Highly Sensitive and Specific Detection of Staphylococcal Enterotoxins SEA, SEG, SEH, and SEI by Immunoassay
Abstract
:1. Introduction
2. Results
2.1. Production and Characterization of 79 Monoclonal Antibodies Directed against SEA, SEG, SEH, or SEI Toxins
2.2. Development of Four Sandwich Enzyme Immunoassays for the Specific Individual Detection of SEA, SEG, SEH, and SEI Toxins
2.2.1. Selection of Best mAb Pairs
2.2.2. Performances of the Immunoassays
2.2.3. Robustness in Complex Matrices
2.3. Development of Immunochromatographic Tests for the Monoplex and Multiplex Detection of SEA, SEB, SEG, SEH, and SEI Toxins
3. Discussion
4. Conclusions
5. Materials and Methods
5.1. Bacterial Cultures
5.2. Recombinant His-Tagged SEA, SEG, SEH, and SEI Production and Purification
5.3. Production of Monoclonal Antibodies against SEA, SEG, SEH, and SEI
5.4. Labeling of Proteins with Biotin
5.5. Evaluation of Polyclonal Response and Screening of mAbs in Hybridoma Supernatants
5.6. Determination of mAb Affinity
5.7. Sandwich Enzyme Immunoassays
5.8. Theoretical Limits of Detection and Quantification
5.9. Repeatability and Reproducibility Assays
5.10. Food and Clinical Sample Preparation
5.11. Immunochromatographic Assays
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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S. aureus Strain | SE Genes | Origin and Source |
---|---|---|
07HMPA50 | Nontoxic coagulase-positive S. aureus | Commercial Camembert 1998 (NRL for CPS, ANSES, France) |
FRI S6 | sea, seb | Reference strain Food Research Institute, Frozen shrimp (M. S. Bergdoll [49]) |
A900322 | seg, sei, sep | Reference strain (Clinical strain from a patient with TSS, NRC for staphylococcus, France) |
16SBCL1204 | seg, sei | Food poisoning outbreak, rice chicken (NRL for CPS, ANSES, France) |
15SBCL1428 | seg, sei | Food poisoning outbreak, cheese (NRL for CPS, ANSES, France) |
FRI 569 | seh | Reference strain Food Research Institute Nasal swab (Y.-C. Su, A.C.L. Wong [31]) |
12CEB51 | seh | Food poisoning outbreak, Pot au feu (NRL for CPS, ANSES, France) |
14SBCL881 | seh | Food poisoning outbreak, mixed salad (NRL for CPS, ANSES, France) |
Target Toxin | mAb Pair | Theoretical LoD (pg/mL) | Theoretical LoQ (pg/mL) |
---|---|---|---|
Lab-made rec. SEA | SEA7/SEA5-biot | 2 | 10 |
Commercial SEA | 3 | 14 | |
Lab-made rec. SEA | SEA7/SEA12-biot | 7 | 35 |
Commercial SEA | 24 | 100 | |
Lab-made rec. SEG | SEG21/SEG26-biot | 6 | 23 |
SEG21/SEG27-biot | 5 | 16 | |
SEG26/SEG27-biot | N.C. | N.C. | |
SEG41/SEG27-biot | 8 | 24 | |
SEG21/SEG28-biot | 30 | 105 | |
SEG26/SEG28-biot | 3 | 9 | |
SEG32/SEG28-biot | 4 | 9 | |
SEG41/SEG28-biot | 4 | 10 | |
Lab-made rec. SEH | SEH1/SEH14-biot | 3 | 11 |
SEH16/SEH14-biot | 13 | 50 | |
SEH19/SEH14-biot | 47 | 135 | |
SEH1/SEH19-biot | 2 | 11 | |
SEH6/SEH19-biot | 148 | 346 | |
SEH11/SEH19-biot | 5 | 22 | |
SEH14/SEH19-biot | 53 | 179 | |
Lab-made rec. SEI | SEI27/SEI26-biot | 63 | 163 |
SEI36/SEI26-biot | 59 | 163 | |
SEI39/SEI26-biot | 34 | 103 | |
SEI44/SEI26-biot | 58 | 160 | |
SEI27/SEI32-biot | 57 | 184 | |
SEI36/SEI32-biot | 41 | 122 | |
