Identification and quantitation of sickle cell hemoglobin with an enzyme-linked immunosorbent assay (ELISA).
The experimental details of ELISA for the identification and quantitation of Hb S are presented; the assay is based upon the passive adsorption of Hb S top a solid phase (polystyrene tubes) and the addition of monospecific rabbit antibodies capable of recognizing the (beta 6 Glu leads to Val) substitution in Hb S. After the addition of alkaline phosphatase-conjugated goat antibody to rabbit IgG and substrate, the yellow color produced by hydrolysis of substrate is measured spectrophotometrically. For the identification and quantitation of Hb S in unknown samples, the hemolysate is added to the Hb S-coated tubes before the addition of antibody to Hb S, thus causing an inhibition of the antigen-antibody reaction as evidenced by an absence or reduction of color formation. With this procedure, there is no cross-reactivity with normal hemoglobins, and the immunoassay has a sensitivity in detecting 50 ng quantities of the abnormal hemoglobin in a 5 microgram hemolysate. The assay can be performed on multiple samples in 1 day and offers many advantages over other techniques currently used for the identification and quantitation of Hb S and other abnormal hemoglobins in the clinical laboratory.
D000755:Anemia, Sickle Cell / Q000175:diagnosis*; D000818:Animals; D000937:Antigen-Antibody Reactions; D004306:Dose-Response Relationship, Immunologic; D004797:Enzyme-Linked Immunosorbent Assay; D006041:Goats; D006441:Hemoglobin A; D006451:Hemoglobin, Sickle; D006801:Humans; D007106:Immune Sera / Q000494:pharmacology; D011817:Rabbits
D006451:Hemoglobin, Sickle; D007106:Immune Sera; D006441:Hemoglobin A