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 integrity 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.
Leder
L
L
Biochemisches Institut, Universität, Zürich, Switzerland.
Wendt
H
H
Schwab
C
C
Jelesarov
I
I
Bornhauser
S
S
Ackermann
F
F
Bosshard
H R
HR
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001667:Binding, Competitive; D003429:Cross Reactions; D003574:Cytochrome c Group / Q000737:chemistry / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D005189:False Positive Reactions; D005287:Ferredoxin-NADP Reductase / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology*; D006736:Horses; D007106:Immune Sera; D016350:Leucine Zippers; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis / Q000737:chemistry / Q000276:immunology*; D011487:Protein Conformation; D011506:Proteins / Q000032:analysis / Q000737:chemistry / Q000276:immunology*; D012680:Sensitivity and Specificity; D013329:Structure-Activity Relationship
D000906:Antibodies; D003574:Cytochrome c Group; D007106:Immune Sera; D010455:Peptides; D011506:Proteins; D005287:Ferredoxin-NADP Reductase