Indirect Competitive Enzyme-linked Immunosorbent Assay Research Articles

Preparation of Monoclonal Antibody and Development of Indirect Competitive Enzyme-Linked Immunosorbent Assay and Fluorescence-Linked Immunosorbent Assay for Detecting 3-Amino-5-methylmorpholino-2-oxazolidinone (AMOZ) in Edible Animal Tissue.

To monitor the illegal used of furaltadone, a highly sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and fluorescence-linked immunosorbent assay (FLISA) based on a monoclonal antibody (mAb) were developed for the detection of 3-amino-5-methylmorpholino-2-oxazolidinone (AMOZ), the major metabolite of furaltadone in animal tissues. The highly specific mAb, which was very sensitive to a nitrophenyl derivative of AMOZ (2-NP-AMOZ) with IC50 values of 0.11 and 0.09 ng/mL for ic-ELISA and FLISA, respectively, was selected for the development of immunoassays. For both the ic-ELISA and FLISA for AMOZ-spiked experiments, acceptable recovery rates of 81.1–105.3% and coefficients of variation of 4.7–9.8% were obtained. In addition, results from both ic-ELISA and FLISA methods for spiked samples’ data showed excellent correlation coefficients ranging from 0.9652 to 0.9927. Meanwhile, the proposed ic-ELISA and FLISA for thirty spiked samples were confirmed by standard LC-MS/MS with high correlation coefficients of 0.9911 and 0.9921, respectively. These results suggest that the developed ic-ELISA and FLISA are valid and cost-effective tools for high-throughput monitoring methods for AMOZ residues in animal tissues.

Development of an Immunoassay for the Detection of Copper Residues in Pork Tissues.

The presence of high concentrations of copper (Cu) residues in pork is highly concerning and therefore, this study was designed to develop a high-throughput immunoassay for the detection of such residues in edible pork tissues. The Cu content in the pork samples after digestion with HNO3 and H2O2 was measured using a monoclonal antibody (mAb) against a Cu (II)–ethylenediaminetetraacetic acid (EDTA) complex. The resulting solution was neutralized using NaOH at pH 7 and the free metal ions in the solution were chelated with EDTA for the immunoassay detection. An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) method was developed for Cu ion analysis. The half maximal inhibitory concentration of the mAb against Cu (II)–EDTA was 5.36 ng/mL, the linear detection range varied between 1.30 and 27.0 ng/mL, the limit of detection (LOD) was 0.43 μg/kg, and the limit of quantification (LOQ) was 1.42 μg/kg. The performances of the immunoassay were evaluated using fortified pig serum, liver, and pork samples and had a recovery rate of 94.53–102.24%. Importantly, the proposed immunoassay was compared with inductively coupled plasma mass spectroscopy (ICP-MS) to measure its performance. The detection correlation coefficients of the three types of samples (serum, pork, and liver) were 0.967, 0.976, and 0.983, respectively. Thirty pork samples and six pig liver samples were collected from local markets and Cu was detected with the proposed ic-ELISA. The Cu content was found to be 37.31~85.36 μg/kg in pork samples and 1.04–1.9 mg/kg in liver samples. Furthermore, we detected the Cu content in pigs with feed supplemented with tribasic copper chloride (TBCC) and copper sulfate (CS) (60, 110, and 210 mg/kg in feed). There was no significant difference in Cu accumulation in pork tissues between the TBCC and CS groups, while a remarkable Cu accumulation was found for the CS group in liver at 210 mg/kg, representing more than a two-fold higher level than seen in the TBCC group. Therefore, the proposed immunoassay was found to be robust and sensitive for the detection of Cu, providing a cost effective and practical tool for its detection in food and other complicated samples.

Production of highly sensitive monoclonal antibody and development of lateral flow assays for phallotoxin detection in urine.

