Among the metabolites produced by Staphylococcus aureus, and other staphylo-coccal spp., enterotoxins (5,16,27) present the greatest foodborne risk to the health of consumers. Staphylococcal enterotoxins are basic proteins produced by certain Staphylococcus strains in a variety of environments, including food substrates. These structurally-related, toxicologically similar proteins are produced primarily by Staphylococcus aureus, although S. intermedius and S. hyicus also have been shown to be enterotoxigenic (1). Normally considered a veterinary pathogen (36,40), S. intermedius was isolated from butter blend and margarine in a food poisoning outbreak (15,29). A coagulase negative S. epidermidis was reported to have caused least one outbreak (17). These incidents support testing staphylococci other than S. aureus for enterotoxigenicity, if they are present in large numbers in a food suspected of causing a food poisoning outbreak.

When large numbers of enterotoxigenic staphylococci grow in foods, they may elaborate enough toxin to cause food poisoning after the foods are ingested. The most common symptoms of staphylococcal food poisoning, which usually begin 2-6 h after contaminated food is consumed, are nausea, vomiting, acute prostration, and abdominal cramps. To date, 8 enterotoxins (types A, B, C1, C2, C3, D, E, and H) have been identified as distinct serological entities. Current methods to detect enterotoxins use specific polyclonal or monoclonal antibodies (33,42,43).

The threshold amount of enterotoxin for causing illness in humans is not known. However, information from food poisoning outbreaks (16,25) and human challenge studies (24) indicates that individuals experiencing illness probably consumed at least 100 ng of enterotoxin A, the serotype most frequently involved in foodborne staphylococcal illness (20). The microslide gel double diffusion technique requires at least 30-60 ng of enterotoxin per gram of food. Chromatographic purification and concentration are used to achieve this toxin concentration so that the serological assay can be performed (4).

The microslide method is approved by AOAC International (4) and is the current standard for evaluating new methods. Other methods used for food extracts should be at least as sensitive as the microslide method, which requires concentrating extracts from 100 g food in as much as 600 ml to about 0.2 ml. Less sensitive methods are inadequate.

Techniques such as radioimmunoassay (RIA), agglutination, and enzyme-linked immunosorbent assay (ELISA), require less concentration of the food extracts ; thus, they save time and are more sensitive. Latex agglutination (16) appears promising as a serological tool for identifying staphylococcal enterotoxins. Several ELISA methods (26,28,30,32,37,38,39) have been proposed for the identification of enterotoxins in foods, but, except for a polyvalent ELISA (7,9), their specificity has not been studied extensively. Among ELISA methods, the "double antibody sandwich" ELISA is the method of choice, because reagents are commercially available in polyvalent and monovalent formats for both toxin screening and serotype specific identification(22). An automated enzyme-linked fluorescent immunoassay (ELFA) has been developed and is commercially available. This method has undergone specificity and sensitivity evaluations and has proven to be an effective serological system for the identification of staphylococcal enterotoxin in a wide variety of foods (14). Other methods, which have been used in the identification of the staphylococcal enterotoxins and may have application in foods, are the T-cell proliferation assay (35), and polyacrylamide gel electrophoresis (PAGE) combined with Western blotting (2).

Examining staphylococci isolated from foods for enterotoxin production helps establish potential sources of enterotoxin in foods. Of the methods developed for laboratory testing of enterotoxin production, the semisolid agar procedure (19) is approved by AOAC International. It is simple to perform and requires minimal, routine laboratory equipment. Another simple approach is the use of pH 5.5 brain heart infusion (BHI) broth (14). The major problem with identifying enterotoxins in foods is that minute concentrations are sufficient to cause food poisoning. Pasteurization and thermal processing may render most toxin serologically unreactive. Consequently, false negatives may result, if detection methods lack sufficient sensitivity to detect active toxin (6).

This chapter presents a technique for the routine culturing of suspect staphylococci, procedures for the extraction of enterotoxin from foods and selected serological methods (Microslide gel double diffusion precipitation test, two manual ELISAs [Tecra^TM, Transia^TM], an automated qualitative "enzyme-linked fluorescent immunoassay" [ELFA^TM, Vidas^TM], and sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]-immunoblotting) for the identification of staphylococcal enterotoxin from isolates and from foods.

Recommended for routine analysis of foods for staphylococcal enterotoxin is the use, initially, of two different polyvalent ELISA kits. If results from different polyvalent ELISA methods yield conflicting results, retest using another method (e.g., another polyvalent ELISA method or the SDS-polyacrylamide gel electrophoresis-immunoblot assay for S. aureus enterotoxin A). Methods were developed to restore serological activity to heat-altered toxin in extracts of heat-processed foods (3,10,11,12,18,41,44). However, current toxin detection assays (described above) are sensitive enough to detect unaltered toxin that may persist after heat without such treatment (2).

1. Special equipment and materials

1. Refrigerated cabinet or cold room. The carboxymethyl cellulose (CMC) column extraction is performed at about 5°C, primarily because the column is allowed to run overnight. Storing food materials and extracts in a cold room or cabinet eliminates the need for a refrigerator.
2. Waring blender or Omnimixer. Grind foods into slurry for adequate extraction of enterotoxin. An Omnimixer (DuPont) is convenient for grinding food directly into stainless steel centrifuge tubes.
3. pH meter. The pH during extraction and the pH of buffers used in the extraction are important. Make adjustments within ± 0.1 pH unit.
4. Refrigerated centrifuge. Food extracts are centrifuged at relatively high speeds at 5°C in a refrigerated centrifuge, such as a Sorvall RC-2B, which can reach 20,000 rpm. The lower the centrifuge speed, the more difficult is the clarification of extracts.
5. Carboxymethyl cellulose (CMC). The extract is partially purified by absorption onto CMC, Whatman CM 22, 0.6 meq/g (H. Reeve Angel, Inc., 9 Bridewell Place, Clifton, NJ). Soluble extractants are removed by this step.
6. Centrifuge tubes. Use 285 ml stainless steel centrifuge bottles (Sorvall No. 530).
7. Magnetic stirrer. A magnetic stirrer keeps test samples agitated during pH adjustments, dialysis, etc.
8. Filter cloth. At various stages in the procedures, food is filtered through several layers of coarse material such as cheesecloth placed in a funnel. Wetting cheesecloth before placing it in the funnel reduces adherence of food to cloth. The coarse material allows rapid flow with efficient removal of food particles, the chloroform layer, etc.
9. Chromatographic tube (with stopcock or rubber tube attachment with finger clamp). Enterotoxin in food is partially purified by using CMC, with elution in a chromatographic tube. For this purpose, a 19 mm id column, e.g., chromaflex, plain with stopcock, size 234 (Kontes Glass Co., Vineland, NJ) is recommended.
10. Polyethylene glycol (PEG). Food extracts are concentrated with PEG (Carbowax 20,000; Union Carbide Corp., Chemical Division, 230 North Michigan Ave., Chicago, IL 60638).
11. Lyophilizer. The extract is finally concentrated by freeze-drying, which conveniently reduces the volume to 0.2 ml and completely recovers the extract.
12. Dialysis tubing. Cellulose casing of 1-1/8 inches flat width and an average pore diameter of 48 Angstrom units is used (12,000-14,000 mol wt exclusion).
13. Separatory funnels. Separatory funnels of various sizes are needed for CHCl₃ extractions and with the chromatographic column.
14. Glass wool. Glass wool makes ideal plugs for chromatographic columns.
15. Chloroform. Food extract is treated with CHCl₃ (several times in some instances) to remove lipids and other substances that interfere with concentration of extract to small volumes.

Note: Chloroform is hazardous. Wear gloves, avoid contact with skin, and perform extraction in a chemical fume hood.

