Hybridoma WXX-1 capable of secreting anti-lomefloxacin monoclonal antibody and application thereof
A monoclonal antibody, hybridoma cell line technology, applied in the field of immunochemistry, can solve the problems of relying on instruments and equipment, time-consuming, etc.
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Embodiment 1
[0016] Example 1: Synthesis of Immunogen and Coating Gen
[0017] Synthesis of immunogen: First, bovine serum albumin (Bovine Serum Albumin, BSA) was cationized with EDEA. Dissolve 200mg BSA and 14mg EDEA in 10 mL PBS, adjust its pH to 6.5; dissolve 18.2mg carbodiimide (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, EDC) in 200 μL PBS , slowly drop it into the BSA solution, adjust the pH to 7.5, and stir at room temperature for 2h. Dialysis removes EDEA not bound to BSA, yielding BSA-EDEA.
[0018] BSA-EDEA is also coupled with the small molecule lomefloxacin by the carbodiimide method. Dissolve 10 mg of lomefloxacin, 15 mg of EDC, and 9 mg of N-hydroxysuccinimide (NHS) in 5 mL of 0.05M MES at pH 4.7, and stir at room temperature for 6 h. Then the activated solution was slowly added dropwise to the EDEA-BSA solution (0.3 μmol EDEA-BSA dissolved in 10 mL, 0.05 M, pH 9.6 carbonate buffer solution), and reacted at room temperature for 3 h. The reaction liquid after the react...
Embodiment 2
[0020] Example 2: Mouse immunization
[0021] The immunogen was diluted to an appropriate concentration with physiological saline, emulsified with an equal volume of Freund's adjuvant, and injected at multiple points on the back of 6-8 week-old BALB / c mice. For the first immunization, complete Freund's adjuvant was used at a dose of 100 μg. The following four booster immunizations used incomplete Freund's adjuvant, the immunization dose was 50 μg, and the immunization interval was 21 days. After the third immunization, blood was collected from the tail vein of the mice, and the sera were tested by indirect ELISA. After the fifth immunization, the mice with high titer and inhibition were screened for intraperitoneal immunization, and the spleens of the mice were taken out three days later for cell fusion.
Embodiment 3
[0022] Example 3: Cell Fusion and Screening
[0023] Mouse splenocytes and tumor cells were fused under the action of PEG 1500 to form hybridoma cells. Detection was carried out on the seventh day after fusion, and the cell lines with high titer and good recognition of lomefloxacin were screened for subcloning, and so on, 5 times of subcloning were performed to obtain pure cell lines.
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