| Objective:To optimize the testing conditions of protein microarray technology of detecting?-Lg and Lf in milk simultaneously,establish the corresponding detection method and evaluate the application of this method.Methods:Experimental researches were conducted to select the mouse monoclonal antibody,optimize the point probe,choose the concentration of point probe and detection antibody,determine consistency of the spot,choose the blocking solution,determine the biometric limits and detection limits,establish the S-type curve and judge the linear range,establish the standard curve and regression equation via the double antibody sandwich method of protein chip to detect the antigen for the selected antibody and antigen of ?-Lg and Lf respectively.The testing conditions of protein chip technology to detect ?-Lg and Lf simultaneously were optimized and determined.On the basis of the conformed experimental conditions,the accuracy,the precision,the methodological control experiment and the detection time limitation research were carried out to estimate the protein microarray method for quantitatively combined measurement of bovine milk ?-Lg and Lf.results:The results of mouse monoclonal antibody selecting experiment showed that the selected ?-lactoglobulin antigen,lactoferrin antigen can be combined with the corresponding antibodys on the chip,While the ?-Lg mouse monoclonal antibody 66#and Lf mouse monoclonal antibody 75#had the best specificity.the point probe optimization experiment showed that the results of the mouse monoclonal antibody of?-lactoglobulin and lactoferrin as the fixed probes were better than the results of rabbit polyclonal antibody as the fixed probes.The fixed concentration of ?-Lg mouse monoclonal antibody 66#was 0.5 mg/mL,and the titer of ?-Lg rabbit polyclonal antibody was 1:2000;The fixed concentration of lactoferrin monoclonal antibody 75#was 0.5 mg/mL,and the titer of lactoferrin was 1:2000.Spot consistency experiment showed that:To ensure the ideal spotting effect of ?-Lg monoclonal antibody 66#and Lf mouse monoclonal antibody 75#at the same time,the spotting pin must be pre-43 points and the maximum number of single sampling after pre-pointing should not exceed 44 points.1%WB protein-free blocking solution was selected as the blocking agent.When the ?-lactoglobulin and the lactoferrin were detected by chip simultaneously,the biometric detection limit and lower detection limit of ?-Lg was 33.52 ng/mL and 5.41 ng/mL,3.60 ng/mL and 0.96 ng/mL of Lf.?-Lg antigen concentration in the range of 134.06-4290.00 ng/mL and Lf antigen concentration in the range of 14.40-460.75 ng/mL had linear relationship with the detection signal value,and five points were chosen in the linear range to establish a standard curve for simultaneous detection of two proteins.The dilution recovery of ?-Lg was between 50%and 112.7%,and the recovery ranged from 86%to 107%.The dilution recovery of Lf was between 106.5%and 144.8%,and the recovery ranged from 83.7%to 128.5%.There was no statistical difference between the results of ?-Lg and Lf by protein chip method and ELISA.The correlation coefficients of two methods detecting ?-Lg and Lf were 0.743 and 0.773,respectively,and the correlation coefficients were statistically significant.The within-lot coefficient of variation of ?-Lg was in the range of 7.6%to 11.6%,while the between-lot coefficient of variation was in the range of 14.9%to 33.5%.The within-lot coefficient of variation of Lf ranged from 4.9%to 14.2%,while the between-lot coefficient of variation ranged from 24.8%to 38.3%.Within 10 days after the completion of the antigen-antibody reaction,there were no statistically statistical differences among the sample concentrations by protein chip technology.Conclusion:The protein chip detection conditions for P-lactoglobulin and lactoferrin in milk were optimized,and a protein chip technique for simultaneous detecting of milk?-lactoglobulin and lactoferrin was established. |
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