阅读说明：本技术 一种水胺硫磷单克隆抗体及其在胶体金检测试纸的应用 (Isocarbophos monoclonal antibody and application thereof in colloidal gold detection test paper ) 是由 盛相国 于 2019-11-27 设计创作，主要内容包括：本发明涉及一种水胺硫磷单克隆抗体,其抗体重链可变区氨基酸序列如SEQ ID NO:1所示,其轻链可变区氨基酸序列如SEQ ID NO:2所示；以及一种水胺硫磷单克隆抗体在胶体金检测试纸的应用,包括衬板和依次排列在衬板上的样品垫、金标结合垫、纤维素膜和吸水垫,所述金标结合垫内吸附有水胺硫磷金标抗体,所述纤维素膜上设置有互相平行的隐形检测线和隐形对照线,所述隐形检测线用水胺硫磷半抗原偶联的载体蛋白溶液印制,所述隐形对照线用兔抗鼠IgG抗体印制；无需专业操作人员及任何辅助类仪器,根据反应所显现的条带几分钟内即可判定结果,不仅操作简便、快速、结果直观准确、成本低,并且可以在室外进行检测。(The invention relates to a isocarbophos monoclonal antibody, wherein the amino acid sequence of the heavy chain variable region of an antibody is shown as SEQ ID NO. 1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2; the application of the isocarbophos monoclonal antibody in the colloidal gold test paper comprises a lining plate, and a sample pad, a gold-labeled combination pad, a cellulose membrane and a water absorption pad which are sequentially arranged on the lining plate, wherein isocarbophos gold-labeled antibodies are adsorbed in the gold-labeled combination pad, the cellulose membrane is provided with an invisible detection line and an invisible contrast line which are parallel to each other, the invisible detection line is printed by using a carrier protein solution coupled with isocarbophos hapten, and the invisible contrast line is printed by using a rabbit anti-mouse IgG antibody; the result can be judged within a few minutes according to the strip shown by the reaction without professional operators and any auxiliary instruments, so that the method is simple and convenient to operate, quick, intuitive and accurate in result and low in cost, and can be used for outdoor detection.)

1. The isocarbophos monoclonal antibody is characterized in that: the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2.

2. The application of the isocarbophos monoclonal antibody in the colloidal gold test paper is characterized in that: the kit comprises a lining plate, and a sample pad, a gold-labeled binding pad, a cellulose membrane and a water absorption pad which are sequentially arranged on the lining plate, wherein isocarbophos gold-labeled antibodies are adsorbed in the gold-labeled binding pad, the cellulose membrane is provided with a stealth detection line and a stealth contrast line which are parallel to each other, the stealth detection line is printed by using a carrier protein solution coupled with isocarbophos hapten, and the stealth contrast line is printed by using a rabbit anti-mouse IgG antibody.

3. The application of the isocarbophos monoclonal antibody in the colloidal gold test paper according to claim 2 is characterized in that: the isocarbophos hapten coupled carrier protein is a product obtained by coupling O-ethyl-O- (1-phenyl-1, 2, 4-triazole-3-yl) -N- (butyrate) thiophosphate with Bovine Serum Albumin (BSA) by an active ester method.

4. The application of the isocarbophos monoclonal antibody in the colloidal gold test paper according to claim 2 is characterized in that: the isocarbophos gold-labeled antibody is a colloidal gold-labeled isocarbophos monoclonal antibody.

5. The application of the isocarbophos monoclonal antibody in the colloidal gold test paper according to claim 2 is characterized in that: the lining plate is made of a non-water-absorbing tough material, the water absorbing pad is a water absorbing filter paper pad or an oil filter paper pad, and the sample pad is a glass fiber cotton pad and/or a nylon membrane pad and/or a polyvinylidene fluoride membrane pad and/or a polyester membrane pad.

Technical Field

The invention relates to the technical field of pesticide residue immunoassay, in particular to a isocarbophos monoclonal antibody and application thereof in colloidal gold test paper.

Background

Isocarbophos (Isocarbophos) is a broad spectrum organophosphorus insecticide used to control pests in stored grains and on various foliar crops, and also to control adult mosquitoes, flies, aquatic larvae and sanitary pests. The action mechanism is to inhibit the activity of acetylcholinesterase. Acetylcholine is a neurotransmitter and excess acetylcholine over-stimulates acetylcholine receptors, leading to death of the target organism. However, some organisms that are not the intended target are also subject to poisoning, including humans, fish, and other organisms.

At present, the detection method of pesticide residue mainly comprises an instrument analysis method, an enzyme-linked immunosorbent assay (ELISA) method and the like. The instrumental analysis methods include High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), Thin Layer Chromatography (TLC), liquid-mass tandem method (HPLC-MS), Gas-mass tandem method (GC-MS), and the like, and although these instrumental analysis methods can achieve higher detection sensitivity, they require complicated and expensive instrumentation and professional operators, and in addition, the sample pretreatment is tedious and time-consuming, the detection cost is High, and the requirements of rapid and online detection are difficult to meet. ELISA (enzyme Linked immunosorbent assay) method is a common immunodetection method, but has the defects of relatively more operation steps, longer detection time, insufficiently visual detection result, incapability of on-line detection and the like. Thus, the application of the ELISA method is greatly limited.

