Genuine and apparent cross‐reaction of polyclonal antibodies to proteins and peptides

Abstract

Antiserum to a native protein may cross‐react with the corresponding denatured protein or with peptides. The cross‐reaction is either a genuine property of the antibodies or caused by antibodies produced against some unfolded protein contaminating the native protein used for immunization. Appropriate conformation‐sensitive immunoassays must be employed to distinguish a genuine from an apparent cross‐reaction. In the present study, we have analyzed critically the cross‐reaction of rabbit antisera against proteins and peptides. We have distinguished between genuine and apparent cross‐reaction with the help of the protein A antibody‐capture ELISA, a new conformation‐sensitive ELISA format. Three systems were analyzed: cross‐reaction of antisera to native yeast and horse cytochrome c with unfolded apo‐cytochrome c; cross‐reaction of antisera to a coiled‐coil leucine‐zipper peptide with a homologous random‐coil peptide obtained by introducing two proline residues into the leucine‐zipper sequence; cross‐reaction of antisera to two peptides that correspond to the N‐terminal and an internal sequence of ferredoxin:NADP+ reductase (FNR), with the native enzyme. The reaction of the anti‐(cytochrome c) sera was clearly due to antibodies produced against unfolded protein, it was an apparent and not a genuine cross‐reaction. Furthermore, the apparently cross‐reactive antibodies to horse cytochrome c did not discriminate against sequence‐related proteins from dog, beef, rabbit and pigeon. In contrast, antibodies to the leucine‐zipper peptide did cross‐react in a genuine way with the homologous random‐coil peptide, that is, the cross‐reactive antibodies do not seem to have been produced against the unfolded form of the leucine‐zipper peptide. Of the two anti‐peptide sera the one against the unstructured and highly accessible N‐terminal segment reacted strongly with the native protein. The second serum against a solvent‐accessible turn‐like sequence of FNR showed apparent cross‐reactivity: antibodies recognizing the native protein were directed against a minor conformational isoform of the free peptide and did not react with the principal form(s) of the free peptide. The generation of cross‐reactive antibodies depends on the conformational stability and intergrity of the immunogen and on the molecular form of its application, i.e., free, polymerized or carrier‐bound. The results clarify the different nature of cross‐reactivity of antisera to proteins and peptides. This knowledge is crucial if antisera are to be used as conformation‐specific probes.

Real‐Time Monitoring of Antigen‐antibody Recognition on a Metal Oxide Surface by an Optical Grating Coupler Sensor

“…The peptide forms a very stable dimeric leucine zipper and was synthesized as described (Leder et al, 1994). mAb 15AF (subclass IgG,) against this peptide was obtained by the standard hybridoma technology (Leder, L. and Bosshard, H. R., unpublished).…”

Immunoinformatics may lead to a reappraisal of the nature of B cell epitopes and of the feasibility of synthetic peptide vaccines

“…3,38,39 For many of these antigens, monoclonal antibodies (Mabs) specific for the native molecule are nowadays available. By using these Mabs together with biosensors based on surface plasmon resonance, a method which avoids some of the pitfalls of the ELISA technique, [40][41][42][43] it should be possible to establish by inhibition assays if the continuous epitopes in the database actually correspond to epitopes of the native antigen that is binding to the Mab.…”

A Structural-Energetic Basis for B-Cell Epitope Prediction

“…Sequences of hexapeptides known to bind antibody raised against MHr [30] are plotted in (B) as dots at the middle positions of their sequences. Apparent crossreactions of antipeptide antibody with protein and antiprotein antibody with peptide can therefore result from protein denaturation [35], but these processes are indistinguishable from the reaction of antipeptide antibody with peptide in terms of epitope and paratope structure. The treatment of epitopes as extended chains in unfolded peptides or surface patches in folded proteins is consistent with the observation that paratopes tend to be groove-like in antipeptide antibody but more planar in antiprotein antibody [29].…”

“…of using peptide immunogens for active immunization or the production of antibody for either passive immunization or antigen detection [33], while antiprotein antibody crossreactive with peptide is the analyte of interest where peptides are used as diagnostic reagents for antibody detection [1]. Genuine crossreaction of antipeptide antibody with protein requires a sequence that elicits antibody production as part of an unfolded peptide and retains its antigenicity even when embedded in a folded protein structure [35], whereas genuine cross-reaction of antiprotein antibody with peptide requires a sequence that occurs as part of a folded protein epitope and yet can bind antibody raised against the intact epitope even as an unfolded peptide fragment thereof [29,35]. In this sense, epitopes of peptides are similar to those of unfolded protein in that their antigenicity depends only on their sequence, as opposed to those of folded protein for which native conformation is essential [34].…”