Establishment of preparation and rapid detection method for polyclonal antiserum of DDT (dichlorodiphenyltrichloroethane) metabolite DDA (2,2-bis(4-chlorophenyl)acetic acid)

A polyclonal antiserum, DDT technology, applied in chemical instruments and methods, derived from serum immunoglobulins, measuring devices, etc., can solve the problem that it is not suitable for on-site analysis, difficult to meet the needs of large-scale sample detection and analysis, and the pretreatment process is cumbersome and complicated. and other problems, to achieve the effect of rapid detection, low detection cost and low cost

Inactive Publication Date: 2011-11-09
NATIONAL MARINE ENVIRONMENTAL MONITORING CENTRE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the conventional analysis methods of the organochlorine pesticide DDT and its metabolites mainly include gas chromatography combined with mass spectrometry or electron capture detector methods (GC/MS, GC/FLD), which not only require e...

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0007] Embodiment 1 (preparation embodiment)

[0008] Antiserum Preparation Method of Specific Anti-DDA Polyclonal Antibody

[0009] 1. Preparation of DDA-carrier protein conjugates

[0010] Dissolve 2,2-bis(4-chlorophenyl)acetic acid (DDA) in dimethyl sulfoxide, add water-soluble carbodiimide (EDC), N-hydroxysuccinimide (NHS), 25°C After reacting for 2 hours, bovine serum albumin (BSA) or ovalbumin (OVA) dissolved in 50 mM carbonate buffer (pH 9.6) containing KCL was added, and the reaction was stirred overnight at 25°C. The reaction mixture was dialyzed against PBS for 3 days at 4°C, and the solution was changed every 12 hours. After aliquoting the dialyzate, store it at -20°C. Prepare DDA-BSA and DDA-OVA conjugates for use.

[0011] 2. Preparation of anti-DDA polyclonal antiserum

[0012] The prepared 2,2-bis(4-chlorophenyl)acetic acid-bovine serum albumin conjugate (DDA-BSA) was used as immunogen to immunize New Zealand white rabbits. For the first immunization, 400 ...

Embodiment 2

[0013] Embodiment 2 (application embodiment)

[0014] Method for using the enzyme-linked immunosorbent assay kit for detecting 2,2-bis(4-chlorophenyl)acetic acid (DDA)

[0015] 1. Pretreatment of shellfish and other samples

[0016] Weigh 20g of shellfish homogenate, add 5ml of water (depending on the water content, add water to make the total water about 20ml), add 40ml of acetone, shake for 30min, add 6g of calcium chloride, and shake well. Add 30ml of petroleum ether, shake for another 30min, and let stand to separate layers. Take 35ml of the supernatant, dehydrate it with anhydrous sodium sulfate, concentrate in a rotary evaporator to nearly dryness, dilute to 5ml with petroleum ether, add 0.5ml of concentrated sulfuric acid to purify, shake for 0.5min, centrifuge at 3000r / min for 15min, and again in Concentrate to near dryness in a rotary evaporator, dissolve the extract completely with 5ml PBS, and take the supernatant for analysis.

[0017] 2. Detection

[0018] DDA...

Embodiment 3

[0019] Embodiment 3 (application embodiment)

[0020] Application of indirect competitive enzyme-linked immunosorbent assay for detection of 2,2-bis(4-chlorophenyl)acetic acid

[0021] 1. DDA-specific polyantibody titer and optimal working concentration experiment

[0022] After the start of immunization, blood was collected from the ears of the rabbits at the fourth, fifth, sixth and seventh weeks respectively, and the serum titer (titer) was determined by direct ELISA method. After immunization for 4 times, the titer can reach more than 1.2 million. Through the square array titration test, in the direct ELISA method, according to the absorbance value of the polyantibody serum diluted in different times, the absorbance value ranges from 1.0 to 1.5, and the optimal dilution ratio of the coated antigen is 1:2000; the sheep anti-rabbit enzyme The optimal dilution ratio of the labeled secondary antibody is 1:12000, the optimal dilution ratio of the serum determined by direct EL...

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Abstract

The invention relates to a detection technology of organochlorine pesticide DDT (dichlorodiphenyltrichloroethane) metabolites, specifically relates to an anti-organochlorine pesticide DDT metabolite 2,2-bis(4-chlorophenyl)acetic acid (DDA) polyclonal antiserum prepared by synthesizing an antigen through protein coupling, and relates to a preparation method of a rapid detection kit capable of analyzing the DDA content in an environmental sample by utilizing the binding of a polyclonal antibody and a DDA antigen. An enzyme-linked immunosorbent assay kit for the rapid detection of organochlorine pesticide DDT metabolite 2,2-bis (4-chlorophenyl)acetic acid comprises a 96-well enzyme label plate detachable slat, an enzyme-labeled second antibody, a Tween-20-containing buffer salt washing liquid, a substrate liquid and a stopping liquid. A synthetic protein conjugate coated antigen is coated on the 96-well enzyme label plate by utilizing the prepared anti-serum containing anti-DDA-specific polyclonal antibody. The kit provided by the invention has the advantages of simple pre-treatment, easy operation process, low cost and price and the like, is suitable for on-site, rapid and large-batch sample detection, and has a detection limit less than 21 ng/ml.

Description

Technical field [0001] The present invention relates to the detection technology of organochlorine pesticide DDT metabolites, in particular to a method for preparing anti-organochlorine pesticide DDT metabolite 2,2-bis(4-chlorophenyl)acetic acid (DDA) by using protein coupling to synthesize antigens The polyclonal antiserum relates to the preparation method of a fast and convenient detection kit for analyzing and detecting the DDA content in environmental samples by using the polyclonal antibody combined with the DDA antigen. Background technique [0002] DDT was widely used as a broad-spectrum insecticide in the 1950s. This kind of organic synthetic insecticides containing chlorine atoms has extremely high fat solubility and stability, can be enriched in high concentrations in organisms, degrades slowly in the environment, and belongs to persistent organic pollutants. The two main decomposition products of DDT in sediments are 2,2-bis(4-chlorophenyl)-1,1-dichloroethylene (...

Claims

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Application Information

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IPC IPC(8): C07K16/44C07K16/06G01N33/543
Inventor 刘仁沿许道艳梁玉波
Owner NATIONAL MARINE ENVIRONMENTAL MONITORING CENTRE
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