also be combined with the charged probe (41) and / or probe E: \ PATENT \ PK-001 〇8'pk-001-〇827 \ pk-001 -0827.doc2003 / 6/12 14 42). Because the polyelectrolyte is designed to combine the probe (41) and / or the probe combination (42) with the B-piece correction signal, it generally needs to avoid electrolysis f as a silk cloth, which is fixed on the surface of the membrane (23). The opposite 'heart (41) and / or probe combination (42) can be detected by the user to a calibration test for immediate detection. Therefore, the polyelectrolyte can be applied to the porous membrane (23) by some methods. This polyelectrolyte cannot be dispersed on the basis of the porous membrane (23). The polyelectrolyte usually forms ions and / or co-interacts with the functional groups existing on the surface of the porous membrane (23), so it retains undine. Although it is not necessary to form a covalent bond between the polyelectrolyte and the porous membrane, a permanent electrolyte is required. For example, in the specific implementation of 财, the 来 -shaped wire electrolyte drunk 醉 is first formed in the solution, and then directly made the porous membrane of the belly ⑻. Various solutions (eg organic solutions, water, temples) can be utilized to form solutions. Being utilized, monomer polymerization was initially performed using tritium, electron beam light shot 'free silk cooperation, and the like. In some examples, such as monomer polymerization, it forms a covalent bond with the functional groups of certain porous membranes (23), and thus fixes the polyelectrofluoride generated. For example, in the specific embodiment, the acetamide monomer may be present in some porous membranes (such as the surface of nitrocellulose fibers). In a specific embodiment, the polyelectrolyte can be formed until the porous membrane is used (23 ). If t = mass, first make the timing solvent, the water phase and the surface axis. After that, the poly-mass solution Eriya Porous Membrane (23) 'and then dry. After drying, the polyelectrolyte can, as described above, dove on For example, in specific embodiments, n-alcoholamines can form ionic bonds with negatives present on the surface of some porous membranes (such as nitrocellulose). In addition, polyelectrolytes can also use a variety of known The technology is connected to the porous membrane (23). For example, some of the implementation methods can be used, such as electrolyzed and electrolyzed samples. These materials are disclosed in the patent No. Η '㈣ and 3772076' Keim It is fully integrated here and sold by π / '^ ~ 1 KymeneTM logo design. Kymene ™ 450: Multi ======== W Kong Vocational and Technical Division. In-hedron Wei Cai The temperature should be reduced to E: \ PATENT \ PK-001 08 \ pk-001 -〇82Tvpk-0〇l -〇827.doc2003 / 6 / l 2 15 587166 12 ° C, and the time of paternal connection is in the range of 10 to 600 seconds. Mandatory Γ: first '1, non-dispersed immobilized polyelectrolyte technology on the porous membrane (23) is disclosed above, above ^: What other non-reversible polyelectrolyte compounds can be used in the present invention. In fact, the Lai secret demonstration '_, which can be made in the present invention. Paste, here-this brown rt ^ 32)-generally can provide any number of Different calibration zones, so users can measure the concentration of analytes in neutrons. In more specific embodiments, for example, the area (32) includes two or more correction areas (such as lines, points, etc.) for different corrections. Person: = body :: 'two f: positive zone (25)' (26), and (27) are formed-«: line = in the square: the heart can be in the position of a line, its parallel test The sample flows through the test: In the specific embodiments, as shown in the fourth B diagram, three correction areas (25a), (26a), or (27a) may be located at the positions of the formation points, which are parallel In the test sample flowing through the test flow, the user can compare the correction signal with the detection signal in less time, because the mother-to-calibration area generates approximately _ correction signals. In many cases) '(26 ) And / or (27) can be installed on the membrane (23) with the binding agent (47) without any amount, so each calibration zone (25), (26) and (27) is in the probe or probe combination. (42) Different signal strengths. Combination in each correction zone = 47) The total number of shao can be used in different size correction zones and / or there are various concentrations in each correction zone = mixture (47) volume and diversity. In general, the binding agent wave in the calibration zone ranges from 0.01% to 25% of the solution weight. If necessary, too many probe molecules can be used in the test (20), so each correction zone 125) '(26) and (27) all reach and predict the potential signal strength first. Therefore, in the correction, (25) '(26) and (27) the number of probes (4 " can be ordered first, because the correction: (25)' (26) and (27) the number of binding agents (47) Predetermined and known concentration. Bichon can be formed on the calibration area (25), (26) and (27) intensity and calibration line (M) to calculate the score E: \ PATE ^ PK.〇0 | 〇8 \ pk-〇〇, .〇827 \ pk-〇01.