Radiolabeling of the biologically active peptide 163-171 of human interleukin-1 beta.

N-succinimidyl[2,3-3H]propionate was used for the radiolabeling of the biologically active peptide fragment 163-171 of human interleukin-1 beta (VQGEESNDK). Suitable reaction conditions were studied to obtain useful labeling of the molecule. A mixture of mono- (70%) and bi- (30%) propionyl derivatives was obtained with a total 3H specific activity of 87 Ci/mmol of peptide. The conditions for an efficient chromatographic separation of labeled peptide from unreacted reagents and by-products were established. The labeled peptide maintained the same biological activity as that of the corresponding unlabeled molecule, indicating that the labeling procedure did not alter the biological characteristics of the peptide. This thus allows the use of the radiolabeled peptide for receptor binding studies.

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.

Studies of the antigenic structure of two cross-reacting proteins, pertussis and cholera toxins, using synthetic peptides.

Peptide fragments of pertussis toxin subunit 1 (PT-S1) have been synthesized in order to investigate their antigenic and immunogenic activity, and to evaluate their possible use as components of a new vaccine. Two peptides (sequence 73-82, EAERAGRGTG and sequence 107-116, YVDTYGDNAG) were selected for their predictable exposure on the surface of the molecule, and a third (8-18, YRYDSRPPEDV) for its homology with the sequence 6-16 of cholera toxin subunit A (CT-A 6-16) (YRADSRPPDEI). Antipeptide polyclonal antibodies produced in rabbits, were tested in different immunoassays for their ability to interact with toxin proteins. All of them proved interactive with recombinant PT-S1 (rPT-S1); CT-A interact not only, as expected, with anti 8-18 antibodies, due to the high homology between the two toxins in this region, but also, unexpectedly, with anti 107-116 antibodies, in spite of the lack of homology of this peptide with the entire CT. We also found a direct cross-reactivity between the two toxins: anti PT and anti rPT-S1 antibodies interacted with CT-A, whereas anti CT antibodies did not recognize PT. Antipertussis antibodies also recognized the peptide 8-18, which therefore represents at least a part of an antigenic determinant of the toxin, while no interaction could be evidenced between anti-cholera antibodies and any of the peptides, thus demonstrating important differences in the antigenic structures of the two toxins. None of the antipeptide antibodies examined showed protective activity against the toxins in a Chinese hamster ovary (CHO) cell test.