| ELISA is becoming an optimal method that could determine the quantum of organic compound in organism because of its speediness, specificity and sensitivity. The purpose of our research was to establish a set of complete assay determined quantum of metallothionein in swine' s body.1. the polyclonal antiserum in mice of MT which was distilled from swine was prepared : After the rarefied MT-II was coupled to carrier protein BSA by means of GD, the protein-hapten conjugates was acted as man-made antigen. Kun Ming mice were injected subcutaneous by the antigen with the carrier protein of BSA. And each group had the lever of MT in antigen were corresponding 8ug/mouse,16ug/mouse,32ug/mouse, 64ug/mouse,128ug/mouse in the first immunity. The quantum of MT in antigen is doubling in the latter boosting immunity in turn. The antigen had been emulsified with Freund' s complete adjuvant in the earlier 3 times and with Freund' s incomplete adjuvant in the latter 3 times. The two batches of mouse were boosted with the same condition except different time between two immunities, one was 10 days, and the other was 2 weeks. After the antiserum titters being determined by the double immunodiffeoion in two dimensions assay , we had these results: The level of MT in antigen was 16-32ug/mouse and the level of MT was doubling in progression, the time between two boosting immunities is 10 days, the impact is fine, which could make the antiserum titers of a majority of mice attach to 1 :16.2.The antibody was purified and identified: The antiserum whose titer attach to 1 I 16 was condensed to get Y-globulin with ammonium sulphate precipitation assay, and then the y -globulin was purified to IgG on a DEAE-Sephadex A50 column. We found when the pH value of washing buffer was 7. 4, the first apex of curve corresponded the solution of purified IgG whose purification was identified with discontinuous on different concentration polyacrylamide electrophoresis. After being conjugated with HRP, the IgG which is the specifically antibody to MT was identified with direct ELISA.3. Establishment of ELISA: There are two assays which were the indirect competitive ELISA and the indirect uncompetitive ELISA, and they were used to determined MT in the sample of swine .the result showed: the result on determined with the indirect uncompetitive ELTSA were erratic, and the samesample of OD value is much discrepant, and this assay is fit for determining the nature of MT. However, the result on MT which was determined by the indirect competitive ELISA were steady and this assay is fit to determine the quantum of MT, and it is comparntively a fine method. Using the method of chessboard titration chose the proper reaction for the indirect competitive ELISA: the carrier antigen of concentration is 6. 25ug/ml; the first antibody (mouse-antiswine) of suitable concentration is 50ug/ml; and the immunoglobulin-HRP(goat-antimouse) of diluent concentration is 1/8000;the reaction of time is 3hr. After liner regression analysis, a linear equation : y=105x2(22.32880-0.39583x), coefficient of correlation: R=0. 9592, and the sensitivity of assay to detect the rabbit MT is 4. Ing/ml. Intra-assay coefficient of variation and Inter-assay coefficient of variation are 9. 9760%-14. 3858%, which showed that this assay has fine stability. It proved that the establishment in our experimentation of indirect competitive ELISA has highly specificity, sensitivity, stability and repetitiveness. And it is fit for determining a big batch samples, and it is an effective method for determining the quantum of MT.
|
PDF Full Text Request