Nonspecific reactions of a commercial enzyme-linked immunosorbent assay kit (TECRA) for detection of staphylococcal enterotoxins in foods.

A staphylococcal enterotoxin visual immunoassay kit (TECRA) has recently become commercially available. Since the kit is an enzyme-linked immunosorbent assay system equipped with polyvalent antisera against staphylococcal enterotoxin types A to E (SEA to SEE) and the test is simple and rapid to perform (4 h), it has been widely used for screening purposes. In this study, the sensitivity of the kit for detection of SEA, SEB, and SEC in ham, cheese, and mushrooms was similar to those of kits based on an enzyme immunoassay and reversed passive latex agglutination: 0.75 to 1.0 ng of SEA per ml, 0.5 to 0.75 ng of SEB per ml, and 1.0 to 1.25 ng of SEC per ml. However, the TECRA kit showed nonspecific reactions with food samples contaminated by microorganisms other than Staphylococcus aureus, such as Enterobacter agglomerans, Enterobacter cloacae, Proteus mirabilis, Pseudomonas aeruginosa, and Serratia marcescens. The substance contributing to the false-positive results differed from true staphylococcal enterotoxins in that it was (i) heat labile (completely inactivated by heating for 2 min at 100 degrees C, whereas true staphylococcal enterotoxins were inactivated by about 10% with this treatment), (ii) lower in molecular weight than staphylococcal enterotoxins, and (iii) not bound to a copper chelate Sepharose gel (all of the substance remained in the unbound wash fraction, whereas staphylococcal enterotoxins were quantitatively bound to the gel). The problem of false-positive results with the TECRA kit could be resolved by heat treatment (2 min at 100 degrees C) or by cleanup procedures involving metal chelate affinity chromatography with copper chelate Sepharose for 4 h before use of the TECRA kit.     

Simple solutions to false-positive staphylococcal enterotoxin assays with seafood tested with an enzyme-linked immunosorbent assay kit (TECRA).

The TECRA kit, a commercial staphylococcal enterotoxin visual immunoassay kit, is an enzyme-linked immunosorbent assay system which utilizes polyvalent antisera against staphylococcal enterotoxin types A to E. The test is simple and rapid to perform (4 h) and has therefore been widely used for screening purposes. In this study, the TECRA kit produced a number of false-positive reactions with seafood; 25% of 218 samples of seven types of seafood gave false-positive results, particularly shellfish such as mussels (85%), clams (32%), oysters (23%), winkles (20%), and squid (13%). Some nonshellfish samples also gave false-positive results with the TECRA kit (smelt [20%] and trout [10%]). The substance contributing to the false-positive results differed from true staphylococcal enterotoxins in that it was: (i) heat labile, being completely inactivated by heating for 3 min at 70 degrees C, compared with 5% inactivation of true staphylococcal enterotoxins by the same heat treatment, (ii) in a selective reaction with normal rabbit or calf serum (nonspecific reactions were completely abolished by these sera, whereas staphylococcal enterotoxins were not affected), and (iii) incapable of binding to a copper-chelate Sepharose gel (all of the substance remained in the unbound wash fraction, whereas staphylococcal enterotoxins were quantitatively bound to the gel). The false-positive reactions occurring with seafood were not associated with substances produced by microorganisms, since the bacterial isolates from the samples did not give positive results with the TECRA kit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Statistical analysis and quality control in radioimmunoassays for staphylococcal enterotoxins A, B, and C.

The objective of these studies was to set up a reliable radioimmunoassay (RIA) for staphylococcal enterotoxins A, B, and C (SEA, SEB, and SEC) in a food system. Significant differences (95% confidence limits) were obtained between the 0- and 1-ng/ml enterotoxin standards, so the sensitivity of the RIAs was 1 ng/ml. Polystyrene tubes coated with anti-SEB and stored at 4 degrees C were unstable. The percentage of iodinated SEB bound to these tubes decreased at a rate of 0.33%/day, in contrast to the rate of 0.07%/day obtained with tubes prepared the day before the analyses. Satisfactory precision and maximum sensitivity were obtained by using six replicates for each sample and freshly coated tubes. The antisera used for coating the tubes were reused four times and were frozen between coatings. The process of drum drying mashed potatoes containing 1 mug of SEB per g of mashed potatoes inactivated 83% (wt/wt) of the SEB. Statistical quality control parameters were used to insure that RIAs were performing reliably with a sensitivity of 1 ng/ml. Over 450 samples of potato flakes and granules, which represented different production lots from 12 different manufacturers, were examined for SEA, SEB, and SEC. No enterotoxins were detected.     