Phallotoxins, toxic cyclopeptides found in wild poisonous mushrooms, are predominant causes of fatal food poisoning. For the early and rapid diagnosis mushroom toxin poisoning, a highly sensitive and robust monoclonal antibody (mAb) against phallotoxins was produced for the first time. The half-maximum inhibition concentration (IC50) values of the mAb-based indirect competitive ELISAs for phallacidin (PCD) and phalloidin (PHD) detection were 0.31ngmL-1 and 0.35ngmL-1, respectively. In response to the demand for rapid screening of the type of poisoning and accurate determination of the severity of poisoning, colloidal gold nanoparticle (GNP) and time-resolved fluorescent nanosphere (TRFN) based lateral flow assays (LFA) were developed. The GNP-LFA has a visual cut-off value of 3.0ngmL-1 for phallotoxins in human urine sample. The TRFN-LFA provides a quantitative readout signal with detection limit of 0.1ngmL-1 in human urine sample. In this study, urine samples without pretreatment were used directly for the LFA strip tests, and both two LFAs were able to accomplish analysis within 10min. The results demonstrated that LFAs based on the newly produced, highly sensitive, and robust mAb were able to be used for both rapid qualitative screening of the type of poisoning and accurate quantitative determination of the severity of poisoning after accidental ingestion by patients of toxic mushrooms.

Synthesis of hapten, production of monoclonal antibody, and development of immunoassay for ribavirin detection in chicken.

Ribavirin (RBV) is an effective antiviral drug, whose use is prohibited in animal husbandry worldwide. In this work, a novel immunizing hapten of RBV, named Hapten 4, was designed by comparing the conformational and electronic properties of RBV and haptens based on computational chemistry. Hapten 4 was synthesized and conjugated with carrier proteins to produce monoclonal antibody (mAb). The obtained mAb 4C3 for RBV exhibited an IC50 value of 6.24ng/ml in an indirect competitive enzyme-linked immunosorbent assay (icELISA) and displayed no cross-reaction with five other antiviral drugs, including amantadine. The applicability of the developed icELISA was verified in chicken, with a calculated limit of detection of 4.23µg/kg. The recoveries in spiked chicken were 79.2%-107.3% with a coefficient of variation less than 15.9%. The results indicated that the produced antibody from the new hapten was reliable and would be useful for RBV screening in chicken. PRACTICAL APPLICATION: RBV is a broad-spectrum antiviral drug, which is commonly used illegally in poultry farms. A high-affinity mAb 4C3 against RBV was produced and used to develop icELISA with acceptable sensitivity and accuracy. The constructed icELISA has excellent performance for detecting RBV residues in chicken.

Design of Hapten Synthesis and Antibody Characterization of G-group Aflatoxins

Food and feed contamination with Aflatoxins pose a serious threat to human health and animal husbandry development and has caused widespread concern, among them, G-group Aflatoxins as the main pollutant has attracted more and more attention. In order to establish a rapid, sensitive, specific and efficient immunoassay method for G-group aflatoxins, this study aimed to designed to synthesize 3 immunogens and coating antigens and identified by UV and SDS-PAGE. Then used to immunize Balb/c mice with prepared of three immunogen the titers were determined by indirect ELISA and the sensitivity was determined by competitive indirect ELISA (icELISA), the specificity was assessed by the cross-reaction test (CR). The results of UV and SDS-PAGE showed that the three immunogens and the corresponding coated antigens were successfully synthesized and the best one was SA method among three synthesis methods of G-group AF artificial antigen and its conjugation ratio of AFG1 to BSA was about 5.64∶1. The immune efficacy of SA method was the best, its AFG1 pAb had high titers of 1∶(6.4×103) by indirect ELISA, a good sensitivity with the 50 % inhibition concentration(IC50) of 13.6 μg/kg－1 to AFG1 by blocking ELISA and a high CR to AFG2 of 82.19 %, it showed high specificity for other aflatoxins. The experimental results not only obtained the ideal G group aflatoxin antibody, but also established a substance and technology foundation for G group aflatoxin immunization methods, and can be referenced in the similar tests.

Fragment antigen-binding (Fab) antibody-based lateral flow immunoassay for rapid and sensitive detection of potent phytoestrogen, deoxymiroestrol.

