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Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting arsanilic acid, nitarsone and Carbarsone

An enzyme-linked immunosorbent reagent and monoclonal antibody technology, applied in the field of veterinary drug residue analysis and immunology, can solve the problems of inability to monitor multiple organic arsine drug residues, and achieve the effects of saving analysis times, simple processing methods, and easy operation.

Inactive Publication Date: 2012-11-07
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This product can only be used for the detection of roxarsine (500μg / L delineation), and cannot be used for residue monitoring of various organic arsine drugs

Method used

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  • Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting arsanilic acid, nitarsone and Carbarsone
  • Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting arsanilic acid, nitarsone and Carbarsone
  • Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting arsanilic acid, nitarsone and Carbarsone

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Preparation of immunogen and coating

[0041] 1.3 Preparation of Diazotized Arsanilic Acid-Bovine Serum Albumin Conjugate (DIA-ASA-BSA)

[0042] Weigh 21.7 mg of arsenic acid and dissolve it in 1 mL of 0.1 mol HCl, adjust pH=1~2, and slowly add 100 mg / mL NaNO at 4°C 2 The solution was until the starch potassium iodide test paper turned blue, and the ice bath was protected from light for 45 minutes. At the end of the reaction, neutralize excess NaNO with a small amount of 50 mg / mL sulfamic acid ammonia. 2 . Dissolve 70 mg BSA in 10 mL of carbonate buffer (accurately weigh Na 2 CO 3 1.59g, NaHCO 3 2.93g, dissolve in a small amount of deionized water, and dilute to 1000 mL. ), adjust pH=9~10 with 1M NaOH, slowly add the first step reaction solution dropwise in an ice bath, and continuously adjust the pH value to 8~9, and react at 4°C overnight. After the reaction was completed, dialysis was performed with normal saline at 4° C. for 3 days, and the dialys...

Embodiment 2

[0047] Example 2 Preparation of monoclonal antibodies

[0048] 2.1 Animal immunization

[0049] Balb / C mice (purchased from the Experimental Animal Center of Hubei Academy of Medical Sciences) were immunized with the diazotized assanilic acid-bovine serum albumin conjugate (DIA-ASA-BSA) prepared in Example 1. The immunization procedure is to take a protein solution containing 50-100 μg of DIA-ASA-BSA conjugate and mix it with an equal amount of adjuvant and then inject it into mice to produce specific serum.

[0050] 2.2 Cell fusion and cloning

[0051] Referring to Yang Hanchun's "Animal Immunology", DIA-ASA-BSA immunogen was used to immunize Balb / C mice (purchased from the Laboratory Animal Center of Hubei Provincial Center for Disease Control and Prevention). After emulsification with complete adjuvant, the mice were injected subcutaneously at multiple points on the back of the mice, and then boosted immunization every 2 weeks, and then replaced with incomplete adjuvant e...

Embodiment 3

[0056] Example 3 Establishment of the Indirect Competitive ELISA Detection Method for Arsanilic Acid

[0057] 3.1 Preparation of reagents (reagents used in this example were prepared by the following methods unless otherwise noted)

[0058] Phosphate buffer: NaCl 8.0g, KH 2 PO 4 0.2g, Na 2 HPO 4 .12H 2 O 2.9g, KCl 0.2g, add double distilled water to 1000mL, adjust pH to 7.4;

[0059] Coating solution: take Na 2 CO 3 1.59g, NaHCO 3 2.93g, add three distilled water to 1000mL, adjust the pH value to 9.6;

[0060] Washing solution: NaCl 8.0g, KH 2 PO 4 0.2g, Na 2 HPO 4 .12H 2 O 2.9g, KCl 0.2g, Tween 20 0.5mL, add double distilled water to 1000mL, adjust pH to 7.4;

[0061] Blocking solution: 1 g of ovalbumin was dissolved in 100 mL of phosphate buffer;

[0062] Substrate solution A: 3, 3', 5', 5-tetramethylbenzidine (TMB) 200mg, absolute ethanol 100mL, add double distilled water to 1000mL;

[0063] Substrate solution B: Na 2 HPO 4 14.6g, 9.3g citric acid, 6....

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Abstract

The invention discloses a monoclonal antibody capable of identifying arsanilic acid, nitarsone and Carbarsone. The monoclonal antibody is secreted by a hybridoma cell line 4F2, which is preserved in China Center for Type Culture Collection, and has a preservation number of CCTCC NO:C201248. The invention also discloses an enzyme-linked immunosorbent assay method and a reagent kit detecting residues of arsanilic acid, nitarsone and Carbarsone. Compared with prior art, the monoclonal antibody prepared by the invention can simultaneously identify arsanilic acid, nitarsone and Carbarsone; the invention of the enzyme-linked immunosorbent assay method and kit for can detect residues in animal feed or edible animal tissue by one time, and have advantages of simpleness, rapidness, sensitivity and accuracy; besides, the sample treatment method has advantages of simpleness, easy operation, and little harm on body health.

Description

technical field [0001] The invention belongs to the technical field of veterinary drug residue analysis and immunology, and in particular relates to a monoclonal antibody capable of recognizing assanilic acid, nifesinic acid and carbachosine, and a monoclonal antibody for detecting assanilic acid, nifesinic acid and carbachosine Residual enzyme-linked immunosorbent assay (ELISA) and kits. Background technique [0002] Arsanilic acid, nifediarsinic acid and carbachol are organic arsenic veterinary drugs, with the basic structure of phenylarsinic acid, broad-spectrum antibacterial, and have strong inhibitory and killing effects on a variety of intestinal pathogens. It has good effects on pig diarrhea, chicken coccidiosis and blackhead disease, and is widely used as a feed additive for pigs and chickens in animal husbandry. [0003] Organic arsines are compounds with very low toxicity, low absorption rate, fast metabolism and excretion, and less residues in tissues. Types of ...

Claims

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Application Information

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IPC IPC(8): C07K16/44G01N33/577
Inventor 袁宗辉冯亮彭大鹏王玉莲陈冬梅潘源虎王涓朱永利
Owner HUAZHONG AGRI UNIV
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