Anti‐idiotype and anti‐anti‐idiotype antibodies generated from polyclonal antibodies against aflatoxin b1

Polyclonal anti‐idiotype antibodies (anti‐id, pAb2) for aflatoxin were generated from mice ascites after immunization with affinity‐purified rabbit polyclonal antibody (pAbl) against aflatoxin B₁ (pAFB₁). After affinity chromatography purification, pAb2 were subjected to various analyses, and were used to generate anti‐anti‐id antibodies (pAb3) in the ascites of Balb/c mice. ELISA analyses revealed that the anti‐id antibodies bound specifically to the original pAb1, but not to other types of monoclonal antibodies or normal mouse IgG. The inhibition of binding of AFB₁ to pAb1 by pAb2 was demonstrated in both regular and biotin‐avidin amplified enzyme‐linked immunosorbent assay (ELISA) systems in which AFB₁‐bovine serum albumin (BSA) was coated on to the microtiter plate. The concentrations causing 50% inhibition (ID₅₀) of the binding of pAbl to AFB₁‐BSA by pAb2 were found to be 6.5 and 1.7 μg/assay respectively. Inhibition of the binding of pAb1 to the solid‐phase pAb2 by free AFB₁ (ID₅₀ = 0.63 μ/assay) was also demonstrated in the ELISA. pAb3 were found to have similar characteristics to the original pAb1. In the direct ELISA, the ID₅₀ of binding of AFB₁‐horseradish peroxidase to the solid‐phase pAb3 by AFB₁ was found to be 0.20 ng ml^‐1. In the indirect ELISA, the ID₅₀ of the binding of pAb3 to the solid‐phase AFB₁‐BSA by AFB₁ was found to be 1.84 ng ml^‐1 Analysis of a naturally contaminated corn sample for AFB₁ by the pAb3‐based ELISA gave a result similar to that obtained from the regular pAb1‐based ELISA (19.7 and 21.8 μg kg^‐1 respectively).     

Rapid determination of triazine contamination of fresh water supplies by immunoassay

A highly sensitive, competitive, enzyme‐linked immunosorbent assay (ELISA) was developed for the detection of five triazine herbicides in water samples. Low detection limits were achieved without the need to concentrate samples prior to analysis by ELISA. The limit of detection for ametryn, prometryn and prometon was 0–05 ng ml～’ while atrazine and propazine had a detection limit of 0.2 ng ml^‐1. The concentrations of analyte required to reduce zero standard absorbances by 50% (IC₅₀) far ametryn, prometryn, prometon, propazine and atrazine were 0.18, 0.18, 0.26, 0.48 and 0.37 ng ml^‐1 respectively. De‐ethylatrazine and simazine had respective ICsos of 4.0 and > 25.0 ng ml^‐1. Inter‐assay coefficients of variation were generally less than 10% and recoveries of triazines from fortified water samples were in the range 90–115% for all five triazine herbicides. The results demonstrate the ability of an enzyme immunoassay to detect five triazine herbicides at concentrations below or very close to the maximum concentration allowed under EC guidelines in drinking water (0.1 ng ml^‐1) for individual pesticides.     

Competitive ELISA employing monoclonal antibodies specific for dothistromin

Dothistromin (DOTH) is a fungal toxin occurring in Pinus radiata needles contaminated with Dothistroma pini. Monoclonal antibodies (MAbs) of high affinity were prepared against DOTH and incorporated into competitive ELISAs. MAbs were secreted by hybri‐doma prepared from mice immunized with DOTH conjugated, through the aromatic ring of the anthraquinone, to bovine serum albumin. DOTH could be quantitated at 2–60 ng ml^‐1 (0.1–2 ng/assay) and at 8–300 ng ml^‐1 (0.4–15 ng/assay) using peroxidase‐labelled MAb 10C12A5 and MAb 6A2D4 respectively in indirect competitive ELISA. The corresponding limits of detection of DOTH were < 300 pg and 1 ng ml^‐1 respectively. The furan ring was an important structure in the epitope recognized by both MAbs.     

