Indirect Competitive ELISA Method Research Articles

Preparation of Anti-Zearalenone IgY and Development of an Indirect Competitive ELISA Method for the Measurement of Zearalenone in Post-Fermented Tea.

Post-fermented tea (PFT) is one of the most commonly consumed beverages worldwide. Rapid microbial growth and significant changes in the microbial composition of PFT during processing and storage pose a potential risk of contamination with mycotoxins such as zearalenone (ZEN). Screening for ZEN contamination in a simple, rapid, and inexpensive manner is required to ensure that PFT is safe for consumption. To monitor ZEN in PFT, ZEN was conjugated with bovine serum albumin to prepare egg yolk immunoglobulins (IgY). A specific indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on IgY was developed and validated. ZEN was extracted with acetonitrile and water (50:50, v/v) containing 5% acetic acid and purified using a mixture of primary and secondary amines and graphitized carbon black to remove matrix interference from the PFT samples. Under optimal conditions, the linear range of this assay was 13.8-508.9 ng mL-1, the limit of detection was 9.3 ng mL-1, and the half-maximal inhibitory concentration was 83.8 ng mL-1. Cross-reactivity was negligible, and the assay was specific for ZEN-related molecules. The recovery rate of ZEN in the control blanks of PFT samples spiked with a defined concentration of ZEN of 89.5% to 98.0%. The recovery and accuracy of the method were qualified for PFT matrices. No significant differences were evident between the results of the actual PFT samples analyzed by high-performance liquid chromatography and ic-ELISA. The collective data indicate that the developed ic-ELISA can be used for the rapid and simple detection of ZEN in PFT products.

Structural Property, Immunoreactivity and Gastric Digestion Characteristics of Glycated Parvalbumin from Mandarin Fish (Siniperca chuaisi) during Microwave-Assisted Maillard Reaction.

This study was aimed to investigate the structural and immunological properties of parvalbumin from mandarin fish during the Maillard reaction. The microwave-assisted the Maillard reaction was optimized by orthogonal designed experiments. The results showed that the type of sugar and heating time had a significant effect on the Maillard reaction (p < 0.05). The SDS-PAGE analysis displayed that the molecular weight of parvalbumin in mandarin fish changed after being glycated with the Maillard reaction. The glycated parvalbumin was analyzed by Nano-LC-MS/MS and eleven glycation sites as well as five glycation groups were identified. By using the indirect competitive ELISA method, it was found that microwave heating gave a higher desensitization ability of mandarin fish parvalbumin than induction cooker did. In vitro gastric digestion experiments showed that microwave-heated parvalbumin was proved to be digested more easily than that cooked by induction cookers. The microwave-assisted Maillard reaction modified the structure of parvalbumin and reduced the immunoreactivity of parvalbumin of mandarin fish.

Development of an indirect competitive ELISA method based on ORF2 detecting the antibodies of novel goose astrovirus

Goose astrovirus (GAstV) characterized by articular and visceral gout, is an emerging pathogen with a wide distribution on mainland China, leading to serious economic losses in the goose-raising industry. Because vaccines to prevent GAstV infections are not available currently, early diagnosis is critical when treating symptomatic geese and in preventing GAstV transmission. In this context, a highly sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on a monoclonal antibody (mAb) to detect GAstV-specific antibodies from geese was developed, and the detections were optimized. A series of experiments proved that the ic-ELISA shows excellent diagnostic performance and discriminatory power with high sensitivity and specificity. The ic-ELISA for GAstV detection was applied on 67 field serum samples, and comparing the detection results with the virus neutralization test verified the accuracy of the ic-ELISA. The correlation coefficient between the ic-ELISA and the virus neutralization test was 80%, demonstrating the proposed ic-ELISA method could be a useful and effective tool for the diagnostic, serological epidemiological investigation and immune monitoring of the GAstV in goose-producing regions.

