Preparation And Identification Of Monoclonal Antibodies Againest FSH

FSH is a kind of glycoprotein hormones, it is serected by endocytoplasmic reticulum of the gonadotropic cell in anterior pituitary. Accquiring monoclonal antibodies againest FSH has considerable significance to studying the physiological function of FSH. Conjugating FSH to BSA by EDC transformed FSH into FSH-BSA complex. FSH-BSA complex was used to immune Balb/c mice.Indirect ELISA with FSH in coating the micro-wells was used to determine the antibody valency. SP2/0 myelomas cells were chosen for cell fusion. Keep SP2/0 myelomas cells in the bloom of proliferation before cell fusion. Highly purified polyethylene 4000(PEG 4000) was adopted for cell fusion. The results are as follows:1 Potency of the mice immunzed by FSH-BSA complex all exceeded 1:2.5×104, without empathema.2 Indirect ELISA method is established: HRP-labelled goat anti mouse IgG diluted by PBST (pH 7.4, 0.1 mol/L) in l:1 200 was choosed as secondary antibody, coated antigen 10μg/mL FSH dissolved in carbonate buffer solution (CBS, pH 9.6, 0.05 mol/L), 1.0% dried skim milk as blocking agent, and 1.0% TMB as a substrate, 2 mol/L H2SO4 as stop buffer.3 Small clones emerge seven days after confusion. When cells have paved 80% of the whole wells at twelve days, screen positive cells using indirect ELISA.Then proliferate the positive cells.The efficiency of confluent cells was 36.45%,the rate of positive clone was 17.71%.4 Subclone the positive cells by limiting dilution for three times to get identical cells, we got two positive hybridoma cells named G3F8 and B4A5 with indirect tiers of 1:5×104 and 1:104. The antibody-serecting ability of the two hybridoma cells keep stable after serial subcultivation and resuscitation several times.5 The antibody secreting ability of two hybridoma cells keep stable after frozening and subcloning several times.