An appraisal of polystyrene-(ELISA) and nitrocellulose-based (ELIFA) enzyme immunoassay systems using monoclonal antibodies reactive toward antigenically distinct forms of human C-reactive protein.
The purpose of this study was to compare and contrast two enzyme immunoassay systems: the enzyme-linked immunosorbent assay (ELISA), which utilizes polystyrene microtiter plates as the adsorptive surface and the enzyme-linked immunoflow assay (ELIFA), which utilizes nitrocellulose membranes. The principal parameter under scrutiny was the denaturing or unfolding effects caused by the interaction of the protein with the adsorptive surfaces in each assay system. These effects were monitored by utilizing two conformationally distinct forms of human C-reactive protein (CRP), the native form of CRP and a denatured form (M-CRP), with a corresponding panel of monoclonal antibodies (MAbs) specific to either CRP or M-CRP. The results show that the ELIFA system was less sensitive than the ELISA system but that the ELIFA assay can be completed in less time than the ELISA. Also, adsorption of native CRP to the polystyrene surface in the ELISA system resulted in conformational changes of the adsorbed native CRP protein such that M-CRP reactive determinants were available for binding with anti-M-CRP MAbs, whereas native CRP adsorbed to the nitrocellulose membrane in the ELIFA system resulted in very limited conversion of CRP to M-CRP reactive epitopes. These results have important implications for development of immunoassays and screening of MAbs for proteins whose conformations may be affected by adsorption to various surfaces.
Shields
M J
MJ
Department of Cell, Molecular and Structural Biology, Northwestern University Medical School, Chicago, IL 60611.
Siegel
J N
JN
Clark
C R
CR
Hines
K K
KK
Potempa
L A
LA
Gewurz
H
H
Anderson
B
B
D000911:Antibodies, Monoclonal; D002097:C-Reactive Protein / Q000032:analysis* / Q000276:immunology; D002118:Calcium / Q000494:pharmacology; D003101:Collodion; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D011137:Polystyrenes; D011487:Protein Conformation; D011489:Protein Denaturation
D000911:Antibodies, Monoclonal; D011137:Polystyrenes; D003101:Collodion; D002097:C-Reactive Protein; D002118:Calcium