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.     

Rapid and sensitive sandwich enzyme-linked immunosorbent assay for detection of staphylococcal enterotoxin B in cheese

A rapid and sensitive screening sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the detection of staphylococcal enterotoxin B (SEB) in cheese by using a highly avid anti-SEB antibody (Ab) as the capture Ab (CAb) and as the biotinylated Ab conjugate. The glutaraldehyde fixation method for the immobilization of CAb on polystyrene dipsticks was superior to the adsorption fixation and the adsorption-glutaraldehyde fixation methods. The glutaraldehyde fixation method resulted in a higher surface-saturating CAb concentration as evaluated by the peroxidase saturation technique and by the ability of the CAb-coated dipstick to discriminate between positive and negative controls (index of discrimination). Of nine blocking agents used alone or in pairs, lysine-human serum albumin, bovine serum albumin, human serum albumin, and gelatin effectively saturated available sites on the CAb-coated dipsticks without causing interference with the antigen-Ab reactions. The addition of 1% polyethylene glycol to the diluent of the biotinylated anti-SEB Ab conjugate improved the detection of SEB. A concentration of 4% polyethylene glycol allowed a 5-min reaction time for the streptavidin-biotin-horseradish peroxidase conjugate. Cheddar cheese homogenate reduced the sensitivity of the SEB assay; however, the sensitivity was restored when 1.6% (wt/vol) of either a nonionic detergent (Mega-9) or two zwitterionic detergents (Zwittergent 3-10 and 3-12 detergent) was added to the diluent. By using the rapid sandwich ELISA, a minimum of 0.5 to 1.0 ng of SEB per ml was detected within 45 min. The whole procedure for the analysis of the cheddar cheese samples was completed within 1 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid and sensitive sandwich enzyme-linked immunosorbent assay for detection of staphylococcal enterotoxin B in cheese.

A rapid and sensitive screening sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the detection of staphylococcal enterotoxin B (SEB) in cheese by using a highly avid anti-SEB antibody (Ab) as the capture Ab (CAb) and as the biotinylated Ab conjugate. The glutaraldehyde fixation method for the immobilization of CAb on polystyrene dipsticks was superior to the adsorption fixation and the adsorption-glutaraldehyde fixation methods. The glutaraldehyde fixation method resulted in a higher surface-saturating CAb concentration as evaluated by the peroxidase saturation technique and by the ability of the CAb-coated dipstick to discriminate between positive and negative controls (index of discrimination). Of nine blocking agents used alone or in pairs, lysine-human serum albumin, bovine serum albumin, human serum albumin, and gelatin effectively saturated available sites on the CAb-coated dipsticks without causing interference with the antigen-Ab reactions. The addition of 1% polyethylene glycol to the diluent of the biotinylated anti-SEB Ab conjugate improved the detection of SEB. A concentration of 4% polyethylene glycol allowed a 5-min reaction time for the streptavidin-biotin-horseradish peroxidase conjugate. Cheddar cheese homogenate reduced the sensitivity of the SEB assay; however, the sensitivity was restored when 1.6% (wt/vol) of either a nonionic detergent (Mega-9) or two zwitterionic detergents (Zwittergent 3-10 and 3-12 detergent) was added to the diluent. By using the rapid sandwich ELISA, a minimum of 0.5 to 1.0 ng of SEB per ml was detected within 45 min. The whole procedure for the analysis of the cheddar cheese samples was completed within 1 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

The use of dacron plates for dot enzyme-linked immunosorbent assay (dot-ELISA).

Dacron (polyethylenetherephthalate) is proposed as a matrix for dot-ELISA procedures, as an alternative to nitrocellulose. Plates of dacron were partially hydrazinolyzed and hydrazide groups introduced were converted to azide groups. The derivative dacron-antigen was covalently linked on to the plates through these azide groups. The derivative dacron-antigen was exaustively washed according to CROOK and antigen was still fixed onto the plates. Protein F1A purified from Yersinia pestis was used as a model. Titration of sera from immunized and non immunized rabbits against this protein was carried out by employing the dot-ELISA method. No significant difference was observed using dacron-antigen and nitrocellulose-antigen preparations. However, both procedures showed to have a significant better performance in comparison with the passive hemagglutination method. The specificity and reproductibility of the dot-ELISA assay using both preparations showed a similar behaviour. Nitrocellulose preparation was stable at 4 degrees C, 28 degrees C and -20 degrees C for 90 days, whereas the dacron-antigen derivative was stable only when stored at 4 degrees C. Dacron-antigen derivative could be re-used when the spot developing was proceeded using 4-chloro-1-naphtol as substrate.     

