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

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 kno