A rapid ELISA for screening aflatoxin B1 in animal feed and feed ingredients in Indonesia

To facilitate a sustainable aflatoxin management system in Indonesia, a simple, rapid and effective Enzyme Linked Immunosorbent Assay (ELISA) test for screening aflatoxin B₁ (AFB₁) in animal feed and feed ingredients was developed. Anti-AFB₁ polyclonal antibodies were produced against AFB₁-BSA and AFB₁-KLH immunogens. Using AFB₁ conjugated to horseradish peroxidase (HRP) as an enzyme marker in a direct competitive ELISA, an IC₅₀ of 0.85±0.15 µg/kg and a detection limit (IC₁₅) of 0.18±0.06 µg/kg of AFB₁ were achieved. The assay was highly specific to AFB₁ with very little cross reaction with other aflatoxin congeners (AFB₂ 0.9%, AFG₁ 3.1% and AFG₂ 1.2%) and metabolites. The ELISA was tolerant to methanol (up to 60%) and pH (pH 7.2-9.6) without significantly affecting the overall performance and was not affected by interferences from the animal feed and corn samples. Satisfactory recovery results were obtained from the spike and recovery study (77-97% recovery for 10-252 µg/kg). A pilot survey conducted on corn and animal feed samples collected from the local feed factories and poultry shops indicated that significant amounts of corn and animal feeds were contaminated by aflatoxin B₁ greater than the MRL (50 µg/kg).     

Anomalous aflatoxin B1 recoveries from whole peanuts and brazil nuts measured by enzyme‐linked immunosorbent assay

Aflatoxin B ₁ extraction solvent (acetonitrile‐water, 1:1) was shown to be acceptable when performing an ELISA using a dilution of 1 in 20 of nut extracts in assay buffer in order to prevent reductions in assay sensitivity due to nut matrix interferences. An extraction period of 30 min using acetonitrile‐water (1:1) was shown to be efficient for the removal of aflatoxin B ₁ from peanuts. The extractions were performed immediately after spiking. This dilution was used when analyzing both raw and roasted peanuts, which are finely ground, whole without testa, or only the testa (for this last, only roasted samples were studied). A significant reduction in aflatoxin B ₁ recoveries was observed when samples were left to stand overnight after spiking. This suggests that either the aflatoxin B ₁ molecule establishes strong bonds with some of the components of the peanut surface, or that substance(s) (non‐enzymic) existing at this surface will react with the aflatoxin B ₁ molecule, causing it to break down, or modify it to give products that cannot be recognized using the ELISA procedure.     

Rapid monitoring of dicyclohexyl phthalate in foods using the direct competitive ELISA

A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) has been developed for the quantitative detection of the dicyclohexyl phthalate (DCHP) in some liquid food. Specific polyclonal antisera to DCHP was raised, using the hapten-bovine serum albumin conjugates as the immunogen. The conjugate of horseradish peroxidase (HRP) with antibody was used as the detectable probe to provide the direct measurement of the antigen and analyte, which was synthesised by a modified glutaraldehyde method. Under the optimised assay, the quantitative working range was from 0.1 to 100 ng mL^?1 (R ²=0.9989), with a limit of detection (LOD) of 0.03 ng mL^?1, and a recovery of 96.6–112.4%. The specificity and accuracy of developed method were evaluated. The cross-reactivities of antibody with structurally related phthalate esters were less than 10%. Results obtained indicated that the dc-ELISA was a sensitive, low expense method to improve the routine monitoring of trace constituents in some liquid food.     

Cloning of a gene associated with aflatoxin B1 biosynthesis in Aspergillus parasiticus

Summary A cosmid library was constructed by inserting genomic DNA isolated from a wild-type aflatoxin-producing strain of Aspergillus parasiticus (SU-1) into a cosmid vector containing an homologous nitrate reductase (niaD) gene as a selectable marker. One cosmid was isolated which complemented an aflatoxin-deficient, nitrate-nonutilizing mutant strain, A. parasiticus B62 (nor-1, niaD), to aflatoxin production. Deletion and complementation analyses showed that, a 1.7 kb BglII-SphI DNA fragment isolated form this cosmid was responsible for renewed aflatoxin production. Northern hybridization analyses revealed that the major RNA transcribed from this DNA fragment, was 1.4 kilonucleotides in size. Genetic complementation, proved to be a useful strategy for cloning a gene associated with aflatoxin biosynthesis in A. parasiticus.     