Statistical analysis and quality control in radioimmunoassays for staphylococcal enterotoxins A, B, and C.

The objective of these studies was to set up a reliable radioimmunoassay (RIA) for staphylococcal enterotoxins A, B, and C (SEA, SEB, and SEC) in a food system. Significant differences (95% confidence limits) were obtained between the 0- and 1-ng/ml enterotoxin standards, so the sensitivity of the RIAs was 1 ng/ml. Polystyrene tubes coated with anti-SEB and stored at 4 degrees C were unstable. The percentage of iodinated SEB bound to these tubes decreased at a rate of 0.33%/day, in contrast to the rate of 0.07%/day obtained with tubes prepared the day before the analyses. Satisfactory precision and maximum sensitivity were obtained by using six replicates for each sample and freshly coated tubes. The antisera used for coating the tubes were reused four times and were frozen between coatings. The process of drum drying mashed potatoes containing 1 mug of SEB per g of mashed potatoes inactivated 83% (wt/wt) of the SEB. Statistical quality control parameters were used to insure that RIAs were performing reliably with a sensitivity of 1 ng/ml. Over 450 samples of potato flakes and granules, which represented different production lots from 12 different manufacturers, were examined for SEA, SEB, and SEC. No enterotoxins were detected.     

On the cross-reactivity of staphylococcal enterotoxins A, B, and C.

Strong cross-reactions were demonstrated for staphylococcal enterotoxins B (SEB) and C1 (SEC1) by antigen-binding capacity and by competitive binding ability. Both SEB and SEC1 combined completely with the heterologous antibody although requiring four times as much antiserum as the homologous enterotoxin and both displaced about one-third of the other enterotoxin from a heterologous antigen-antibody system. It is proposed that one of the three major antigenic determinants of these enterotoxins possesses a significant similarity but probably not an identity of structure. SEB and SEC1 did not combine with antiserum to enterotoxin A nor inhibit the reaction of SEA with anti-SEA. SEA had no intrinsic binding capacity for anti-SEB or anti SEC1 nor did it inhibit the binding of either enterotoxin to its own antibody. Affinity chromatography was employed to demonstrate that a small apparent binding of SEA to anti-SEB was due to antibody to SEA in the anti-SEB serum and that an almost complete displacement of SEC1 binding to anti-SEC1 was caused by contaminating SEC (about 0.01%) in preparations of enterotoxin A.     

Evaluation of a commercial enzyme immunoassay kit (RIDASCREEN) for detection of staphylococcal enterotoxins A, B, C, D, and E in foods.

The RIDASCREEN SET kit (R-Biopharm GmbH, Darmstadt, Germany), a commercial staphylococcal enterotoxin (SE) visual immunoassay kit, was evaluated for its efficacy. The kit utilizes monovalent capture antibodies against SE types A to E (SEA to SEE); therefore, it simultaneously detects and identifies the enterotoxin types. The major advantages of the kit are (i) a high degree of specificity (except for naturally occurring peroxidases, food compositions or ingredients and microbiological products due to growth of nonstaphylococcal microorganisms did not cause false-positive results; additionally, no cross-reactions among reagents of the kits were observed), (ii) excellent sensitivity (minimum detectable limits were 0.20 to 0.30 ng of SEs per ml of extracts of ham, salami, and mushroom and 0.30 to 0.35 ng of SEs per ml of cheese extracts, or 0.50 to 0.75 ng of SEs per g of foods such as noodles, ham, salami, cheese, and turkey), (iii) simplicity (the kit enabled direct assay of SEs in food extracts without the need for lengthy extraction or concentration procedures), (iv) rapidity (it took less than 3 h to complete the analysis of individual enterotoxin types SEA to SEE), and (v) its semiquantitative results (optical density values could be read against a standard curve to estimate the amount of SE in the extract). The RIDASCREEN kit is a convenient, rapid, and reliable tool for the detection and identification of SEs in foods.     

Detection of enterotoxigenicity of Staphylococcus aureus strains: a comparative use of the modified Ouchterlony precipitation test, reversed passive latex agglutination test, and avidin-biotin ELISA.

