Enzyme immunoassays for the detection of mycotoxins in plant-based milk alternatives: pitfalls and limitations.

Plant-based milk alternatives (PBMAs) are a potential source of mycotoxin uptake. To ensure food safety, simple and rapid testing methods of PBMAs for mycotoxins are therefore required. This study investigated the applicability of enzyme immunoassay (EIA) methods for direct testing of PBMAs without sample extraction. Mycotoxin analyses included aflatoxin B1 (AFB1), sterigmatocystin (STC), ochratoxin A (OTA), deoxynivalenol (DON), and T-2/HT-2-toxin (T-2/HT-2). It was found that the PBMA matrix negatively affected the EIA to varying degrees, thus affecting the reliability of the results. A dilution of PBMAs of at least 1:8 was necessary to overcome matrix interference. This resulted in calculated detection limits of 0.4 µg/L (AFB1), 2 µg/L (STC), 0.08 µg/L (OTA), 16 µg/L (DON), and 0.4 µg/L (T-2/HT-2). After analysis of 54 PBMA products from German retail stores, positive results in at least one test system were obtained for 23 samples. However, most positive results were near the calculated detection limit. Control analyses of selected samples by LC-MS/MS for AFB1, STC, and OTA qualitatively confirmed the presence of trace amounts of STC in some samples, but quantitative agreement was poor. It was concluded that the high diversity of ingredients used in PBMAs led to a highly variable degree of sample matrix interference even in a 1:8 dilution. Since the use of higher dilutions conflicts with the need to achieve low detection limits, the application of EIA for routine mycotoxin analysis in PBMA for mycotoxins requires further study on the development of a feasible sample preparation method.

Occurrence and risk assessment of sterigmatocystin in agricultural products and processed foods in Korea.

Sterigmatocystin (STC), a carcinogenic mycotoxin, is known to be produced during the biosynthetic pathway of aflatoxin B1. STC in various foods was determined by LC-MS/MS and its risks were assessed. The analytical method was validated in different food categories, and the performance was acceptable based on the criteria of AOAC. A total 1,135 samples (613 agricultural products and 522 processed foods) were analysed, and STC was detected in 46 samples, indicating a detection rate of 4.1%. STC was found in the range of 0.08-10.07 ng/g, and the detection rates of STC were 3.9% in agricultural products and 4.2% in processed foods. The exposure to STC by average consumption of foods was estimated to 0.09 ng/kg b.w./day. The margin of exposure (MOE) approach was applied to assess the risk of STC, and MOE for the whole population was over 1 × 106. Exposure to STC from the consumption of foods distributed in Korea is unlikely to cause human health problems.

A New Protocol for the Detection of Sterigmatocystin-producing Aspergillus Section Versicolores Using a High Discrimination Polymerase.

Aspergillus section Versicolores species, except Aspergillus sydowii, produce a carcinogenic mycotoxin sterigmatocystin (STC). Since these fungi are found in varied environmental milieu including indoor dust and food products, our aim was to develop a sensitive and convenient assay to detect STC producing fungal strains. We made use of a high discrimination DNA polymerase (HiDi DNA polymerase), for single nucleotide polymorphism (SNP)-based PCR amplification. Using specific primer pairs based on the SNPs between A. sydowii and other strains of Aspergillus section Versicolores, we succeeded in amplifying the genomic DNA all target strains except A. sydowii. These results confirm that the SNP-based PCR amplification technique, using a high discrimination DNA polymerase, was a reliable and robust screening method for target fungal strains.

Development and validation of an analytical method for the analysis of Sterigmatocystin in roasted coffee beans and black pepper using liquid chromatography-tandem mass spectrometry.

Sterigmatocystin (STC) is a toxic and potentially carcinogenic fungal toxin found in a variety of food commodities. This study describes the development of an analytical method to determine STC in roasted coffee beans and black pepper using ultra performance liquid chromatography (UPLC) coupled with triple quadrupole tandem mass spectrometry (MS/MS). 13C18-STC was used as internal standard. STC was extracted with a mixture of acetonitrile/water, diluted with a buffer, followed by purification with a solid-phase extraction and an immunoaffinity column prior to the UPLC-MS/MS analysis. Two multiple reaction monitoring (MRM) transitions were employed, one for quantification and one for confirmation of STC. The UPLC-MS/MS analytical method was validated with respect to selectivity, linearity, sensitivity, accuracy, precision, recovery, and stability. Calibration curves were linear over a concentration range 25-2,500 pg mL-1 with correlation coefficients (r) > 0.998. The method limit of quantification for STC in roasted coffee beans and black pepper was 0.10 µg kg-1. The accuracy and precision of the analytical method were acceptable within 15% at all quality control levels. This method was suitable to determine STC levels because of its selectivity, precision, and accuracy. The method was successfully applied to roasted coffee beans and black pepper samples.

