Isolation of high-affinity single-chain antibodies against Mycobacterium avium subsp. paratuberculosis surface proteins from sheep with Johne's disease

Abstract

Johne's disease, caused by infection with Mycobacterium avium subsp. paratuberculosis, causes significant economic losses to the livestock farming industry. Improved investigative and diagnostic tools-necessary to understand disease processes and to identify subclinical infection-are much sought after. Here, we describe the production of single-chain antibodies with defined specificity for M. avium subsp. paratuberculosis surface proteins. Single-chain antibodies (scFv) were generated from sheep with Johne's disease by cloning heavy-chain and lambda light-chain variable regions and expressing these in fusion with gene III of filamentous phages. Two scFv clones (designated SurfS1.2 and SurfS2.2) were shown to be immunoreactive against M. avium subsp. paratuberculosis surface targets by flow cytometry, and immunoblotting identified specificity for a 34-kDa proteinase-susceptible determinant. Both antibodies were cross-reactive against Mycobacterium avium subsp. avium but nonreactive against Mycobacterium bovis or Mycobacterium phlei cells and were shown to be capable of enriching M. avium subsp. paratuberculosis cells by a factor of approximately 10(6)-fold when employed in magnetic bead separation of mixed Mycobacterium sp. cultures. Further, magnetic bead separation using SurfS1.2 and SurfS2.2 was capable of isolating as few as 10(3) M. avium subsp. paratuberculosis cells from ovine fecal samples, indicating the diagnostic potential of these reagents. Finally, inclusion of SurfS1.2 or SurfS2.2 in in vitro broth culture with M. avium subsp. paratuberculosis indicated that surface binding activity did not impede bacterial growth, although colony clumping was prevented. These results are discussed in terms of the potential use of single-chain phage display monoclonal antibodies as novel diagnostic reagents.

FIG. 1.

BstNI fingerprinting of scFv genes from the first subset of an ovine M. avium subsp. paratuberculosis-specific phage-displayed antibody. Clones were selected for reactivity against M. avium subsp. paratuberculosis cells via three rounds of panning against M. avium subsp. paratuberculosis cell lysate and confirmed positive by ELISA. The selected antibody genes were amplified by PCR, digested for 3 h with BstNI, and analyzed by electrophoresis after resolution through a 4% (wt/vol) agarose gel. Lane 1, negative control; lane 2, clone SurfS2.2; lane 4, SurfS1.2; lanes 3 and 5 to 9, other scFv clones.

FIG. 2.

Purification of two selected scFv clones and antigen recognition in M. avium subsp. paratuberculosis cell lysate. (A) Purification of scFv His-tagged fusion proteins SurfS1.2 (lane 1) and SurfS2.2 (lane 2). scFv were expressed using the vector pAK500 in JM83 E. coli host cells for 18 h at 25°C. The cells were lysed, the supernatant collected, and the fusion protein purified by immobilized metal affinity chromatography. Approximately 1% of purified scFv from a 200-ml culture was resolved by 10% SDS-PAGE and stained with Coomassie-brilliant blue. (B) Immunoblotting of M. avium subsp. paratuberculosis lysate and detection with SurfS1.2 or SurfS2.2. Approximately 20 μg of mycobacterial proteins were either digested with proteinase K (+) or remained untreated (−) and resolved on a 10% SDS-PAGE gel, and the products were transferred to nitrocellulose and probed with peroxidase-conjugated anti-His antibodies. The figure depicts strong binding of both antibodies to a proteinase-susceptible 34-kDa determinant.

FIG. 3.

Flow cytometry to detect Mycobacterium sp. surface antigen recognition by selected scFv clones SurfS1.2 and SurfS2.2. Data represent fluorescence intensity histograms generated by the surface binding of scFv clones SurfS1.2 and SurfS2.2 to different Mycobacterium sp. targets. Each histogram depicts 30,000 acquired mycobacterial targets. Red lines represent signals generated using SurfS1.2 (A) or SurfS2.2 (B); black lines represent signals from coculture of bacteria with a non-target-specific scFv. Note the specific signal generated against M. avium subsp. paratuberculosis targets only. (C) M. avium subsp. paratuberculosis (M. ptb) cells were stained with either SurfS1.2 (red line) or SurfS2.2 (green line) or with both antibodies (blue line). Controls were stained with an irrelevant scFv (dashed black line). No increase in the fluorescence intensity was observed by the addition of both scFv simultaneously over the signal observed using either antibody alone.

