TRANSIA^® PLATE Staphylococcal Enterotoxins

Background

Food poisoning with staphylococcal enterotoxins (SE) produced by coagulase-positive staphylococci (mainly Staphylococcus aureus) is a major health concern, causing nausea, vomiting, abdominal cramps and diarrhea. Outbreaks of SE in a variety of foods have been reported.^1-4 There are seven major SE toxins: SEA, SEC, SEC_1,2,3, SED, and SEE, which can be detected by most enzyme-immunoassay (EIA) kits.

The TRANSIA PLATE Staphylococcal Enterotoxin (SET) EIA contains specific capture and enzyme-conjugated detector antibodies to correctly detect all of these pertinent SE toxins at very low concentrations. The assay is designed to be highly selective, with no known cross reactivity with other protein toxins.

Principle of the Assay

The TRANSIA PLATE Staphylococcal Enterotoxin EIA consists of strips of 8 polystyrene wells that can be mounted into a 96-well format. The wells are coated with a mixture of monoclonal antibodies that capture all the Staphylococcal Enterotoxins A, B, C₁, C₂, C₃, D, and E. A sample is added to each well and if any SE are present, they will bind to the capture antibody specific for that SE. Once the plate is washed to remove any unbound material, a second mixture of monoclonal antibody-horse radish peroxidase conjugates is added. For those wells with SE present, the appropriate conjugate will bind to a different epitope on the SE. The plate is again washed to remove unbound material and a ready-to-use substrate/chromogen mixture is added which is converted by any enzyme present into a blue compound. An acid-based stop solution is added that turns the color to yellow/orange, which is read at 450 nm.

General Sample Preparation Flowchart

AOAC^® Performance Tested Method Validation Summary
PTM Certification # 031803
PTM Certified March 2018
Kit Components

• Microtiter Plate, 96 wells (8 wells x 12 strips)
• Negative Control
• Positive Control
• Washing Buffer
• Conjugate
• Substrate
• Stop Solution

AOAC Performance tested method study was done to determine the probability of detection (POD) for the toxins in various relevant food matrices.

TRANSIA PLATE Staphylococcal Enterotoxins EIA was validated according to the ISPAM Guidelines for Validation of Qualitative Binary Chemistry Methods⁶ and the AOAC INTERNATIONAL Methods Committee Guidelines for Validation of Microbiological Methods for Foods and Environmental Surfaces⁷ to determine the probability of detection (POD) for staphylococcal enterotoxins SEA, SEB, SEC₁, SEC₂, SEC₃, SED and SEE in raw milk cheese, liquid infant formula, eclairs, ready-to-eat ham, and canned mushrooms. The cheese and infant formula were tested with and without the dialysis/concentration protocol suggested in ISO standard 19020.⁸ The Performance Tested Methods^SM (PTM) validation has two main parts:  Method developer studies and the Independent Laboratory study. For this validation, the Method Developer studies include inclusivity/exclusivity studies (Tables 1 and 2 respectively), applicable matrix studies (Table 3), and a product consistency and stability study (Table 4). The Independent Laboratory study includes a matrix study of the infant formula using both the direct and the dialysis/concentration methods (Table 3).

