Anti-idiotypic antibodies to delineate epitope specificity of anti-amiloride antibodies.
Amiloride and related compounds have found widespread use as cation transport inhibitors. We have previously raised a series of polyclonal anti-amiloride antibodies using different amiloride-protein conjugates as immunogens, where amiloride was coupled to protein either through its guanidino moiety or through its 5-aminopyrazinyl moiety. The anti-amiloride antibodies recognized distinct sites on amiloride, and the site of attachment of amiloride to carrier protein was a critical factor in determining which part of the amiloride molecule was recognized by the anti-amiloride antibody. The specificity of binding of amiloride analogues to these polyclonal anti-amiloride antibodies mimicked the specificity of binding of amiloride analogues to selected isoforms of the epithelial Na+ channel or the Na+/H+ exchanger, suggesting that antigen binding site of these antibodies might be similar in structure to amiloride binding sites on selected Na+ transport proteins. We previously generated monoclonal anti-idiotypic antibodies RA2.4 and RA6.3 by an auto-anti-idiotypic approach, using amiloride coupled to albumin through the guanidinium moiety (amiloride-A1). We have now raised a series of monoclonal anti-idiotypic antibodies, T6, T26, T40, and T181, using amiloride coupled to keyhole limpet hemocyanin through the 5-aminopyrazinyl moiety (amiloride-A5) as an immunogen with the same auto-anti-idiotypic approach. These monoclonal anti-idiotypic antibodies recognized both polyclonal anti-amiloride-A1 and anti-amiloride-A5 antibodies, suggesting that idiotype-anti-idiotype interaction was not epitope restricted.(ABSTRACT TRUNCATED AT 250 WORDS)
Lin
C
C
Department of Medicine, University of Pennsylvania, Philadelphia.
Musch
M
M
Meo
P
P
Zebrowitz
J
J
Chang
E
E
Kleyman
T R
TR
D000584:Amiloride / Q000031:analogs & derivatives / Q000276:immunology* / Q000494:pharmacology; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology; D001692:Biological Transport; D002460:Cell Line; D002645:Chickens; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D007130:Immunoglobulin Idiotypes / Q000276:immunology*; D007421:Intestine, Small / Q000378:metabolism; D008871:Microvilli / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism
D000906:Antibodies; D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D000939:Epitopes; D007130:Immunoglobulin Idiotypes; D017923:Sodium-Hydrogen Exchangers; D000584:Amiloride; D012964:Sodium; C039614:ethylisopropylamiloride