2. Reagents
1. 0.2 M NaH₂PO₄ · H₂O
2. 0.2 M Na₂HPO₄
3. H₃PO₄ (0.005 M, 0.05 M)
4. Na₂HPO₄ (0.005 M, 0.05 M)
5. NaCl (crystal)
6. 1 N (or 0.1 N) NaOH
7. 1 N (or 0.1 N) HCl
3. Preparation of materials and reagents

1. Polyethylene glycol (PEG). Prepare 30% (w/v) PEG 20,000 mol wt solution by adding 30 g PEG for each 70 ml distilled water. Cut dialysis tubing (1/8 inch flat width) long enough to accommodate food extract to be concentrated. Soak tubing in 2 changes of distilled water to remove glycerol coating. Tie one end of tubing with 2 knots close together. Fill tube with distilled water and test for leaks by squeezing filled sac while holding untied end tightly closed. Empty sac and place it in distilled water until use.
2. Sodium phosphate buffer solutions

1. Phosphate buffer, pH 5.7, 0.2 M (stock). Prepare solution by adding 0.2 M NaH₂PO₄•H₂O (27.60 g in 1 liter water) to 0.2 M Na₂HPO₄ (28.39 g in 1 liter water) to final pH of 5.7.
2. 0.005 M Phosphate buffer. Dilute 0.2 M, pH 5.7 buffer (stock) with water (1 + 39). Adjust pH to 5.7 with 0.005 M H₃PO₄.
3. 0.2 M Phosphate buffer, pH 6.4 (stock). Add 0.2 M Na₂HPO₄ to 0.2 M NaH₂PO₄ to pH 6.4.
4. 0.05 M Sodium phosphate-NaCl buffer, pH 6.5. Add NaCl (11.69 g/liter) to pH 6.4, 0.2 M solution (stock) to give 0.2 M NaCl, pH about 6.3. Dilute with water (1 + 3), and adjust to pH 6.5 with 0.05 M H₃PO₄ or 0.05 M Na₂HPO₄.
3. Reservoir (separatory funnel). Attach about 60 cm latex tubing to stem of separatory funnel of appropriate size and attach other end of tube to glass tubing inserted through No. 3 rubber stopper to fit chromatographic column. Suspend separatory funnel from ring stand above chromatographic tube.
4. Carboxymethy1 cellulose (CMC) column. Suspend 1 g CMC in 100 ml 0.005 M sodium phosphate buffer, pH 5.7, in 250 ml beaker. Adjust pH of CMC suspension with 0.005 M H₃PO₄. Stir suspension intermittently for 15 min, recheck pH, and readjust to 5.7 if necessary. Pour suspension into 1.9 cm chromatographic tube, and let CMC particles settle. Withdraw liquid from column through stopcock to within 1 inch of surface of settled CMC. Place loosely packed plug of glass wool on top of CMC. Pass 0.005 M sodium phosphate buffer, pH 5.7, through column until washing is clear (150-200 ml). Check pH of last wash; if not 5.7, continue washing until pH is 5.7. Leave enough buffer in column to cover CMC and glass wool to prevent column from drying out.
4. Extraction and chromatographic separation of enterotoxin from food (see Fig. 1 - Scheme).

   Note: This procedure and other procedures that may generate aerosols of pathogenic microorganisms should be performed in an approved biohazard hood.

   Grind 100 g food in Waring blender at high speed for 3 min with 500 ml 0.2 M NaCl. Use Omnimixer for smaller quantities. Adjust pH to 7.5 with 1 N NaOH or HCl if food is highly buffered, and 0.1 N NaOH or HCl if food is weakly buffered (e.g., custards). Let slurry stand for 10 to 15 min, recheck pH, and readjust if necessary.

   Transfer slurry to two 285 ml stainless steel centrifuge bottles. Centrifuge at 16,300 x g for 20 min at 5°C. Lower speeds with longer centrifuge time can be used, but clearing of some foods is not as effective. Separation of fatty materials is ineffective unless food is centrifuged at refrigeration temperature. Decant supernatant fluid into 800 ml beaker through cheesecloth or other suitable filtering material placed in a funnel. Re-extract residue with 125 ml of 0.2 M NaCl by blending for 3 min. Adjust pH to 7.5 if necessary. Centrifuge at 27,300 x g for 20 min at 5°C. Filter supernatant through cheesecloth, and pool filtrate with original extract.

Place pooled extracts in dialysis sac. Immerse sac in 30% (w/v) PEG at 5°C until volume is reduced to 15-20 ml or less (usually overnight). Remove sac from PEG and wash outside thoroughly with cold tap water to remove any PEG adhering to sac. Soak in distilled water for 1-2 min and in 0.2 M NaCl for a few min. Pour contents into small beaker.

Rinse inside of sac with 2-3 ml 0.2 M NaCl by running fingers up and down outside of sac to remove material adhering to sides of tubing. Repeat rinsing until rinse is clear. Keep volume as small as possible.

Adjust pH of extract to 7.5. Centrifuge at 32,800 x g for lO min. Decant supernatant fluid into graduated cylinder to measure volume. Add extract with < to = volume of CHCl₃ to separatory funnel. Shake vigorously 10 times through 90 degree arc. Centrifuge CHCl₃ extract mixture at 16,300 x g for 10 min at 5°C. Return fluid layers to separatory funnel. Draw off CHCl₃ layer from bottom of separatory funnel, and discard. Measure volume of water layer and dilute with 40 volumes of 0.005 M sodium phosphate buffer, pH 5.7. Adjust pH to 5.7 with 0.005 M H₃PO₄ or 0.005 M Na₂HPO₄. Place diluted solution in 2 liter separatory funnel.

Place stopper (attached to bottom of separatory funnel) loosely into top with liquid from separatory funnel. Tighten stopper in top of tube and open stopcock of separatory funnel. Let fluid percolate through CMC column at 5°C at 1-2 ml/min by adjusting flow rate with stopcock at bottom of column so that percolation can be completed overnight. If all liquid has not passed through column overnight, stop flow when liquid level reaches glass wool layer. If all liquid has passed through overnight, rehydrate column with 25 ml distilled water.

After percolation is complete, wash CMC column with 100 ml 0.005 M sodium phosphate buffer (1-2 ml/min); stop flow when liquid level reaches glass wool layer. Discard wash. Elute enterotoxin from CMC column with 200 ml 0.05 M sodium phosphate buffer, pH 6.5 (0.05 M phosphate-0.05 M NaCl buffer, pH 6.5), at flow rate of 1-2 ml/min at room temperature. Force last of liquid from CMC by applying air pressure to top of chromatographic tube.

Place eluate in dialysis sac. Place sac in 30% (w/v) PEG at 5°C and concentrate almost to dryness. Remove sac from PEG and wash. Soak sac in 0.2 M phosphate buffer, pH 7.4. Remove concentrated material from sac by rinsing 5 times with 2-3 ml 0.01 M sodium phosphate buffer, pH 7.4-7.5. Extract concentrated solution with CHCl₃. Repeat CHCl₃ extractions until precipitate is so lacy that it falls apart in CHCl₃ layer in cheesecloth.

Place extract in short dialysis sac (about 15 cm). Place sac in 30% (w/v) PEG, and let it remain until all liquid is removed from inside sac (usually overnight). Remove sac from PEG and wash outside with tap water. Place sac in distilled water for 1-2 min. Remove contents by rinsing inside of sac with 1 ml portions of distilled water. Keep volume below 5 ml. Place rinsings in test tube (18 x 100 mm) or other suitable container and freeze-dry. Dissolve freeze-dried test sample in as small an amount of saline as possible (0.1-0.15 ml). Check for enterotoxins by microslide method.