Development of an ELISA for the N‐dealkylated s‐triazines: Application to environmental and biological samples

A sensitive and rugged ELISA for the detection of the N‐dealkylated metabolites of atrazine and simazine was developed and applied to environmental and biological samples. The limits of detection were approximately 10 ng ml^‐1 for water samples, 40 ng ml^‐1 for human urine when analyzed without any sample preparation and approximately 45 ng g^‐1for solvent‐extracted soil. This assay was tolerant to changes in pH, salt and solvent effects. Comparison of the results from this ELISA with nominal values for a series of tap water samples spiked in a blind fashion resulted in a good correlation coefficient between the values found and those spiked. However, the ELISA systematically underestimated concentrations. Examination of the source of this error revealed that the dealkylated triazine concentrations of the samples declined with time. The assay was used to monitor the presence and loss of dealkylated triazines in a variety of samples. These data indicated that caution must be used in the analysis and interpretation of the presence of these N‐dealkylated metabolites.     

Competitive Elisa for quantifying small Amounts of aflatoxin B1

An indirect ELISA was developed to quantify aflatoxin B₁ (AFB₁). The detection limit was 0.025 ng ml^‐1. The test used polyvinyl chloride (PVC) plates, activated with AFB₁ bound to bovine serum albumin (BSA). Polyclonal antibodies were raised in rabbits against AFB₁‐BSA. The specific anti‐AFB₁antibodies were recovered from the crude antiserum by affinity chromatography from a column containing immobilized BSA on Nylon 6–6. Goat anti‐rabbit IgG antibodies bound to peroxidase were used to detect the rabbit IgG anti‐AFB₁ antibodies bound to PVC plates. The colour developed by the subsequent enzyme conversion of the substrate was detected by spectrophotometry. The developed colour gave clear absorbance differences at varying doses of AFB₁. Cross‐reactivity with aflatoxin B₂, aflatoxin G₁ and aflatoxin G₂ was measured, showing percentages of 5.43, 64.5 and 5.07 respectively.     

Monoclonal antibodies specific for the organophosphate pesticide azinphos‐methyl

2‐(2‐Mercapto‐5‐methyl‐1,3,2‐dioxaphosphorinan‐5‐yl,2‐sulphide)methoxyacetic acid has been synthesized and used to prepare an azinphos hapten and protein conjugates. Monoclonal antibodies of high affinity against the pesticide azinphos‐methyl were prepared from mice immunized with the hapten‐ovalbumin conjugate. The best monoclonal antibody could be used in an indirect enzyme‐linked immunosorbent assay (ELISA) to quantify azinphos‐methyl at concentrations from 50 pg ml^‐1 to 2 ng ml^‐1 (2.5–100 pg/assay). The corresponding limit of detection of the pesticide was less than 40 pg ml^‐1. Only the closely related pesticide azinphos‐ethyl showed any appreciable cross‐reactivity in this assay.     

Enzyme immunoassays for picloram detection: A comparison of assay formats

Two direct enzyme immunoassays for picloram (4‐amino‐3,5,6‐trichloro‐2‐pyridinecarboxylic acid) detection were developed. The assay using IgG isolated from a rabbit antipicloram antiserum had a working range from 5 to 5000 ng ml^?1 with a mean I₅₀ value of 180 ng mh^?1 and a limit of quantification of 10 ng ml^?1. The assay using a monoclonal anti‐picloram antibody had a working range from 1 to 200 ng mh^?1 with a mean I₅₀ value of 18 ng mh^?1 and a limit of quantification of 5 ng ml^?1. The direct assays were compared to an existing monoclonal antibody‐based indirect enzymes immunoassay for accuracy and precision of picloram determinations in fortified ground and surface waters. In both matrices, the monoclonal antibody‐based indirect enzyme immunoassay was shown to be the superior format in terms of greater precision and equal or greater accuracy.     

Combined Immunomagnetic Separation and Detection of Salmonella enteritidis in Food Samples

A model system for immunochemical detection of Salmonella enteritidis has been developed, using immunomagnetic separation (IMS) with both commercially‐available and laboratory‐prepared antibodies, followed by an enrichment stage and end‐point detection by ELISA. IMS alone gave an average of 77% recovery of cells artificially inoculated into the food, and a range of 20–110% recovery, depending on food type. The combined model system (IMS‐ELISA) enables detection of either < 10 cells m^?1 whole egg extract (using overnight enrichment after IMS), or > 10 cells ml^?1 (3 h enrichment after IMS). Cross‐reactivity of antibodies with Citrobacter was decreased by using two different immunochemical steps: IMS with monoclonal antibody‐coated beads, and a ‘sandwich’ ELISA with polyclonal capture antibody and monoclonal detector antibody. Discussion is presented on the best food preparation method, optimal substrate type for the ELISA and on the potential of IMS.     