Table 1. Validation of inclusivity of Staphylococcal Entertoxin EIA

No.	Organism	Source	Toxin ID (if known)a	Resultb
1	Staph. aureus	FDA, SA38c	SEC	+
2	Staph. aureus	FDA, SA310	SEA, SEB	+
3	Staph. aureus	FDA, SA312	SEA, SEB	+
4	Staph. aureus	FDA,SA316	SEA, SEB	+
5	Staph. aureus	FDA,SA327	SEB	+
6	Staph. aureus	FDA, SA332	SEA, SEB	+
7	Staph. aureus	FDA, SA333	SEA, SEB	+
8	Staph. aureus	FDA, SA334	SEA, SEB	+
9	Staph. aureus	FDA, SA340	SED	+
10	Staph. aureus	FDA, SA344	SEC	+
11	Staph. aureus	FDA, SA346	SEA, SEB	+
12	Staph. aureus	FDA, SA347	SEA, SEB	+
13	Staph. aureus	FDA, SA354	SEA, SEB	+
14	Staph. aureus	FDA, SA357	SED	+
15	Staph. aureus	FDA, SA214	SEA, SEB	+
16	Staph. aureus	FDA,SA228	SEC	+
17	Staph. aureus	FDA, SA245	SEA, SEB	+
18	Staph. aureus	FDA, SA247	SEA, SEB	+
19	Staph. aureus	FDA, SA254	SEA, SEB	+
20	Staph. aureus	FDA, SA261	SEA, SEB	+
21	Staph. aureus	FDA, SA4	SED	+
22	Staph. aureus	BCS, St1d	SEA	+
23	Staph. aureus	ATCC, 13301e	SEA	+
24	Staph. aureus	BCS, St35	SEB	+
25	Staph. aureus	BCS, St36	SEB	+
26	Staph. aureus	BCS, St41	SEB	+
27	Staph. aureus	BCS, St43	SEA	+
28	Staph. aureus	BCS, St45	SED	+
29	Staph. aureus	BCS, St46	SED	+
30	Staph. aureus	BCS, St47	SEC	+
31	Staph. aureus	BCS, St50	SEA	+
32	Staph. aureus	BCS, St51	SEB	+
33	Staph. aureus	BCS, St52	SEA	+
34	Staph. aureus	BEI, NR-45906f	SEB	+
35	Staph. aureus	BEI, NR-45914	SEA, SED	+
36	Staph. aureus	BEI,  NR-45915	SEC, SED	+
37	Staph. aureus	BEI,  NR-45917	SEA, SEC, SEE	+
38	Staph. aureus	BEI,  NR-45918	SEA	+
39	Staph. aureus	BEI,  NR-45920	SEB	+
40	Staph. aureus	BEI,  NR-45924	SEA, SEB	+
41	Staph. aureus	BEI,  NR-45926	SEA, SEB	+
42	Staph. aureus	BEI,  NR-45930	SED	+
43	Staph. aureus	BEI,  NR-46062	SEC	+
44	Staph. aureus	ATCC, BAA-1750		+
45	Staph. aureus	ATCC, BAA-1754		+
46	Staph. aureus	ATCC, BAA-1758		+
47	Staph. aureus	ATCC, BAA-1761		+
48	Staph. aureus	ATCC, BAA-1762		+
49	Staph. aureus	ATCC, BAA-1766		+
50	Staph. aureus	ATCC, BAA-1769		+
51	Staph. aureus	ATCC, BAA-1771		+
52	Staph. aureus	ATCC, BAA-1747		+
53	Staph. aureus	ATCC, 27664	SEE	+
54	Staph. aureus	ACC, 19095	SEC	+

^a determined using pcr (Tallent paper, FDA, BEI) or individual toxin test (BCS)
^b + is above threshold, - is below threshold
^c U.S. Food and Drug Administration
^d BioControl Systems culture collection
^e American Type Culture Collection
^f BEI Resources

Inclusivity was ascertained using Staphylococcus aureus strains known to produce toxins.⁵ 54 strains of Staphylococcus aureus were tested that are known to express at least one of the target Staphylococcal Enterotoxins. All were detected. Some collections identified the individual toxin(s) expressed and the inclusivity testing included representatives of each of the five major groups: A, B, C, D, E (Table 1).