〇827d〇c2〇3 / 6/12 16 The amount of the precipitate (4〇) in the test sample. The comparison step takes place, and it can be read Take equipment to help or use other technologies. Calibration and test samples can indicate the same situation at the same time, so it provides accurate quantitative results and increased sensitivity. Test (20) can also be used for semi-quantitative detection. In particular, When the multiple correction areas (25), (26), and (27) provide signal strength ranges, the detection area (3) can be compared with the signal strength of the correction areas (25), (26), and (27). Based on the intensity range, the detection area (31) decreases, and the general concentration range of the analyte (40) can be detected. If necessary, the signal ratio between the detection area (31) and the correction area (25), (26), and (27) can be compared with the known analyte concentration range _ analyte concentration edge to make a plane _ to generate a calibration curve, as shown in the section Five Figures π. "Detection" does not know the amount of the test sample, and the signal ratio can then be converted to the calibration curve mentioned by the lock as the analyte song. However, when a fluorescent agent is used to detect the amount of the analyte in the test sample, the receiver, the receiving device can be used to measure the amount of photochemical generated in the detection (31) and the calibration area (3), and therefore Proper comparison to detect the amount of analyte in the test sample. In addition to the composites described above, the overflow channel inspection (20) may also include additional composites. For example, referring to Figures 1-3, the inspection (20) may also include a wick pad (28). The wick art (28) receives the liquid moving through the entire porous membrane (23). As is known in the art, wicks (28) can help promote capillary action and liquid flow through the film (23). Although the specific examples of the various tests are described above, it must be known that the tests of the present invention generally have any desired structure and do not include all the above-mentioned complexes. Further, other known compounds are not mentioned in this system, and can be added to the present invention. By way of example, the various tests ... are described in U.S. Patent No. 5,395,754, Lambotte et al .; 56 Issue 81, Jou et al., And 6194220, Malick et al., All of which are incorporated herein. In addition, it is also known that the argument test mentioned in the present invention is also relevant. Competitive inspection techniques and structural know-how. For example, in the specific embodiment, the overflow channel test (20) is disclosed above, and it is illustrated in the first to third figures that it can be simply modified to form a competitive test using a probe conjugate (42), which includes a special binding object ( 9〇) Similarly for the analyte (40), as a result, the analyte (々ο) and the probe conjugate (, 42) will compete for the quantified capture agent (45) in the detection region (31). In general, because the knife precipitate (40) is not bound, it will quickly pass through the porous membrane and occupy a larger binding position in the detection region (3). Any unbound probe conjugate (42) will axis to the calibration zone (32) its EAPATENT \ PK-〇〇 | 〇8 \ pk-〇〇 | .〇827 \ pk-001-0827.doc2003 / 6/12 γη t It can be combined with a binding agent (47). The signal generated in the calibration area (32) can be compared to the signal generated in the detection area (3!), Where the relative content of the analyte in the test sample is the ratio of the strength of the detection signal to the opposite and the direct ratio of the strength of the correction signal. Identical 'In another embodiment, a competitive assay can be formed using a capture agent (M), which is also directed against the analyte (40). Therefore, therefore, in this specific embodiment, the probe conjugate (42) is initially bound to the analyte to form a triple complex M). The unbound probe community (42) and the triple complex (milk) then move to the detection zone (31). The unbound probe conjugate (42) is combined with the pedicure agent (45). Any residual unbound probe conjugate ⑹ and triple complex (49) will move to the calibration area (32), where it competes for the quantified binding agent (4. Therefore, the number generated in the calibration area (U) can be compared with that in the detection The comparison of the acoustic signal is generated in the area (31), where the relative content of the analyte in the test sample is the ratio of the inverse of the detection signal intensity and the direct ratio of the correction signal intensity. The present invention is better understood by the following examples. Example 1 The ability of the calibration zone to effectively utilize a variety of polyelectrolytes was demonstrated in the sandwich test. Initially, two samples of porous porx membranes made of nitrocellulose were laminated on a 30 cm-long polyelectrolyte. The solution was then dissolved in the membrane sample. Polyelectrolyte was dissolved in the membrane sample using a plastic pipette tip, or a dissolver such as a beaker. After making ethanolamine, the membrane was dried at 37 ° C for 1 hour. Polyelectrolyte tested Solubility: Polyelectrolyte net charge supplier trademark test concentration range (% by weight) _Polyionine Sigma-Aldrich Sigma 2-25 _ Inducing ethanolamine Sigma-Aldrich 〇Aldrich 2-25 Epoxy propane Sigma-Aldrich Aldrich 2-25 Dimethyl-aluminum chloride Sigma-Aldrich Aldrich 2-25 Polyisoprene heptamethyl 2ethylridine Induce Amphoteric Polymer Source No 2-25 E: \ PATENT \ PK -OOt 08 \ pk-001 -0827 \ pk-001 -0827.doc2003 / 6/12 18 Polyethylene sodium methacrylate