Detection of Aeromonas hydrophila in food with an enzyme-linked immunosorbent assay

A microtitration plate, antibody capture, enzyme-linked immunosorbent assay was developed for the detection of Aeromonas hydrophila serotype O : 11 (highly virulent strains). The assay utilizes a detector antibody which shows no cross-reactions with Aeromonas strains other than serotype O : 11 or non- Aeromonas competing organisms. The detector antibody is mixed with the sample and incubated for 1 h, microcentrifuged and the supernatant fluid (unadsorbed antibody) titred in a microtitre plate coated with A. hydrophila cells from serotype O : 11. All the A. hydrophila strains from serotype O : 11 tested reacted strongly with the detector antibody. Also by culturing and performing the immunoassay with the detector antibody we established and quantified the presence of A. hydrophila O : 11 in different foods.     

Development of an enzyme-linked immunosorbent assay specific to Sudan red I

To obtain antibodies to develop an enzyme-linked immunosorbent assay (ELISA) for the analysis of Sudan red I, haptens were designed and synthesized via four different strategies: (i) attachment of a spacer at the para position of the benzene ring, (ii) attachment of a spacer at the naphthol part, (iii) attachment of a spacer at the hydroxyl group of the Sudan red I molecule, and (iv) use of a fragment of the target molecule. A total of 10 haptens were used to generate immunogens, coating antigens, and polyclonal antibodies. One of the heterologous ELISAs developed exhibited an IC₅₀ of 1.6 ng/ml, a limit of detection (LOD) of 0.03 ng/ml, and a dynamic range between 0.1 and 14 ng/ml. The assay had 13% cross-reactivity with Para red and negligible cross-reactivity with other structure-related compounds. This ELISA was much more specific than those published previously. This assay was used to determine Sudan red I residues in tomato sauce and chili powder samples after simple pretreatment. The results were validated by comparison with high-performance liquid chromatography (HPLC). The average recoveries of Sudan red I by ELISA and HPLC were in ranges of 70-97% and 82-114%, respectively, indicating suitability of the developed ELISA for screening of Sudan red I in foods.     

A background-free assay for cells forming antibody to glucose oxidase coupled with an enzyme-linked immunosorbent assay (ELISA) to quantitate antibody secretion

We describe an immunocytochemical assay for cells forming antibody to glucose oxidase (GO). The method is specific in that only cells containing intracytoplasmic antibody capable of binding the immunogen (GO binding cells; GOBC) are stained. The method is sensitive because there is no GO activity in mammalian tissues. This lack of background readily permits detection of one GOBC among 10(6) nucleated lymphohemopoietic cells. The technique is reliable because purified chemicals are used. Although it is not possible to determine the Ig class of antibody formed by an individual cell, as can be done with the hemolytic plaque assay, the amount and class of secreted antibody to GO can be quantitated by an indirect enzyme-linked immunosorbent assay (ELISA), which is also described. GO is immunogenic and stimulates the formation of large numbers of GOBC in the popliteal lymph nodes after injection with adjuvant into the footpads of mice, but 1-mg doses injected IV or IP are lethal because of its enzymatic activity, which causes hypoglycemia and methemoglobinemia.     

Development of an improved preparation and an enzyme-linked immunosorbent assay for anti-neuroexcitation peptide (ANEP)

Anti-neuroexcitation peptide (ANEP) is a novel recombinant peptide obtained from the venom of the Chinese scorpion Buthus martensii Karsch. However, the expression of recombinant ANEP in Escherichia coli results in the formation of insoluble aggregates known as inclusion bodies. Here, we describe a novel method for the preparation of ANEP which maximizes the yields of recombinant peptide in a soluble and active form. A non-fusion expression plasmid pNJUTRX-1-ANEP-His(6) encoding recombinant ANEP with a His(6)-tag at its C-terminus was constructed and transformed into E. coli strain BL21 (DE3). The expressed ANEP was almost in soluble form and accounted for about 12% of the total cellular proteins. The recombinant ANEP in the cell lysate was purified to homogeneity by His Bind affinity chromatography. This effective method solved the problem of a lack of sufficient active peptide which, until now, has hampered the further research and development. In order to develop an immunoassay method for ANEP, polyclonal rabbit antiserum was raised against the prepared ANEP and purified by protein A affinity chromatography. It was confirmed that the antibody reacted with recombinant ANEP by both Western blotting and ELISA results. Using purified antibody, the immunoassay method was developed.     

