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Staphylococcal Enterotoxin Test Kit for the detection of staphylococcal enterotoxins A, B, C and D in food samples or culture filtrates by reversed passive latex agglutination

INTRODUCTION
Staphylococcal food poisoning is caused by eating foods contaminated with enterotoxins produced during the growth of certain strains of Staphylococcus aureus. Reports on the assay of these toxins by reversed passive latex agglutination (RPLA) have been published. ^1,2,3The technique of reversed passive latex agglutination (RPLA) enables soluble antigen such as bacterial toxins to be detected in an agglutination assay.
In a standard agglutination assay, soluble antibody reacts with particulate antigen such as bacterial cells. However, in a REVERSED agglutination assay the antibody, which is attached to particles, reacts with the soluble antigen. The particles (in this case, latex) do not themselves play a part in the reaction and they are therefore PASSIVE. The cross-linking of the latex particles by the specific antigen/antibody reaction results in the visible LATEX AGGLUTINATION reaction.
The SET-RPLA test kit is based upon the reports by Shingaki et al.¹ and Oda et al.⁴ It was developed under the guidance of the Tokyo Metropolitan Research Laboratory of Public Health.
The SET-RPLA test may be used to detect staphylococcal enterotoxins in a wide variety of foods and to give a semi-quantitative result. The test may also be used to demonstrate enterotoxin production in isolates of S. aureus grown in culture. It should be noted that coagulase-negative staphylococci have been isolated which also produce enterotoxin in staphylococcal food poisoning.⁵

PRINCIPLE OF ASSAY
Polystyrene latex particles are sensitised with purified antiserum taken from rabbits, immunised individually with purified staphylococcal enterotoxins A, B, C and D. These latex particles will agglutinate in the presence of the corresponding enterotoxin. A control reagent is provided which consists of latex particles sensitised with non-immune rabbit globulins. The test is performed in V-well microtitre plates. Dilutions of the food extract or culture filtrate are made in five rows of wells, a volume of the appropriate latex suspension is added to each well and the contents mixed. If staphylococcal enterotoxins A, B, C or D are present, agglutination occurs, which results in the formation of a lattice structure. Upon settling, this forms a diffuse layer on the base of the well. If staphylococcal enterotoxins are absent or at a concentration below the assay detection level, no such lattice structure can be formed and, therefore a tight button will be observed.
The diluent provided contains sodium hexametaphosphate, which has been shown to reduce the incidence of non-specific reactions with components of food matrices.⁶

PRECAUTIONS
This product is for in vitro diagnostic use only.
Do not freeze.
Reagents with different lot numbers should not be interchanged.
Reagents and diluent contain 0.1% sodium azide as a preservative. Sodium azide may react with lead or copper plumbing to produce metal azides which are explosive by contact detonation. To prevent azide accumulation in plumbing, flush with copious amounts of water immediately after waste disposal.

STORAGE
The SET-RPLA Kit must be stored at 2-8° C. Under these conditions the reagents will retain their reactivity until the date shown on the kit box. After reconstitution, the enterotoxin controls should be stored at 2^-8° C. Under these conditions, the reconstituted enterotoxin controls will retain the reactivity for 3 months, or until the date shown on the kit box, whichever is the sooner.

SAMPLE PREPARATION
Food Matrices
A wide range of foods may be tested for staphylococcal enterotoxins; the extraction procedure may, however, require modification for particular foods. The main requirement is to achieve a non-turbid, fat-free extract. A low dilution factor is desirable for optimum sensitivity, but if the nature of the food dictates a greater dilution during extraction, a reduced sensitivity will result.
To gain a representative sample of a batch, a series of 10g portions are collected from different locations within the batch (see T.P.I., U.S.D.A. sampling plans or equivalent).

Culture Filtrates
Staphylococci from either clinical sources or food matrices may be recovered and identified using suitable techniques described in standard textbooks.

METHOD OF USE
Materials required but not provided
Blender or homogeniser
Microtitre plates (V-well) and lids
Fixed or variable pipette and tips (25ml)
Centrifuge capable of generating 900g (typically 300 rpm in a small bench top centrifuge)
Membrane filtration unit using low protein-binding disposable filters with a porosity of 0.2mm-0.45mm (such as Millipore SLGV)
Tryptone Soya Broth (CM129)
Sodium chloride solution (0.85%)
Sodium hypochlorite solution (>1.3% w/w)
25ml dropper (optional)
25ml diluter (optional)
Micromixer (optional)
Moisture box (optional)

Components of Kit
TD901 Latex sensitised with anti-enterotoxin A. Latex suspension sensitised with specific antibodies (rabbit lgG) against staphylococcal enterotoxin A.
TD902 Latex sensitised with anti-enterotoxin B. Latex suspension sensitised with specific antibodies (rabbit lgG) against staphylococcal enterotoxin B.
TD903 Latex sensitised with anti-enterotoxin C. Latex suspension sensitised with specific antibodies (rabbit lgG) against staphylococcal enterotoxin C.
TD904 Latex sensitised with anti-enterotoxin D. Latex suspension sensitised with specific antibodies (rabbit lgG) against staphylococcal enterotoxin D.
TD905 Latex control. Latex suspension sensitised with non-immune rabbit globulins.
TD906 Staphylococcal enterotoxin A control
TD907 Staphylococcal enterotoxin B control.
TD908 Staphylococcal enterotoxin C control.
TD909 Staphylococcal enterotoxin D control.
TD910 Diluent. Phosphate buffered saline containing bovine serum albumin and sodium hexametaphosphate.
Instruction leaflet

