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PET-RPLA TOXIN DETECTION KIT

Code: TD0930

A kit for the detection of enterotoxin type A of Clostridium perfringens in faecal samples or culture filtrates by reversed passive latex agglutination.

INTRODUCTION
Food poisoning can result from eating foods contaminated with Clostridium perfringens. The ingested cells multiply in the patient’s intestines and produce spores. The production of enterotoxin is associated with this spore-forming process. It is, therefore, important to detect the enterotoxin in faecal specimens obtained from the patient or in the culture fluid of the bacterial isolates.
The direct detection of Cl.perfringens enterotoxins in faeces is the most reliable method due to the much larger amount of toxin formed in vivo. The culture method is less reliable due to problems in encouraging Cl. perfringens to produce sufficient toxin in artificial media.
Reversed passive latex agglutination (RPLA) has been reported to be a reliable method for the detection of Cl.perfringens enterotoxin.¹ 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 immunised² with purified Cl. perfringens enterotoxin. These latex particles will agglutinate in the presence of Cl. perfringens 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 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 Cl. perfringens enterotoxin is present, agglutination occurs due to the formation of a lattice structure. Upon settling, this forms a diffuse layer on the base of the well. If Cl. perfringens enterotoxin is absent or at a concentration below the assay detection level, no such lattice structure can be formed. A tight button will therefore 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 PET-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 control should be stored at 2-8°C. Under these conditions, the reconstituted enterotoxin control will retain its reactivity for 3 months, or until the date shown on the kit box, whichever is the sooner.

SAMPLE PREPARATION
The direct detection of Cl. perfringens enterotoxin in faeces is the most reliable method and it should be followed unless insufficient faecal materials is available. It is much quicker than the cultural method, taking approximately 24 hours to obtain a result from sampling the faeces.
If the culture method is to be used for diagnosis of Cl. perfringens food poisoning it should be noted that Cl. perfringens is a common inhabitant of the bowel. It is recommended that at least 6 colonies are separately tested to ensure a higher probability of detecting an enterotoxin-producing strain.
There is no single culture procedure which is suitable for all enterotoxin-producing strains of Cl.perfringens. Any culture method used may fail due to insufficient toxin production. This may be due to inadequate growth, poor spore formation or low-level toxin production.
It should also be noted that some enterotoxin-positive strains may actually be killed by heat treatment and will not, therefore, produce enterotoxin in the second medium. It is recommended that each culture is checked for viability after heating.
It is advisable to test the particular cultural method used with a known enterotoxin-producing strain such as Cl. perfringens NCTC 8239 or Cl. perfringens ATCC® 12917.

METHOD OF USE
Materials required but not provided.
Microtitre plate (V-well) and lid.
Fixed or variable pipette and tips (25ml)
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 0.2mm-0.45mm (such as Millipore SLGV)
Culture media for proportion of enterotoxin production of Cl perfringens strains (a suitable medium is that developed by Duncan and Strong³ modified by Harmon and Kautter¹ (see Appendix)).
Sodium hypochlorite solution (>1.3%w/w)
25ml dropper (optional)
25ml diluter (optional)
Micromixer (optional)
Moisture box (optional)

Components of the Kit
Instruction Leaflet
TD931 Sensitised latex. Latex suspension sensitised with specific antibodies (rabbit lgG) against Cl. perfringens enterotoxin.
TD932 Latex control. Latex suspension sensitised with non-immune rabbit globulins.
TD933 Enterotoxin control Dried Cl. perfringens enterotoxin.
TD934 Diluent. Phosphate buffered saline containing bovine serum albumin.

Toxin Extraction or Production
Extraction from Faeces
To one volume of faeces, add a similar volume of phosphate buffered saline (Oxoid BR14A).
Homogenise completely and centrifuge at 1300g for 20 minutes at 4°C then membrane filter using a 0.2mm - 0.45mm low protein binding filter. The filtrate is then used as the test sample.
Production of Enterotoxin Culture Fluid
Culture the isolated organism in Cooked Meat Medium (Oxoid CM81) at 37°C for 18-20 hours, then inactivate by heating at 75°C for 20 minutes. Subculture to a medium designed for promotion of enterotoxin production ^{3 4} .
Modified Duncan and Strong Medium ^3,4

Autoclave at 121°C for 15 minutes.
Add a sufficient amount of filter-sterilised 0.66M sodium carbonate to increase the pH to 7.8 ± 0.1.
Inoculate 16 to 18ml of the medium with 0.8ml of the Cooked Mead Medium culture (taken from the base of the tube). NOTE: If the dilution rate is greater than 1:20, the Cooked Meat Medium constituents may be transferred in amounts large enough to inhibit spore formation.
Incubate at 37°C for 24 hours. After incubation either centrifuge at 900g for 20 minutes at 4°C and use the supernatant as the test sample or membrane filter using a 0.2mm-0.45mm 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 enterotoxin control add 0.5ml of diluent (TD934) 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 2 such rows.
Using a pipette or dropper, dispense 25ml of diluent in each well of the 2 rows except for the first well in each row.
Add 25ml 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 25ml and perform doubling dilutions along each of the 2 rows. Stop at the 7th well to leave the last well containing diluent only.
Add 25ml of sensitised latex to each well of the first row.
Add 25ml 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 from 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 solutions (>1.3% w/w).
Dispose of toxin controls and culture extracts in hypochlorite solutions (>1.3%w/w).

INTERPRETATION OF RESULTS
The agglutination pattern should be judged by comparison with the following illustration:

Results classified as (+), (++) and (+++) are considered to be positive.
In some cases, non-specific agglutination may be observed. If the faecal specimen or culture filtrate reacts with the sensitised latex to a dilution greater than that seen with the latex control, the test result should be regarded as positive. The last well in all rows should be regarded as invalid.

LIMITATIONS OF THE TEST
The sensitivity of this test in detecting the enterotoxin is approximately 2ng/ml.
Enterotoxin present at concentrations lower than this will, therefore, give negative results.
This kit is not intended for the detection of enterotoxins of other types of Clostridium perfringens.

REFERENCES
1. Harmon, S M and Kautter, D A (1986). J. Food Prot. 49: 523.
2. Sakaguchi, G. Vemura, T. and Riemann, H.P. (1973). J Appl. Microbiol, 26: 762.
3. Duncan, C.L. and Strong, D H (1968). J. Appl. Microbiol. 1 : 82
4. Harmon, S.M. and Kautter, D.A. (1986). J. Food Protection 49: 706.