The isocarbophos colloidal gold test strip provided by the invention does not need professional operators and any auxiliary instruments, can judge the result within a few minutes according to the strip shown by the reaction, is simple and convenient to operate, is rapid, has intuitive and accurate result and low cost, and can be used for outdoor detection.

Disclosure of Invention

The invention aims to: provides a isocarbophos monoclonal antibody and application thereof in colloidal gold test paper.

The technical scheme of the invention is as follows: the isocarbophos monoclonal antibody has the antibody heavy chain variable region amino acid sequence shown in SEQ ID No. 1 and the light chain variable region amino acid sequence shown in SEQ ID No. 2.

The invention also provides an application of the isocarbophos monoclonal antibody in the colloidal gold test paper, which comprises a lining plate, and a sample pad, a gold-labeled binding pad, a cellulose membrane and a water absorption pad which are sequentially arranged on the lining plate, wherein isocarbophos gold-labeled antibodies are adsorbed in the gold-labeled binding pad, the cellulose membrane is provided with a stealth detection line and a stealth contrast line which are parallel to each other, the stealth detection line is printed by using a carrier protein solution coupled with isocarbophos hapten, and the stealth contrast line is printed by using a rabbit anti-mouse IgG antibody.

Further: the isocarbophos hapten coupled carrier protein is a product obtained by coupling O-ethyl-O- (1-phenyl-1, 2, 4-triazole-3-yl) -N- (butyrate) thiophosphate with Bovine Serum Albumin (BSA) by an active ester method.

Further: the isocarbophos gold-labeled antibody is a colloidal gold-labeled isocarbophos monoclonal antibody.

Further: the lining plate is made of a non-water-absorbing tough material, the water absorbing pad is a water absorbing filter paper pad or an oil filter paper pad, and the sample pad is a glass fiber cotton pad and/or a nylon membrane pad and/or a polyvinylidene fluoride membrane pad and/or a polyester membrane pad.

Compared with the prior art, the invention has the advantages that:

1) strong specificity and high sensitivity. The colloidal gold test strip is prepared on the basis of a colloidal gold labeled high-affinity monoclonal antibody, has strong specificity and high sensitivity, and the minimum detection amount can reach 2 ng/mL.

2) Simple and fast. When the colloidal gold test strip is used, no other auxiliary instrument is needed, the field operation can be realized, only the detection sample is added into the sample pad, and the detection result can be judged within 3 to 5 minutes.

3) The result display is visual, intuitive and accurate. The detection result of the colloidal gold test strip is judged by the color development condition on the cellulose membrane. If the sample contains the substance to be detected, the substance to be detected and the gold-labeled antibody are combined to form an antigen-gold-labeled antibody compound, and the antigen-gold-labeled antibody compound continuously moves upwards, and the positive or weak positive of the detected sample is represented when the invisible detection line T line does not develop color or the color development is weak; and if the sample to be detected does not exist in the sample, the gold-labeled antibody continuously moves upwards, and a clear red line is displayed when the gold-labeled antibody meets the T line of the invisible detection line, namely the detection sample is negative. The presence or absence of the color of the invisible control line C indicates the effectiveness or ineffectiveness of the test strip. The result judgment is visual, intuitive, accurate, simple and clear.

4) Saving cost, wide application range and convenient popularization. The cost for detecting by using the colloidal gold test strip is greatly reduced compared with the cost for detecting by using an instrument and an ELISA kit. Moreover, the test strip has wide application range, can meet the requirements of personnel at different levels, is convenient to popularize and apply, and has wide market prospect and obvious economic and social benefits.

The isocarbophos colloidal gold test strip provided by the invention does not need professional operators and any auxiliary instruments, can judge the result within a few minutes according to the strip shown by the reaction, is simple and convenient to operate, is rapid, has intuitive and accurate result and low cost, and can be used for outdoor detection.

Drawings

The invention is further described with reference to the following figures and examples:

FIG. 1 is a schematic view of a structure of a test strip for testing colloidal gold isocarbophos;

FIG. 2 is a schematic cross-sectional view of a test strip of colloidal gold isocarbophos;

wherein: 1: a lining plate, 2: sample pad, 3: gold-labeled bond pad, 4: cellulose film, 5: invisible detection line, 6: stealth control line, 7: absorbent pad, 8-1: sample immersion end protective film, 8-2: handle end protective film, 9: marking a line;

FIG. 3 is a schematic diagram showing the result determination of a test strip for testing colloidal gold isocarbophos;

wherein, A is a negative sample detection result, B is a weak positive sample detection result, C is a strong positive sample detection result, and D and E are test strip failure indications.

Detailed Description