Generation of monoclonal antibodies to human acylphosphatase (muscular isoenzyme) and application in solid-phase immunoassay.
Monoclonal antibodies to human acylphosphatase (muscle isoenzyme) were generated by an improved hybridoma technique. Immunization consisted of four antigen administrations in an overall period of 15 weeks. After cell fusion and repeated subcloning of positive lines, seven monoclonal antibodies with good affinity and specificity were selected. These antibodies were characterized for their affinity constant and immunoreactivity. The latter was determined using peptides generated by CNBr cleavage of the antigen. One of the selected antibodies had an affinity constant such that it could be used to develop a competitive enzyme-linked immunosorbent assay. In our test, the antigen that was coated on the matrix, and the free one, competed for the antibody-horseradish peroxidase conjugate. No cross-reactivity with the erythrocyte iso-enzyme was found, and the test showed a limit in sensitivity of 0.32 ng/ml of antigen. We expect that the enzyme immunoassay could be useful for clinical application.
Degl'Innocenti
D
D
Department of Biochemical Sciences, University of Florence, Italy.
Berti
A
A
Stefani
M
M
Ramponi
G
G
D017766:Acid Anhydride Hydrolases; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D001709:Biotechnology; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007124:Immunoenzyme Techniques; D007527:Isoenzymes / Q000276:immunology; D009132:Muscles / Q000201:enzymology; D010744:Phosphoric Monoester Hydrolases / Q000276:immunology*; D000097024:Acylphosphatase
D017766:Acid Anhydride Hydrolases; D000911:Antibodies, Monoclonal; D007527:Isoenzymes; D010744:Phosphoric Monoester Hydrolases; D000097024:Acylphosphatase