Antibody library of bacteriophages and applications in immunoassay of pesticide residue
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A phage antibody library and phage technology, applied in the phage antibody library and its application fields
Inactive Publication Date: 2008-10-22
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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Embodiment 1
[0108] Construction of phage antibody library
[0109] Material:
[0110] Phagemid pCANTAB5E, E.coli TG1 freeze-dried strain, M13K07 helper phage are all products of Pharmacia. The kits SV Total RNA Isolation System; ReverseTranscription System; various restriction endonucleases and ligases were purchased from Promega. Immunogen milbemycin oxime (A 4 、A 3 )-BSA (MILO-BSA) laboratory preservation, female 7-week-old Balb / c mice were purchased from the Comparative Medicine Center of Yangzhou University.
[0111] Primer design
[0112] Design multiple groups of degenerate primers in the light and heavy chain framework regions FR1 and FR4 of the antibody molecule as follows: and add linking peptides (Gly 4 Ser) 3 Partial sequence and restriction site of (Linker), wherein, V L F primer contains NotI restriction site, V H Primer B contains a SfiI restriction site. V H F primer contains part of Linker sequence, V L Primer B contains part of the Linker sequence, and the two ...
Embodiment 2
[0154] Taking milbemycin pesticide as an example to screen soluble scFv
[0155] (1) Enrichment and screening of MILO-BSA phage antibody
[0156] (1) Coat the immunotube with 4ml purified MILO-BSA as the antigen (100μg / ml), the coating solution is PBS (pH7.4), coat the plate overnight at 4°C, wash the immunotube 3 times with PBS, 5min each time.
[0157] (2) Fill the immunotube with 2% MPBS and block at 37°C for 2 hours.
[0158] (3) Remove the blocking solution, add the amplified phage antibody, and incubate at 37°C for 2h.
[0159] (4) To remove unbound phage antibodies, wash the immunotube 10 times with PBST buffer (10 times in the first round, and 20 times in each subsequent round).
[0160] (5) Add 500 μl of 1 mg / ml trypsin into the immunotube, incubate at 37°C for 30 minutes, and repeat pipetting to elute the specifically bound phage antibody.
[0161] (6) Infect 3.5 ml of E.coli TG1 in the logarithmic growth phase with 500 μl of the eluate, and bathe in water at 37° ...
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Abstract
The invention relates to a phage antibody library which assembles an ScFv gene fragment between Restriction Enzyme cutting sites of SfiI and NotI of a pCANTAB5E carrier. The phage antibody library is characterized in that the ScFv gene fragment can be affinitive and enriched with antigens of 16-membered macrolide agricultural chemicals to form a soluble single-chain antibody and the phage antibody library is applied to immunoassay of pesticide residue. The phage antibody library has the advantages that the antibody with high affinity can be obtained without animal immunization, the test period is short, the antibody library is the phage antibody library which takes small molecular milbemycin oxime of a 16-membered macrolide generic structure as immunogen to construct, the antibody library theoretically can directly obtain a specific antibody library of 16-membered macrolide compound through screening, the screening flux is high, the efficiency is high, the specificity is strong, and the affinity selecting range is wide, so that the phage antibody library has wide application prospect in the aspects such as agricultural chemical antibody preparation, testing technique development and the like.
Description
Technical field: [0001] The invention relates to a phage antibody library and its application, especially an antibody library capable of detecting the small molecule specificity of sixteen-membered macrolide pesticides and its application in the immunoassay of pesticide residues, belonging to a Display the antibody library constructed by the technology and use it in the field of pesticide residue immunoassay. Background technique: [0002] Trace pesticide immunoassay technology is a widely researched and widely used pesticide residue screening and determination technology at home and abroad. It integrates the high sensitivity of the assay and the strong specificity of the antibody reaction. It has the advantages of simplicity, rapidity, high sensitivity, strong specificity, and less sample requirement. It has achieved fruitful results in the trace detection of pesticides. Among them, the antibody preparation of small molecule pesticides is the core content, which is current...
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