1. Equipment and materials
1. Test tubes, 25 x 100 and 20 x 150 mm
2. Petri dishes, 15 x 100 and 20 x 150 mm, sterile
3. Bottles, prescription, 4 oz
4. Microscope slides, pre-cleaned glass, 3 x 1 inch (7.62 x 2.54 cm)
5. Pipets, sterile, 1, 5, and 10 ml, graduated
6. Centrifuge tubes, 50 ml
7. Sterile bent glass spreaders
8. Electrical tape, 0.25 mm thick, 10.1 mm wide, available from Scotch Branch, 3M Co., Electro-Products Divisions, St. Paul, MN 55011.
9. Templates, plastic (Fig. 2)
10. Silicone grease, high vacuum, available from Dow Corning Corp., Midland, MI 48640
11. Sponges, synthetic
12. Wooden applicator sticks
13. Glass tubing, 7 mm, for capillary pipets and de-bubblers
14. Pasteur pipets or disposable 30 or 40 1 pipets, available from Kensington Scientific Corp., 1165-67th St., Oakland, CA 94601
15. Staining jars (Coplin or Wheaton)
16. Desk lamp
17. Incubator, 35 ± 1°C
18. Hot plate, electric
19. Sterilizer (Arnold), flowing steam
20. Blender and sterile blender jars
21. Centrifuge, high speed
22. Timer, interval
2. Media and reagents
1. Brain heart infusion (BHI) + 0.7% (w/v) agar (M23)
2. Agar, bacteriological grade, 0.2% (w/v)
3. Gel diffusion agar, 1.2% (w/v) (R28)
4. Baird-Parker medium (M17)
5. Nutrient agar, slants (M112)
6. Distilled water, sterile
7. Butterfield's phosphate-buffered dilution water (R11)
8. 0.2 M NaCl solution, sterile (R72)
9. Physiological saline solution, sterile (antisera diluent) (R63)
10. Thiazine Red R stain (R79)
11. Slide preserving solution (R69)
12. No. 1 McFarland standard (R42)
13. Antisera and reference enterotoxins (Toxin Technology Inc., 7165 Curtiss Ave., Sarasota, FL 34231)
3. Preparation of materials and media

1. BHI with 0.7% (w/v) agar. Adjust BHI broth to pH 5.3; add 7 g agar per liter broth [0.7 % (w/v)], and dissolve with minimal boiling. Dispense 25 ml portions into 25 x 200 mm test tubes and autoclave 10 min at 121°C. Just before use, aseptically pour sterile medium into standard petri dishes.
2. No. 1 McFarland standard. Prepare turbidity standard No. 1 of McFarland nephelometer scale (31). Mix 1 part 1% (w/v) BaCl₂ with 99 parts 1% (v/v) H₂SO₄ in distilled water.
3. 1.2% (w/v) Gel diffusion agar for gel diffusion slides. Prepare fluid base for agar in distilled water as follows: NaCl 0.85% (w/v); sodium barbital 0.8% (w/v); merthiolate 1:10,000 (crystalline), available from Eli Lilly and Co., Terre Haute, IN. Adjust pH to 7.4. Prepare agar by adding 12 g per liter Noble special agar (Difco) [final concentration 1.2 %(w/v)]. Melt agar mixture in Arnold sterilizer (steamer) and filter while hot, in steamer, through 2 layers of grade filter paper (Whatman No. 1 or equivalent). Dispense small portions (15-25 ml) into 4 oz. prescription bottles. (Remelting more than twice may break down purified agar.)
4. Thiazine Red R stain. Prepare 0.1% (w/v) solution of Thiazine Red R stain in 1.0% (v/v) acetic acid.
5. Preparation of slides. Wrap double layer of electrician's plastic insulating tape around both sides of glass slide, leaving 2.0 cm space in center. Apply tape as follows: Start piece of tape (9.5-10 cm long) 0.5 cm from edge of undersurface of slide and wrap tightly around slide twice. Wipe area between tapes with cheesecloth soaked with 95% ethanol, and dry with dry cheesecloth. Coat upper surface area between tapes with 0.2% (w/v) agar in distilled water as follows: Melt 0.2% (w/v) bacteriological grade agar; maintain at 55°C or higher in screw-cap bottle. Hold slide over beaker placed on hot plate adjusted to 65-85°C and pour or brush 0.2% (w/v) agar over slide between 2 pieces of tape. Let excess agar drain into beaker. Return agar collected in beaker to original container for reuse. Wipe undersurface of slide. Place slide on tray and dry in dust-free atmosphere (e.g., incubator). NOTE: If slide is not clean, agar will roll off without coating it uniformly.
6. Preparation of slide assembly. Prepare plastic templates as described by Casman et al. (21) (see Fig. 2 for specifications). Spread thin film of silicone grease on side of template that will be placed next to agar, i.e., side with smaller holes. Place 0.4 ml 1.2% (w/v) gel diffusion agar, melted and cooled to 55-60°C, between tapes. Immediately lay silicone-coated template on melted agar and edges of bordering tapes. Place one edge of template on one of the tapes and bring opposite edge to rest gently on the other tape. Soon after agar solidifies, place slide in prepared petri dish (C-7, below). Label slide with number, date, or other information.

7. Preparation of petri dishes for slide assemblies. Maintain necessary high humidity by saturating 2 strips of synthetic sponge (about 1/2" inch wide x 1/2" inch deep x 2-1/2 inches long) with distilled water and placing them in each 20 x 150 mm petri dish. From 2 to 4 slide assemblies can be placed in each dish.
8. Recovery of used slides and templates. Clean slides without removing tape; rinse with tap water, brush to remove agar gel, and boil 15-20 min in detergent solution. Rinse about 5 min in hot running water, and boil in distilled water. Place slides on end, using test tube rack or equivalent, and place in incubator to dry. If slides cannot be uniformly coated with hot 0.2% (w/v) agar, they are not clean enough and must be washed again. Avoid exposing plastic templates to excessive heat or plastic solvents when cleaning. Place templates in pan, pour hot detergent solution over them, and let soak 10-15 min. Use soft nylon brush to remove residual silicone grease. Rinse sequentially with tap water, distilled water, and 95% ethanol. Spread templates on towel to dry.
9. Directions for dissolving reagents used in slide gel. Enterotoxins and their antisera are supplied as lyophilized preparations. Rehydrate antisera in physiological saline. Rehydrate reference enterotoxins in physiological saline containing 0.3% (w/v) proteose peptone, pH 7.0, or physiological saline containing 0.37% (w/v) BHI broth, pH 7.0. Preparations should produce faint but distinct reference lines in slide gel diffusion test. Lines may be enhanced (E-3, below).
4. Procedure for enumeration and selection of staphylococcal colonies

To examine foods, use procedures described for detecting coagulase- positive staphylococci (see Chapter 12). Test isolates for enterotoxigenicity as described in E, below. To examine food in a suspected staphylococcal food poisoning outbreak, however, the following method is recommended:

1. Enumeration of staphylococci/g. Blend food with sterile 0.2 M NaCl solution for 3 min at high speed (20 g food in 80 ml 0.2 M NaCl, or 100 g in 400 ml, or whatever amount gives 1:5 dilution). Prepare decimal dilutions as follows: 10 = 1 part 1:5 dilution plus 1 part Butterfield's buffer; prepare dilutions 10^-2, 10^-3, 10^-4, 10^-5, 10^-6. Place 0.1 ml portion of each dilution onto prepared Baird-Parker Agar and spread with sterile bent glass rod. Incubate plates inverted at 35°C for 48 ± 2 h. Count plates at dilution having 30-300 well-distributed colonies. Calculate staphylococci/g: total count x dilution factor of slurry x 10.

2. Enumeration of enterotoxigenic staphylococci/g. Note any variation in type or amount of pigment or other morphological characteristics produced by colonies. Count number of colonies in each group type, and record. Transfer two or more colonies from each type to nutrient agar slants or comparable medium. Test for enterotoxigenicity as described in E, below. Calculate enterotoxigenic staphylococci/g as follows: number of enterotoxigenic staphylococcal colonies x dilution factor of slurry x 10.

NOTE: To determine presence of enterotoxin producers in food, add enough 0.2 M NaCl to slurry (1:5 dilution) to obtain 1:6 dilution, e.g., add additional 100 ml of 0.2 M NaCl to 1:5 dilution of slurry containing food and 400 ml of 0.2 M NaCl.

3. Production of enterotoxin. Of the methods described by Casman and Bennett (19) for production of enterotoxin, cultivation of staphylococci on semisolid BHI agar (pH 5.3) is simple and does not require special apparatus. Add loopful of growth from nutrient agar slants to 3-5 ml sterile distilled water or saline.

Turbidity of suspension should be equivalent to No. 1 on McFarland nephelometer scale (approx. 3.00 x 10⁸ organisms/ml). Using sterile 1.0 ml pipet, spread 4 drops of aqueous culture suspension over entire surface of BHI agar plate with sterile spreader and incubate at 35°C. Good surface growth is obtained after 48 h incubation, when pH of culture should have risen to 8.0 or higher. Transfer contents of petri dish to 50 ml centrifuge tube with wooden applicator stick or equivalent. Remove agar and organisms by high speed centrifugation (10 min at 32,800 x g). Examine supernatant for presence of enterotoxin by filling depots in slide gel diffusion assembly (see E, below).

