Competitive Enzyme-linked Immunosorbent Assay Method Research Articles

Unveiling connections: bisphenol A and vitamin D dynamics in breast milk among healthy lactating mothers

ABSTRACT Bisphenol A (BPA) has negative impact on human health. This study aims to explore the association between the levels of BPA and 25(OH) vitamin D in breastmilk. BPA exposure and vitamin D sources were recorded on the study form. Breastmilk 25(OH) vitamin D was measured by the competitive enzyme-linked immunosorbent assay method, and BPA was analyzed by fluorescence detector high pressure liquid chromatography technique. Multiple logistic regression analysis were performed for association. Among 70 lactating mothers, the median 25(OH) vitamin D level was 12.6 ng/mL. In 92.9% of breastmilk samples, detectable levels of BPA were found, with a median BPA concentration of 2.56 ng/mL. However, both univariate and multivariate analyses failed to reveal any association between breastmilk BPA and 25(OH) vitamin D levels. This lack of association in healthy lactating mothers, highlighting the need for further research to comprehensively understand the intricate dynamics of these factors.

Sandwich Enzyme-linked Immunosorbent Assay (ELISA) to Quantify Monoclonal Antibody (B[a]P-13) for Herbal Medicine Products

Abstract: Sandwich enzyme-linked immunosorbent assay (ELISA) to quantify monoclonal antibody (B[a]P-13) Background: Only a few studies have focused on the analysis using specific antibodies in the sandwich ELISA method to each B[a]P in herbal medicine products. In contrast to the sandwich ELISA method, many competitive ELISA methods using specific antibodies such as benzo[a]pyrene monoclonal antibody (B[a]P-13) and a goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, horseradish peroxidase (HRP) were developed. Introduction: The objective of this study was to develop and validate the method for the response of the benzo[a]pyrene monoclonal antibody (B[a]P-13) and goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody (HRP) to prepare the immunogen and its application to detect the benzo[a]pyrene in various herbal medicine products. Methods: This research method includes preparation of B[a]P-protein conjugates, sampling and extraction procedure for herbal medicines, sandwich ELISA procedure, evaluation of cross-reactivity for determination, matrix effect of the organic solvents, correlation of benzo[a]pyrene detection ELISA compared to HPLC-FLD in herbal medicine products. Results: The sandwich ELISA method for B[a]P was validated in linearity (R2 > 0.99), the limit of detection (LOD) (0.080.19 μg/kg) and limit of quantification (LOQ) (0.240.57 μg/kg), accuracy (95.58117.06 %), and precision (3.8010.26 %). The cross-reactivity (CR) was found for B[a]P (100%), CHR (39%), B[b]F (27%), and B[a]A (41%). As a solvent, acetonitrile (MeCN) was used to express the normalized sandwich ELISA calibration curves with benzo[a]pyrene monoclonal antibody (B[a]P-13). The antigen-antibody binding in sandwich ELISA was decreased about 10 times with increasing the salt content (0.0060.18 mol/L phosphate to 20400 mmol/L). The pH range from 6 to 9 was not considered to affect the performance of the sandwich ELISA. Correlation of B[a]P detection in herbal medicines with ELISA compared to HPLC-FLD expressed good correlation (R2 = 0.991) and the slope of the graph for the ELISA (B[a]P-equivalents μg/kg) value divided by the HPLC-FLD (B[a]P μg/kg) value was 0.7292. Conclusion: Therefore, sandwich ELISA method using benzo[a]pyrene monoclonal antibody (B[a]P-13) could be an alternative screening method for detection of B[a]P in herbal medicine products.