Development of a polyclonal antibody-based indirect competitive ELISA for determination of sterigmatocystin in wheat and corn flours.

Sterigmatocystin (STC) is a toxic secondary metabolite produced by more than 50 fungal species, including Aspergillus flavus, A. parasiticus, A. nidulans, and A. versicolor. The Joint FAO/WHO Expert Committee on Food Additives concluded that sterigmatocystin is genotoxic and carcinogenic with the critical effect determined to be carcinogenicity. The present study describes a simple method to prepare hapten and immunogens in order to generate polyclonal antibodies against this metabolite. We developed a sensitive and specific polyclonal antibody-based competitive indirect enzyme-linked immunosorbent assay (ciELISA) for monitoring STC in wheat and corn flours without the need for derivatisation of STC or clean-up of samples by immunoaffinity chromatography for quantification. The half inhibitory concentration (IC50) of the established method was 4.52 ± 0.81 ng mL-1, with the limit of detection (IC10) being 0.19 ± 0.04 ng mL-1 in wheat and corn flour matrices with the coefficient of variation of less than 22%.The assay was very specific to STC and showed no cross-reactivity with its analogue structures. The ELISA allowed for up to 5% methanol without significant influence on the IC50 value. Validation of the assay was performed by spiking STC into a blank flour matrix and the recoveries were in the range of 75.3 % to 104.5 % with a coefficient of variation less than 15%. A small retail survey was conducted by purchasing wheat (n = 8) and corn flours (n = 2) from local grocery stores. All of these retail samples were negative for STC using the developed ELISA method and were confirmed by LC-MS/MS. We demonstrated a rapid, simple, and reliable method for screening STC in wheat and corn flours.

Sterigmatocystin: A mycotoxin to be seriously considered.

Sterigmatocystin is a carcinogenic compound that affects several species of crops and several species of experimental animals. The sterigmatocystin biosynthetic pathway is the best known and most studied. The International Agency for Research on Cancer classifies sterigmatocystin in the Group 2B. Three groups of analytical methods to determine sterigmatocystin in food can be found: chromatographic, ELISA immunoassays and chemical sensors. In addition, sterigmatocystin is a precursor of aflatoxin B1 in those cases where cereals and/or food are contaminated with fungi capable of producing aflatoxins. Chemical structures of sterigmatocystin and aflatoxin B1 are similar. These mycotoxins are pathogens of animals and cereals, producing a major economic impact on biotechnology and agricultural and food industries. This review summarizes different aspects related to sterigmatocystin such as its biosynthesis, toxicological studies and analytical methods for its determination.

Development and in-house validation of a rapid and simple to use ELISA for the detection and measurement of the mycotoxin sterigmatocystin.

Sterigmatocystin (STG) is a highly toxic secondary fungal metabolite structurally closely related to the well-known carcinogenic aflatoxins. Its presence has been reported in grains and grain-based products as well as in other foodstuffs like nuts, green coffee beans, spices, beer and cheese. Due to the lack of suitable data on the occurrence of STG, in 2013, the European Food Safety Authority (EFSA) could not characterise its risk for human health and recommended that more data on STG in food and feed needed to be collected. In order to provide a new tool for the specific detection of STG, a competitive enzyme-linked immunosorbent assay (ELISA) was developed, optimised and validated in this study based on a sensitive monoclonal antibody specific to STG with no cross-reactivity with aflatoxins. The sample preparation method for rice, wheat and maize was based on a modified QuEChERS (quick, easy, cheap, effective, rugged and safe) approach. The assay was validated for the detection of STG in rice, wheat and maize in accordance with the guidelines for validation of semi-quantitative screening methods included in Commission Regulation (EU) 519/2014. The screening target concentration (STC) was set at 1.5 µg/kg. The cutoffs for rice, wheat and maize were 1.2, 1.2 and 1.3 µg/kg and the false suspected rates were 0.34, 1.15 and 0.78%, respectively. Good correlation was found between the results obtained by the STG ELISA and LC-MS/MS method for naturally contaminated rice samples. This validated method can be applied as a sensitive and high-throughput screening for the presence of STG in a range of agricultural commodities. Graphical abstract A new enzyme-linked immunosorbent assay based on an antibody specific to sterigmatocystin for the detection of this mycotoxin in corn, wheat and rice.