SEI39/SEI32-biot | 41 | 131 | |
SEI44/SEI32-biot | 30 | 95 |
Capture/Tracer mAb Pair | |||||||
---|---|---|---|---|---|---|---|
SEA7/SEA5-biot | SEG41/SEG27-biot | SEH1/SEH19-biot | SEI27/SEI26-biot | ||||
Lab-Made Recombinant SEA | Commercial SEA | Lab-Made Recombinant SEG | Lab-Made Recombinant SEH | Lab-Made Recombinant SEI | |||
Calculated estimation using fitting curves | Theoretical LoD | (pg/mL) | 2.0 ± 1.3 (n = 20) | 5.4 ± 0.7 (n = 5) | 0.21 ± 0.09 (n = 12) | 0.59 ± 0.26 (n = 12) | 3.0 ± 1.5 (n = 12) |
(fM) | 70.7 ± 46.0 (n = 20) | 199.4 ± 25.8 (n = 5) | 7.4 ± 3.2 (n = 12) | 22.4 ± 9.9 (n = 12) | 114.9 ± 57.4 (n = 12) | ||
Theoretical LoQ | (pg/mL) | 6.0 ± 3.0 (n = 20) | 17.3 ± 6.1 (n = 5) | 0.66 ± 0.16 (n = 12) | 1.73 ± 0.72 (n = 12) | 10.1 ± 5.1 (n = 12) | |
(fM) | 212.1 ± 106.0 (n = 20) | 638.8 ± 225.3 (n = 5) | 23.4 ± 5.7 (n = 12) | 65.7 ± 27.4 (n = 12) | 386.7 ± 195.3 (n = 12) | ||
Experimental evaluation | Experimental LoD | (pg/mL) | 4.1 (100%, n = 28) | 12.3 (100%, n = 5) | 0.41 (100%, n = 17) | 1.37 (100%, n = 17) | 4.1 (94.1%, n = 17) |
(fM) | 144.9 (100%, n = 28) | 454.2 (100%, n = 5) | 14.5 (100%, n = 17) | 52.1 (100%, n = 17) | 157.0 (94.1%, n = 17) | ||
Experimental LoQ | (pg/mL) | 12.3 (100%, n = 28) | 37.0 (100%, n = 5) | 1.23 (100%, n = 17) | 4.12 (94.1%, n = 17) | 37.0 (100%, n = 17) | |
(fM) | 434.8 (100%, n = 28) | 1366.3 (100%, n = 5) | 43.6 (100%, n = 17) | 156.6 (94.1%, n = 17) | 1416.8 (100%, n = 17) |
Target Toxin | Quality Control Standard Concentration | Accuracy (bias) | Repeatability (CV) | In-House Reproducibility (CV) |
---|---|---|---|---|
Commercial SEA | 25 pg/mL (0.92 pM) | 101.3% | 14.1% | 15.9% |
50 pg/mL (1.85 pM) | 97.0% | 6.1% | 13.8% | |
100 pg/mL (3.69 pM) | 93.3% | 6.4% | 7.2% | |
300 pg/mL (11.08 pM) | 97.1% | 4.6% | 4.7% | |
1000 pg/mL (36.93 pM) | 96.7% | 5.2% | 5.5% | |
Lab-made recombinant SEA | 25 pg/mL (0.88 pM) | 95.0% | 8.2% | 9.5% |
50 pg/mL (1.77 pM) | 95.5% | 7.0% | 8.0% | |
100 pg/mL (3.53 pM) | 91.5% | 6.8% | 6.7% | |
300 pg/mL (10.60 pM) | 92.1% | 7.9% | 7.9% | |
1000 pg/mL (35.35 pM) | 91.3% | 9.6% | 8.9% | |
Lab-made recombinant SEG | 3 pg/mL (0.11 pM) | 90.7% | 6.9% | 8.2% |
5 pg/mL (0.18 pM) | 91.6% | 8.3% | 9.4% | |
10 pg/mL (0.35 pM) | 90.5% | 6.5% | 8.3% | |
30 pg/mL (1.06 pM) | 93.0% | 6.5% | 8.9% | |
60 pg/mL (2.12 pM) | 94.7% | 6.9% | 9.8% | |
Lab-made recombinant SEH | 5 pg/mL (0.19 pM) | 87.1% | 9.1% | 10.6% |
10 pg/mL (0.38 pM) | 89.0% | 7.1% | 12.6% | |
30 pg/mL (1.14 pM) | 88.0% | 6.1% | 7.6% | |
100 pg/mL (3.80 pM) | 89.9% | 5.4% | 6.3% | |
300 pg/mL (11.4 pM) | 88.3% | 7.0% | 7.8% | |
Lab-made recombinant SEI | 30 pg/mL (1.15 pM) | 88.8% | 8.6% | 8.8% |
50 pg/mL (1.91 pM) | 90.8% | 6.4% | 7.8% | |
100 pg/mL (3.83 pM) | 89.9% | 5.1% | 6.8% | |
300 pg/mL (11.49 pM) | 88.9% | 5.1% | 6.1% | |
1000 pg/mL (38.29 pM) | 88.3% | 3.7% | 5.3% |
mAb pair | Target SE | Measured SE Concentrations (ng/mL eq. Recombinant SE) in Culture Supernatant from Strain: | |||||||
---|---|---|---|---|---|---|---|---|---|
07HMPA50 | FRI S6 | A900322 | 16SBCL1204 | 15SBCL1428 | FRI 569 | 12CEB51 | 14SBCL881 | ||
