Generation of a Monoclonal Antibody Specific for Hb G-Philadelphia [α₂68(E17)Asn→Lysβ₂] and Development of an Immunoassay

Abstract

A murine hybridoma was generated which secreted a monoclonal antibody (Mab) that specifically recognized the α₂68(E17)Asn→Lysβ₂ 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 microtitre plate. The ascites fluid containing the Hb G-Philadelphi a Mab was added to the wells followed by goat anti-mouse IgG conjugated with horseradish peroxidase. After the addition of substrate (tetramethylbenzi-dine), 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-βthal, 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.