Detection of Atrazine Residue in Food Samples by a Monoclonal Antibody-based Enzyme-linked Immunosorbent Assay

Atrazine（AT,2-chloro-4-ethylamino-6-isopropyl-amino-s-triazine）has been detected in ground water in several areas of the United States for many years,as well as in China,wherein the growth rate of its gross

Development of an Indirect Enzyme-Linked Immunosorbent Assay for Seromonitoring Contagious Bovine Pleuropneumonia Using Recombinant Lipoprotein LppQ of Mycoplasma mycoides subsp mycoides SC as Antigen

Mycoplasma mycoides subsp mycoides SC （MmmSC） is the etiological agent of contagious bovine pleuropneumonia （CBPP）. The lipoprotein LppQ encoded by lppQ gene is specific to MmmSC and is found in the type strain and in field strains isolated in Europe, Africa, and Australia, as well as in vaccine strains. No serological cross-reactions were observed with the related mycoplasmas of the Mycoplasma mycoides cluster. The N-terminal domain of the mature lipoprotein LppQ is hydrophilic, and it induces a strong, specific, early, and persistent immune response in naturally and experimentally infected animals. Mycoplasma-specific TGA （Trp） codons are utilized as stop codons in most other organisms. The lppQ N-terminal fragment from MmmSC HVRI X strain, the Chinese strain for CF antigen production, was mutated with one-step overlapping extension PCR. Sequence analysis confirmed the successful mutation from A to G in codon 198 in the lppQ gene. The fragment containing the mutation site was subcloned into the pET32a expression vector. The recombinant protein with molecular weight of 42 kDa was purified using the Ni-NTA His.Bind purification kit, with a purity of up to 95%. Western blot indicated that the standard positive serum of CBPP could react with the recombinant protein. The purified protein was diluted to 0.35 μg mL^-1, and coated to microtiter enzyme-linked immunosorbent assay （ELISA） plates. Indirect ELISA reaction conditions were optimized. The value of P/N was determined to be 4.8 （0.934/0.193）, the sensitivity to be 95.8% （46/48）, and the specificity to be 98.9% （161/163）. 3 817 cattle serum samples from three different provinces were detected by the indirect ELISA and CFT. The Kappa value is 0.63, which is middle or high agreement between the two methods.

A Comparison of Enzyme-Linked Immunosorbent Assay versus Multiplex Methodology Using an <i>in Vitro</i>Model of Pulmonary Hypertension and Inflammation

Enzyme-linked immunosorbent assay (ELISA) is the most widely used method for measuring a single cytokine. Recent developments in cytokine quantification such as multiple arrays measure multiple cytokines simultaneously. Although good correlations between ELISA and multiplex methods have been observed, side by side comparisons are limited. In the present study we hypothesized that ELISA and Luminex techniques are comparable in detecting cytokines in culture medium when pulmonary artery smooth muscle cells (PASMC) are exposed to stress. Primary human PASMC were cultured in modular chambers and exposed to 21% FiO2 and peak inspiratory and positive end expiratory pressure of 24 and 8 cmH2O respectively, and 95% FiO2. At 24 hours, culture medium was collected and assayed for interleukin-6 (IL-6) and IL-8 by quantitative ELISA and by Human Cytokine 25-Plex Panel using a Luminex 200 analyzer. A comparative analysis of agreement between our ELISA and Luminex data was detailed for control and stress conditions using the Bland-Altman plot analysis. Each assay resulted in comparable increased (p < 0.001) levels of IL-6 and IL-8 as compared to control in response to oxidative and biophysical stress. The Bland-Altman analysis demonstrated that 95% of the differences between ELISA and Luminex values were within ±1.96 SD from the mean difference indicated by the 95% limits of agreement for the measurements of IL-6 and IL-8. There was no systematic bias as a function of inflammation level. We conclude that in this cell culture model, ELISA and Luminex are comparable in detecting the levels of IL-6 and IL-8 in the culture medium. If measurements of multiple cytokines are demanded and the amount of sample is limited, Luminex multi-analyte profiling technology is accurate and sensitive.

