Double-Antibody Radioimmunoassay for Staphylococcal Enterotoxin C2

A sensitive double-antibody radioimmunoassay for staphylococcal enterotoxin C₂ is described. The assay procedure employs anti-rabbit gamma globulin, prepared in goats, to precipitate the antigen-antibody complex of enterotoxin C₂ and anti-enterotoxin C₂. The test is sensitive to 100 pg of enterotoxin.     

A rapid, colourimetric assay for cytotoxin activity in Campylobacter jejuni

Abstract Cell extracts and culture supernates of Campylobacter jejuni NCTC 11168 and three isolates from faecal samples from patients with enteritis were tested for cytotoxic activity on HeLa and Vero cells using a sensitive and rapid dye reduction assay which represents a simple assay for cytotoxin activity that can be assessed visually or spectrophotometrically in the wells of microplates. The assay was as sensitive as trypan blue exclusion and did not require the use of radioisotopes. A low level of cytotoxin activity, compared to that produced by a control verotoxin 2-producing Escherichia coli strain, was detected in cell extracts of all four strains, but no activity was detected in culture supernates. Production of an enterotoxin was evaluated by reverse passive latex agglutination with anti-cholera toxin antibody, a procedure which also represents a rapid and simple assay for this toxin. No enterotoxin activity was detected in cell extracts or culture supernates from any of the isolates.     

Protein A insensitive ELISA detection of staphylococcal enterotoxin B

A sandwich enzyme-linked immunosorbent assay to detect staphylococcal enterotoxin B was developed using rat monoclonal antibodies as capture antibodies and as a biotinylated conjugate. This test was sensitive, less than 1 ng/ml of enterotoxin B was detected and interference by protein A was prevented by the use of rat monoclonal antibodies of the IgG2a isotype which were insensitive to protein A even at concentrations greater than 1000 ng/ml.     

Detection of Staphylococcal Enterotoxin A,B, and C in Milk By an Elisa Procedure

Enterotoxigenic reference strains of Staphylococcus aureus were cultivated in sterile whole and skim milk for 18 h at 37°G. Staphylococcal enterotoxin A, B, and C were detected directly in the milk by an enzyme linked immunosorbent assay (ELISA), sensitive down to 1 ng/ml. Enterotoxins in the range of 1 ng–20 µg/ml milk were detected without any concentration or extraction. Skim and whole milk were almost identical as medium for enterotoxin production.     

反向间接胶乳凝集试验法检测粪便A型产气荚膜梭菌肠毒素的结果及评价

本文以１０种５２株供试菌分别与７个不同年龄组的健康人粪便混合,共配成３６４份模拟标本,采用反向间接胶乳凝集（ＲＰＬＡ）试验法与生物学试验法（小鼠致死试验、豚鼠皮肤血管透性因子试验,Ｖｅｒｏ细胞毒性试验）检测各标本中的Ａ型产气荚膜梭菌肠毒素（简称Ｃｐ－Ｅｎｔ）。除产气荚膜梭菌之外的其他菌种培养液２３８份标本（３４株）,ＲＰＬＡ与生物学试验结果完全一致,均为阴性。产气荚膜梭菌１２６份标本（１８株）中有７０份标本的ＲＰＬＡ同生物学诸法完全一致地检出了Ｃｐ－Ｅｎｔ．有１株７份标本（ＣｐＮＣＴＣ８７９７）的ＲＰＬＡ为阳性,而各生物学试验却均为阴性,该菌株经ＰＣＲ检查证明确为肠毒素原性产气荚膜梭菌,表明ＲＰＬＡ比生物学试验法更灵敏。有５株（ＣｐＮＣＴｃ８２３８,ＣｐＮＣＴＣ１０６１１,ＣｐＮＣＴＣ１０６１４,ＣｐＮＣＴＣ１０６１２,ＣｐＬ－５２）３５份标本ＲＰＬＡ与各生物学试验结果均为阴性,但经ＰＣＲ检吉证明该５株菌均为肠毒素原性产气荚膜梭菌,后经超声波破碎菌体提取物对其中部分菌株进行试验的结果仍然显示了ＲＰＬＡ与生物学法的一致性。有２株（ＣｐＮＣＴＣ８６８６,ＣｐＮＣＴＣ８４４９）１４份标本的所有结果均为阴性,ＰＣＲ     

A highly sensitive enzyme-linked immunosorbent assay for Clostridium perfringens enterotoxin

A highly sensitive enzyme-linked immunosorbent assay (ELISA) for quantitation of Clostridium perfringens enterotoxin (CPE) by a sandwich method with polystyrene beads was elaborated. The ELISA was very sensitive with a detection limit of 1 pg/ml of CPE. Clostridium perfringens culture fluid did not interfere with the assay. This ELISA may be useful for the mass screening for Cl. perfringens producing small amounts of CPE.     

