479 Journal of Food Protection, Vol. 50, No. 6, Pages 479-484 (June 1987) Copyright1" International Association of Milk, Food and Environmental Sanitarians Monoclonal Antibodies Directed Against the Flagellar Antigens of Listeria Species and Their Potential in EIA-Based Methods JEFFREY M. FARBER* and JOAN I. SPEIRS Bureau of Microbial Hazards, Food Directorate, Health Protection Branch, Health and Welfare Canada, Tunney's Pasture, Ottawa, Ontario, Canada K1A 0L2 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/50/6/479/1651168/0362-028x-50_6_479.pdf by guest on 23 September 2021 (Received for publication March 10, 1987) ABSTRACT Enzyme immunoassay (EIA) methods have been widely used in food microbiology for detection of micro­ Monoclonal antibodies directed against antigens of Listeria organisms and their toxins (1,9,16). The EIA is a rapid spp. were produced. Three main classes of immunoglobulins and sensitive test which is simple and inexpensive. The were found that reacted with Listeria strains containing either antibody used in the EIA is critical to success of the pro­ the A, B, or C flagellar antigen. These antibodies reacted with Listeria monocytogenes, Listeria welshimeri, Listeria seeligeri, cedure, and must be very specific. This is especially true Listeria ivanovii and Listeria innocua, but not Listeria grayi, for Listeria, because it is known to cross-react with many Listeria murrayi or Listeria denitrificans. The monoclones gram-positive as well as gram-negative bacteria tested did not cross-react with any of the 30 non-Listeria cul­ (14,15,19). In an effort to develop a rapid EIA procedure tures examined, including Staphylococcus aureus and Strep­ for detection of Listeria spp. in foods, our initial goal tococcus faecalis. Cheese and milk samples naturally-contami­ was to develop a monospecific antibody which would nated with L. monocytogenes were found to be positive for Lis­ react only with Listeria spp. teria within two working days after initiation by using the monoclonal antibodies in an enzyme immunoassay. MATERIALS AND METHODS Organisms Listeria monocytogenes is a pathogen of growing con­ L. monocytogenes serotypes 3a, 3b, 3c were obtained from cern to the food industry. It has caused at least four Dr. J. Rocourt, Institut Pasteur, France; strain V7 (serotype Via) major foodborne outbreaks in the last 6 years (3,5,8,18), from Dr. J. Lovett, FDA, Cincinnati, Ohio; and serotype Vic and its presence in dairy products has recently resulted (19112), 4c (19116), 4d (19117), 4e (19118), 5 {Listeria ivanovii; 19119), Listeria murrayi (25401), Listeria grayi in recalls of semi-soft cheeses, ice cream, sherbet and (19120) and Listeria denitrificans (14870) from the ATCC ice-milk products. (ATCC numbers in parentheses) in Rockville, MD. All other There is no one official method currently being used cultures (including non-Listeria strains) of Listeria (L. to isolate Listeria from foods. The cold enrichment pro­ monocytogenes serotype Vib (82-464), 4a (82-131), 4b (81- cedure (6), in which samples are kept at 4°C and subcul- 861), Listeria innocua (82-26), Listeria welshimeri and Listeria tured at weekly intervals for up to 6 months, still appears seeligeri were from collections maintained at the Health Protec­ to be the most sensitive method, although because of the tion Branch in Ottawa. Non-Listeria culture tested in this study length of time required, it is quite impractical. Thus there included: Staphylococcus aureus, Staphylococcus epidermidis, is a crucial need to develop a rapid and sensitive method Streptococcus faecalis, Streptococcus faecium, Streptococcus for detection of Listeria spp. in foods. durans, Streptococcus lactis, Streptococcus pyogenes, Strep­ tococcus faecalis subsp liquefaciens, Streptococcus faecalis A fluorescent-antibody technique for rapid identifica­ subsp. zymogenes, Streptococcus sanguis, Streptococcus tion of L. monocytogenes in meat and milk has been de­ salivarius, Bacillus cereus, Bacillus subtilis, Lactobacillus bre- scribed (10). However, because of cross-reactions of the vis, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus antibody with micrococci, false-positive reactions oc­ helveticus, Lactobacillus plantarum, Lactobacillus acidophilus, curred when examining milk samples. A more promising Corynebacterium glutamicum, Rhodococcus equi, Erysipelothrix automated fluorescent-antibody technique for rapid de­ rhusiopathiae, Brochothrix thermosphacta, Escherichia coli, tection of L. monocytogenes in raw milk, uses flow Citrobacter freundii, Providencia sp., Shigella sonnet, Sal­ cytometry to permit the rapid characterization of Listeria, monella newport, Proteus rettgeri, and Pseudomonas based on parameters such as surface antigenicity, nucleic aeruginosa. acid content and cell morphology (4). However, this technique relies on commercially available polyclonal Preparation of flagellar extract antibodies which have proven to be non-specific (10). L. innocua strain 82-26 was transferred from a tryptose