Monoclonal antibody to the gamma chain of human fetal hemoglobin used to develop an enzyme immunoassay.
A monoclonal antibody (mAb) that recognizes the gamma chain of human fetal hemoglobin (Hb F) has been produced by cell hybridization techniques. The mAb reacts with Hb F (alpha 2 gamma 2), Hb Bart's (gamma 4), and Hb Kenya (gamma-beta hybrid), but does not cross-react with Hb A (alpha 2 beta 2) or Hb A2 (alpha 2 delta 2). We describe a direct enzyme-linked immunoassay (ELISA) for measurement of Hb F, in which hemoglobins from standards or from unknown hemolysates are covalently bound to the wells of microtiter plates. The antigen is quantified by addition of the gamma-specific mAb, followed by anti-mouse IgG conjugated with horseradish peroxidase, and incubation with the substrate, tetramethylbenzidine. Absorbances at 630 nm are directly proportional to the amount of Hb F present in the standards or samples. Results for Hb F in 53 hemolysates agreed well with values obtained by "high-performance" liquid chromatography, RIA, alkali denaturation, and magnetic affinity immunoassay. This ELISA can detect a 0.5% proportion of Hb F in 1 h and offers distinct advantages over other techniques currently in use.
Moscoso
H
H
Department of Cell and Molecular Biology, Medical College of Georgia, Augusta 30912-2100.
Shyamala
M
M
Kiefer
C R
CR
Garver
F A
FA
D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D002851:Chromatography, High Pressure Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D005319:Fetal Hemoglobin / Q000032:analysis* / Q000276:immunology / Q000592:standards; D006801:Humans; D007118:Immunoassay; D011863:Radioimmunoassay
D000911:Antibodies, Monoclonal; D005319:Fetal Hemoglobin