Identification of new B cell epitopes in the sera of rheumatoid arthritis patients using a random nanopeptide phage library

Abstract

A random nanopeptide phage library was used to screen a pool of immunoglobulin fractions obtained from rheumatoid arthritis (RA) patients. After three rounds of panning, random individual phages were selected by their capacity to react with individual sera from RA patients. By sequencing the inserts corresponding to the peptides displayed on the surface of the phages, we found that phages displaying particular peptides were overrepresented in the selected libraries. The peptides displayed by these phages were: pep1 = Ala-Asp-Gly-Gly-Ala-Gln-Gly-Thr-Ala; pep2 = Pro-Gly-Pro-Ser-Arg-Ala-His-Phe-Leu; pep3 = Leu-Ser-Ser-Arg-Glu-Pro-Gln-Ala-Arg; pep4 = Arg-Leu-Thr-Arg-Glu-Leu-Tyr-Ala-Gln and pep5 = Tyr-Thr-Gln-Lys-His-Gln-Ala. The percentage of sera positive for pep1 was higher in RA patients as compared to the normal adults (p < 0.0004) and the reacting antibody was mainly of IgG isotype. The specificity of binding to the phage displaying pep1 was confirmed by competition experiments using both isolated phages and a synthetic peptide. Interestingly, a mutated phage displaying only Ala-Asp-Gln-Gly-Thr-Ala had no significant reactivity with the sera, indicating that the amino acids (Gly-Gly-Ala) of pep1 are the vital for the binding. Taken together this study demonstrates that it is possible to select specific ligands from a random phage library using sera from RA patients. In addition, this approach could be useful for identifying peptide antigens that might be part of causitive agents in autoimmune diseases.

Novel Phage Display-Based Subtractive Screening To Identify Vaccine Candidates ofBrugia malayi

“…After washing the wells with phosphate buffered saline containing 0.1% Tween 20 (PBST), nonspecific sites were blocked with 5% bovine serum albumin (BSA) for 1 h at 37°C. For iterative screening, 100 l of T7Select library (containing 10 11 PFU/ml) was first added to wells coated with NEN sera and incubated for 1 h at room temperature. Unbound phages were removed from the wells and transferred to wells coated with CP sera.…”

“…Conversely, random peptide phage libraries could also be used to identify linear epitopes or part of a larger antigenic determinant of infectious agents (15) and to identify new B-cell epitopes using serum antibodies that are important in autoimmune diseases (10). Folgori et al (11) used this approach to screen a random peptide library with human sera containing antibodies against hepatitis B virus and identified mimotopes that elicited antigen-specific immune responses in mice.…”

A comparative study of antibody expressions in primary biliary cirrhosis and autoimmune cholangitis using phage display

“…Thus, phage display could provide a means of discerning traces of an earlier etiologic agent that could have initiated an ongoing autoimmune disease. Disease-associated motifs have been sought by phage display in various autoimmune diseases, multiple sclerosis, 41 rheumatoid arthritis, 42 diabetes mellitus, 43 and idiopathic thrombocytopenic purpura. 44 However, these studies have used smaller numbers of patients, or pooled IgG, and although disease-associated motifs have been discerned, in no instance so far has the motif identified a response to a cryptic agent.…”

Specific and Randomly Derived Immunoactive Peptide Mimotopes of Mycobacterial Antigens

“…This is particularly important, since many antigenic determinants may not exist as discrete entities and could be "conformational" in nature; i.e., a certain pattern rather than a definite structure could play the role of an antigenic determinant or immunomodulator. In some cases, such as cancer, rheumatoid arthritis and AIDS, in which the precise antigen or immunogenic epitopes were not known, differential phage display library screenings have been done with sera from patients and healthy individuals (15,33,46).…”