FIG. 4.

Influence of extrinsic stressors or the intrinsic growth phase of M. avium subsp. avium (M. a.a.) on the subsequent patterns of antibody binding by scFv clones SurfS1.2 and SurfS2.2. (A and B) Live M. avium subsp. avium cells were subjected to pH stress (pH 4 and pH 10, 2 h), heat stress (52°C, 15 min) or oxidative stress (0.2 mM and 2.0 mM H₂O₂, 2 h) prior to incubation with scFv clones SurfS1.2 and SurfS2.2. Patterns of surface antigen expression were examined by flow cytometry, and data were expressed as units of fluorescence intensity due to the binding of SurfS1.2 (A) or SurfS2.2 (B). The asterisk indicates a significant increase in signal generation by pH 4-treated M. avium subsp. avium cells exposed to SurfS2.2 in comparison to nontreated control cells (P < 0.05). (C, D) M. avium subsp. avium cells were harvested at different phases of the growth cycle and labeled with scFv clone SurfS1.2 (C) or SurfS2.2 (D). Green lines represent antibodies incubated with fresh cultures (incubated for 4 days), red lines represent antibodies incubated with late-phase cultures (3-week incubation), and black lines represent signal obtained using a non-target-specific antibody. Note the down-regulated surface binding by SurfS2.2 in late-phase cultures (D).

FIG. 5.

Effect on bacterial growth of the inclusion of SurfS1.2 and SurfS2.2 with M. avium subsp. avium cells. One-week-old cultures of M. avium subsp. avium were harvested from flasks and further cultured for 48 h in the presence of PBS (control) (A), an irrelevant scFv (B), or 5 μg/ml of SurfS1.2 (C) or SurfS2.2 (D). Cell colony morphology was examined visually under a phase-contrast microscope (A to D). Note the prevention of bacterial cell clumping by the inclusion of antibodies. All bacterial cell suspensions were then resuspended by vigorous pipetting, and the OD₆₀₀ was assessed (E) (data refer to mean OD ± standard errors of the means for 1/100 to 1/400 dilutions of the cultures).

FIG. 6.

Use of scFv antibodies to isolate M. avium subsp. paratuberculosis cells from mixed Mycobacterium sp. samples. Magnetic beads were coated with SurfS1.2 antibodies and used to separate mycobacterial cells from a mixture comprising M. phlei and M. avium subsp. paratuberculosis cells at a ratio of 100:1. To differentiate, M. phlei cells were labeled with fluorescein isothiocyanate (green) and M. avium subsp. paratuberculosis cells with TRITC (red), and cells were examined at ×400 magnification using a UV microscope. (A) The arrow indicates a single M. avium subsp. paratuberculosis cell among several M. phlei cells in the preseparation mixture. (B) After immunomagnetic separation, M. avium subsp. paratuberculosis cells were highly enriched, with only a few M. phlei cells present (arrowheads). (C) Direct binding of M. avium subsp. paratuberculosis cells to SurfS1.2-bearing magnetic beads (the arrowhead identifies a single unbound M. phlei cell).

FIG. 7.

Recovery and identification of IS900-positive cells from M. avium subsp. paratuberculosis-spiked fecal samples, using scFv antibodies as the primary purification method. Magnetic beads were coated with SurfS1.2 or SurfS2.2 monoclonal antibodies and employed as the primary purification method to isolate target bacterial cells from ovine fecal samples that had been spiked with various numbers of M. avium subsp. paratuberculosis cells. A monoclonal antibody with no specificity for M. avium subsp. paratuberculosis was used as a control. Following purification, captured cells were subjected to PCR amplification for the IS900 conserved region. The figure represents an ethidium bromide-stained gel bearing the PCR-amplified products. Lane 1, unspiked fecal sample; lane 2, 10⁴ M. avium subsp. paratuberculosis cells alone (positive control); lanes 3 to 5, fecal sample spiked with 10⁴ to 10² M. avium subsp. paratuberculosis cells.