Detection of staphylococcal enterotoxins A, B, C, D, and E in foods by radioimnunoassay, using staphyloccal cells containing protein A as immunoadsorbent.

A sensitive radioimmunoassay utilizing Staphylococcus aureus cells containing protein A as a coprecipitant was developed for the detection and quantitation of staphylococcal enterotoxins A, B, C, D, and E in a variety of foods. The enterotoxins were extracted from the foods by a simple and rapid procedure. The sensitivity of the assay is 1.0 ng or less of enterotoxin per g of food.     

Improvements in the microtitre GM1 ganglioside enzyme-linked immunosorbent assay for Escherichia coli heat-labile enterotoxin

A variant of the microtitre G_M1-ELISA for Escherichia coli heat-labile enterotoxin was studied. The test was improved by both reducing the assay time from 2½ d to 8 h and by determining the most appropriate G_M1 coating concentration. Coating the plates with >3 μg of G_M1/ml yielded a maximal sensitivity and ensured a linear relationship between the enterotoxin concentration and the extinction observed when using the final assay-procedure. Thus an optimal accuracy was obtained. This ELISA was 4- to 8-times more sensitive than the Vero cell monolayer assay. The sensitivity of this ELISA and of the Chinese hamster ovary cell monolayer assay were identical.     

Detection of Staphylococcus enterotoxin B using fluorescent immunoliposomes as label for immunochromatographic testing

Staphylococcus enterotoxin B (SEB) is one of several toxins produced by the gram positive bacterium Staphylococcus aureus. SEB is a major cause of food poisoning and represents a significant biological threat with regard to bioterrorism. A rapid, sensitive, and specific method is required to monitor food and water in cases of both natural and intentional contamination by this toxin. This report presents an improved immunochromatographic test (ICT) using immunoliposomes as label for the detection of SEB. For the first time in an ICT, the signal generated by the sulforhodamine B encapsulated into immunoliposomes was measured by fluorescence, allowing a 15-fold increase in sensitivity compared with that for visual detection of colored labels. The ICT was completed within 30 min, providing a limit of detection close to 20 pg/ml in buffer and showing no cross-reactivity with the other major toxin of the bacterium, Staphylococcus enterotoxin A. This sensitivity was retained when analyzing SEB spiked in various alimentary matrices, mimicking contaminated foods. Due to the use of fluorescent immunoliposomes as label, the present assay offers the inherent simplicity and speed of a dipstick assay while providing detection of low levels of SEB in real samples.     

Development of a highly sensitive bead-ELISA to detect bacterial protein toxins

A highly sensitive sandwich enzyme-linked immunosorbent assay to detect bacterial toxins was developed. Fab' of anti-toxin IgG was conjugated with horseradish peroxidase by the maleimide method and tetramethylbenzidine was used as substrate. As the solid phase, a 6.5 mm diameter polystyrene bead was used and this was coated with the anti-toxin IgG. The entire assay could be completed within 3.5 hr. The sensitivity of this bead-ELISA was found to be quite high with various bacterial toxins: less than 20 pg/ml for thermostable direct hemolysin of Vibrio parahaemolyticus, less than 60 pg/ml for Shiga toxin, less than 20 pg/ml for VT2 (Shiga-like toxin II) of Escherichia coli, less than 200 pg/ml for heat-labile enterotoxin of E. coli, and less than 6 pg/ml for cholera enterotoxin.     

