Hybridoma cell strain secreting metronidazole monoclonal antibody and application thereof
A hybridoma cell line and metronidazole single technology, applied in the field of immunochemistry, can solve the problems of long time, high cost, professional and complex pretreatment technology, etc., and achieve the effect of high sensitivity
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Embodiment 1
[0046] Example 1: Synthesis of Metronidazole Hapten
[0047] (1) First weigh metronidazole (MNZ, 40g, 233.9mmol) and dissolve it into anhydrous N,N-dimethylformamide (DMF, 400mL), then add N,N-dimethylformamide to the above solution Formamide dimethyl acetal (DMF-DMA, 400 mL) and trifluoroacetic acid (TFA, 2 mL) were reacted with magnetic stirring at 100 °C under nitrogen protection for 48 h. After the reaction was completed, vacuum rotary evaporation was concentrated to obtain a black color. Solid compound 1 (37 g, 69.9%).
[0048]
[0049] (2) Compound 1 (37 g, 163.7 mmol) and triethylamine (TEA, 33.1 g, 322.4 mmol) were first dissolved in dichloromethane (370 mL), and then added dropwise to the above at 0°C under nitrogen protection. Acetyl chloride (19.3 g, 245.6 mmol) was added to the solution, and the reaction was kept under magnetic stirring at room temperature overnight. After the reaction was completed, the above solution system was washed with water and saturate...
Embodiment 2
[0055] Example 2: Synthesis of Metronidazole Complete Antigen
[0056] The preparation method of metronidazole immunization antigen comprises the following steps:
[0057] (1) The metronidazole hapten MNZ-COOH (16.2 mg, 0.06 mM) of formula 1 was dissolved in N,N-dimethylformamide (DMF, 1 mL) solution, and then dissolved in N,N- 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 34.3 mg) in dimethylformamide (DMF, 0.2 mL) and dissolved in MES (0.1 M, N-hydroxysuccinimide (NHS, 20.7 mg) in pH=4.0, 0.2 mL), and magnetically stirred at room temperature for 1 h to obtain solution A.
[0058] (2) Dissolve the carrier protein in carbonate buffer (CBS, 0.05M, pH=9.6) to obtain solution B. In this study, bovine serum albumin BSA (67mg, 0.001mM) was selected.
[0059] (3) At room temperature, slowly add solution A dropwise to solution B kept magnetic stirring, and use sodium hydroxide (0.5M) to maintain the pH of the solution between 8.5-9, and keep the magnetic stirrin...
Embodiment 3
[0067] Example 3: Preparation of Metronidazole Hybridoma Cell Line 1A10
[0068] (1) Mice immunization: Select healthy 6-8 week old Balb / C female mice for immunization. BALB / c mice were immunized by subcutaneous injection on the back after mixing and emulsification of complete metronidazole antigen with an equal amount of Freund's adjuvant. Complete Freund's adjuvant was used for the first immunization, and incomplete Freund's adjuvant was used for booster immunizations, with an interval of 21 days between each immunization. Blood was collected 7 days after the third immunization, and ic-ELISA was used to determine the serum titer and inhibition of mice. Mice with high titers and good inhibition were selected. 21 days after the fifth immunization, the immunization was performed by intraperitoneal injection, and the immune dose was required. Halved and without any adjuvants.
[0069] (2) Cell fusion and subcloning: The mouse spleen cells and mouse myeloma cells were fused by ...
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