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Enzyme linked immunosorbent assay kit for detecting diethoxy organophosphorus pesticide based on nano antibody

An organophosphorus pesticide and diethoxy technology, which is used in biological testing, measuring devices, material inspection products, etc., can solve the problems of easy deactivation of enzyme preparations, expensive instruments, poor stability of antibodies, etc., and achieves low price and high effect. Good and stable effect

Active Publication Date: 2018-09-28
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

(2) There are many kinds of organophosphorus pesticide residues in the sample
Although the instrument method has high accuracy, the instrument is expensive, the detection cost is high, and it is not universal; the enzyme preparation in the enzyme inhibition method is easily inactivated; the immunoassay method based on the antibody is fast, sensitive, and high-throughput. Advantages, but antibodies are often poorly stable and easily inactivated under extreme conditions

Method used

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  • Enzyme linked immunosorbent assay kit for detecting diethoxy organophosphorus pesticide based on nano antibody
  • Enzyme linked immunosorbent assay kit for detecting diethoxy organophosphorus pesticide based on nano antibody
  • Enzyme linked immunosorbent assay kit for detecting diethoxy organophosphorus pesticide based on nano antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Construction of anti-diethoxy organophosphorus pesticide nanobody immune library

[0051] 1. Coupling diethoxyorganophosphorus pesticide hapten H1 with ovalbumin OVA (albumin) and keyhole limpet hemocyanin KLH (keyhole limpet heocyanin) by active ester method to prepare diethoxyorganophosphorus pesticide complete antigen H1 -OVA and H1-KLH. 500 μg of H1-KLH was emulsified with an equal volume of complete Freund's adjuvant, and multi-point immunization was performed subcutaneously on the neck of Bactrian camels. Booster immunization was performed every 2 weeks, each time 500 μg of H1-KLH was emulsified with an equal volume of Freund's incomplete adjuvant for immunization, and venous blood was collected one week after each immunization. The serum titer was determined by indirect competitive ELISA method, and the blood sample with the best serum inhibition was taken for lymphocyte separation and RNA extraction.

[0052] 2. RNA was extracted according to the Tr...

Embodiment 2

[0061] Example 2: Screening and identification of six types of diethoxyorganophosphorus pesticide nanobodies

[0062] 1. Use H1-OVA as the coating antigen, 100 μl per well, and incubate overnight in a 37°C water bath. The coating concentration gradient is 10, 5, 1, 0.5 μg / ml. After 12 hours, wash the plate twice with PBST, add 120 μl of 5% skimmed milk powder to block for 3 hours, and dry at 37°C for later use. Add 100 μl of phage antibody library (approximately 10 11 pfu) into KLH (1 mg / ml, 100 μl) wells, and shake at room temperature for 1 hour to remove antibodies that non-specifically adsorb KLH. Then transfer to antigen-coated wells, shake at room temperature for 1 hour, suck out unbound phage, and wash the plate with PBST 5 times, 8 times, 15 times, 15 times. The phage antibody adsorbed in the wells of the plate was eluted with 100 μl of eluent (triethylamine, 100 mM), and the eluted product was neutralized with 50 μl of Tris-HCl (pH 7.4). 10 μl was taken out for tit...

Embodiment 3

[0070] Example 3: Soluble expression and identification of nanobodies against diethoxyorganophosphorus pesticides

[0071] The VHH-pComb3xss plasmid was extracted by an extraction kit, and then introduced into competent Escherichia coli BL21DE3 by chemical transformation. Take a single clone for PCR identification and sequencing, and confirm that the inserted fragment is the target fragment. Cultivate the BL21DE3 colony containing the target fragment of the nanobody to logarithmic phase OD 600 When the value is 0.5, add 1 mM IPTG and induce expression at 37°C for 12 hours. The next day, the bacteria were obtained by centrifugation. Then the periplasmic cavity proteins were extracted by sucrose osmotic pressure method, and the soluble nanobodies in the periplasmic cavity were recovered after one-step Ni column purification.

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Abstract

The invention discloses an enzyme linked immunosorbent assay kit for detecting a diethoxy organophosphorus pesticide based on a nano antibody. The enzyme linked immunosorbent assay kit contains the nano antibody as a detection antibody, and an amino acid sequence of the nano antibody is shown as SEQ ID NO.1. The kit is the enzyme linked immunosorbent assay kit, and the enzyme linked immunosorbentassay kit comprises an enzyme label plate containing organophosphorus pesticide antigen, a horseradish peroxidase-labelled secondary antibody solution, a diethoxy organophosphorus pesticide standard solution, substrate liquid, a substrate buffer solution, a stopping solution, and condensation washing liquid. The expression level of the diethoxy organophosphorus pesticide based on the nano antibodyis high, the stability is strong, the kit can be used for detecting a plurality of the diethoxy organophosphorus pesticides, realizes broad spectrum specific identification of the diethoxy organophosphorus pesticide, has the advantages of accurate detection result, good effect, good stability, simple operation, high sensitivity, and low cost, and can accurately detect the residues of the diethoxyorganophosphorus pesticide in an agricultural product.

Description

technical field [0001] The invention relates to the technical field of diethoxy organophosphorus pesticide detection, and more specifically, relates to an ELISA kit for detecting diethoxy organophosphorus pesticide based on a nanobody and a method for using the same. Background technique [0002] Pesticides are of great significance to today's agricultural harvest and high yield, and are being widely used in the field of crop pest control. Since organophosphorus pesticides replaced organochlorine pesticides in the 1970s, the amount of organophosphorus pesticides in my country has accounted for 70% of the total amount of pesticides and insecticides. Organophosphorus pesticides are a class of insecticides that can inhibit the activity of cholinease. They are widely used in agricultural production because of their strong insecticidal effect, wide application range and low price. Although the Chinese government has strengthened the supervision and monitoring of pesticide use in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/6854
Inventor 王弘张玉琪徐振林杨金易张瑾如沈玉栋肖治理孙远明
Owner SOUTH CHINA AGRI UNIV
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