Pueraria candollei is an ingredient of Thai herbal medicine, dietary supplements, and cosmetics. The in vitro and in vivo studies of this plant supported anti-osteoporotic activity and used for hormone replacement therapy. Deoxymiroestrol shows the most potent phytoconstituent in tuberous root of P. candollei with estrogenic activity. The quality controls are important for good agricultural practice (GAP) and good manufacturing practice (GMP) of plant-derived raw materials. The rapid detection of lateral flow immunoassay (LFIA) using colloidal gold is simply method, easy visualize detection and produce less waste than conventional chromatographic detection. In this study, LFIA for qualitative detection of deoxymiroestrol using antigen-binding fragment antibody (Fab) was developed. The result showed that the developed LFIA displays specific detection of deoxymiroestrol. Cross reactivity of this method was analyzed with miroestrol, isomiroestrol and methylisomiroestrol which showed 39.97%, 7.71% and 5.72%, respectively. After optimal condition, limit of detection (LOD) for deoxymiroestrol is 250ng/ml. Plant samples were applied to strip test compare with indirect competitive ELISA using polyclonal antibody to confirm the application of LFIA. The results of LFIA method were comparable with those from ELISA. This developed lateral flow immunoassay can apply to detect deoxymiroestrol for the rapid testing. The developed method can use for quality control in plant samples as deoxymiroestrol is biomarker compound in P. candollei.

Generation of recombinant antibodies by mammalian expression system for detecting S-metolachlor in environmental waters

Current immunoassays for herbicide detection are usually based on polyclonal or monoclonal antibodies (MAbs) raised in animals. The mammalian expression system allows the procurement of specific and highly sensitive antibodies, avoiding animal immunization. In this study, S-metolachlor-specific IgG vectors bearing either Thosea asigna virus 2A or internal ribosome entry site (S-T2A or S-IRES) and single-chain variable fragment (scFv) vectors were designed and expressed. The recombinant antibodies (RAbs) were characterized by indirect competitive enzyme-linked immunosorbent assays (icELISA). The results showed that full-length RAbs exhibited significantly better performance than scFv, and both bicistronic vectors expressed antibodies of correct size, while RAb S-T2A elicited a higher yield than RAb S-IRES. Further analyses showed that RAb S-T2A and RAb S-IRES exhibited comparable reactivities and specificities to the parental MAb, with IC50 values of 3.44, 3.89 and 3.37 ng/mL, respectively. Finally, MAb- and RAb-based icELISAs were established for the determination of S-metolachlor in environmental waters. The recoveries were in the range of 73.0–128.1%, and the coefficients of variation were mostly below 10%. This article describes the production of RAbs for S-metolachlor from mammalian cells for the first time and paves the way to develop RAb-based immunoassays for monitoring herbicide residues in the environment.

High sensitivity and specificity Enzyme-Linked immunosorbent assay for detection of Acetaminophen in herbal tea and liquor

The acidified structure of acetaminophen (ACA) and 3-Oxo-3-(phenylamino) propanoic acid (3-O-3) were used to couple bovine serum albumin (BSA) to prepare haptens, respectively. A highly sensitive and specificive competitive indirect enzyme-linked immunosorbent assay (ciELISA) method was developed for the detection of acetaminophen using 3-O-3-BSA for New Zealand white rabbit immunization to produce antibodies. The IC50 was 6.66 ng/mL, the detection limit was 0.32 ng/mL, and the quantitative detection range was 0.91-48.54 ng/mL. Comparing the performance of ACA-BSA antibody and 3-O-3-BSA antibody recognition of acetaminophen, it was found that the carbonyl site of 3-O-3 plays a major role in antigen-antibody recognition, while para-phenolic hydroxyl does not play an important role in the recognition process. The study provided theoretical guidance for the preparation of acetaminophen recognition antibodies, and established a rapid detection method for acetaminophen.

