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

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 a...