Influence of the peptide insolubilization method on detection of anti-peptide antibodies in ELISA. Evaluation of nonspecific interactions.
Different methods of peptide insolubilization in solid phase were compared in ELISA, to verify the influence of the peptide antigen presentation in the interaction with related antibodies. Our studies were performed using as model the peptide fragment 163-171 of human Interleukin 1 beta, and polyclonal or monoclonal anti-peptide antibodies. It was found that the peptide, N-terminally linked to a protein carrier before the adsorption on microtiter wells, interacted with specific polyclonal and monoclonal antibodies with high sensitivity and specificity. In contrast the recognition of similar random conjugates, prepared using a bivalent cross-linking reagent or the peptide covalently linked to poly-L-Lysine-pretreated wells, was hampered generally by very high levels of nonspecific binding. On the other hand, the free peptide adsorbed directly to the solid phase interacted with antibodies with very low sensitivity and specificity. Nonspecific interactions were found in particular between peptides and hyperimmune sera or nonrelated monoclonal antibodies. On the contrary pre-immune sera and normal mouse immunoglobulins never showed significant interactions with any of peptides. This nonspecificity was also overcome when N-terminally linked peptide-protein conjugates were used for the assay.
Presentini
R
R
Sclavo Research Center, Siena, Italy.
Perin
F
F
Ancilli
G
G
Boraschi
D
D
Volpini
G
G
Antoni
G
G
D000818:Animals; D000906:Antibodies / Q000032:analysis*; D000911:Antibodies, Monoclonal; D000941:Antigens; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007375:Interleukin-1 / Q000276:immunology; D053583:Interleukin-1beta; D010446:Peptide Fragments / Q000276:immunology; D010455:Peptides / Q000276:immunology*; D012995:Solubility
D000906:Antibodies; D000911:Antibodies, Monoclonal; D000941:Antigens; D007375:Interleukin-1; D053583:Interleukin-1beta; D010446:Peptide Fragments; D010455:Peptides; C052994:interleukin-1beta (163-171)