Gas Chromatography-Mass Spectrometry for Metabolite Profiling of Japanese Black Cattle Naturally Contaminated with Zearalenone and Sterigmatocystin.

The objective of this study was to evaluate the metabolic profile of cattle fed with or without zearalenone (ZEN) and sterigmatocystin (STC)-contaminated diets using a gas chromatography-mass spectrometry metabolomics approach. Urinary samples were collected from individual animals (n = 6 per herd) from fattening female Japanese Black (JB) cattle herds (23 months old, 550-600 kg). Herd 1 had persistently high urinary ZEN and STC concentrations due to the presence of contaminated rice straw. Herd 2, the second female JB fattening herd (23 months old, 550-600 kg), received the same dietary feed as Herd 1, with non-contaminated rice straw. Urine samples were collected from Herd 1, two weeks after the contaminated rice straw was replaced with uncontaminated rice straw (Herd 1N). Identified metabolites were subjected to principal component analysis (PCA) and ANOVA. The PCA revealed that the effects on cattle metabolites depended on ZEN and STC concentrations. The contamination of cattle feed with multiple mycotoxins may alter systemic metabolic processes, including metabolites associated with ATP generation, amino acids, glycine-conjugates, organic acids, and purine bases. The results obtained from Herd 1N indicate that a two-week remedy period was not sufficient to improve the levels of urinary metabolites, suggesting that chronic contamination with mycotoxins may have long-term harmful effects on the systemic metabolism of cattle.

Development and validation of a simple and fast method for simultaneous determination of aflatoxin B1 and sterigmatocystin in grains.

In this study, a simple, fast, sensitive and reliable method was developed for the simultaneous determination of aflatoxin B1 (AFB1) and sterigmatocystin (STC) in rice, wheat, maize, oat, rye and barley grains based on a modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) method and liquid chromatography-tandem mass spectrometry (LC-MS/MS). AFB1 and STC were extracted using 95% acetonitrile in water and cleaned up using N-Propylethane-1,2-diamine (PSA). The method was validated according to the SANCO/12571/2013 regulation of the European Union (EU). Recoveries at three spiked levels of 0.5, 2.5 and 5.0µg/kg ranged from 77.7% to 119.7% and the relative standard deviations (RSDs) were between 1.3% and 12.8%. Limits of detection (LOD) were 0.03 and 0.02µg/kg for AFB1 and STC, respectively. The limit of quantification (LOQ) was 0.5µg/kg for both AFB1 and STC. The proposed method was successfully applied to determine AFB1 and STC levels in ten non-processed rice samples from Guangdong province in China.

A highly specific competitive direct enzyme immunoassay for sterigmatocystin as a tool for rapid immunochemotaxonomic differentiation of mycotoxigenic Aspergillus species.

A simplified method to produce specific polyclonal rabbit antibodies against sterigmatocystin (STC) was established, using a STC-glycolic acid-ether derivative (STC-GE) conjugated to keyhole limpet haemocyanin (immunogen). The competitive direct enzyme immunoassay (EIA) established for STC had a detection limit (20% binding inhibition) of 130 pg ml-1 . The test was highly specific for STC, with minor cross-reactivity with O-methylsterigmatocystin (OMSTC, 0·87%) and negligible reactivity with aflatoxins (<0·02%). STC-EIA was used in combination with a previously developed specific EIA for aflatoxins (<0·1% cross-reactivity with STC and OMSTC), to study the STC/aflatoxin production profiles of reference strains of Aspergillus species. This immunochemotaxonomic procedure was found to be a convenient tool to identify STC- or aflatoxin-producing strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The carcinogenic mycotoxin sterigmatocystin (STC) is produced by several Aspergillus species, either alone or together with aflatoxins. Here, we report a very simple and straightforward procedure to obtain highly sensitive and specific anti-STC antibodies, and their use in the first ever real STC-specific competitive direct enzyme immunoassay (EIA). In combination with a previous EIA for aflatoxins, this study for the first time demonstrates the potential of a STC/aflatoxin EIA pair for what is branded as 'immunochemotaxonomic' identification of mycotoxigenic Aspergillus species. This new analytical tool enhances analytical possibilities for differential analysis of STC and aflatoxins.