Comparison of the Tecra VIA kit, Oxoid BCET-RPLA kit and CHO cell culture assay for the detection of Bacillus cereus diarrhoeal enterotoxin

Two commercial serological kits (Oxoid BCET-RPLA and Tecra VIA) and a Chinese hamster ovary (CHO) cell cytotonicity assay for the detection of Bacillus cereus diarrhoeal enterotoxin were compared. Eleven B. cereus strains and one enterotoxigenic B. thuringiensis strain were evaluated. Both kits and the CHO cell assay yielded positive toxin responses for cell-free culture filtrates from eight out of 11 diarrhoeal enterotoxigenic strains. An emetic enterotoxin producing strain was negative with all three assays. Two B. cereus strains were negative using the BCET-RPLA kit, but positive with the Tecra VIA kit and CHO cell assay. The BCET-RPLA indicated significant levels of enterotoxin after samples were boiled, whereas the CHO cell and Tecra assays were negative. Overall, the cell culture assay was the most sensitive. However, the Tecra VIA kit provided similar results and was better suited for the rapid detection of B. cereus diarrhoeal enterotoxin.     

Nature and reactivity of staphylococcal enterotoxin A monoclonal antibodies

Monoclonal antibodies from four clones (C5, C3, B2II, and B2I) directed against staphylococcal enterotoxin A were tested by the indirect enzyme-linked immunosorbent assay and double-gel immunodiffusion (micro-Ouchterlony) assay for the nature of heavy and light chain types. The reactivities of monoclonal antibodies were also tested by indirect enzyme-linked immunosorbent assay with various levels of purified staphylococcal enterotoxin A and various levels (dilutions) of monoclonal antibodies and saturation analysis-competitive indirect enzyme-linked immunosorbent assay. The heavy-chain isotype of monoclonal antibodies was found to be an unspecified subclass of immunoglobulin G1, and the light chain was the kappa type. Monoclonal antibodies from all of the clones exhibited high reactivity and nearly the same affinity to staphylococcal enterotoxin A in saturation analysis-competitive enzyme-linked immunosorbent assay. Purified immunoglobulin G from B2I yielded very high absorbance (1.2) at 405 nm with 1 ng of staphylococcal enterotoxin A as the coating antigen in the enzyme-linked immunosorbent assay. Monoclonal antibodies from B2I also neutralized the biological activity of staphylococcal enterotoxin A when tested by the kitten bioassay.     

Detection of staphylococcal enterotoxins by enzyme-linked immunosorbent assays and radioimmunoassays: comparison of monoclonal and polyclonal antibody systems.

Murine monoclonal antibodies reactive with at least one of the serological types of staphylococcal enterotoxin were examined for use in assay systems for the detection of enterotoxin at the level of 1.0 ng of enterotoxin per ml. An antibody sandwich enzyme-linked immunosorbent assay was devised for each toxin type by identifying an effective antibody pair. One antibody (the coating antibody) was coated onto a polystyrene plate and removed the enterotoxin from the test solution; the second antibody (the probing antibody) was conjugated to horseradish peroxidase and detected the captured toxin. Enterotoxins A and E could be detected in the same system by the use of cross-reacting monoclonal antibodies. All subtypes of enterotoxin C could be detected in one assay system. Two effective systems were described for each of types B and D. Each of these systems, when compared with the homologous enterotoxin-specific polyclonal rabbit antibody systems, was found to compare favorably. The monoclonal enzyme-linked immunosorbent assay systems for the detection of enterotoxins A and C2 were examined for a variety of food extracts; no abnormal interference could be detected from these extracts. The monoclonal antibody systems were also compared with the homologous enterotoxin-specific polyclonal serum for the detection of enterotoxin by the competitive radioimmunoassay (RIA). Single monoclonal antibodies generally did not perform as well in the RIA as did the homologous toxin-specific polyclonal serum. However, pools of monoclonal antibodies were prepared that approached the sensitivity and precision of the polyclonal system for the detection of each toxin by the RIA.     

Detection of staphylococcal enterotoxins by enzyme-linked immunosorbent assays and radioimmunoassays: comparison of monoclonal and polyclonal antibody systems