Sandwich ELISA for detecting urinary Survivin in bladder cancer

Objective： Survivin as a tumor marker in the diagnosis of bladder cancer has not been completely confirmed yet and there are few reports about using Survivin enzyme-linked immunosorbent assay （ELISA） kit to detect the urine of bladder cancer patients. This study aimed to develop a Survivin ELISA and validate its value in the detection of bladder cancer. Methods： Through square matrix titration, different combinations of coating antibody and detecting antibody, a Survivin ELISA was constructed. This assay was evaluated according to intra-assay precision, inter-assay precision and minimum detectable dose （MDD）. Survivin levels were detected and analyzed in 102 bladder cancer patients and 102 healthy people by established ELISA. Then cutoff value was defined according to the analysis of receiver operating characteristic （ROC） curve. The sensitivity and specificity of detection were calculated on the basis of cutoff value to diagnose bladder cancer patients. Furthermore, the value of Survivin expression detected by ELISA among different clinicopathological characteristics of patients was also compared. Results： Through optimization of different conditions, intra-assay precision was 8.39%, inter-assay precision 8.57% and MDD 0.0625 ng/mL in this assay. When the optical density at 450 nm （OD 450 ） was 0.09, it could get the optimized diagnostic cutoff value. According to this value, the sensitivity and specificity of diagnosis in bladder cancer patients were 70.6% and 89.2%, respectively. The associations between patients＇ clinical variables and OD 450 were not significant except tumor numbers in patients. Conclusions： This experiment has preliminarily developed a Survivin ELISA and confirmed Survivin as a biomarker which owned a practical and significant value in the diagnosis of bladder cancer.

液相色谱-质谱联用法(LC-MS/MS)和酶联免疫法(ELISA)对体内25(OH)D3水平的测定

目的同时用液相色谱-质谱联用法(LC-MS/MS)和酶联免疫法(ELISA)检测患者体内25(OH)D3水平,分析两者结果的差异。方法随机选取50例住院患者,对同一血清样本分别用LC-MS/MS法和ELISA法测定25(OH)D3水平,同时用LC-MS/MS法测定25(OH)D2的水平。结果 LC-MS/MS法测定的维生素D3的均数为14.99±6.51 ng/mL,酶联免疫法测定的均数为20.91±9.70 ng/mL,两者的相关系数为0.725(P<0.01),线性相关方程为维生素D3(LC-MS/MS法)=4.829+0.486×维生素D3(ELISA法)。LC-MS/MS法组25(OH)D3浓度高于20 ng/mL的比例17%,酶联免疫法组为52%,LC-MS/MS法组的25(OH)D2和25(OH)D3总浓度高于20 ng/mL的为24%。25(OH)D2占25(OH)D总量的8.4%。结论 LC-MS/MS法测定的维生素D3的数值明显低于ELISA法,两者正相关性较高,可经方程互换。酶联免疫法低估了体内维生素D的缺乏,检测25(OH)D3的同时需测定25(OH)D2浓度。

Simple and sensitive determination of sparfloxacin in pharmaceuticals and biological samples by immunoassay

Plasma quinolone concentrations are not routinely measured in clinical practice.However,in order to optimize quinolone treatment,monitoring of plasma concentrations could sometimes be useful particularly in critically ill patients.In this study,anti-sparfloxacin antibody was obtained by immunizing rabbits with sparfloxacin conjugated with bovine serum albumin using isobutyl chloroformate method.After the assay procedure was optimized,the standard curve of sparfloxacin was established.The practical measuring range of the competitive ELISA extended from 5 ng/mL to 2 mg/mL.The recovery rates and coefficients of variation for rat plasma,urine and tissues were 87.7-106.2% and 4.8-15.3%,respectively.To demonstrate the potential of the ELISA,a preliminary pharmacokinetics and tissue distribution study of sparfloxacin in rats and quantitative analysis of sparfloxacin in several pharmaceuticals were performed and compared with high-performance liquid chromatography(HPLC).The experimental data indicated that the proposed method would be a valuable tool in therapeutic drug monitoring(TDM) for sparfloxacin.

