15.5.10 (c) SET standard.

—Contains purified SET A with 1 g/L sodium

AOAC Official Method 2007.06 azide and protein stabilizers.
VIDAS SET 2 for Detection of Staphylococcal (d) SET positive control solution.—Contains purified SET A
Enterotoxins in Selected Foods with 1 g/L sodium azide and protein stabilizers.
Immunoassay Method (e) SET negative control solution.—Contains TRIS buffered saline
First Action 2007 (150 mmol/L) Tween pH 7.6 with 1 g/L sodium azide.
(f) SET 2 concentrated extraction buffer.—Contains 2.5 mol/L
Caution: Some reagents in the kit contain 1 g/L concentrations TRIS, 10 g/L Tween, 10 g/L MIT, pH 8.0. Diluted extraction buffer
of sodium azide. Check local regulations prior to
can be stored up to 3 months at 2–8°C.
disposal. Disposal of these reagents into sinks with
(g) Master lot entry (MLE) card.—To calibrate the test.
copper or lead plumbing should be followed
(h) Package insert.
immediately with large quantities of water to prevent
(i) NaOH.—1 N.
potential hazards.
(j) HCl.—5 N.
See Table 2007.06 for the results of the interlaboratory study C. Apparatus
supporting acceptance of the method. (a) VIDAS or mini-VIDAS automated immunoassay
A. Principle system.—Available from bioMérieux.
(b) Balance.—Weight range and tolerance of the balance should
The VIDAS Staph enterotoxin II (SET 2) test is an enzyme-linked
be ±0.1 g.
fluorescent immunoassay (ELFA) used on the automated VIDAS
instrument for the specific detection of Staphylococcal enterotoxins (c) Pipet with disposable tips.—Calibrated to dispense 500 mL.
(SET) A, B, C1, C2, C3, D, and E. The solid-phase-receptacle (SPR) (d) Blender and blender jars.
serves as the solid phase as well as the pipetting device for the assay. (e) Centrifuge and centrifugation tubes (50 mL).
Reagents for the assay are ready-to-use and predispensed in the sealed (f) Syringes.—10 mL.
reagent strips. (g) Absorbent cotton.
The instrument performs all of the assay steps automatically. The (h) pH paper.—Strip paper with 3 color bands and a precision at
user places the sample extract into the reagent strip. Then the sample least equal to 0.5 pH unit.
is cycled in and out of the SPR for a specific length of time. SET D. VIDAS SET 2 Procedure
present in the sample will bind to the anti-SET monoclonal Extraction and concentration protocols are food-type specific and
antibodies, which are coated on the interior of the SPR. Unbound are described in the method. The concentrated extracts are tested
s a m p l e c o m p o n e n t s a r e w a s h e d a w a y. A l k a l i n e immediately after preparation. The VIDAS or mini-VIDAS must be
phosphatase-labeled antibodies are cycled in and out of the SPR and readied by entering the master lot data and calibrating the
will bind to any SET captured on the SPR wall. Further wash steps instrument. A 500 mL aliquot of sample is then added to the reagent
remove unbound conjugate. strip. Standards and controls are analyzed in the same run. The strips
During the final detection step, the substrate and SPRs are inserted into the instrument and the assay is initiated
(4-methyl-umbelliferyl phosphate) is cycled in and out of the SPR. according to the operator’s manual.
The bound enzyme conjugate catalyzes the hydrolysis of this When the assay is completed, the results are analyzed
substrate into a fluorescent product (4-methyl-umbelliferone), the automatically by the instrument and a test value is generated. This
fluorescence of which is measured at 450 nm. test value is compared to a threshold and each result is interpreted as
SET are among the most common causes of food poisoning. positive or negative and a printed report is generated.
These heat-stable toxins have been found in meat, poultry, canned
E. VIDAS SET 2 Assay
mushrooms, dairy products, eggs, mayonnaise, and other foods. The
threshold amount of enterotoxin for causing illness in humans is not (a) For complete instructions, see the VIDAS or mini-VIDAS
known. However, information from food poisoning outbreaks and operator’s manual.
human challenge studies indicate that individuals experiencing (b) Enter factory master calibration curve data into the
illness probably consumed at least 100 ng enterotoxin A, the instrument using the MLE card.
serotype most frequently involved in foodborne staphylococcal (c) Remove the kit reagents and materials from refrigerated
illness. Ingestion of SET may cause gastrointestinal irritation and storage and allow them to come to room temperature for at least
other adverse effects. The positive control and standard contain 30 min.
purified SET A. Handle these reagents and food extracts with great (d) Use one VIDAS SET 2 reagent strip and one VIDAS SET 2
care. Use protective devices such as gloves, coat, and glasses. SPR for each sample, control, or standard to be tested. Reseal the
storage pouch after removing the required number of SPRs.
B. Reagents
(e) Enter the test code by typing or selecting “SET 2” on the
Items are available as the VIDAS SET 2 test kit from bioMérieux, instrument. If the standard is to be tested, identify the standard by
Inc. (595 Anglum Rd, Hazelwood, MO 63042, USA). Each kit “S1” and test in duplicate. If the positive control is to be tested,
contains sufficient materials and reagents for 30 tests. Store the SET identify it by “C1.” If the negative control is to be tested, identify it
2 kit at 2–8°C until use. by “C2.”
(a) Anti-SET-coated SPRs. Note: The standard must be tested upon receipt of a new lot of
(b) Reagent strips.—Containing prewash solution, wash buffer, reagents and then every 14 days. The relative fluorescence value
alkaline phosphatase-labeled anti-SET antibodies, and (RFV) of the standard must fall within the set range provided with
4-methyl-umbelliferyl phosphate. the kit.

