Antigen Mimotope Mimicking Ochratoxin A and Its Application
An ochratoxin and mimic epitope technology, used in recombinant DNA technology, DNA/RNA fragments, material testing products, etc., can solve the health and environmental threats of inspectors, restrict the application and promotion of immunological detection methods, and be expensive and cause cancer It can achieve the effect of good effect, cost saving and high application value
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Embodiment 1
[0017] Example 1. Affinity panning and identification of OTA antigen mimotope
[0018] 1) Affinity panning of OTA antigen mimic epitopes: the specific method is: dilute anti-OTA monoclonal antibody with 10 mM PBS (pH 7.4), and coat a 96-well microtiter plate with a final concentration of 100 μg / mL, at 4°C Incubate overnight. The next day, after washing 10 times with TBST (50 mM NaCl, pH 7.5 containing 0.1% Tween-20 (v / v) ), add 300 μl of blocking solution (3% BSA-PBS) and incubate at 4°C for 2 hours. After 2 hours, the blocking solution was discarded, washed 5 times with TBST, and 100 μl of phage peptide library (phage display dodecapeptide library, purchased from NEB Company) was added to each well, and the phage stock solution was diluted 10 times with TBS, about 1.0×10 11 pfu), shake and react for 1 hour at 22-26°C. Unbound phages were discarded, washed 10 times with TBST, bound phages were eluted with 0.2 M Glycine-HCl (pH 2.2), and immediately neutralized with 15 μl 1 ...
Embodiment 2
[0022] Example 2. Sequencing of the gene encoding the mimotope of the OTA antigen and determination of its amino acid sequence
[0023]The phages identified by indirect competition ELISA displaying OTA antigen mimic epitopes were amplified, and the DNA sequencing templates of the phages were extracted. The brief process is as follows: For phage amplification, after the first step of centrifugation, transfer 800 µl of the phage-containing supernatant to a new centrifuge tube. Add 200 µl PEG / NaCl to precipitate the phage. After centrifugation, resuspend the pellet in 100 µl iodide buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 4 M NaI), add 250 µl absolute ethanol to precipitate DNA, centrifuge and wash with 70% ethanol Precipitation (DNA sequencing template). The pellet was finally resuspended in 20 µl of sterilized water, and 2 µl was taken for agarose gel electrophoresis analysis; 5 µl of the phage template was used for DNA sequencing, and its -96 gIII sequencing primer was: 5...
Embodiment 3
[0024] Example 3. Application of OTA antigen mimotope as a competitive antigen in ELISA
[0025] (1) Sample extraction
[0026] Weigh 5g of the sample (cereals and related foods), add 25ml of 60% methanol-PBS solution, shake at 200 rpm for 5 minutes; filter the extract with No. 1 filter paper, take 1ml of the filtrate and add 4ml of PBS ( Phosphate buffer, pH = 7.2) After mixing, it is the sample extract, ready for use.
[0027] (2) Coating and sealing
[0028] Dilute the anti-OTA monoclonal antibody with 10 mM PBS (pH 7.4), coat the microtiter plate with 10 µg / mL, and incubate overnight at 4°C. The next day, after washing 3 times with PBST (10 mM PBS, 0.05% Tween-20 (v / v)), block with PBS containing 3% skimmed milk powder, incubate at 37°C for 1 hour, then wash the plate 6 times with PBST stand-by.
[0029] (3) Establishment of standard curve
[0030] Take out the strips treated in step (2), and put 50 µl of phage displaying OTA antigen mimic epitopes into each well (1.0...
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