negative Sigma-Aldrich Aldrich 2-25 Laminated film is then cut into small pieces of semi-test paper. A kind of cellulose A fiber wick pad (MilliPore Co ·) is attached to one end of the semi-test paper. The other end of the membrane is embedded with a plurality of probes and probe combination suspensions. In particular, the following probes are tested: Probe color particle size (microns) Net electricity Who Suppliers Varicolored Carboxylate Latex Beads Blue 0.3 Positive Bang's Laboratory, Inc. Fluorescent Carboxylate Latex Beads Red 0.2 Positive Molecular Probes, Inc. Green 0.5 Yellow 1.0 Acid Red 37 Red None, Positive Sigma-Aldrich Semi-Test Paper Also embedded in the probe conjugate suspension. In particular, the probes mentioned above are anti-CRP Mab, anti-LH Mab and use known techniques Polyalbumin (Anti_Pab) binding. For example, 100 millimeters of 0.5 micron Laoguang carboxylated microparticles (commercially available from Molecular pr.bes, inc.) 蕞 first buffered saline solution (PBS), 凊 washed twice, and then twice Suspended in 2000 liters of pBS. In suspension, 5 mg of carbodiether was added and the mixture was mixed for 1 hour. The microspheres were washed twice with boric acid buffer and washed again. The microspheres were resuspended in 185 milliliters of humic acid buffer. 15 ml of a_LH monoclonal antibody (9.7 * g / ¾L) was then added to the suspension, and the mixture was allowed to react for 3 hours. After that, 200 ml of a 1 M ethanolamine aqueous solution was added to the reaction and mixed for 20 minutes. The microspheres were then washed twice with PBS and stored in pbs. The probe and probe combination contain water and a% monoheterooxetamidine (a non-ionic surfactant described by Sigma Aldnch Tween20). The resulting probe concentration ranged from 5 to 5 mg / mL and the concentration of probe conjugates ranged from 0.2 to 10 mg / mL. After the clock, a separate calibration line for each sample is followed to detect if the probe / probe combination is visually detectable. E: \ PATENT \ PK-00I 08 \ pk-00I-0827 \ pk-00 | -0827.doc2003 / 6 / I2 J9 Polyionine, polyethanolamine, polydimethylamine-epoxypropane, and poly_ Propylene dimethyl-gasification shows that the probe is almost completely captured, and when the capture dose is larger than the probe and the probe knot, the film is combined. In the above polyelectrolyte towel, transethanolamine is effective in capturing C »Best (when the number of S probes and probe combinations is less than the number of tests, a small number of spies or probe figures spread over the correction line); the quality of the line (the shape of the line and the clear edges); and after ϋ minutes Residual line shape). In addition, the amphoteric polyisoprene-bN-methyl 2-acetamidine is induced by direct capture of charge and probe complexes. Similarly, negatively charged polyethylenimine _Sodium directly captures positively-charged opalescent green, such as the dyed carboxylated amine-terminated latex particles of Bang, s Labomtory, Inc, and their binding. It also makes a good control / correction line. Interesting, some poly The electrolyte also captures the probe and heterozygous conjugates of the same electrode. Paste, poly6 alcohols, and silk-terminated latex particles are bound to the membrane. To form a control / correction line. However, trapped particles with the same charge, such as polyelectrolyte, tend to spread their wealth _ larger than her particles and charged electric charges. Example 2 The present invention controls the correction of polyethanolamine in the calibration area. A proof of the ability to capture by a semi-test paper sandwich test. Initially, a sample of porous ipore sx membrane made of nitrocellulose was laminated on a support plate with a length of 3:30. The aqueous solution of the polyethanolamine solution was then transferred by plastic. The dropper tip was dissolved in the film at 6%, 2%, and 7.4%. After the application of polyethanolamine, the film was dried at 3r > c for 1 hour. The laminated film was then cut into small test paper shapes. A cellulose fiber wick The pad (Mmip⑽c〇) im at the one end of the half-test paper. The other end of the membrane is embedded with a variety of probes and seeds as described in the example, combined with fresh objects. After the money material is coated with the nazi and / sad child combined with the ride 1G minutes, each The decomposition correction line of each sample was then observed, and the silk / probe combination was visually detectable. Observed, it was detected that when there were too many blue latex particles (03μη1, Bang, Laboratory, Inc), it was collected by 7.4%. Ethanolamine solution formation Polyethanolamine formed the correction line showing a stronger intensity correction line. In addition, it also produced when the red fluorescent green particles touched the combination of the [7.4% polyethanolamine solution and the correction line formed from the 16% polyethanolamine. A calibration line showing greater intensity is used. Example 3 E: \ PATENT \ PK-00I 08 \ pk-001-0827 \ pk-00I-0827.doc2003 / 6/12 2〇 The polyethanolamine of the present invention is calibrated in the calibration area The ability to use a semi-test strip sandwich test was proven. Initially, samples of Milipore SX porous membrane made of nitrocellulose were laminated on a support plate 30 cm in length. A 7.4% aqueous solution of polyethanolamine solution was dissolved in a Mmpore grade membrane to form a