Comparison of radioimmunoassay and enzyme-linked immunosorbent assay for determining aflatoxin M1 in milk

Using a highly specific antibody against aflatoxin M1, a radioimmunoassay (RIA) and an enzyme-linked immunosorbent assay (ELISA) were developed for the quantitation of M1 in milk. RIA was sensitive in the range of 5-50 ng per assay but was subject to interference by whole milk. Extraction and cleanup were therefore necessary for the detection of M1 in milk at 0.5 ng/mL. An ELISA procedure was developed by using an aflatoxin M1-carboxymethyl-horseradish peroxidase conjugate as the ligand. Competitive assays revealed that this system was relatively more sensitive for M1 than for B1, and had a much lower degree of cross-reactivity for aflatoxins B2, G1, G2, B2a, and aflatoxicol. As low as 0.25 ng M1/mL in artificially contaminated milk (raw, whole, skim) could be detected by ELISA in 3 h without extraction or cleanup. Because of its simplicity, sensitivity, and specificity, ELISA is the preferred method for monitoring aflatoxin M1 in milk.     

Improved enzyme-linked immunosorbent assay for aflatoxin B1 in agricultural commodities

An improved enzyme-linked immunosorbent assay (ELISA) for aflatoxin B1 in cornmeal and peanut butter was developed. Aflatoxin B1 in cornmeal and peanut butter samples was extracted with 70% methanol in water containing 1% dimethylformamide diluted with assay buffer to a final concentration of 7.0% methanol, and directly subjected to an ELISA procedure that took less than 1 h for quantitative analysis and less than 30 min for screening tests. Analytical recoveries for 5-100 ppb B1 added to the cornmeal and peanut butter were 91 and 95.4%, respectively. The interwell and interassay coefficient of variation was 10% or less at the 20 ppb level and above. Agreement for B1 levels in more than 30 naturally contaminated corn, mixed feed, and peanut butter samples was excellent between the ELISA data and the data obtained from different independent laboratories using TLC or other analytical methods.     

Direct competitive enzyme-linked immunosorbent assay for sulfamethazine residues in milk

A direct competitive enzyme-linked immunosorbent assay (ELISA) is described for the detection and estimation of sulfamethazine residues in milk. Samples are cleaned up rapidly by acidifying and centrifuging the milk, adjusting the supernatant liquid to pH 7.0, and centrifuging again. The supernate is then assayed using set points to estimate sulfamethazine levels in the sample in the range of 1 ppb to 1 ppm. Multiple samples of milk can be screened in 1.5-2 h by this ELISA method.     

Improved enzyme-linked immunosorbent assay for determination of soy protein in meat products

An indirect, competitive enzyme-linked immunosorbent assay (ELISA) has been developed for quantitation of soy protein in meat products. The methodology allows rapid aqueous extraction of meat samples into a liquid form suitable for assay. The assay is highly specific for soy protein and is designed to measure soy protein levels between 1 and 10% of the wet weight of the sample. Standardized, stabilized reagents for carrying out the procedure are commercially available in a kit. The analysis, including sample preparation, can be completed within a workday, and the actual immunoassay in less than 60 min.     

Determination of aflatoxin M1 in milk and milk products contaminated at low levels

A new method is described for the determination of aflatoxin M1 in milk and dairy products by thin layer chromatography. The main characteristic is the extraction system using an alkaline solution. Lipids are removed by centrifuging at low temperatures, and the aflatoxins are then extracted with CHCl3. The method has 2 options: Technique II (detection limit 0.02 ppb) requires cleanup on a chromatographic column; this is not necessary in Technique I (detection limit 0.1 ppb). The recovery rate in both techniques is over 92.8% in milk and yoghurt. This method may also be used for other aflatoxins. Because of the advantages of the method, Technique II is recommended for aflatoxin M1 control in milk, where a low detection limit is necessary. Technique I is proposed for experimental aflatoxin production studies in dairy products, which require analysis of a large number of samples but which do not require a very low detection limit.     

Rapid monoclonal antibody-based enzyme-linked immunosorbent assay for detection of Listeria in food products

A rapid diagnostic test for the detection of Listeria in food products has been created. This test, known as Listeria-Tek, uses 2 monoclonal antibodies specific for Listeria in an enzyme-linked immunosorbent assay (ELISA) format. The test requires only 40 h of broth enrichment with no culturing on solid media. It is extremely simple to perform and easy to interpret, and is at least as sensitive and accurate as the best of the culture methods. The test can be used with dairy products, meat products, and environmental samples. The ELISA test is safely performed on the open bench of the laboratory because no live cultures, no radioactivity, no phage, etc., are necessary. There is no need for special licenses or reserved laboratory space, and no waste disposal problems are encountered. If necessary, one technician could easily perform hundreds of assays per day. A printed data sheet is available for permanent records.     

