COMPARISON of ANALYTICAL SENSITIVITIES of THREE ENZYME IMMUNOASSAY PROCEDURES FOR the DETECTION of SALMONELLA LIPOPOLYSACCHARIDE

The analytical sensitivities of three different enzyme linked immunoassays (ELISA), two competitive and a capture format were assessed. the assay systems employed monoclonal antibodies to Salmonella lipopolysaccharide (LPS) outer core epitopes to detect crude LPS antigens from Salmonella typhimurium. the most sensitive ELISA was the capture procedure, being capable of detection 1.3 ng/ml of LPS. This technique, however also gave the greatest between-test variation and as a result, the lowest amount that could be detected with a 95% confidence limit was actually 12.8 ng/ml and it took the longest time to perform (3 h, 30 min). A competitive ELISA using limiting monoclonal antibody to compete between solid phase antigen and soluble antigen in the sample, ranked second in sensitivity, and can detect 2.8 and 3.8 ng/ml of LPS when tested with two different monoclonal antibodies. However, because of the slight between test variation, the actual sensitivities that could be detected with a 95% confidence limit were 3.1 and 4.6 ng/ml, respectively. This test takes approximately 1 h and 30 min to perform.
The classical type of competitive assay, employing a labelled antigen, was the least sensitive being capable of detecting 5.8 ng/ml if the LPS was conjugated with horseradish peroxidase and 16.0 ng/ml if alkaline phosphatase was used as a label. to account for the between-test variation, the sensitivities with a 95% confidence limit were 8.6 and 18.7 ng/ml for the respective assays, which take 2 h and 15 min to perform.
These sensitivities compare favorably with those published for similar assays, but all of the procedures were judged insufficiently sensitive for direct use on food samples to be tested for the presence of Salmonella species. However, the assays would be quite suitable for demonstration of Salmonella sp. after an enrichment procedure.     

Detection and serogroup differentiation of Salmonella spp. in food within 30 hours by enrichment-immunoassay with a T6 monoclonal antibody capture enzyme-linked immunosorbent assay.

We previously described an antigen capture enzyme-linked immunosorbent assay which makes use of monoclonal antibody T6, which recognizes an epitope on the outer core polysaccharide of Salmonella lipopolysaccharide molecules that is common to almost all Salmonella serovars. In this paper, we show that this assay can detect between 10(5) and 10(7) Salmonella cells per ml even in the presence of excess Escherichia coli. A total of 153 of 154 (99%) serogroup A to E strains and 51 of 78 (71%) serogroup F to 67 strains were reactive as determined by this assay. This corresponds to a detection rate of approximately 98% of all salmonellae known to affect humans. None of the 65 strains of non-Salmonella bacteria tested positive. Taking advantage of the O-factor polysaccharides also present on the antigen captured by the immobilized T6 antibody, we showed that 136 of 154 Salmonella serogroup A to E strains (88%) were correctly differentiated according to their serogroups by use of enzyme conjugates of a panel of O-factor-specific monoclonal antibodies. We evaluated this assay for the detection and serogroup differentiation of salmonellae directly from enrichment cultures of simulated food, eggs, pork, and infant formula milk. All 26 samples which had been contaminated with Salmonella spp. were detected by T6 (100% sensitivity), with only one false-positive result from 101 samples not contaminated by Salmonella spp. (99% specificity). The detection time was substantially reduced to between 17 and 29 h, depending on the enrichment methods used. Since there were no false-negative results, we concluded that this enrichment-immunoassay method can afford rapid screening for Salmonella spp. in food samples.     

Resolution of optical isomers by chiral high-performance liquid chromatography: separation of dihydrodiols and tetrahydrodiols of benzo[a]pyrene and benz[a]anthracene

A competitive enzyme-linked immunoassay (CELIA) for human serum angiotensin-1-converting enzyme (ACE) was developed. The sensitivity was amplified by using a secondary antibody and an avidin biotin-conjugated horseradish peroxidase complex as the enzyme-labeled reagent. This configuration was compared to three other configurations for an indirect CELIA, and was found to be the most sensitive. A sensitivity of 39 ng/ml ACE was achieved with intraassay and interassay coefficients of variation of 6.3 and 8%, respectively. CELIA will detect ACE in human serum without interference from either pharmacological or endogenous ACE inhibitors. In normal human volunteers, ACE values obtained using CELIA correlated well with values obtained by enzymatic assay.     