Development and validation of an indirect competitive ELISA for quantification of recombinant staphylokinase in rabbit plasma: Application to pharmacokinetic study

The relatively short circulatory half-life (2–3 min) of staphylokinase is a major drawback in the development of SAK- (staphylokinase) based thrombolytic drug. A rapid and sensitive method, based on indirect competitive ELISA, was developed and validated for quantitative determination of SAK in rabbit plasma. The dynamic range of the assay varied between 0.41 ± 0.16 μg/L and 9.03 ± 0.38 μg/L (R² = 0.98) for SAK in rabbit plasma. There were no dilution linearity issues apparent with this assay. The precision (% CV) ranged from 4.6–9.7% for the intraassay and from 17.1–19.3% for interassay. This validated method was successfully employed for evaluation of various pharmacokinetic parameters of SAK in rabbit.     

Production of the class-specific antibody and development of direct competitive ELISA for multi-residue detection of organophosphorus pesticides

Based on the common structure of organophosphorus pesticides, O,O-dimethylphosphorothioate, two haptens were synthesised with different spacer arms. The class-specific polyclonal antisera were prepared from the conjugates of haptens and keyhole limpet hemocyanin. Under optimum conditions, a homologous direct competitive enzyme-linked immunosorbent assay (ELISA) method was developed for multi-residue detection of 23 organophosphate pesticides. The most sensitive IC₅₀ values of the multi-residue analysis were estimated as 0.25, 0.65 and 0.80 mg L^?1 for methyl parathion (PM), fenitrothion and fenthion, respectively. Furthermore, when the developed ELISA was applied to detection of PM in water samples, adequate spike recoveries in the range from 70.3 to 131% were obtained. It provided a useful multi-residue determination method of organophophorus pesticides.     

ELISA determination of aflatoxin levels in whole nuts

An indirect, double antibody, microtitration plate ELISA technique was used to assay aflatoxin B, in samples of peanuts, Brazil nuts, almonds, hazelnuts and walnuts. The minimum dilutions of nut extracts required to prevent interferences were of 1 in 10 for almonds; of 1 in 20 for Brazil nuts and peanuts; and of 1 in 50 for hazelnuts and walnuts. The ELISA procedure was validated for aflatoxin B, detection in the shelled nuts referred to above by determining the percentage of this toxin recovered from artificially spiked nut samples. The results confirmed that this technique can be used for aflatoxin B₁ determination in these nuts. The comparative studies undertaken using naturally contaminated peanut samples also led to the conclusion that this ELISA protocol can be recommended as an alternative to the already adopted thin‐layer chromatography (TLC) method. It was also observed that placing peanut samples under a UV light is not an advisable method of ascertaining aflatoxin B₁ contamination of these nuts, as levels of 0.6 μg g^‐1 were not detectable.     

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.     

Determination of Aflatoxin B 1 in Highly Contaminated Peanut Samples Using HPLC and ELISA

The precision, accuracy, detection limit and peanut matrix influence of an ELISA were analysed in the determination of aflatoxin B 1 . The assay was performed on two different reference samples: peanut extract and peanut paste, that were spiked with known amounts of aflatoxins. The lower detectable level was 0.5 w g kg -1 . The average intra-assay precision expressed as coefficient of variation (CV) was 11.7% for concentrations between 2.54 and 901 w g kg -1 and the average inter-assay precision was 29.7% for the same range of concentrations. The average accuracy measured by a recovery assay in samples that contained only aflatoxin B 1 was 107%. The correlation between the ELISA and high-performance liquid chromatography (HPLC) applied to 28 peanut samples artificially contaminated with Aspergillus flavus and A. parasiticus spores showed a high correlation ( r = 0.977, P < 0.0001). The detection limits and matrix influence associated with our ELISA procedure were more sensitive than those reported for other ELISA procedures due to specific antiserum treatment.     