A sensitive and practical ELISA for analyzing naringenin in pummelo and herb samples

Naringenin, a flavonoid compound found in pummelo, is a key biological active compound in some traditional Chinese medicines, including Citri reticulatae pericarpium, Citri reticulatae pericarpium viride, Aurantii fructus immaturus, and Aurantii fructus. These Chinese medicinal preparations are the peels or immature fruits of certain citrus species. Aiming at detecting naringenin in complex matrices such as pummelo and traditional Chinese medicines, we put forward a sensitive and practical indirect competitive enzyme-linked immunosorbent assay (icELISA) based on anti-naringenin monoclonal antibodies (anti-Nar-mAbs). The median inhibitory concentration (IC50) was 4.43 ng/mL, and the working range was 1.15–15.81 ng/mL. The findings of the icELISA for the analysis of naringenin in pummelo and herb samples had a good correlation with the ultra performance liquid chromatography (UPLC) methodology and showed good accuracy and reproducibility. These data demonstrated that the developed icELISA is reliable, accurate, and suitable for detecting naringenin in pummelo and traditional Chinese medicines.

Preparation and Characterization of Monoclonal Antibodies with High Affinity and Broad Class Specificity against Zearalenone and Its Major Metabolites.

This study aimed to detect and monitor total Zearalenone (ZEN) and its five homologs (ZENs) in cereals and feed. The monoclonal antibodies (mAbs) with a high affinity and broad class specificity against ZENs were prepared, and the conditions of a heterologous indirect competitive ELISA (icELISA) were preliminarily optimized based on the ZEN mAbs. The immunogen ZEN-BSA was synthesized using the oxime active ester method (OAE) and identified using infrared (IR) and ultraviolet (UV). The coating antigen ZEN-OVA was obtained via the 1,4-butanediol diglycidyl ether method (BDE). Balb/c mice were immunized using a high ZEN-BSA dose with long intervals and at multiple sites. A heterologous indirect non-competitive ELISA (inELISA) and an icELISA were used to screen the suitable cell fusion mice and positive hybridoma cell lines. The ZEN mAbs were prepared by inducing ascites in vivo. The standard curve was established, and the sensitivity and specificity of the ZEN mAbs were determined under the optimized icELISA conditions. ZEN-BSA was successfully synthesized at a conjugation ratio of 17.2:1 (ZEN: BSA). Three hybridoma cell lines, 2D7, 3C2, and 4A10, were filtered, and their mAbs corresponded to an IgG1 isotype with a κ light chain. The mAbs titers were between (2.56 to 5.12) × 102 in supernatants and (1.28 to 5.12) × 105 in the ascites. Besides, the 50% inhibitive concentration (IC50) values were from 18.65 to 31.92 μg/L in the supernatants and 18.12 to 31.46 μg/L in the ascites. The affinity constant (Ka) of all of the mAbs was between 4.15 × 109 and 6.54 × 109 L/mol. The IC50 values of mAb 2D7 for ZEN, α-ZEL, β-ZEL, α-ZAL, β-ZAL and ZAN were 17.23, 16.71, 18.27, 16.39, 20.36 and 15.01 μg/L, and their cross-reactivities (CRs, %) were 100%, 103.11%, 94.31%, 105.13%, 84.63%, and 114.79%, respectively, under the optimized icELISA conditions. The limit of detection (LOD) for ZEN was 0.64 μg/L, and its linear working range was between 1.03 and 288.55 μg/L. The mAbs preparation and the optimization of icELISA conditions promote the potential development of a rapid test ELISA kit, providing an alternative method for detecting ZEN and its homologs in cereals and feed.

Screening of anti-idiotypic domain antibody from phage library for development of Bt Cry1A simulants

Anti-idiotypic antibody technique is a new approach for the rapid development of insecticidal protein. In this study, anti-Cry1A polyclonal antibodies were used as antigen to screen the anti-idiotypic antibody that can simulate Cry1A toxins from a phage display human domain antibody (DAB) library. After four rounds of panning, five positive clones that have binding activities with anti-Cry1A polyclonal antibodies were obtained. Indirect competitive ELISA (IC-ELISA) results showed that the positive clone D6 showed significant inhibition for the binding of Cry1A toxins with anti-Cry1A polyclonal antibodies, and the inhibition ratio increased with the increase of D6 content. While, B3, F4, G5, C7 and the controls showed no obvious inhibition to Cry1A toxins. The results suggest that D6 is the “β” subtype anti-idiotypic antibody, which can simulate Cry1A toxins and competitive binding with anti-Cry1A polyclonal antibodies. Meanwhile, D6 had certain binding activity with the brush border membrane vesicles (BBMV) of p. xylostella, which was the receptor of Cry1A toxins. The results of bioassay showed that D6 had certain insecticidal activity, and the lethal concentration of 50% (LC50) was 976 ng/cm2. This study provides basic materials and experience for the development of Cry toxin simulants.