A monoclonal antibody to aflatoxin B1: detection of the mycotoxin by enzyme immunoassay

A monoclonal antibody (McAb) was produced after fusion of mouse (X63.Ag8.6.5.3) myeloma cells with spleen cells isolated from female Balb-c/NZB F1 hybrid mice immunized with aflatoxin B₁ (oxine)-keyhole limpet haemocyanin conjugate. The hybridoma cell line producing antibody specific for aflatoxin B₁ (AFB₁) was grown in tissue culture and as an ascites tumour. The ascitic fluid gave suitably high dilution titres (1:800 000) by enzyme immunoassay and was conjugated to horseradish peroxidase by a two-step procedure with glutaraldehyde. The conjugate was used to develop a direct competitive enzyme-linked immunosorbent (ELISA) assay for AFB₁. The sensitivity of the ELISA was 0–2 ng/ml with a working range up to 10 ng/ml for AFB₁. The specificity of the McAb was determined and it was shown not to cross-react significantly with any of the metabolites tested. This McAb and the direct competitive ELISA described may prove of use in the detection of AFB₁ in foods and feeds.     

Highly sensitive solid-phase enzyme immunoassay for terminal deoxynucleotidyl transferase

A highly sensitive and specific solid-phase enzyme immunoassay system for terminal deoxynucleotidyl transferase (TdT, EC 2.7.7.31) has been developed by the use of monospecific antibody against calf thymus TdT and β-d-galactosidase from Escherichia coli as label. The immunoassay system was composed of solid phase (polystyrene beads) with immobilized F(ab′)₂ antibody fragments and the antibody Fab′ fragments labeled with β-d-galactosidase. The minimum detectable concentration of calf TdT was 0.1 ng/ml (0.01 ng/assay), making it more sensitive than the radioimmunoassay or enzyme immunoassay methods that use alkaline phosphatase as label, as reported previously. The assay system cross-reacted with human TdT, and TdT in neoplastic cells or sera from leukemic patients was successfully detected by the present immunoassay method.     

Immunological analysis of methamphetamine antibody and its use for the detection of methamphetamine by capillary electrophoresis with laser-induced fluorescence

An accurate, simple and rapid immunoassay is demonstrated for the detection of methamphetamine in urine by capillary electrophoresis (CE) with laser-induced fluorescence (LIF). An aminobutyl derivative of methamphetamine was conjugated with proteins, and used as an immunogen to produce antibodies for the assay. The methamphetamine derivative was also labeled with fluorescein isothiocyanate (FITC) to compete with free methamphetamine in the sample for the antibody binding site. Levels of free and antibody-bound FITC-labeled methamphetamine were monitored by performing CE–LIF using an untreated fused-silica column. This competitive immunoassay used antiserum instead of purified antibody or antibody fragment, yet was found to have good precision with a sensitivity of lower than 20 ng/ml. Various antibodies were also screened, and cross-reactivity of anti-MA antibody with methamphetamine analogues were also investigated. The results indicate that CE–LIF-based immunoassay is a powerful tool for the screening and characterization of antibody and may have possible applications in the detection of abused drugs in urine.     

Elaboration of a rapid method of determining the sensitivity of Brucella to antibiotics]

Optimal conditions were developed for determination of antibiotic sensitivity in Brucella by using enzyme immunoassay directly in the primary cultures of the material tested. The Brucella concentration in the material tested should be not lower than 1.10(6) microbial cells/ml and the time of culture incubation be 24 hours at 37 degrees C. The obligatory condition is to use a liquid medium, i.e. the Albimi broth with 1% glucose. To inhibit the foreign microflora it is recommended to use polymyxin B and amphoglucamine in a concentration of 3 microgram/ml. The use of enzyme immunoassay was shown that it was possible to determine the antibiotic sensitivity of Brucella in practice.     

Enzyme immunoassay for the macrocyclic trichothecene roridin A: production, properties, and use of rabbit antibodies

Antisera against roridin A were prepared by using a roridin A-hemisuccinate derivative coupled to human serum albumin as the immunogen. Antibodies could be detected in the sera of the immunized rabbits as early as 4 weeks after the initial exposure. After one booster injection at week 14, high antibody titers were measured over a period of 21 weeks. The specificity and sensitivity of the antibodies were tested by using roridin A-hemisuccinate coupled to horseradish peroxidase as an enzyme-linked toxin in a competitive assay with a double-antibody solid phase. The assay was most specific for the tested macrocyclic trichothecenes, and the relative cross-reactivities with roridin A, roridin J, verrucarin A, satratoxin H, and satratoxin G were 1, 0.41, 0.15, 0.15, and 0.07, respectively. When 16 nonmacrocyclic trichothecenes were tested, only diacetylverrucarol (0.0015) and verrucarol (0.0005) showed minor cross-reactivity. The sensitivity of the enzyme immunoassay for the detection of roridin A was in the range of 5 to 50 ng/ml (0.16 to 1.6 ng per assay).     