Use of a monoclonal antibody to estrone-3-glucuronide in an enzyme-linked immunosorbent assay (ELISA)

A direct urinary ELISA for estrone-3-glucuronide has been produced following cloning and characterisation of a monoclonal antibody to the above estrogen metabolite. The ELISA follows our established pattern of absorbing a thyroglobulin conjugate, to which estrone-3-glucuronide has been coupled, to the wells of a microtitre plate using guanidine hydrochloride. A competition reaction between either standards/samples and the adsorbed hormone compete for antibody combining sites. The assay is completed by addition of an anti-mouse Ig-peroxidase complex and read at 492 nm following additions of O-phenylenediamine substrate in under 4 h. The correlation between urinary "total estradiol" and "total estrone and estradiol" is very good and, in conjunction with our ELISA for pregnanediol glucuronide, has allowed for the improved clinical management of infertile and subfertile women.     

Dot enzyme-linked immunosorbent assay (dot ELISA) for antibodies to Encephalitozoon cuniculi

A dot-ELISA procedure was developed to detect antibodies against Encephalitozoon cuniculi. Sera from 84 rabbits, 22 dogs, 18 squirrel monkeys and 200 mice were tested by dot-ELISA and most also were tested by immunofluorescence. Comparison of the two tests showed that there was excellent agreement of the results (Kappa values greater than or equal to 0.74) in all four species. Dot-ELISA is a simple, quantitative, rapid alternative to immunofluorescence when large numbers of serum samples must be evaluated.     

Development of an improved preparation and an enzyme-linked immunosorbent assay for anti-neuroexcitation peptide (ANEP)

Anti-neuroexcitation peptide (ANEP) is a novel recombinant peptide obtained from the venom of the Chinese scorpion Buthus martensii Karsch. However, the expression of recombinant ANEP in Escherichia coli results in the formation of insoluble aggregates known as inclusion bodies. Here, we describe a novel method for the preparation of ANEP which maximizes the yields of recombinant peptide in a soluble and active form. A non-fusion expression plasmid pNJUTRX-1-ANEP-His₆ encoding recombinant ANEP with a His₆-tag at its C-terminus was constructed and transformed into E. coli strain BL21 (DE3). The expressed ANEP was almost in soluble form and accounted for about 12% of the total cellular proteins. The recombinant ANEP in the cell lysate was purified to homogeneity by His Bind affinity chromatography. This effective method solved the problem of a lack of sufficient active peptide which, until now, has hampered the further research and development. In order to develop an immunoassay method for ANEP, polyclonal rabbit antiserum was raised against the prepared ANEP and purified by protein A affinity chromatography. It was confirmed that the antibody reacted with recombinant ANEP by both Western blotting and ELISA results. Using purified antibody, the immunoassay method was developed.     

Development of an enzyme-linked immunosorbent assay for vitellogenin of Morelet's crocodile (Crocodylus moreletii)

The purpose of this study was to develop an immunoassay for vitellogenin in Morelet's crocodile (Crocodylus moreletii). Blood was collected from wild-caught crocodiles in Belize. Plasma samples from adult females taken during the breeding season were used for vitellogenin purification and samples from adult males were used for comparison. No differences were detected between males and females for plasma total protein concentration, as measured by Coomassie assay. However, denaturing polyacrylamide gel electrophoresis (SDS-PAGE) revealed that female plasma contained a 210-kDa protein, presumably the vitellogenin monomer, that was absent in adult male plasma. The identity of the putative vitellogenin was confirmed by its cross-reactivity in Western blots with a vitellogenin antiserum that was generated against a conserved vitellogenin peptide sequence. Crocodile vitellogenin was purified by two successive rounds of DEAE chromatography. The purified protein had an apparent molecular mass of 450 kDa, as determined by gel filtration chromatography, and 210 kDa on SDS-PAGE. An indirect enzyme-linked immunosorbent assay (ELISA) was then developed for C. moreletii vitellogenin. The detection limit of the assay was 20.0 ng/mL. The intra- and inter-assay coefficients of variation were 5.3% and 9.8%, respectively. The recovery of vitellogenin diluted into male plasma was 94.7%. The ELISA assay revealed that vitellogenin levels of adult female plasma during the breeding season ranged from 1.8 to 3.1 mg/mL with a mean of 2.5+/-0.25 mg/mL. No vitellogenin was detected in adult male plasma. Induction of vitellogenin in Morelet's crocodile may be a useful model system for field studies of crocodile reproduction and for investigations of endocrine disruption in this species.     

Quantification of two cytochrome P-450 isoenzymes by an enzyme-linked immunosorbent assay (ELISA)

An enzyme linked immunosorbent assay (ELISA) using monoclonal and polyclonal antibodies has been developed to quantify individual cytochrome P-450 isoenzymes in microsomal preparations, namely UT-A and PB-B. This very sensitive method can be used for the rapid processing of large quantities of determinations and requires only limited amounts of antibodies.     