single calibration line, and an anti-c-reactive protein (anti-CPR) monoclonal antibody (Mab A5804, mg / 毫克 L, (Available from BiosPaciflc, Inc.) was dissolved on the membrane to form a calibration curve. The membrane was dried at 37 ° C for one hour. The laminated film was then cut into small test strips. A cellulose fiber wick pad (MmipOrcC〇.) Is adhered to one end of the film. '-A half test paper was applied to the control well, which contained Tween 20, anti-c-reactive protein (anti_CRp) Mab bound to blue latex particles (anti-CPR Mab particles) and water, while the other half test paper was applied to the test well. Contains C-reactive protein (CRP), Tween 20, anti-CRP Mab bound to blue latex particles (anti-CPR Mab particles), and water. The mixture in each well moves along the test strip to the calibration line, calibration line, and wick pad of the test strip. On the test wells coated with the test paper and the mixture, the CRp analyte sulfur_CRp Mab particles were captured on the calibration line. At the same time, Ren Yuliu's unbound anti-CRP Mab particles were captured by the polyethylene solution on the calibration line. Therefore, after 10 minutes, the blue line appears on the calibration line and the calibration line. Similarly, the test paper and the mixture were smeared on the control well, and all anti-CRp Mab particles were captured on the calibration line. As a result, a blue line appears only on the calibration line. Example 4 The ability of the polyethanolamine of the present invention to correct the use of a full test strip sandwich inspection in the inner calibration zone is overexposed. Two samples of Miiipore sx porous membrane made of nitrocellulose were taken and laminated on a support plate with a length of 30 cm. A cellulose fiber wick pad (Millipore Co. ·) was adhered to one end of the film, while a cellulose fiber sample pad (Miiiiporec ..) was adhered to the other end of the film. A glass fiber bonded pad (MilliporeCo.) Is also located immediately adjacent the film pad of the sample pad. A 7.4% aqueous solution of polyethanolamine was dissolved in MiUporesx membrane to form a single positive line. The binding pad was loaded with anti-C-reactive protein (anti_cPR) monoclonal antibody (Mab A5804, 1 g / ml 'available from BiosPacific, Inc.) to form a calibration line. The film was dried at 37¾ for 1 hour. The sample pad of the test paper is then applied to the control well, which contains only phosphate buffered saline (PBS), E: \ PATENT \ PK-001 08 \ pk-001 -0827 \ pk-001 -0827.doc2003 / 6/12 21 587166 At the same time, samples of other test strips were recorded in test wells, which contained c-reactive protein (CRp), 6% 6% Tween 20, and water. The mixture from each well moves along the test strip to the calibration line, calibration line, and wick pad of the test strip. The test paper and the mixture were smeared on the test well. The CRP analyte was captured on the calibration line by the anti-CRp lion particles, and the _any_unbound anti-CRP Mab particle recorded B_ solution was captured on the school spring. Therefore, after 10 minutes, the blue line appears on the calibration line and the calibration line. In the same way, the test paper and the mixture were smeared on the manhole, and the whole CRP Mab grains were captured on the school money. As a result, a blue line appears only on the calibration line. ° Example 5 The ability of the polyethanolamine of the present invention to calibrate a semi-test strip sandwich test in the internal calibration zone was demonstrated. Initially, samples of Milipore SX porous membranes made of nitrocellulose were laminated on a support plate 30 cm in length. A 7.4% polyethanolamine solution, a leech solution, was dissolved on the Milipore SX membrane to form a single calibration line, and an anti-β-lutein (anti ^ -LH) monoclonal antibody (Mab, 1 mg / ml, from Fitzgerald Industrues Int'I, (Available from Inc.) dissolved in the membrane to form a calibration curve. The film was dried for 1 hour at ye. The laminated film was then cut into small test strips. A cellulose fiber wick pad (MmiporeC.) Is adhered to one end of a half test paper. One half of the test strip was applied to the control well, which contained Tween 20, anti-a-LH Mab bound to blue latex particles (anti-a-LH Mab beads) and water, while the other half of the test strip was applied to The test well contained β-lutein (LH), Tween 20, anti-α-lutein (anti-a-LH) Mab bound to blue latex beads (anti-a-LH Mab particles), and water. The mixture in each well moves along the test strip to the calibration line, calibration line, and wick 塾 of the test strip. The test paper and the mixture were smeared on the test well. The LH analyte bound anti-α-LH Mab particles were captured on the calibration line with anti β-LH Mab particles, while any remaining unbound anti_a_LH Mab particles were captured by the polyethanolamine solution on the calibration line. . Therefore, after 10 minutes, the blue line appears on the calibration line and the calibration line. Similarly, the test paper and the mixture were applied to the control well, and all anti-a_LH Mab particles were captured on the calibration line. As a result, a blue line appears only on the calibration line. Example 6 The ability of the polyethanolamine of the present invention to correct a full test paper sandwich inspection in the inner calibration area is E: \ PATENT \ PK-001 08 \ pk-001 -0827 \ pk-001 -0827.doc2003 / 6/12 22 587166 = : A kind of 7.4% polyethanolamine solution made from acid cellulose is dissolved in the membrane to form a single calibration solution.