ã 2007 AOAC INTERNATIONAL

 Table 2007.06. Interlaboratory study results for the detection of Staphylococcal enterotoxins in selected foods by the VIDAS SET 2 assay
95% 95%
Toxin Toxin Total Total Detection Confidence Confidence False
a b c d
Matrix type level, ng/g Replicates/lab te s te d p o s i ti v e rate , % interval LOD/g S p e c i fi c i ty interval p o s i ti v e , %
e
Cooked chicken A 0 .5 6 114 111 9 7 .4 9 2 .5 0 – 9 9 .4 5 0 .2 5 — — —
0 .2 5 6 114 112 9 8 .2 9 3 .8 1 – 9 9 .7 9 — — —
0 6 114 0 — — — 100 9 7 .4 1 – 1 0 0 0
e
Ham B 0 .5 6 108 106 9 8 .1 9 3 .4 7 – 9 9 .7 8 0 .2 5 — —
e
0 .2 5 6 108 107 99 9 4 .9 5 – 9 9 .9 8 — —

ã 2007 AOAC INTERNATIONAL

0 6 108 0 — — — 100 9 7 .4 1 – 1 0 0 0
e
P o ta to s a l a d C1 0 .5 6 108 108 100 9 7 .2 6 – 1 0 0 0 .2 5 — —
e
0 .2 5 6 108 107 9 9 .1 9 4 .9 5 – 1 0 0 — —
0 6 108 0 — — — 100 9 7 .2 6 – 1 0 0 0
e
Pasteurized liquid whole milk D 0 .5 6 102 101 99 9 4 .6 0 – 9 9 .9 8 0 .5 — —
e
0 .2 5 6 102 87 8 5 .2 7 6 .9 1 – 9 1 .5 3 — —
0 6 102 0 — — — 100 9 7 .2 6 – 1 0 0 0
e
Canned mushrooms E 0 .5 6 114 114 100 9 7 .4 1 – 1 0 0 0 .2 5 — —
e
0 .2 5 6 114 114 100 9 7 .4 1 – 1 0 0 — —
0 6 114 0 — — — 100 9 7 .4 1 – 1 0 0 0
a
Detection rate was defined as 100 ´ the total number positive test portions divided by the total number of test portions.
b
LOD/g = limit of detection of the assay per gram; for milk, the units are LOD/mL.
c
Specificity rate was defined as 100 ´ the total number of analyzed negative test portions divided by the total number of known negative test portions.
d
Incidence of false-positives is 100 – specificity rate.
e
— = Not determined.
 Thoroughly mix the standard, controls, and samples before use. (b) Blend at high speed to obtain a homogeneous suspension. If
(f) Pipet 500 mL of sample, standard, or control into the sample the suspension is too dense, add an additional 25 mL distilled water
well of the reagent strip. and reblend or restomach.
Note: Test samples and controls singly and standard in duplicate. (c) Adjust pH to 4.0 with 5 N HCl.
(g) Insert the SPRs and reagent strips into the instrument. Verify (d) Let stand for 15 to 30 min at 18–25°C.
that the color labels with the assay code on the SPRs and reagent (e) Centrifuge the suspension for 15 min at 3000–5000 ´ g at
strips match. 18–25°C.
(h) Initiate the assay as directed in the operator’s manual. The (f) Using the plunger, pump the supernatant liquid through
instrument performs all assay steps and data interpretation moistened absorbent cotton placed in a syringe.
automatically and the assay will be completed within ca 70 min. (g) Check the filtrate pH and adjust to between 7.5 and 8.0 with
(i) After the assay is completed, remove the SPRs and reagent 1 N NaOH.
strips from the instrument and dispose of properly. (h) If a precipitate forms after pH adjustment, centrifuge an
(1) General extraction protocol.
aliquot for 15 min at 3000–5000 ´ g at 18–25°C.
(a) To 25 g food, add 25 mL reconstituted extraction buffer.
