Development And Primary Application Of Hybridoma Cell Strain Secreting Monoclonal Antibody Against Strepto- Mycin

Streptomycin(SM) was conjugated with bovine serum albumin(BSA), human serum albumin(HSA), ovalbumin(OVA) and keyhole limpet hemocyanin(KLH) separatedly with EDC method to form artificial antigens. Mice inoculated with different immunogens above produced antibodies against streptomycin. The conjugants were identified by UV spectrum method. The rate of conjugation were 22:1 (SM-BSA), 26:1(SM-HSA), 29:1(SM-OVA) and 495:1(SM-KLH). The results of the indirect Enzyme-linked Immunosorbent Assay(ELISA) showed the titers of the first three conjugatants(SM-BSA,SM-HSA and SM-OVA) were above 1:3200. The titers of SM-KLH lineared from 1:400 to 1:12800.Indirect Enzyme-linked Immunosorbent Assay(ELISA) method was developed to detect antibodies against streptomycin. The optimum condition of the ELISA are set as follows: 0.5ug mL~-1 of SM-HSA(streptomycin-human serum albumin) conjugation used as a coated antigen dissolved in carbonate buffer solution(CBS,pH 9.6,0.05mol L~-1), 0.5 % glutin in PBS(pH 7.4,0.0 lmol L~-1) as a blocking agent post-coating, horse radish heroxidase-labeled sheep anti-mouse IgG(HRP-anti IgG) diluted by PBS(pH 7.4,0.lmol L~-1) in 1:1000 as a secondary antibody, and TMB (3,3',5,5'-tetramethyl benzidine dihydrochloride) as a sbustrate. The specificity and repetitiveness of the ELISA was primely proved by the blocking test. The ELISA was conducted to detect the antisera of mouse and presented good results.One clone which can stably secrete the McAbs against streptomycin was obtained after fuse and named cell strain 1B6. The ELISA titers of the McAbs in cell supernatant and ascite were 1:12800 and 1:819200, respectively. The McAb was categorized into IgGl/ k. subtype and the rate of cross reactivity with other antibiotics were below 0.01%. The cell strain 1B6 also can stably secrete the McAbs after twenty-six setial passages and freezing ang recicfing three times in six months.