Improved sensitivity of the anti-microcystin-LR ELISA using phage-displayed alpha-type anti-idiotypic nanobody

Anti-idiotypic antibodies (Ab2) are valuable tools that can be used for a better understanding of molecular mimicry and the immunological network. In this work, we showed a new application of a phage-displayed alpha-type Ab2 (Ab2α) to improve the sensitivity of an enzyme-linked immunosorbent assay (ELISA) detecting cyanobacterial toxin microcystin-LR (MC-LR). A monoclonal antibody (mAb) against MC-LR was used as an antigen to isolate binders in a camelid nanobody library. After three rounds of panning, three unique clones with strong binding against anti-MC-LR mAbs were isolated. These clones could specifically bind to anti-MC-LR mAbs without influencing mAbs binding with MC-LR, meaning these clones were Ab2αs. Based on the signal amplification effect of phage coat proteins and the non-competitive nature of Ab2α, a novel competitive ELISA method for MC-LR was established with a phage-displayed Ab2α. It showed that the phage-displayed Ab2α greatly enhanced the ELISA signal and sensitivity of the method was improved 3.5-fold to the conventional one. Combining with the optimization of pre-incubation time, the optimized ELISA decreased its limit of detection (LOD) from 4.5 ng/mL to 0.8 ng/mL (5.6-fold improvement). This new application of Ab2α may potentially be employed to improve the sensitivity of immunoassays for other environmental pollutants.

The Effect of Moringa Leaf Extract on Malondialdehyde Levels in Male Wistar Rats

Excessive physical activity will result in body fatigue. This causes oxidative stress that occurs due to an imbalance between oxidants and antioxidants which can then cause damage characterized by MDA. Moringa leaf extract is an antioxidant from outside the body that is useful in reducing fatigue. This study aimed to decrease MDA levels in male Wistar rats by Moringa leaf extract. We gave swimming exercises to tire the mice. It will increase the levels of MDA in their blood. Fatigue can be characterized by a variety of biological signs. The difference in our research lies in the biological marker used as an indicator of fatigue, namely MDA levels. The sample used in this study consisted of 4 groups to be given treatment, with each group consisting of 5 rats. MDA levels were tested using the competitive ELISA (enzyme-linked immunosorbent assay) method. One-way ANOVA test was used to find out where the difference was, with 95% correct confidence for this test, and if p < 0.05, a significant difference was obtained. The lowest mean MDA levels were in the treatment of Moringa extract at 300 mg/kg in the pre-test, the first week, and the second week, 1.82 ± 0.20, 1.24 ± 0.03, and 0.86 ± 0.12, respectively. The results of the one-way ANOVA test in this study showed differences in each treatment group with p= 0.000. The results of the examination of MDA levels at each treatment time showed a difference between before the treatment, the first week, and the second week.

Development of indirect competitive ELISA for determination of dehydroabietic acid in duck skin and comparison with the HPLC method

Defeathering with rosin results in rosin residue in duck skin, which may present as potential risk to human health. Dehydroabietic acid (DHAA) is a major component of rosin. An indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed for determination of DHAA in duck skin. A set of parameters was optimized, including coating antigen concentration, dilution of antiserum, dilution of HRP-IgG antibody, incubation time, and temperature for antigen reaction with antiserum. The indirect competitive ELISA yielded an excellent specificity against DHAA with low cross-reactivity toward other resin acids. The limit of detection and the working concentration range of DHAA in duck skin were 16.4 ng/g and from 40 to 8,060 ng/g, respectively. The indirect competitive ELISA was applied to the determination of DHAA in duck skin samples spiked with DHAA at different contents, and recoveries were found between 78.2 and 97.2%. Finally, DHAA contents in 32 duck samples were quantified by the indirect competitive ELISA and high performance liquid chromatography-fluorescence detector (HPLC-FLD) method. No significant difference was found between DHAA concentrations from indirect competitive ELISA and HPLC-FLD method for all samples, which indicated the indirect competitive ELISA established in this article was of the same accuracy as the HPLC-FLD method. The indirect competitive ELISA was simple, rapid, and reliable, which could be used to identify the duck carcasses defeathered with rosin in the market.

Determination of the Mycelium and Antigens of a Number of Micromycetes in Soil Extracts via Enzyme-Linked Immunosorbent Assay

Sixteen variants of test systems (two-site sandwich and competitive enzyme-linked immunosorbent assay (ELISA)) were studied to determine the concentration of microfungi, some of which are known opportunistic pathogens for humans, in soil extracts. The correlation of the amount of fungal biomass (the length of the growing mycelium) with the concentrations of antigens detected by the two-site sandwich ELISA method and the competitive ELISA method was evaluated. It has been shown that the methods are highly effective in the case of long incubation (more than 5 days) of soil samples and are ineffective for short-term incubation, because there was no correlation between hypha length and antigen concentration during the first day of cultivation. The perspectivity of ELISA methods for the detection of some soil microfungi from genera Aspergillus (Asp. flavus, Asp. fumigatus, Asp. niger), Fusarium (F. solani, F. poae), and others (Alternaria alternata, Phoma lingham, Mucor hiemalis) was shown. The competitive ELISA method was more efficient than two-site sandwich ELISA.