The avidin-biotin enzyme-linked immunosorbent assay (ELISA), reversed passive latex agglutination (RPLA) test, and the modified Ouchterlony precipitation test (MOPT) were compared in detecting enterotoxin production by Staphylococcus aureus strains. A total of 1015 strains isolated from human beings, animals, and foods were tested for staphylococcal enterotoxins A (SEA), B (SEB), and C (SEC). Of these, 495 (48.8%), 467 (46.0%), and 204 (20.1%) were classified as enterotoxigenic by the ELISA, RPLA test, and MOPT, respectively. The difference in the number of strains classified as enterotoxigenic by the ELISA and RPLA test was not significant (P > or = 0.05; chi 2), but both tests detected significantly (P < 0.001; chi 2) more enterotoxigenic strains than the MOPT. The combined use of the three assay systems classified 258 (25.4%), 278 (27.4%), and 263 (25.9%) of 1015 strains tested as positive for SEA, SEB, and SEC, respectively. However, the three systems were all positive in only 29.1% of SEA-producing strains, 32.0% of SEB-producing strains, and 25.1% of SEC-producing strains. The MOPT was negative when the corresponding ELISA and RPLA test were positive (46.9% for SEA, 43.5% for SEB, and 40% for SEC); the RPLA test was negative when the corresponding ELISA was positive (10.5% for SEA, 15.5% for SEB, and 25.5% for SEC); and the ELISA was negative when the RPLA test was positive (13.6% for SEA, 9.0% for SEB, and 9.5% for SEC). All factors considered, the RPLA test appears most suitable for quantitatively screening large numbers of strains for staphylococcal enterotoxins.     

Detection of antibodies to staphylococcal enterotoxins in the serum and milk of healthy goats

A study was made of the presence of antibodies (Ab) to staphylococcal enterotoxins A to E (SEA-SEE) in the serum and milk of 133 healthy goats, using a competitive ELISA method. Antibodies to some enterotoxins were detected in 83 sera (62.4%) and in 41 (30.8%) milk samples. In serum, antibodies to all SE types were detected, the most frequent being antibodies to SEA (24.8%). Milk contained antibodies to SEA, SEB and SEC, the latter being the most frequent (24.8%). A statistical study was performed to correlate the number of animals harbouring antibodies to a given enterotoxin with the presence in these animals of staphylococci producing that enterotoxin.     

Large-scale purification of staphylococcal enterotoxins A, B, and C2 by dye ligand affinity chromatography

A simple method for the purification of staphylococcal enterotoxins A (SEA), B (SEB), and C2 (SEC2) from fermentor-grown cultures was developed. The toxins were purified by pseudo-affinity chromatography by using the triazine textile dye "Red A" and gave overall yields of 49% (SEA), 44% (SEB), and 53% (SEC2). The purified toxins were homogeneous when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but isoelectric focusing of the preparations revealed the microheterogeneity associated with these toxins. The SEA and SEB preparations each consisted of two isoelectric forms with pI values of 7.3 and 6.8 (SEA) and 8.9 and 8.55 (SEB); in contrast, SEC2 contained five different isoelectric forms, with pI values ranging between 7.6 and 6.85. The pattern of elution of the isoelectric forms from the column indicated a cationic-exchange process involved in the binding of toxin to Red A. Such a method forms the basis of a high-yielding, rapid means of purifying the staphylococcal enterotoxins that can easily be adapted to large-scale production.     

Production and characterization of anti-peptide monoclonal antibodies with specificity for staphylococcal enterotoxins A and B

A synthetic peptide containing selected epitopes from staphylococcal enterotoxin A (SEA) and enterotoxin B (SEB) was used to produce monoclonal antibodies (Mabs) to respective enterotoxins in a single fusion procedure. The peptide inhibited the reaction of polyclonal anti-SEA or anti-SEB antisera with their homologous enterotoxin, thus showing that the chosen epitopes are part of the antibody-inducing enterotoxin sequences. Two Mabs, Mab-A and Mab-B, reacted with both the peptide and with either SEA or SEB. Used in a double antibody sandwich ELISA, the Mabs were able to quantitate the native SEA or SEB toxins at nanogram levels.     

Large-scale purification of staphylococcal enterotoxins A, B, and C2 by dye ligand affinity chromatography.

A simple method for the purification of staphylococcal enterotoxins A (SEA), B (SEB), and C2 (SEC2) from fermentor-grown cultures was developed. The toxins were purified by pseudo-affinity chromatography by using the triazine textile dye "Red A" and gave overall yields of 49% (SEA), 44% (SEB), and 53% (SEC2). The purified toxins were homogeneous when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but isoelectric focusing of the preparations revealed the microheterogeneity associated with these toxins. The SEA and SEB preparations each consisted of two isoelectric forms with pI values of 7.3 and 6.8 (SEA) and 8.9 and 8.55 (SEB); in contrast, SEC2 contained five different isoelectric forms, with pI values ranging between 7.6 and 6.85. The pattern of elution of the isoelectric forms from the column indicated a cationic-exchange process involved in the binding of toxin to Red A. Such a method forms the basis of a high-yielding, rapid means of purifying the staphylococcal enterotoxins that can easily be adapted to large-scale production.     

