Research paper
Single-chain Fv antibody with specificity for Listeria monocytogenes

https://doi.org/10.1016/j.jim.2004.04.001Get rights and content

Abstract

Single chain antibodies (scFv) exhibiting specific binding to Listeria monocytogenes strains were isolated from a pool of random scFvs expressed on the surface of filamentous bacteriophages. Positive selection (panning) using L. monocytogenes was used to enrich for phage clones with the desired binding affinity, and negative selection using L. innocua and L. ivanovii was used to remove phages expressing cross-reactive antibody fragments. A single phage clone, P4:A8, was selected using two independent panning schemes. A rapid assay was devised to determine phage antibody binding specificity and was used to develop a selectivity profile for individual phage clones. The P4:A8 clone was screened against a panel of bacteria consisting of eight strains of L. monocytogenes, one each of the other six species of Listeria and nine other relevant bacterial species. A collection of individual clones from the penultimate panning was also screened against a subset of the panel of bacteria. The selectivity profiles indicate that multiple clones, including P4:A8, exhibit binding to one or more strains of L. monocytogenes without cross-reactivity toward any other species in the panel. This is the first report of a species-specific antibody for viable cells of L. monocytogenes (i.e., the ability to bind to L. monocytogenes without cross-reactivity toward any other species of Listeria).

Introduction

Modern food production and distribution systems permit nationwide distribution of large quantities of food in a few days. These systems provide consumers with consistent, low-cost, high quality foods, but may also be a conduit for exposing large populations to pathogens introduced inadvertently, or by deliberate acts of bioterrorism, into foodstuffs. There is intense interest in rapid assays which can detect harmful levels of pathogens in hours rather than days as required by conventional culture methods. Listeria monocytogenes, the only species of Listeria which is pathogenic in humans, is of particular interest because it is able to survive and grow at low temperatures and because the mortality rate for infected individuals is much higher than for other common food-borne pathogens.

Virtually all rapid assay methods make use of the high affinity and selectivity of antibody–antigen interactions. Antibodies are frequently used for isolation and concentration of pathogens from the sample matrix prior to detection. Immunomagnetic beads Skjerve and Olsvik, 1991, Luk and Lindberg, 1991, Fratamico et al., 1992, Gehring et al., 1996 are very widely used, and related approaches such as immunoaffinity columns (Brewster, 2003), and immunofiltration membranes Paffard et al., 1996, Gehring et al., 1998 have also been developed. The purified, concentrated bacteria may be detected immediately, or cultured to further increase the bacterial concentration, establish viability, or provide confirmation by conventional methods. Antibody used for this purpose must bind to the surface of intact, viable cells. Many methods utilize labeled antibodies as signal generation reagents (Feng, 1996), exploiting such diverse detection modes as absorbance (Wyatt et al., 1993), epifluorescence microscopy (Tortorello and Gendel, 1993), fluorescence (Tu et al., 2001), amperometry Hadas et al., 1992, Brewster and Mazenko, 1998, and electrochemiluminescence Yu and Bruno, 1996, Crawford et al., 2000. Antibody for detection purposes may be directed against surface as well as internal antigens.

Although researchers have developed a wide variety of innovative and effective approaches for rapid pathogen detection, their application has been restricted to the few target bacteria for which appropriate antibodies are available (E. coli O157:H7 and Salmonella spp.). Development of rapid assays for important pathogens such as L. monocytogenes and Campylobacter has been thwarted by the lack of specific, high affinity antibody. The importance of L. monocytogenes as a food-borne human pathogen has led to extensive efforts to develop a species-selective antibody Nannapaneni et al., 1998a, Nannapaneni et al., 1998b, Mattingly et al., 1988, Curiale et al., 1994, Bubert et al., 1994, Bhunia, 1997. Hundreds of publications over the past two decades have focused on this task, and numerous approaches have been pursued to produce a polyclonal or monoclonal antibody with the appropriate selectivity (Bhunia, 1997). Success has been elusive, suggesting that L. monocytogenes either lacks surface epitopes which are both unique and antigenic, or that such epitopes are not effectively processed or presented during in-vivo antibody production and maturation. Antibody phage display techniques (Winter et al., 1994) do not rely on antigen processing and presentation, and may provide a route to success where conventional techniques have failed.

Phage display has proven a useful tool for the isolation of antibody fragments with desired specificities. The technique involves the display of a library of single-chain antibody (scFv) fragments on the surface of filamentous phages followed by selection of the desired recombinant phages by means of specific binding to an antigen of interest. Although phage display has advantages over conventional polyclonal and monoclonal antibody production, few reports (Benhar et al., 2001) have employed this technique for the selection of reagents for the detection of food-borne pathogens.

Phage display also provides several approaches for rational improvement of antibody affinity and selectivity. Given a set of phage clones with known affinity and selectivity profiles, selection strategies can be designed to isolate clones with optimal properties from the existing library of clones. By correlating affinity and selectivity data with DNA sequence information, it is feasible to design and construct novel sequences that express antibodies with the desired properties.

We present here the results of the first stage in this process of generating a species-specific immunological reagent for the detection of L. monocytogenes in food. Using live L. monocytogenes cells to select phage antibodies and cells of other Listeria spp. to remove cross-reacting phages, a single phage antibody clone was isolated. This phage antibody bound some strains of L. monocytogenes without cross-reactivity toward any other species of Listeria. This is the first report of a species–specific antibody for viable cells of L. monocytogenes.

Section snippets

Materials

Water was deionized in-house with a Nanopure water treatment system (Barnstead, Dubuque, IA). The Griffin.1 library (Human Synthetic VH+VL scFv Library) and the phage host strain Escherichia coli TG1 were the gift of the Centre for Protein Engineering (Cambridge, UK). Bacteriological media and agar were from Difco (Detroit, MI, USA). All other chemicals used were of reagent grade.

Bacterial cultures and media

E. coli TG1 was used to propagate phages. E. coli, Salmonella typhimurium, and Pseudomonas putida were grown in

Pannings

Phage antibody selections were conducted as described above using the scheme shown in Fig. 2. An increase in phage affinity was observed with sequential rounds of panning against whole cells of L. monocytogenes with a dramatic increase after the fourth panning (Fig. 3A). When the phage pool from the third panning was panned against L. ivanovii and L. innocua to subtract phage displaying antibody fragments bound to cell surface antigens that were not unique to L. monocytogenes, a modest drop in

Discussion

Conventional techniques have failed to produce antibodies with the specificity and avidity required for rapid, selective assays for the detection of L. monocytogenes and other important pathogens. Phage display provides promise of reaching this goal. The specific goals for this stage of our research were to develop suitable panning protocols and methodology for assaying binding affinity that would be applicable to a wide range of bacterial species and to apply these methods to select and

Acknowledgements

The authors would like to thank Dr. Arun Bhunia, Dr. Pina Fratamico, Dr.George Somkuti, Dr. Robert Mandrell and Mr. Marshall Reed for providing bacterial strains as well as The Centre for Protein Engineering at the University of Cambridge (UK) for the generous gift of the Griffin.1 library. We also acknowledge the excellent technical assistance of Ms. Ly Nguyen in conducting many of the experiments.

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