agar JOURNAL OF FOOD PROTECTION, VOL. 50, JUNE 1987 480 FARBER AND SPEIRS (Difco Laboratories, Detroit, MI) plate to 5-ml of tryptose tories, Mississauga, Canada). After incubation for 1 h at am­ phosphate broth (TPB; Difco Laboratories), which was incu­ bient temperature, 0.1 ml of hybridoma culture supernatant bated at 22°C for 8 h. Using this broth as inoculum, 50 flasks fluid was added and the plates incubated at 37°C for 1 h in (500-ml Erlenmeyer) containing TPB (125 ml) plus 0.6% yeast 5% C02 in humid air. After washing the wells three times with extract and 0.2% glucose were inoculated and incubated for 3 PBS-Tween, 0.1 ml of peroxidase goat anti-mouse G + A + M d at 22°C. The cell suspensions were then centrifuged (10,200 (Zymed; supplier, Dimension Laboratories Inc., Mississauga, X g, 20 min) and each pellet resuspended in about 7 ml of Canada), diluted 1:1000 in PBS-FCS, was added and the plates 0.01 M phosphate buffered saline solution, pH 7.0 (PBS). The were incubated for 1 h at ambient temperature. The wells were resuspended pellets were pooled and then divided equally into washed three times with PBS-Tween and once with citrate buf­ two sterile 250-ml Erlenmeyer flasks. To remove flagella (22), fer pH 5.0 before addition of 0.1 ml of substrate consisting cells suspended in 85-90 ml of PBS were blended for 30 s in of 0.04% o-phenylenediamine (Sigma Chemical Co.) and a Polytron homogenizer (model PT 10-35; Brinkmann Instru­ 0.012% H202 in citrate buffer. After 15-30 min the reaction ments Canada, Ltd.) at low speed. The deflagellated cells were was blocked with addition of 0.05 ml of 2.5 M H2S04. Plates then centrifuged first at 3300 X g for 30 min and then at were read at 492 nm. Immunoglobulin classes were determined 16,000 x g for 20 min to remove remaining cells and debris. by coating wells with Zymed goat anti-mouse G + A + M and Downloaded from http://meridian.allenpress.com/jfp/article-pdf/50/6/479/1651168/0362-028x-50_6_479.pdf by guest on 23 September 2021 The flagella were then recovered by centrifuging at 40,000 x using KPL (Kirkegaard and Perry Laboratories Inc., Gaith- g for 3 h in a Beckman model L8-70M ultracentrifuge ersburg, MD) peroxidase goat anti-mouse IgG (-y), IgA (a) or (Beckman Instruments Canada Inc., Toronto, Ontario). Pellets lgM (|x) as the detection antibody. were resuspended in sterile distilled water overnight at 4°C. Dot and blot assays. Dry nitrocellulose membranes (NCM) This fraction is referred to as semi-purified flagellar material. were obtained from either Bio-Rad Laboratories, Mississauga, Further purification was effected by running the semi-purified Canada, or as Sartorius 11306 cellulose nitrate, from BDH flagellar preparation through a DEAE-Sephacel column (Phar­ Chemical Ltd., Toronto, Canada. The NCM were either laid macia Fine Chemicals AB, Uppsala, Sweden). The proteins onto the surface of inoculated Modified MacBride's Agar were fractionated on the column by stepwise increases of NaCl (MMA; 13) or rolled over colonics lying on the surface of hy- concentration (0.1 M, 0.2 M, 0.4 M, 0.8 M,) in PBS (12). drophobic-grid membrane filters (HGMF; supplies as IsoGrad by QA Laboratories, Toronto, Canada). NCM were immersed Electron microscopy in 3% gelatin-tris buffered saline solution, pH 7.5 (TBS block­ Presence and purity of flagellar material was followed by ing solution) for 30 min with gentle agitation and transferred examining material under the electron microscope. Samples to undiluted hybridoma culture supernatant fluids containing were negatively stained by mixing with 3% potassium phos- Listeria anti-flagellar antibodies for 1 h. The membranes were photungstate, pH 7.5 and then were examined using a Siemens washed twice (10 min each) in 0.05% Tween 20-TBS, then Elmiscope 102 at 80 keV. placed in Zymed peroxidase goat anti-mouse G + A + M or KPL peroxidase goat anti-mouse IgG (-y), IgA (a) or lgM (|x) or Monoclonal antibody production HRP-Protein A (Bio-Rad Laboratories) for 1 h. The membranes BALB/c mice were immunized by intraperitoneal (200 |xg), were washed twice, then placed in HRP color development sol­ subcutaneous (200 |xg), and foot pad injections (25 |xg) of the ution consisting of 0.05% 2-chloro-l-naphthol, 0.015% H202 semi-purified flagellar preparation (1 mg/ml) in Freund com­ and 16% methanol in TBS (2). All reactions were carried out plete adjuvant. A further dose (200 ]xg) in Freund incomplete at room temperature. To terminate the reaction, membranes adjuvant was given by intraperitoneal and subcutaneous injec­ were rinsed in distilled water and air-dried. tion at day 14. On day 21, the mice received an intravenous Electrophoretic separation and immunoblotting injection of the same antigen; 3 d later the spleens were re­ SDS electrophoresis was performed on Phast System with moved. The spleen lymphocytes were mixed with SP2/0 cells Phast Gel"' gradient media 10-15, and SS Buffer Strips (Phar­ (GM 3569A, Institute for Medical Research, Camden, NJ) in macia AB, Uppsala, Sweden).