Generation of a monoclonal antibody specific for Hb G-Philadelphia [alpha 2(68)(E17)Asn----Lys beta 2] and development of an immunoassay.

Abstract

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.

• Authors: F A Garver, H Moscoso, S Swamy, C R Kiefer
• Journal: Hemoglobin (Hemoglobin) Vol. 12 Issue 2 Pg. 125-36 ( 1988) ISSN: 0363-0269 [Print] England
• PMID: 3384705 (Publication Type: Journal Article, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Antibodies, Monoclonal
• Hemoglobins, Abnormal
• hemoglobin G Philadelphia

Topics

• Animals
• Antibodies, Monoclonal (biosynthesis, immunology)
• Antibody Specificity
• Enzyme-Linked Immunosorbent Assay (methods)
• Hemoglobins, Abnormal (analysis, immunology)
• Humans
• Mice
• Radioimmunoassay