5. Slide gel diffusion test. To prepare record sheet, draw hole pattern of template on record sheet, indicate contents of each well, and number each pattern to correspond with number on slide.

1. Addition of reagents (Fig. 3). Place suitable dilution of antienterotoxin (antiserum) in central well and place homologous reference enterotoxin in upper peripheral well (if diamond pattern is used); place test material in well adjacent to well containing reference enterotoxin(s). If bivalent system is used, place other reference toxin in lower well. Use reference toxins and antitoxins (antisera), previously balanced, in concentrations that give line of precipitation about halfway between their respective wells.

Figure 3. Arrangement of antiserum (antisera) and homologous reference enterotoxins for assay of test preparation(s) for presence of 2 serologically distinct enterotoxins (simultaneously (bivalent detections system) or for assay of dilutions of a test preparation system (monovalent detection system).

Adjust dilutions of reagents to give distinct but faint lines of precipitation for maximum sensitivity (see C-9, above). Prepare control slide with only reference toxin and antitoxin. Fill wells to convexity with reagents, using Pasteur pipet (prepared by drawing out glass tubing of about 7 mm outside diameter) or disposable 30 or 40 1 pipet. Remove bubbles from all wells by probing with fine glass rod. Make rods by pulling glass tubing very fine, as for capillary pipets; break into 2-1/2 inch lengths and melt ends in flame. It is best to fill wells and remove bubbles against a dark background. Insert rods into all wells to remove trapped air bubbles that may not be visible. Before examination , keep slides in covered petri dishes containing moist sponge strips at room temperature for 48-72 h or at 37°C for 24 h

2. Reading the slide. Remove template by sliding it to one side. If necessary, clean by dipping slide momentarily in water and wiping bottom; then stain as described below. To examine, hold slide over source of light and against dark background. Identify lines of precipitation through their coalescence with reference line of precipitation (Fig. 4). Excessive concentration of enterotoxin in test material will inhibit formation of reference line, and test material must then be diluted and retested. Figure 5, diagram A, shows typical precipitate line inhibition caused by enterotoxin excess by test preparation reactant arrangement in Fig. 3. Figure 6 shows typical line formation of diluted preparation. Occasionally, atypical precipitate patterns may be difficult for inexperienced analysts to interpret. One of the most common atypical reactions is formation of lines not related to toxin but caused by other antigens in test material. Examples of such patterns are shown in Fig. 7.

Fig. 4. Microslide gel diffusion test as bivalent detection system. Antisera to staphylococcal enterotoxins A and B are in well 1; known reference enterotoxins A and B are in wells 3 and 5, respectively, to produce reference lines of A and B; test preparations are in wells 2 and 4. Interpret 4 reactions as follows: (1) No line development between test preparations and antisera--absence of enterotoxins A and B; (2) coalescence of preparation line from well 4 with enterotoxin A reference line (intersection of test preparation line with enterotoxin B reference line)-- absence of enterotoxins A and B in well 2, presence of enterotoxin A and absence of enterotoxin B in well 4; (3) presence of enterotoxin A and absence of enterotoxin B in both test preparations; and (4) absence of enterotoxins A and B in test preparation in well 2, presence of enterotoxins A and B in well 4.

Fig. 5. Effect of amount of staphylococcal enterotoxin in test preparation on development of reference line of precipitation. Diagram A demonstrates inhibition (suppression) of reference line when 10 and 4 µg enterotoxin/ml, respectively, are used. Diagrams B-E show precipitate patterns when successively less enterotoxin (test preparation) is used. Diagram F shows typical formation of reference line of precipitation observed in slide test control system.

Fig. 6. Microslide gel double diffusion tests as monovalent detection system in which varying dilutions of test preparation are assayed for the presence of staphylococcal enterotoxin.

Fig. 7. S. aureus enterotoxin: Precipitate patterns in microslide gel diffusion test demonstrate nonspecific (atypical) lines of precipitation caused by other antigens reacting with antienterotoxin antibodies. In pattern 1, test preparation in well 4 produces atypical reaction indicated by nonspecific line of precipitation (lines of nonidentity with enterotoxin references A and B) which intersects with enterotoxin reference lines. In pattern 2, both test preparations (wells 2 and 4) are negative for enterotoxins A and B but, produce nonspecific lines of precipitation, which intersect enterotoxin A and B reference lines of precipitation.

3. Staining of slides. Enhance lines of precipitation by immersing slide 5-10 min in Thiazine Red R stain, and examine. Such enhancement is necessary when reagents have been adjusted to give lines of precipitation that are only faintly visible. Use staining procedure described by Crowle (23), modified slightly, when slide is to be preserved. Rinse away remaining reactant liquid by dipping slide momentarily in water and immersing it for 10 min in each of the following baths: 0.1% (w/v) Thiazine Red R in 1% (v/v) acetic acid; 1% (v/v) acetic acid; 1% (v/v) acetic acid; and 1% (v/v) acetic acid containing 1% (w/v) glycerol. Drain excess fluid and dry slide in 35°C incubator to store as permanent record. After prolonged storage, lines of precipitation may not be visible until slide is immersed in water.

1. General precautions

• For raw or fermented foods and culture fluids from staphylococcal growth in laboratory media, check after extraction or collection of the culture fluid to determine if the test preparation contains peroxidase, which could interfere with the proper interpretation of results. To determine peroxidase presence, add 50 µl of sample to 50 µl of ELISA kit substrate reagent in an untreated microtiter plate (no antibody to staphylococcal enterotoxin) and let stand 10 min. If color changes to blue (or bluish-green), the sample contains intrinsic peroxidase, which must be inactivated. If sample remains colorless (or original color), analyze it for enterotoxin by ELISA. For inactivation of intrinsic peroxidase, prepare a 30% (w/v) solution of sodium azide and add 1 ml of this solution (30% w/v sodium azide) to 4 ml of test sample (final sodium azide concentration 6% (w/v)). Mix sample with azide solution, add extra sample additive, and let stand 1-2 min at room temperature. Retest sample for peroxidase presence (50 µl sodium azide-treated sample with 50 µl ELISA kit substrate reagent), as described above. If reaction is colorless (or original color), proceed with ELISA to identify enterotoxin in the peroxidase-inactivated sample. CAUTION: Use appropriate safety waste containers for disposal of preparations containing sodium azide, a hazardous material.

  When examining processed foods with obvious can defects which might result in the growth of organisms that produce peroxidase, test the extract for peroxidase production and inactivate as described above before testing for staphylococcal enterotoxin.

2. Procedures

Note: Raw food (e.g., vegetables), see, General Precautions, above. Follow directions under 5. Other Foods, below

1. Milk and milk powder. Reconstitute milk powder (25 g) by mixing with 125 ml 0.25 M Tris, pH 8.0. Treat reconstituted milk powder in same way as fluid milk. For milk samples (5.0 ml), ensure that pH is in range 7-8; then add 50 µlsample additive (in TECRA^TM kit). For clearer extract, adjust pH to 4.0 with concentrated HCl. For milk samples (50 ml), ensure that pH is in range 7-8; then add 50 µl sample additive (in kit). Centrifuge sample for at least 10 min at 1000-3000 x g. Decant extract and pump about 5.0 ml through syringe containing wetted absorbent cotton into polypropylene tube. Readjust pH to 7.0-8.0 (use pH paper), add 50 µl additive (in kit), and mix thoroughly.
2. Dehydrated food ingredients. Add 125 ml 0.25 Tris, pH 8, to 25 g of food, and homogenize in blender for about 3 min at high speed. Centrifuge sample for about 10 min at 1000-3000 x g and collect extract. Remove plunger from plastic syringe containing prewetted absorbent cotton and carefully pump solution through, collecting eluate. Take 5 ml of eluate; adjust pH to 7.0-8.0; then add 50 µl of sample additive, and mix thoroughly.
3. Cheeses. Add 50 ml water to 25 g of cheese and homogenize for about 3 min at high speed in blender. Adjust pH to 4 (pH paper) with concentrated HCl. Centrifuge sample for about 10 min at 1000-3000 x g. Remove plunger of plastic syringe containing prewetted cotton, and place 5.0 ml of extract into syringe; insert plunger and carefully pump solution through, collecting eluate. Take 5 ml of eluate, and add NaOH to adjust pH to 7.0-8.0; add 50 µl of sample additive, and mix thoroughly.
4. Other foods. Prepare foods other than those described above as follows: Add 50 ml 0.25 M Tris, pH 8, to 25 g of food and homogenize for about 3 min at high speed in blender. Centrifuge sample for about 10 min in bench centrifuge at 1000-3000 x g. Remove plunger from plastic syringe containing prewetted absorbent cotton and place 5 ml of extract into syringe; insert plunger and carefully pump solution through, collecting eluate in polypropylene tube. Take 5 ml of eluate; adjust pH, if necessary, to 7.0-8.0; add 50 µl of sample additive, and mix thoroughly.
NOTE: Prepare food extracts immediately before testing.

