Identification and Quantification of Hb C With an Enzyme-Linked Immunosorbent Assay

Abstract

A highly specific enzyme-linked immunosorbent assay (ELISA) was developed for the rapid identification and quantification of hemoglobin C in hemolysates. The procedure involves coating the surface of microtiter wells with Hb C and then addition of monospecific rabbit antibodies that recognize the unique β6 GLU→LYS substitution in Hb C. Next, an antibody to rabbit γ-globulin conjugated with alkaline phosphatase is added, followed by substrate; a yellow color is formed due to the enzymatic hydrolysis of the substrate, which can be measured spectrophotometrically. for quantification purposes, a hemolysate containing Hb C is introduced just prior to the addition of the Hb C antibody. This results in blocking the attachment of the anti-Hb C to the Hb C coated to the plastic surface. Upon addition of anti-rabbit γ-globulin conjugate and substrate, there is a consequent reduction or elimination of color formation. Since the degree of diminution of color formation is dose-dependent, standard curves can be developed for quantification of Hb C in unknowns. of the total hemoglobin, the amounts of Hb C in heterozygotes averaged 27.3 ± 5.7% by ELISA and 25.1 ± 3.9% by radioimmunoassay (RIA). In SC individuals the corresponding values were 30.2 ± 10.1% by ELISA and 24.7 ± 10.9% by RIA. In homozygotes, Hb C values averaged 83.2 ± 4.2% by ELISA and 85.0 ± 6.6% by RIA. Subjects with Hb Cβ⁺ -thalassemia had 66.5 ± 3.7% Hb C as measured by ELISA and 63.5 ± 9.1% as determined by RIA. The ELISA procedure offers distinct advantages for Hb C identification and quantification over other techniques in parameters such as specificity, sensitivity, and rapidity.