Development of a sandwich ELISA for the detection of bovine herpesvirus type 1

Objective:To develop a standard enzyme-linked immunosorbent assay（ELJSA） for the detection of bovine herpesvirus type 1（BHV-1）.Methods:The assay was based on hyperimmune rabbit and guinea pig antisera raised against purified BHV-1.Polyethylene glycol precipitation and sucrose density gradient methods were adopted for viral concentration and purification.Antisera were raised using Freund’s adjuvant followed by extraction of IgG of high purity.Results: Optimum antisera dilutions as determined by titrations were chosen as 14 000,whereas the conjugate was used at 1:2 000 dilution.Using 95 clinical specimens,the ELISA test showed a sensitivity and specificity of 91.90%and 93.10%,respectively when compared to PCR.The cutoff value was fixed at 0.15490) and a P/N ratio of】1.30 indicated a significant positive reaction. Conclusions:The results have demonstrated that this ELISA could efficiently detect BHV-1 and can be used as an important diagnostic tool.

Determnation of ochratoxin A in grain by monoclonal antibody－based enzyme－linked immunosorbent assay

The simple rapid and sensitive enzyme-linked immunosorbent assay (ELISA) methods, di-rect and indirect ELISA, for quantitation of ochratoxin A in cereal had been developed by theutilization of monoclonal antibody on immunomicroplate. Direct FLIAS was found to be less timeconsuming than indirect ELISA. For direct FLISA, recovery of 1 -500 ppb OA added to wheat was78.9-100.0% and rice was 88.9- 120.0%. For indirect EI.IAS, recovery of 1-500 ppb OA addedto wheat was 79.0- 110.0% and rice was 82.0 120.0%. The minimal detection level for OA was Ippb. Analyses of 31 samples that caused humanintoxicant for OA showed that the ELISA resultsagreed wtll with those obtained by thin-layer chromatogrdphy.

Microcystin-LR detection based on indirect competitive enzyme-linked immunosorbent assay

Microcystins(MCs)are a group of closely related toxic cyclic heptapeptides produced by common cyanobacte-ria,which cause lots of accidents and threatens human health.In this paper,an indirect competitive enzyme-linked immu-nosorbent assay(ic-ELISA)was established and used to detect microcystin-LR(MC-LR)in drinking and surface waters.The concentration of coating antigen was 5 mg/mL,the dilution of monoclonal antibody MC10E7 was 1:3000,the dilution of enzyme tracer(goat anti-mouse IgG-peroxidase)was 1:3000,the standard concentration of MC-LR ranged from 0.001 mg/L to 30 mg/L,and o-phenylenediamine was used as substrate.The assay showed high relativity with high performance liquid chromatography(HPLC)with a correlation coefficient of more than 99%.The relative standard deviation was less than 10%,the detection limit was achieved down to 0.01 mg/L and up to 5.1 mg/L.The quantitative detection range was from 0.03 mg/L to 3 mg/L,and the antibody had high specificity for[4-arginine]microcystins.It performed well in spite of the influence of the real samples.

食品中丙烯酰胺检测方法的研究进展

丙烯酰胺是在食品高温加工过程中产生的小分子有机化合物,具有致癌性。从样品提取、衍生化、净化、富集等前处理过程以及对检测器的选择出发,总结国内外近年来用于检测丙烯酰胺的方法,如从传统的气相色谱、液相色谱及其联用技术,到新兴开发的分子印迹技术、酶联免疫吸附和生物传感器等新检测技术。并根据其适用范围和操作条件,对各分析方法的优点和不足进行讲述,最后对将来丙烯酰胺检测方法发展新思路提供策略和依据。

乙型肝炎表面抗原不同检测方法的优势比较分析

目的：比较化学发光微粒子免疫分析法（CMIA）和酶联免疫吸附法（ELISA）用于乙型肝炎（乙肝）表面抗原检测的优势效果。方法选取2011年3月～2013年4月采用ELISA法与CMIA法对386例患者进行了乙肝表面抗原检测的对比研究。结果 CMIA法检测乙肝表面抗原的阳性率为51.3%，ELISA法阳性率为43.5%，CMIA法阳性率明显更高，与ELISA法比较差异有统计学意义（P〈0.05）；CMIA法的灵敏度与特异性均明显高于ELISA法（P〈0.05）。结论与ELISA法比较，CMIA法对乙肝表面抗原检测在阳性率、灵敏度以及特异性等方面均存在有比较明显的优势，故建议将CMIA法作为临床检测乙肝表面抗原的首选方法而推广应用。

Level of circulating PD-L1 expression in patients with advanced gastric cancer and its clinical implications