3. Proceed to desired assay protocol.
1. Test 200 µl sample extract for TECRA kit
2. Test 500 µl for VIDAS or Transia kits

Visual ELISA: Polyvalent (Types A-E) Screening for Determining
Enterotoxigenicity and Identifying Staphylococcal Enterotoxins in Foods

This visual immunoassay provides a rapid (4 h), sensitive (1.0 ng or greater per ml or g), specific screening test for the simultaneous identification of staphylococcal enterotoxins A-E. However, this kit cannot be used to distinguish among specific toxin serotypes. The ELISA is performed in a "sandwich" configuration. The kit is commercially available as TECRA^TM (TECRA Diagnostics, 28 Barcoo St., NSW, P.0. Box 20, Roseville, 2069, Australia) and is distributed by International Bioproducts Inc., 14796 N.E. 95th St., Redmond, WA 98052. This method has been adopted "First Action" by AOAC International (13).

1. Special equipment and supplies

Materials supplied in kit:

1. Anti-SET antibody coated Removawells (48 or 96 wells)
2. Removawell holder for securing wells
3. Instruction booklet methods manual
4. Color comparator
5. Protocol sheet

Materials/equipment supplied by user:

1. Absorbent cotton
2. Pipets, 50-200 µl; 5-20 µl
3. Tips, plastic
4. Incubator, 35-37°C
5. Plastic film wrap or sealable plastic container
6. Omnimixer, Waring blender (or equivalent) for preparation of food extracts
7. pH paper (range 0-14)
8. Centrifuge and Centrifuge cups
9. Plastic squeeze bottle (500 ml)
10. Disposable plastic syringes (25 ml)
11. Microplate shaker (optional)
12. Microplate reader (optional, but dual wavelength is recommended)
13. Polypropylene tubes (12 x 75 mm)
14. Polyethylene glycol (PEG, 15,000 - 20,000 mol wt)
15. Dialysis tubing (12,000 - 14,000 mol wt exclusion)
16. Balance
17. Beakers (250 ml)
2. Reagents

Materials supplied in kit:

1. Wash concentrate
2. Sample additive
3. Positive control; negative control
4. Conjugate diluent; conjugate, lyophilized
5. Substrate diluent; substrate, lyophilized
6. Stop solution

Reagents supplied by user:

1. Tris buffer (0.25 M; 30.28 g TRIS/liter, pH 8.0)
2. Sodium hydroxide solution (1.0 N NaOH)
3. Hydrochloric acid
4. Deionized or distilled water
5. Sodium hypochlorite
3. Preparation of materials and reagents
1. BHI with 0.7% (w/v) agar,
2. Syringe type filter (for foods). Prepare disposable plastic syringe (0.25 ml) by inserting plug of 0.5 cm thick absorbent cotton. Pump about 5.0 ml distilled water through to ensure tight packing. Do this just before filtering 5 ml of food extracts for treatment with additive provided in kit.
3. Reconstitution of wash solution. Dilute wash concentrate (as per kit directions) with distilled or deionized water in reagent bottle to 2 liters. Use this "wash solution" for washing wells and for diluting positive control when required.
4. Reconstitution of conjugate. Add conjugate diluent to conjugate and rehydrate at room temperature by gently mixing. This solution is referred to as "reconstituted conjugate."
5. Reconstitution of substrate. Add substrate diluent to substrate. Be sure contents have dissolved and are at room temperature before use.
4. General Precautions

• Note expiration date of kit. This is the last date on which product should be used. Prepare all reagents carefully and enter date of reconstitution on outside label of box. Use reconstituted kit within 56 days. Refrigerate all components (2-8°C) when not in use. DO NOT FREEZE.

• Components in SET visual immunoassay are intended for use as integral unit. Do not mix components of different batch numbers.

• Use a new pipet tip for each sample. Take care not to cross-contaminate wells. If plastic troughs are used to dispense conjugate and substrate, they must always be kept separate.

• Use positive and negative controls with each assay.

• Prepare trough containing 2% (v/v) sodium hypochlorite to be used for disposal of all samples that contain toxin.

• Store unused Removawell strips in pouch provided and re-seal with resealing strip after each use.

5. Laboratory production of toxin by suspect staphylococci (see Production of Enterotoxin, above).
6. Enterotoxin testing by polyvalent visual ELISA (Fig. 8)

   Secure desired number of anti-SET antibody-coated Removawells in holder provided. Allow 1 well for each food sample, 1 well for negative control, and 1 well for positive control. Additional wells are required if optional positive (food) and negative controls are prepared. Fill each well with wash solution and let stand 10 min at room temperature (20-25°C). Empty wells by quickly inverting holder; remove residual liquid by firmly striking holder face-down on paper towel several times.

   Transfer 200 µl aliquots of controls and samples (food extracts or culture fluids) into individual wells; record position of each sample on sample record sheet (original provided in kit). Gently tap holder containing test wells to ensure homogeneous distribution and contact of test material with walls of wells. Agitation of wells on microtiter plate shaker for 30 s is optional. To prevent evaporation, cover wells with plastic film or plate sealers (Dynex Technologies, Inc., 14340 Sullyfield Circle, Chantilly, VA 20151-1683) and incubate 2 h at 35-37°C. Wash well liberally with wash solution from squeeze bottles as follows: Press Removawells firmly into holder. Quickly invert holder, emptying contents into trough containing 2% (v/v) sodium hypochlorite. Remove residual liquid by firmly striking holder face-down on paper towel several times. Completely fill each well with wash solution. Repeat liberal washing 2-3 more times. Finally, empty wells.

   

   Figure 8. Typical double antibody "sandwich" ELISA scheme.

   Add 200 µl reconstituted enzyme conjugate into each well. Cover tray and incubate 1 h at room temperature (20-25°C). Empty wells and wash them thoroughly 5 times, as above. Empty wells and remove residual liquid as described above.

   Add 200 µl reconstituted substrate to each well. Leave at room temperature (20-25°C) for at least 30 min until positive control reaches absorbance greater than 1.0 or color darker than panel No. 4 on Color Comparator. Color development tends to concentrate around edge of wells. For accurate results, tap sides of plate gently to mix contents before reading. Add 20 µl of stop solution to each well. Tap sides of plate gently to mix contents. Assay is now complete. Determine results visually or with microtiter tray reader.

7. Interpretation of ELISA results
1. Visual observation. Place tray holding wells on white background; then compare individual test wells with Color Comparator provided in kit. Positive toxin control (and positive food control, if used) should give strong green color, indicating that all reagents are functional. If negative control is significantly darker than "negative" panels on Color Comparator, washing step was probably inadequate and assay must be repeated.

   Sample is considered positive when the following criteria are met:

   (1) negative control is within negative range on Color Comparator, and

   (2) sample has green (or blue) color greater than negative range on Color Comparator.

   Sample is considered negative for enterotoxin when the following criteria are met:

   (1) negative control is within negative range on Color Comparator, and

   (2) sample is colorless or has color within negative range on Color Comparator.

