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The most preferred method for the detection of foot-and-mouth disease (FMD) viral antigen and identification of viral serotype is the enzyme-linked immunosorbent assay (ELISA). Diagnostic tests with high sensitivity are necessary both to distinguish infected vaccinated animals and execute disease control programs for the identification of the carrier animals. The current strategies for the detection of FMD virus are mainly based on the capture antibody (sandwich) ELISA test. The usage of laying pullets as an animal bioreactor for the production of specific egg yolk antibodies (IgY) has increased in recent years due to its high yield, affinity, low price, and quick production turnover. The present study aimed to produce a concentrated and purified IgY polyclonal antibody to design a capture antibody ELISA kit against the FMD virus (FMDV) serotype A. At first, laying hens were immunized with inactivated FMDV serotype virus, and then, on days 14, 21, and 28 following vaccination, the eggs and sera were collected. Afterward, the IgY polyclonal antibodies were extracted and purified from the chicken egg yolk using a polyethylene glycol 6000-ethanol precipitation procedure. Extracts were filtered, purified by ion exchange chromatography, and dialyzed. The purified IgY concentration, estimated by Bradford assay, confirmed its presence by SDS-PAGE and Western blot and also its specific immune reaction by Ouchterlony double immunodiffusion and Dot blot tests. Moreover, for achieving the optimum concentration of antigen/antibody (sera) in sandwich ELISA, a checkerboard titration test was set up based on indirect ELISA results. Eventually, 119 previously confirmed samples (including 80 positive and 39 negative) by both real-time polymerase chain reaction (quantitative PCR, qPCR) and a commercial ELISA kit were used for evaluation of the sensitivity and accuracy of our developed Capture antibody ELISA kit. In this manner, the sensitivity and specificity of our designed kit were 100% and 98%, respectively. Accordingly, the present developed capture ELISA kit based on IgY had high sensitivity and specificity for FMD virus detection and it could be used in the future for both commercial detecting and serotyping applications., Competing Interests: The authors declare that there is no conflict of interest.

Background: The coronavirus disease 2019 (COVID-19) was first reported in December 2019 in Wuhan, Hubei Province of China. As long as the 27th of December 2021, approximately 280 million people have been infected with coronavirus, resulting in more than 5,418,421 deaths worldwide. Since the beginning of the COVID-19 pandemic, different methods were introduced for diagnosing coronavirus-infected patients and evaluating the immune response, following the vaccination., Objective: The current study aimed to compare the level of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) specific IgG in a group of patients who recovered from COVID-19, measured by three different enzyme-linked immunosorbent assay (ELISA) kits., Methods: This cross-sectional study was conducted on sera from patients who recovered from a real-time reverse transcriptase-polymerase chain reaction (RT-PCR)-confirmed COVID-19 in Birjand, South Khorasan, Iran. SARS-CoV-2 anti-nucleocapsid (N) and spike (S) protein IgG levels were measured using commercial ELISA kits. Comparison between groups was made using one-way ANOVA and Tukey post hoc tests., Results: The mean titer of anti-N IgG was significantly higher for the PishtazTeb Diagnostics kit than the Ideal Tashkhis Atieh kit (p<0.05). There was no correlation between the titer of anti-N IgG (PishtazTeb Diagnostics and Ideal Tashkhis Atieh) and anti-S IgG (Chemobind Company) antibodies., Conclusion: This study indicates that the domestic ELISA kits have variable but acceptable sensitivity for detecting SARS-CoV-2 specific IgG antibodies.

Quality control is very important during the development of 3-valent (16/18/58), 9-valent (6/11/16/18/31/33/45/52/58), and 15-valent human papillomavirus (HPV) vaccines (6/11/16/18/31/33/35/39/45/52/56/58/59/68). All 3-valent, 9-valent, and 15-valent HPV vaccines contain the HPV16 antigen; therefore, a detection method that can specifically identify HPV16 in vaccines is urgently required. This study aimed to develop and characterize monoclonal antibodies to assemble a highly specific HPV16 detection kit. The HPV16 L1 pentameric protein developed as an immunogen was used to prepare monoclonal antibodies. From the pool of prepared monoclonal antibodies, we selected 4G12 and 5A6 to screen and evaluate their subtypes, specificity, neutralizing activity, serum competition, binding affinity, and gene sequencing. After these characterizations, an enzyme-linked immunosorbent assay kit for these monoclonal antibodies was developed, and excellent quality was demonstrated in the assessment of linearity, repeatability, and specificity. The developed detection kit has great potential for wide use in clinical testing and quality control in vaccine production processes., (© 2022 International Union of Biochemistry and Molecular Biology, Inc.)

