Specific Competitive Enzyme-linked Immunosorbent Assay 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.

A Sensitive and Specific Competitive Enzyme-Linked Immunosorbent Assay for Serodiagnosis of COVID-19 in Animals.

In addition to human cases, cases of COVID-19 in captive animals and pets are increasingly reported. This raises the concern for two-way COVID-19 transmission between humans and animals. Here, we developed a SARS-CoV-2 nucleocapsid protein-based competitive enzyme-linked immunosorbent assay (cELISA) for serodiagnosis of COVID-19 which can theoretically be used in virtually all kinds of animals. We used 187 serum samples from patients with/without COVID-19, laboratory animals immunized with inactive SARS-CoV-2 virions, COVID-19-negative animals, and animals seropositive to other betacoronaviruses. A cut-off percent inhibition value of 22.345% was determined and the analytical sensitivity and specificity were found to be 1:64–1:256 and 93.9%, respectively. Evaluation on its diagnostic performance using 155 serum samples from COVID-19-negative animals and COVID-19 human patients showed a diagnostic sensitivity and specificity of 80.8% and 100%, respectively. The cELISA can be incorporated into routine blood testing of farmed/captive animals for COVID-19 surveillance.

Quantification of Blood Caffeine Levels in Patients With Parkinson's Disease and Multiple System Atrophy by Caffeine ELISA.

Caffeine is considered to be a neuroprotective agent against Parkinson's disease (PD) and is expected to offer a blood-based biomarker for the disease. We herein investigated the ability of this biomarker to discriminate between PD and neurodegenerative diseases. To quantify caffeine concentrations in serum and plasma, we developed a specific competitive enzyme-linked immunosorbent assay (ELISA). To validate the diagnostic performance of the assay, we conducted a case control-study of two independent cohorts among controls and patients with PD and multiple system atrophy (MSA). Parallelism, recovery rate, and intra- and inter-assay precision of our assay were within the standard of acceptance. In the first cohort of 31 PD patients, 18 MSA patients and 33 age-matched controls, serum caffeine levels were significantly lower in PD patients than in Controls (p = 0.018). A similar trend was also observed in the MSA group, but did not reach the level of significance. In the second cohort of 50 PD patients, 50 MSA patients and 45 age-matched controls, plasma caffeine levels were significantly decreased in both PD and MSA groups compared to Controls (p < 0.001). This originally developed ELISA offered sufficient sensitivity to detect caffeine in human serum and plasma. We reproducibly confirmed decreased blood concentrations of caffeine in PD compared to controls using this ELISA. A similar trend was observed in the MSA group, despite a lack of consistent significant differences across cohorts.

Proteoglycan remodeling is accelerated in women with angina pectoris and diffuse myocardial fibrosis: the iPOWER Study

Abstract Background Women with angina and no obstructive coronary artery disease (CAD) have an unfavourable prognosis, possibly due to coronary microvascular disease and diffuse myocardial fibrosis (DMF). In DMF myocardial extracellular matrix (ECM) proteins are actively remodeled by matrix metalloproteinase (MMP). Purpose We investigated MMP-mediated degradation of the protegoglycans biglycan and versican in women with angina pectoris and possible DMF assessed by cardiac magnetic resonance T1 mapping. Methods Seventy-one women with angina pectoris and no obstructive CAD were included. Asymptomatic age-matched women served as controls (n=32). Versican and biglycan were measured in serum by specific competitive enzyme-linked immunosorbent assays. T1 mapping was performed by cardiac magnetic resonance with gadolinium measuring T1 and extracellular volume (ECV). Results Both biglycan and versican levels were higher in symptomatic women compared with controls; 31.4 ng/mL vs. 16.4 ng/mL (p&amp;lt;0.001) and 2.1 ng/mL vs. 1.8 ng/mL (p&amp;lt;0.001), respectively (Figure 1) and were moderately correlated to global ECV (r2=0.38, p&amp;lt;0.001 and r2=0.26, p=0.015 respectively). Conclusion Turnover of biglycan and versican was increased in symptomatic compared to asymptomatic women and associated to ECV, supporting a link between angina with no obstructive CAD and fibrotic cardiac remodeling. The examined biomarkers may prove to be suitable for monitoring active ECM remodeling. Figure 1. Levels of BGM and VCANM Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): This work was supported by The Danish Heart Foundation, the Danish Research Fund (Den Danske Forskningsfond) and by University of Copenhagen.