Serological survey for Lyme disease in sika deer (Cervus nippon yesoensis) by enzyme-linked immunosorbent assay (ELISA).

Seventy-six wild sika deer (Cervus nippon yesoensis) from areas endemic for Borrelia burgdorferi during 1988 to 1989 had the IgG antibody to the bacteria in enzyme-linked immunosorbent assay. The percentage of seropositive deer was 25.0% to strain HO14 and 22.4% to strain HP3, respectively. Specific IgG antibody titers were high in summer but low in winter. In summer, seropositive deer were 75.0%. Similar results were obtained in serum specimens obtained monthly from 4 farmed deer. It was suggested that the sika deer could be one of the wild reservoirs for B. burgdorferi in Hokkaido, Japan. The transmission risk of B. burgdorferi is considered to be greatest during late spring to early summer.     

Development of an enzyme-linked immunosorbent assay for thiacloprid in soil and agro-products with phage-displayed peptide

A monoclonal antibody (3A5) that can recognize thiacloprid was produced, and a linear 8-residue peptide phage library was constructed. Six phage-displayed peptides were isolated from the linear 8-residue peptide phage library and a cyclic 8-residue peptide phage library. A phage enzyme-linked immunosorbent assay (ELISA) was developed to detect thiacloprid using a phage-displayed peptide. Under the optimal conditions, the half-maximal inhibition concentration (IC₅₀) and the limit of detection (IC₁₀) of the developed phage ELISA were 8.3 and 0.7 μg/L, respectively. Compared with the conventional ELISA, the sensitivity was improved more than 3-fold. The cross-reactivity (CR) was less than 0.08% for the tested structural analogues and was regarded as negligible. The recoveries of thiacloprid ranged from 80.3% to 116.3% in environmental and agricultural samples, which conformed to the requirements for residue detection. The amount of thiacloprid detected by phage ELISA in the samples was significantly correlated with that detected by high-performance liquid chromatography. The current study indicates that isolating phage-displayed peptides from phage display libraries is an alternative method for the development of a sensitive immunoassay and that the developed assay is a potentially useful tool for detecting thiacloprid in environmental and agricultural samples.     

Detection and distribution of scorpion (Heterometrus bengalensis) venom in rabbit tissues by enzyme-linked immunosorbent assay (ELISA).

ELISA was carried out to detect distribution of scorpion venom in experimental animal tissues. The venom content of different tissues was in the order, liver greater than kidney greater than spleen greater than lung greater than heart greater than diaphragm greater than brain. Tissue distribution of venom antigen in the envenomental subject by ELISA will provide a better approach for serotherapy.     

In vitro production of feline IgG: quantification by an enzyme-linked immunosorbent assay (ELISA)

We report here on the development of a sensitive and convenient enzyme-linked immunosorbent assay (ELISA) for feline IgG by using commercially available reagents and optimizing their concentrations. The reagents employed include goat anti-cat IgG antibody and alkaline phosphatase-conjugated goat anti-cat IgG antibody. The assay described is sensitive, reproducible, and highly specific for feline IgG. The assay was applied for the measurement of feline IgG synthesized and secreted in vitro by peripheral blood mononuclear cells cultured with or without a polyclonal B-cell activator. The amounts of secreted IgG in the supernatants measured by an ELISA correlated well with the numbers of IgG-secreting cells which were induced upon stimulation with pokeweed mitogen and determined by a reverse hemolytic plaque assay.     

Arctic charr (Salvelinus alpinus) vitellogenin: development and validation of an enzyme-linked immunosorbent assay

Vitellogenin (Vtg) was isolated from plasma of estradiol-17 beta-treated Arctic charr males by double precipitation with MgCl2-EDTA and distilled water, followed by ion-exchange chromatography. The monomeric form of Vtg, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 158 kDa. The purified Vtg was used to raise a polyclonal antibody for Vtg (AbVtg), and the specificity of the AbVtg was assessed by Western blot analysis. No cross-reactivity was observed with plasma from control males. Using this AbVtg, a competitive enzyme-linked immunosorbent assay was developed. The detection limit of the assay was 2 ng ml-1, and the intra- and inter-assay variations determined from plasma samples were 8.6 and 13.3%, respectively. The assay was validated by quantification of Vtg in plasma samples obtained during a reproductive cycle of Arctic charr. Vtg of females increased gradually from 3 mg ml-1 in early March to a peak value of 22 mg ml-1 in late August, followed by a rapid drop to 2 mg ml-1 at the time of spawning in mid-October. The temporal changes in plasma Vtg of females correlate well with the reproductive cycle. Vtg was undetectable in males, except on some sampling dates during July-September when minute amounts (3-13 micrograms ml-1) were detected in some individuals.