Objective：The programmed cell death-1 receptor/programmed cell death-1 ligand （PD-1/PD-L1） pathway plays a crucial role in tumor evasion from host immunity.This study was designed to evaluate the association between circulating PD-L1 expression and prognosis in patients with advanced gastric cancer.Methods：Totally 80 advanced gastric cancer patients and 40 health controls from Beijing Cancer Hospital were enrolled in the present study.Circulating PD-L1 expression was tested by enzymelinked immunosorbent assay （ELISA）.The associations between the expression level of PD-L1 and clinicopathological features and prognosis were analyzed statistically.Results：Expression of PD-L1 in advanced gastric cancer patients was significandy up-regulated compared with health people （P=0.006）.The expression of PD-L1 was significantly correlated with differentiation and lymph node metastasis （P=0.026 and P=0.041,respectively）.Although we didn＇t find significant difference in all advanced gastric cancer patients with different PD-L1 expression,the adenocarcinoma patients with higher up-regulated PD-L1 expression had much better prognosis than low expression patients （65.6％ vs.44.7％,P=0.028）.Conclusions：PD-L1 was elevated in advance gastric cancer patients and may play an important role in tumor immune evasion and patients prognosis.

Highly Sensitive and Specific Monoclonal Antibody-Based Serological Methods for Rice Ragged Stunt Virus Detection in Rice Plants and Rice Brown Planthopper Vectors

Rice ragged stunt virus（RRSV） is a serious rice disease in Asia, causing serious yield losses on rice. The capsid protein（CP） gene of the major outer capsid protein of RRSV was expressed in Escherichia coli BL21（DE3） using the pMAL-C2 X expression vector. The recombinant protein was used as the immunogen to immunize BALB/c mice. A hybridoma cell line 8A12 secreting monoclonal antibody（MAb） against RRSV was obtained by fusing mouse myeloma cells（Sp 2/0） with spleen cells from the immunized BALB/c mice. Western blot analysis showed that the MAb 8A12 can specifically react with RRSV CP. Using the MAb, an antigen-coated-plate enzyme-linked immunosorbent assay（ACP-ELISA）, a dot enzyme-linked immunosorbent assay（dot-ELISA）, and immunocapture-RT-PCR（IC-RT-PCR） assay were developed to detect RRSV. The established ACP-ELISA, dot-blot ELISA and IC-RT-PCR methods could detect RRSV in infected rice tissue crude extracts with dilutions of 1：40 960, 1：1 280 and 1：655 360（w/v, g mL-1）, respectively. The ACP-ELISA and dot-blot ELISA methods could detect RRSV in infected insect vector crude extracts with dilutions of 1：12 800 and 1：1 600（an individual planthopper μL-1）, respectively. The field survey revealed that Rice ragged stunt disease occurs on rice in Hainan, Yunnan, Guangxi, Sichuan, Guizhou, Fujian, Hunan, Jiangxi and Zhejiang in China.

Development of Anti-Isoproturon Polyclonal Antibody

A competitive enzyme-linked immunosorbent assay （ELISA） suitable for the determination of the urea herbicide isoproturon, 3-（4-isopropylphenyl）-l,l-dimethylurea, in food and environmental samples was developed. Two haptens named 1-（3- carboxypropyl）-3-（4-isopropylphenyl）-1-methylurea （hapten 4C） and 1-（5-carboxypentyl）-3-（4-isopropylphenyl）-1- methylurea （hapten 6C） were synthesized. The haptens were coupled to bovine serum albumin （BSA） and ovalbumin （OVA）, respectively, using the N-hydroxysuccinimide reaction. The hapten 6C-BSA conjugate was used as the immunogen, with which a high-titer anti-isoproturon polyclonal antibody （pAb） was successfully obtained by immunization of New Zealand white rabbits. The hapten 4C-OVA conjugate was used as coating antigen and a method of the indirect competitive ELISA for isoproturon was established. The haptens were confirmed with TLC, IR, and 1H NMR. The conjugation molar ratios of hapten 4C to OVA and hapten 6C to BSA were 36：1 and 46：1, respectively, as calculated by a UV spectrophotometry. The highest titer of the anti-isoproturon sera determined by a non-competitive indirect ELISA procedure was 1.6 × 10^5. The optimal concentrations of the coating antigen and the dilution of the anti-isoproturon sera used in the ELISA were 0.1 mg·L^-1 and 1.0 × 10^5, respectively. The concentration of isoproturon that inhibits 50% of antibody-antigen binding （IC50） was 0.07 mg·mL^-1. The cross-reactivities of six urea herbicides including chlorbromuron, fluometuron, monolinuron were lower than 0.1%. Isoproturon is a small molecule without immune activity and active functional group for attaching to carrier protein. To produce an antibody against isoproturon with high titer and high specificity is the most important step in the development of an immunochemical method for the determination of isoproturon in food and environmental samples. The two haptens synthesized in this study have carboxyl groups and accommodate different lengths of spacer arms, and the phenyl and isopropyl groups are fully exposed. An anti-isoproturon polyclonal antibody with high titer and high specificity was successfully obtained by immunization of rabbits with the conjugate of the hapten attached to the protein carrier.