Ttl^cXTi Biogenesis, inc-m#> ..泉。膜在37C餘1小時。層壓膜接著切成小半試紙狀 燈芯墊（臟㈣Co.)黏附於半試紙的一個末端。 戚陳減、准 -個半試驗抹缝制井，其包含_紅色魏齡結合抗白蛋白原 多株抗體’同時其他半執塗抹·試井包含2()微升白蛋白原（在碎酸 缓衝鹽溶液G.2毫克/毫升），㈣壯色螢級粒與抗自蛋自賴朱抗體 及4〇毫升2%Τ_η 20水溶液。每個井中的混合物沿半試紙移動至檢量 線，校正線及試紙的燈芯墊。 試紙與混合物塗抹綱試井上，自蛋自原分析物錄i線上以抗白蛋 白原多株抗體粒捕捉。白蛋白原結合粒穿過檢量線且在校正線上捕捉。因 此，10分鐘後，紅色線僅在校正線上出現。同樣的，試紙與混合物塗抹於 L制井上，抗白蛋白原多株抗體粒首先在檢量線上捕捉，且接著一些殘餘 株粒在校正線上捕捉。如結果，紅色線在檢量線及校正線上出現。 例子七 本發明内校正區校正一種夾層檢驗的能力被證明。最初，由硝酸纖維 素製成的Milipore SX多孔膜樣品層壓於30公分長度的一支撐板。聚乙醇 胺水樣溶液接著溶解於膜上（1X，10x，及1〇〇χ稀釋的7·4%聚乙醇胺溶 液）形成不同濃度的三條分别校正線。在使用聚乙醇胺後，膜在37艺乾燥 1小時。 一種纖維素纖維燈芯塾（Millipore Co·)黏附於膜的一個末端。膜的 其他末端嵌入多個探子及探子結合物懸浮物。特别的，下列探子被測試： 探子 顏色 微粒大小 (微米） 淨電荷 供應商 杂色羧化乳膠 珠粒 藍色 0.3 正 Bang’s Laboratory，Inc· E:\PATENT\PK-001 08\pk-001 -0827\pk-001 -0827.doc2003/6/12 23 螢光羧化乳膠 _ 珠粒 紅色 0.5 正 Molecular Probes, Inc. 檢驗亦嵌入探子結合物懸浮物。特别的，上述所提的探子與抗反應 蛋白單株抗體（anti-CRP Mab )，抗-黄體素單株抗體（anti_LH Mab )及使 用已知技術的抗白蛋白原多株抗體（anti-Pab)結合。舉例，1〇〇毫升〇5 微米螢光銳微粒（❾Mdeoilai: Prcbesjne.麟）最初6雜麟鹽溶液 (PBS )清洗2次，接著二次懸浮於毫升pBS中。懸浮物，添加5毫 克碳二_且混合物混合丨小時。微球體以織触清洗2次，且再清洗。 微球粒二次懸浮於185毫升的硼酸緩衝液中。15毫升的a_LH單株抗體(9·7 耄克/耄升）接著添加致懸浮物，且允許混合反應3小時。之後，2〇〇毫升 1Μ的乙醇胺水樣溶液添加至反應混合2〇分鐘。微球體接著此用pBs清洗, 2次，且儲存於PBS中。 探子及探子結合物包含水及丨.6%單服酸氧乙烯三梨醇聚合（一種 由Sigma-Aldnch “Tween2〇 “購得的非離子表面活性劑）。產生的探子濃度 範圍從0.001至5毫克/毫升且探子結合物的濃度範圍微Μ至1〇毫克鴒 升。 在5分鐘之後，分開的校正線接著注意來偵測若探子/探子結合物爲視 覺可偵測。包含1X稀釋液的線展現高信號強度，同時包含ΙΟΟχ稀釋液的 線展現較低的信號強度。 例子八 …本發明内校正區校正一種半試紙夾層檢驗的能力被證明。最初，由硝 維素製成的Mil_eSX纽膜樣品層壓於％公分長度的—支撐板。 7·4%聚乙醇胺溶液水樣溶液（1χ，1〇χ，及1〇〇χ稀釋）接著溶解於膜上形 成不同濃度的三條分别校正線。 抗-c-反應蛋白（anti-CPR)單株抗體（MabA58〇4，丨毫克/毫升，由 biosPaafic，lnc·購得）溶解於膜上形成檢量線。膜在於乾燥^小時。一 種纖維素纖維燈芯塾（Millipore Co·)黏附於膜的一個末端。層壓膜接著切 成小半試紙狀。燈純崎馳末端塗抹包含c·反應蛋白（CRp)， 2〇 ’ antl-CRP Mab結合至藍色乳膠珠粒（a‘CpR廳珠粒），及水。物 E:\PATENT\PK-001 08\pk-001 -0827\pk-001 -0827.doc2003/6/12 24 587166 質混合沿半試紙移動至檢量線，校正線，及試紙的燈芯墊。 CRP分析物由抗·CRp Mab珠粒在檢量線上捕捉，同時任何殘留未結 合抗-CRP Mab珠粒由校正線捕捉。因此，5分鐘後，一個藍色線在檢量線 上出現，同時三條藍線在校正區出現。包含lx稀釋液的線展現高信號強 度’同時包含l〇〇x稀釋液的線展現較低的信號強度。 例子九 本發明内校正區校正一種半試紙夾層檢驗的能力被證明。最初，由硝 酸纖維素製成的HF09002多孔膜樣品層壓於30公分長度的一支撐板。0J4 %(校正#1 )，0.64%(校正#2 )，及1.4%(校正#3 )聚乙醇胺水樣溶液（lx， 10x，及ΙΟΟχ稀釋樣品）接著溶解於膜上形成不同濃度的三條分别校正線。 抗心反應蛋白Unti-CPR)單株抗體（Mab A5804，1毫克/毫升，由 BiosPacific，Inc·購得）溶解於膜上形成檢量線。膜在37。〇乾燥1小時。一 種纖維素纖維燈芯墊（Millipore Co·)黏附於膜的一個末端。層壓膜接著切 成小半試紙狀。 义豆心塾相對薄膜末端塗抹包含C-反應蛋白(CRP )，Tween 20，anti-CRP Mab結合至藍色乳膠珠粒（anti_CPR Mab珠粒），及水。此測試物亦包含 不同的C-反應蛋白濃度。特别的’溶液包含〇ng，，5.4ng，及54ng 的 CRP。 物質混合沿半試紙移動至檢量線，校正線，及試紙的燈芯墊。CRp分 析物由抗-CRP Mab珠粒在檢量線上捕捉，同時任何殘留未結合抗_CRp Mab珠粒由校正線捕捉。因此，每一個樣品，一個藍色線在檢量線上出現， 同時三條藍線在校正區出現。包含1.4%聚乙醇胺溶液展現高信號強度， 同時包含0.14%聚乙醇胺溶液展現較低的信號強度。以分析爲基礎，其被 偵測校正線#1包含〇.54ng的CRP，校正線#2包含5.4ng的CRP，且校正 線#3包含54ng的CRP 〇 因此’當測試未知的測試樣品，CRP濃度可由比較檢量線及三條校正 線偵測。特别的，當檢量線強度爲可偵測有校正線#2及#3強度問的強度， CRP濃度爲5.4至54ng間。同樣的，當檢量線強度爲可偵測有校正線#1 及#2強度間的強度，CRP濃度爲0.54至5.4ng間。進一步，檢量線強度爲 ΕΛΡΑΤΕΝΤλΡΚ-ΟΟ 1 08\pk-001 -0827\pk-001 -0827.doc2003/6/12 25 可偵測有小於校正線#1強度的強度，CRP濃度爲小於054ng，同雜 強度爲輸w大於校正_強度_度，CRp濃度敲於5岭 校正線強度亦可由儀器測量，如檢驗讀取機。舉例 細所示）使職正_細的賴，及其⑽濃度^曲^ 的鱗方程式可輸M義財，其可_设度來_在測試樣品 例子十 一本發明内校正區校正—種半試紙央層檢驗的能力被翻。最初，由確 酸纖維素製成的SHF G75多孔膜樣品層壓於3〇公分長度的_支撐板。多 種濃度的CelQu_ Η·接著溶解於膜上形成不同濃度的三條分别校正 線。特别的，使用的濃度爲μ CelQuat⑧關〇/百萬溶液（鹏）（校正線 #1 ) ’ 5ppm (校正線#2 )，及 2〇ppm (校正線#3 )。 抗-β利用荷爾蒙(anti+LH )單株抗體(Mab，i毫克/毫升，由FitzgeraW hdiistnes IntUnc.購得）溶解於膜上形成檢量線。膜在37。〇乾燥丨小時。 一種纖維素纖維燈芯墊（Minip〇re c〇.)黏附於膜的一個末端。層壓膜接著 切成小半試紙狀。 、 k心墊相對薄膜末端塗抹包含Tween 20，anti-a-黄體素（⑽) Mab結合至藍色乳膠珠粒（ αηΗ_α4ΗΜΑ珠粒），及水。混合物亦包含多 樣Ρ-黄體素（LH )濃度。特别的，濃度測試爲〇ppm，2〇ppm，1〇〇ppm， 其符合包含Ong，2〇ng，及i〇〇ngLH的測試濃度。 物貝混合沿半試紙移動圭檢量線，校正線，及試紙的燈芯塾。分 析物由抗-α-LH Mab珠粒在檢量線上捕捉，同時任何殘留未結合抗-a_LH Mab珠粒由校正線捕捉。因此，每一個樣品，一個藍色線在檢量線上出現， 同時二條監線在校正區出現。包含2〇 ppm CelQuat® H-100溶液展現高信 號強度，同時包含2.5??111€啦_(1)仏100溶液展現較低的信號強度。以 为初爲基礎’其被偵利校正線#1包含2Ung的LH且校正線#3包含l〇〇ng 的LH。然而，使用儀器可讀取線強度，其偵測校正線#卜椏，及把有1， 2，4的線性強度。 校正線強度亦可由儀器測量，如檢驗讀取機。舉例，一個校正曲線（如 E:\PATENT\PK-001 08\pk-001 -0827\pk-001 -0827.doc2003/6/12 2β 587166 第七圖所示）使用校正線#1至#3的線強度，及其LH濃度形成。i校正曲 線產生的數學方程式可輸入儀器中。包含未知濃度的測試樣品接著塗抹形 成上述之薄膜。使用儀器，其可以偵測1·5的檢波信號強度。如結果，其 偵測在未知測試樣品中的LH濃度爲36ng。 例子十一 本發明内校正區校正一種半試紙夾層檢驗的能力被證明。最初，由硝 酸纖維素製成的HF 12〇多孔膜樣品層壓於30公分長度的一支撐板。多種 很度的 CelQuat® H-100 (從 National Starch & Chemical，Inc.購得的纖維素 衍生物）接著溶解於膜上形成不同濃度的三條分别校正線。特别的，使用 的濃度爲2·5 CelQuat® H-100/百萬溶液（ppm)(校正線#i )，5ppm (校正 線#2 )，及2〇ppm (校正線#3 )。 白蛋白原,（1毫克/毫升，購自Biogenesis，Inc·)溶解於膜上形成檢量 線。膜在37¾乾燥1小時。一種纖維素纖維燈芯墊（Mimp〇re c〇.)黏附 於膜的一個末端。層壓膜接著切成小半試紙狀。 燈芯墊相對薄膜末端塗抹包含30毫升2%Tween 20，10毫升與抗白 蛋白原多株抗體結合的紅色螢光微粒，及水。混合物在嶙酸缓衝鹽溶液中 亦包含多種白蛋白原濃度。特别的，測試濃度爲〇毫克，75毫克，及125 毫克。 其可察覺三條校正線轉換不同的紅色強度，其中強度校正線#3最高且 線#1最低。在競爭檢驗中的檢量線強度與測試白蛋白原濃度有相反的比 例。當其不是白蛋白原，由檢量線及三條校正線捕捉結合物。與增加的白 蛋白原抗原數量，檢量線變成較不強烈。 線強度接著由螢光讀取機讀取，且產生校正曲線。結果如表一所示。 奉一：白蛋白檢量及校正線強度 信號強度 _校正#1 1 1 1 —_校正#2 10 10 10 ΕΛΡΑΤΕΝΊΛΡΚ-001 〇8\pk-〇〇|.〇827\pk-001-0827.doc2003/6/12 27 587166Ttl ^ cXTi Biogenesis, inc-m # > .. The membrane was left at 37C for 1 hour. The laminated film was then cut into small test strips, and a wick pad (dirty co.) Was adhered to one end of the half test strip. Qi Chenjian, quasi-half test wiped wells, which contain _ red Weiling combined anti-albumin polyclonal antibody 'while other semi-permanent smear · test wells contain 2 () microliters of albuminogen (in broken acid Buffered saline solution G. 2 mg / ml), strong color fluorescein grade granules, anti-self egg self-reliance antibody and 40 ml 2% T_η 20 aqueous solution. The mixture in each well moves along the test strip to the calibration line, calibration line, and wick pad of the test strip. The test paper and the mixture were smeared on the outline test wells, and captured from the egg and the original analyte record i-line with anti-albumin