Detection of aflatoxins,trichothecenes, ochratoxin a and zearalenone by test strip enzyme immunoassay: A rapid method for screening cereals for mycotoxins

The development of test strip enzyme immunoassays (EIA) for the rapid detection of a number of mycotoxins is described. Monoclonal or polyclonal antibodies against aflatoxin B₁ (AFB₁), aflatoxin M₁ (AFM₁), ochratoxin A (OA), T‐2 toxin, diacetoxyscirpenol (DAS), 3‐acetyldeoxynivalenol (3‐AcDON), roridin A (RA) and zearalenone (ZEA) were immobilized on a nylon membrane. Using the corresponding toxin‐horseradish peroxidase conjugate in a direct competitive assay, dot colour development of toxinpositive test strips was visually discernible from that of the negative control. The results of the visual evaluation were compared with that of instrumental reflectance measurements of the test strips. The visual detection limits of the test strip assays for mycotoxins were at 0.6 ng ml^?1 (AFB₁) 0.2 ng ml^?1 (AFM₁), 2.0 ng ml^?1 (OA), 1.0 ng ml^?1(T‐2 toxin), 0.2 ng ml^?1 (DAS), 10.0 ng ml^?1 (3‐AcDON), 15.0 ng ml^?1 (RA), and 5.0 ppb (ZEA) respectively. Utilizing a simple extraction procedure, AFB₁, OA, T‐2 toxin, and ZEA in spiked corn samples were detected by test strip EIA at levels of 15 ng g^?1, 100 ng g^?1, 20 ng g^?1 and 80 ng g^?1 respectively.     

Detection of aldrin/dieldrin in milk by competitive enzyme‐linked immunosorbent assay (ELISA)

Polyclonal antibodies raised against 6,7‐dihydro‐6‐carboxyaldrin can be used to detect aldrin and dieldrin. These analytes are preferentially fat soluble. An immunoassay is described which provides a method for detecting these pesticides in milk, a fat‐rich matrix. The enzyme‐linked immunosorbent assay (ELISA) can detect aldrin/dieldrin in milk in the range 1 ng ml^‐1‐5 μg ml^‐1 simply and reliably. The detection range differs in skimmed and semi‐skimmed milk and in cream, reflecting the differences in fat content between these samples.     

Anti‐idiotype and anti‐anti‐idiotype antibodies for aflatoxin from laying hens

Anti‐idiotype (anti‐id) antibodies (IgY2)for aflatoxin (AF) were obtained from the egg yolks of laying hens immunized with affinity‐purified rabbit polyclonal anti‐aflatoxin B₁ (AFB₁) carboxymethyloxime‐bovine serum albumin (BSA) antibodies (pAb1). The IgY2 were affinity purified and then subjected to various analyses. Inhibition of the binding of pAbl to the solid‐phase AFB₁‐BSA by IgY2 and the binding of pAb1 to the solid‐phase IgY2 by free AFB₁ were demonstrated in a biotin‐avidin amplified enzyme‐linked immunosorbent assay (ELISA) system. The concentration of IgY2 causing 50% inhibition (ID₅₀) of the binding of pAb1 to AFB₁‐BSA was found to be 2.45 μg/assay. The ID₅₀ concentration of the binding of pAb1 to IgY2 by free AFB₁ was found to be 0.30 μg/assay. Inhibition of the binding of AFB₁‐horseradish peroxidase (HRP) to the solid‐phase pAbl by IgY2 (ID50 = 9.65 μg/assay) was also demonstrated in the direct ELISA. Egg yolk anti‐anti‐id antibodies (IgY3) were obtained by immunizing laying hens with rabbit pAb2 against anti‐AFB₃‐hemisuccinate‐BSA monoclonal antibody. IgY3 was subjected to affinity chro‐matography purification with Sepharose gel armed with AFB₂‐carboxymethytoxime, and then subjected to various analyses. ELISA analysis revealed that IgY3 has characteristics similar to other anti‐AFB antibodies induced in various experimental animals. In the direct ELISA, the ID₅₀ of the binding of AFB₁‐HRP to solid‐phase lgY3 by AFB₁ was found to be 0.12 ng ml^‐1. In the indirect ELISA, the ID₅₀ of the binding of IgY3 to solid‐phase AFB₁‐BSA by AFB₁ was found to be 2.2 ng ml^‐1. The IgY3‐based ELISA analysis showed higher sensitivity than that of the egg yolk antibodies directly against AFB‐protein conjugates (IgY1). A good correlation was found for the data obtained from IgY3‐based and pAb1‐based ELISAs in the analysis of AFB in the fungal culture filtrates.     