Enzyme immunoassay for the macrocyclic trichothecene roridin A: production, properties, and use of rabbit antibodies.

Antisera against roridin A were prepared by using a roridin A-hemisuccinate derivative coupled to human serum albumin as the immunogen. Antibodies could be detected in the sera of the immunized rabbits as early as 4 weeks after the initial exposure. After one booster injection at week 14, high antibody titers were measured over a period of 21 weeks. The specificity and sensitivity of the antibodies were tested by using roridin A-hemisuccinate coupled to horseradish peroxidase as an enzyme-linked toxin in a competitive assay with a double-antibody solid phase. The assay was most specific for the tested macrocyclic trichothecenes, and the relative cross-reactivities with roridin A, roridin J, verrucarin A, satratoxin H, and satratoxin G were 1, 0.41, 0.15, 0.15, and 0.07, respectively. When 16 nonmacrocyclic trichothecenes were tested, only diacetylverrucarol (0.0015) and verrucarol (0.0005) showed minor cross-reactivity. The sensitivity of the enzyme immunoassay for the detection of roridin A was in the range of 5 to 50 ng/ml (0.16 to 1.6 ng per assay).     

COMPETITIVE ENZYME IMMUNOASSAY FOR THE RAPID IDENTIFICATION OF SALMONELLA

A competitive enzyme immunoassay using a murine monoclonal antibody M105 directed against a genus-specific epitope in the Salmonella lipopolysaccharide was used to identify over 200 strains of Salmonella submitted to the National Laboratory for Enteric Pathogens. The immunoassay rapidly identified 208 strains of Salmonella representative of subspecies I, II, III_a, III_b, IV, and V, including 89 different serotypes from 26 O serogroups. The competitive enzyme immunoassay did not give positive results with 3 strains of Citrobacter freundii and 4 strains of Escherichia coli which were submitted to our laboratory as suspect Salmonella.     

Entwicklung eines hochempfindlichen Enzymimmuntests zum Nachweis von Ochratoxin A

Antibodies against ochratoxin A were produced in rabbits after immunization with an ochratoxin A-keyhole limpet hemocyanine conjugate. The immunogen was found to be very efficient, and high antibody titers were detected in the sera of all immunized rabbits. In a competitive enzyme immunoassay using ochratoxin A-horseradish peroxidase as the labelled antigen, low levels of ochratoxin A in buffer solution could be detected. The mean standard curve detection limit and 50% inhibition level of the optimized assay were at 15 pg/ml and 50 pg/ml, respectively. Relative cross-reactivity of ochratoxin B was found to be 2%. With these characteristics, this novel enzyme immunoassay should be useful for the routine detection of ochratoxin A in food and in biological samples at levels well below 1 ng/g.     

Does polymyxin B nonapeptide increase outer membrane permeability in antibiotic supersensitive enterobacterial mutants?

Abstract The outer-membrane-disorganizing peptide (polymyxin B nonapeptide; PMBN) was able to sensitize even "antibiotic supersensitive" enterobacterial mutants to hydrophobic antibiotics. This resulted in an extreme sensitivity. The mutants included the "deep rough" lipopolysaccharide mutants, as well as the acrA mutant of Escherichia coli and the "class A, B, and C mutants of Salmonella typhimurium . Sensitization factors of approx. 30 or more were found for most antibiotics. Even minimum inhibitory concentrations as low as approx. 0.5 ng/ml (rifampicin), 1.5 ng/ml (erythromycin), 2 ng/ml (fusidic acid), 6 ng/ml (novobiocin), and 30 ng/ml (clindamycin) were achieved in the presence of 30 μg/ml of PMBN. The finding indicates that the mechanisms which mediate the increase in hydrophobic diffusion are different but synergistic in the mutants and in the PMBN-grown cells.     

An indirect enzyme immunoassay for the mycotoxin citrinin.