Table 2. Validation of exclusivity of Staphylococcal Entertoxin EIA

No.	Organism	Source	Resulta
1	Staph. aureus	BCS, St2b	-
2	Staph. saprophyticus	BCS, St5	-
3	Staph. xylosus	BCS, St8	-
4	Staph. aureus	ATCC, 6538c	-
5	Staph. epidermis	ATCC, 12228	-
6	Staph. epidermis	ATCC, 14990	-
7	Staph. pseudointermedius	ATCC, 49444	-
8	Staph. xylosus	ATCC, 29971	-
9	Staph. aureus	ATCC, 25923	-
10	Staph. epidermis	BCS, St21	-
11	Staph. aureus	BCS, St23	-
12	Staph. warnerii	BCS, St24	-
13	Staph. warnerii	BCS, St25	-
14	Staph. saprophyticus	BCS, St26	-
15	Staph. epidermis	BCS, St32	-
16	Staph. aureus	BCS, St38	-
17	Staph. xylosus	BCS, St53	-
18	Staph. haemolyticus	BCS, St54	-
19	Staph. carnosus	BCS, St56	-
20	Staph. lentis	BCS, St59	-
21	Bacillus lichenforms	ATCC, 14580	-
22	Bacillus cereus	ATCC, 14579	-
23	Bacillus subtilis	ATCC, 6051	-
24	Bacillus cereus	ATCC, 13061	-
25	E. coli, stx 1, 2	ATCC, 43895	-
26	E. coli, stx 1, 2	ATCC, 43894	-
27	E. coli, stx 1, 2	ATCC, 700840	-
28	E. coli, stx 1, 2	ATCC, BAA-179	-
29	E. coli, stx 1, 2	ATCC, BAA-183	-
30	E. coli, stx 1, 2	ATCC, 51434	-

+ is above threshold, - is below threshold
^b BioControl Systems culture collection
^c American Type Culture Collection

Exclusivity was determined using 30 strains of a variety of Staphylococcus spp. that are known to not produce toxins.⁵ Also included are E. coli strains that produce Shiga toxins and Bacillus strains. All were negative using the TRANSIA PLATE Staphylococcal Enterotoxin EIA kit (Table 2).

Matrix Studies

Eclairs: The eclairs were inoculated directly into the cream filling in order to simulate what would be the most likely culprit to harbor staphylococcal enterotoxin. After mastication and centrifugation, there were four distinct layers. The breading precipitated to the bottom while the chocolate formed a layer just above the breading. The aqueous layer was next, with a solid layer of lipid on top. It was necessary to carefully extract 1 mL from the aqueous layer without disturbing the chocolate filling layer. The pH was consistently between 7.0 and 7.5 and the samples were tested directly. TRANSIA PLATE Staphylococcal Enterotoxin EIA was able to detect some of the SEA at 0.2 ng/g of eclair, with 4/20 positive samples. At the next higher tested level, 0.25 ng/g, there were 17/20 positives. Inoculation at 0.30 ng/g yielded 8/10 positives (Table 3).

Ham: The sliced deli ham was inoculated with both SEB and SEE. After mastication and centrifugation, the pH of all sample supernatants was around 6.5 and they were carefully titrated to 7.2 ± 0.2. Inoculation with SEB at 0.05 ng/g of ham yielded 0/20 positives whereas an increase in the inoculation level to 0.10 ng/g yielded 11/20 positives. Doubling the concentration again, to 0.20 ng/mL yielded 10/10 positives. (Table 3).

The inoculation with SEE at 0.10 ng/g yielded 7/20 positives. Inoculation at 0.15 ng/g yielded 20/20 positives. At 0.20 ng/g there were 10/10 positives (Table 3).

Canned Mushrooms: The canned mushrooms were inoculated with both SEC₁ and SED. These were blended as is without adding any liquid, and 25 g portions were aliquoted into 50 mL conical tubes. The samples were inoculated and mixed on a rotating wheel for 15 min. After allowing these to sit at room temperature for one additional hour, they were centrifuged. The pH of the all supernatants was around 6.5 and was carefully titrated to 7.2 ± 0.2. Inoculation with SEC1 at 0.03 ng/g of mushrooms plus liquid yielded 5/20 positives. Increasing the level of SEC1 to 0.05 ng/g yielded 14/20 positives.

