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Toxin Detection Kits

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Code: TD0940

A kit for the detection of staphylococcal toxic shock syndrome toxin in culture filtrates by reversed passive latex agglutination.

Todd et al.1 were the first to describe the disease known as toxic shock syndrome. Staphylococcus aureus strains isolated from patients with this syndrome have been reported to produce a toxin, toxic shock syndrome toxin-one (TSST-1)1,2,3.The TST-RPLA test for the detection of TSST-1 in the culture fluid of Staphylococcus aureus was developed under the guidance of the Tokyo Metropolitan Research Laboratory of Public Health.
The 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.

Polystyrene latex particles are sensitised with purified antiserum taken from rabbits immunised4 with purified TSST-1. These latex particles will agglutinate in the presence of TSST-1. A control reagent is provided which consists of latex sensitised with non-immune rabbit globulins.
The test is performed in V-well microtitre plates. Dilutions of the culture filtrate are made in two rows of wells, a volume of the latex suspension is added to each well and the contents mixed. If TSST-1 is 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 TSST-1 is absent or at a concentration below the assay detection level, no such lattice structure can be formed. Therefore, a tight button will be observed.

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.

The TSE-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 TST control should be stored at 2-8°C. Under these conditions, the reconstituted TST control will retain its reactivity for 3 months, or until the date shown on the kit box, whichever is the sooner.

Staphylococcus aureus for testing may be recovered from swabs, etc. and identified by suitable techniques described in standard textbooks.
If Staphylococci are recovered from body sites, such as the vagina, which are normally colonised with staphylococci, it is recommended that several colonies are separately tested to ensure a higher probability of detecting TSST-1 producing strains.

Materials required but not provided
Microtitre plates (V-well) and lids
Fixed or variable pipette and tips (25l)
Centrifuge capable of generating 900g (typically 3000rpm in a small bench top centrifuge) or membrane filtration unit using low protein-binding disposable filters with a porosity of 0.2m 0.45m (such as Millipore SLGV).
Brain Heart Infusion Broth (Oxoid CM225)
Sodium hypochlorite solution (>1.3% w/w)
25l dropper (optional)
25l diluter (optional)
Micromixer (optional)
Moisture box (optional)

Components of the kit
TD941 Sensitised latex -
Latex suspension sensitised with specific antibodies (rabbit IgG) against staphylococcal TSST-1.
TD942 Latex Control - Latex suspension sensitised with non-immune rabbit globulins.
TD943 TST Control - Dried toxic shock syndrome toxin.
TD944 Diluent - Phosphate buffered saline containing bovine serum albumin.

Toxin Production in Culture Fluid
Inoculate the isolated into Brain Heart Infusion Broth (CM225). Incubate at 37° C for 18 to 24 hours, preferably with shaking. After growth, either centrifuge at 900g for 20 minutes at 4° C and use the supernatant as the test sample or membrane filter using a 0.2m 0.45m low protein-binding filter and use the filtrate as the test sample.

The reconstituted toxin control will agglutinate the sensitised latex. The use of the toxin control will provide a reference for the positive patterns illustrated below (see Interpretation of Test Results). The control should be used from time to time only to confirm the correct working of the test latex. The toxin control is 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 TST control, add 0.5ml of diluent (TD944) to the 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 2 such rows.
Using a pipette or dropper, dispense 25l of diluent in each well of the 2 rows except for the first well in each row.
Add 25l of test sample to the first and second well of each row
Using a pipette or diluter and starting at the second well of each row, pick up 25l and perform doubling dilutions along each of the 2 rows. Stop at the 7th well to leave the last well containing diluent only.
Add 25l of sensitised latex to each well of the first row.
Add 25l of latex control to each well of the second row.
To mix the contents of each well, rotate the plate by micromixer or agitate by hand. Take care that no spillage occurs form the wells.
To avoid evaporation, either cover the plate with a lid or place the plate in a moisture box. 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 solution (>1.3%w.w).
Dispose of toxin controls and culture extracts in hypochlorite solution (>1.3%w/w)

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 control latex 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.

The sensitivity of this test in detecting TSST-1 is approximately 2ng/ml in the extract. TSST-1 present at concentrations lower than this will, therefore, give negative results.
The production of TSST-1 by a Staphylococcus aureus is isolated from an individual does not constitute a diagnosis of toxic shock syndrome. Strains producing TSST-1 have been isolated from healthy individuals, although with a lower frequency that from patients with toxic shock syndrome.

Todd, J., Fishaut, M., Kapral, F. and Welch, T. (1978). Lancet ii: 116.
2. Cohen, M.L. and Falkow, S. (1981). Science 211: 842
3. Bergdoll, M.S. and Schievert. P.M. (1984) Lancet ii: 691
4. Igarashi, H., Fujkawa, H., Usami, H., Kawabata S. and Morita, T. (1984). Infection and Immunity44: 175.

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