An indirect competitive enzyme immunoassay using rabbit antisera could detect citrinin in buffer solutions at 1 to 13 ng/ml (0.05 to 0.65 ng per assay). Cross-reactivity with austdiol, alternariol, ochratoxin A, and deoxynivalenol was < 0.1% relative to citrinin. Recovery of citrinin added to wheat flour at 200 to 2,000 ng/g was 89 to 104%, with a coefficient of variation of 6.9 to 13%.     

快速同时检测玉米赤霉烯酮和脱氧雪腐镰刀菌烯醇的研究

目的：利用稀土离子作为示踪剂,建立DON/ZEN双标记间接竞争时间分辨荧光免疫分析方法同时检测DON、ZEN。 方法：以DON BSA、ZEN-BSA共包被于固相微孔板,与DON/ZEN标准或样品中的DON、ZEN竞争结合抗DON多抗、抗ZEN单抗,然后分别用稀土离子Eu3+-羊抗兔IgG及Sm3+ 羊抗鼠IgG进行示踪检测,并对建立DON/ZEN-双标记TRFIA进行方法学的考核。结果：DON/ZEN-双标记TRFIA检测灵敏度,DON为0.2 ng/ml、ZEN为0.7 ng/ml,检测范围为：DON 0.2~100 ng/ml,ZEN 0.7~50 ng/ml,批内、批间变异率均小于10%。不同样品添加回收实验表明玉米、小麦样品中DON平均回收率分别为102.8%、98.8%,ZEN平均回收率分别为94.2%、95.7%。DON/ZEN-双标TRFIA检测时,DON与ZEN不相互干扰,该方法特异性好。玉米样品检测结果表明,DON/ZEN双标记TRFIA与单标记DON -TRFIA、ZEN-TRFIA试剂盒结果高度相关,具有较好的一致性,两者检测DON的结果相关系数为0.9760,检测ZEN结果的相关系数为0.9695,结论：DON/ZEN-双标记TRFIA灵敏度高,检测范围宽,重复性、稳定性好,一次检测可同时得到DON、ZEN两个结果,是一种简便、快速、经济、稳定、可进行大批量样品筛查的检测方法。     

Development of enzyme-linked immunosorbent assay for determination of polybrominated diphenyl ether BDE-121

Our interests are in the development of immunoassay-based fast scanning methods for persistent organic pollutants. To develop the immunoassay method of polybrominated diphenyl ether (PBDE), a model compound of PBDE, 2,3′,4,5′,6-pentabromodiphenylether (BDE-121), has been chosen to develop its antibody and the competitive indirect enzyme-linked immunosorbent assay (ELISA) is developed. The hapten of BDE-121 containing reactive carboxylic acid was synthesized and conjugated to carrier proteins (bovine serum albumin [BSA] and ovalbumin [OVA]). Anti-BDE-121 polyclonal antibody was then developed in rabbits as a result of immunization with the BDE-121–BSA conjugate. The optimal amount of coating antigen BDE-121–OVA conjugate and the dilution of antiserum needed in the ELISA were determined with the checkerboard method, and the effects of the properties of PBST (phosphate-buffered saline and Tween 20) buffer (pH and salt concentration) and chemical solvent (types and concentrations) on the ELISA were investigated to achieve a rapid robust assay with high sensitivity. Under the optimized conditions, the developed indirect ELISA shows a linear detection range from 1.74 to 84.1 ng/ml, with an IC₅₀ value of 8.07 ng/ml and a detection limit of 0.644 ng/ml. In total, 11 kinds of compounds were tested for calculating the cross-reactivity, which was less than 8% for nearly all of them. Real samples were analyzed by the proposed immunoassay and gas chromatography/mass spectrometry (GC/MS).     

DETECTION OF CLOSTRIDIUM BOTULINUM TYPE E TOXIN BY MONOCLONAL ANTIBODY ENZYME IMMUNOASSAY

Botulinum type E toxin is a well recognized causative agent of seafood botulism poisoning. Underprocessing or postretort recontamination of preserved seafoods has resulted in sporadic cases of botulism. Currently, laboratory mice are being used to detect this toxin. However, it requires three to six days to obtain final results. A rapid method using monoclonal antibody (Mab) enzyme immunoassay was therefore developed. Hybridomas secreting specific Mab against the type E epitope were generated by fusion of SP/20-Ag 14 myeloma cells with spleen cells from BALB/c mice immunized with botulinum type E neurotoxoid. Five potent, stable hybridomas were selected, cloned, propagated, and preserved in liquid nitrogen as cell lines. Immunoglobulin subisotyping showed these Mabs belonged to the IgG subclasses. No cross-reaction was observed with culture supernatants of C. botulinum types A, B, and F or with crude toxins extracts of type C and D. Large quantities of Mabs were produced in ascites fluids, harvested, and affinity purified. A Mab-based biotin-avidin amplified double sandwich enzyme-linked immunosorbent assay allowed detection of type E toxin in inoculated seafoods at levels equivalent to 1–10 MLDs/ml (5–10 pg/ml).     