Histamine detection in fish samples based on indirect competitive ELISA method using iron-cobalt co-doped carbon dots labeled histamine antibody

Due to complex matrixes and specific reagent deficiency, the rapid detection of histamine is still a challenge to date. Based on the high peroxidase-like activity of iron-cobalt co-doped carbon dots, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was established for histamine detection using the mimic enzyme labeled with histamine antibody (His-Ab). Through the competitive binding of the labeled His-Ab to solid-phase and sample antigens, histamine content was detected with a linear range of 2.5–150 μg mL−1. The detection limit based on 3σ/K was 0.50 mg kg−1, which was much lower than those of commercial His-kit and HPLC methods. The ic-ELISA method was applied to histamine detection in fish samples with the recovery of (103.4 ± 0.5)%, which was in accord with those of commercial His-kit and HPLC methods. The results indicated that the established ic-ELISA method was suitable for rapid detection of histamine in fish samples with high accuracy, sensitivity and stability.

A novel peptide shows excellent anti-HIV-1 potency as a gp41 fusion inhibitor

Fusion inhibitors of HIV prevent the virus from entering into the target cell via the interaction with gp41, which stops the process of spatial rearrangement of the viral envelope protein. A series of peptides have been designed and screened to obtain a highly potent novel sequence. Among them, CT105 possesses the most potent anti-viral ability at low nanomolar IC50 values against a panel of HIV-1 pseudoviruses from A, B, C and A1/D subtypes, whereas T20 shows much weaker potency. CT105 also shows excellent inhibitory activity at 260 pico molar IC50 against HIV-1 replication. As a fusion inhibitor, CT105 has a strong ability to interrupt gp41 core formation. The terminal half-life of CT105 possesses 1.72-fold longer than that of T20 as determined by developing an indirect competitive ELISA method. The results suggest that this artificial peptide CT105 could be a favorable architype for further optimization and modification.

Changes in abscisic acid levels in embryonic axis of Saraca asoca seeds during maturation and artificial dehydration

Abscisic acid (ABA) plays an important role in many cellular processes, including seed development, dormancy, germination and stress responses. Investigation on changes in endogenous ABA content of Saraca asoca seeds during development and artificial dehydration by indirect competitive ELISA method explains their influence on viability. The low ABA levels towards later stages of seed development validates non-orthodox nature of species. About 37% germination was observed in immature seeds of 60 days after anthesis onwards, which possess comparatively higher ABA content of 0.92 nmol g−1 fr. wt. In the course of seed maturation gradual loss of ABA was noticed with concurrent increase in germination. Artificial seed drying in open laboratory condition for five different periods, viz., 48, 96, 144, 192 and 240 h registered 49.7, 48.4, 46.9, 45.2 and 42.8% moisture contents, respectively, in fresh and mature seeds as control which possess moisture content of 51.6 with 100% germination. Dehydration of seeds results the significant increase of abscisic acid level in embryonic axis but the viability was declined simultaneously. So the present study deals with the influence of abscisic acid in desiccation tolerance during maturation and dehydration of recalcitrant seeds of Saraca asoca.

Investigation of diethylstilbestrol residue level in human urine samples by a specific monoclonal antibody.

Diethylstilbestrol (DES) is used as a kind of animal feed additive and affects people's health through the food chain. The purpose of this study is to detect the residue level of DES in 576 human urine samples directly. DES-BSA was used to immunize Balb/c mice. The monoclonal antibody was produced by hybridoma that was screened through cell fusion techniques. Finally, we developed the indirect competitive ELISA method to analyze 576 human urine samples from Zhejiang Province, China. The IC50 of this method was 3.33ng/mL. The LOD and LOQ were 0.16 and 0.54ng/mL. Linear range of the standard curve was from LOD to 12.50ng/mL. There was no cross-reactivity with two kinds of estrogens and two structural analogs with DES. Five hundred seventy-six urine samples were analyzed by the indirect competitive ELISA method, and the detection rate was 98.78%. The mean concentration and geometric mean were 4.70 and 3.50ng/mL. The indirect competitive ELISA method based on monoclonal antibody was sensitive and reliable for the detection of DES in human urine samples. The results warned us to pay more attention to human health and food safety.

