McAb And PcAb Preparation Of PMetMbaseA And Evaluation Of Its Sandwich-ELISA For Meat Color

By animal immunity and monocloning technology,pMetMbaseA was used to prepare its high specific monoclonal antibodies(McAb)on mice and polyclonal antibodies(PcAb)on rabbits.Based on the matrix double antibodies,one sandwich-ELISA for measuring pork pMetMbaseA level was established to evaluate and improve the identification of pork quality.1 Preparation of pMetMbaseA-McAb and pMetMbaseA-PcAbAdult male BALB/c mice and female New Zealand rabbits were selected to be immunized with purified pMetMbaseA preparation,respectively.3A5 and 4F7(two strains of hybirdoma cells)were achieved with monocloning technique.As their titers reaching over 1×105,the ascites were collected and purified as corresponding McAbs.The rabbit antiserum titers reached 2×105,concentrated and purified as the polyclonal antibodies(PcAb).2 Choice of pMetMbaseA-sandwich ELISA for pork color3A5·McAb and 4F7·McAb were individually crossed with PcAb and four kinds of sandwich-ELISA models were established.Compared with the experimental determination,the appropriate conditions of model 2 were 2.00μg/mL 3A5 McAb/4℃overnight,1%casein/37℃/1h,4.00μg/mL pMetMbaseA/37℃/1.5h,2μg/mL PcAb/37℃/1.5h,1:4000(V:V)2nd Ab/37℃/1h.The total recovery rate was 101.08%(P>0.05)and the detection base line was 15.1 ng/mL;and the appropriate conditions of model 4 were 2.00μg/mL 4F7·McAb/3h/37℃,1%casein/37℃/1.5h,4.00μg/mL pMetMbaseA/37℃/1h,2μg/mL PcAb/37℃/1h,1:4000(V:V)2nd Ab/37℃/0.75h:the total recovery rate was 102.22%(P>0.05)and the detection base line was 30.9ng/mL.Altogether,the pork monitoring effect of the mode 2 was the best.3 Evaluation of detection effect of pMetMbaseA sandwich-ELIS A for pork colorMeasurement of the pork color sensory as references,this ELISA method was used to evaluate experimental effect of pork color.Research showed that the measuring levels of color sensory evaluation could be determined the rough differences in 4℃ storage only about 5d-7d.While the pMetMbaseA sandwich-ELISA could identify the pork’ freshness in 24h accurately;and greatly improved the level of pork color measurement.It was also found that the content of pMetMbaseA significantly declined 2.00-6.00ng/mg less than 24h in 4℃(P<0.05).The ELISA technique can accurately identify the pork quality during different kept periods in frozen conditions,-20℃ or-70℃(P<0.05).The content of pMetMbaseA could be more accurately distinguished pork quality.The discriminative power of the sandwich-ELISA was significantly higher than that of sensory evaluation and instrument method.Therefore,the pMetMbaseA sandwich-ELISA established in this paper was as one evaluation method for pork color with high sensitivity,accuracy and specificity.