Determination of sterigmatocystin in grain using gas chromatography-mass spectrometry with an on-column injector.

Sterigmatocystin (STC) is a mycotoxin produced by several species of Aspergillus and other fungi. STC is a precursor of the carcinogen aflatoxin B1 and exerts carcinogenic, teratogenic, and mutagenic effects. A reliable GC-MS-based analytical method using on-column injection was developed and validated to determine STC content in grain. In this method, STC was extracted with acetonitrile (84%), and the filtered extract was diluted with phosphate buffer before immunoaffinity column (IAC) cleanup. After elution and N2 evaporation, the sample containing STC was dissolved in acetone and injected into the GC-MS system without derivatization. Separation of STC was carried out by a capillary column (0.25 mm i.d.×30 m, 0.25 µm) with a deactivated pre-column (0.53 mm i.d.×0.6 m). The matrix effect was investigated in maize, wheat, and rice, and an insignificant matrix effect was observed after IAC cleanup. The calibration curve was linear in the range of 20-300 pg (equivalent to 8-120 µg/kg in grain) with a coefficient of determination (R2) of 0.999, and the relative standard deviation of repeatability (RSDr) was less than 10 %. The limit of detection (LOD) of the method was 6 pg (equivalent to 2.4 µg/kg in grain), and the limit of quantification (LOQ) was 20 pg (equivalent to 8 µg/kg). The method described in this report is sensitive, reliable, and can be used to monitor STC contamination in grain.

Development of an analytical method for the determination of sterigmatocystin in grains using LCMS after immunoaffinity column purification.

The mycotoxin sterigmatocystin (STC) is produced mainly by some Aspergillus and Penicillium fungi; it naturally contaminates cereals, peanuts, and products derived from these crops, and is both mutagenic and carcinogenic. As an intermediate of aflatoxin (AF) biosynthesis, its structure is similar to that of AF. Although immunoaffinity columns (IACs) are a popular approach to sample clean-up, no IAC is commercially available for STC, but a commercially available IAC for AF shows cross reactivity to STC. We here developed a new method for analyzing STC in grains using such an IAC and liquid chromatography mass spectrometry (LCMS), and validated this method using six different grains. The STC limit of detection (signal-to-noise ratio, S/N = 3) was 2.5 pg (1.0 µg/kg in the product), and the calibration curve was linear in the range of 7.5-375 pg (3.0-150 µg/kg in the product). The within-day recovery of STC from samples spiked with STC at 5.0 and 50 µg/kg was 83.2-102.5% and the RSDr (relative standard deviation of repeatability) of these samples was 1.9-6.5%; the RSDr of STC-pretreated grain samples was 3.1-14.0%. Average recovery of STC from samples spiked with STC in the range of 5.0-100 µg/kg STC was 83.2-102.5%, with an RSDr of 0.24-6.5%; the RSDr of STC-pretreated grain samples was 2.4-14.0%. In an intermediate precision study, the average STC recovery from STC-spiked samples by three analysts was 95.2-107.5%, with RSDRi (intermediate precision) of 4.0-7.1%; the RSDRi of the STC-pretreated samples was 4.8-10.4%. Thus, the proposed method was effective for STC analysis in grains, and holds potential for a novel application of a commercial IAC, intended for AFs, in STC analysis.

Stability of sterigmatocystin during the bread making process and its occurrence in bread from the Latvian market.

Sterigmatocystin (STC) is a carcinogenic and mutagenic mycotoxin produced by fungi of many Aspergillus species. The aim of this research was to test the stability of STC during the bread making process and to check bread samples from the Latvian market for STC contamination, using a previously developed electrospray positive ionisation (ESI(+)) liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Wheat grain naturally contaminated with STC was used for bread baking. STC was found to be stable during the bread-making process. In the food survey 17% of the analysed breads were positive for STC, with concentration levels of 2-7 µg kg(-1). One out of six rye bread samples, one out of nine rye-wheat bread samples and three out of 14 wheat bread samples were contaminated with STC. Four out of five contaminated samples contained whole grains as the main ingredient. We conclude that whole grain bread may be a possible source of STC, although even STC-positive bread samples identified in this study contained quite low toxin levels.