Occurrence of gonadtropins like substance in the thoracic ganglion mass of the mud crab, Scylla paramamosain (Crustacea: Decapoda: Brachyura)

The identification and localization of vertebrate follicle stimulating hormone （FSH） and luteinizing hormone （LH） in crustaceans may further elucidate the regulation mechanisms in arthropod repro-duction. Using immunocytochemical staining techniques, this study has localized vertebrate FSH-like and LH-like substances in neurons in the subesophageal and thoracic ganglia from the thoracic ganglion mass （TGM） of Scylla paramamosain （Crustacea： Decapoda： Brachyura）. Enzyme-linked immunosorbent assay （ELISA） has shown that the concentrations of both FSH-and LH-like sub-stances increased markedly in the TGM during the vitellogenic stage compared with that in the previtellogenic stage. These results indicate that substances resembling the vertebrate FSH and LH are present in S. paramamosain, and they may be involved in the development of the ovary as well as in ovulation.

Application of Current Hapten in the Production of Broad Specificity Antibodies Against Organophosphorus Pesticides

Diethylphosphono acetic acid （DPA） was used as a current hapten to generate broad specificity polycolonal antibodies against a group of organophosphorus pesticides. Six New Zealand white rabbits were immunized with immunogens synthesized by the active ester method （AEM） or 1-ethyl-3-（3-dimethylaminopropyl）-carbodimide method （EDC）. The titers of antisera reached 25 600 by AEM and 6 400 by EDC, respectively. Polyclonal antibodies raised against DPA were screened and selected for the competitive indirect enzyme-linked immunosorbent assay （CI-ELISA）. A CI-ELISA for DPA was developed with a detection limit of 3.536 ng mL^-1and an I50 value of 0.182 μg mL^-1. The assay specificity was evaluated by obtaining competitive curves for several structurally related compounds as competitors. The antiserum showed high affinities to chlorpyrifos, diazinon, omethoate, parathion-ethyl and profenofos with I50 of 0.12, 0.15, 0.21, 0.88, 0.97 and 2.5 μg mL^-1, respectively. The results indicate that the assay could be a screening tool for quantitation and semiquantitation determination of the above former five organophosphorus pesticides.

Clinicopathological features of hypoxia-inducible factor-1α and vascular endothelial growth factor expression in patients with lung cancer

Objective The aim of the study was to investigate the clinicopathological characteristics of hypoxiainducible factor-1α(HIF-1α) and vascular endothelial growth factor(VEGF) expression in patients with lung cancer.Methods Cancerous and noncancerous tissues were collected post-operation from 115 patients with lung cancers by the self-control method. Total RNA was extracted from the lung tissues. The status of tissue HIF-1α expression and intercellular distribution was observed by immunochemistry using a tissue microarray. The expression levels of circulating HIF-1α and VEGF were detected by enzyme-linked immunosorbent assay(ELISA).Results The expression of serum HIF-1α [(138.3 ± 28.8) μg/L] in the group of patients with lung cancer was significantly higher(P < 0.01) than that in the group of patients with pneumonia [(58.8 ± 14.5) μg/L] and the control group of patients ((24.1 ± 3.3) μg/L)There was a strong positive correlation of serum HIF-1α levels(r = 0.937, P < 0.01) with serum VEGF levels. The specific concentration of total RNA [(1.52 ± 1.14) μg/mg wet lung tissues] in the cancerous tissues was significantly higher(t = 8.494, P < 0.001) than that in the noncancerous tissues ((0.58 ± 0.33) μg/mg)The clinicopathological features of HIF-1α expression in lung cancer tissues revealed a significant relationship between positive HIF-1α expression and patient sex(χ~2 = 4.494, P = 0.034), tumor size(χ~2 = 4.679, P = 0.031), differentiation degree(χ~2= 8.846, P = 0.012), and presence of lymphatic node metastasis(χ~2= 6.604, P = 0.037).Conclusion Abnormal HIF-1α expression in lung cancer is closely related with nucleic acid metabolism and angiogenesis, and it may be helpful in the diagnosis and identification of lung cancer.