An enhanced open sandwich immunoassay by molecular evolution for noncompetitive detection of Alternaria mycotoxin tenuazonic acid

Open sandwich enzyme-linked immunosorbent assay (OS-ELISA), a novel noncompetitive immunoassay format, has shown great potential in rapid detection for small molecules compared with traditional competitive format. Here, an enhanced OS-ELISA towards the mycotoxin tenuazonic acid (TeA) was developed for the first time based on heavy chain variable region (VH) and light chain variable region (VL) from the hybridoma cells (3F10) producing anti-TeA monoclonal antibody (mAb). The established OS-ELISA exhibited a limit of detection of 0.08 ng/mL, and was 13 times more sensitive than mAb-based indirect competitive ELISA (ic-ELISA). The proposed assay was also applied to detect TeA contents in juice, flour and tomato ketchup samples with satisfactory recoveries of 87.6%–111.3%. Finally, the great accuracy of the established OS-ELISA method was validated by the standard ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS).

Development of a group-specific antibody-based immunoassay method for simultaneously detecting sildenafil-like adulterants in herbal spirit drinks

ABSTRACT Phosphodiesterase type 5 (PDE-5) inhibitors are commonly used to treat erectile dysfunction. There is a problem with synthesis and illegal use of a wide range of analogues of the licenced drugs and a simple class-wide analytical method is required. In this work, based on structural modelling, we developed an immunological method using norneovardenafil as a hapten as it contains only the general sub-structure and the common features of sildenafil-like adulterants, such as hydrophobic centres, hydrogen-bond donor atoms and hydrogen-bond acceptor atoms. Thus theoretically it could induce production of antibody which could recognise multiple sildenafil-like adulterants. By immunising rabbits, a group-specific polyclonal antibody was obtained with the desired broad-spectrum molecular recognition performance against sildenafil-like adulterants. Then, an indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed for the detection of sildenafil-like adulterants in herbal spirit drinks. Under the optimised conditions, the icELISA method showed broad linear ranges for acetildenafil, sildenafil and vardenafil respectively of 0.7 to 27.7 μg/kg, 1.0 to 70.7 μg/kg and 1.5 to 22.7 μg/kg, with half-maximal inhibition concentration (IC50) values of 4.5 μg/kg, 8.3 μg/kg and 5.7 μg/kg, respectively. For eleven herbal spirit drinks, there was good agreement between total levels of sildenafil-like adulterants measured by icELISA and levels of each of four individual adulterants determined by LC-MS/MS. In short, the developed icELISA can be employed for rapid and simple screening for adulteration of herbal spirit drinks with sildenafil-like compounds.

Development of a sensitive indirect competitive enzyme-linked immunosorbent assay for high-throughput detection and risk assessment of aflatoxin B1 in animal-derived medicines

Animal-derived medicine is an important part of traditional Chinese medicine (TCM). Studies have shown that many animal-derived medicinal products are susceptible to contaminate of aflatoxins, nevertheless, the rapid detection for animal-derived medicine is prone to be ignored. Here we developed a sensitive indirect competitive enzyme-linked immunosorbent assay (icELISA) for rapid screening of aflatoxin B1 (AFB1) in ground beetle, cockroach, silkworm and earthworm. The sensitivity of the icELISA method was significantly enhanced. The IC50 for the four animal-derived medicinal samples ranged from 0.092 to 0.135 ng mL−1; the limit of detection (LOD) was 0.008–0.020 ng mL−1. To obtain high accuracy, the extraction solution and time were evaluated. By using this method, a total of 138 samples were investigated, and the detection rates of AFB1 in ground beetle and earthworm samples were 26.6% and 16.7%, respectively. The result was validated by liquid chromatography combined with tandem mass spectrometry, and an excellent correlation was observed between the two datasets, with a R2 value of 0.999. Our results indicate that the proposed method can be used for the rapid detection of AFB1 in animal-derived medicine. Furthermore, the quantitative risk assessment was conducted for ground beetle and earthworm based on the results, demonstrating that the intake of AFB1 in ground beetle had a slight threat to the risk of cancer.