Development of an enzyme‐linked immunosorbent assay (ELISA) for the detection of porcine pepsin as a milk clotting enzyme

An enzyme‐linked immunosorbent assay (ELISA) for the detection of porcine pepsin in milk‐clotting enzyme preparations has been developed. The assay is capable of detecting porcine pepsin in the range 1 μgto 1 mg ml^‐1 without enhancement or modification. The specificity of the technique was studied by inhibition assay. Slight cross‐reactions with bovine rennet and Mucor miehei rennet occurred at high concentrations (1.0 mg ml^‐1). The ELISA used in this investigation appears to provide a quick, sensitive and specific method for the detection of porcine pepsin and has potential applications in the dairy industry.     

Development of high‐sensitive enzyme immunoassays for gliadin quantification using the streptavidin‐biotin amplification system

Optimization of three enzyme immunoassays of very high sensitivity using three antiprolamin monoclonal antibodies (MAbs) (13B4, 11C4 and 12A1) is presented here. These MAbs are specific for those prolamins toxic for coeliac patients, as determined by immunoblotting analysis. Biotinylated MAbs were used in two of the assays. In a competitive ELISA, the binding of each biotinylated MAb to a gliadin‐coated solid phase was inhibited by gliadin in the fluid phase. The best result was obtained using the biotinylated MAbl3B4 (detection limit: 20 ng ml^?1). With regard to capture ELISA, we tested the performance of the three MAbs. In this sandwich ELISA, the MAb used for antigenic capture was the same as that used as secondary biotinylated antibody. The MAbl2Al had the best performance (detection limit: 1 ng ml^?1). The use of biotin‐labelled gliadin in a quantitative immunoassay with a detection limit of 5 ng ml^?1 is also reported. This assay involves an antigenic capture using the MAbl2Al followed by a competition between biotinylated and non‐biotinylated gliadin. We have found the use of the streptavidin‐biotin interaction as signal amplification system to be very useful. This technique, as far as we know, has not been previously reported for gliadin quantification.     

Enzyme immunoassay for the detection of streptomycin and dihydrostreptomycin in milk

Polyclonal antisera against streptomycin were prepared by using a streptomycin‐oxime derivative coupled to bovine serum albumin for the immunization of rabbits. The specificity and sensitivity of these antibodies were tested in a competitive assay using a streptomycinenzyme conjugate (prepared by coupling a streptomycin‐hydrazone derivative to horseradish peroxidase) in a double antibody solid phase technique. The only detectable cross‐reactivity of the assay system with other aminoglycoside antibiotics and other substances similar in structure was shown to be with dihydrostreptomycin of about 148.7%. The detection limit in buffer solution was 0.6 ng ml^‐1 for streptomycin and 0–4 ng ml^‐1 for dihydrostreptomycin. Employing rapid sample preparation procedures, streptomycin and dihydrostreptomycin were detected in milk at levels as low as 6 and 0.8 ng ml^‐1respectively.     

Optimization of a competitive ELISA with polyclonal antibodies for quantification of prolamins in foods

The optimization of a sequential competitive ELISA for the quantification of prolamins in foods is described in this article. The assay was developed using polyclonal antibodies obtained by the hyper‐immunization of rabbits with commercial gliadin. The ELISA developed in this way showed a very high degree of detectability (detection limit, 1 ng ml^‐1), as well as the ability to discriminate between prolamins harmful to coeliac individuals from non‐toxic prolamins. The influence of the solvent used for extraction of the samples on the detection capability of the test was also studied. The assay proved to be useful for the evaluation of gliadins in processed foods including meat products. The assay was applied to many types of foods and was compared with a commercial kit approved by the Association of Official Analytical Chemists.     