The aim was to evaluate the use of a bovine procalcitonin (PCT) ELISA kit (Cusabio, China) for assessing PCT in bovine milk samples. Validation was performed by using 10 plasma and corresponding milk samples from mastitic cows. The limit of detection (LOD) was calculated. The coefficient of variation (CV%) of the readings of five plasma samples measured five times in the same plate (intra-assay) and the CV% of the same five samples read five times in three separate plates was evaluated. Parallelism was determined by serial twofold dilutions of five plasma and corresponding milk samples. Milk samples were analyzed with and without centrifugation. Regarding plasma PCT, the method presented an inter- and intra-CV < 23.7% and parallelism had very good recovery values. The ELISA kit studied can measure bovine plasma PCT concentrations. The kit antibodies fail in binding PCT in milk samples because all centrifuged milk samples showed a lower LOD than blank samples. Only three uncentrifuged milk samples showed measurable PCT concentrations. Due to these results, the commercial ELISA kit investigated could not be employed for the detection of PCT in milk samples.

Determination of serum or plasma progesterone (P4) concentrations is important to recognize pregnant and non-pregnant ewes, and also to predict the number of carried lambs. The 2 most common methodologies for the detection of plasma P4 are radioimmunoassay (RIA) and enzyme immunoassay (EIA). RIA is very expensive, and not all laboratories are equipped to perform this test; EIA is commercially available for human use, but only a few companies produce species-specific kits, which are expensive. We verified for ovine plasma a less expensive and easily available ELISA kit (DiaMetra) designed to quantify P4 in humans. Pools of ovine and human plasma were used to compare repeatability, accuracy, sensitivity, and stability of P4 measured by the DiaMetra kit. Repeatability data were within 15%, and accuracy values were ~90% for both plasma matrices. Stability data showed a loss of <20% for freeze-thaw and <30% for 30-d storage. All parameters were acceptable under international guidelines for method validation. The human ELISA kit was used successfully to quantify plasma P4 in 26 ewes during pregnancy until delivery. P4 concentrations were also correlated with the number of carried lambs.

Background: Serosurveys provide the prevalence of infection and over time will reveal the trends. The present study was conducted to estimate the seroprevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among healthcare workers (HCWs) and to analyse various characteristics (risk factors) associated with SARS CoV-2 infection., Methods: Eight government designated Corona virus disease -19 (COVID-19) hospitals were selected based on the hospital admission of patients with COVID-19 and the local epidemiological situation in the region. Multistage population proportion to size sampling was performed for the selection of HCWs. Serosurvey was conducted using the enzyme-linked immunosorbent assay-based IgG antibody test (COVID KAVACH). Bivariate and multiple logistic regression was performed to find out the factor/factors associated with the positive antibody test., Results: Out of 3255 HCWs that participated in the study, data of 3253 were analysed. The seroprevalence was 19.7% (95% confidence interval: 18.5-21.3%). Factors associated were location, category of HCWs, male sex, previously tested positive by the molecular test, training on infection prevention and control, personal protective measures, handwashing technique, close contact with a patient confirmed with COVID-19, use of personal protective equipment and symptoms in the last 30 days. However, in multiple logistic regression, only location, category, previously tested positive by the molecular test and symptoms in the last 30 days were statistically significant., Conclusion: HCWs are vulnerable to SARS-CoV-2 infection. One in five HCWs had detectable antibodies. The presence of antibodies among HCWs may help in their placement and triage. HCWs may be advised to report early in case of any symptoms of COVID-19. Preventive measures may be targeted based on the location, with particular emphasis on ancillary workers and nurses., Competing Interests: The authors have none to declare., (© 2021 Director General, Armed Forces Medical Services. Published by Elsevier, a division of RELX India Pvt. Ltd.)

