Peroxidase/monoclonal antibody conjugates for the study of hemoglobinopathies.
Monoclonal antibodies (mAbs) to normal human hemoglobins (Hbs) A and F and to variant Hbs C and G-Philadelphia were conjugated to horseradish peroxidase (HRP) and used in qualitative or quantitative enzyme-linked immunosorbent assays (ELISAs). Conjugates with output molar HRP/IgG ratios close to 2.0 had higher avidity for the cognate antigens than those with ratios above or below 2.0. The analytical sensitivities of the conjugates ranged from 0.2 to 4 ng of hemolysate containing the target hemoglobin, and it was not related to the input or the output HRP/IgG ratios. The overall imprecision for the qualitative ELISA was below 8%, and the accuracy for the identification of Hbs C and G-Philadelphia was 100% as compared with established methods. Quantitative determinations of HbA based upon direct dose-response curves showed an analytical sensitivity of 1% and an imprecision < or = 11%. The most significant application of the HbA assay was in the differential diagnosis of hemoglobinopathies associated with partial or total suppression of HbA synthesis. Competitive dose-response curves for the HRP/anti-gamma conjugate allowed the quantification of HbF in the clinically significant range of 0.5-10%, with an imprecision < or = 12%. It is concluded that the incorporation of HRP/mAb conjugates into the ELISA technique offers a simpler, more rapid, yet specific alternative for the measurement of hemoglobins.
Moscoso
H
H
Department of Immunology and Microbiology, Medical College of Georgia, Augusta 30912-2400.
Kiefer
C R
CR
Shyamala
M
M
Garver
F A
FA
D000328:Adult; D000911:Antibodies, Monoclonal / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005319:Fetal Hemoglobin / Q000032:analysis; D006441:Hemoglobin A / Q000032:analysis; D006453:Hemoglobinopathies / Q000175:diagnosis; D006455:Hemoglobins, Abnormal / Q000032:analysis* / Q000276:immunology; D006735:Horseradish Peroxidase; D006801:Humans; D007700:Kinetics; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D006455:Hemoglobins, Abnormal; D006441:Hemoglobin A; D005319:Fetal Hemoglobin; D006735:Horseradish Peroxidase