Monocyte chemoattractant protein-1 production by intestinal myofibroblasts in response to staphylococcal enterotoxin a: relevance to staphylococcal enterotoxigenic disease

Food poisoning due to staphylococcal enterotoxins A and B (SEA and SEB) affects hundreds of thousands of people annually. SEA and SEB induce massive intestinal cytokine production, which is believed to be the key factor in staphylococcal enterotoxin enteropathy. MHC class II molecules are the major receptors for staphylococcal enterotoxins. We recently demonstrated that normal human subepithelial intestinal myofibroblasts (IMFs) express MHC class II molecules. We hypothesized that IMFs are among the first cells to respond to staphylococcal enterotoxins and contribute to the cytokine production associated with staphylococcal enterotoxin pathogenesis. We demonstrated here that primary cultured IMFs bind staphylococcal enterotoxins in a MHC class II-dependent fashion in vitro. We also demonstrated that staphylococcal enterotoxins can cross a CaCo-2 epithelial monolayer in coculture with IMFs and bind to the MHC class II on IMFs. IMFs responded to SEA, but not SEB, exposure with 3- to 20-fold increases in the production of proinflammatory chemokines (MCP-1, IL-8), cytokines (IL-6), and growth factors (GM-CSF and G-CSF). The SEA induction of the proinflammatory mediators by IMFs resulted from the efficient cross-linking of MHC class II molecules because cross-linking of class II MHC by biotinylated anti-HLA-DR Abs induced similar cytokine patterns. The studies presented here show that MCP-1 is central to the production of other cytokines elicited by SEA in IMFs because its neutralization with specific Abs prevented the expression of IL-6 and IL-8 by IMFs. Thus, MCP-1 may play a leading role in initiation of inflammatory injury associated with staphylococcal enterotoxigenic disease.     

Detection rate of enterotoxigenic Staphylococcus aureus isolated from children with intestinal disbacteriosis

Detection rate of enterotoxigenic Staphylococcus aureus isolated from faeces of 62 children aged from 3 months old to 7 years old with intestinal dysbacteriosis was studied by indirect hemagglutination assay and enzume immunoassay. It was shown that strains of S.aureus producing staphylococcal enterotoxin A (SEA) are prevailed (40.3%) in children with disturbances of intestinal microflora while staphylococcal enterotoxin B (SEB)-producing strains were detected in 20.9% of children. Amount of produced enterotoxin varied for SEA from 125 ng/ml to 2000 ng/ml and for SEB--from 125 ng/ml to 250 ng/ml. Inverse dependence of detection rate of enterotoxin-producing strains in faeces on age of children was established. The most number of enterotoxigenic strains of S.aureus was detected in infants. These data point to expediency of determination of enterotoxin-producing ability of S. aureus strains isolated from children with dysbacteriosis as measure of danger of this microorganism for children's health and indication for adequate actions for its elimination.     

Production of enterotoxin A by supposedly nonenterotoxigenic Staphylococcus aureus strains

The production of staphylococcal enterotoxins A (SEA) and B (SEB) was studied by inoculating six well-defined staphylococcal collection strains into cow's, goat's, or sheep's milk (individually or as a 50% mixture of cow's + goat's or cow's + sheep's), into brain heart infusion, and into a medium generally used to enhance the synthesis of enterotoxins (3+3 medium). Four of the strains used are considered to be SEB producers, another is considered an SEA producer, and the remaining strain is nonenterotoxigenic but produces large quantities of staphylococcal protein A. Staphylococcal protein A masked the results in most cases. Only one strain secreted exclusively SEB, while the other three SEB producers synthesized SEA in different amounts. We conclude that enterotoxin production depends on the natural substrate and may differ from the results obtained when the strain is grown on cellophane over agar to determine its toxigenicity.     

Production of enterotoxin A by supposedly nonenterotoxigenic Staphylococcus aureus strains.

The production of staphylococcal enterotoxins A (SEA) and B (SEB) was studied by inoculating six well-defined staphylococcal collection strains into cow's, goat's, or sheep's milk (individually or as a 50% mixture of cow's + goat's or cow's + sheep's), into brain heart infusion, and into a medium generally used to enhance the synthesis of enterotoxins (3+3 medium). Four of the strains used are considered to be SEB producers, another is considered an SEA producer, and the remaining strain is nonenterotoxigenic but produces large quantities of staphylococcal protein A. Staphylococcal protein A masked the results in most cases. Only one strain secreted exclusively SEB, while the other three SEB producers synthesized SEA in different amounts. We conclude that enterotoxin production depends on the natural substrate and may differ from the results obtained when the strain is grown on cellophane over agar to determine its toxigenicity.     