The administration of nitrofurans to livestock to treat or prevent animal diseases has been banned in the EU for the production of food of animal origin. The corresponding marker residues are tissue-related metabolites AMOZ, AHD, SEM, and AOZ. The MRPL (minimum required performance limit)/RPA (Reference point for action) was set at 1 µg kg -1 in the EU. Thus, all the laboratories involved in the control of nitrofuran metabolites must detect at least at this analytical limit of performance. The objectives of the work reported here were to evaluate the performance of ELISA kits from two different manufacturers (R-Biopharm, Germany; Europroxima, the Netherlands) for the individual screening of the four nitrofuran metabolites (AOZ, AMOZ; AHD; and SEM) in aquaculture products (fish, shrimps), and then to validate the kits according to the European Decision EC/2002/657 and to the European guideline for the validation of screening methods. The false positive rates were below 9 % for the kits from both manufacturers. The detection capabilities CCβ determined were all below the current RPA (1 µg/kg). However, regarding the updated RPA at 0.5 µg/kg that shall apply in 2022, the AMOZ and SEM kits from R-Biopharm and the SEM kit from Europroxima will not be able to reach it.

Colistin is a polypeptide antibiotic mainly used in porcine and poultry to treat gastrointestinal infections. It has been included by the World Health Organisation (WHO) in the list of critically important human antibiotics of high priority for antimicrobial resistance since 2017. Therefore, it is necessary to develop specific and sensitive screening methods for this molecule. Screening for colistin with immunoassays is an interesting alternative to LC-MS/MS screening methods. The performance of three commercially available ELISA kits was evaluated in poultry and porcine muscles for the detection of colistin in regards to its European maximum residue limit (MRL) (150 µg/kg). The applicability of the three ELISA kits to the detection of colistin at or below the MRL in porcine and poultry muscles was demonstrated. The detection capabilities (CCβ) of two kits were or lower than or equal to the MRL (150 µg/kg). The lowest detection capability (30 µg/kg) was achieved with the third ELISA kit. The specificity of the three kits was very satisfactory (false positive rates 0%). The three kits are very specific for the detection of colistin (colistin A and B) and polymyxin B.

A total of 328 agricultural product samples highly suspected to be contaminated, from flour companies, feed companies, and livestock farms throughout China, were surveyed for deoxynivalenol (DON) contamination using a self-assembly enzyme-linked immunosorbent assay (ELISA) kit. An ELISA kit for DON was developed with a 4.9 ng mL -1 limit of detection (LOD) in working buffer and a 200 ng g -1 LOD in authentic samples. The DON contamination detection rate was 88.7%, concentrations ranged from 200.9 to 6480.6 ng g -1 , and the highest DON contamination was found in distillers' dried grains with solubles with an average of 3204.5 ng g -1 . Wheat bran and wheat were found to be the most commonly contaminated samples, and the corn meal samples had the lowest average DON level. This ELISA kit is a powerful alternative method for the rapid, sensitive, specific, accurate, and high-throughput determination of DON and can meet the maximum requirement levels. This survey suggests that DON contamination in the Chinese market is serious, and the contamination risk deserves attention. Essential preventive measures should be implemented to ensure food safety and human health.

Background: HPV 18 is one of the most prevalent oncogenic types, only second to HPV 16, and included in the licensed vaccines on the market. In this study, we describe the production and characterization of a panel of monoclonal antibodies (mAb) to HPV18., Methods: The immunocompetence of 1B1 and 4C2 mAbs for HPV L1 protein was evaluated by SDS-PAGE analysis, neutralization assays, affinity identification, and ELISA. The 1B1 and 4C2 genes were sequenced and analyzed. Finally, the detection kit with the two mAbs was assessed for linearity, repeatability and specificity., Results: Both mAbs specifically recognized HPV18 L1 and virus-like particles (VLPs). These mAbs are conformation-neutralizing antibodies that have high affinity and type specificity. Based on these characteristics of these mAbs, we developed an ELISA kit for specifically detecting HPV 18 antigen. We showed that this kit displayed good linearity, repeatability and sensitivity for detecting HPV18 L1 pentamer and HPV18 VLP., Conclusions: We characterized two monoclonal neutralizing antibodies for HPV L1 protein, and developed an ELISA kit for specifically detecting HPV 18 antigen. This newly developed kit can be used to monitor the potency of HPV vaccines throughout the entire production process as well as preliminary analysis of HPV18 infections.