Application of experimental design techniques to optimize a competitive ELISA

This paper describes the application of experimental design techniques to optimize a sensitive ELISA for a hapten molecule with a calibration range of 0–1000 pg/ml. Ten factors that were expected to affect the assay performance were initially screened, followed by factorial experiments to delineate the effects of the critical factors identified at the screening stage. Assay performance was evaluated using a unique rating system based on standard curve reproducibility, assay detection limits and the use of desirability functions. This rating system allowed multiple responses to be evaluated simultaneously. It was found that the substrate incubation time and enzyme label lot played an important role, while dilutions of the enzyme label and the anti-hapten antibody showed significant interaction. These observations were in good agreement with optimal assay conditions based on historical data collected over a period of two to three years. Application of experimental design techniques enabled us to confirm the significance of the factors affecting the assay within a three month period, with a minimum number of experiments. In addition, information on interaction between factors were determined.     

Inter-laboratory studies for the evaluation of ELISA kits for the detection of chloramphenicol residues in milk and muscle

The use of chloramphenicol in veterinary medicine was banned in the EU in 1994. As the Community Reference Laboratory for antibiotic residues in food of animal origin, one of our functions is to organize inter-laboratory studies. A first inter-laboratory study for the analysis of chloramphenicol (CAP) in milk by ELISA kits was organized in 2001 and a second one for the detection of CAP in pig muscle by ELISA kits in 2002. These studies were intended to allow participants to control their CAP ELISA methods when used routinely and also to compare the performances of various ELISA kits for the detection of chloramphenicol in milk (commercial or in-house kits). In 2001, 15 participants received ten randomly coded frozen milk samples (four blank samples and six spiked milk samples from 0.5 to 5.0 μg/l). In 2002, 20 participants received eight randomly coded frozen muscle samples (two blank samples and six incurred muscle samples from 2.1 to 6.5 μg/kg). They were asked to analyse each sample in triplicate with the ELISA kit of their choice. Different kits from different suppliers were used and compared in the two studies. The results of the two inter-laboratory studies on ELISA kits were satisfactory regarding qualitative results. The global rates of false compliant results of 2.2% for milk and 0.0% for pig muscle samples were lower than 5% whichever kit was used. The global rates of false non-compliant results (16.7% and 10% for milk and muscle respectively) were also satisfactory. The distribution of false non-compliant results depends on the kind of kit used and on the detection limit for milk as well as for muscle. Moreover the sample preparation was very important to avoid false non-compliant results. Finally, this study demonstrates that ELISA kits for chloramphenicol in milk and muscle globally show good repeatability and accuracy. So these kits could be considered as suitable tests for screening purposes.     

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.     

Quantitative measurement of HBeAg in chronic hepatitis B: A comparison between a radioimmunoassay a fluorescence ELISA and a chemiluminescence ELISA

The presence of the hepatitis B e antigen (HBeAg) in peripheral blood of chronic hepatitis B patients is a widely accepted marker of active replication of the hepatitis B virus. HBeAg determination during interferon therapy is a useful guide for the therapeutic regimen. The aim of the study was to compare the suitability of an HBeAg radioimmunoassay (RIA, Abbott Laboratories, North Chicago, IL, USA), the IMx-HBeAg assay (IMx, Abbott Laboratories) and the HBeAg/anti-HBe Amerlite assay (Amerlite, Johnson & Johnson Clinical Diagnostics, Cardiff, UK) for semiquantitative monitoring of HBeAg during therapy. HBeAg levels in serum samples obtained before and during interferon therapy were measured using an in-house standard calibrated against the Paul Ehrlich Institute HBeAg reference preparation (PEI standard). When serial dilutions of pretreatment serum samples were assayed by the three methods, radioimmunoassay was found to be highly sensitive although it had a very limited working range (0.5 to 12 PEI U/ml). A broader linear working range was observed for Amerlite (0.5 to 50 PEI U/rnl) and the IMx assay (0.5 to 100 PEI U/ml). The intra-assay and interassay variations did not differ significantly. Since the IMx assay was less susceptible to sample variation and had a broad working range, semiquantitative measurement of HBeAg in one diluted and one undiluted sample by this assay may justifiably be introduced as routine procedure. Routine semiquantitative HBeAg measurement may improve individual dose adjustments and thus the success of interferon therapy. © Wiley-Liss, Inc.     

