A suggested guiding panel of seromarkers for efficient discrimination between primary and secondary human hepatocarcinoma.

Although alpha-fetoprotein (AFP) is a golden diagnostic marker for hepatocellular carcinoma (HCC), its value is debatable. Differentiation between primary and secondary hepatocarcinomas (HC) relying on AFP is confusing, does not exceed 20 % in the later. To find alternative markers other than AFP to differentiate between primary and secondary HC from colorectal carcinoma (CRC) and breast (BC) and lung cancers (LC), 60 individuals were recruited: group 1, healthy volunteers; group 2, with primary; and group 3, with secondary HC. Carcinoembryonic antigen (CEA), total glycosaminoglycans (TGAGs), total sialic acid (TSA), free glucosamine (FGA), leucine aminopeptidase (LAP), 5'-nucleotidase (5'-NU) activities, and AFP were estimated in sera, in addition to liver histology. CEA, TGAGs, TSA, and FGA were elevated in secondary HC among CRC primary cancers, while LAP, 5'-NU activities, and AFP were elevated in primary HCC. We concluded that a new panel can be used to differentiate primary from secondary HC better than AFP, speculating the primary cancer. AFP, LAP, and 5'-NU predominated in primary, while CEA, TGAGs, TSA, and FGA, in secondary HC. Elevation of 5'-NU, LAP, TGAGs, TSA, and FGA to CEA indicated that primary source of HC is CRC. Association of TGAGs, TSA, and FGA only to CEA indicated that the primary cancer is breast. Elevation of TGAGs, TSA, and FGA, with other normal parameters, indicated that the primary cancer is lung. A guiding table is recommended in the oncology laboratory, for management and follow-up, and having more expected level of sensitivity than AFP.

Production of a hybridoma cell line secreting retinoic acid-specific monoclonal antibody.

A stabilized hybridoma cell line secreting anti-retinoic acid monoclonal antibodies of subclass IgG1 with kappa chains was produced by fusing NS-1 myeloma cells with the spleen cells from BALB/c female mice immunized with all-trans-4-oxoretinoic acid-oxime-chicken IgG conjugate. The antibody titer of mice ascitic fluid ranged from 1/12,800 to 1/25,600, as determined by competitive indirect enzyme-linked immunosorbent assay (ELISA). 50% inhibition dosage of all-trans-retinoic acid at a 1/20,000 dilution of mice ascitic fluid was 6.6 ng/ml, as determined by ELISA. The anti-retinoic acid monoclonal antibody was generated in mice ascitic fluid and purified by protein G affinity chromatography. Cross-reactivity of the monoclonal antibody was determined at 0.1 microgram/ml concentration of retinoids and indicated high specificity to both all-trans-retinoic acid (86% inhibition) and 13-cis-retinoic acid (87% inhibition), and strong cross-reactivity with 4-oxoretinoic acid (77%) and 4-oxoretinoic acid oxime (109%). Specificity was confirmed by the horseradish peroxidase-linked immunostaining method and immunoradioassay. The affinity constant of the monoclonal antibody, K, was determined to be 3.6 X 10(9) l/mol. A calibration curve for retinoic acid using the monoclonal antibody to retinoic acid was developed; the detection limit for all-trans-retinoic acid is 1 ng/ml in the competitive indirect ELISA. The antibody counteracts the effect of retinoic acid on growth inhibition and differentiation in HL-60 cells.

Polyspecific and autoreactive IgA secreted by hybridomas derived from Peyer's patches of vomitoxin-fed mice: characterization and possible pathogenic role in IgA nephropathy.

A total of 122 immunoglobulin (Ig)A-producing hybridoma clones were isolated from the Peyer's patches of vomitoxin-fed BALB/c mice and the resultant antibodies were characterized for their antigenic specificity and pathogenic potential. When reactivity was tested against a panel consisting of DNA, sphingomyelin, thyroglobulin, collagen, casein, cardiolipin and bovine serum albumin conjugates of phosphorylcholine, inulin and trinitrophenol that were representative of self and non-self antigens, approximately 95% of the monoclonal IgAs bound to at least one of the panel antigens and 80% bound to more than one antigen. The polyspecificity of some of the monoclonal IgAs was further suggested by demonstrating the capacity of one antigen to inhibit binding of monoclonal IgA to another antigen. Protein staining and Western blotting of gradient native polyacrylamide gels, indicated that trimeric IgA predominated in the isolated monoclonal IgAs. Repeated injections of mice with representative monoclonal IgAs induced microhaematuria in three of four of the clones tested but not IgA deposition in the kidney glomerulus. In addition, three of the four monoclonal IgAs caused IgG and C3 deposition in the kidney mesangium. These and previous results suggest that dietary vomitoxin promotes the polyclonal activation and expansion of IgA-secreting B cells at the Peyer's patch level and that resultant polyspecific, autoreactive IgA may contribute to kidney pathogenesis.

