Selection of Ceratitis capitata (Diptera: Tephritidae) specific recombinant monoclonal phage display antibodies for prey detection analysis

Abstract

Several recombinant antibodies against the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), one of the most important pests in agriculture worldwide, were selected for the first time from a commercial phage display library of human scFv antibodies. The specificity and sensitivity of the selected recombinant antibodies were compared with that of a rabbit polyclonal serum raised in parallel using a wide range of arthropod species as controls. The selected recombinant monoclonal antibodies had a similar or greater specificity when compared with classical monoclonal antibodies. The selected recombinant antibodies were successfully used to detect the target antigen in the gut of predators and the scFv antibodies were sequenced and compared. These results demonstrate the potential for recombinant scFv antibodies to be used as an alternative to the classical monoclonal antibodies or even molecular probes in the post-mortem analysis studies of generalist predators.

Figure 1. Indirect enzyme-linked immunosorbent assay (ELISA) response to rabbit polyclonal serum raised against total soluble protein extract of C. capitata (Cc) and D. melanogaster (Dm).

Absorbance values (±SE) at 492 nm are displayed for different dilutions of the rabbit polyclonal serum and different protein concentrations (10 µg/ml, 1 µg/ml and 0.1 µg/ml) combinations of C. capitata and D. melanogaster total soluble protein extracts. For each serum dilution, different letters indicates significant differences among means (Tukey’s test: P<0.05).

Figure 2. Indirect ELISA with selected monoclonal recombinant antibodies raised against total soluble protein extracts from C. capitata, D. melanogaster, S. nonagrioides and P. cribata.

Absorbance values (±SE) at 492 nm are displayed for the selected monoclonal recombinant antibodies and total soluble protein extracts (10 µg/ml) combinations. Three percent skimmed milk in PBS was used as the negative control. The values obtained are compared with those determined for the rabbit polyclonal serum. Notice that the rabbit polyclonal serum was not tested against S. nonagrioides and P. cribata extracts.

Figure 3. Sequence alignments of the VH and Vκ regions encoded by the selected clones.

Panel A. Sequence alignment of the VH region. Black color indicates no differences among sequences (TJCc2: J2, TJCc22: J22, TJCc31: J31, TJCc63: J63, TJCc64: J64, TJCc65: J65, TJCc67: J67 and TJCc72: J72). Lighter colors indicate higher differences among the sequences for the nucleotides located in the same position. Panel B. Sequence alignment of the Vκ region. Black color indicates no differences among sequences (TJCc2: J2, TJCc22: J22, TJCc31: J31, TJCc63: J63, TJCc64: J64, TJCc65: J65, TJCc67: J67 and TJCc72: J72). Lighter colors indicate higher differences among the sequences for the nucleotides located in the same position. The first 25 and 26 nucleotides of the TJCc sequences were not sequenced.

Figure 4. Indirect ELISA with the monoclonal recombinant antibody TJCc2.

Absorbance values (±SE) at 492 mm obtained by indirect ELISA using different amounts of recombinant antibody TJCc2 against different protein concentrations (5 µg/ml, 2.5 µg/ml and 1 µg/ml) of C. capitata (Cc) and 10 µg/ml of D. melanogaster (Dm) total soluble protein extracts. Abreviations: pfu (plaque forming units) refers to the number of phage particles capable of forming plaques per ml. For each antibody dilution, different letters indicates significant differences among means (Tukeýs test: P<0.05).

Figure 5. Western blot analysis.

(A) Soluble protein extracts separated on 12.5% SDS-PAGE and stained with Coomassie blue. Lane 1, molecular weight markers (molecular weight (MW) is shown in kDa); lane 2, C. capitata extract (70 µg); lane 3, D. melanogaster extract (135 µg). (B) Western blot for total soluble protein extracts (4.5 µg each) using a rabbit polyclonal serum. Lane 1, C. capitata; lane 2, D. melanogaster. (C) Western blot for total soluble protein extracts (13.5 µg each) using the monoclonal recombinant antibody TJCc2. Lane 1, C. capitata; lane 2, D. melanogaster.