Method Description

Microwells are precoated with the Haemophilus influenzae type B (HIB) capsular polysaccharide antigen conjugated to human serum albumin. The calibrators, controls, and diluted patient specimens are added to the wells and antibodies recognizing the HIB antigen bind during the first incubation. After washing the wells to remove all unbound proteins, purified peroxidase-labeled rabbit antihuman IgG (gamma chain specific) conjugate is added. The conjugate binds to the captured human antibody and the excess unbound conjugate is removed by a further wash step. The bound conjugate is visualized with 3,3',5,5' tetramethylbenzidine (TMB) substrate, which gives a blue reaction product, the intensity of which is proportional to the concentration of antibody in the specimen. Phosphoric acid is added to each well to stop the reaction. This produces a yellow end point color, which is read at 450 nm.(Madore DV, Anderson P, Baxter BD, et al. Interlaboratory study evaluating quantitation of antibodies to Haemophilus influenzae type B polysaccharide by enzyme-linked immunosorbent assay. Clin Diagn Lab Immunol. 1996;3[1]:84-88. doi: 10.1128/cdli.3.1.84-88.1996.; Bennett JE, Dolin R, Blaser MJ, eds. Mandell, Douglas, and Bennett's Principles and Practice of Infectious Diseases. 9th ed. Elsevier; 2020)