Enzyme Amplification, A Method Applied to Provide An ELISA for Studying Anti-Hib-Ps Antibodies in Children

ABSTRACT

PURPOSE: Enzyme immunoassay (EIA) is now a widely used technique. We have described the application of enzyme amplification (sensitive ELISA) in the field of immunoassays of pediatric population. There are two issues with the sensitive ELISA. First is that one can minimize the serum volume, an important concern for pediatricians. The second is the problem of background signal. We demonstrate that it is possible to develop EIAs of high sensitivity and detectability with using very small volume of infant's sera immunized with Hib-PRP vaccine. METHODS: Monoclonal Abs HG11, HP6016, HK2, and KL1 specific for human IgG1, IgG2, C , and C were used. The mAb OAK-1 specific for a subfamily of V I L chains (V Ia), the mAbs KB13 and B12 specific for human V II and V III L chains were also used respectively. Adults were immunized with Hib-CRM vaccine. Immune serum was obtained 4 to 8 wk after immunization. Twenty infants received Hib-CRM vaccine at 2, 4, and 6 month of age and blood samples were obtained at 7 month old. The amount of anti-Hib-PS Ab expressing a V subgroup or V was determined by sandwich type immunoassays using conventional substrate. The amount of the enzyme immobilized to the well was determined with para-nitrophenyl phosphate substrate. A standard ELISA was performed but different substrate (lyophilized NADPH) and amplifier (alcohol dehydrogenase and diaphorase) were used to develop color in final step for enzyme amplification method. RESULTS: We get the dose-response curves obtained using the conventional and amplified detection methods in the anti-PRP Ab assay. The sensitivities of the two assay methods were compared. We can increase the sensitivities four to sixteen folds and minimize the infant's sera volume to perform varing anti-PRP antibody assays. To obtain the advantages of increased sensitivity, any background is minimized by using noncontaminated reagents. CONCLUSIONS: It is possible to develop EIAs of high sensitivity and detectability with using very small volume of infant's sera with using enzyme amplification system (sensitive ELISA).