SEA7/SEA5-biot | SEA | <LoD | 0.18 ± 0.02 | <LoD | LoD | <LoD | <LoD | <LoD | <LoD |
SEB27/SEB26-biot | SEB | <LoD | 13400 ± 2263 | <LoD | <LoD | <LoD | <LoD | <LoD | <LoD |
SEG41/SEG27-biot | SEG | <LoD | <LoD | 2.6 ± 0.9 | 5.3 ± 1.6 | 3.3 ± 0.4 | <LoD | <LoD | <LoD |
SEH1/SEH19-biot | SEH | <LoD | <LoD | <LoD | <LoD | <LoD | 103.6 ± 41.2 | 43.9 ± 14.4 | 82.7 ± 33.5 |
SEI27/SEI126-biot | SEI | <LoD | <LoD | 8.1 ± 7.7 | 1.9 ± 0.6 | 5.7 ± 0.1 | <LoD | <LoD | <LoD |
Context | Sample | EN ISO 19020 | Enzyme Immunoassays | Expected Results | ||||
---|---|---|---|---|---|---|---|---|
(Qualitative Detection) | ||||||||
Vidas SETII (TV(a)) | Ridascreen SET Total (AU(b)) | SEA (pg/g) | SEG (pg/g) | SEH (pg/g) | SEI (pg/g) | |||
Certified reference materials [42,43,44] | IRMM-359a cheese | not detected | not detected | <LoD (c) | <LoD (c) | <LoD (c) | <LoD (c) | SEA, SEG, SEH, and SEI not detected |
Vidas SET II: SEA to SEE not detected | ||||||||
<LoD (d) | <LoD (d) | <LoD (d) | <LoD (d) | Ridascreen ST: SEA to SEE not detected | ||||
IRMM-359b cheese | 1.35 TV (positive) | 0.96 AU (positive) | 38.4 (c) | <LoD (c) | <LoD(c) | <LoD (c) | 42 pg SEA/g (interval: 29–59 pg/g) | |
Vidas SET II: 1.14 TV (0.47–1.53 TV) | ||||||||
41.8 (d) | <LoD (d) | <LoD(d) | <LoD (d) | Ridascreen ST: 0.61 AU (0.28– 1.11 AU) | ||||
IRMM-359c cheese | 2.08 TV (positive) | 2.03 AU (positive) | 115.4 (c) | <LoD (c) | <LoD (c) | <LoD (c) | 102 pg SEA/g (interval: 81–145 pg/g) | |
Vidas SET II: 1.97 TV (1.10–2.42 TV) | ||||||||
110.5 (d) | <LoD(d) | <LoD (d) | <LoD (d) | Ridascreen ST: 1.36 AU (0.45–2.31 AU) | ||||
Samples from French CPS NRL collection | Emmental cheese 55 | 1.45 TV (positive) | 0.92 AU (positive) | 123.3 (c) | <LoD (c) | <LoD (c) | <LoD (c) | 80 pg SEA/g (spiking concentration) |
Emmental Cheese 469 | 1.45 TV (positive) | 0.92 AU (positive) | 185.4 (c) | <LoD (c) | <LoD (c) | <LoD (c) | 80 pg SEA/g (spiking concentration) | |
Morbier cheese 08BAC553 | 1.06 TV (positive) | 0.66 AU (positive) | <LoD (c) | <LoD (c) | <LoD (c) | <LoD (c) | Natural contamination with 180 pg SED/g |
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Féraudet Tarisse, C.; Goulard-Huet, C.; Nia, Y.; Devilliers, K.; Marcé, D.; Dambrune, C.; Lefebvre, D.; Hennekinne, J.-A.; Simon, S. Highly Sensitive and Specific Detection of Staphylococcal Enterotoxins SEA, SEG, SEH, and SEI by Immunoassay. Toxins 2021, 13, 130. https://doi.org/10.3390/toxins13020130
Féraudet Tarisse C, Goulard-Huet C, Nia Y, Devilliers K, Marcé D, Dambrune C, Lefebvre D, Hennekinne J-A, Simon S. Highly Sensitive and Specific Detection of Staphylococcal Enterotoxins SEA, SEG, SEH, and SEI by Immunoassay. Toxins. 2021; 13(2):130. https://doi.org/10.3390/toxins13020130
Chicago/Turabian StyleFéraudet Tarisse, Cécile, Céline Goulard-Huet, Yacine Nia, Karine Devilliers, Dominique Marcé, Chloé Dambrune, Donatien Lefebvre, Jacques-Antoine Hennekinne, and Stéphanie Simon. 2021. "Highly Sensitive and Specific Detection of Staphylococcal Enterotoxins SEA, SEG, SEH, and SEI by Immunoassay" Toxins 13, no. 2: 130. https://doi.org/10.3390/toxins13020130