Enzyme-linked immunosorbent assay for screening aflatoxin B1 in cottonseed products and mixed feed: collaborative study

A joint AOAC/IUPAC (International Union of Pure and Applied Chemistry) interlaboratory study of an enzyme-linked immunosorbent screening assay (ELISA) for aflatoxins was conducted in laboratories in Canada, France, Japan, South Africa, Switzerland, The Netherlands, Tunisia, and the United States. Twenty-eight samples of raw and roasted peanuts, corn, whole cottonseed, cottonseed meal, ammoniated cottonseed meal, and poultry feed containing various quantities of natural aflatoxins and supplemented when appropriate with aflatoxin B1 were distributed to participating laboratories for testing. The assay is based on conjugation of pure aflatoxin B1 to an enzyme and the competition between this conjugate and (free) aflatoxins in the product for aflatoxin-specific antibodies coated onto microtiter well walls. After a wash step to remove all unbound aflatoxins, a substrate, added to each well, is catalyzed from a colorless to a green solution by any bound enzyme-conjugated aflatoxin B1 present. The intensity of the color decreases as the amount of free aflatoxin B1 in the product increases. Overall correlation was good between ELISA and thin-layer chromatographic (TLC) results for cottonseed products and mixed feed. Variable results were reported for corn and peanut product samples. Although some positive samples (greater than 15 ng/g) of cottonseed products and mixed feed were reported to contain less than 15 ng/g by visual determination, a review of data for absorbance measurements showed that the contamination level was close to the greater than or equal to 15 ng/g standard and would not have been reported as negative under routine screening.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of pyrethroid residues in agricultural products by an enzyme-linked immunosorbent assay

To determine cypermethrin and permethrin in agricultural products, a competitive enzyme-linked immunosorbent assay (ELISA) method was employed. The matrix interferences were minimized by direct dilution of the extracts. No further cleanup was needed. A minimum matrix effect with a 1:10 dilution of white wine for cypermethrin and a 1:200 dilution of red and white wines, fruits, and vegetables for permethrin was found when phosphate-buffered saline containing 40% methanol was employed as the diluent. Good recoveries of spiked levels were observed. The mean percentage recoveries of cypermethrin spiked in white wine and permethrin spiked in red and white wines were 99.7, 74, and 78%, respectively. The mean percentage recoveries of permethrin spiked in apple, banana, cucumber, lettuce, onion, and peach were 99.2, 105, 70.2, 97.5, 94.4, and 89.4%, respectively. Validation of the ELISA method with permethrin-spiked lettuce and peach was carried out using gas chromatography with mass spectrometry, resulting in a good recovery and correlation.     

Evaluation of enzyme-linked immunosorbent assay of cleanup for thin-layer chromatography of aflatoxin B1 in corn, peanuts, and peanut butter

A simple, rapid enzyme-linked immunoassay (ELISA) was used to evaluate the performance of each step (extraction, filtration, solvent partition, and silica gel column chromatography) of a solvent-efficient thin-layer chromatographic (TLC) method which is undergoing interlaboratory collaborative study for the determination of aflatoxin B1 in corn, raw peanuts, and peanut butter. The apparent average recoveries using the ELISA method were about 30 to 50% higher than those using the TLC method if only the amount of B1 added to the samples was used in the calculations. After the cross-reaction of the antibody with other aflatoxins added to the samples was considered, the amounts recovered approached the levels of aflatoxins added in all 3 commodities tested. With no cleanup treatment, ELISA recoveries at aflatoxin B1 levels above 7.5 ng/g were 84, 79, and 103% for corn, raw peanuts, and peanut butter, respectively. The coefficients of variation were between 5.2 and 25.2%. With each cleanup step in the TLC method, ELISA detected a progressive decrease in recovery from 150.5 to 105.3% (before correction for the presence of other aflatoxins) or from 93.5 to 65.4% (after correction for other aflatoxins) of B1 added to the samples. The ELISA data support the conclusion obtained from previous studies that cleanup treatments were not necessary in the ELISA. When large amounts of other aflatoxins are present, an understanding of the cross-reactivity of antibody with other aflatoxins in the ELISA is essential for final interpretation of the data.     

Development and application of solvent-free extraction for the detection of aflatoxin M1 in dairy products by enzyme immunoassay

The official methods for the quantification of aflatoxin M1 in dairy products (cheese and yogurt) include extraction into dichloromethane or chloroform, evaporation of the solvent, partitioning of the reconstituted residue with hexane, and subsequent analysis. To secure a rapid and inexpensive screen for aflatoxin M1 contamination, a sensitive competitive ELISA, using a rabbit polyclonal antibody, was developed for measuring aflatoxin M1 in milk and used in a comparative study for measuring the extraction efficiency of aflatoxin M1 in aqueous or organic solvent buffers using yogurt samples. An aqueous sodium citrate solution was found to be suitable for extracting aflatoxin M1, thus eliminating the need for organic solvents. The citrate extraction proved to be efficient (recovery ranged from 70 to 124%) in fortified samples of very different kinds of dairy products, including yogurt and six types of cheese. Fourteen yogurt and cheese samples were extracted with citrate solution and analyzed by ELISA. A good correlation was observed (y=0.95x-0.59, r2=0.98) when the data were compared with those obtained through the official method, across a wide range of aflatoxin M1 contaminations (10-200 ng/kg).     