Enhanced Mulberroside A Production from Cell Suspension and Root Cultures of Morus alba Using Elicitation

Morus alba L. has been used in Asian traditional medicine as an anti-inflammatory, anti-asthmatic, anthelmintic and as a whitening agent in cosmetic products. Mulberroside A is the major active compound from M. alba root bark. In this study, cell suspension and root cultures of M. alba were established, and the effect of the elicitors on the enhancement of mulberroside A production in M. alba was investigated. The cell suspension and root cultures of M. alba were exposed to elicitors and then mulberroside A contents were determined by an indirect competitive ELISA method. High levels of mulberroside A were obtained by addition of 100 and 200 μM salicylic acid with 24 h exposure time in cell suspension cultures (37.9 ± 1.5 and 34.0 ± 4.7 mg/g dry wt., respectively). Furthermore, addition of yeast extract at 2 mg/mL with 24 h exposure time can significantly increase mulberroside A contents from both cell suspension (3.2-fold) and root cultures (6.6-fold). Mulberroside A contents from both cell suspension and root cultures after treatment with elicitors are similar or higher than those found in the intact root and root bark of several years old M. alba. These results indicate that mulberry tissue cultures using the elicitation method are interesting alternative sources for mulberroside A production.

Erratum for: A Sensitive and Specific Indirect Competitive Enzyme-Linked Immunosorbent Assay for Detection of Paeoniflorin and Its Application in Pharmacokinetic Interactions between Paeoniflorin and Glycyrrhizinic Acid.

This study aimed to develop an indirect competitive enzyme-linked immunosorbent assay based on monoclonal antibodies against paeoniflorin to study the effects of different doses of glycyrrhizinic acid on the pharmacokinetics of paeoniflorin in mice. An anti-paeoniflorin monoclonal antibody was produced from a hybridoma created through a fusion of splenocytes immunized with paeoniflorin-bovine serum albumin and conjugated with the hypoxanthine-aminopterin-thymidine-sensitive mouse myeloma cell line SP2/0. The resultant antibody was used to develop and validate a rapid, specific and sensitive, indirect competitive enzyme-linked immunosorbent assay for the measurement of paeoniflorin (linear range 4.8–312.5 ng · mL−1). The intraday and interday precision values of the indirect competitive ELISA method were well within the recommended range (≤ 10 %), and the recovery rate was, on average, 101.13 %. Pharmacokinetic parameters obtained from mouse blood samples at various intervals following the oral administration of paeoniflorin and glycyrrhizic acid at three doses (1 : 0.3, 1 : 1, 1 : 3, respectively) demonstrated that the highest dose of glycyrrhizic acid inhibits the absorption of paeoniflorin.

A Sensitive and Specific Indirect Competitive Enzyme-Linked Immunosorbent Assay for Detection of Paeoniflorin and Its Application in Pharmacokinetic Interactions between Paeoniflorin and Glycyrrhizinic Acid.

This study aimed to develop an indirect competitive enzyme-linked immunosorbent assay based on monoclonal antibodies against paeoniflorin to study the effects of different doses of glycyrrhizinic acid on the pharmacokinetics of paeoniflorin in mice. An anti-paeoniflorin monoclonal antibody was produced from a hybridoma created through a fusion of splenocytes immunized with paeoniflorin-bovine serum albumin and conjugated with the hypoxanthine-aminopterin-thymidine-sensitive mouse myeloma cell line SP2/0. The resultant antibody was used to develop and validate a rapid, specific and sensitive, indirect competitive enzyme-linked immunosorbent assay for the measurement of paeoniflorin (linear range 4.8-312.5 ng · mL(-1)). The intraday and interday precision values of the indirect competitive ELISA method were well within the recommended range (≤ 10 %), and the recovery rate was, on average, 101.13 %. Pharmacokinetic parameters obtained from mouse blood samples at various intervals following the oral administration of paeoniflorin and glycyrrhizic acid at three doses (1 : 0.3, 1 : 1, 1 : 3, respectively) demonstrated that the highest dose of glycyrrhizic acid inhibits the absorption of paeoniflorin.

