A general anti-pyrinomycin monoclonal antibody hybridoma cell line and its application
A hybridoma cell line and monoclonal antibody technology, which is applied in the field of food safety immunology detection, can solve the problems of expensive instruments, complicated operations, and cumbersome processing, achieve good sensitivity, simple preparation methods, and meet the requirements of dose dependence. Effect
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Embodiment 1
[0018] Example 1 Obtainment of Monoclonal Cell Line B and Identification of Anti-Pilimycin Monoclonal Antibody Produced
[0019] 1. Acquisition of monoclonal cell line B
[0020] 1. Preparation of PIR-KLH, the complete antigen of pyrilimycin: 2 mg of PIR was dissolved in water, 5.2 mg of sodium periodate was added, and activated at room temperature for 40 min. Another 7 mg of KLH was dissolved in 2.5 mL, 0.01 M, pH 9.6 CBS solution, the above-mentioned activated pyrilimycin solution was slowly added dropwise to the KLH solution, stirred at room temperature for 4 h, and dialyzed at 4 °C for three days. Store at -20°C in aliquots.
[0021] 2. Preparation of pirinomycin-coated antigen PIR-OVA: 6 mg of PIR was dissolved in DMF, 10 mg of CDI was dissolved in DMF, and the two solutions were mixed and activated at 37°C for 50 min. Dissolve 15 mg of OVA in 3 mL, 0.01 M, pH 9.6 CBS solution, add the activated solution dropwise into the protein solution, react overnight, dialyze at 4°...
Embodiment 2
[0029] Example 2 Preparation and Identification of Monoclonal Antibody
[0030] 1. Obtain and purify antibody ascites: select 10-week-old BALB / c mice, and 7-14 days before cell inoculation, pre-inject liquid paraffin 1mL / mouse intraperitoneally. Adjust the concentration of monoclonal cell line B to 2.0×10 with normal saline 6 cells / mL, the monoclonal cell line B was inoculated intraperitoneally, the ascites was purified by the octanoic acid-ammonium sulfate method, and the obtained monoclonal antibody was stored at -20°C.
[0031] 2. Antibody subtype identification:
[0032] Use the monoclonal antibody subtype detection kit (Southern Biotech Cat No. 530005) to identify the immunoglobulin subtype of the antibody obtained in step 1. The specific method is: use PIR-OVA to coat 1 μg / mL enzyme in 96 wells Combine the plates (0.1mL per well), overnight at 4°C, discard the coating solution, wash 3 times, add blocking solution (PBS containing 1% BSA) at 0.2mL / well, incubate at 37°C...
Embodiment 3
[0035] Example 3 Application of the Indirect Competitive ELISA Method Established Using the Antibody Secreted by the Monoclonal Cell Line B
[0036] The monoclonal antibody prepared by the monoclonal cell line B through the in vivo ascites was applied to the addition and recovery test of pyrilimycin ELISA, and the specific steps were as follows:
[0037] (1) 1 μg / mL PIR-OVA diluted with carbonate buffer solution (CBS) was used as the coating material to coat the 96-well ELISA plate, 100 μL per well, after coating for 2 hours at 37°C, wash with PBST washing solution Plate three times, 250 μL per well each time, 3 min each time, and pat dry;
[0038] (2) Block with CBS containing 0.01% gelatin, 200 μL per well, block at 37°C for 2 h, wash the plate three times with PBST washing solution, 250 μL per well for 3 min each time, and pat dry;
[0039] (3) Prepare 0, 0.625, 1.25, 2.5, 5, 10, 20, and 40 μg / L standard solutions of pirinomycin in phosphate buffered saline (PBS) containin...
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