Development and validation of a monoclonal antibody-based competitive ELISA for detection of antibodies against porcine epidemic diarrhoea virus (PEDV)

Porcine epidemic diarrhoea virus (PEDV), belongs to the genus Alphacoronavirus in the family Coronaviridae and causes severe diarrhoea, vomiting, dehydration and high mortality in seronegative newborn piglets. Thus, a precise and rapid diagnosis of PEDV infection is important for the application of control measures to limit viral dissemination. In this investigation, a monoclonal antibodies (MAbs)-based competitive enzyme-linked immunosorbent assay (ELISA) for detecting antibodies against PEDV was developed and validated. The diagnostic performance of the test was evaluated by receiver operating characteristic (ROC) analysis using a panel of 829 known sera collected from different commercial pig farms, with or without a history of PED presence and from experimentally infected pigs. The competitive ELISA showed excellent diagnostic performance and discriminatory power with high sensitivity (Se) and specificity (Sp) values (Se = 96.5%, 95% IC 94.1–98.1; Sp = 98.2%, 95% IC 96.3–99.3). Moreover, this competitive ELISA method has other properties, such as high feasibility of testing sera without pre-treatment and automatic and instrument-mediated revealing, that makes it appropriate for large-scale screenings of affected pig farms in endemic regions or for monitoring plans in PEDV-free areas.

Biomimetic ELISA detection of malachite green based on molecularly imprinted polymer film.

A highly selective and sensitive enzyme-linked immunosorbent assay (ELISA) was developed for the detection of malachite green (MG) using a molecularly imprinted polymer (MIP) film as bionic antibody. The MIP film, based on the self-polymerization of dopamine, was fabricated on the surfaces of a 96-well microplate. It showed specific recognition for MG in aqueous solution. A direct competitive ELISA method was established with the sensitivity reaching 10.31μgL-1 and the detection limit being 0.3μgL-1. The cross-reactivity of two structural analogues to MG was less than 10%. The average recovery tested by MG standard spiking was 88.8% for bass and 90.4% for water, and the relative standard deviations were less than 3.6%. All the above results indicated that the developed method could be used to detect MG in fish and water samples rapidly, specifically and accurately.

Evaluation of oxidative stress marker in patients with human papillomavirus infection

Introduction and Objective: Human papillomavirus (HPV) unenveloped viruses with double-stranded circular DNA cause infection in skin epithelial cells and mucosal surfaces. HPV infection has been detected in more than 70% of cervical cancers throughout the world. Among the effective factors in the progression of HPV infection are oxidative stress and oxidative markers, and the oxidative stress during viral infection can be due to the host immune responses to viral proteins or as a result of viral genes expression. This study aims to compare the mean serum level of oxidative malondialdehyde (MDA) marker and the mean of 8-Hydroxyguanosine (8- OhdG) in two groups of healthy people and patients with HPV infection. Methods: This is a descriptive, cross-sectional study in which 30 patients with HPV infection and 35 healthy people (as the control group) were selected using convenience sampling method. After obtaining written consent from the subjects, the researchers took fasting blood samples and morning urine samples from them. Serum MDA was measured using the spectrophotometric method, and 8-OHdG was measured using the competitive enzyme-linked immunosorbent assay method. Finally, the data were analyzed using SPSS version 17. Results: The results of this study showed that the mean and standard deviation of serum MDA concentration have increased significantly in patients with HPVs infection (4.56 ± 1.64 nM) than in the control group (1.64 ± 0.37 nM). The mean and standard deviation of urinary concentration (8-OHDG) in patients with HPV infection was 14.61 ± 1.39 ng/ml while it was 9.66 ± 1.74 ng/ml in the control group. It was significantly higher in people who had high-risk HPV infection than in the group with low-risk HPV (P