Murine monoclonal antibodies reactive with staphylococcal enterotoxins A, B, C2, D, and E

By fusion of mouse spleen cells immunized with five different staphylococcal enterotoxins (SEA, SEB, SEC2, SED, and SEE) with myeloma cells, we obtained 15 hybridomas producing monoclonal antibodies (mAbs). Four mAbs were reactive with both SEA and SEE, whereas 8 mAbs were reactive with SEB and SEC2. One mAb reacted with SEA, SED, and SEE. The other two mAbs were found to be reactive with all five serotypes of SEs. The mAbs specific for five serotypes of SEs were found to be most reactive with SED, reactive with SEA, and slightly less reactive with SEB, SEC2, and SEE. Those mAbs with specificities for all serotypes of SEs may be valuable to prepare immunoadsorbent(s) for isolation of SEs and to detect SEs in foods and clinical specimens involved in outbreaks of staphylococcal food poisoning.     

Crystal structure of the superantigen staphylococcal enterotoxin type A.

Staphylococcal enterotoxins are prototype superantigens characterized by their ability to bind to major histocompatibility complex (MHC) class II molecules and subsequently activate a large fraction of T-lymphocytes. The crystal structure of staphylococcal enterotoxin type A (SEA), a 27 kDa monomeric protein, was determined to 1.9 A resolution with an R-factor of 19.9% by multiple isomorphous replacement. SEA is a two domain protein composed of a beta-barrel and a beta-grasp motif demonstrating the same general structure as staphylococcal enterotoxins SEB and TSST-1. Unique for SEA, however, is a Zn2+ coordination site involved in MHC class II binding. Four amino acids including Ser1, His187, His225 and Asp227 were found to be involved in direct coordination of the metal ion. SEA is the first Zn2+ binding enterotoxin that has been structurally determined.     

Bindings of toxic shock syndrome toxin-1 and staphylococcal enterotoxins A, B, and C to rabbit spleen cells

Toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxins (SE) A, B, and C were studied on binding to rabbit spleen cells. The toxins showed remarkable mitogenic effects on the cells. Among them, SEA and TSST-1 had much stronger mitogenic activities than SEB and SEC. Binding study showed that labeled TSST-1 and SEA bound considerably to cells, but that labeled SEB or SEC was not observed to bind at a detectable level under the same conditions as TSST-1 and SEA. Competitive binding analysis between toxins to cells proved that TSST-1 and SEA clearly competed with each other in binding. Scatchard plots for TSST-1 and SEA in binding were linear at the doses used. The Scatchard analysis for TSST-1 and SEA gave a dissociation constant of 2.5 X 10(-9) M and 7.6 X 10(-8) M and the number of binding sites per cell of 5.3 X 10(3) and 1.0 X 10(5), respectively.     

Raising Antibodies to Staphylococcal Enterotoxins A and B

A method is described for raising specific antibodies to staphylococcal enterotoxins A (SEA) and B (SEB) by intravenous inoculation of rabbits with small doses of enterotoxin diluted in sterile physiological saline. A course of six injections over a period of two weeks was given on four occasions. After the third course, 3/5 rabbits given SEA had a titre ± 1/50 and 4/6 given SEB had a titre of 1/50. Titres were not appreciably enhanced by the fourth series of injections. Only minor non-specific reactions which would require no absorption were found at the end of the series. It is believed that the findings in this study would be reproducible and that the method is likely to be suitable for raising antisera to other enterotoxins. Reference is also made to preliminary experiments in which latex sensitized with SEA or SEB was inoculated intravenously into rabbits.     

A Novel ELISA Format for the Rapid and Sensitive Detection of Staphylococcal Enterotoxin A

Staphylococcal food poisoning is one of the leading causes of bacterial food poisoning each year. Detection kits for staphylococcal enterotoxins are commercially available and the assays can require from one and a half to twenty-four hours to complete with detection limits ranging from 0.5 to 2 ng enterotoxin per gram of food. We have successfully demonstrated a microsphere-packed capillary (MPC) ELISA for the detection of staphylococcal enterotoxin A (SEA) and have compared it to two commercially available kits. The MPC assay detected a lower amount of SEA in ham, chicken, cheese, and bean sprouts than either of the two commercially available kits. In addition, the novel MPC assay was completed in less than ten minutes, as compared to three and twenty-four hours for the two commercially available kits. This research also demonstrated that the MPC ELISA can contain integrated positive and negative controls and has the potential to simultaneously detect and identify multiple enterotoxins.