Development of an enzyme-linked immunosorbent assay for the insecticide thiamethoxam

An enzyme-linked immunosorbent assay (ELISA) was developed for the neonicotinoid insecticide thiamethoxam, 3-(2-chlorothiazol-5-ylmethyl)-5-methyl-4-nitroimino-1,3,5-oxadiazinane. Three antisera were raised from rabbits immunized with the hapten-KLH conjugate. On the basis of the computational analysis of hapten candidates, the hapten with a spacer arm on the thiazolyl ring of thiamethoxam was synthesized to elicit thiamethoxam-specific antisera. The hapten was 3-[2-(2-carboxyethylthio)-5-ylmethyl]-5-methyl-4-nitroimino-1,3,5-oxadiazinane. Antisera were characterized with indirect competitive ELISA. Cross-reactivity and effects of organic solvents, pH, and ionic strengths were evaluated. The antiserum was specific for thiamethoxam and tolerant of up to 5% acetonitrile and 5% acetone. Various ionic strengths and pH values in the tested ranges had negligible effect on the assay performance. Under the optimized conditions, the half-maximal inhibition concentration (IC(50)) and the limit of detection were approximately 9.0 and 0.1 microg/L of thiamethoxam, respectively. ELISA analysis of stream and tap water samples showed an excellent correlation with the fortification levels.     

Development of an enzyme-linked immunosorbent assay for the pyrethroid cypermethrin

A competitive enzyme-linked immunosorbent assay (ELISA) for the detection of cypermethrin was developed. Two haptens, the trans- and cis-isomers of 3-[(+/-)-cyano-3-(2,2-dichloroethenyl)-2,2-dimethylcyclopropanecarbonyloxy]methyl]phenoxyacetic acid, were conjugated with thyroglobulin as immunogens. Four antisera were generated and screened against six different coating antigens. The assay that was the most sensitive for cypermethrin was optimized and characterized. The IC(50) for cypermethrin was 13.5 +/- 4.3 microg/L, and the lower detection limit (LDL) was 1.3 +/- 0.5 microg/L. This ELISA had relatively low cross-reactivities with other major pyrethroids, such as deltamethrin, phenothrin, resmethrin, fluvalinate, and permethrin. Methanol was found to be the best organic cosolvent for this ELISA, with an optimal sensitivity observed at a concentration of 40% (v/v). The assay parameters were unchanged at pH values between 5.0 and 8.0, whereas higher ionic strengths strongly suppressed the absorbances. To increase the sensitivity of the overall method, a C(18) sorbent-based solid-phase extraction was applied to various domestic and environmental water samples. The water samples, fortified with cypermethrin, were analyzed according to this method. Good recoveries and correlation with spike levels were observed.     

Development of an enzyme-linked immunosorbent assay for the insecticide imidacloprid

Enzyme-linked immunosorbent assays (ELISAs) were developed for imidacloprid, a neonicotinoid insecticide. Haptens were designed in such ways that spacer arms were introduced on either the pyridinyl or the imidazolidinyl ring of imidacloprid. Two sets of polyclonal antibodies were raised from rabbits immunized with two different immunogens and were characterized with an indirect ELISA format. Cross-reactivities and effects of organic solvents on the assays were evaluated. One set of antibodies shows approximately equal cross-reactivities to imidacloprid and its major metabolites with half-maximum inhibition concentrations (I(50)) of 73-88 ppb. Another is specific to imidacloprid with an I(50) of 35 ppb. The assay was initially applied to the analysis of imidacloprid in fortified water, coffee cherry, and bean extracts.     