In this work, the chronoamperometry-based redox cycling of 3,3',5,5'-tetramethylbenzidine (TMB) was performed by using interdigitated electrode (IDE). The signal was obtained from two sequential chronoamperometric profiles: (1) with the generator at the oxidative potential of TMB and the collector at the reductive potential of TMB, and (2) with the generator at the reductive potential of TMB and the collector at the oxidative potential of TMB. The chronoamperometry-based redox cycling (dual mode) showed a sensitivity of 1.49 μA/OD, and the redox cycling efficiency was estimated to be 94% (n = 10). The sensitivities of conventional redox cycling with the same interdigitated electrode and chronoamperometry using a single working electrode (single mode) were estimated to be 0.67 μA/OD and 0.18 μA/OD, respectively. These results showed that the chronoamperometry-based redox cycling (dual mode) could be more effectively used to quantify the oxidized TMB than other amperometric methods. The chronoamperometry-based redox cycling (dual mode) was applied to immunoassays using a commercial ELISA kit for medical diagnosis of the human hepatitis B virus surface antigen (hHBsAg). Finally, the chronoamperometry-based redox cycling (dual mode) provided more than a 10-fold higher sensitivity than conventional chronoamperometry using a single working electrode (single mode) when applied to a commercial ELISA kit for medical diagnosis of hHBsAg.

The purpose of this study was to develop a portable surface plasmon resonance (SPR) bioanalyzer for the sensitive detection of Escherichia coli O157:H7 in comparison with an enzyme-linked immunosorbent assay (ELISA). The experimental setup mainly consisted of an integrated biosensor and a homemade microfluidic cell with a three-way solenoid valve. In order to detect Escherichia coli O157:H7 using the SPR immunoassay, 3-mercaptopropionic acid (3-MPA) was chemisorbed onto a gold surface via covalent bond for the immobilization of biological species. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were used as crosslinker reagents to enable the reaction between 3-MPA and Escherichia coli O157:H7 antibodies by covalent -CO-NH- amide bonding. The experimental results were obtained from the Escherichia coli O157:H7 positive samples prepared by 10-, 20-, 40-, 80-, and 160-fold dilution respectively, which show that a good linear relationship with the correlation coefficient R of 0.982 existed between the response units from the portable SPR bioanalyzer and the concentration of Escherichia coli O157:H7 positive samples. Moreover, the theoretical detection limit of 1.87 × 10³ cfu/mL was calculated from the positive control samples. Compared with the Escherichia coli O157:H7 ELISA kit, the sensitivity of this portable SPR bioanalyzer is four orders of magnitude higher than the ELISA kit. The results demonstrate that the portable SPR bioanalyzer could provide an alternative method for the quantitative and sensitive determination of Escherichia coli O157:H7 in field., Competing Interests: The authors declare no conflict of interest.

The recent discovery of irisin has generated considerable interest in the scientific community. However, many studies on the biochemistry and biology of this intriguing hormone yielded controversial results in humans, which were mostly attributable to a number of drawbacks in the methods used for its detection and measurement., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Aim: A quantitative ELISA kit for the detection of human secreted CD306/LAIR-2 was developed using monoclonal and polyclonal antibodies which were raised against a highly purified recombinant human secreted CD306/LAIR-2., Methods: Anti-human secreted CD306/LAIR-2 monoclonal and polyclonal antibodies were raised by immunizing mouse or rabbit with recombinant human secreted CD306/LAIR-2. The monoclonal antibody was purified by protein G affinity, whereas the polyclonal antibody was purified by protein A affinity. The best match pair of antibodies were found and used to develop a double antibody sandwich ELISA kit for the detection of human secreted CD306/LAIR-2 in human samples., Results: A human secreted CD306/LAIR-2 ELISA kit was formulated with highly purified recombinant human secreted CD306/LAIR-2, highly specific monoclonal and polyclonal antibodies. This kit realized the quantitative measurement of recombinant human CD306/LAIR-2 and natural CD306/LAIR-2 in human serum samples., Conclusions: The developed human secreted CD306/LAIR-2 ELISA kit is a reliable quantitation immunoassay kit., (Copyright © 2014 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.)

