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TST-RPLA TOXIN DETECTION KIT
STAPHYLOCOCCAL TOXIC SHOCK SYNDROME
Code: TD0940
A kit for the detection of staphylococcal toxic shock syndrome toxin in culture filtrates by reversed passive latex agglutination.
INTRODUCTION
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.
PRINCIPLE OF ASSAY
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.
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 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.
SAMPLE PREPARATION
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.
METHOD OF USE
Materials required but not provided
Microtitre plates (V-well) and lids
Fixed or variable pipette and tips (25µl)
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 0.2µm – 0.45µm (such as Millipore SLGV).
Brain Heart Infusion Broth (Oxoid CM225)
Sodium hypochlorite solution (>1.3% w/w)
25µl dropper (optional)
25µl 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 0.2µm
– 0.45µm low protein-binding filter and use the filtrate as the test sample.
Control
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 25µl of diluent in each well of the 2 rows
except for the first well in each row.
Add 25µl 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 25µl and perform doubling dilutions along each of the 2 rows. Stop at the
7th well to leave the last well containing diluent only.
Add 25µl of sensitised latex to each well of the first row.
Add 25µl 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)
INTERPRETATION 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 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.
LIMIATIONS OF THE TEST
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.
REFERENCES
1. 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.