A further doubling of the SEC₁ to 0.10 ng/g yielded 10/10 (Table 3). The inoculation with SED resulted in similar sensitivities, with 0.025 ng/g yielding 17/20 positives, while 0.04 ng/g yielded 20/20. At 0.05 ng/g, there were 10/10 positives (Table 3).

Infant Formula: The extraction of SEC2 from infant formula was done using two methods. The first method tested the formula directly without any extra handling. Using this extraction method yielded 0/20 positives at 0.05 ng/mL, 11/20 positives at 0.075 ng/mL and 10/10 positives at 0.10 ng/mL. When the dialysis/concentration method outlined in ISO 19020:2017 was used, the sensitivity improved some. Inoculation at 0.015 ng/mL yielded 3/20 positives and 0.03 ng/mL yielded 20/20 positives.

This extraction was also done at an Independent Laboratory and yielded very similar results. Using the direct extraction method yielded 2/20 positives at 0.05 ng/mL, 14/20 positives at 0.075 ng/mL, and 10/10 positives at 0.10 ng/mL. Using the dialysis/concentration method resulted in 2/20 positives at 0.01 ng/mL, 9/20 positives 0.02 ng/mL, and 10/10 positives at 0.03 ng/mL (Table 3).

Raw Milk Cheese: The extraction of SEC3 from raw milk cheese was also done using two methods. Because the aqueous portion of the extraction of cheese is rather viscous and difficult to test directly, it was subjected to acidification to precipitate the proteins. Once this mixture was centrifuged to clarify the liquid and its pH was adjusted to neutral, it was easier to test. Using this extraction method yielded 6/20 positives at 0.10 ng/g of cheese, 14/20 positives at 0.15 ng/g, and 10/10 positives at 0.20 ng/g. When this acidified cheese extract was subjected to the dialysis/concentration protocol,⁶ the sensitivity improved about 3–4 fold to give 7/20 positives at 0.03 ng/g, 14/20 positives at 0.04 ng/g, and 10/10 positives at 0.05 ng/g (Table 3).

Robustness Study

Table 3. Results of Enterotoxin extractions

Matrix/ Spiking Toxin	Extraction Procedure	Contamination Level	Number of replicates	Number of positives	POD	95% CI
Chocolate Elairs SEA	Direct	0 ng/g	10	0	0.00	0.00, 0.28
		0.20 ng/g	20	4	0.20	0.08, 0.42
		0.25 ng/g	20	17	0.85	0.64, 0.95
		0.30 ng/g	10	8	0.80	0.49, 0.94
Cooked ham SEB	Direct	0 ng/g	10	0	0.00	0.00, 0.28
		0.05 ng/g	20	0	0.00	0.00, 0.16
		0.10 ng/g	20	11	0.55	0.34, 0.74
		0.25 ng/g	10	10	1.00	0.72, 1.00
Cooked ham SEE	Direct	0 ng/g	10	0	0.00	0.00, 0.28
		0.10 ng/g	20	7	0.35	0.18, 0.57
		0.15 ng/g	20	20	1.00	0.84, 1.00
		0.20 ng/g	10	10	1.00	0.72, 1.00
Mushrooms SEC1	Direct	0 ng/g	10	0	0.00	0.00, 0.28
		0.03 ng/g	20	5	0.25	0.11, 0.47
		0.05 ng/g	20	14	0.70	0.48, 0.85
		0.10 ng/g	10	10	1.00	0.72, 1.00
Mushrooms SED	Direct	0 ng/g	10	0	0.00	0.00, 0.28
		0.025 ng/g	20	17	0.85	0.64, 0.95
		0.04 ng/g	20	20	1.00	0.84, 1.00
		0.05 ng/g	10	10	1.00	0.72, 1.00
Infant formula SEC2	Direct	0 ng/g	10	0	0.00	0.00, 0.28
		0.05 ng/g	20	0	0.00	0.00, 0.16
		0.075 ng/g	20	11	0.55	0.34, 0.74
		0.10 ng/g	10	10	1.00	0.72, 1.00
	Dialysis	0 ng/g	10	0	0.00	0.00, 0.28
		0.015 ng/g	20	3	0.15	0.05, 0.36
		0.03 ng/g	20	20	1.00	0.84, 1.00
		0.04 ng/g	10	10	1.00	0.72, 1.00
Infant formula SEC2a	Direct	0 ng/g	10	0	0.00	0.00, 0.28
		0.05 ng/g	20	2	0.10	0.03, 0.30
		0.075 ng/g	20	14	0.70	0.48, 0.85
		0.10 ng/g	10	10	1.00	0.72, 1.00
	Dialysis	0 ng/g	10	0	0.00	0.00, 0.28
		0.01 ng/g	20	2	0.10	0.03, 0.30
		0.02 ng/g	20	9	0.45	0.26, 0.66
		0.03 ng/g	10	10	1.00	0.72, 1.00
Raw milk cheese SEC3	Direct	0 ng/g	10	0	0.00	0.00, 0.28
		0.10 ng/g	20	6	0.30	0.15, 0.52
		0.15 ng/g	20	14	0.70	0.48, 0.85
		0.20 ng/g	10	10	1.00	0.72, 1.00
	Dialysis	0 ng/g	10	0	0.00	0.00, 0.28
		0.03 ng/g	20	7	0.35	0.18, 0.57
		0.04 ng/g	20	14	0.70	0.48, 0.85
		0.05 ng/g	10	10	1.00	0.72, 1.00

