Abstract: Sandwich ELISAfor the dtection of Rotavirus(RV)as experimental model,the classical immune procedures were changed from double incubation system(DIS)into single incubation system(SIS) in which RVsample(Ag) and HRPlabeled anti-RV antiboty(Ab^*) were added together into microwell coated by anti-RVantibody(Ab).After coincobation,the multilayer complex Ab-Ag-Ab* was formed on the inner surface of microwell.It was illustrated theroically that the enzyme amount in mutilayer complex Ab-Ag-Ab* was linearly related to the concentration of Ag binding site of RVin sample when the amount of Ab and Ab^* were saturated and their affinites to Ag were assumed to be same.Anew method termed chemiluminescence single incubation multilayer immune technique(CL-SIMIT) was established on this porpose,in which HRPcatalyzing luminol-p-iodopenol-H₂O₂ reaction was as its detection system.Experiments showed that CL-SIMITcould acheive 4-fold sensitivity of CL-ELISAand 16-fold sensitivity of ELISA,and higher specificity than CL-ELISAtested with several uncorrelational antigens.Detection of 32 samples with ELISA,CL-ELISA,CL-SIMITshowed that the positive ratios were 12/32,12/32,17/32 respectively,while the results of CL-SIMITaccorded well with the clinical dignosis results(18/32).

Key words: Rotavirus, Enzyme-linked immunosorbent assay, Chemiluminescence, Horseradish peroxidase