Development of a double-recombinant antibody sandwich ELISA for quantitative detection of epsilon toxoid concentration in inactivated Clostridium perfringens vaccines

Abstract

Background: Epsilon toxin (ETX) causes a commonly fatal enterotoxemia in domestic animals. Also, ETX causes serious economic losses to animal husbandry. In this study, we selected several clones against ETX using repertoires displayed on filamentous phage. Anti-ETX specific clones were enriched by binding to immobilized antigen, followed by elution and re-propagation of phage. After multiple rounds of binding selection, ELISA analysis showed that most isolated clones had high affinity and specificity for ETX.

Results: Two recombinant monoclonal antibodies against ETX were isolated by phage display technology. B₁ phage VH antibody isolated from DAb library and G₂ soluble scFv antibody isolated from Tomlinson I + J libraries have been applied as the capture and detection antibodies for developing an ETX sandwich ELISA test, respectively.

Conclusions: Designed ETX sandwich ELISA could be a valuable tool for quantitative detection of ETX in inactivated commercial vaccines against enterotoxemia.

Keywords: Clostridium perfringens; Epsilon toxin; Phage display; Recombinant antibody; Sandwich ELISA.

Fig. 1

a. Screening of clones by polyclonal phage ELISA. Epsilon toxoid was coated to plastic and then detected with an anti-c-Myc antibody. The coating was carried out in duplicate; the mean value is presented, the error bars indicat the standard deviation of the two values. 5% MPBS buffer was coated as a negative control. S₁, S₂, S₃ = selection rounds 1, 2 and 3. b. Screening of clones by monoclonal phage ELISA of Tomlinson I + J and DAb libraries. Individual phage clones from the second and third rounds of selection were tested by monoclonal phage ELISA against purified epsilon toxoid. Phages were applied as follows; S₂: 32 clones picked at random after round 2 of selection; S₃: 36 clones picked at random after round 3 of selection. OD at 450 nm was measured after 10 minutes.

Fig. 2

Schematic illustrations of double-recombinant antibody sandwich ELISA for quantitative measurement of ETX. (1) Well is coated with the B₁ phage VH antibody isolated from the DAb library and the uncoated surface is blocked by 3% BSA/PBS. (2) The C. perfringens epsilon toxoid binds to the coated B₁ phage VH antibody. (3) G₂ soluble scFv antibody selected from Tomlinson I + J libraries as the detector antibody binds to epsilon toxoid. (4) HRP-conjugated monoclonal anti-Polyhistidine antibody as the conjugate antibody binds to hexahistidine tag fused to G₂ scFv

Fig. 3

The standard curve of the double-recombinant antibody sandwich ELISA for epsilon toxoid with R² = 0/997. Each point is the mean ± standard deviation (n = 2)