Occurrence of aflatoxins in wheat flour specified for sangak bread and its reduction through fermentation and baking

This study was conducted as a screening survey to determine the occurrence of total aflatoxins (TAF) levels in sangak flour samples provided from flour factories and bakeries and to assess the effect of commercial fermentation and baking practices on the most contaminated flour samples. In addition, the effect of fermentation by yeast (Saccharomyces cerevisiae) and a mixture of yeast and a lactobacillus strain (Lactobacillus plantarum LA7) in reducing aflatoxins in prepared sangak breads were compared. The competitive enzyme-linked immunosorbent assay method was employed to determine toxin levels in the samples. With contamination levels ranging from 6.68 to 34.47 ng/g, the majority of the tested samples were not considered safe. Mean aflatoxin contamination of examined samples was 18.06 ng/g. Considering the limits for TAF in wheat flour set by ISIRI and EC (15 ng/g), 56.7% of the samples were unacceptable. No significant correlation was found between ash content and TAF levels (P>0.05). A significant de...

Development of A Monoclonal Antibody‐Based Competitive Elisa Method for Quantitative Detection of Dextran

AbstractTo solve processing problems caused by bacterial activity in sugar industry, a sensitive method for dextran detection is expected. In this study, monoclonal antibodies (mAbs) against dextran were prepared using dextran T40 conjugated with bovine serum albumin as immunogens. Basing on the mAbs, a competitive enzyme‐linked immunosorbent assay (ELISA) method to measure dextran quantitatively was developed. Results demonstrated that this competitive ELISA method had a calibration curve with correlation coefficient of 0.9986, IC50 value of 204.2 ng/mL and detection value ranging from 26.3 ng/mL to 1174.9 ng/mL. In addition, cross‐reaction assay suggested the method had no significant cross‐reaction with dextran‐related substances such as β‐glucan, starch, sucrose, sugar and glucose. Besides, spike recovery assay indicated this quantitative method for dextran detection had a perfect accuracy.Practical ApplicationsWith wide detection range, high specificity and good calibration curve, this developed competitive ELISA method will be reliable to detect dextran quantitatively in sugar industry, and even may be commercially applied into kit development.

Biomimetic Enzyme-linked Immunosorbent Assay Using a Hydrophilic Molecularly Imprinted Membrane for Recognition and Fast Determination of Trichlorfon and Acephate Residues in Vegetables

This study describes the development of a new direct competitive enzyme-linked immunosorbent assay method with a hydrophilic imprinted film as biomimetic antibody for determining multi-pesticide residues. The imprinted film, which can selectively recognize trichlorfon and acephate, was synthesized directly on the surface of a 96-well plate using 4-(dimethoxyphosphorothioylamino) butanoic acid as the template molecule. This film exhibited antibody-like recognition ability, fast adsorption–desorption dynamics and good stability. Under optimal conditions, the sensitivity (IC50) of the biomimetic enzyme-linked immunosorbent assay (BELISA) method was 12.0 mg/L for trichlorfon and 30.0 mg/L for acephate. The limit of detection (LOD, IC15) was 8.0 μg/L for trichlorfon and 12.0 μg/L for acephate. The developed method was applied to the determination of the trichlorfon and acephate in the spiked asparagus and cucumber samples with recoveries ranging from 72.1 to 92.0 % for trichlorfon and 70.0 to 85.0 % for acephate.

Occurrence of tetracyclines, sulfonamides, fluoroquinolones and florfenicol in farmed rainbow trout in Iran

This study was carried out to determine the occurrence of tetracyclines, sulfonamides, fluoroquinolones and florfenicol residues in rainbow trout muscle samples (n = 138) obtained from Iranian trout farms. Concentrations of the antibiotics were determined using competitive enzyme-linked immunosorbent assay method. At measurable levels, 63.1%, 16.7%, 19.6% and 40.6% of the samples contained the residues of tetracyclines, sulfonamides, fluoroquinolones and florfenicol, respectively. The detected range of concentrations for positive samples was 1.43–101.4 µg/kg for tetracyclines, 4.03–90.4 µg/kg for sulfonamides, 6.75–99.8 µg/kg for fluoroquinolones and 0.11–172.6 µg/kg for florfenicol. The residues of the antibiotics in trout samples did not exceed the maximum residue levels recommended by the Institute of Standards and Industrial Research of Iran. However, the high occurrence of tetracyclines and florfenicol in the samples is alarming and could be a potential hazard for public health. Further investigations should be performed to determine the residues of antibiotics in other farmed fish species in Iran.

