Study On Immunoaffinity Column Test Penicillinse In Milk

Penicillinse is a kind of beta-lactan enzyme that catalyze penicillin beta-in acid and produce penicilloic acid.At present,in majority market of products enterprise in milk increase Penicillinse,achieved the degeneration penicillin class goal,reduced the penicillin class to remain the examination to limit.But the penicillinse stored up in the human body is harm and is forbids the use of food additive.Therefore,the fast recognition research to milk is imperative.The research selects Penicillinse TEM-1 as the factor , immunes health experimental rex rabbit,prepares specificity antiserum which aim to Penicillinse,uses the indirect ELISA law to appraise the preparation specificity antiserum,and purify the specificity antiserum,uses the highest price specificity antiserum to preparation immunoafinity chromatography , establishes the fast recognition method to penicillinse in milk.The concrete contents and results are as follows:1.Use penicillinse TEM-1 as the antigen,according to the routine immunization method immunity health experimantel rex rabbit,prepares specificity antiserum which aim to Penicillinse,Simultaneously establishes the indirect ELISA method,in which the work density of the covering antigen is 10μg/ml,the work density of negative serum is 1:2000 time of dilution,the best dilution multiples of HRP-the goat anti-rabbit IgG is 1:2000,the best covering condition of antigen is overnight at 4℃,the best reaction time of serum is one hour at 37℃,the best reaction time of他the enzymed 2nd antibody is one hour at 37℃,the best time of the substrate function is 20min.The most highly effective price of the specificity antiserum aim to penicillinse is 1:6.4×10~4.2.Raise specificity antiserum aims to penicillinse with the saturated ammonlum sulfate,then further purify IgG with DEAE-sephadex A50,after use 10% PAGE-SDS electrophoresis appraisal for goal protein.Simultaneously couple purified IgG and CNBr-Sepharose 4B , obtain the coupling rate is 92% , continue to prepare immunoafinity chromatography.3.Establish capillary vessel electrophoresis examination method to examin penicillin,protract the standard curve and the linear equation is y=2698x-2330.2,R2=0.9991.The lowest examination limits is 2μg/mL.Establish penicillin and penicillinse correspondence relations standard curve , the linear equation is y=-1071.1x+1126.1,R~2=0.9699.Obtain immunoafinity chromatography column dynamic capacity is 2041ng/mg , certainly the colum paired capacity is 410.98μg/mL.Separately increase 5mL 200μg/mL,300μg/mL and 400μg/mL penicillinse standard driving fluid,elute with 5mL methyl alcohol,examined the highly effective capillary vessel electrophoresis,determine continuously 6 times,obtain its return-ratio is 91%,93.33% and 95.5%,the coefficient of variation is 5.12%,3.31% and 3.31%.Simultaneously obtain the penicillinse density in the milk is 0.378μg/mL.The results above show:The research was successfully prepared penicillinse specificity serum , established penicillinse specific resistance ELISA detection method.Successfully prepared specific absorption of penicillinse immune affinity chromatography colum , and analyzed affinity chromatography colum efficiency.Successfully established method of penicillinse by high performance capillary electrophoresis.Milk sample processed by affinity chromatography colum,detected by high performance capillary electrophoresis.The result is good.