FIG 1.

Biopanning and identification of affinity peptides from TiLV convalescent-phase serum by phage-displayed peptide library. (A) Schematic representation of the strategy of biopanning for serum-specific affinity peptides. TiLV convalescent-phase serum was used as the target, and three rounds of affinity panning were performed using phage display 12-mer peptide libraries. After biopanning, immunopositive phage clones were further identified by DNA sequencing and ELISA. (B) Monitoring the output/input ratios of phage in sequential rounds of biopanning. (C) The corresponding amino acid sequences of 22 randomly selected phage clones after DNA sequencing and the frequency of occurrence of each sequence. Seventeen of the 22 phage clones were sequenced normally. After obtaining the sequencing results, we merged the phage clones with the same sequence for subsequent ELISA determination. (D) Determination of the reactivity of selected phage clones with TiLV convalescent-phase serum by ELISA. When the OD₄₅₀ (phage clone) was ≥ 2.1 × OD₄₅₀ (control), the reaction was considered immunopositive, and the clone was used for subsequent peptide synthesis. According to the OD₄₅₀ (from high to low values), the immunopositive clones were named Pep1, Pep2, Pep3, Pep4, Pep5, and Pep6. (E) Variation curves of the reactions between different concentrations of synthetic peptides and TiLV convalescent-phase serum by ELISA. The synthetic peptides were serially diluted, and the reactivity with serum antibody (1:100 diluted) was measured by absorbance at 450 nm. Each dilution was tested in 3 replicates, and the values in the curve are the mean absorbance ± standard deviation (SD). (F) Synthetic peptides competitively inhibited the reaction of TiLV convalescent-phase serum with TiLV. Each well was coated with 6.0 × 10⁴ copies of TiLV, serum was diluted 1:100, and the concentration of synthetic peptide was 100 μg/mL. The values shown are means ± SD with 5 replicates. Statistical significance was tested by one-way ANOVA and Tukey’s multiple-comparison tests. Significant differences (P < 0.05) are indicated by different lowercase letters.