Hybridoma cell strain secreting phosphate bis (1, 3-dichloro-2-propyl) ester monoclonal antibody, monoclonal antibody and application
A hybridoma cell line and monoclonal antibody technology, applied in the field of detection, can solve the problems of high cost, cumbersome and time-consuming operation, and inability to be widely used
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Embodiment 1
[0028] Example 1 Hapten Conjugation
[0029] Bis(1,3-dichloro-2-propyl) phosphate protein conjugates, that is, bis(1,3-dichloro-2-propyl) phosphate and bovine serum albumin (BSA) are chemically coupled in The combined antigen (abbreviated as B-BSA) was coupled by sodium periodate method.
[0030] first day:
[0031] (1) Weigh 5 mg of BDCIPP and dissolve it in 1 mL of distilled water to obtain a BDCIPP solution.
[0032] (2) Add 0.2 mL of newly prepared 0.1M (mol / L) NaIO to the above BDCIPP solution 4 Aqueous solution, stirred at room temperature for 20 minutes in the dark.
[0033] (3) Put the solution in step (2) into a dialysis bag, and dialyze against 1 mM (mmol / L) sodium acetate buffer solution with pH 4.4, overnight at 4° C. to obtain a hydroformylated BDCIPP solution.
[0034] the next day:
[0035] (4) Add 20 μL of 0.2M pH9.5 carbonate buffer to increase the pH of the formaldehyde-BDCIPP solution in step (3) to 9.0-9.5, then immediately add 10 mg of BSA, and gently...
Embodiment 2
[0042] Embodiment 2 animal immunization
[0043] The experimental animals were female 6-week-old mice of Balb / c strain. Immunization process: B-BSA was mixed with Freund's complete adjuvant, emulsified, and subcutaneously injected into multiple points for immunization. The dosage of B-BSA is 0.1mg / time / only. Complete Freund's adjuvant was used for the first immunization, and incomplete Freund's adjuvant was used for the second immunization. The interval between each immunization was 3 weeks, a total of 3 immunizations. After the third immunization, recall stimulation of the mice before cell fusion was performed, and 0.1 mg of antigen B-BSA was dissolved in 0.5 mL of PBS buffer and injected intraperitoneally. Three days after recall stimulation, cell fusion and hybridoma construction were performed.
Embodiment 3
[0044] Embodiment 3 hybridoma cell line construction
[0045] Prepare feeder cell suspension one day before cell fusion: one mouse can obtain 5×10 6 pcs - 8×10 6 peritoneal macrophages, if mouse thymocytes are used as feeder cells, the cell concentration is 5×10 6 cells / mL, mouse splenocytes are 1×10 6 cells / mL, mouse fibroblasts (3T3) 1×10 5 cells / mL, both 100 μL / well.
[0046] (1) Take the logarithmic growth of mouse myeloma cells SP2 / 0, centrifuge at 1000rpm for 5 minutes, discard the supernatant, suspend the cells with incomplete culture medium (RPMI1640) and count them, get the required number of cells, use incomplete The culture medium (RPMI1640) was washed twice.
[0047] (2) Simultaneously prepare immune splenocyte suspension and wash twice with incomplete culture medium.
[0048] (3) Mix mouse myeloma cells and splenocytes at a ratio of 1:10 or 1:5, and wash once with incomplete culture medium in a 50mL plastic centrifuge tube at 1200rpm for 8 minutes. Discard ...
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