Preparation method of small molecule compound antibody based on high-throughput sequencing and hybrid hybridoma technology
A technology for cloning antibodies and nucleic acid molecules, which is applied in the fields of immunology and genetic engineering, can solve the problems of difficulty in obtaining rabbit myeloma cells, limited application, and inability to establish and promote the technology, and achieve the problem of limited source of rabbit tumor cells and high sensitivity , high stability effect
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[0047]Furthermore, the present invention provides a method for preparing monoclonal antibodies based on high-throughput sequencing technology.
[0048] Furthermore, the present invention provides the application and promotion of a monoclonal antibody expressed based on the variable region sequence of the antibody in the field of small molecule detection.
[0049] Further, the present invention is realized through the following technical solutions:
[0050] The preparation method of ractopamine immunogen (RAC-BSA) and coating former (RAC-OVA) comprises:
[0051] Hapten 1 was covalently linked to BSA / OVA by the glutaraldehyde method to prepare the immunogen and coating agent respectively as follows: 20 mg of Hapten 1 was mixed with 40 mg of BSA or OVA in 5 mL of PBS (pH 7.4). Glutaraldehyde (1 mL, 0.5% solution) was added dropwise to the mixture, stirred at room temperature for 3 h, then dialyzed against PBS for 3 days, and stored at 20° C. until use.
[0052] The immunization...
Embodiment 1
[0062] The immunization of embodiment 1 rat and rabbit
[0063] The concentration of the immunogen (RAC-BSA) was adjusted to 1 mg / mL with PBS buffer, and the concentration of the immunogen was determined by the Bicinchoninic Acid method.
[0064] Take 5 New Zealand white rabbits, numbered RAC-BSA-1, RAC-BSA-2, RAC-BSA-3, RAC-BSA-4 and RAC-BSA-5 respectively. Take 5 Wistar rats, numbered RAC-BSA-6, RAC-BSA-7, RAC-BSA-8, RAC-BSA-9 and RAC-BSA-10. The preparation method of the first immunoemulsified preparation: Take 4.0 mL of immunogen with a concentration of 1 mg / mL, then mix it with an equal volume of Freund's complete adjuvant and emulsify it. The preparation method of boosted immunoemulsification preparation: Take 4.0 mL of immunogen with a concentration of 1 mg / mL, then mix and emulsify with an equal volume of Freund's incomplete adjuvant. The emulsified adjuvant prepared above was injected into the neck and back of rabbits and rats by multi-point dispersed injection (the...
Embodiment 2
[0065] Example 2 Screening of Rat and Rabbit Antisera
[0066] The titer and sensitivity of the antiserum obtained one week after immunization were tested. The method used to measure antiserum titer is indirect ELISA; the method used to measure sensitivity is indirect competition ELISA.
[0067] The steps of indirect ELISA are as follows:
[0068] Dilute the coated RAC-OVA to 0.1 μg / mL with carbonate buffer (pH 9.6), coat the ELISA plate with an amount of 100 μL / well, incubate at 37° C. for 2 h, and then wash the plate 3 times with PBST solution. The ELISA plate was then blocked with 2% skimmed milk powder (150 μL / well), incubated at 37° C. for 1 h, washed three times with PBST solution, and patted dry. The antiserum was serially diluted with PBS, starting from a dilution of 1:4000, with a gradient of 2, a total of 8 dilutions. Add 50 μL of PBS buffer and 50 μL of serially diluted antiserum solution to the coated ELISA plate, incubate at 37°C for 30 min, then wash with PBST...
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