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F S Chu

Publications and source records attributed to F S Chu.

122 records · Page 7Linked to original sources
PubMedDate not supplied

Enzyme-linked immunosorbent assay for T-2 toxin metabolites in urine.

A direct competitive enzyme-linked immunosorbent assay (ELISA) for determination of total T-2 toxin metabolites in urine was developed. The assay involves coating anti-3-acetyl-neosolaniol-hemisuccinate-bovine serum albumin conjugate (anti-3-Ac-NEOS-HS-BSA) antibody to the ELISA plate and using 3-Ac-NEOS-HS-peroxidase as the enzyme marker. Competitive ELISA revealed that the antibody had good cross-reactivity with acetyldiacetoxyscirpenol (Ac-DAS), T-2 tetraol tetraacetate, 3'-OH-Ac-T-2, 3-Ac-NEOS, and 3,4,15-triacetyl-12,13-epoxytrichothec-9-en-8-one (Ac-T-2-8-one), but less cross-reactivity with Ac-T-2 toxin and T-2 toxin. All metabolites of T-2 toxin in urine were converted to T-2 tetraol tetraacetate (T-2-4ol-4Ac) by acetylation of the sample extract before ELISA. To test the ELISA accuracy, a radioimmunoassay (RIA) was performed simultaneously. The linear portion of the standard curve of this direct ELISA for T-2-4ol-4Ac was 0.2-2.0 ng/mL, which was 10 times more sensitive than RIA. The minimum detection level for T-2-4ol-4Ac was 0.02 ng/mL (0.4 pg/assay) in the absence of urine sample. The overall analytical recoveries for T-2 toxin, HT-2, T-2-4ol, 3'-OH-HT-2, NEOS, and a mixture of these 5 toxins added to the urine samples in the ELISA at concentrations of 0.05 and 0.2 ng/mL were 87 and 94%, respectively.

Animals↗
PubMedDate not supplied

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.

Aflatoxin B1↗
PubMedDate not supplied

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.

Aflatoxin B1↗
PubMedDate not supplied

Enzyme-linked immunosorbent assay for deoxynivalenol in corn and wheat.

The availability of antibody against deoxynivalenol (DON) triacetate (Tri-Ac-DON) has enabled development of a direct enzyme-linked immunosorbent assay (ELISA) and an indirect ELISA for DON in corn and wheat. In both assays, DON is extracted from the sample with acetonitrile-water, reacted with acetic anhydride to form Tri-Ac-DON, and diluted in phosphate buffer for analysis. Direct ELISA was found to be the more sensitive procedure. Fewer interferences are evidenced, and the assay is less time consuming than is indirect ELISA. For direct ELISA, recovery of 10-1000 ppb DON added to corn and wheat was 100% (SD 15, CV 15%) and 102.1% (SD 12.2, CV 11.9%), respectively. For indirect ELISA, overall recovery of 10-1000 ppb DON added to wheat was 121.5% (SD 39.5, CV 32.5%); in the higher concentration range (500-1000 ppb), recovery was 105% (SD 18, CV 17%). The minimal detection level for DON was around 10 ppb. Analysis of 7 naturally contaminated samples for DON showed that the ELISA results agreed well with those obtained by radioimmunoassay and thin-layer chromatography.

Acetylation↗
PubMedDate not supplied

Simultaneous analysis of T-2 toxin and HT-2 toxin by an indirect enzyme-linked immunosorbent assay.

An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of HT-2 toxin in the presence or absence of T-2 toxin is described. In the indirect ELISA, the relative cross-reactivities of antibodies against T-2 toxin (anti-T-2) with T-2 toxin and HT-2 toxin were 1 and 0.1, whereas anti-HT-2 cross-reactivities with T-2 toxin and HT-2 toxin were 0.33 and 1, respectively. Using such relationships, a formula was established that could be used to calculate the individual toxin concentration in a mixed sample after experimentally analyzing for T-2 and HT-2 toxins in the 2 indirect ELISAs. This method was tested by analyzing urine samples spiked with HT-2 toxin alone and samples spiked with both T-2 toxin and HT-2 toxin. A cleanup protocol for treatment of urine samples before ELISA was also established. The overall analytical recovery of HT-2 toxin when it was added at concentrations of 0.1-10 parts per billion (ppb) to the urine samples was ca 89%. When both T-2 and HT-2 toxins were added to the urine samples at equal concentrations of 0.5 to 5.0 ppb, their recoveries were 112 and 109%, respectively.

Antibody Specificity↗
PubMedDate not supplied

Instability of some trichothecenes in methanol.