Sterigmatocystin: occurrence in foodstuffs and analytical methods--an overview.

Sterigmatocystin (STC) is a mycotoxin produced by fungi of many different Aspergillus species. Other species such as Bipolaris, Chaetomium, Emiricella are also able to produce STC. STC producing fungi were frequently isolated from different foodstuffs, while STC was regularly detected in grains, corn, bread, cheese, spices, coffee beans, soybeans, pistachio nuts, animal feed and silage. STC shows different toxicological, mutagenic and carcinogenic effects in animals and has been recognized as a 2B carcinogen (possible human carcinogen) by International Agency for Research on Cancer. There are more than 775 publications available in Scopus (and more than 505 in PubMed) mentioning STC, but there is no summary information available about STC occurrence and analysis in food. This review presents an overview of the worldwide information on the occurrence of STC in different foodstuffs during the last 40 years, and describes the progress made in analytical methodology for the determination of STC in food.

A novel biosensor for sterigmatocystin constructed by multi-walled carbon nanotubes (MWNT) modified with aflatoxin-detoxifizyme (ADTZ).

Sterigmatocystin, ST, is carcinogenic mycotoxin with toxicity second to aflatoxins, contaminated in foods- and feeds-stuff widely. A three-electrode system was employed to examine the response character of the covalently united ADTZ-MWNTs electrode to ST, and the results indicated that an oxidation peak of ST was observed at about +400 mv, the linear detection range of ST was from 4.16 x 10(-5) mg/ml (0.13 microM) to 1.33 x 10(-3) mg/ml (4.29 microM) with the detection limit at 0.13 microM. Compared to the corresponding results obtained from the MWNTs modified electrode that ADTZ was directly sediment (adsorbed) on it, the sensitivity of ours had been improved by two orders of magnitude, which could provide some important data to further research.

[The primary study on the detection of sterigmatocystin by biologic enzyme electrode modified with the multiwall carbon nanotubes].

Sterigmatocystin (ST), the secondary metabolite of many kinds of filamentous fungi, is a potent carcinogen structurally related to the aflatoxins (AFT). With similar chemical structure, sterigmatocystion behaves much the homogeneous properties to aflatoxins, both of these mycotoxins exhibit similar biological properties due to their bisfuranoid structure. Since the common, and even heavier pollution, found in foods and feeds-stuff, sterigmatocystion is more harmful than aflatoxins. The reported detection methods of sterigmatocystion included the Thin-layer Chromatography, the High-Performance-Liquid Chromatography, the Enzyme-Linked Immunosorbant Assay and the PCR detection to the toxic gene, however studies about both easy and inexpensive electro-chemical methods have not been found. Our previous studies had discovered that Sterigmatocystin (ST) exist similar sensitivity towards aflatoxin-detoxifizyme (ADTZ), which we had isolated from a fungus, as aflatoxin does. In this work, the preliminary study on electrochemical analysis and determination of ST with triplet electrode enzyme-biosensor system (Ag/AgCl as the reference electrode, Pt and Au as the pair and work electrode, respectively) was carried out. Multiwall-carbon-nanotube (MWNT) had been used to increase the electron transportation on electrode. In the research, the Au electrode was modified by MWNT-immobilized ADTZ, and then the voltammertric behavior of ST was studied by means of cyclic voltammogram analysis and different pulse analysis. Autoprobe CP Research Atomic Force Microscope and TECNAI 10 Transmission Electron Microscope, had been used to detect the MWNT as well as the surface of MWNT-modified ADTZ. The voltammertric behavior of ST was studied by means of cyclic voltammogram analysis and different pulse analysis. The results show that the red-ox peak potential of ST is at the point of -600 mV, the linear detection range is from 8.32 x 10(-5) to 66.56 x 10(-5) mg/mL, the detection limit is at 8.32 x 10(-5) mg/mL, and the response time is 10 seconds. This study provided a good basic work for further research.

