Recombinant single-chain variable fragment antibodies directed against Clostridium difficile toxin B produced by use of an optimized phage display system

Abstract

Recombinant antibody cloning and phage display technologies were used to produce single-chain antibodies (scFv) against Clostridium difficile toxin B. The starting material was the mouse B cell hybridoma line 5A8, which generates a monoclonal antibody against the toxin. The integrated cloning, screening, and phage display system of Krebber et al. (J. Immunol. Methods 201:35-55, 1997) allowed us to rapidly obtain toxin B-binding scFv sequences derived from the hybridoma cell line. The best candidate scFv sequences, based on preliminary enzyme-linked immunosorbent assay (ELISA) screening data were then subcloned into the compatible expression vector. Recombinant single-chain antibodies were expressed in Escherichia coli. A 29-kDa band was observed on polyacrylamide gel electrophoresis as predicted. The expressed product was characterized by immunoblotting and detection with an anti-FLAG antibody. The toxin B-binding function of the single-chain antibody was shown by a sandwich ELISA. The antibody was highly specific for toxin B and did not cross-react with material isolated from a toxin B-negative C. difficile strain. The sensitivity of the soluble single-chain antibody is significantly higher than the original monoclonal antibody based on ELISA data and could detect a minimum of 10 ng of toxin B/well. Competitive ELISAs established that the affinity of the 5A8 parent antibody and the best representative (clone 10) of the single-chain antibodies were similar and in the range of 10(-8) M. We propose that recombinant antibody technology is a rapid and effective approach to the development of the next generation of immunodiagnostic reagents.

FIG. 1.

Agarose gels showing the sequence of cloning of the variable heavy and light chain antibody genes from the hybridoma line 5A8. Lanes 1, 4, and 9 are the DNA ladders. Lanes 2 and 3 are the light and heavy chain DNAs, respectively. Lanes 5 and 6 are assembly PCR products (the complete scFv DNA). Lanes 7 and 8 are two different samples of the recombinant expression cassette after SfiI digestion. After amplification from cDNA, the V_H and V_L DNA appeared on the 1% agarose gel as sharp bands at ca. 400 bp. The V_H and V_L were then combined into a single-chain fragment through the following assembly PCR, which is shown in lanes 5 and 6 as a 750-bp band. This scFv product was further cloned into the phage display vector pAK100 to form the single-chain antibody phage display cassette. As shown in lanes 7 and 8, the display cassette is comprised of two fragments after SfiI digestion, one is pAK100 DNA, which is about a 4-kb band on the gel, and another is the scFv insert, which is a 750-kb band on the gel as predicted.

FIG. 2.

ELISA signal from phage pools as a function of the round of the biopanning cycle. Phage antibodies after 0, 1, 2, and 3 rounds of panning were tested in parallel for their antibody activity against 50/50 serially diluted toxin B or CRP, starting from 1 μg/well. The bound phage was detected with an anti-m13 antibody. The CRP antigen was coated on the plates in the same way as toxin B. Binding levels to wells coated with toxin B or CRP for pooled phage after rounds 0 (rows 1 and 2), 1 (rows 3 and 4), 2 (rows 5 and 6), and 3 (rows 7 and 8) are shown.

FIG. 3.

Performance of single clones screened for binding with toxin B by phage ELISA. Each column represents the ELISA signal from an individual clone isolated after the indicated number of pannings. The columns are arranged in the order in which the clones were isolated: clones 1 to 20, before panning; clones 21 to 26, after the second panning; clones 27 to 31, after the third panning. Three clones, the highest representative from each panning, were set aside for further study.

FIG. 4.

Immunoblot of scFv antibody detected with an anti-FLAG antibody. Inserts were grown in the expression vector pAK400. Cells were harvested and samples were extracted, separated by PAGE, and transferred to nitrocellulose membranes. They were blocked, reacted with peroxidase-coupled antibody, and visualized with a bioluminescence kit. Lane 1, uninduced with IPTG. Lane 2, blank vector. Lane 3, molecular mass marker (value indicated at the left, in kilodaltons). Lane 4, whole-cell extract from clone 10. Lane 5, periplasm extract from clone 10. Lane 6, whole-cell extract from clone 6. Lane 7, periplasm extraction from clone 6.

FIG. 5.

Sandwich ELISA of equivalent amounts of soluble scFv antibodies compared with 5A8 hybridoma supernatant. Lines are as indicated on the figure. Antibody concentrations were constant, as indicated in Methods and Materials. The toxin B concentration was serially diluted.

FIG. 6.

Competition ELISA to determine relative affinity for scFv clone 10 and 5A8 IgG. The relative affinities were determined by inhibition ELISA with toxin B as the competing antigen.