2. Absorbance measurement with microtiter tray reader. Read absorbance (A) of samples at 414 ± 10 nm, using microtiter tray reader. Prepare dual wavelength reader blank against air, and set second "reference" wavelength at 490 ± 10 nm. Typical wavelength settings could be A_405-490 or A_414-492 for peroxidase-based systems such as the described ELISA. Prepare single wavelength instrument blank on well containing 200 µl of substrate (provided in the kit) or water. Absorbance of positive toxin control should be at least 1.0, indicating that all reagents are functional. If absorbance of negative control is greater than 0.200, washing of wells was probably inadequate and assay must be repeated. Refer to Troubleshooting Guide in kit.

   Sample is considered positive if absorbance is > 0.200.

   Sample is considered negative if absorbance is <= 0.200.

   Generally, culture fluids that contain toxin have absorbance readings significantly greater than 0.200. Some strains of S. aureus produce intrinsic peroxidase, which can be inactivated with sodium azide.

1. Recommended controls

1. Positive toxin control. Prepare by making 1:100 dilution of positive control solution (in kit) in wash solution (50 µl to 5 ml wash solution, as per kit directions) in a polypropylene tube. Run positive control whenever assay is performed to indicate that all reagents are functional and that assay has been conducted correctly. Discard unused diluted toxin control into sodium hypochlorite solution.
2. Negative toxin control. Use negative control solution provided in kit. No dilution of negative control solution is necessary. Use 200 µl of all controls.
3. Positive food control (optional). Add aliquot of positive control provided in kit to known enterotoxin-negative food product to serve as positive food control. Extract and assay sample under same conditions as suspect sample.
4. Negative food control (optional). Use same type of food as suspect food, but which is known to be toxin-free. Prepare negative food control in exactly the same manner as suspect food. This control will ensure that washing of wells was adequate and that no food components will interfere with test results. Extract and assay sample under same conditions as suspect sample.
2. Serotype toxin confirmation

If confirmation of serotype by the AOAC method is necessary, use CHCl₃ to extract the food extract as previously described, and proceed with remaining steps in procedure. Analyze chromatographed eluate with microslide gel double diffusion test. A faster and more sensitive option would be confirmation by SDS-PAGE-Western blotting described in this Chapter.

This kit can be purchased from biomerieux Vitek, Inc.,545 Anglum Dr., Hazelwood, Missouri 63042-2395.

1. Special equipment

Materials and reagents supplied in kit:

1. 30 SET Reagent Strips

The SET Reagent Strip (refer to the table below) is a polypropylene strip of 10 wells covered with a foil seal and paper label. The first well of the strip is for the sample. The last well of the strip, an optically clear cuvette, is for the fluorometric determination. The eight wells in the center of the strip contain the various reagents for the assay. (See description of reagent strip below).




DESCRIPTION OF THE STAPH ENTEROTOXIN REAGENT STRIP
Wells	Reagents
1	Sample Well: 0.5 ml of food extract is placed into the well
2	Pre-Wash Solution (0.4 ml): TBS - Tween with 0.1% (w/v) sodium azide
3-4-5-7-8-9	Wash Solution (0.6 ml): TBS - Tween with 0.1% (w/v) sodium azide
6	Conjugate (0.4 ml): alkaline phosphatase labeled polyclonal antibodies with 0.1% (w/v) sodium azide
10	Cuvette with substrate (0.3 ml): 4-methyl-umbelliferyl phosphate with 0.1% (w/v) sodium azide
The name of the test, the lot number, and the expiration date of the kit are included on a bar code which is printed on the SET Reagent Strip. The test identification, lot number and calibration parameters are both clearly indicated in the kit's specification sheet and printed with a bar code.

2. 30 SET SPRs

The interior of the SET SPR is coated at the time of manufacture with anti-enterotoxin antibodies.

3. 1 Bottle Standard (3 ml)

Purified staphylococcal enterotoxin B (5 ng/ml) with 0.1% (w/v) sodium azide and protein stabilizers.

CAUTION: HANDLE WITH CARE!

4. 1 Bottle Positive Control (6 ml)

Purified staphylococcal enterotoxin B (5 ng/ml) with 0.1% (w/v) sodium azide and protein stabilizers. Control range indicated on the vial label. CAUTION: HANDLE WITH CARE!

5. 1 Bottle Negative Control (6 ml)

TRIS buffered saline (TBS) - Tween with 0.1% (w/v) sodium azide.

6. 1 Bottle Concentrated Extraction Buffer (55 ml)

2.5 mol/1 TRIS - 1% (w/v) Tween with 1% (w/v) sodium azide.

MATERIALS REQUIRED BY USER BUT NOT PROVIDED IN KIT:

1. Pipette which will dispense a minimum of 0.5 ml.
2. Tips, Plastic, to deliver 500 µl.
3. Centrifugation/filtration tubes (bioMerieux Product Number: 30550) or plastic syringes (20 ml)- optional.
4. Omni mixer, Waring blender (or equivalent) for preparation of food extracts.
5. pH paper (range 0-14).
6. Centrifuge.
7. Centrifuge cups.
8. Polyethylene glycol (15,000-20,000 mol wt).
9. Sodium hydroxide solution (1.0 N Na0H).
10. Hydrochloric acid.
11. Sodium hypochlorite.
12. Dialysis tubing, flat width 32 mm or comparable.
2. General Precautions, recommendations and considerations.

1. WARNINGS AND PRECAUTIONS

1. Routinely clean and decontaminate the VIDAS instrument. See the VIDAS Operator's Manual for the appropriate procedures.
2. Reagents contain 0.1% (w/v) sodium azide which could react with lead or copper plumbing to form explosive metal azides. If liquid containing sodium azide is disposed of in the plumbing system, flush drains with large volumes of water to avoid build-up.
3. The positive control and standard bottles contain purified staphylococcal enterotoxin. Handle with great care and use protective gloves. Consult physician immediately if ingested.
4. Handle all kit components as potentially biohazardous material. Dispose of all used components and other contaminated material by acceptable procedures for potentially biohazardous material.
2. STORAGE AND HANDLING

1. Store the VIDAS SET Kit at 2-8°C.
2. Do not freeze reagents.
3. Return unused components to 2-8°C.
4. The indicator on the desiccant in the resealable SPR storage pouch should be blue. Do not use the remaining SPRs in the pouch if the indicator is pink. Completely reseal the pouch after removing SPR's; this will maintain their stability.
5. When stored appropriately, all kit components are stable until the expiration date printed on the label. Do not use any kit components beyond the expiration date.
3. LIMITATIONS OF THE ASSAY

1. Do not mix reagents or disposables of different lot numbers.
2. Bring the reagents to room temperature before inserting them into the VIDAS.
3. Mix the standard, controls and samples well before use to ensure reproducibility.
4. Improper sample processing or storage may yield incorrect results.
4. SPECIFIC PERFORMANCE CHARACTERISTICS

Staphylococcal enterotoxins A, B, C1, C2, C3, D, E are detected by the VIDAS SET Assay at the sensitivity of at least 1 ng/ml.

3. Procedures for Preparation of Controls and Extraction of Enterotoxins from Suspect Foods.

In addition to the food extraction procedures described here, a greater variety of food extraction procedures are described by the kit manufacturer. Prepare food extracts immediately before testing.

1. Recommended controls

1. Positive toxin control.

Dispense 500 µl of control reagent provided in the kit. Run positive control whenever assay is performed to indicate that all reagents are functional and that the assay has been conducted correctly.

2. Negative toxin control.

Use negative control solution provided in kit. No dilution of negative control solution is necessary. Add 500 µl of negative control reagent to test strip.

3. Positive food control (optional).

Add aliquot of positive control provided in kit to known enterotoxin-negative food product to serve as positive food control. Extract and assay sample under same conditions as suspect sample.

4. Negative food control (optional).

Use same type of food as suspect food, but which is known to be toxin-free. Prepare negative food control in exactly the same manner as suspect food. This control will ensure that washing of wells was adequate and that no food components will interfere with test results. Extract and assay sample under same conditions as suspect sample.

2. Extraction of toxin from foods.
1. see section on Extraction of Enterotoxins from Foods for ELISA Assays, above.
4. ASSAY PROCEDURE, VIDAS

Important: A standard must be run in duplicate for every lot of kits. The result is stored in the computer and automatically used for assay analysis. A standard may be run with each SET work list, or a stored standard result (stored in the computer) may be used. See the VIDAS Operator's Manual for complete instructions.