Analytical methods for the determination of sterigmatocystin in cheese, bread and corn products using HPLC with atmospheric pressure ionization mass spectrometric detection.

Methods have been developed for the determination of sterigmatocystin in bread, maize and cheese using HPLC linked to mass spectrometry (MS) atmospheric pressure chemical ionization for detection and concurrent confirmation of sterigmatocystin at levels down to less than 5 micrograms/kg. Candidate extraction methods were initially checked for recovery and reproducibility by spiking commodities at a level of 200 micrograms/kg of sterigmatocystin and using HPLC with post-column derivatization. Recovery was found to be greater than 90% for bread and maize, and 75% for cheese. Mass spectrometer conditions for detecting sterigmatocystin were established by injecting solutions directly into the mass spectrometer. Extracts of bread, maize grits and cheese prepared by the candidate extraction methods were examined using HPLC/MS using samples spiked at a level of 20 micrograms/kg of sterigmatocystin. Results for bread and maize samples showed that the extraction procedure recovered more than 90% of added sterigmatocystin and produced extracts free of interference from co-extractives, with limits of detection of less than 2 micrograms/kg for both commodities. The HPLC/MS results for cheese extracts gave lower average recoveries of 55%. These results were also more variable. However, the apparent limit of detection for sterigmatocystin in cheese was still about 4 micrograms/kg.

[Determination of sterigmatocystin in cancerous tissues, blood and urine in patients with liver and stomach cancer].

Sterigmatocystin (ST) was determined with modified Southern-Western blot and indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) for 28 specimens of cancerous tissues, 13 of blood and 20 of urine in 14 patients with liver and stomach cancer. Results showed DNA-ST adduct was detected in 14 specimens of cancerous tissues and/or pericancerous tissues. ST values were higher in four of 13 patients (65-113 micrograms/kg), as compared only in one of 14 healthy persons (68 micrograms/kg). And, ST values all were very low in urine, with a maximum of 13 micrograms/kg.

Mycotoxins and fungi in wheat harvested during 1990 in test plots in the state of São Paulo, Brazil.

Wheat from two cultivars with contrasting characteristics were harvested in ten experimental plots located in wheat producing areas of the State of São Paulo, Brazil. The samples (10 of each cultivar) were analyzed by a gas-chromatographic method for deoxynivalenol (DON), nivalenol (NIV), diacetoxyscirpenol (DAS), toxins T-2 (T-2) and HT-2, T-2 tetraol, T-2 triol, and by a thin-layer chromatographic method for zearalenone (ZEN), aflatoxins B1, B2, G1, G2, ochratoxin A and sterigmatocystin. No mycotoxins were detected in 13 samples. DON was found in four samples (0.47-0.59 microgram/g), NIV in three samples (0.16-0.40 microgram/g), T-2 in two samples (0.40, 0.80 microgram/g), DAS in one sample (0.60 microgram/g), and ZEN in three samples (0.04-0.21 microgram/g). The wheat samples were also examined for the incidence of fungi. Alternaria, Drechslera, Epicoccum and Cladosporium were the prevailing genera. Among the Fusarium spp., F. semitectum was present in 19 samples and F. moniliforme in 18 samples. No F. graminearum was isolated in the samples.

[Preparation and identification of monoclonal antibody against sterigmatocystin].

Compound antigen, bovine serum albumin-sterigmatocystin (BSA-ST) or hemocyanin sterigmatocystin (H-ST) was prepared by converting ST to its hemiacetal group in ST and taking ST conjugated to BSA or H. Balb/c mice were immunized with BSA-ST, and then their spleen cells were isolated and fused with SP2/0 murine myeloma cells. A hybridoma cell strain that could stably secrete monoclonal antibody against ST (McAb-ST) was selected, and both of their ascites and serum were rich in McAb-ST induced by cells of this strain. McAb-ST was identified as Ig-Gi with a molecular weight of 168,000 kd (53,000 for heavy chain and 31,000 for light one), and with an affinity constant of H-ST reaction of 1.29 x 10(9) mol/L measured by indirect competitive enzyme-linked immunosorbent assay (IC-ELISA). Relative cross reaction of McAb-ST with ST analogues was less than 2%. The minimal 0.5 pg/25 microliters of ST can be detected with IC-ELISA using McAb-ST and ST simultaneously, with a linear range of 0.1-10 ng/ml.