Human health risk assessment related to blue-green algae mass development in the Kuibyshev Reservoir

Health risk assessment related to water quality and microcystin contamination of water in the Kuibyshev Reservoir was conducted in August 2012 during the period of algal bloom. The health risk during recreational activity was estimated for potential exposure to extracellular microcystins. Microcystin content in water measured by the indirect competitive ELISA method was in the range from 0.21 to 26.96 μg L−1. The results of the present study show that the health risk can reach a dangerous level even at 1 h of exposure due to the high concentration of extracellular microcystins in the water.

Contamination level of aflatoxin B1 in lotus seeds rapid screening by indirect competitive ELISA method

A simple and cost-effective indirect competitive enzyme-linked immune sorbent assay (ic-ELISA) was developed to rapidly screen the content of aflatoxin B1 (AFB1) in lotus seeds, and the results were confirmed by ultra-fast liquid chromatography-tandem mass spectrometry( UFLC-MS/MS). Matrix-matched calibration expressed a good linearity ranging from 0. 171 to 7. 25 µg · L(-1) for AFB, with R2 > 0.978. The medium inhibitory concentration( IC50 ) for AFB1 was 1.29 µg · L(-1), the recovery for AFB1 was 74.73% to 126.9% with RSD < 5%, and the limit of detection (IC10) was 0.128 µg · L(-1). The developed ic-ELSIA method was applied to rapid analysis of AFB, in 20 lotus seeds samples and the results indicated that the contents of AFB, in samples 1-15 were in the range of 1. 19- 115. 3 µg · kg(-1) and in 40% of the samples exceeded the legal limit(5 µg · kg(-1)), while the contamination rate of AFB, in samples 16-20 was 40%. Pearson correlation coefficient(r) reached 0.997 for AFB1 content in the samples detected by ic-ELSIA and UFLC-MS/MS methods. The results proved that the developed ic-ELISA method is simple, sensitive and reliable, and can be used for rapid and high-throughput screening of AFB1 in lotus seeds

ELISA for Detection of Soya Proteins in Meat Products

Indirect competitive ELISA method for the detection of soya proteins in meat products was developed. The detection limit of the method is 0.5% of the weight of added soya protein. A total of 131 meat product samples such as salamis or sausages from the Czech Republic market were investigated for the presence of soya proteins. Soya proteins were detected in 84% of the investigated samples without any declaration on the package of the product. The use of vegetable additives, namely soya in meat products in the market of the Czech Republic is very frequent and the restriction of its usage by legislation relates only to some kinds of durable products and ham (Act 264/2003 Coll.). The need for sensitive inspecting methods for soya protein detection is not only associated with the economic aspect (adulteration), but mainly with consumer health protection in case of allergy to soya proteins.

Immunochemical characterization of alkaline-soluble polysaccharide, P-1, from the kernels of Prunus mume Sieb. et Zucc.

Polyclonal antibodies against P-1, a pectic polysaccharide fraction extracted with 0.5 M NaOH from the kernels of Prunus mume and consisted of arabino-galacturonan, and I-3, the partial acid (0.1 M trifluoroacetic acid) hydrolysate of P-1, were prepared in Japanese white rabbits. Competitive ELISA experiments strongly suggested that anti P-1 and anti I-3 antibodies were different but P-1 and I-3 cross-reacted with each other to recognize a partly similar epitope structure. The reactivities of polysaccharide fractions from the raw flesh of P. mume, and the kernels of apricot and peach extracted with either water or sodium hydroxide were examined using both antisera by the indirect competitive ELISA method. The polysaccharide fractions extracted with sodium hydroxide solutions had the reactivities but not those extracted with cold and hot water. These facts suggested that the similar structure of polysaccharides to P-1 was present in the flesh of P. mume and the kernels of apricot and peach. However, neither pectin of apple nor citrus had reactivity with each antiserum. P-1 would be different in chemical structure from a commercially available pectin, a water-soluble polysaccharide from apple and citrus.