1. Remove the VIDAS Staph enterotoxin kit from the refrigerator and allow it to come to room temperature (approximately 30 minutes).
2. Remove necessary components from the kit and return all unused components to storage at 2-8°C.
3. In the space provided, label the SET Reagent Strips with the appropriate sample identification numbers.
4. Enter the appropriate assay information to create a work list. Type "SET" to enter the assay code, and enter the number of tests to be run. If a standard is being tested, type "S" ("S" then "1" on mini VIDAS) for the sample ID. The standard may be run in any position of the work list. bioMerieux recommends running the standard in duplicate. See the VIDAS Operator's Manual for a complete explanation.
5. Pipette 0.5 ml of standard, control or sample into the center of the sample well of the SET Reagent Strip.
6. Load the SET Reagent Strips and the SET SPRs into the positions that correspond to the VIDAS section indicated by the work list. Check to make sure the color labels with the three letter assay code on the SPRs and the Reagent Strips match.
7. Dispose of all used SPRs and Reagent Strips in appropriate biohazard containers.
5. QUALITY CONTROL

A positive and negative control are provided to validate kit performance.

Test the positive and negative controls with each new lot or shipment to ensure that assay performance has remained unimpaired throughout shipping and storage. Test the controls as specified by your laboratory's regulatory guidelines. Controls are provided in ready-to-use form and must be thoroughly mixed and pipetted directly into the sample well of a reagent strip.

The expected positive control value will be: included in the range indicated on the vial label. If the results from testing the controls do not fall within this range, do not report sample results. NOTE: if the standard is out of range, the test value can be recalculated with another standard. See the VIDAS Operator's Manual for complete information.

6. INTERPRETATION OF RESULTS

Two instrument readings for fluorescence in the Reagent Strips's optical cuvette are taken for each specimen tested. The first reading is a background reading of the cuvette and substrate before the SPR is introduced into the substrate. The second reading is taken after the substrate has been exposed to the enzyme conjugate remaining on the interior of the SPR. The background reading is subtracted from the final reading to give a Relative Fluorescence Value (RFV) for the test result. A test value is generated for each sample by forming a ratio from the RFV of the sample to that of a standard. Test values from test samples and control samples are compared to a set of thresholds stored in the computer. The table below shows the thresholds and the interpreted results.

Thresholds and Interpretations
Test Value Threshold	Interpretation
< 0.13	Negative
>0.13	Positive

A report is printed that records the type of test performed, the sample identification, the date and time, the lot number and expiration date of the reagent kit being used and each sample's RFV, test value and interpreted result.

Results with test values less than the low threshold indicate sample without detectable enterotoxin. Samples with test values greater than (or equal to) the high threshold are reported as positive.

Invalid results are reported when the background reading is above a pre-determined cut-off (indicating low-level substrate contamination). In this case, repeat the assay with the original sample.

An invalid result is also seen if there is no standard available for the lot number of the sample test strip. In this case, run a standard in duplicate in SET strips with the same lot number as the invalid sample test. The sample test result can then be recalculated using the new stored standard. See the VIDAS Operator's Manual for complete information.

7. Serotype toxin confirmation

If confirmation of serotype by the AOAC method is necessary, use CHCl₃ to extract the food extract as previously described, and proceed with remaining steps in procedure. Analyze chromatographed eluate with microslide gel double diffusion test. A more sensitive and faster option is the SDS-PAGE-Western blotting method described in this Chapter.

This kit is produced by Transia-Diffchamb S.A. Lyon, France and is distributed by Idetek, Inc., Sunnyvale, CA. in the U.S.A.

1. Special equipment

Materials and reagents supplied in kit:

• Bladed tubes sensitized by a mixture of monoclonal antibodies specific for enterotoxin A, B, C, D and E, packed with a dehydrating agent into a plastic bag.

• Reference ST 714B contains 10 tubes.

• Reference ST 724B contains 20 tubes.

• Reference ST 744B contains 40 tubes.

• Vial 1: Negative control: ready to use.

• Vial 2: Positive control (concentrated 50X): mixture of staphylococcal enterotoxins A, B, C, D and E at 10 ng/ml. To be diluted to 1/50th before use.

  CAUTION: use gloves to handle.

• Vial 3: Washing buffer (concentrated 30X): to be diluted to 1/30th with distilled water.

• Vial 4: Conjugate: mixture of monoclonal and polyclonal anti-staphylococcal enterotoxin antibodies conjugated to peroxidase: ready to use solution.

• Vial 5: Substrate.

• Vial 6: Chromogen.

• Vial 7: Stopping solution: ready to use.

Equipment and reagents not provided in kit:

Equipment

1. Scales and weighing vessels.
2. homogenizer, mixer or stomacher.
3. Gloves.
4. Graduated pipettes.
5. Magnetic bars and magnetic stirrer.
6. Centrifuge tube.
7. Filter paper (Whatman or equivalent).
8. Laboratory centrifuge (1500 x g minimum).
9. Vortex mixer.
10. pH-meter or pH paper (range 0-14).
11. Clean glassware.
12. Test tube rack.
13. 100-1000 µl micropipettes and fitting tips.
14. Eppendorf type multipette, 5 and 2.5 ml tips for multipette.
15. Shaker (about 60 rpm).
16. 1 liter beaker.
17. Plastic squeeze bottle.
18. Absorbent paper.
19. Basin or another container: bleach- or soda-resistant.
20. Microcuvettes or unsensitized microtiter plastic wells.
21. Dialysis tubes (cut off threshold 12,000-14,000 Da)
22. Spectrophotometer for tubes or microcuvettes, with 450 nm filter (optional to microtiter plate reader).
23. Unsensitized (antibody free) removawell strips (plastic wells, Immulon 2® Removawell Strips cat. #011-010-6302, Dynatech Laboratories, Inc.) or equivalent.
24. Microtiter plate reader with 450 nm filter.
2. Reagents
1. Polyethylene glycol, mol wt 15,000-20,000
2. Decontamination solutions
3. TRIS buffer, 0.25 M, pH 8.0

• To prepare the samples:

1. Distilled water.
2. Extraction buffer: 0.25 M TRIS Buffer, pH 8.0.
3. for pH adjustment: NaOH 6N and HCl 6N.
4. For a possible concentration of the sample through dialysis: Polyethylene glycol (15,000-20,000 minimum) at 30% (w/v) in distilled water.

• To decontaminate materials and reagents:

  -   Chlorine bleach or 1N soda solution.

3. Reagent preparation

1. Extraction buffer:

For preparation of 1 liter, add 30.28 g TRIS hydroxymethy - aminomethane to approx. 800 ml. of distilled H₂0.Adjust to pH 8.0 and adjust volume to 1 liter.

2. pH adjustment:
1. Sodium hydroxide solution (NaOH) 6N: dissolve 240 g in 1 liter of distilled water.
2. Hydrochloric acid solution (HC1) 6N: dissolve 218,76 g in 1 liter of distilled water.
3. Polyethylene glycol solution:

Dissolve 30 g of polyethylene glycol (PEG) in 100 ml of distilled water or use dry flakes of PEG

4. Decontamination:

Bleach: dilute 50 ml of concentrated bleach in 950 ml of water.

NaOH 1N: dissolve 40 g in 1 liter of distilled water.

4. General Precautions and Recommendations.
1. Store the kit at 2-8°C when not in use.
2. Read the instructions for use entirely before using the kit.
3. Enterotoxins represent a potential risk or food poisoning. The use of gloves is highly recommended when performing the test.
4. Decontaminate all materials and reagents that have been in contact with staphylococcal enterotoxins with bleach or NaOH.
5. Do not pipette reagents by mouth.
6. Strictly follow incubation times.
7. In case of contact of any reagent with eyes or skin, rinse immediately with plenty of water.
8. A safety data sheet is available on request to the manufacturer.
5. Procedures for preparation of controls and the extraction of enterotoxins from suspect foods.

In addition to the food extraction procedures described here, a greater variety of food extraction procedures are presented in manufacturer's directions.

