| Objective: Quantitative analysis on the influence of the activity of antibody and antigen,and DNA by the treatment of SDS in Western Blot and general reagent of immunohistochemistry pre-treatment Steps,which may cause the loss of experimental information and errors in the antigen and antibody reaction,and try to find alternative reagents.Methods:1.Quantitative analysis of the effects of SDS on serum antigen was detected by double immunodiffusion to observe the precipitating line and determine the antigen titer.2.Quantitative analysis of the effects of SDS on IgA,IgM,IgG was detected by immunoturbidimetry.3.Quantitative analysis of the effects of SDS on HBsAg was detected by electrochemiluminescence immunoassay.4.The effect of SDS treatment on the structure of immunoglobulin antigen was analyzed by immunofixation electrophoresis.5.Quantitative analysis of the impact on particulate antigens of immunohistochemistry pre-treatment steps by agglutination reaction.6.Quantitative analysis of the effect of SDS and ethanol on hepatitis B surface antibody by Enzyme linked immunosorbent assay(ELISA).Effects of 0.1% SDS on typhoid antibody and ovalbumin antibody in different types of patients by ELISA.7.Quantitative analysis of the impact of SDS and ethanol on DNA by Fluorescent quantitative PCR.8.Comparison of the inhibitory effects of N-Dodecyl ?-D-maltoside(DDM)and SDS on antibody and DNA to discuss DDM instead of SDS in protein extraction.Results:1.The results of the double immunodiffusion showed: antigen titer was 1:4 by treatment of SDS at 37?.No precipitation line was found at 100?.The antigen titer was 1:256 in the control group.2.The results of the immunoturbidimetry showed: the levels of IgA,IgM,IgG were slightly increased by treatment of SDS at 37?,however,these levels were decreased at 100?,inhibition rate of IgA and IgG were 90%,IgM was 30%.3.The results of the electrochemiluminescence immunoassay showed: the inhibition rate of HBsAg was 70% by treatment of SDS at 37?,and reached 96% at 100?.4.The results of the immunofixation electrophoresis showed: immunoglobulin antigen structure was destroyed by SDS.5.The results of the agglutination reaction showed: the inhibition rate of antigen was 72.2% by immunohistochemistry pre-treatment steps.Compared with the control group,the results of treating by each reagent alone showed that inhibition rate was about 70% by treatment of more than 75% ethanol,was about 20% by formalin,was about 10% by xylene.6.The results of ELISA showed: The median inhibitory concentration of SDS on HBsAb was 0.92%(3.2×10-2mol/L)and 30% ethanol had no obvious inhibitory effect on HBsAb.Typhoid antibody and ovalbumin antibody were inhibited by 0.1% SDS and the inhibition was no significant difference in different types of patients.7.The results of fluorescent quantitative PCR showed: the Ct values of GAPDH and CRP increased with the increase of SDS and ethanol concentration,and no amplification curve was observed by treatment of 0.05% SDS.8.The median inhibitory concentrations of SDS and DDM on HBsAb were 0.92%(3.2×10-2mol/L)and 1.90%(3.7×10-2mol/L),respectively.The resulting gene could not be amplified by the treatment concentration,SDS was 0.05%(1.7×10-3mol/L),DDM was about 5%(1.0×10-1mol/L).Conclusion:1.SDS and immunohistochemistry pre-treatment steps can inhibit antigen,antibody and DNA,which may result in the loss of information in antigen antibody reaction and the error of the results.2.The inhibition of DDM in DNA is less than SDS and DDM is superior to SDS. |
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