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The Enzyme Linked Immunosorbant Assay (ELISA) test of potato leaves collected from different areas within three (3) selected divisions in Khyber Pakhtunkhwa, Pakistan was carried out in 2016. The region-wise results showed the over all incidence of potato virus Y (PVY) to be highest in all the three divisions on potato crop followed by the incidence of potato leaf roll virus (PLRV). The PVYand PLRV incidence in Malakand divisionwas noted by 62.7 and 44.6 %, respectively. In Peshawar division the incidence of PVY was 55.4% and that of the PLRV was 40.7% and the incidence of PVY and PLRV was 50.7 and 37.3%, respectively in Hazara. The field-wise results of Malakand region showed the highest incidence of PVY and PLRV in Kalam was found by 70 and 50%) followed by Miandam (62 and 46 %) and the lowest incidence was recorded in Malamjaba (56 and 38%). In Peshawar the highest incidence of PVY and PLRV wasnotedon potato crop in Warmarh (66 and 46%) followed by Regi (52 and 32%) and the lowest was noted in Pul Dheri Mehra (48 and 44%). In the Hazara potato crop in Kaghan had 62 and 54% PVY and PLRV incidence followed by Bagnotar (48 and 36%) and the lowest incidence was noted in Kalanderabad (42 and 22%), respectively.Results obtained by this study indicating that both PVY and PLRV are major viruses of potato crop in selected potato growing areas of KPK province which could pose serious threat to the potato crop, development of resistance cultivar against vector insect could be recommended. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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

Confirming detected SARS-CoV-2-specific antibodies is necessary to reveal immune response in COVID-19 convalescent subjects as well as to conduct population studies by screening for specific antibodies to assess rate of COVID-19 prevalence. With this purpose St. Petersburg Pasteur Institute was the first in Russia to develop the ELISA kit for the quantitative determination of human IgG to the SARS-CoV-2 nucleocapsid (N-CoV-2-IgG PS). Arbitrary units (AU/ml) were used to assess the level of antibodies. The data shown in AU/ml were recalculated later to the international units (BAU/ml) in accordance with established the First WHO International Standard for anti-SARS-CoV-2 human Immunoglobulin. Comparing the data of the N-CoV-2-IgG PS calibration curve with those of the First WHO International Standard for anti-SARS-CoV-2 human Immunoglobulin revealed a complete inter-assay association (r = 0.999, R2 = 0.997) allowing to find that 1BAU/ml = 5.97 AU/ml. The aim of the study was to characterize the SARS-CoV-2 protein N Human IgG Quantitative ELISA Kit (N-CoV-2-IgG PS), compare quantitative and qualitative data of ELISA kits, assess a correlation between the binding antibodies to SARS-CoV-2 N proteins and the neutralizing antibodies against SARS-CoV-2. The data of correlation analysis of the 83 COVID-19 convalescent blood plasma samples a significant relationship between the antibodies quantitative values and titers SARS-CoV-2-specific antibody (r = 0.8436, R2 = 0.7802) as well as a moderate relationship between antibody concentration and positivity index (r = 0.6648, R2 = 0.3307), assessed by Chaddock scale. Comparing concentration of N-protein binding antibodies with neutralizing antibody titers level uncovered data consistency obtained by quantitative and virus microneutralization assays (r = 0.7310, R2 = 0.6527) used in parallel to analyze 80 blood plasma samples obtained from COVID-19 patients and convalescents. AUC under the ROC curve comprised 0.701 (P 0.0001) evidencing about a satisfactory informative value for N-CoV-2-IgG PS compared with microneutralization assay. In addition, the efficacy of the N-CoV-2-IgG PS was 95%, while the positive and negative prognostic value was 97% and 87%, respectively. The data obtained confirmed a correlation between N-protein binding antibody level and neutralizing antibody titer. Checking inter-assay agreement evidenced about acceptance for informativeness and efficacy of using N-CoV-2-IgG PS, thereby confirming an opportunity to apply the Kit to screen for SARS-CoV-2 N protein-specific IgG antibody level and assess seroprevalence in diverse population cohorts.

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. [ABSTRACT FROM AUTHOR]

The enzyme-linked immunosorbent assay (ELISA), as a universal method for the determination of Microcystins, is of great significance for the rapid detection of Microcystins pollution. This study aimed to propose a simplified validation method for Microcystins ELISA kit by summarizing related documents and guidelines. After summarizing and clarifying from 20 validation parameters, 11 parameters were selected to simplify the validation of Microcystins ELISA kit. In addition, the acceptable range and validation details of each parameter were analyzed. The results indicated that the coefficient of determination of the Microcystin-LR standard curve was higher than 0.99. The concentration of quality control samples was within control limits. The accuracy of spiked and proficient samples was within 70%–130%. The variability of intra-assay, inter-assay, and reproducibility was less than 11, 15 and 21%, respectively. The LOD and LLOQ were 0.002 μg/L and 0.05 μg/L, respectively. When the concentration of Microcystins exceeded 5 μg/L, it was recommended to dilute the samples to the working range before detection. The specificity was estimated with seven Microcystin analogues and three amino acids, indicating that the cross-reactivity was less than 30%. These results revealed that the ELISA kit was satisfactory for detecting Microcystins in water. HIGHLIGHTS 20 validation parameters were summarized from related regulatory documents and studies.; 11 parameters were selected for Microcystins ELISA kit validation after the clarification and simplification of summarized parameters.; Acceptance range and validation details of selected parameters were investigated.; A simplified method for Microcystins ELISA kit validation was proposed.;

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. [ABSTRACT FROM AUTHOR]

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.

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.

Simple Summary: Mastitis is one of the major economic and animal welfare problems on dairy farms. The gold standard test for mastitis diagnosis is milk culture, but bacteria are not always isolated (only in 11–44% of milk samples from clinical cases of mastitis) and sometimes a positive culture can result from a contamination of the milk. Procalcitonin is a new biomarker which may lead to an early detection of inflammation due to bacterial infection. In humans, procalcitonin concentration has also been evaluated in milk in addition to plasma. The authors aimed to evaluate the possible application of a commercially available ELISA kit for bovine procalcitonin for the assessing of procalcitonin in bovine milk samples. Plasma and milk samples from cows with mastitis were collected to measure procalcitonin concentrations by using a bovine procalcitonin ELISA kit. Our results showed that the ELISA kit tested can be employed to assess bovine procalcitonin in plasma but not for analyzing milk samples. 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. [ABSTRACT FROM AUTHOR]