Broad-specificity ELISA with a heterogeneous strategy for sensitive detection of microcystins and nodularin

A highly sensitive and broadly specific competitive indirect enzyme-linked immunosorbent assay (ciELISA) method was developed for the simultaneous detection of nine microcystins (MCs) and nodularin (NOD) using MC-LR-keyhole limpet hemocyanin (KLH) for New Zealand white rabbit immunization to produce antibodies. The MC-LR-bovine serum albumin (BSA) and NOD-BSA coating antigens were compared and heterogeneous coating strategy was found to significantly improve the sensitivity of detection, as evident from the appropriate structure. Comparison of the half-maximum inhibitory concentration (IC50) with MC-LR and MC-LR-BSA coating techniques (0.29 ng/mL) revealed the superior performance of 0.054 ng/mL for NOD-BSA coating. NOD-BSA was selected as the coating antigen, because it showed ultrahigh sensitivity for the detection of MC-LR with a limit of detection (LOD) of 0.0016 ng/mL, which was below the maximum residue level (MRL) of 1 ng/mL. In addition, high reproducibility, good stability, and acceptable spiked sample detection, as validated by liquid chromatography tandem mass spectrometry (LC-MS/MS), indicated the possible application of this method for the analysis of MCs and NOD in water sample.

Development of specific enzyme-linked immunosorbent assay for yellowtail kingfish (Seriola lalandi) follicle stimulating hormone using recombinant gonadotropins

We developed a specific competitive enzyme-linked immunosorbent assay (ELISA) for yellowtail kingfish (Seriola lalandi) follicle stimulating hormone (FSH). We previously produced a full-length single chain recombinant yellowtail kingfish FSH using the Pichia pastoris expression system. We used the same method to produce the β subunit of the hormone, against which polyclonal antibodies were raised in rabbits. We first confirmed immunoreactivity of the polyclonal antibodies with the recombinant full length FSH and FSHβ as well as plasma and pituitary FSH of sexually immature and mature yellowtail kingfish by Western blot analysis. We then developed a precise and reproducible ELISA for yellowtail kingfish FSH and validated the assay in plasma and pituitary extracts. The intra- and inter-assay coefficients of variation was <2.2% and 10.2%, respectively. The sensitivity of the assay was 78 pg/ml. For further validation of the assay, we measured the plasma FSH in immature yellowtail kingfish treated with increasing doses (blank, 50, 100 and 150 µg/kg) of kisseptin2-10 peptide from a previous study. The dose response observed in treated females was not significant, however the increased plasma FSH levels coincided with the significantly higher estradiol levels we previously reported in the treated groups. We assessed the applicability of the assay in measuring circulating FSH in other species. We observed parallelism between the linearized FSH standard curve and displacement curves of serially diluted plasma from Atlantic bluefin tuna (Thunnus thynnus) and tilapia (Oreochromis niloticus). We also observed similar parallelism with full length recombinant giant grouper (Epinephelus lanceolatus) FSH. The ELISA we developed for yellowtail kingfish FSH will be useful in understanding the reproductive biology of the species as well as enhancing its aquaculture.

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

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

Circulating miR-421 Targeting Leucocytic Angiotensin Converting Enzyme 2 Is Elevated in Patients with Chronic Kidney Disease

Background: Decreased levels of leucocytic angiotensin converting enzyme 2 (ACE2) relate to atherosclerosis in patients with chronic kidney disease (CKD). Recently, micro RNA 421 (miR-421) was found to target and down-regulate ACE2 in human cardiac myofibroblasts. In this study, we investigated the correlation between serum levels of miR-421 and ACE2 transcripts in circulating leukocytes of healthy individuals (NP), CKD (3–5) and haemodialysis (HD) patients. Furthermore, we tested the possible interaction between miR-421 and 3′-UTR of ACE2 under normal and uremic milieu. Methods: The levels of circulating miR-421, serum Ang1–7 and expression of leucocytic ACE2, ACE, MASR, AT1R and AT2R were investigated in 16 CKD3–5 (76 ± 10 years), 32 HD patients (65 ± 13 years) and 23 NP (51 ± 5 years) by employment of specific primers, TaqMan and competitive enzyme-linked immunosorbent assay assays. Interaction between miR-421 and ACE2–3′-UTR was investigated on THP-1 cells by employment of normal and uremic sera, reporter vectors and miR-421 inhibitor. Effects of uremic toxins indoxyl sulphate, p-cresol and p-cresyl sulphate on ACE2 and miR-421 levels were investigated in THP-1 monocytes. Results: The levels of serum miR-421 were significantly elevated, while Ang1–7 was significantly decreased in CKD3–5 and HD patients as compared with NP. Serum Ang1–7 correlated positively with leucocytic ACE2 (r² = 0.213, p < 0.001). We found a significant and inverse correlation between the levels of circulating miR-421 and the expression of leucocytic ACE2 (r² = 0.223, p < 0.0001). Further significant and positive correlations could be demonstrated between miR-421 and the transcripts of leucocytic AT1R (r² = 0.094, p < 0.05) or eGFR (r² = 0.231, p < 0.0001) or CRP (r² = 0.092, p < 0.01). We found no correlations between miR-421 and ACE or AT2R or MASR expression. Treatment with miR-421 or uremic serum led to noticeable decrease of reporter 3′UTR-ACE2. Anti-miR-421 treatment resulted in the up-regulation of ACE2 protein. All uremic toxins tested were able to significantly elevate miR-421 levels and decrease the monocytic ACE2 transcripts. Conclusions: Uremic patients show an enhanced expression of serum miR-421 as compared to healthy individuals. A strong association of circulating miR-421 with decreased transcripts of ACE2 may contribute to the low expression of the enzyme in leukocytes of CKD patients, further supporting the development of atherosclerotic events.