Murine monoclonal antibodies reactive with at least one of the serological types of staphylococcal enterotoxin were examined for use in assay systems for the detection of enterotoxin at the level of 1.0 ng of enterotoxin per ml. An antibody sandwich enzyme-linked immunosorbent assay was devised for each toxin type by identifying an effective antibody pair. One antibody (the coating antibody) was coated onto a polystyrene plate and removed the enterotoxin from the test solution; the second antibody (the probing antibody) was conjugated to horseradish peroxidase and detected the captured toxin. Enterotoxins A and E could be detected in the same system by the use of cross-reacting monoclonal antibodies. All subtypes of enterotoxin C could be detected in one assay system. Two effective systems were described for each of types B and D. Each of these systems, when compared with the homologous enterotoxin-specific polyclonal rabbit antibody systems, was found to compare favorably. The monoclonal enzyme-linked immunosorbent assay systems for the detection of enterotoxins A and C2 were examined for a variety of food extracts; no abnormal interference could be detected from these extracts. The monoclonal antibody systems were also compared with the homologous enterotoxin-specific polyclonal serum for the detection of enterotoxin by the competitive radioimmunoassay (RIA). Single monoclonal antibodies generally did not perform as well in the RIA as did the homologous toxin-specific polyclonal serum. However, pools of monoclonal antibodies were prepared that approached the sensitivity and precision of the polyclonal system for the detection of each toxin by the RIA.     

Improvements in the microtitre GM1 ganglioside enzyme-linked immunosorbent assay for Escherichia coli heat-labile enterotoxin

A variant of the microtitre GM1-ELISA for Escherichia coli heat-labile enterotoxin was studied. The test was improved by both reducing the assay time from 2 1/2 d to 8 h and by determining the most appropriate GM1 coating concentration. Coating the plates with greater than or equal to 3 micrograms of GM1/ml yielded a maximal sensitivity and ensured a linear relationship between the enterotoxin concentration and the extinction observed when using the final assay-procedure. Thus an optimal accuracy was obtained. This ELISA was 4- to 8-times more sensitive than the Vero cell monolayer assay. The sensitivity of this ELISA and of the chinese hamster ovary cell monolayer assay were identical.     

γ Irradiation of Staphylococcal Enterotoxin B

The inactivation of enterotoxin B by γ irradiation was studied by use of single-and double-gel-diffusion assay techniques. Enterotoxin B (99+% purity) was suspended either in 0.04 m Veronal buffer (pH 7.2) or in milk, dispensed and heat-sealed in borosilicate glass vials, and irradiated essentially at 21 to 26 C with a cobalt-60 source. Parallel titrations of irradiated enterotoxin B in Veronal buffer were made by use of gel-diffusion and cat assay procedures to establish the relative sensitivity of these two assay procedures to irradiated enterotoxin. Results were identical. A dose of 5 Mrad was required to reduce an enterotoxin B concentration of 31 μg/ml in Veronal buffer to less than 0.7 μg/ml. When milk was used as a vehicle, a dose of 20 Mrad was needed to inactivate a 30 μg/ml concentration of enterotoxin B to less than 0.5 μg/ml. With Veronal buffer and milk as vehicles, the D values (dose required to inactivate 90%) for enterotoxin B inactivation were 2.7 and 9.7 Mrad, respectively.     

Staphylococcal Enterotoxin C: Solid-Phase Radioimmunoassay

A solid-phase radioimmunoassay test employing 125I-labeled enterotoxin C and polystyrene tubes coated with specific antibody was used for the detection and quantitation of entertoxin C in condensed milk, cheddar cheese, custard, and ham salad. The assay was sensitive to 1 to 10 ng of toxin per g of food; nonspecific inhibitions were 16% or less.     

Production of monoclonal antibodies to staphylococcal enterotoxin A.

Spleen cells from mice immunized with staphylococcal enterotoxin A were successfully fused with NS-1 mouse myeloma cells. Two of the four clones studied produced monoclonal antibodies to staphylococcal enterotoxin A in growth medium which showed titers of greater than 10(6) to 10(7) when tested by the indirect enzyme-linked immunosorbent assay. These monoclonal antibodies showed reactivity with enterotoxins A and E in the enzyme-linked immunosorbent assay. However, the reactivity was higher with enterotoxin A than with enterotoxin E. Nanogram quantities of crude staphylococcus enterotoxin A from Staphylococcus aureus growth were detected by the monoclonal antibodies in electroimmunoblots via autoradiography.     

Rapid Solid-Phase Radioimmunoassay for Staphylococcal Enterotoxin A

A rapid solid-phase radioimmunoassay for staphylococcal enterotoxin A is described. The assay procedure requires 3 to 4 h for completion by using a competitive inhibition system in which the antibody is attached to bromacetyl cellulose particles. It is accurate to a level of 0.01 mug of enterotoxin A/ml in a variety of media such as ham, milk products, crab meat, custard, etc. No significant interference was found with any media or food product tested.