Development of an enzyme linked immunosorbent assay for fast screening of cimaterol residues in various matrices

IntroductionInterest in methods to improve the efficiency of animal production has focused on betaadregenic agonists such as cimaterol in recent years. Cimaterol has been recognised as a repartitioning agent that improves carcass composition by increasing muscle deposition and reducing fat. It also has the ability to accelerate livestock growth. In human however, they are generally used as anti-asthmatic agent. Therefore, excess cimaterol residues in livestock products may pose a genuine risk for human health. This has led to the prohibition of cimaterol usage in every stage of food production. To know whether food matrices contain cimaterol residue, one must need to test it. ObjectiveTo develop a sensitive and specific Enzyme-linked Immunosorbent Assay (ELISA) for the detection of cimaterol in various matrices. MethodsThe antiserum was raised in rabbits, employing cimaterol-diazo-BSA (CIM-BSA) as antigen. The ELISA test is a solid phase direct competitive assay using horseradish peroxidase conjugate as the competing entity. The total incubation time of the assay is 45 minutes. Results & DiscussionThe concentration of cimaterol to decrease tracer binding by 50% (IC50 value) in a direct competitive ELISA method was found to be 2.9ng/mL. The antibody is specific with low crossreactivity towards other beta-agonists tested such as propanolol (11.7%), clenbuterol (11.4%), mapenterol (7.3%), metoprolol (5.3%), and atenolol (4.6%). The antibody has cross-reactivity of < 1% to other drugs such as terbutaline, ractopamine, timolol and isoxsuprine. The limits of detection (LOD) of the ELISAfor swine urine, chicken muscle, shrimp tissue, milk and animal feed were determined as 0.33 ng/mL, 0.30 ng/mL, 0.30 ng/mL,1.18ng/mL, and 0.28 ng/mL respectively. ConclusionA sensitive and specific ELISA was developed to detect the beta 2-adregenic agonist cimaterol in various matrices such as urine (swine), muscle (chicken), tissue (shrimp), milk and animal feed.

Evaluation of Salivary Cortisol and Anxiety Levels in Myofascial Pain Dysfunction Syndrome

BackgroundMyofascial pain dysfunction syndrome (MPDS), otherwise called myofascial pain is one of the most common temporomandibular disorders, which in turn is the most common cause of orofacial pain of non-dental origin. Its etiology is multifactorial and still poorly understood. Psychological factors have been shown to play a role in the etiology. The aim of the study was to evaluate the association between anxiety and salivary cortisol levels in patients with myofascial pain.MethodsTwenty patients suffering from myofascial pain were recruited as the study group. The same number of age and sex matched healthy individuals were taken as the control group. The salivary samples collected between 9-9:15 am from both groups were analyzed for cortisol levels with the competitive enzyme-linked immunosorbent assay method. Anxiety levels of 40 patients were measured using Hamilton's anxiety scale.ResultsThe mean serum cortisol level of the MPDS group showed a highly significant difference (p < 0.001) from the controls. The mean anxiety scores of the MPDS group showed a highly significant difference (p < 0.001) from the controls. A positive correlation was found between anxiety and the salivary cortisol levels in MPDS patients.ConclusionsThese findings suggest that anxiety plays a vital role in the etio-pathogenesis of MPDS; thus, besides pharmacological treatment, psychological support is also needed.

Development of an enzyme-linked immunosorbent assay for octylphenol

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the quantitative analysis of octylphenol. A linear carboxylated analogue of 4-nonylphenol was synthesised and characterised as hapten. The hapten was coupled to the carrier protein to produce a specific antibody. An indirect competitive ELISA method was established based on the polyclonal antibody, which exhibited an IC50 value of 51 ng/mL for octylphenol. A monoclonal antibody was produced with an IC50 value of 76 ng/mL for octylphenol. The immunoassay used with the monoclonal antibodies was applied to analyse water samples from Tai Lake in China.

