Rapid detection of staphylococcal enterotoxins in foods with a modification of the reversed passive latex agglutination assay

A modification of the reversed passive latex agglutination kit assay for detection of staphylococcal enterotoxins by centrifugation of microtitration plates reduces the incubation time of the assay from 20–24 h to 4 h. Enterotoxins can therefore be detected in foods within the working day.     

Evaluation of the reversed passive latex agglutination (RPLA) test kits for detection of staphylococcal enterotoxins A, B, C, and D in foods

Four lots of the SET-RPLA kit (Denka Seiken Co. Ltd., Tokyo), a commercial reverse passive latex agglutination test kit for the detection of staphylococcal enterotoxins A, B, C, and D in foods, have been evaluated for their efficacy. The kits showed high specificity and sensitivity with a detection limit of 0.75 ng enterotoxin/g of food. The test is simple, is completed within 24 h, and does not require complicated extraction or concentration procedures nor expensive equipment. In addition, the assay is semiquantitative. However, as in any other immunological system, routine verification of the specificity of the latex reagents against standard enterotoxins and toxin-free food extracts is necessary.     

Rapid latex agglutination test for detection of staphylococcal enterotoxins A to E that uses high-density latex particles

A rapid reversed passive latex agglutination method that uses high-density latex particles for the detection of staphylococcal enterotoxins (SE) A to E was developed. It took 3 h for incubation, much less than the 16 h needed with a customary latex agglutination test for SE detection such as a commercial test kit (SET-RPLA; Denka Seiken Co. Ltd., Tokyo, Japan). The rapid test was shown to be highly specific and sensitive for SE detection (detection limit, about 0.5 ng of SE per ml), comparable to the SET-RPLA test. The rapid test was also efficient in SE detection in foods and culture supernatants of staphylococcal strains, similar to the SET-RPLA test. This showed that a rapid test with high-density latex particles is fully reliable for use.     

Rapid latex agglutination test for detection of staphylococcal enterotoxins A to E that uses high-density latex particles.

A rapid reversed passive latex agglutination method that uses high-density latex particles for the detection of staphylococcal enterotoxins (SE) A to E was developed. It took 3 h for incubation, much less than the 16 h needed with a customary latex agglutination test for SE detection such as a commercial test kit (SET-RPLA; Denka Seiken Co. Ltd., Tokyo, Japan). The rapid test was shown to be highly specific and sensitive for SE detection (detection limit, about 0.5 ng of SE per ml), comparable to the SET-RPLA test. The rapid test was also efficient in SE detection in foods and culture supernatants of staphylococcal strains, similar to the SET-RPLA test. This showed that a rapid test with high-density latex particles is fully reliable for use.     

Enzyme-linked immunosorbent assay for detection of staphylococcal enterotoxins in foods.

An enzyme-linked immunosorbent assay (ELISA) was developed for detection of staphylococcal enterotoxins in foods. The "double-antibody sandwich" protocol combines parts of several procedures reported previously. Horseradish peroxidase was conjugated to antibody specific for an enterotoxin, and the antibody-enzyme conjugate was assayed with a 2,2'-azino-di-(3-ethylbenzthiazoline sulfonic acid)-H2O2 substrate solution. Enterotoxins were added to a variety of foods that were representative of those implicated in staphylococcal food poisoning outbreaks. Extracts of the foods were assayed by the ELISA and radioimmunoassay. Enterotoxin levels below 1 ng/g of food were consistently detectable by the ELISA. These results compared favorably with those of the radioimmunoassay. Experiments confirmed the interference of protein A in double-antibody sandwich ELISAs. Although protein A interference has not been demonstrated to be a problem in food extracts, we suggest a screen for protein A interference in which immunoglobulin G from nonimmunized rabbits is used. All of the known staphylococcal enterotoxins could be detected by this method. Analysis of a food product for entertoxin by the ELISA can be completed in an 8-h working day.     

Detection of Clostridium difficile toxin A by reversed passive latex agglutination.

A reversed passive latex agglutination (RPLA) assay for detecting Clostridium difficile toxin A is presented. Purified monoclonal antibody (mAb 37B5) was used for latex sensitization. The culture supernatants of 93 strains of C. difficile were tested by RPLA assay and the results compared with those of a commercially available latex agglutination test, PCR and cytotoxin assay with Vero cells. There was agreement between RPLA, cytotoxicity and PCR assays, but 29 strains were positive in the RPLA assay while 35 were positive in the cytotoxicity test and PCR using primer pair NK3-NK2 directed to the nonrepeating portion of the C. difficile toxin A gene. The 6 cytotoxic but RPLA-negative strains were demonstrated to be toxin A-negative/toxin B-positive strains in the PCR assay by using primer pair NK11-NK9 directed to the repeating portion of the C. difficile toxin A gene. There were no cross-reactions with culture supernatants of the other clostridial strains except for two strains of C. sordelli that produced hemorrhagic toxin (which is immunologically related to C. difficile toxin A).     

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.     

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.     