Enzyme-linked immunosorbent assay of aflatoxin B1 in naturally contaminated corn and cottonseed

Naturally contaminated corn and cottonseed samples were screened for aflatoxin B1 (AFB1) by a direct competitive enzyme-linked immunosorbent assay (ELISA). Samples were blended 5 min in an extraction solvent of methanol-water-dimethylformamide (70 + 29 + 1) and filtered. Filtrates were assayed by direct competition between AFB1 in the corn and cottonseed samples and AFB1-peroxidase conjugate for binding to specific antibody adsorbed to a solid phase microtiter plate. Standard curves prepared using the extract of AFB1-free corn and cottonseed samples, and extraction solvent only, showed negligible interference by the sample extract in the performance of ELISA. The AFB1 content in corn and dehulled cottonseed samples as determined by ELISA ranged from 7 to 422 micrograms/kg and 7 to 3,258 micrograms/kg, respectively. When ELISA estimates of AFB1 in corn were compared with values obtained by thin layer chromatography (CB method), the correlation coefficient (n = 10) was 0.95. Average interassay and subsample coefficients of variation for ELISA in corn were 21.4 and 22.0%, respectively. When ELISA estimates of AFB1 in cottonseed were compared with values obtained by liquid chromatography (Pons method), the correlation coefficient (n = 15) was 0.96. Using this ELISA, 36 duplicate sample extracts can be screened for AFB1 in less than 2 h.     

Use of soybean peroxidase in chemiluminescent enzyme-linked immunosorbent assay

A procedure for the production of conjugates of soybean peroxidase (SbP) oxidized by sodium periodate and anti-mouse IgG antibody (Ab) was optimized. A sandwich chemiluminescent enzyme-linked immunosorbent assay (ELISA) for determination of mouse IgG using SbP and specific Ab was developed, and SbP-catalyzed oxidation of luminol was carried out in the absence of any enhancer. Comparison of conjugates produced by labeling Ab by soybean and horseradish peroxidases in the chemiluminescent ELISA showed that in the case of SbP a rate of emission decay formed through luminol oxidation was significantly lower. Application of the soya enzyme allowed the development of the immunoassay with improved sensitivity and a wider linear range.     

Development of an enzyme-linked immunosorbent assay (ELISA) for residue analysis of the fungicide azoxystrobin in agricultural products

A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) was developed for residue analysis of azoxystrobin in garden crops, for which the maximum residue limits (MRLs) are 0.5-50 mg/kg in Japan. For hapten synthesis, an ethyl carboxyl group was introduced to the 4-position of the 2-cyanophenoxy group in azoxystrobin, and its cyano group was changed to a methyl group. An anti-azoxystrobin monoclonal antibody was prepared from mice immunized with hapten-keyhole limpet hemocyanin conjugate. The dc-ELISA using prepared antibody showed 50-250-fold higher sensitivity compared to the MRLs. The working range of the dc-ELISA was 10-200 ng/mL. The dc-ELISA showed high specificity to azoxystrobin. When methanol extracts from nine kinds of garden crops spiked with azoxystrobin ranging near the MRLs were analyzed, the determined results by the dc-ELISA agreed well with the results of their controls. In addition, azoxystrobin spiked in garden crops homogenates was satisfactorily extracted by methanol solution and easily analyzed. The recovery rate of dc-ELISA was 96-109% and correlated well with the results obtained by HPLC analysis.     

Development of an enzyme-linked immunosorbent assay for the pyrethroid insecticide cyhalothrin

A competitive enzyme-linked immunosorbent assay (ELISA) was developed for detection of the pyrethroid insecticide cyhalothrin. Three haptens with an amine or propanoic acid terminus were synthesized and then conjugated with bovine serum albumin to give immunogens. Eight polyclonal antisera produced by rabbits were screened for titers and affinity using three different coating antigens. The antiserum CWB-C had the highest affinity with cyhalothrin and a low affinity with fenvalerate, fenpropathrin, deltamethrin, and fluvalinate. The half-maximum inhibition concentration for cyhalothrin was 37.2 microg/L, and the limit of detection was 4.7 microg/L. The recoveries of different concentrations of cyhalothrin (0.1-2500 microg/L) from fortified tap water, well water, and wastewater samples as determined with the ELISA were 81-114%.     

Development of an enzyme-linked immunosorbent assay for the organophosphorus insecticide bromophos-ethyl

A competitive enzyme-linked immunosorbent assay (ELISA) was developed for the quantitative detection of the organophosphorus insecticide bromophos-ethyl. Four bromophos-ethyl derivatives (haptens) were synthesized and were coupled to carrier proteins through the pesticide thiophosphate group to use as an immunogen or as a coating antigen. Rabbits were immunized with either one of two haptens coupled to bovine serum albumin for production of polyclonal antibodies, and the sera were screened against one of the haptens coupled to ovalbumin. Using the serum with highest titer, an antigen-coated ELISA was developed, which showed an IC(50) of 3.9 ng/mL with a detection limit of 0.3 ng/mL (20% inhibition). An antibody-coated ELISA using an enzyme tracer was also developed, which showed an IC(50) of 6.5 ng/mL with a detection limit of 1.0 ng/mL (20% inhibition). The antibodies showed negligible cross-reactivity with other organophosphorus pesticides except with the insecticides bromophos-methyl and chlorpyrifos in the antibody-coated assay only. Recoveries of bromophos-ethyl from fortified crop and water samples ranged from 82 to 128% and from 95 to 127%, respectively.