Screening of specific serum biomarker of ankylosing spondylitis from a random peptide library

Abstract

Objective To screen the specific serum biomarker of ankylosing spondylitis (AS)from phage random peptide library of 12 amino acids with the serum IgG of AS patients. Methods Phage random peptide library was first immunoscreened with purified IgG from healthy individuals to adsorb non-specific phages, and microtiter wells were coated with purified IgG of AS patients, then the above-treated phages were screened 3 rounds according to the process of adsorption, elution and amplification. Positive clones obtained were selected and detected by phage-ELISA and some of them were sequenced. The binding test of positive clones with the serum sample from 50 AS patients, 30 systemic lupus erythematosus (SLE) patients,30 rheumatoid arthritis (RA) patients, 30 osteoarthritis (OA) as well as 50 healthy individuals were detected using phage-ELISA. The correlations were compared among the absorbance (A) value, erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) in AS patients. Results When serum IgG of AS patients was used as ligand for screening of the phages which were not bound to healthy individuals, the radio of output to input increase from 2.2×10-5% to 1.9×10-2% after 3 rounds screening. At the third round of screening, 20 clones were selected for reacting with antibodies from AS patients. 17 of them were proved to specifically react with the sera of AS patients. The results of sequences indicated that these 7 clones sequenced came from the same one, and the sequence inserted in the coat protein Ⅲ was deduced to be LALPPLAPNHHH named AS1. When AS1 reacted with the serum samples from 50 AS patients and 140 controls by ELISA, the A value was 0.767±0.250 in AS group, 0.491±0.250 in SLE group, 0.445±0.194 in RA group, 0.302±0.113 in OA group, 0.294±0.150 in healthy control group. There were statistical significances of A values among 5 groups (P<0.01 )or between the AS group and the control group (P<0.01). And positive rates of the positive clones in AS group, SLE group, RA group, OA group and healthy control group were 92.0%, 56.7%, 50.0%, 13.3% and 14.0% respectively. There was significant difference (χ2=77.418, P<0.01). Positive phage clone AS1 can be used to diagnose AS patients with 100% sensitivity, specificity of 80%, positive predictive value of 83.3%, and negative predictive value of 100% and accuracy of 90%. The A values of AS1 reaction with the sera of AS patients showed positive correlation with ESR and CRP. The correlation coefficients were 0.165 (P=0.448) and 0.259 (P=0.270). Conclusions The short peptide AS1 screened from the phage random peptide library of 12 amino acids has antigenicity and can react with sera of AS patients. These findings indicate that AS1 could be one of candidate molecules of AS-specific serum markers. Key words: Spondylitis; ankylosing; Peptide library; Immanogiobulin G; Biologicalmarkers; Enzyme-linked immunosorbent assay