Screening Of Specific Antibody Binding Peptides And Applied Research In Serology Detection Of Mycobacterium Tuberculosis

Objective To screen the specific antibody binding peptides of tuberculosis from phage-displayed random peptide libraries with purified IgG of tuberculosis serum, provide the basis for the development of serological detection reagent of tuberculosis.Methods strategy 1: Purified IgG mixture of 12 tuberculosis patients'serum was used as solid ligand to screen the binding peptide from the Ph.D.-7 peptide library, according to the biopanning process of absorption, elution and amplification. Purified IgG mixture of 12 health people serum was used as the molecule of counter selection during the second and third selection. Phages were enriched after 3 rounds of screening, then 20 single phages separately eluted by IgG of tuberculosis and health people were randomly selected on each direction of the determination plates and amplified. The single chains DNA were extracted as template for sequencing. Strategy 2: Purified IgG of health people serum was used as the molecule of counter selection through the selection, then use the purified IgG of tuberculosis to screenthe binding peptide. And the positive clone was identified. The combination abilities of selected clones to IgG of tuberculosis and health people were tested by indirect enzyme linked immunosorbent assay (ELISA), Serum samples, from 47 patients with tuberculosis and 37 health people vaccinated with BCG, were verified by positive phage clones using phage-ELISA. The verifying results of 24 serum samples used for panning were separately statistic. Establish the indirect ELISA system after the peptides were synthesized in vitro, then diagnose 147 tuberculosis serum and control serum(22 other pulmonary disease serum and 135 health serum).Results After the panning, remarkable enrichment of phages that can specifically bind with target molecules were observed. Single phages were randomly selected for sequencing analysis and 20 sequences were obtained. 20 phage clones with different sequences were amplified and detected with indirect ELISA and single phage H(12), T6, T15, T18 showed higher affinity with IgG of tuberculosis(S/N≥2.1)and was identified as the positive clone. It was found that, in indirect ELISA with each single phage, difference of the results of 47 tuberculosis patients serum and 37 healthy individuals serum was significant. The results of 12 tuberculosis patients serum and 12 health individuals serum was significant,too. ELISA results of synthesized peptide H(12), T15 and T18 to 147 tuberculosis serum and 157 control serum are as follows: Sensitivities are 59.5%, 45.6%, 59.2%; specificities are 95.5%,96.2%å'Œ94.9%.Conclusion By using phage-displayed random peptide libraries, we obtained the specific antibody binding peptides of tuberculosis, which showed specific binding activity with IgG of tuberculosis. It can provide a basis for the establishment of a new serological detection method of tuberculosis with peptide.