Localization of sequence-determined neoepitopes and neutrophil digestion fragments of C-reactive protein utilizing monoclonal antibodies and synthetic peptides.
We recently described 17 anti-CRP mAb, seven to native- (or conformational) and 10 to neo- (or sequence-determined) epitopes, including several anti-neo-CRP mAb specific for CRP peptide 199-206. In the present study, four new anti-native- and four new anti-neo-CRP mAb were generated and characterized by ELISA reactivity with native and modified human and rabbit CRP, as well as binding to pronase fragments of human CRP in Western blots. Assays with 17 synthetic CRP peptides identified anti-neo-CRP mAb specific for peptides 1-16, 14-24 and 137-152, respectively. The anti-neo-CRP mAb were reacted with fragments obtained by digesting CRP with multiple additional enzymes, including Staphylococcal V8 protease, trypsin, elastase, plasmin, thrombin and alpha-chymotrypsin. Native CRP was remarkably resistant to enzymic digestion, particularly in the presence of calcium, but was readily cleavable upon denaturation. Twenty-three informative fragments served to further distinguish mAb reactivity with at least four additional neo-CRP epitopes, which presumptively included residues in the regions of amino acids 22-45, 41-61, 114-121 and 130-138, respectively. The eight epitopes identified corresponded well with predicted regions of CRP antigenicity. In addition, at least six distinct native or conformation-determined epitopes were delineated. Reactivity of the anti-neo-CRP mAb with fragments of CRP generated by PMN enzymes indicated that regions sensitive to cleavage by neutrophil enzymes are located at approximately 3, 10 and 16 kD from the amino terminus of the CRP subunit. We expect that the anti-CRP mAb described and mapped herein will be useful tools for the elucidation of CRP structure and function.
Ying
S C
SC
Rush Medical College, Chicago, Illinois.
Shephard
E
E
de Beer
F C
FC
Siegel
J N
JN
Harris
D
D
Gewurz
B E
BE
Fridkin
M
M
Gewurz
H
H
D000911:Antibodies, Monoclonal / Q000276:immunology*; D002097:C-Reactive Protein / Q000032:analysis / Q000276:immunology*; D002118:Calcium / Q000494:pharmacology; D000939:Epitopes / Q000032:analysis*; D006801:Humans; D009504:Neutrophils / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis* / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D002097:C-Reactive Protein; D002118:Calcium