Fumonisins Veratox. A new rapid quantitative ELISA for determination of fumonisin in food and feed.

Polyclonal antibodies against fumonisin B1 were produced by immunizing sheep with fumonisin B1-keyhole limpet hemocyanin as an immunogen. A quantitative competitive enzyme-linked immunosorbent assay was developed whereby free fumonisins or sample extract containing fumonisins and enzyme-labelled fumonisin competed for binding to the solid phase-bound antibodies. The color intensity of wells, formed by substrate reaction with the enzyme, was inversely related to FB1 concentration. Detection limits for the assay were 0.1 ng/mL fumonisin B1 and concentrations of fumonisins B1, B2, and B3 required for 50% binding inhibition were 5.5, 23 and 18 ng/mL, respectively. For food and feed analyses, samples were extracted with 70% methanol and dilution of the extracts were used directly for ELISA. ELISA results were compared to HPLC analyses by a reference laboratory and the correlation (r value) between ELISA and HPLC was 0.967. The assay may be used to quantitate fumonisins in food and feed within 30 minutes.

Natural Occurrence of Fumonisins in Rice with Fusarium Sheath Rot Disease.

Twenty samples of rough rice (Oryza sativa) (unpolished kernels) collected during the 1995 harvest season from Arkansas (seven samples) and Texas (13 samples) were obtained from rice fields known to include plants with symptoms of Fusarium sheath rot putatively caused by Fusarium proliferatum. Samples were analyzed for fumonisin B1 (FB1) at three laboratories using three different extracting solvents by high-performance liquid chromatography (HPLC) or enzyme-linked immunosorbent assay (ELISA) methods. Forty percent of the samples were positive for FB1 at levels ≤4.3 µg/g by HPLC. The same samples contained FB1 at ≤3.6 µg/g when measured by an ELISA method. Most samples that were positive for FB1 were positive for fumonisin B2 (FB2) and fumonisin B3 (FB3) by HPLC at levels ≤1.2 µg/g. Very good agreement was obtained among the two laboratories using HPLC methods and the third using ELISA. Shelling of the unpolished rice results in hull and brown rice fractions. In a sample that contained 4.3 µg/g in whole kernels, the fumonisin level was very high in hulls (≤16.8 µg/g) and low in brown rice (≤0.9 µg/g). Milling of brown rice results in bran and white rice fractions. Fumonisins were found in bran at a level of ≤3.7 µg/g but were below the level of detection by HPLC in white rice. The presence of fumonisins (FB1, FB2, and FB3) was confirmed by fast atom bombardment/mass spectrometry. This is the first report of fumonisins in naturally contaminated rice in the United States.

Simultaneous screening of fumonisin B1, aflatoxin B1, and zearalenone by line immunoblot: a computer-assisted multianalyte assay system.

A line immunoblot assay was developed for the simultaneous screening of fumonisin B1 (FB1), aflatoxin B1 (AFB1), and zearalenone (ZEA). Monoclonal antibodies for each of these toxins were immobilized as multiple lines on nitrocellulose membrane strips and sectored into hydrophobic compartments to minimize use of reagents. A modified enzyme-linked immunosorbent assay was conducted whereby free mycotoxins and horseradish peroxidase-labeled mycotoxins competed for binding to the nitrocellulose-bound antibodies. Color intensity of lines formed by a precipitating substrate was inversely related to mycotoxin concentration. Detection limits for the individual mycotoxins, as determined by visually comparing the color intensity of positive strips to negative controls, were 500, 0.5, and 3 ng/mL for FB1, AB1, and ZEA, respectively. Line density was quantitatively assessed also by using a camera, video monitor, and microcomputer equipped with a video-digitizing board. The assay could be used to determine range values for the various mycotoxins in extracts of spiked corn in less than 30 min, and values could be recorded on the microcomputer hard disk. This combination of line immunoblot assay and image analysis, termed computer-assisted multianalyte assay system, should be applicable to the simultaneous screening of multiple mycotoxins and other agricultural residues in food.

Direct fermentation of potato starch to ethanol by cocultures of Aspergillus niger and Saccharomyces cerevisiae.

