Preparation And Preliminary Application Research Of Monclonal Antibody Against Streptomycin

Streptomycin(SM),a type of important aminoglycoside antibolitic,is widely used in treatment and prevention of animal diseases Since it is of stable quality, broad-spetrum,simple maching process and good curative effect special for tubercle bacillus.But,its acute side-effect in ear and kidney has brought people's attention to SM residue in animal food.In oder to find a quick,high efficient, sensitive and specific means to detection of SM residue,we prepared monoclonal antibody against SM and developed the CiELSA method .SM and carrier proteins (BSA and OVA) interacted for 12 hours in 37℃ pH9.6 carbonate buffer in order to condensate aldehyde group of SM and amino group of carrier proteins.The reaction product was dialysed for 3 days by pH 7.4 0.01mol/l PBS and identified by UV scan and maltol reaction.The results showed that SM and carrier proteins condensate successfully .the protein content of immunogen(SM-BSA) and coating antigen(OVA-BSA) is 1.651mg/ml and 0.540mg/ml respectively.The BALB/C mice were immunized by SM-BSA and were selected by indirect ELISA to detect the antiserum.Utilized cell's integration technology,merged the spleen cell of selected mouse and SP2/0 bone myeloma cell by 50%-PEG-1500 ,adopted indirect ELISA and CiELISA technology,utilized the clone technology of limit dilution to clone and screen through 3 times,screened 3 kinds of hybridization tumour cell that can secrete the monlclonal antibody steadily.Though identification of supernatant fluid from cell culture, the hypotype of the monoclonal antibody were lgG2b.For obataining a quantity of antibody,we injected hybriddoma cells named 3C₁₂ into mice to produce ascites which was purified.Then protein content,titer and purity of purified ascites were measured by UV scan, indirect ELISA and SDS-PAGE .The results showed that protein content was 1.451 mg/ml,that titer was 1:51200 and that purity was high.Applying purified monoclonal preliminarily to CiELISA ,we developed CiELISA and determined working contents of coating antigen and monoclonal antibody which were 5ug/ml and 1:8000 ,and optimized reaction conditions.Throgh analysis of CiELISA ,we drawed that the standard curve equation was y=81-20.41x ;LOD is 1.0ng/ml; IC₅₀ was 33.03ng/ml ;coefficient of association...