| Prolactin is peptide hormone that secreted by prolactin cells in pituitary In human and animal reproductivitive physiology the main function was to promote the growth of galactophore, launch and maintain lactation milk. In livestock, particularly in sheep production, the high level of prolactin in ewe post partum will inhibit ovarian function, the performance is that there is no follicle development and ovulation in ovulation.and can not hybridizate and pregnant during lactation, so the lamb interval of production was estended. In sheep production, the most effective way of improving the breeding efficiency is to reduce lamb interval as much as possible. The most effective measures to shorten the lamb interval is to reduce the level of prolactin promote and restore ovarian activity on the base of early ablactation and improving nutritional status of ewes and so on. Researchers have been using bromocriptine effectively reduce the level of prolactin, and promote the recovery of ovarian function ewes and heat. Another way of decreasing the level of prolactin rapidly is polyclone antibodies and monoclonal antibodies.We use prolactin and Frund adjuvant as antigen with routine program to immune BALB/C mice,Results of Indirect ELISA showed that antibody titer of serum was between 1:8000 and 1:10 000 after three immunizations on 1 d, 15 d and 29 d respectively. Three days later after fourth strengthened immunization, spleen cells can be used for fusion.In order to select hybridomas that secret positive antibodies, the serum of immunized BALB/C mice was used as posotive control, serum of unimmunized BALB/c mice was used as negetive control, We constituted Indirect ELISA to detect antibody against PRL successfully and the results as follows: concentration of antigen was 100 ng/mL; confining liquid was 1% BSA. Optimal dilution of enzyme enzyme labelled antibody was 1:10000, action time of enzyme labeled antibody was 90 mins at 37℃, and action time of substrate was 30 mins at 37℃. This experiment improved that the method specificity, better repeatability. The method was used to detect serum antibody of immuned mice and hybridoma supernatants.40 % of highly purified polyethylene glycol 4 000(PEG4 000) was used as fusion adjunt,, The SP2/0 myloma cells that were in logarithmic growth phase and immuned spleen cells were fused at room tempreture with 40% PEG4 000. The entire fusion process was controlled in 1 min. pacely. And then use 8 ml medium to dilute PEG. After centrifuged and washed the hybridomas was cultured with HAT medium. Every 3 to 4 days half liquid was changed. In the first three days after the cell fusion, we found the small clone, the hybridoma cells covered the 20%, holes after seven days of cell fusion, with the established Indirect ELISA, We screened the positive holes with the established indirect ELISA and found cloning rate of 69%, positive clone's rate of 23%.Positive cells was clonied with limited dilution after filtering. Monoclonal antibodies were prepared.in the way of ascites. Hybridomas were identified by karyotype analysis. Through cell fusion, We obtained five hybridoma cell lines that secreted positive antibodies and named AA5B1, AB6C8, BB3C1, CA6E4, CE2H6. The antibody titer of cell culture supernatant was between 1:100 and 1:500, antibody titer of ascites was between 1:10 000 and 1:40 000. Atlas of karyotype analysis showed the chromosome number were between 92 and 103 and conformed that the hybridomas were fused by SP2/0 myloma cells and spleen cells.The hybridoma cell lines passaged 16 generations continuously after frozen within five months and still have the ability of secreting antibody against PRL steadily.
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