Characterization of nine monoclonal antibodies against the Escherichia coli cyclic AMP receptor protein.
Nine hybridoma clones producing antibodies against the Escherichia coli cAMP receptor protein (CRP) have been isolated. Five of the monoclonal antibodies (Class I) had a much higher affinity for native CRP while the remaining four (Class II) bound equally well to native or denatured CRP. Using native N-terminal CRP cores, it was shown that none of the Class I monoclonal antibodies cross-reacted with the 15,000-Da CRP core, and only two bound to the 18,800-Da CRP core. The positions of the antigenic determinants for the Class II monoclonal antibodies were found by Western blotting analysis to reside in the N-proximal region of CRP. Only one monoclonal antibody strongly inhibited cAMP binding by CRP, and this was accompanied by a consequent strong inhibition of both lac DNA binding and abortive initiation by RNA polymerase. Each of the Class I monoclonal antibodies inhibited abortive initiation, and four of these antibodies also blocked the binding of cAMP X CRP to the lac DNA fragment. One Class I and one Class II monoclonal antibody bound to the cAMP X CRP X DNA complex. Two of the Class II monoclonal antibodies were without apparent effect on any of the assays used.
Li
X M
XM
Krakow
J S
JS
D000818:Animals; D000911:Antibodies, Monoclonal; D003429:Cross Reactions; D000242:Cyclic AMP / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis; D004926:Escherichia coli / Q000032:analysis*; D005260:Female; D051379:Mice; D008970:Molecular Weight; D011953:Receptors, Cyclic AMP / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D011953:Receptors, Cyclic AMP; D004247:DNA; D000242:Cyclic AMP