Evaluation of IL-6 and IL-8 in Tear Fluid of Sulfur Mustard Gas-Exposed Patients with Eye Lesions

The purpose of this study is to compare the levels of IL-6, 8 in tear fluids of people exposed to mustard gas in the war between Iraq and Iran who had the chronic dry-eye symptoms compared to the normal group. In this study, 25 of the patients who were exposed to mustard gas and had chronic dry eye symptoms were compared to 25 patients as control group, consisting of 25 people who had common chronic dry eye symptoms with blepharitis and 25 healthy people as normal group. The levels of IL-6 and IL-8 in tear fluid of people of these three groups were assessed by enzyme-linked immunosorbent assay (ELISA). The results for levels of IL-6 (P = 0.002) and IL-8 (P = 0.001) in tear fluid of patients in comparison with normal group show a significant increase. The differences were considered statistically significant at P < 0.05. The effect of exposure to mustard gas on eyes of chemical-injured veterans destroyed meibomian glands which paved the way for evaporative type of dry-eye. As a result, the cited ILs in the tear fluid of these patients increased and resulted in later eye-impairments.

Do Occupational Factors Render Army Service a High-Leptospirosis-Risk Occupation?

Objective: Leptospirosis is a known zoonotic disease which is closely related to certain high-risk occupational group. This study aimed to identify the risk factors associated with seropositive leptospirosis among army personnel. Methodology: A cross sectional study was conducted involving 616 army personnel using a self-administered questionnaire. The questionnaire consisted of sociodemographic, occupational and environmental data as well as recreational activities history. Serum sample for leptospirosis was taken for screening with IgM Enzyme-linked Immunosorbent Assay (IgM ELISA) test. Positive tests were confirmed by Microscopic Agglutination Test (MAT). Results: All respondents were male, Malays and married with the mean age of 29.28 (7.15) years old. The mean duration of working was 11.50 (7.75). Majority of the respondents were non-officer and riflemen, smokers (455 (73.9%), stayed inside the camp and engaged in recreational activities. The mean duration of employment was 11.50 (7.75) years. The overall seroprevalence of leptospirosis was 16.2% (95% CI: 13.32, 19.15) and the predominant pathogenic serovar identified was Terengganu (38.2%). Those who had lower educational levels were more protective for positive leptospirosis compared to those who have only primary education (OR: 0.32;95% CI: 0.14, 0.76;p = 0.009). Conclusion: Despite being named as one of the high-risk occupations for leptospirosis, this study found no significant occupational factors related to leptospirosis seropositivity among army personnel.

Aflatoxin Contamination of Garri Sold in Some Selected Markets in Benue State, North Central, Nigeria

The presence of aflatoxin-producing fungi in foods consumed by humans and animals has often resulted in the health hazards and even death. Aflatoxin contaminations of garri sold in some markets of Benue State, Nigeria, were studied, to ascertain the health implications on the consumers. Sixty garri samples comprising of 30 white garri and 30 yellow garri respectively were studied. The garri samples were ground to a particle size of 250 μm using a sterile blender. The total aflatoxin was extracted using 70% (v/v) methanol. The total aflatoxin concentration was detected and quantified using the Enzyme-Linked Immunosorbent Assay technique. The results showed that overall, 76.67% of the white garri samples and 80% of the yellow garri samples were aflatoxin positive. The total aflatoxin concentration in white garri ranged from 0.3 μg/kg to 2.4 μg/kg and 0.2 to 2.4 μg/kg in yellow garri respectively. The total mean aflatoxin across the States recorded was 2.96 μg/kg in white garri and 3.07 μg/kg in yellow garri. All the aflatoxin-positive garri samples of both the white and yellow garri were within the NAFDAC permissible aflatoxin level. Even though the aflatoxins are within the approved standard consumable limits, the continuous consumption of these doses over a long period of time could lead to the accumulation of these toxins in the body. These may eventually constitute a toxic health challenge.