Stability of 8 trichothecenes stored in methanol at room temperature was studied by thin layer chromatography. Results indicate that the trichothecenes which bear an acetoxy group at both C3 and C4 are very susceptible to methanolysis. In the early stage, the acetoxy group at C3 is most favorable for the transesterification. All trichothecenes tested were transformed to several products after prolonged (22 days) exposure to methanol.

Chromatography, Thin Layer↗
PubMedDate not supplied

Radioimmunoassay of deoxynivalenol in wheat and corn.

With the availability of antibody against deoxynivalenol triacetate (DON-triacetate), a radioimmunoassay (RIA) for DON in wheat was developed. DON is extracted from the sample with acetonitrile-water (84 + 16), defatted with hexane, and then reacted with acetic anhydride to form DON-triacetate. The reaction mixture is loaded onto a C-18 cartridge to remove excess reagents and impurities. Acetylated DON is eluted from the cartridge with 50% methanol in water, and then analyzed by radioimmunoassay utilizing antiserum against DON-triacetate and tritiated DON-triacetate. Overall recovery for DON added to wheat between 50 and 5000 ppb was 86% with a standard deviation of 7% and coefficient of variation of 8%. The limit of detection for DON was about 20 ppb. Analysis of 12 naturally contaminated wheat, corn, and mixed feed samples for DON revealed that RIA results agreed well with thin layer chromatographic analyses performed by other laboratories.

Acetylation↗
PubMedDate not supplied

Indirect enzyme-linked immunosorbent assay for saxitoxin in shellfish.

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of saxitoxin (STX). Antibodies against STX were demonstrated in rabbits 5 weeks after immunizing with STX-bovine serum albumin (STX-HCHO-BSA). In the ELISA, STX-HCHO-BSA or polylysine-STX was coated onto the microtiter plate, followed by incubation with standard toxin and anti-STX antibody. The amount of antibody bound to the solid phase was determined by incubation with goat anti-rabbit IgG peroxidase conjugate and a reaction with chromogenic substrate. Competitive indirect ELISA revealed that the anti-serum did not cross-react with either carbamoyl-neo-STX-sulfate or tetrodotoxin. The antibodies for STX cross-reacted with decarbamoyl-STX and neo-STX about 56% and 16% as much as they did with STX, respectively. The lower detection limits for STX, decarbamoyl-STX, and neo-STX in this system were about 25, 45, and 156 pg per assay, respectively. When STX added to clams or mussels was assayed, the detection limit for STX was about 50-100 ppb, and recoveries were in the range of 86.8-107%.

Animals↗
PubMedDate not supplied

Direct enzyme-linked immunosorbent assay for determining aflatoxin M1 at picogram levels in dairy products.

Protocols for detecting picogram quantities of aflatoxin M1 in dairy products were established. Milk samples were subjected to a reverse phase Sep-Pak C18 cartridge treatment before analysis by an enzyme-linked immunosorbent assay (ELISA) according to previously published procedures. M1 in yogurt, brick cheddar, and ripened Brie cheese was extracted by a modified Pons method, subjected to a normal phase silica cartridge treatment, and analyzed by ELISA. The detection limits for M1 in milk, yogurt, cheddar, and Brie were 10, 10, 50, and 25 ppt (ng/kg), respectively. Recovery for M1 added to these products was in the range 70-110%. Good agreement was found for M1 levels in several naturally contaminated milk samples analyzed by both ELISA and liquid chromatography.

Aflatoxin M1↗
PubMedDate not supplied

Enzyme-linked immunosorbent assay of ochratoxin A in wheat.

An enzyme-linked immunosorbent assay (ELISA) was developed for the quantitation of ochratoxin A amended to wheat. Ochratoxin A conjugated to horseradish peroxidase (HRP) was used as an enzyme marker in the assay. At toxin levels below 30 ppb, a cleanup treatment was necessary for ELISA. Among 3 cleanup methods tested (solvent partition, Sep-Pak cartridge treatment, solvent partition and cartridge treatment), reverse phase cartridge treatment was the most simple and effective. In the analysis, ochratoxin A was extracted from wheat with methanol. The methanolic extract was diluted with water to a final 10-15% methanol content, and then passed through a cartridge. Ochratoxin A was eluted from the cartridge with 85% methanol which was then concentrated. The final solution, in 0.1M, pH 7.5 sodium phosphate-Tween 20 buffer and 5% methanol, was then subjected to ELISA. ELISA allowed minimal detection of the toxin in wheat at the 1-2 ppb level after cleanup. Recoveries of toxin added to wheat samples in the 1.0-30 ppb range were 85-90% with standard deviations of 10-15%.

Enzyme-Linked Immunosorbent Assay↗
PubMedDate not supplied

Assessment of immunochemical methods for the analysis of trichothecene mycotoxins in naturally occurring moldy corn.