Inter-laboratory studies for the evaluation of ELISA kits for the detection of chloramphenicol residues in milk and muscle

The use of chloramphenicol in veterinary medicine was banned in the EU in 1994. As the Community Reference Laboratory for antibiotic residues in food of animal origin, one of our functions is to organize inter-laboratory studies. A first inter-laboratory study for the analysis of chloramphenicol (CAP) in milk by ELISA kits was organized in 2001 and a second one for the detection of CAP in pig muscle by ELISA kits in 2002. These studies were intended to allow participants to control their CAP ELISA methods when used routinely and also to compare the performances of various ELISA kits for the detection of chloramphenicol in milk (commercial or in-house kits). In 2001, 15 participants received ten randomly coded frozen milk samples (four blank samples and six spiked milk samples from 0.5 to 5.0 μg/l). In 2002, 20 participants received eight randomly coded frozen muscle samples (two blank samples and six incurred muscle samples from 2.1 to 6.5 μg/kg). They were asked to analyse each sample in triplicate with the ELISA kit of their choice. Different kits from different suppliers were used and compared in the two studies. The results of the two inter-laboratory studies on ELISA kits were satisfactory regarding qualitative results. The global rates of false compliant results of 2.2% for milk and 0.0% for pig muscle samples were lower than 5% whichever kit was used. The global rates of false non-compliant results (16.7% and 10% for milk and muscle respectively) were also satisfactory. The distribution of false non-compliant results depends on the kind of kit used and on the detection limit for milk as well as for muscle. Moreover the sample preparation was very important to avoid false non-compliant results. Finally, this study demonstrates that ELISA kits for chloramphenicol in milk and muscle globally show good repeatability and accuracy. So these kits could be considered as suitable tests for screening purposes.     

A novel signal amplification technology for ELISA based on catalyzed reporter deposition. Demonstration of its applicability for measuring aflatoxin B(1)

In an earlier communication we have described a novel signal amplification technology termed Super-CARD, which is able to significantly improve antigen detection sensitivity in conventional Dot-ELISA by approximately 10⁵-fold. The method utilizes hitherto unreported synthesized electron rich proteins containing multiple phenolic groups which, when immobilized over a solid phase as blocking agent, markedly increases the signal amplification capability of the existing CARD method (Bhattacharya, R., Bhattacharya, D., Dhar, T.K., 1999. A novel signal amplification technology based on catalyzed reporter deposition and its application in a Dot-ELISA with ultra high sensitivity. J. Immunol. Methods 227, 31.). In this paper we describe the utilization of this Super-CARD amplification technique in ELISA and its applicability for the rapid determination of aflatoxin B₁ (AFB₁) in infected seeds. Using this method under identical conditions, the increase in absorbance over the CARD method was approximately 400%. The limit of detection of AFB₁ by this method was 0.1 pg/well, the sensitivity enhancement being 5-fold over the optimized CARD ELISA. Furthermore, the total incubation time was reduced to 16 min compared to 50 min for the CARD method. Assay specificity was not adversely affected and the amount of AFB₁ measured in seed extracts correlated well with the values obtained by conventional ELISA.     

Direct coupling of an atrazine analogue to nicrotiter plates for a competitive immunoassay for s‐triazines

Commercially available maleic anhydride‐activated enzyme‐linked immunosorbent assay (ELISA) plates were used to immobilize atrazine in the form of aminohexyl‐atrazine (AHA). The immobilized atrazine was used to create a competitive s‐triazine assay. The assay based on the pre‐activated ELISA plates was compared with previously used assays where the binding of atrazine to the plate was indirect through an AHA‐dextran conjugate. Atrazine, in the form of AHA, bound quickly and reliably to the activated ELISA wells, and a competitive immunoassay was established with similar effectiveness to an assay based on a carrier conjugate. The ELISA format in general has many advantages to the end user, including the need for only small sample volumes, rapid assays and ease of automation. The availability of ELISA plates which spontaneously immobilize small amine‐containing ligands such as AHA might open up new possibilities for assay development. Moreover, it allows the presentation of small antigens without a carrier protein.     

An improved indirect competitive Elisa for aflatoxin m1 in milk powders using novel monoclonal antibodies

Among three newly prepared monoclonal anti‐aflatoxin M₁ antibodies, named AM.1, AM.2 and AM.3, both AM.1 and AM.3 possessed high specificity and sensitivity towards aflatoxin M₁, while AM.2 exhibited lower specificity. Employing AM.3 and horseradish peroxidase‐labelled second antibody, an improved ELISA was developed with a detection limit of 1.0 pg ml^‐1 of aflatoxin M₁ in solution. The contents of aflatoxin M₁ in powdered milks suspended in water were assayed by the indirect ELISA. The detection limit was 5 pg g^‐1dry weight, and no clean‐up procedures were required. The reliability of the present ELISA with monoclonal antibody AM.3 was confirmed with reference powdered milks for aflatoxin M₁. A limited ELISA survey showed the presence of aflatoxin M₁ in commercial milk powders sampled in France, the US and Thailand at levels of 30–418 pg g^‐1, and it was confirmed by an improved HPLC analysis.     