DEVELOPMENT AND EVALUATION OF A RAPID ENZYME-LINKED IMMUNOFILTRATION ASSAY (ELIFA) AND AN ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) FOR THE DETECTION OF STAPHYLOCOCCAL ENTEROTOXIN B

An enzyme-linked immunofiltration assay (ELIFA) and a microtitre plate enzyme-linked immunosorbent assay (ELISA) were developed and compared for their ability to detect staphylococcal enterotoxin B (SEB). The double antibody capture format was used for both assays. Factors which improved the sensitivity of the ELIFA system were (1) addition of casein and thimerosal to the antigen dilution buffer; (2) addition of polyethylene glycol (MW 6000) to the detection and conjugate antibody dilution buffers; and (3) washing with diethanolamine buffer prior to addition of the substrate/chromogen. The ELIFA system had a turnaround time of approximately 1 h and a detection limit of 1 ng/mL of purified SEB. The ELISA had a total turnaround time of 21 h, or 3 h using plates pre-coated overnight with the capture antibody. The detection limit of the ELISA for purified SEB was 0.05 ng/mL. The detection limit of SEB in cheese samples spiked with purified enterotoxin and subjected to a simple extraction procedure was 1 ng/mL and 0.1 ng/mL of extract, with the ELIFA and the ELISA, respectively.     

A monoclonal antibody to T-2 toxin

A specific, high affinity monoclonal antibody to T-2 toxin of Fusarium has been produced. The monoclonal antibody was conjugated to horse-radish peroxidase and employed to develop a direct competitive enzyme-linked immunosorbent assay (ELISA) for the toxin. The sensitivity of the ELISA was 10 ng/ml with a working range up to 500 ng/ml. The antibody cross-reacted with HT-2 toxin (25%) but did not bind to any other trichothecene tested.     

A NEW RAPID TECHNIQUE FOR SALMONELLA ANTIGEN DETECTION USING A PARTICLE CONCENTRATION FLUORESCENCE IMMUNOASSAY (PCFIA)

A fluorescent assay employing polystyrene beads coated with antibody to a common structural antigen (CSA) of salmonellae has been developed to detect the presence of Salmonella. This sensitive technique is very fast, relatively simple and with further testing may be suitable for screening food products as well as clinical diagnostics. Comparison was made with a commercially available Salmonella antigen capture enzyme immunoassay (EIA). As little as one single colony-forming unit (CFU) could be detected by fluorescence after only 16 h of enrichment. the procedure requires only 1 h to run and uses proportionately smaller volumes of sample and reagents than EIA.     

Evaluation of the fluorescence polarization assay and comparison to other serological assays for detection of brucellosis in cervids

The complement fixation test (CFT), competitive enzyme immunoassay (CELISA), indirect enzyme immunoassay (IELISA) and fluorescence polarization assay (FPA) were evaluated for the detection of antibodies to Brucella abortus and Brucella suis biotype 4 in caribou (Rangifer tarandus caribou), elk (Cervus elapus), red deer (Cervus elapus), and reindeer (Rangifer tarandus tarandus). When combining the data the FPA and the CELISA were determined to be the most suitable tests for serodiagnosis of Cervidae. The overall actual sensitivity of the CFT and the IELISA was 100%. The overall actual sensitivity for the CELISA and FPA was 99%. The overall relative specificity of the CFT (including treatment of anti-complementary data as positive or negative for analysis), the CELISA, the IELISA and the FPA were 65%, 93%, 99%, 99%, and 99%, respectively. The specificities of the buffered plate agglutination test (BPAT), the CFT, the CELISA, the FPA and the IELISA for 55 elk vaccinated with B. abortus strain 19 and tested 4 mo post vaccination were 14%, 31%, 51%, 84%, and 2%, respectively. The FPA is the diagnostic test of choice because it has sensitivity and specificity values comparable to the CELISA; it has the capability to distinguish vaccinal antibody and antibody resulting from exposure to cross-reacting organisms such as Yersinia enterocolitica 0:9 from antibody to Brucella spp. in most cases; it is technically simple to do; it is adaptable to field use and it is relatively inexpensive.