| Chemiluminescent immunoassay (CLIA) is one of the advanced immunoassay of non-radioisotopic immunoassay because of its high sensitivity,wide dynamic range,high accuracy,stable labeled protein,full automation and extensive application field. CLIA using acridinium ester derivatives as chemiluminescent label has advantages of low background,high signal-to-noise ratio,no need of catalyst and simple luminescence system.Different aspects associating the DMAE-NHS-based CLIA were studied in this thesis,including synthesis of acridinium ester (DMAE-NHS),chemiluminescent characteristics of DMAE-NHS,labelling antibody or streptavidin with DMAE-NHS,two-site sandwich chemiluminescent immunoassay (CLIA) for TSH,two-site sandwich chemiluminescent immunoassay involved biotin-streptavidin system(BAS-CLIA) for TSH,and competitive chemiluminescent immunoassay using biotin-streptavidin system(B AS-CLIA) for TLVThe desired acridinium ester,DMAE-NHS,was synthesized according to the reference method with some modifications. The products were identified by IR,NMR,MS and elemental analysis. In our method,K.OH was used in place of NaOH to synthesize benzyl ester of 3,5-dimethyl-4-hydroxybenzoic acid,2',6'-dimethyl-4'-(n-succinimidyloxycarbonyl) phenyl-acridinium-9-carboxylate was purified on a silica gel column with chloroform/ethylacetate(4:l,v/v) as eluent and further purified by triturating with hexane/acetone(2:l,v/v).The luminescence produced by DMAE-NHS is a flash light with maximumemission at 0.4s and decay half-time of 0.9s. The luminescence intensity is 6.11x10 cps/mol,which is affected by the composition of trigger and surfactant. Chemiluminescent characteristics of labeled antibody are similar to the DMAE-NHS. Specific luminescence activity of the labeled antibody is l.SxlO1 cps/mol with an average incorporation ratio of 2.4 mole of DMAE-NHS per mole of antibody. The stability of DMAE-NHS and labeled antibody was tested under various pH values andtemperatures. In aqueous solution,the stability of DMAE-NHS decreases with the increase of pH value and temperature due to its decomposition by hydrolysis. The labeled antibody stored in neutral media is more stable than that in acidic or basic media. There is no significant loss of chemiluminescence and immunological activity of DMAE-NHS and labeled antibody stored in pH 7.0 phosphate buffer at 4C for six months.A two-site sandwich CLIA for TSH and a two-site sandwich BAS-CLIA for TSH were developed. In the two-site sandwich CLIA for TSH,two anti-TSH monoclonal antibodies were involved,one was coated on microwells and another was labeled with acridinium ester (DMAE-NHS). The assay sensitivity is 0.01mlU/L. Intrassay and interassay coefficients of variation are 4.29-6.71 and 4.36-6.14%,respectively. Average recovery is 98%. Correlation coefficients and correlation equations of the assay with TSH IRMA and Ciba Corning are 0.993,0.986,Y=0.20 + 0.92X and Y=-0.16+0.92X,respectively. The two-site sandwich BAS-CLIA for TSH employs the same two monoclonal antibodies. Differently,one of the monoclonal antibodies was biotinylated instead of being labeled directly by DMAE-NHS,and DMAE-NHS labeled streptavidin was used as tracer. The assay sensitivity of the two-site sandwich BAS-CLIA for TSH was higher than the former.A competitive CLIA for serum TTj was also developed. In this assay,the biotinylated T4-BSA conjugate competes with the T4 in the serum to bind the limited amount of anti-T4 McAb. The amount of the McAb-Ti-BSA-biotin complex was determined after a reaction with DMAE-NHS labeled streptavidin.In conclusion,the DMAE-NHS has many advantageous performances in CLIA. The assay methods established for TSH and TT4 are efficient and reliable. |
PDF Full Text Request