The Clinical Practice and Best Aerosol Delivery Location in Intubated and Mechanically Ventilated Patients: A Randomized Clinical Trial.

This randomized clinical trial (RCT) is aimed at exploring the best nebulizer position for aerosol delivery within the mechanical ventilation (MV) circuitry. This study enrolled 75 intubated and MV patients with respiratory failure and randomly divided them into three groups. The nebulizer position of patients in group A was between the tracheal tube and Y-piece. For group B, the nebulizer was placed at the inspiratory limb near the ventilator water cup (80 cm away from the Y-piece). For group C, the nebulizer was placed between the ventilator inlet and the heated humidifier. An indirect competitive enzyme-linked immunosorbent assay (ELISA) was used to measure salbutamol drug concentrations in serum and urine. The serum and urine salbutamol concentrations of the three groups were the highest in group B, followed by group C, and the lowest in group A. Serum and urine salbutamol concentrations significantly differed among the three groups (P < 0.05). It was found that the drug was statistically significant between group differences for groups B and A (P = 0.001; P = 0.002, respectively) for both serum and urine salbutamol concentrations. There were no significant differences observed among the other groups. It was found that the drug concentrations were the highest when the nebulizer was placed 80 cm away from the Y-piece, while the location between the tracheal tube and the Y-piece with the higher frequency of nebulizer placement was the location with the lowest drug concentration.

Quantum dot-labelled antibody based on fluorescence immunoassays for the determination of arsanilic acid in edible pork and liver

ABSTRACT Arsanilic acid (ASA) residue, which is the most common contaminant in edible animal tissues such as pork and liver, has caused environmental and food-safety concerns. In this study, direct and indirect competitive fluorescence-linked immunosorbent assays (dc-FLISA and ic-FLISA) incorporating quantum dots (QDs) as the fluorescent label were developed for the first time to detect ASA residues in edible pork and animal liver. Monoclonal antibodies against ASA and rabbit anti-mouse antibody were conjugated to orange QDs with excitation wavelengths at 450 nm, and the QD-Abs served as detection probes. The limits of detection for dc-FLISA and ic-FLISA were 0.11 ng/mL and 0.001 ng/mL, respectively. QD-FLISA was used to analyse spiked samples; recoveries ranged from 80.2%-91.2% in dc-FLISA and 82.5%-91.2% in ic-FLISA, and the coefficients of variations (CV) were less than 12%. Compared with conventional indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), the QD-FLISA described here was more sensitive and accurate in the analysis of ASA residues in animal tissues. Moreover, the results of QD-FLISA correlated well with HPLC. These results indicate that dc-FLISA and ic-FLISA are sensitive and reliable for detection of ASA residues in edible animal tissues.

Lateral flow immunoassay for the simultaneous detection of fipronil and its metabolites in food samples

We developed a sensitive and rapid lateral flow immunochromatographic (LFI) assay for the simultaneous detection of fipronil and its metabolites in eggs and cucumbers using gold nanoparticle (GNP)-labeled monoclonal antibodies (mAbs). Anti-fipronil mAbs (1B6) were produced using two haptens and identified by heterologous indirect competitive enzyme-linked immunosorbent assay (icELISA) with half maximal inhibitory concentration (IC50) and limit of detection (LOD) values of 0.46 ± 0.07 and 0.05 ± 0.01 ng mL−1, respectively. The developed LFI strip showed high sensitivity and specificity in the detection of fipronil with cut-off and visual limit of detection (vLOD) values of 10 and 0.25 ng mL−1, respectively. Furthermore, the application of LFI in the detection of fipronil-spiked egg and cucumber samples was validated by liquid chromatography tandem mass spectrometry (LC-MS/MS). Our developed LFI assay is suitable for detection of fipronil and its metabolites in real samples.

