Te. Anthony et Ec. Azmitia, MOLECULAR CHARACTERIZATION OF ANTIPEPTIDE ANTIBODIES AGAINST THE 5-HT1A RECEPTOR - EVIDENCE FOR STATE-DEPENDENT ANTIBODY-BINDING, Molecular brain research, 50(1-2), 1997, pp. 277-284
Differential immunohistochemical labeling is often observed using diff
erent antibodies against the same protein. Two polyclonal antipeptide
antibodies against the 5-HT1A receptor have been generated by our grou
p. The S1A-170 (aa 170-186) and 258 (aa 258-274) are specific for site
s in the second extracellular loop and third intracellular loop, respe
ctively [E.C. Azmtia, I. Yu, H.M. Akbari, N. Kheck, P.M. Whitaker-Azmi
tia and D.R. Marshak, Antipeptide antibodies against the 5-HT1A recept
or, J. Chem. Neuroanat., 5 (1992) 289-298]. Comparison of the labeling
patterns of these two antibodies and other antipeptide antibodies aga
inst the 5-HT1A receptor revealed that although similar populations of
cells were labeled, individual antibodies favor certain staining patt
erns. Immunocytochemistry and western blotting results of transfected
cell lines and brain tissue revealed the following: (1) both the S1A-1
70 and S1A-258 are specific for the 5-HT1A receptor when used for immu
nocytochemistry in transfected HEK-293 and COS-1 cells; (2) when expre
ssed in cultured cell lines, the 5-HT1A receptor is differentially gly
cosylated dependent on cell type, and the S1A-258 is specific for only
certain species on immunoblots; and (3) the S1A-258 and L5B7 [M. Riad
, S. El Mestikawy, D. Derge, H. Gozlan, and M. Hamon, Visualization an
d quantification of central 5-HT1A receptors with specific antibodies,
Neurochem. Int., 4 (1991) 413-423] label common bands at 40 and 70 kD
a on immunoblots of hippocampal proteins, but show opposite staining i
ntensities. These results provide evidence for the immunocytochemical
specificity of both the S1A-170 and S1A-258 and suggest that the discr
epancies noted in immunohistochemistry may be due in part to different
molecular conformations. (C) 1997 Elsevier Science B.V.