Detection of porcine pepsin in rennet mixtures used in cheese making by ELISA

An ELISA for the detection of porcine pepsin has been developed. Porcine pepsin was detected using this assay at concentrations of ≥ 1 μg ml^‐1. Other cheese rennetting agents, such as bovine rennet or Mucor miehei rennet did not cross‐react in the assay either separately or in mixtures. The ELISA was used for the determination of porcine pepsin at concentrations of 1% (10 μg ml^‐1) and over in combination with bovine rennet/M. miehei rennet mixtures. The effects on the assay of samples which had been subjected to a pH range between 5.0 and 7.0 (which may be encountered during the storage of rennet mixtures and in cheese making procedures), were tested. Similarly pre‐incubation of porcine pepsin in the presence of NaCl up to 10%, conditions encountered in certain salty cheese preparations, had no subsequent effect on the sensitivity of the assay when such samples were tested. Milk proteins showed no cross‐reactivity with the anti‐porcine pepsin and increasing concentrations of casein to 75 mg ml^‐1 caused no significant inhibition     

Development and application of immunochromatographic tests for the detection of staphylococcal enterotoxin E

Two rapid immunochromatographic assay formats for the detection of Staphylococcus aureus enterotoxin serotype E (SEE) were developed. For both tests, polyclonal antibodies against SEE were immobilized on a membrane strip representing the test zone, while second anti‐SEE antibodies conjugated to dyed latex particles were employed as markers in sandwich immunoassay. In a two‐step test format, sample and labelled antibodies were preincubated prior to immunochromatography, whereas in a one‐step test the labelled antibodies were integrated in the test pad and activated by addition of the sample solution. In the presence of SEE in a sample solution, colour development at the antibody‐coated zone occured within 20 min. The visual detection limits for SEE in buffer solution were at 5 ng ml^?1 (two‐step test) and at 10 ng ml^?1 (one‐step test), respectively. Both assays were specific for SEE and showed no cross‐reaction with serotype A, B, C, and D enterotoxins. When the two‐step test format was used for the identification of SEE in S. aureus culture broth, the detection limit was found to be 10 ng ml^?1. Culture supernatants of 10 enterotoxigenic strains of S. aureus were analysed with the immunochromatographic two‐step test and with a microtitre plate enzyme immunoassay (EIA) test kit for enterotoxins A‐E. Results obtained by the immunochromatographic test (presence or absence of SEE) were in excellent agreement with those of the microtitre EIA.     

Development of a polyclonal antibody-based indirect competitive enzyme-linked immunosorbent assay to detect dufulin residue in water,soil and agricultural samples

Dufulin (DFL) is a potent, new-generation agrochemical that protects crops, such as tobacco, from viruses. Herein, for the first time, we developed a sensitive and specific polyclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) to detect DFL residues in samples. The assay showed high sensitivity and specificity to DFL, with a half maximal inhibitory concentration (IC₅₀) of 48.5?ng?ml^?1 and limit of detection (IC₂₀) of 2.5?ng?ml^?1. The average recovery of DFL in different spiked samples, such as tobacco, rice, tomato, cucumber, soil and water, was in the range of 70.7–138.8%. A statistically significant correlation was observed between our developed ELISA and previously established methodology of high-performance liquid chromatography with a diode array detector. These results indicate our assay is a potentially useful analytical tool for rapid detection of DFL residue in actual samples.     

Rapid detection of streptomycin and Dihydrostreptomycin in milk by enzyme‐linked immunofiltration assay

Using a membrane‐based immunochemical technique, a simple and rapid method for the detection of streptomycin and dihydrostreptomycin in milk has been developed. This enzyme‐linked immunofiltration assay has a detection limit in milk of 2 ng ml^‐1 for dihydrostreptomycin and 5 ng ml^‐1 for streptomycin. No sample preparation was needed, and the total assay procedure was completed within 10 min.     

Development of an ELISA specific for Listeria monocytogenes using a polyclonal antibody raised against a cell extract containing internalin B

We have developed a new enzyme-linked immunosorbent assay (ELISA) that is specific to the foodborne pathogenic micro-organism Listeria monocytogenes. It is based on an antibody raised against an L. monocytogenes cell preparation optimized for extraction of internalin B. Only in a sandwich ELISA format was the protein A-purified antibody specific to L. monocytogenes. In a competitive ELISA format, the antibody recognizes other Listeria species. The sandwich ELISA shows no recognition of L. innocua, L. ivanovii, L. welshimeri, L. seeligeri, or L. grayii. It has a minimum detectable level for L. monocytogenes of log₁₀?6.37 cfu ml^?1 in pure culture, is reproducible, and is unaffected by the presence of high numbers (approximately log₁₀?8.0 cfu ml^?1) of the other Listeria species. Possible reasons for the format-dependent specificity are discussed. When the ELISA was applied to milk samples inoculated with L. monocytogenes reference material (5 cfu ml^?1), there was a strong response to the enrichment cultures. The new assay may prove useful in detection of L. monocytogenes in enrichment cultures of food samples.