Toxin Extraction or Production
Extraction from Food Matrices
Blend 10g of sample with 10ml of sodium chloride solution (0.85%) in a blender or homogeniser.
Centrifuge the blended sample at 900g at 4° C for 30 minutes. NOTE: If a refrigerated centrifuge is not available, cool the sample to 4° C before centrifugation.
Filter the supernatant through a 0.2mm-0.45mm low protein-binding membrane filter. Retain the filtrate for assay of toxin content.
Production of Enterotoxins in Culture Fluids
Inoculate the isolated organism into Tryptone Soya Broth (CM129) and incubate at 37° C for 18-24 hours, preferably with shaking.
After growth, either centrifuge at 900g for 20 minutes at 4° C or membrane filter using a 0.2mm low protein-binding filter. Retain the filtrate for assay of toxin content.

Control
Each reconstituted toxin control will cause agglutination with its respective sensitised latex. The use of the toxin controls will provide references for the positive patterns illustrated below (see interpretation of Test Results). The controls should be used from time to time only to confirm the correct working of the test latex. The toxin controls are not provided at a specified level and therefore must not be used as a means of quantifying the level of toxin detected in the test sample.

Assay Method
Working Reagents
The latex reagents and diluent are ready for use. The latex reagents should be thoroughly shaken before use to ensure a homogeneous suspension. To reconstitute the control reagents, add 0.5ml of diluent (TD910) to each vial. Shake gently until the contents are dissolved.
Arrange the plate so that each row consists of 8 wells. Each sample needs the use of 5 such rows.
Using a pipette or dropper, dispense 25ml of diluent in each well of each of the 5 rows.
Using a pipette or dropper, dispense 25ml of test sample to the first well of each of the 5 rows.
Using a pipette or diluter and starting at the first well of each row, pick up 25ml and perform doubling dilutions along each of the 5 rows. Stop at the 7th well to leave the last well containing diluent only.
To each well in the first row, add 25ml of latex sensitised with anti-enterotoxin A.
To each well in the second row, add 25ml of latex sensitised with anti-enterotoxin B.
To each well in the third row, add 25ml of latex sensitised with anti-enterotoxin C.
To each well in the fourth row, add 25ml of latex sensitised with anti-enterotoxin D.
To each well in the fifth row, add 25ml of latex control.
To mix the contents of each well, rotate the plate by micromixer or agitate by hand. Take care that no spillage occurs from the wells.
To avoid evaporation, cover the plate with a lid. Placing the plate in a moisture box is an acceptable alternative. Leave the plate undisturbed on a vibration-free surface at room temperature for 20-24 hours. It will help subsequent reading of the test if the plate is placed on black paper for the duration of this incubation.
Examine each well in each row for agglutination, against a black background.
Centrifuge tubes, membrane filters, microtitre plates, lids and pipette tips should be sterilised by autoclaving at 121° C or disinfected before disposal in hypochlorite solutions (>1.3% w/w).
Dispose of culture extracts, food extracts, samples and toxin controls in hypochlorite solutions (>1.3% w/w).

INTERPREATION OF TEST RESULTS
The agglutination pattern should be judged by comparison with the following illustration.

Results classified as (+), (++), and (+++) are considered to be positive.
Results in the row of wells containing latex control should be negative. In some cases, non-specific agglutination may be observed. In such cases the results should be interpreted as positive, provided that the reaction with sensitised latex is positive to a higher dilution of test sample than that seen with the latex control. The last well in all rows should be negative. If positive patterns are observed in some of these wells, the reaction should be regarded as invalid.
NOTE: Certain staphylococcal strains are known to produce more than one enterotoxin.

LIMITATIONS OF THE TEST
The sensitivity of this test in detecting the enterotoxins has been reported to be 0.5ng/ml in the test extract. When a food extract is made with a dilution ration of 1 : 1 with diluent, the sensitivity is, therefore, 1ng/g of food matrix. The detection limit will vary according to any extra dilution conditions dictated by the type of food matrix. Concentration of the enterotoxin in the food extract can be effected by a variety of methods, such as ultrafiltration. Production in culture of SET’s depends on the growth conditions. A positive result obtained by the culture demonstrates the production of one or more SET under those circumstances; it does not imply the in vivo production of toxins to those levels.

REFERENCES
1. Shingaki,M et al. (1981). Ann. Rep. Tokyo Metro. Lab. Public Health 32: 128.
2. Oda. T., et al. (1979) Ann. Rep. Fukuoka City Lab Hyg. 4 : 33.
3. Park, C and Szabo, R. (1986). Can. J. Microbiol. 32 : 723.
4. Oda, T. (1978) Jap. J. Bacteriol. 33: 743.
5. Crass, B and Bergdoll, M (1986). J Clin. Microbiol. 23: 43.
6. Rose, S., Bankes, P. and Stringer, M. (1989) Int. J Food Microbiol. 8: 65-72