multiple strain antibody particles. The albuminogen binding particles cross the calibration line and are captured on the calibration line. Therefore, after 10 minutes, the red line appears only on the calibration line. Similarly, the test paper and the mixture were smeared on the L-made wells, and the anti-albumin prokaryotic antibody particles were first captured on the calibration line, and then some residual strains were captured on the calibration line. As a result, a red line appears on the calibration and calibration lines. Example 7 The ability of the correction zone in the present invention to correct a sandwich test has been demonstrated. Initially, samples of Milipore SX porous membranes made of nitrocellulose were laminated on a support plate 30 cm in length. The aqueous solution of polyethanolamine was then dissolved in the membrane (1X, 10x, and 100x diluted 7.4% polyethanolamine solution) to form three calibration lines with different concentrations. After using polyethanolamine, the film was dried at 37 ° C for 1 hour. A cellulose fiber wick (Millipore Co ·) is attached to one end of the film. Multiple probes and probe conjugate suspensions are embedded at the other end of the membrane. In particular, the following probes were tested: Probe color particle size (micron) Net charge supplier Varicolored carboxylated latex beads blue 0.3 positive Bang's Laboratory, Inc · E: \ PATENT \ PK-001 08 \ pk-001 -0827 \ pk-001 -0827.doc2003 / 6/12 23 Fluorescent Carboxylated Latex_ Bead Red 0.5 Positive Molecular Probes, Inc. test also embeds probe conjugate suspensions. In particular, the above-mentioned probes and anti-reactive protein monoclonal antibodies (anti-CRP Mab), anti-progestin monoclonal antibodies (anti_LH Mab), and anti-albumin polyclonal antibodies (anti-Pab) using known techniques Combined. For example, 100 ml of 05 micron fluorescent sharp particles (❾Mdeoilai: Prcbesjne. Lin) was first washed twice with 6 lin salt solution (PBS), and then suspended twice in ml of pBS. In suspension, 5 mg of carbon dicarbonate was added and the mixture was mixed for 1 hour. The microspheres were washed twice with weaving touch, and then washed again. The microspheres were resuspended in 185 ml of boric acid buffer. 15 ml of a_LH monoclonal antibody (9.7 g / g) was added to the suspension and allowed to react for 3 hours. After that, 200 ml of a 1 M ethanolamine aqueous solution was added to the reaction and mixed for 20 minutes. The microspheres were then washed with pBs twice, and stored in PBS. Probes and probe conjugates contain water and a .6% monooxyethylene sorbitan polymer (a nonionic surfactant commercially available from Sigma-Aldnch "Tween20"). The concentration of probes produced ranges from 0.001 to 5 mg / ml and the concentration of probe conjugates ranges from microM to 10 mg 鸰 L. After 5 minutes, separate calibration lines are followed to detect if the probe / probe combination is visually detectable. The line containing the 1X dilution showed high signal intensity, while the line containing the 100 × dilution showed low signal intensity. Example 8… The ability of the correction zone to correct a half-strip sandwich inspection in the present invention was demonstrated. Initially, a sample of Mil_eSX button film made of nitrate was laminated on a support plate of a cm length. A water sample solution of 7.4% polyethanolamine solution (1x, 10x, and 100x dilutions) was then dissolved in the membrane to form three calibration lines with different concentrations. An anti-c-reactive protein (anti-CPR) monoclonal antibody (MabA5804, mg / ml, commercially available from biosPaafic, lnc ·) was dissolved on the membrane to form a calibration curve. The film was left to dry for ^ hours. A cellulose fiber wick (Millipore Co ·) was attached to one end of the membrane. The laminated film was then cut into small test strips. The light pure Sakichi end smear contains c · reactive protein (CRp), 20 'antl-CRP Mab bound to blue latex beads (a'CpR hall beads), and water. Object E: \ PATENT \ PK-001 08 \ pk-001 -0827 \ pk-001 -0827.doc2003 / 6/12 24 587166 The mass mixture moves along the half test strip to the calibration line, calibration line, and wick pad of the test strip. CRP analytes are captured on the calibration line by anti-CRp Mab beads, while any remaining unbound anti-CRP Mab beads are captured by the calibration line. Therefore, after 5 minutes, a blue line appears on the calibration line and three blue lines appear on the calibration area. The line containing the lx dilution showed a high signal intensity 'while the line containing the 100x dilution showed a lower signal intensity. Example 9 The ability of the correction zone in the present invention to correct a half-test strip sandwich test was demonstrated. Initially, a sample of HF09002 porous membrane made of cellulose nitrate was laminated on a support plate 30 cm in length. 0J4% (Calibration # 1), 0.64% (Calibration # 2), and 1.4% (Calibration # 3) polyethanolamine aqueous solution (1x, 10x, and 100x diluted samples) are then dissolved in the membrane to form three strips of different concentrations, respectively. Calibration line. Anti-cardiac response protein (Unti-CPR) monoclonal antibody (Mab A5804, 1 mg / ml, commercially available from BiosPacific, Inc.) was dissolved on the membrane to form a calibration curve. The membrane is at 37. 