(5) Dairy product protocol without TCA precipitation.—This
(b) Blend at high speed to obtain a homogeneous suspension.
protocol includes an acidification step, which precipitates milk
(c) Let stand for 15 min at 18–25°C. proteins that may interfere in the test. The SET remain soluble at this
(d) Centrifuge the blended sample for 15 min at 3000–5000 ´ g at pH. After centrifugation, readjustment of the extract to neutral pH is
18–25°C. Pump the supernatant liquid through moistened absorbent necessary for an optimum antigen-antibody reaction.
cotton placed in a syringe, using the plunger. (a) For liquid products, adjust pH of 25 mL sample to between
(e) Check the filtrate pH and adjust to between 7.5 and 8.0 if 3.5 and 4.0 with 5 N HCl. Proceed to step f.
necessary, using 1 N NaOH. (b) For solid products, add 40 mL distilled water prewarmed to
(2) Liquid food protocol. 38 ± 2°C to 25 g food.
(a) Dilute the concentrated food product as indicated by the (c) Blend at high speed to obtain a homogeneous suspension.
manufacturer. For ready-to-drink products, go to step (b).
(d) Let stand for 30 min at 18–25°C.
(b) In the case of a precipitate, centrifuge the diluted product for
(e) Check the pH and adjust to between 3.5 and 4.0 using 5 N
15 min at 3000–5000 ´ g at 18–25°C and pump the supernatant
HCl.
liquid through moistened absorbent cotton placed in a syringe, using
(f) Centrifuge the suspension for 15 min at 3000–5000 ´ g at
the plunger.
(c) Check the filtrate pH and adjust to between 7.5 and 8.0 if 18–25°C.
necessary, using 1 N NaOH. (g) Recover the supernatant liquid and adjust the pH to between
(3) Canned food protocol. 7.5 and 8.0 with 1 N NaOH.
(a) Blend at high speed the whole canned food or a representative (h) Centrifuge the suspension for 15 min at 3000–5000 ´ g at
aliquot to obtain a homogeneous suspension. 18–25°C and filter if necessary. Recover the filtrate.
(b) To 25 g suspension, add 25 mL reconstituted extraction F. Assay Results
buffer.
The test value is calculated by the instrument and is equal to the
(c) Proceed as described in General Extraction Protocol from
sample RFV/standard RFV. A “negative” result has a test value less
step b.
than the threshold (0.13) and indicates that the sample does not
(4) Raw meat products, seafood, and delicatessen meats
contain SET or contains SET at a concentration below the detection
protocol.
limit. A “positive” result has a test value equal to or greater than the
This protocol includes an acidification step, which precipitates
some proteins that may interfere in the test. The SET remain soluble threshold (³0.13) and indicates that the sample is contaminated with
at this pH. After centrifugation, readjustment of the extract to SET. If the background reading is above a predetermined cutoff,
neutral pH is necessary for an optimum antigen-antibody reaction. then the result is reported as invalid.
(a) To 25 g food, add 25 mL distilled water. Reference: J. AOAC Int. (future issue).

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