Mechanism of the Reduced IgG/IgE Binding Abilities of Glycated β-Lactoglobulin and Its Digests through High-Resolution Mass Spectrometry.

Glycation between proteins and reducing sugars is the common chemical modification in food protein, and many studies have focused on the allergenicity of the glycated protein. However, a systemic study on the allergenicity change of its digests is lacking. In this work, we explored the change rule of the digestibility and allergenicity of glycated β-Lg during in vitro gastrointestinal digestion and interpreted the mechanism using high-resolution mass spectrometry. Glycation with arabinose increased the resistance of β-Lg to digestive enzyme, with a low hydrolysis value. Indirect competitive ELISA showed that the IgG/IgE binding rates of β-Lg were reduced after glycation and further reduced after digestion, in comparison with the digests of unglycated β-Lg. There are two reasons for this phenomenon. On the one hand, 11 glycated sites were determined in the lowest allergenicity arabinose-β-Lg conjugation (Ara-β-Lg), which was distributed in the IgG and IgE linear allergic epitopes of β-Lg. On the other hand, glycation masking linear allergenic epitopes had a more significant effect on reducing allergenicity in comparison to digestive enzyme hydrolysis. These results indicated that the allergenicity of Ara-β-Lg in the human body might be lower than that of unglycated β-Lg.

English

BACKGROUND Ochratoxins include a family of toxic secondary metabolites produced by several species of fungi of the genus aspergillus and penicillium. Mycotoxins1 are important environmental pollutants that can be generated in a variety of food and feed components. Nowadays, most mycotoxins can be identified at an acceptable level in terms of legal, veterinary and medical needs. We wanted to compare the egg yolk total antioxidant capacity relativities to mycotoxins ochratoxins (OTA) to determine surges in side effects of human consumption. METHODS Samples were tested by the indirect competitive enzyme-linked immunosorbent assay (ELISA) test kits according to the kit’s instructions. Finally, the collected data was analysed using SPSS software and descriptive statistics (contamination ratio, mean and standard deviation) and one-way analysis of variance. RESULTS Ochratoxin in all egg yolk samples of the studied 34 brands showed a minimum of 23.17 ppb and a maximum of 252.80 ppb. OTA residues indicate high risk levels to consumers. CONCLUSIONS According to the results, the transfer of mycotoxin metabolites to eggs has been in greater possibilities; therefore, control of mycotoxin contamination in laying hen diets and surveys in food and feed in intense risks is recommended to avoid the presence of mycotoxins in eggs intended for human consumption. KEY WORDS Mycotoxin, Total Antioxidant Capacity (TAC), OTA, Egg Yolk

Heterologous immunoassay strategy for enhancing detection sensitivity of banned dye rhodamine B in fraudulent food

BackgroundThe high quality of antibody (Ab) is critical for an immunoassay; usually, an Ab with low affinity is often regarded as a “bad” one in the immunoassay development. How to use a “bad” Ab to develop a highly sensitive immunoassay is still a huge challenge.MethodsIn this study, a heterologous immunoassay strategy was designed to enhance the sensitivity for the detection of banned dye, rhodamine B (RB), in fraudulent food. The RB Ab could not recognize RB by pairing with homologous coating antigen (Ag). However, the Ab showed unexpected high specificity and sensitivity recognition after being replaced by heterologous coating Ag. Indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed based on the heterologous strategy.ResultsThe detection limit of icELISA for chilli powder, Chinese prickly ash, hot-pot seasoning, and chilli sauce was 0.002 μg/kg, and the recoveries of the four samples ranged from 76.0 to 102.0%, with the coefficient of variation between 3.9 and 18.8%. Parallel experiment for 20 market samples with high-performance liquid chromatography (HPLC) was performed on to confirm the performance of the practical application of the developed icELISA, and the results of the two methods had good correlation. Molecular modeling inferred that the carboxyl group of hapten and its exposure level played an important role in the hapten-Ab recognition.ConclusionsThe proposed icELISA can be used for the surveillance screening of RB in a range of seasoning foods, and the heterologous strategy is an effective approach to enhance the sensitivity in an immunoassay.