〇Dry for 1 hour. A cellulose fiber wick pad (Millipore Co ·) is adhered to one end of the film. The laminated film was then cut into small test strips. The coating of the end of the thin film of the bean sprouts contains C-reactive protein (CRP), Tween 20, anti-CRP Mab bound to blue latex beads (anti_CPR Mab beads), and water. This test substance also contains different C-reactive protein concentrations. A particular 'solution contains 0ng, 5.4ng, and 54ng of CRP. Substance mixing moves along the test strip to the calibration line, calibration line, and wick pad of the test strip. CRp analytes are captured on the calibration line by anti-CRP Mab beads, while any remaining unbound anti-CRp Mab beads are captured by the calibration line. Therefore, for each sample, one blue line appears on the calibration line and three blue lines appear on the calibration area. A solution containing 1.4% polyethanolamine exhibited high signal intensity, while a solution containing 0.14% polyethanolamine exhibited lower signal intensity. Based on the analysis, its detected calibration line # 1 contains 0.54ng CRP, calibration line # 2 contains 5.4ng CRP, and calibration line # 3 contains 54ng CRP. Therefore 'When testing an unknown test sample, CRP Concentration can be detected by comparative calibration line and three calibration lines. In particular, when the intensity of the calibration line is an intensity that can detect the intensity of the calibration lines # 2 and # 3, the CRP concentration is between 5.4 and 54 ng. Similarly, when the intensity of the calibration line is the intensity that can detect the intensity between the calibration lines # 1 and # 2, the CRP concentration is between 0.54 and 5.4 ng. Further, the intensity of the calibration line is ΕΛΡΑΤΕΝΤλΡΚ-ΟΟ 1 08 \ pk-001 -0827 \ pk-001 -0827.doc2003 / 6/12 25 It can detect that the intensity is less than the intensity of the calibration line # 1, and the CRP concentration is less than 054ng. The intensity of the same impurity is greater than the correction w_intensity_degree, and the intensity of the CRp concentration knocking on the 5 ridge correction line can also be measured by an instrument, such as checking a reader. The example is shown in detail.) The professional formula _ fine Lai and its scale equation of concentration ^ Qu ^ can input M Yicai, which can _ set the degree _ in the test sample example 11 correction zone correction in the invention- The ability of semi-test paper central layer inspection was turned over. Initially, a sample of SHF G75 porous membrane made of cellulose acetate was laminated on a 30 cm support plate. CelQu_ Η at various concentrations is then dissolved in the film to form three calibration lines with different concentrations. In particular, the concentrations used are μCelQuat⑧guan / Million solution (Peng) (correction line # 1) '5 ppm (correction line # 2), and 20 ppm (correction line # 3). Anti-beta was dissolved in a membrane using a hormonal (anti + LH) monoclonal antibody (Mab, i mg / ml, commercially available from FitzgeraW hdiistnes IntUnc.) To form a calibration curve. The membrane is at 37. 〇 Drying 丨 hours. A cellulose fiber wick pad (Minipore Co.) is adhered to one end of the film. The laminated film was then cut into small test strips. The end of the k film relative to the end of the film is coated with Tween 20, anti-a-lutein (⑽) Mab bound to blue latex beads (αηΗ_α4ΗΜΑ beads), and water. The mixture also contained various P-lutein (LH) concentrations. In particular, the concentration tests were 0 ppm, 20 ppm, and 100 ppm, which met the test concentrations including Ong, 20 ng, and 100 ngLH. The shell mix moves the calibration line, the calibration line, and the wick of the test strip along the half test strip. The analytes were captured on the calibration line by anti-α-LH Mab beads, while any remaining unbound anti-a-LH Mab beads were captured by the calibration line. Therefore, for each sample, a blue line appears on the calibration line, and two monitoring lines appear in the calibration area. A solution containing 20 ppm CelQuat® H-100 exhibits high signal strength, while a solution containing 2.5 ?? 111 € _ (1) 仏 100 exhibits lower signal strength. On the basis of this, its detection correction line # 1 contains 2Ung of LH and correction line # 3 contains 100ng of LH. However, the intensity of the line can be read using the instrument, which detects the correction line #Bu 桠, and has a linear intensity of 1, 2, and 4. The strength of the calibration line can also be measured by instruments, such as inspection readers. For example, a calibration curve (such as E: \ PATENT \ PK-001 08 \ pk-001 -0827 \ pk-001 -0827.doc2003 / 6/12 2β 587166 as shown in the seventh figure) uses calibration lines # 1 to # 3 The line strength, and its LH concentration are formed. The mathematical equations generated by the i calibration curve can be entered into the instrument. A test sample containing an unknown concentration is then applied to form the film described above. Using the instrument, it can detect the detection signal strength of 1.5. As a result, the LH concentration detected in the unknown test sample was 36 ng. Example XI The ability of the correction zone in the present invention to correct a half-test strip sandwich test was demonstrated. Initially, a sample of HF 120 porous membrane made of cellulose nitrate was laminated on a support plate of 30 cm in length. Various grades of CelQuat® H-100 (cellulose derivatives available from National Starch & Chemical, Inc.) were then dissolved in the film to