Development of a Neo-Epitope Specific Assay for Serological Assessment of Type VII Collagen Turnover and Its Relevance in Fibroproliferative Disorders.

Type VII collagen is the main component of the anchoring fibrils connecting the basement membrane to the underlying interstitial matrix. Mutations in the type VII collagen gene cause dystrophic epidermolysis bullosa. Increased levels of type VII collagen in the skin have been reported in patients with systemic sclerosis (SSc), whereas reduced levels in the airways have been related to asthma. This indicates that type VII collagen plays an important part in upholding tissue integrity and that its remodeling may lead to pathological states. The aim of this study was to investigate the role of type VII collagen remodeling in fibroproliferative disorders. We produced monoclonal antibody targeting a specific fragment of type VII collagen (C7M) released to the systemic circulation and developed a neo-epitope specific competitive enzyme-linked immunosorbent assay (ELISA). Biological relevance was evaluated in serum from patients with SSc or chronic obstructive pulmonary disease (COPD). The C7M ELISA was technically robust and specific for the C7M neo-epitope. Serum C7M levels were significantly elevated in two cohorts of patients with SSc and in patients with COPD as compared with healthy individuals (P < 0.0001). The C7M ELISA enabled quantification of type VII collagen turnover in serum. Elevated serum C7M levels indicated that the turnover rate of type VII collagen was significantly increased in patients with SSc or COPD, suggesting a pathological role. Thus, the C7M ELISA may become useful in future investigations of type VII collagen turnover in fibroproliferative disorders, and it may prove a valuable tool for evaluating novel anti-fibrotic drugs.

Sensitive quantitative analysis of the bitter glycoside amarogentin by specific monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay.

Amarogentin (AG) is a naturally occurring secoiridoid glycoside produced mainly in the plant genera Swertia and Gentiana. Extracts of these plants have a long history of use in Japan as bitter stomachics because of their strong bitterness. Because the AG content directly reflects the potential activity of the extract, the quality control of these plant extracts through the quantitative analysis of AG is important. In the present study, we aimed to develop a quantitative analysis of AG using a monoclonal antibody (MAb) against AG (MAb 1E9) in the plant family Gentianaceae. Surprisingly, production of MAb 1E9 was successfully achieved by immunizing AG–bovine serum albumin (BSA) conjugates into mice although the number of AG bound to BSA was only one. The characterization of MAb 1E9 revealed that it shows high specificity to AG, enabling the development of an icELISA for the specific determination of AG. In addition, the detectable concentration of AG in the developed icELISA ranged from 1.95 to 62.5 ng mL−1 with a limit of detection of 1.28 ng mL−1, which is approximately 30–625 times higher in sensitivity compared with the conventional HPLC method. Validation analysis revealed that the icELISA using MAb 1E9 is precise (intra-assay variation <3.9%, inter-assay variation <8.8%) and accurate (recovery rates of spiked samples were between 91.0% and 106.4%). This method can be used for the quality control of plant extracts using AG as an index.

Development of ic-ELISA and lateral-flow immunochromatographic strip for detection of vitamin B2 in an energy drink and vitamin tablets

ABSTRACT Vitamin B2 (riboflavin) is a water-soluble vitamin that has important roles in human health. In this study, we developed a sensitive and specific monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and lateral-flow immunochromatographic assay (ICA) strip for the rapid detection of vitamin B2. Following routine fusion and selection, the optimum monoclonal antibody against vitamin B2 was obtained. The 50% inhibitory concentration and limit of detection of ic-ELISA were 8.18 and 1.80 ng/mL, respectively. The cut-off value of the lateral-flow ICA strip was 50 ng/mL. The results revealed that our developed methods are suitable for the on-site detection and mass screening of vitamin B2 in food and pharmaceutical products.