^a Performed by Independent Laboratory

Stability Study

The NC values for all three tested kits were quite similar, with the two older kits the same as the newer kit with 95% confidence (Student’s T-test, 2 tail, paired). This shows that the kits are stable for longer than the one year dating that they are given
(Table 4).

Table 4. Stability of Transia Plate Staphylococcal Enterotoxin test components

	Age of Kit (months)		Total samples	Results
Lot 1	2	Positive	10	10/10
		Negative	10	0/10
Lot 2	12	Positive	10	10/10
		Negative	10	0/10
Lot 3	14	Positive	10	10/10
		Negative	10	0/10

References

1. Fetsch A, Contzen M, Hartelt K, Kleiser A, Maassen S, Rau J, Kraushaar B, Layer F, Strommenger B. 2014. Staphylococcus aureus food-poisoning outbreak associated with the consumption of ice-cream. International Journal of Food Microbiology. 1871-6. https://doi.org/10.1016/j.ijfoodmicro.2014.06.017

2. Hennekinne J, De Buyser M, Dragacci S. 2012. Staphylococcus aureusand its food poisoning toxins: characterization and outbreak investigation. FEMS Microbiol Rev. 36(4):815-836. https://doi.org/10.1111/j.1574-6976.2011.00311.x

3. Teague NS, Grigg SS, Peterson JS, Gomez GA, Talkington DF. 2013. Outbreak of Staphylococcal Food Poisoning from a Military Unit Lunch Party MMWR . 62. Center for disease control and prevention: 1026-1027.

4. Kadariya J, Smith TC, Thapaliya D. 2014. Staphylococcus aureusand Staphylococcal Food-Borne Disease: An Ongoing Challenge in Public Health. BioMed Research International. 20141-9. https://doi.org/10.1155/2014/827965

5. Hait J, Tallent S, Bennett R. 2014. J AOAC. 971078-1083..

6. 2013. Official Methods of Analysis 19th Ed., Appendix J. [Internet]. Gaithersburg, MD. : AOAC INTERNATIONAL. Available from: www.eoma.aoac.org/app_j.pdf

7. 2013. Official Methods of Analysis. [Internet]. Available from: http://www.eoma.aoac.org/app_n.pdf

8. ISO 19020:2017(E) (2017) Microbiology of the food chain - Horizontal method for the immunoenzymatic detection of staphylococcal enterotoxins in foodstuffs. [Internet]. Available from: https://www.iso.org/standard/63747