Generation of a monoclonal antibody specific for Hb G-Philadelphia [alpha 2(68)(E17)Asn----Lys beta 2] and development of an immunoassay.
A murine hybridoma was generated which secreted a monoclonal antibody (Mab) that specifically recognized the alpha 2(68)(E17)Asn----Lys beta 2 substitution of Hb G-Philadelphia. Hybridomas were produced by fusion of RBF/DnJ immune splenic lymphocytes with FOX-NY murine myeloma cells and selected in adenine-aminopterin-thymidine (AAT) medium. Culture fluids were screened by ELISA for antibody reacting with Hb G-Philadelphia but not Hb A. One such culture was cloned by limiting dilution, expanded and injected into pristane-primed, cyclophosphamide-suppressed BALB/c mice for ascites production. An enzyme-linked immunoassay was developed by conjugating hemoglobin in hemolysates or purified hemoglobins to the plastic surface of wells of a microtiter plate. The ascites fluid containing the Hb G-Philadelphia Mab was added to the wells followed by goat anti-mouse IgG conjugated with horseradish peroxidase. After the addition of substrate (tetramethylbenzidine), a deep blue color developed, signifying a positive reaction. We analyzed 58 hemolysates (17 adult, 41 cord) containing a G-variant along with 28 control hemolysates (12 cords comprising FA, FAC, FAS, FSS, FCC phenotypes; 16 adults consisting of AA, AS, SS, SC, S-beta thal, AD-Los Angeles phenotypes). Of the 58 hemolysates containing a G-variant, 53 were positive by ELISA and confirmed by radioimmunoassay (RIA). Four of the five hemolysates negative for Hb G-Philadelphia were shown to be Hb G-Montgomery by RIA. None of the control hemolysates were positive. The assay could be completed in 1 hr and represents a technological advance in hemoglobin identification.
Garver
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Department of Cell and Molecular Biology, Medical College of Georgia, Augusta 30912-3331.
Moscoso
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Swamy
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Kiefer
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D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D000918:Antibody Specificity; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006455:Hemoglobins, Abnormal / Q000032:analysis / Q000276:immunology*; D006801:Humans; D051379:Mice; D011863:Radioimmunoassay
D000911:Antibodies, Monoclonal; D006455:Hemoglobins, Abnormal; C031679:hemoglobin G Philadelphia