The analytical performance and evaluation of a kit-based ELISA for the determination of acrylamide in fried potato and corn chip samples are described. The sample homogenate is subjected to clean-up using SPE, followed by analyte derivatization and ELISA detection. Accuracy, precision and linearity of the ELISA procedure have been validated using spiked samples. Analytical recovery ranged from 91.8% to 96.0% with coefficients of variation below 15%. Good linearity over a wide range of dilution and minimal assay drift was observed within a microtiter plate. IC50 value of the calibration curve was 110 ng/mL, with the limit of detection about 5 ng/mL and dynamic range from 10 to 1000 ng/mL. The high specificity of the ELISA was demonstrated by cross-reactivity study using 11 potential cross-reactants. A good correlation between the results obtained from the ELISA and GC-MS within the concentration range 120-1500 μg/kg was found in the chip samples (r=0.992, n=120). The data demonstrate that the evaluated and validated ELISA has a potential utility in a quick, simple and reliable acrylamide screening analysis for the medium- and large-sized food companies, as well as for residue laboratories and the food industry dealing with improving the chemical safety of foods available to the consumer., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Country lacks sensitive and indigenous diagnostic kits for the screening of goats and sheep against Johne's disease. Therefore an indigenous ELISA kit was developed using protoplasmic antigen from native Mycobacterium avium subspecies paratuberculosis 'Bison Type' strain of goat origin (Kit 1). In the present study, kit 1 and two commercial kits (kit 2 and 3) were evaluated with respect to 'Gold Standard' fecal culture in 71 animals (55 goats and 16 sheep). Kit 1 using indigenous antigen (protoplasmic antigen) was sensitive at very low concentration (0.1 μgm / well) as compared to purified commercial protoplasmic antigen (4 μgm / well) used in kit 2, in the Type 1 reactors (strong positive as positive). Screening of 71 animals by fecal culture detected 38.0% animals (goats-40.0%, sheep-31.2%) as positive (clinical shedders of bacilli) from these farm animals. Of the farm animals located at Central Institute for Research on Goats, herds of goat were endemic whereas, sheep flocks were comparatively resistant to Johne's disease. The 29.5 and 61.9, 15.4 and 57.7 and 4.2 and 14.0% animals (goats and sheep) were in the category of sero-reactors type 1 and 2 of the ELISA kits 1, 2 and 3, respectively. In the type 1 sero-reactors, sensitivity and specificity of kit 1, 2 and 3 was 53.7 and 86.0, 17.8 and 86.0 and 3.5 and 94.7%, respectively. Indigenous ELISA test (kit 1) was significantly superior for the screening of goatherds and sheep flocks against JD as compared to commercial ELISA kits (Kit 2 and 3). In comparison to kit 2 and 3, kit 1 had highest sensitivity, comparable specificity and substantial to nearly perfect proportional agreement (Kappa Scores) with respect to 'Gold standard' fecal culture in goats and sheep. Disease being endemic in herds and flocks screened using ELISA kits, Type I sero-rectors had better correlation with fecal culture in comparison to Type II sero-reactors therefore, used for estimation of sero-prevalence. Newly developed Indigenous ELISA kit was simple, inexpensive, sensitive and reliable for screening of goats and sheep population against Johne's disease. The study reports high prevalence of Johne's disease in farm goatherds and sheep flocks, using sensitive tests (fecal culture and ELISA kit). Results of Type 1 reaction in kit 1 were optimally correlated with culture and were good for estimating the sero-prevalence. For controlling Johne's disease in endemic herds initial removal of the animals in strong positive category (Tyep 1 reactors), may help to remove heavy shedders.