Aflatoxin M1 in Milk Products in China and Dietary Risk Assessment

This study was conducted to investigate the occurrence of aflatoxin M1 (AFM1) in milk products in China using the competitive enzyme-linked immunosorbent assay method and to estimate the dietary exposure to this toxin through a probabilistic approach. Based on the exposure assessment results, a quantitative cancer potency formula developed by the Joint Food and Agriculture Organization and World Health Organization Expert Committee on Food Additives was applied to assess the cancer risk. AFM1 was detected in 48.07% of the milk samples and 4.49% of the yoghurt samples. No samples contained AFM1 above the current regulatory limit in China. The simulated AFM1 intake (90% confidence interval) in various sex-age groups ranged from 0.023 (0.021 to 0.023) ng/kg of body weight per day for 30- to 45-year-old men to 0.382 (0.354 to 0.386) ng/kg of body weight per day for 2- to 4-year-old girls at the 99th percentile. The cancer risk of AFM1 to the general population of China was assessed to be 0.129 cancer cases per year per 10(8) persons at the 99th percentile. These results indicate that the health risk associated with AFM1 in milk in China is relatively low.

Development of a new rabbit monoclonal antibody and its based competitive indirect enzyme‐linked immunosorbent assay for rapid detection of sulfonamides

Monoclonal antibodies generally obtained through the classic mouse hybridoma system were requisite for the establishment of various immunoassays. In this study, a new rabbit monoclonal antibody (RabMAb) against sulfonamides (SAs) was first produced via hybridoma technique in rabbit. The related enzyme-linked immunosorbent assay (ELISA) was then developed and applied to real sample analysis. A sensitive competitive indirect ELISA method based on a novel RabMAb for rapid detection of sulfonamides was first established. The obtained half-maximum inhibition concentration (IC(50)) values for four SAs were all below 10 ng mL(-1) , with 0.68 ng mL(-1) sulfathiazole (STZ), 1.11 ng mL(-1) sulfadiazine (SD), 1.15 ng mL(-1) sulfapyridine (SP) and 5.27 ng mL(-1) sulfamethoxazole (SMX). Desirable recoveries when detecting different spiked swine urine and milk samples were achieved ranging from 92.6% to 104.3% and from 61.1% to 81.6%, respectively. The proposed immunoassay with the newly developed RabMAb is capable of detection of four SAs (STZ, SD, SP and SMX) with proven satisfactory performance and is applicable for routine large-scale analysis in practical uses.

Occurrence of several main antibiotic residues in raw milk in 10 provinces of China

In the present study, 199 raw milk samples were analysed for beta-lactams, tetracyclines, sulfonamides and quinolones. They were obtained from collection tanks of milk routes in 10 provinces of China. The occurrence and concentration range of the four antibiotics were investigated by competitive enzyme-linked immunosorbent assay methods. At the detectable level, 0.5%, 47.2% and 20.1% of samples were positive for beta-lactams, quinolones and sulfonamides, respectively. No sample was positive for tetracyclines. Beta-lactams were positive only in Beijing while quinolones and sulfonamides were positive in all provinces. One (0.5%) sample for beta-lactams was above the maximum residue limits (MRLs, regulated by EU, CAC and China). The maximum concentrations for sulfonamides and quinolones were 16.28 µg kg–1 and 23.25 µg kg–1 which were under their maximum residue limits. More precaution should be taken on antibiotic controls because of the high detection rate of some antibiotics.

Development of an Enzyme-Linked Immunosorbent Assay System for the Determination of Asymmetric Dimethylarginine Using a Specific Monoclonal Antibody

We produced a monoclonal antibody (mAb) against N(G),N(G)-dimethyl-L-arginine (asymmetric dimethylarginine: ADMA), an endogenous competitive inhibitor of nitric oxide synthase (NOS), and developed an enzyme-linked immunosorbent assay (ELISA). The competitive ELISA method using the mAb determined 5 nM-100 nM ADMA, and ADMA levels in human plasma and urine were found to be 0.78 µM and 51.3 µmol/g of creatinine respectively.