Development of a polyclonal antibody-based indirect competitive enzyme-linked immunosorbent assay to detect dufulin residue in water, soil and agricultural samples

ABSTRACTDufulin (DFL) is a potent, new-generation agrochemical that protects crops, such as tobacco, from viruses. Herein, for the first time, we developed a sensitive and specific polyclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) to detect DFL residues in samples. The assay showed high sensitivity and specificity to DFL, with a half maximal inhibitory concentration (IC50) of 48.5 ng ml−1 and limit of detection (IC20) of 2.5 ng ml−1. The average recovery of DFL in different spiked samples, such as tobacco, rice, tomato, cucumber, soil and water, was in the range of 70.7–138.8%. A statistically significant correlation was observed between our developed ELISA and previously established methodology of high-performance liquid chromatography with a diode array detector. These results indicate our assay is a potentially useful analytical tool for rapid detection of DFL residue in actual samples.

Chondrocyte activity is increased in psoriatic arthritis and axial spondyloarthritis.

BackgroundPsoriatic arthritis (PsA) and axial spondyloarthritis (axSpA) are chronic inflammatory rheumatic diseases with complex origins. Both are characterized by altered extracellular matrix remodeling in joints and entheses that results in destructive and osteochondral proliferative lesions. There is a need for biomarkers reflecting core disease pathways for diagnosis and disease mapping. Pro-C2 reflects mature cartilage collagen type IIB formation, while C-Col10 represents turnover of type X collagen, which is exclusively expressed by hypertrophic chondrocytes. The objectives of this study were to study cartilage metabolism in axSpA and PsA by assessing Pro-C2 and C-Col10 and to evaluate their diagnostic utility against a healthy reference population.MethodsPatients with PsA (n = 101) or axSpA (n = 110) were recruited consecutively from three rheumatology outpatient clinics. Demographic and clinical disease measures were recorded. Pro-C2 and C-Col10 were quantified in serum by using newly developed and specific competitive enzyme-linked immunosorbent assays based on monoclonal antibodies. One-way analysis of variance and Tukey’s multiple comparison tests were performed on log-transformed data. ROC curve analysis was carried out to evaluate their discriminative power.ResultsPro-C2 levels in serum were significantly increased in both axSpA (median concentration 1.11 ng/ml, 0.67–1.64) and PsA (median concentration 1.03 ng/ml, 0.53–1.47) compared with healthy controls (median concentration 0.30 ng/ml, 0.16–0.41) (p < 0.0001). Pro-C2 did not differ according to treatment. C-Col10 was slightly but equally elevated in the PsA and axSpA groups vs. the control group, but it was significantly lower in patients with axSpA undergoing tumor necrosis factor-α inhibitor (TNFi) treatment. ROC curve analysis revealed AUCs of 0.85 (95 % CI 0.79–0.89) for axSpA and 0.81 (95 % CI 0.75–0.86) for PsA.ConclusionsThese findings indicate that cartilage collagen metabolism was enhanced in the axSpA and PsA groups compared with the healthy control group. The lower C-Col10 level in patients with axSpA undergoing TNFi treatment may reflect that hypertrophic chondrocytes in axSpA are targeted by TNFi. ROC curve analysis showed a diagnostic potential for Pro-C2 in axSpA and PsA.

Development of a Monoclonal Antibody-Based icELISA for the Detection of Ustiloxin B in Rice False Smut Balls and Rice Grains

Rice false smut is an emerging and economically-important rice disease caused by infection by the fungal pathogen Villosiclava virens. Ustiloxin B is an antimitotic cyclopeptide mycotoxin isolated from the rice false smut balls that formed in the pathogen-infected rice spikelets. A monoclonal antibody (mAb) designated as mAb 1B5A10 was generated with ustiloxin B—ovalbumin conjugate. A highly-sensitive and specific indirect competitive enzyme-linked immunosorbent assay (icELISA) was then developed. The median inhibitory concentration (IC50) of the icELISA was 18.0 ng/mL for the detection of ustiloxin B; the limit of detection was 0.6 ng/mL, and the calibration range was from 2.5 to 107.4 ng/mL. The LOD/LOQ values of the developed ELISA used for the determination of ustiloxin B in rice false smut balls and rice grains were 12/50 μg/g and 30/125 ng/g, respectively. The mAb 1B5A10 cross-reacted with ustiloxin A at 13.9% relative to ustiloxin B. Average recoveries of ustiloxin B ranged from 91.3% to 105.1% for rice false smut balls at spiking levels of 0.2 to 3.2 mg/g and from 92.6% to 103.5% for rice grains at spiking levels of 100 to 5000 ng/g. Comparison of ustiloxin B content in rice false smut balls and rice grains detected by both icELISA and high performance liquid chromatography (HPLC) demonstrated that the developed icELISA can be employed as an effective and accurate method for the detection of ustiloxin B in rice false smut balls, as well as rice food and feed samples.