Quantification of Ara h 1 in peanuts: why roasting makes a difference

BACKGROUND: Increased allergenicity of roasted vs. raw peanut has been reported by showing higher IgE binding to roasted peanut extracts. OBJECTIVE: To study the effect of roasting on Ara h 1 quantification in peanut using a specific monoclonal antibody-based ELISA, and to compare the Ara h 1 content from different kernel size peanuts from four runner cultivars. METHODS: Raw or oven-roasted (177 degrees C for 5-30 min) runner peanuts were crushed and extracted at 60 degrees C. Inhibition ELISA was used to study binding of Ara h 1 purified from raw or roasted peanut. Runner peanuts of four different cultivars were collected, shelled, sized and roasted for 15 min at 177 degrees C. Ara h 1 in the extracts was compared by ELISA. RESULTS: Ara h 1 levels were up to 22-fold higher in roasted than in raw peanuts (820 vs. 37 microg/mL, in a representative experiment) with an Ara h 1 peak at 10-15 min of roasting. Inhibition ELISA indicated that this increase was not due to conformational changes in the Ara h 1 monoclonal antibody epitopes. Ara h 1 was found at lower levels in number 1 than in jumbo- and medium-sized peanuts, and no differences were found among cultivars. CONCLUSION: These results suggest that roasting increases the efficiency of Ara h 1 extraction, and/or that the monoclonal antibody binding epitopes were more accessible in roasted peanut. Expression of Ara h 1 is associated with peanut maturity.     

Detection of aldrin and dieldrin in egyptian milk samples using a competitive ELISA

A number of milk samples collected in Egypt from different animal species and at different locations were analyzed for the presence of the organochloride pesticides aldrin and dieldrin. A simple competitive enzyme‐linked immunosorbent assay (ELISA) was used for the detection and quantification of aldrin and dieldrin in milk samples from different species: buffalo, cow, goat, sheep and donkey. Pesticides were detected in 62.5% (10/16) of the buffalo milk samples, 73.33% (11/15) of the cows’ milk samples, 25% (3/12) of the goats’ milk samples, 71.42% (5/7) of the sheeps’ milk samples and 66–66% (2/3) of the donkeys’ milk samples.     

Antineutrophil cytoplasmic antibody measurement: advantages and disadvantages of a capture PR3 ELISA and a direct PR3 ELISA

AIM: To compare the performance of a capture proteinase 3 enzyme linked immunosorbent assay (PR3 ELISA) with a direct PR3 ELISA in the measurement of PR3 antineutrophil cytoplasmic antibodies (ANCA). METHOD: The performance of both assays systems was compared using two sets of sera. Sera from patients (n = 49) suffering from Wegener's granulomatosis (WG) and fulfilling the American College of Rheumatology classification criteria (or a modification of those criteria that allowed for ANCA positivity) were used along with sera from a group of patients (n = 48) considered to have a clinically false positive PR3 ANCA result when measured by routine direct ELISA. RESULTS: Using the assay specific cut-offs, the direct ELISA gave a positive result in 92% on repeat testing and the capture ELISA a positive result in 84% of sera from patients with WG. The capture ELISA was negative in 75% of patients considered to have a false positive PR3 ANCA on initial testing by direct ELISA (27% were negative on repeat testing by direct ELISA). The mean concentration of PR3 ANCA in WG patient sera measured by the capture ELISA was significantly higher than that measured by the direct ELISA. The capture PR3 ELISA had a broader analytical range which was also reflected in PR3 ANCA concentrations measured in serial serum samples from WG patients. CONCLUSION: In this study the direct PR3 ELISA performed better as a screening test for PR3 ANCA compared with the capture PR3 ELISA, mainly because the cut-off for the capture ELISA needed to be set higher to avoid non-specific binding. In contrast, the improved analytical range of the capture ELISA made it a potentially more useful method for monitoring serial PR3 ANCA concentrations. In specific serum samples the capture ELISA was better able to discriminate 'false positive' PR3 ANCA.