The efficacy of various immunochemical methods for simultaneous analysis of different trichothecene mycotoxins in corn samples naturally contaminated with various Fusarium molds was evaluated. Antibodies against either T-2 tetraol tetraacetate (T-2-4ol-4Ac) or deoxynivalenol triacetate (Tri-Ac-DON) were used in the enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) for total type A and total DON-related trichothecenes (TCTCs), respectively. Specific antibodies against T-2 toxin were used in an RIA for analysis of T-2 toxin alone. Analytical recoveries of T-2 toxin analyzed as T-2-4ol-4Ac by ELISA and T-2 toxin by RIA at 10-200 ppb were 83.5-123% and 81.8-110.6%, respectively. T-2 toxin accounts for about 30% of total type A TCTCs by both liquid chromatography (LC)-ELISA and RIA methods. The correlation coefficients (r) of total type A TCTC levels versus T-2 toxin level by RIA was 0.725 (p < 0.0001). A good correlation (r = 0.84 and p = 0.04) also was found for T-2 toxin levels obtained by LC-ELISA and RIA methods. In addition to T-2 toxin, HT-2 toxin, diacetoxyscirpenol, and T-2 tetrol were found in samples containing high levels of type A TCTCs by LC-ELISA. For analysis of DON alone, samples were treated with a Sep-Pak C18 cartridge to remove other acetylated DONs before RIA. Recovery of DON after the C18 cartridge treatment was 90%. Overall analytical recoveries of DON as Tri-Ac-DON (sample with no C18 treatment) and DON alone (with C18 treatment) at 10-500 ppb were 81.3-94% and 72.7-104%, respectively. RIA revealed that DON accounts for about 50% of total DON-related TCTCs. The coefficient of correlation between total DON-related TCTCs and DON levels was 0.84 (p < 0.0001).

Chromatography, Liquid↗
PubMedDate not supplied

Analysis of fumonisins and Alternaria alternata toxin by liquid chromatography-enzyme-linked immunosorbent assay.

Use of a direct competitive enzyme-linked immunosorbent assay (ELISA) as a postcolumn monitoring system after liquid chromatography (LC) is described for analysis of different fumonisin analogs. Without cleanup and derivatization, sample extracts are directly injected into a C18 reversed-phase column and then subjected to LC. Fractions (0.5 mL each) are collected and then analyzed by ELISA. LC using a water-methanol gradient separated the 3 major fumonisins FmB1, FmB2, and FmB3, and as low as 0.1 ng FmB1 could be detected. Recovery of FmB1 added to ground corn (100-1000 ng/g) and then extracted with CH3CN-H2O (1 + 1, v/v) was 78.8%. Analysis of fumonisins in one starch and 14 naturally contaminated corn samples showed that FmB1 was the major fumonisin. Ten samples also were contaminated with FmB2, but only 2 samples were contaminated with FmB3. The method also was used to analyze extracts from cultures of 3 Alternaria alternata (AAL) strains. Both FmB1 and the AAL toxin TA were detected in the culture extracts, and their amounts varied considerably with the cultures tested.

Alternaria↗
PubMedDate not supplied

Immunoaffinity column as cleanup tool for a direct competitive enzyme-linked immunosorbent assay of cyclopiazonic acid in corn, peanuts, and mixed feed.

An immunoaffinity column (IAC) for cyclopiazonic acid (CPA) was prepared by coupling a CPA-specific monoclonal antibody to CNBr-activated sepharose 4B. A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) was used to study the chromatographic behavior of a 0.2 mL gel column with a binding capacity of 4 micrograms CPA/column as well as to evaluate its efficacy as a cleanup tool for analysis of naturally occurring CPA. Sample extract either in buffer solution or in a solution containing up to 35% methanol could be loaded onto the column. After the column is washed with 5 mL deionized water and 5 mL 50% methanol, CPA could be quantitatively eluted with 2 mL 100% methanol. The column could be regenerated at least 10 times by washing with 10 mL equilibrating buffer and then storing in a cold room overnight before reuse. Recoveries of CPA added to corn, peanut, and mixed feed extracts in the range 10-200 ng/g were 88-105, 86-100, and 90-110%, respectively. Detection limits were 2.0, 4.4, and 4.7 ng/g for corn, mixed feed, and peanuts, respectively. Twenty-two peanut samples naturally contaminated with CPA were subjected to both IAC and solvent partition cleanup followed by dc-ELISA. Although a good correlation between data obtained from IAC-dc-ELISA and from SP-dc-ELISA (r = 0.75, p < 0.0001) was obtained, the slope of the linear regression was low (0.67), indicating loss during solvent partition cleanup. The overall data showed that the combination of IAC and dc-ELISA is an effective method for CPA analysis.

Animal Feed↗