High-specificity quantification method for almond-by-products, based on differential proteomic analysis

A highly specific competitive enzyme-linked immunosorbent assay (ELISA) protocol has been developed to identify and classify almond products based on differential proteomic analysis. We applied two-dimensional electrophoresis to compare the differences between almond and apricot kernels to search for almond-specific proteins. The amino acid of apricot Pru-1 was sequenced and aligned to almond Pru-1. One peptide, RQGRQQGRQQQEEGR, which exists in almond but not in apricot, was used as hapten to prepare monoclonal antibody against almond Pru-1. An optimized ELISA method was established using this antibody. The assay did not exhibit cross-reactivity with the tested apricot kernels and other edible plant seeds. The limit of detection (LOD) was 2.5–100μg/g based on different food samples. The recoveries of fortified samples at levels of twofold and eightfold LOD ranged from 82% to 96%. The coefficients of variation were less than 13.0%. Using 7M urea as extracting solution, the heat-treated protein loss ratios were 2%, 5% and 15% under pasteurization (65°C for 30min), baking (150°C for 30min) and autoclaved sterilization (120°C for 15min), respectively.

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.

A monoclonal antibody-based enzyme-linked immunosorbent assay for detection of ustiloxin A in rice false smut balls and rice samples

Ustiloxin A, a cyclopeptide mycotoxin, was isolated from the pathogenic fungus Villosiclava virens that causes rice false smut, a worldwide devastating rice disease. A monoclonal antibody (mAb) 2D3G5 was generated with ustiloxin A–bovine serum albumin conjugate. A highly sensitive and specific indirect competitive enzyme-linked immunosorbent assay (icELISA) was then developed. It possessed a median inhibition concentration (IC50) of 13.8ng/mL and a working range of 2.8–72ng/mL. The mAb 2D3G5 recognized ustiloxin B with the cross-reactivity as 4%. The average recoveries of ustiloxin A from rice false smut balls and peeled rice samples ranged from 92% to 117% and from 92% to 107%, respectively. Comparison of the concentrations of ustiloxin A in rice false smut balls detected by both icELISA and high performance liquid chromatography–photodiode array detection indicated that the developed icELISA was suitable for detection of ustiloxin A in rice food and feed samples.

Enzyme-Linked Immunosorbent Assay for Hyodeoxycholic Acid in Pharmaceutical Products Using a Monoclonal Antibody

Herein is reported an immunochemical approach to determine hyodeoxycholic acid using hybridoma-secreting monoclonal antibodies. The hyodeoxycholic acid-specific antibody was produced by fusing splenocytes immunized with a hyodeoxycholic acid-bovine serum albumin conjugate with a hypoxanthine–aminopterin–thymidine-sensitive mouse myeloma cell line (SP2/0). The antibody was highly specific for hyodeoxycholic acid, with less than 0.05 percent cross-reactivity to over fifty structurally related compounds. The antihyodeoxycholic acid monoclonal antibody was then used to develop a rapid, specific, and sensitive indirect competitive enzyme-linked immunosorbent assay (icELISA) for the determination of hyodeoxycholic acid in pharmaceutical compounds. The linear dynamic range was from 0.48 to 62.5 nanograms per milliliter with an IC50 value of 8 nanograms per milliliter. The icELISA results correlated well with a conventional high-performance liquid chromatography method for the determination of hyodeoxycholic acid (R2 = 0.9982). This study shows that the icELISA method was successfully applied to the quantification of hyodeoxycholic acid in pharmaceutical products.

A highly sensitive and direct competitive enzyme-linked immunosorbent assay for the detection of di-(2-ethylhexyl) phthalate (DEHP) in infant supplies

A sensitive and specific direct competitive enzyme-linked immunosorbent assay (dc-ELISA) was studied in this paper for the detection of di-(2-ethylhexyl) phthalate (DEHP) based on an antigen-coating format.