Direct fermentation of unhydrolyzed potato starch to ethanol by monocultures of an amylolytic fungus, Aspergillus niger, and cocultures of A. niger and Saccharomyces cerevisiae was investigated. Amylolytic activity, rate and amount of starch utilization, and ethanol yields increased several-fold in coculture versus the monoculture due to the synergistic metabolic interactions between the species. Optimal ethanol yields were obtained in the pH range 5 to 6 and amylolytic activity was obtained in the pH range 5 to 8. Ethanol yields were maximal when fermentations were conducted anaerobically. Increasing S. cerevisiae inoculum in the coculture from 4 to 12% gave a dramatic increase in the rate of ethanol production, and ethanol yields of greater than 96% of the theoretical maximum were obtained within 2 days of fermentation. These results indicate that simultaneous fermentation of starch to ethanol can be conducted efficiently by using cocultures of the amylolytic fungus A. niger and a nonamylolytic sugar fermenter, S. cerevisiae.

Immunochemical assay applied to mycotoxin biosynthesis: ELISA comparison of sterigmatocystin production by Aspergillus versicolor and Aspergillus nidulans.

Conventional thin layer and instrumental methods for analyzing mycotoxins and their precursors are time-consuming and make the investigation of mycotoxin biosynthesis particularly difficult. As an alternative, sensitive enzyme-liked immunosorbent assays (ELISAs) can be utilized to analyze for these compounds. In this report, sterigmatocystin production in test tube cultures of Aspergillus versicolor ATCC 18643 and Aspergillus nidulans ATCC 32610 were compared using competitive ELISA. Polyclonal antiserum that was prepared against a sterigmatocystin hemiacetal-bovine serum albumin conjugate exhibited greatest specificity for sterigmatocystin hemiacetal and sterigmatocystin with less reactivity for O-methylsterigmatocystin. The antiserum could be used to detect as little as 50 ng/ml sterigmatocystin in ELISA. Direct ELISA could be performed on diluted culture broth and on mycelial extracts solubilized with N,N-dimethylformamide. Aspergillus versicolor ATCC 18643 produced more sterigmatocystin in SLS medium than in YES medium, and showed maximal levels at between 9 to 12 days incubation. Approximately 75% of sterigmatocystin was detectable in mycelium (254 micrograms/ml culture) compared to the extracellular fraction (87 micrograms/ml culture). Aspergillus nidulans exhibited qualitatively similar patterns of growth and toxigenesis in SLS medium but accumulated maximal levels of only 15 micrograms mycelial sterigmatocystin/ml culture and 5 micrograms extracellular sterigmatocystin/ml broth, respectively.

Production of polyclonal antibodies to the trichothecene mycotoxin 4,15-diacetylnivalenol with the carrier-adjuvant cholera toxin.

The trichothecene mycotoxin 4,15-diacetylnivalenol (DNIV) was conjugated to cholera toxin (DNIV-CT) for use as an immunogen and as an adjuvant for specific antibody production. Repeated intravenous injection of 7.5 micrograms of the conjugate was effective at generating specific antibodies to DNIV in rabbits as determined by enzyme-linked immunosorbent assay (ELISA). When small amounts (1 to 10 micrograms per animal) of DNIV-CT were used to immunize mice, polyclonal antibodies were observed as early as 4 weeks of immunization. The relative affinity of the antibodies to DNIV increased with the immunogen dose in mice. Antibodies were not detectable in either rabbits or mice that were injected with DNIV conjugated to the carrier protein bovine serum albumin or when DNIV-CT was blocked with glutaraldehyde. Competitive ELISA of mouse and rabbit serum revealed that the antibodies were most specific for DNIV but reacted to a small extent with fusarenone-X, deoxynivalenol, and nivalenol. No reactivity was observed with 3- or 15-acetyldeoxynivalenol. The results suggest that specific polyclonal antibodies can be prepared against a trichothecene when CT is used as an adjuvant and carrier protein. DNIV antibodies will be useful for monitoring the compound in food in conjunction with other trichothecene antibodies, detection of DNIV-producing cultures, and investigation of 8-ketotrichothecene biosynthesis.

Immunochemical assessment of mycotoxins in 1989 grain foods: evidence for deoxynivalenol (vomitoxin) contamination.