A latex agglutination assay for specific detection of Panton-Valentine leukocidin

Panton-Valentine leukocidin (PVL) is produced by some isolates of Staphylococcus aureus, and has been associated with the high pathogenic potential of these strains. To rapidly detect the toxin producer strains, we developed a reverse passive latex agglutination (RPLA) reaction assay specific for PVL. By testing 64 S. aureus strains, the assay could detect the 35 pvl-gene-positive strains with 100% specificity and sensitivity. Furthermore, the assay revealed an extensive variation in the amount of PVL produced by the pvl-positive strains.     

Evaluation of a reversed passive latex agglutination test for the detection of Verocytotoxin (VT) expressed by strains of VT-producing Escherichia coli

AIMS: To compare an experimental Reversed-Passive Latex Agglutination (RPLA) with Vero cells for the detection of Verocytotoxin expressed by VT-producing strains of Escherichia coli (VTEC). METHODS AND RESULTS: The RPLA was used alongside a Vero cell tissue culture assay for the detection of VT in bacterial culture supernatant fluids and patients' faecal extracts. CONCLUSION: The RPLA was comparable with the Vero cell assay, although slightly less sensitive. Significance and Impact of the Study: The RPLA test proved to be a simple, rapid and convenient method of detecting VT in bacterial culture supernatant fluids and in the faeces of patients infected with VTEC.     

A 6 h microslide immunodiffusion assay for confirmed detection of staphylococcal enterotoxins

Use of appropriately balanced immunological reagents and incubation of microslides at 45 degrees C resulted in confirmed assay results in 6 h for staphylococcal enterotoxins. The sensitivity of the 45 degrees C-6 h assay was comparable to the 37 degrees C-24 h assay: 0.1 micrograms of staphylococcal enterotoxin A/ml.     

Rapid detection of varicella-zoster viral antibodies by latex agglutination assay: a practical experience for medical and science undergraduates

From 1993 to 1997, laboratory exercises have been conducted using a latex agglutination assay to detect antibodies to varicella-zoster virus (VZV) among 576 medical and science undergraduates aged 18–25 years. Of 295 students who volunteered a past history of chickenpox, there was good correlation with VZV antibody positivity (89.8%). However, despite a history of chickenpox, 30 (10.2%) tested negative for VZV antibodies, suggesting previous misdiagnosis or false negatives caused by the prozoning phenomenon due to unusually high VZV antibody levels. Indeed, out of 22 initially seronegative sera from selected students with a history of chickenpox, 13 sera were subsequently seropositive upon re-testing the sera at higher dilutions. Although 192 students gave a negative history of chickenpox, 22 (11.5%) tested positive for VZV antibodies (of whom 2 were vaccinated, with the rest reflecting previous subclinical infection). Of 89 subjects unsure of their history of chickenpox, 30 (33.7%) were seropositive. Thus elicitation of a history of symptoms of chickenpox may not be a reliable indicator of past VZV exposure. While 55% of this cohort of this cohort of young adults were seropositive, a significant fraction of seronegative individuals (45%) were still susceptible to VZV infection. This practical study exemplifies latex agglutination as a rapid and simple diagnostic technique, and integrates certain key principles of virology, immunology and medicine.     

A latex agglutination assay for specific detection of Panton–Valentine leukocidin

Panton–Valentine leukocidin (PVL) is produced by some isolates of Staphylococcus aureus, and has been associated with the high pathogenicpotential of these strains. To rapidly detect the toxin producer strains, we developed a reverse passive latex agglutination (RPLA) reaction assay specific for PVL. By testing 64 S. aureus strains, the assay could detect the 35 pvl-gene-positive strains with 100% specificity and sensitivity. Furthermore, the assay revealed an extensive variation in the amount of PVL produced by the pvl-positive strains.     

Rapid detection of methicillin resistance in teicoplanin-resistant coagulase-negative staphylococci by a penicillin-binding protein 2' latex agglutination method

Thirty-five clinical isolates of coagulase-negative staphylococci with decreased glycopeptide sensitivity were examined by a penicillin-binding protein (PBP2′) latex agglutination (LA) test and were compared to the detection of the mecA gene by PCR, and oxacillin susceptibility determined minimum inhibitory concentrations. The latex test demonstrated high sensitivity and specificity for detecting methicillin resistance in coagulase-negative staphylococci after PBP2′ induction with oxacillin.     

Development of the reverse passive latex agglutination method for the detection and quantification of the genus Nitrospira in the wastewater treatment process

This report describes a new immunological method for the detection and quantification of Nitrospira populations using the reverse passive latex agglutination (RPLA). The numbers of the genus Nitrospira have been quantified only by molecular biological techniques such as FISH and quantitative PCR to date. Using high-density latex particles and a specific polyclonal antibody, Nitrospira populations in the wastewater treatment process were quantified in the shortest 4 h of incubation. The minimum detectable number of Nitrospira cells was 7.0x10(5) (log(10) 5.85) cells/ml. It is thought that the RPLA method can quantify Nitrospira populations more simply, economically, and speedily than molecular biological techniques or the culture method, because this procedure has a simple protocol and does not require the use of specialized equipment, expensive reagents, or technical skill. Therefore it is applicable for use in the everyday control and maintenance of water quality in wastewater treatment facilities where equipment is not sufficient or in the field.