form three separate calibration lines at different concentrations. In particular, concentrations of 2.5 CelQuat® H-100 / million solution (ppm) (calibration line #i), 5 ppm (calibration line # 2), and 20 ppm (calibration line # 3) were used. Albuminogen, (1 mg / ml, purchased from Biogenesis, Inc.) was dissolved on the membrane to form a calibration curve. The film was dried at 37¾ for 1 hour. A cellulose fiber wick pad (Mimpore c.) Is adhered to one end of the film. The laminated film was then cut into small test strips. The opposite end of the wick pad is coated with 30 ml of 2% Tween 20, 10 ml of red fluorescent particles bound to anti-albumin polyclonal antibodies, and water. The mixture also contained various albuminogen concentrations in a gallate buffer solution. Specifically, the test concentrations were 0 mg, 75 mg, and 125 mg. It can detect that the three correction lines convert different red intensities, among which the intensity correction line # 3 is the highest and the line # 1 is the lowest. The intensity of the calibration curve in the competition test is inversely proportional to the concentration of albumin tested. When it is not proalbumin, the conjugate is captured by a calibration line and three calibration lines. With an increase in the amount of proalbumin antigen, the calibration line becomes less intense. The line intensity is then read by a fluorescent reader and a calibration curve is generated. The results are shown in Table 1. Bong Yi: albumin measurement and correction line intensity signal intensity_correction # 1 1 1 1 —_correction # 2 10 10 10 ΕΛΡΑΤΕΝΊΛΡΚ-001 〇8 \ pk-〇〇 | .〇827 \ pk-001-0827.doc2003 / 6/12 27 587166
一檢量線，偵,信號強度爲20，1〇 ’及G相對白蛋白原量爲〇毫克， 75毫克’及125毛克。校正曲線由數據產生亦顯示於第入圖。使用校正曲 線，可偵測未知濃度白蛋白原存在及含量。 當本發_細揭賴明這祕财補，雜錢此技雛能，到達 先可未知，立即構想出替換，變換及姆的具體實施例。·，本發明的 目的必須確定如附加的具體實施例及任何相等的事物一樣。 圖示元件簡要説明 一 20 sandwich-type flow-through assay 夾層式溢流道檢驗 21 sampling pad 抽樣墊 22 conjugate pad 結合墊 —23 porous membrane 多孔薄膜 24 detection line 檢量線 25 calibration zone 校正區 25a calibration zone 校正區 _ 26 calibration zone 校正區 26a calibration zone 校正區 _27 calibration zone 校正區 _ 27a calibration zone 校正區 28 wicking pad 燈芯墊 29 arrow 箭頭 31 detection zone 檢波區 32 calibration zone 校正區 40 analyte 分析物 __41^ probe 探子 _42 probe conjugate 探子結合物 — 45 —---- capture reagent 捕捉劑 47 ----— . ______ binder 結合劑 — 49 complex 複合物 — 50 ternary complex 三聚物 _ 90 binding member 結合物件 ΕΛΡΑΤΕΝΤΛΡΚ-001 08\pk-001 -0827\pk-001 -0827.d〇c2003/6/12 28 587166 【圖式簡單説明】 弟一圖爲本發明具體實施例俯視圖’顯示有三個校正線在校正區的溢 流道檢.驗； 第二圖爲本發明溢流道具體實施例的透視圖，顯示在包含分析物的測 試樣品後之薄膜片應用至樣品墊； 第二圖説明第二圖顯示的側向檢驗，但與測試樣品穿過檢驗； 第四圖爲本發明另一個具體實施例俯視圖，其中第四A圖顯示平行分 析物，動的校正線且第㈣圖顯示平行分析物流動的校正點； 第五圖顯示爲使用於本發明具體實施例的校正曲線； 第六圖顯示如例子九揭示的CRp偵測校正曲線； 第七圖顯示如例子十揭示的LH偵測校正曲線； 第八圖顯示如例子十-揭示的自蛋喊侧的校正曲線。 E:\PATENT\PK-001 08\pk-001 -0827\pk-001 -0827.doc2003/6/12 29A calibration curve, detection, the signal intensity was 20,10 'and the relative albumin amount of G was 0 mg, 75 mg' and 125 gross grams. The calibration curve generated from the data is also shown in the first plot. Using the calibration curve, the presence and content of albuminogen of unknown concentration can be detected. When this issue _ reveals Lai Ming's secret financial supplement, miscellaneous money and skill can be reached, it may be unknown before it arrives, and specific embodiments of replacement, transformation, and conversion are immediately conceived. · The purpose of the present invention must be determined as if it were an additional specific embodiment and anything equivalent. The illustrated components are briefly described. 20 sandwich-type flow-through assay 21 sampling pad sampling pad 22 conjugate pad conjugate pad—23 porous membrane 24 detection line 25 calibration zone 25a calibration zone Calibration zone_ 26 calibration zone Calibration zone 26a calibration zone Calibration zone_27 calibration zone Calibration zone_ 27a calibration zone Calibration zone 28 wicking pad 29 arrow arrow 31 detection zone Detection zone 32 calibration zone Calibration zone 40 analyte Analyte__41 ^ probe 探子 _42 probe conjugate — 45 —---- capture reagent 47 — — — — ______ binder — 49 complex — 50 ternary complex — 90 binding member ΕΛΡΑΤΕΝΤΛPK -001 08 \ pk-001 -0827 \ pk-001 -0827.d〇c2003 / 6/12 28 587166 [Brief description of the figure] The first figure is a top view of a specific embodiment of the present invention 'shows that there are three calibration lines in the calibration area The overflow channel inspection. Inspection; The second picture shows the overflow of the invention The perspective view of the specific embodiment shows that the thin film sheet after the test sample containing the analyte is applied to the sample pad; the second figure illustrates the lateral inspection shown in the second diagram, but passes through the inspection with the test sample; the fourth diagram is A plan view of another embodiment of the present invention, wherein the fourth diagram A shows a parallel analyte, a moving calibration line and the second diagram shows a calibration point for the parallel analyte flow; the fifth diagram shows a calibration used in a specific embodiment of the present invention Curves. The sixth graph shows the calibration curve of CRp detection as disclosed in Example 9. The seventh graph shows the calibration curve of LH detection as disclosed in Example 10. The eighth graph shows the calibration curve from the egg shout side as disclosed in Example 10. E: \ PATENT \ PK-001 08 \ pk-001 -0827 \ pk-001 -0827.doc2003 / 6/12 29