To assess the potential for mycotoxin contamination of the human food supply following the 1988 U.S. drought, 92 grain food samples were purchased from retail outlets in the summer of 1989 and surveyed for aflatoxin B1, zearalenone, and deoxynivalenol (DON [vomitoxin]) by monoclonal antibody-based competitive enzyme-linked immunosorbent assay (ELISA). Only one sample (buckwheat flour) was found to contain aflatoxin B1 (12 ng/g), whereas zearalenone was found in 26% of the samples at a mean concentration of 19 ng/g. In contrast, the DON ELISA was positive in 50% of the samples at a detection level of 1.0 micrograms/g. Between 63 and 88% of corn cereals, wheat flour/muffin mixes, rice cereals, and corn meal/muffin mixes yielded positive results for DON, whereas 25 to 50% of oat cereals, wheat- and oat-based cookies/crackers, corn chips, popcorn, and mixed-grain cereals were positive for DON. The mean DON content of the positive samples was 4.0 micrograms/g, and the minimum and maximum levels were 1.2 and 19 micrograms/g, respectively. When positive ELISA samples were also analyzed by high-performance liquid chromatography, a strong correlation between the two methods was found. The presence of DON in the two highest samples, corn meal and mixed-grain cereal, which contained 19 and 16 micrograms/g, respectively, was quantitatively confirmed by gas chromatography-mass spectrometry. The results indicated that DON was present in 1989 retail food products at concentrations that exceeded those found in previous market surveys and that have been experimentally associated with impaired animal health.

Generation of antibodies reactive with fumonisins B1, B2, and B3 by using cholera toxin as the carrier-adjuvant.

Murine polyclonal antibodies reactive with fumonisins B1, B2, and B3 were produced after a novel immunization procedure with cholera toxin as both a hapten carrier protein and adjuvant. Immunization of mice with two 7.5-micrograms doses of fumonisin B1-cholera toxin conjugate without adjuvant resulted in the production of fumonisin B1-specific antibodies in all mice within 15 days when intraperitoneal, subcutaneous, and intravenous routes were used. In contrast, conventional immunization procedures with fumonisin B1-bovine serum albumin conjugates with and without Freund's adjuvant were largely ineffective. Fumonisin antibodies could be readily mass-produced in ascites fluid by using cholera toxin as a carrier-adjuvant. A competitive indirect enzyme-linked immunosorbent assay (ELISA) was devised whereby immobilized fumonisin B1-ovalbumin and free fumonisin B1 competed for antibody binding. The detection limit for fumonisin B1 in the ELISA was 100 ng/ml. The antiserum cross-reacted with fumonisins B2 and B3 but not with the hydrolyzed backbone of fumonisin B1 and tricarballylic acid. Concentrations of fumonisins B1, B2, and B3 required for 50% binding inhibition were 260, 300, and 650 ng/ml, respectively. These polyclonal antibodies should find wide usage in the ELISA for fumonisins in foods, feeds, and tissues.

Ochratoxin A concentrations in food and feed from a region with Balkan Endemic Nephropathy.

Balkan Endemic Nephropathy (BEN), a chronic renal disease of unknown aetiology, is found in geographically close areas of Bulgaria, Romania, Serbia, Croatia, Bosnia and Herzegovina, Slovenia, and the former Yugoslav Republic of Macedonia. Ochratoxin A (OTA), a secondary metabolite of Aspergillus and Penicillium species and a natural contaminant of food and feed, is a putative cause of BEN. Some studies have found a geographic covariation between OTA content in food/feed and BEN manifestation; others have not. In May 2000, using a competitive direct ELISA assay for OTA (detection limit 1 microg kg(-1)), we investigated OTA contamination in 165 samples of home-produced food (beans, potatoes, corn, wheat, flour) and feed from households in villages from the BEN region (Vratza district) of north-western Bulgaria. Samples were collected from: (a) BEN villages (n = 8), and therein from BEN households (20), and BEN-free households (16) (within-village controls, WVC households); and (b) BEN-free villages (7) and therein BEN-free households (22) (between-village controls, BVC). BEN households consistently had a higher proportion of OTA-positive samples than WVC households, but similar (for some foods) or lower (for other foods) proportions to BVC households. The proportion of OTA-positive samples was also higher in BVC than in WVC households. Furthermore, BEN households had a similar proportion of OTA-positive samples to the pooled, WVC and BVC, group of households. OTA-exposure estimates, derived from our OTA-concentration findings and the reported average per capita monthly consumption of basic foods in rural Bulgaria, showed the highest OTA intake in BEN households (1.21 microg day(-1)), versus 1.03 microg day(-1) in BVC and 0.71 microg day(-1) in WVC households. These OTA intakes are higher than those in the EU, and are close to the upper limits acceptable to several food-safety organizations. The results indicate that OTA may not alone cause BEN; only synergistically with other environmental toxicants and/or predisposing genotypes may do so.