NOTE: Prepare food extracts immediately before testing.

1. Recommended controls

1. Positive toxin control.

Positive control solution is prepared by adding 10 µl positive control to 500 µl of wash buffer in a polypropylene tube. Run positive control whenever assay is run to verify that all reagents are functional and that assay has been conducted correctly. Discard unused diluted toxin control into sodium hypochlorite solution.

2. Negative toxin control.

Use negative control solution provided in kit. No dilution of negative control solution is necessary. Use 500 µl of all controls.

3. Positive food control (optional).

Add aliquot of positive control provided in kit to known enterotoxin-negative food product to serve as positive food control. Extract and assay sample under same conditions as suspect sample.

4. Negative food control (optional).

Use same type of food as suspect food, but which is known to be toxin-free. Prepare negative food control in exactly the same manner as suspect food. This control will ensure that washing of wells was adequate and that no food components will interfere with test results. Extract and assay sample under same conditions as suspect sample.

2. Extraction of toxin from foods (see Extraction of Enterotoxins from Foods for ELISA Assays, above.)
3. Immunoenzymatic Test, Transia^TM

Recommendation for Use

1. Bring all the reagents and the samples to room temperature (18-25°C) one hour before use.
2. Shake manually or vortex each vial before use.
3. Return the reagents to 2-8°C storage after use.
4. Do not interchange individual reagents between kits of different batch numbers.
5. The washing step is very important: when washing, direct a strong stream to the bottom of the tubes.
6. Incubations of the immunoenzymatic test are to be done under agitation (about 600 rpm).

6. Immunoenzymatic test

   See immunoenzymatic test flow chart (Figure 9)

   

   Figure 9. Scheme for Immunoenzymatic test.

   1. Remove the number of tubes needed from the bag and put them into the test tube rack.

   Allow:

   1 tube for the negative control (Vial 1),
   1 tube for the positive control (Vial 2),
   1 tube per sample.

   2. Return unused tubes to the plastic bag with dehydrating agent and close hermetically.
   3. Identify the tubes with the reference of the sample to test and note their location on the work sheet (ENRCOM 180).
   4. Prepare the washing buffer (Vial 3): Dilute the washing buffer in distilled water 30X, homogenize, and transfer to the plastic squeeze bottle.
   5. Prepare the positive control: dilute 40 µl of the positive control 50X (Vial 2) in 2 ml of reconstituted washing buffer. Mix properly (the preparation of a larger volume than the required volume reduces the errors that may occur from pipetting of a too low positive control volume).

   6. With a micropipette, add the controls and the samples: 500 µl per tube. Change tips each time.
   7. Incubate for 15 min at room temperature (18-25°C) with shaking.

   NOTE: Increasing the first incubation period from 15 to 60 min improves the detection of enterotoxins.

   8. Wash the tubes 3 times: empty the tubes by inverting them over a basin. Fill tubes rigorously with the washing buffer to the bottom of each tube and empty: repeat 3 times. Shake out the tubes with a downward motion on absorbent paper to completely remove any residual buffer.
   9. Add 500 µl conjugate (Vial 4) in all the tubes.
   10. Incubate for 15 min at room temperature (18-25°C) with shaking.
   11. Wash the tubes 5 times as described above. Return the washing buffer to 2-8°C storage.
   12. Prepare the substrate-chromogen mixture (Vial 5 and Vial 6) as follows: for n tubes, mix n x 300 µl of substrate with n x 300 µl of chromogen.
   13. Add 500 µl of the substrate-chromogen mixture to all tubes.

   NB: Separate addition of the substrate and the chromogen can be done: distribute successively 250 µl of substrate (Vial 5) and then 250 µl of chromogen (Vial 6).

   14. Incubate for 30 minutes at room temperature (18-25°C) while shaking.
   15. If results are read with a spectrophotometer or microtiter plate reader, add with a multipipette 500 µl of the stopping solution (Vial 7) to all the tubes.

7. Transia^TM test validation

The optical density of the positive control (PC) has to be higher than or equal to 0.40. The optical density of the negative control (NC) has to be lower than or equal to 0.25. The test can be validated if the optical densities of the controls meet the requirements defined above. If not, start the test again.

8. Interpretation of results.

Spectrophotometric reading

Read the optical densities at = 450 nm against an air blank. If you do not have a spectrophotometer for tubes, read the optical densities after having transferred the contents of the tubes into identified microcuvettes with 1 cm of optical pathway.

Microtiter plate reading

At FDA, a microtiter plate reader set at a wavelength of 450 nm is used to determine the optical density of each sample. This is accomplished by removing 200 µl of the test sample after addition of the stop solution and adding this volume (200 µl) to plain (unsensitized) flat bottom polystyrene microwells (Removawell, Dynatech) designed to fit a Removawell strip Holder (Dynatech Laboratories, Inc.). Place holder containing the sample extracts in microtiter plate reader and determine their absorbances. Record the results from the microtiter plate reader printout on the worksheet.

• Definitions:
  Positivity threshold:

  Optical density of the negative control + 0.20.

  • A sample is considered positive if its optical density is higher than or equal to the positivity threshold.

  • A sample is considered negative if its optical density is lower than the positivity threshold: the tested sample does not contain enterotoxins or does not contain a detectable level by the used method.

  • If the optical density of an extract is slightly lower (between NC + 0.15 and positivity threshold) than the positivity threshold, results can be checked by concentrating the extract through dialysis against 30% (w/v) PEG(cut off threshold 12,000 to 14,000 Da) and by testing again: an increase in the optical density means the presence of enterotoxins in small quantity.

    NOTE: Some food samples containing peroxidase, protein A or endogenous substances may interfere with this method. False positives are rare and inconsistent.

9. Serotype toxin confirmation.

If confirmation of serotype by the AOAC method is necessary, use CHCl₃ to extract as previously described, and proceed with remaining steps in procedures described in BAM. Analyze chromatographed eluate with microslide gel double diffusion test. A faster, more sensitive option would be to confirm with the SDS-PAGE-Western blotting method described in this chapter.

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2. Anderson, J.E., R.R. Beelman and S. Doores. 1996. Persistence of serological and biological activities of staphylococcal enterotoxin A in canned mushrooms. J. Fd. Prot. 59:1292-1299.

3. Anderson, J.E.1996. Survival of the serological and biological activities of staphylococcal enterotoxin A in canned mushrooms. UMI Dissertation Services, Ann Arbor, Michigan.

4. Association of Official Analytical Chemists. 1990. Official Methods of Analysis, 15th ed. AOAC, Gaithersburg, MD.

5. Baird-Parker, A.C. 1990. The staphylococci: An Introduction. J. Appl. bacterial Symp. Suppl. 15-85.

6. Bennett, R.W., and M.R. Berry, Jr. 1987. Serological reactivity and in vivo toxicity of Staphylococcus aureus enterotoxins A and D in selected canned foods. J. Food Sci. 52:416-418.

7. Bennett, R.W., and V. Atrache. 1989. Applicability of visual immunoassay for simultaneous indication of staphylococcal enterotoxin serotype presence in foods. ASM Abstracts, p. 28.

8. Bennett, R.W., D.L. Archer, and G. Lancette. 1988. Modified procedure to eliminate elution of food proteins under seroassay for staphylococcal enterotoxins. J. Food Safety 9:135-143.

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12. Bennett, R.W. 1994. Urea renaturation and identification of staphylococcal enterotoxin. In: R.C. Spencer, E.P. Wright and S.W.B. Newsom (eds.) RAMI-93. Rapid Methods and Automation in Microbiology and Immunology. Intercept Limited, Andover, Hampshire, England.

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44. Van der Zee, H. and K.B. Nagel. 1993. Detection of staphylococcal enterotoxin with Vidas automated immunoanalyzer and conventional assays. In 7th International Congress on Rapid Methods and Automation in Microbiology and Immunology. RAMI-93. Conference Abstracts 1993. PI127, p38.

Hypertext Source: Bacteriological Analytical Manual, 8th Edition, Revision A, 1998. Chapter 13A.
*Author: Reginald W. Bennett

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Hypertext updated by kwg/cjm 2001-OCT-24