Development of Dual Fluorescent Microsphere Immunological Assay for Detection of Pseudorabies Virus gE and gB IgG Antibodies

2020 ◽  
Author(s):  
chihai ji ◽  
Jingyu Wang ◽  
Yuchen Zeng ◽  
Haoming Pan ◽  
Yingfang Wei ◽  
...  
Keyword(s):  
Pseudorabies Virus ◽  
High Specificity ◽  
Antibody Detection ◽  
Economic Losses ◽  
Igg Antibodies ◽  
Wild Type ◽  
Swine Industry ◽  
Igg Antibody ◽  
Fluorescent Microsphere ◽  
Specificity And Sensitivity

Abstract Background Pseudorabies, also known as Aujezsky’s disease, is an acute viral infection caused by pseudorabies virus (PRV). Swine are one of the natural hosts of pseudorabies, therefore, the disease brings huge economic losses to the swine industry. Establishment of a differential diagnosis technique that can distinguish between wild-type infected and vaccinated pigs, and monitor vaccine-induced IgG is crucial for eventual eradication of pseudorabies.Results The aim of this study was to develop a rapid dual detection method for PRV gE and gB protein IgG antibodies with high specificity and sensitivity. PRV gE codons at amino acid residues (aa) 52–238 and gB codons at aa 539–741 were expressed to obtain recombinant PRV gE and gB proteins by pMAL-c5x vector. After purification with Qiagen Ni–NTA agarose affinity, the two proteins were analyzed by SDS-PAGE and immunoblotting assay. Two single fluorescent-microsphere immunoassays (FMIA) were established by coupling two recombinant proteins (gE and gB) with two magnetic microbeads and an effective dual FMIA was developed by integrating the two single assays. Optimal serum dilution for each assay, correlation with other common swine virus-positive sera and comparison with ELISA for two PRV antigens were tested for validation. Compared with ELISA, the specificity and sensitivity were 99.26% and 92.3% for gE IgG antibody detection and 95.74% and 96.3% for gB IgG antibody detection by dual-FMIA.Conclusion We provide a new method for monitoring PRV protective antibody in vaccinated pigs and differentiating wild-type-PRV-infected from vaccinated pigs

scholarly journals Development of a Dual Fluorescent Microsphere Immunological Assay for Detection of Pseudorabies Virus gE and gB IgG Antibodies

Viruses ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 912
Author(s):  
Chihai Ji ◽  
Yingfang Wei ◽  
Jingyu Wang ◽  
Yuchen Zeng ◽  
Haoming Pan ◽  
...  
Keyword(s):  
Pseudorabies Virus ◽  
High Specificity ◽  
Nitrilotriacetic Acid ◽  
Antibody Detection ◽  
Economic Losses ◽  
Igg Antibodies ◽  
Wild Type ◽  
Igg Antibody ◽  
Fluorescent Microsphere ◽  
Specificity And Sensitivity

Pseudorabies, also known as Aujezsky’s disease, is an acute viral infection caused by pseudorabies virus (PRV). Swine are one of the natural hosts of pseudorabies and the disease causes huge economic losses in the pig industry. The establishment of a differential diagnosis technique that can distinguish between wild-type infection and vaccinated responses and monitor vaccine-induced immunoglobulin G(IgG) is crucial for the eventual eradication of pseudorabies. The aim of this study was to develop a rapid dual detection method for PRV gE and gB protein IgG antibodies with high specificity and sensitivity. PRV gE codons at amino acid residues (aa) 52–238 and gB codons at aa 539–741 were expressed to obtain recombinant PRV gE and gB proteins via a pMAL-c5x vector. After purification with Qiagen Ni–nitrilotriacetic acid (NTA) agarose affinity chromatography, the two proteins were analyzed via SDS-PAGE and immunoblotting assays. Two single fluorescent-microsphere immunoassays (FMIAs) were established by coupling two recombinant proteins (gE and gB) to magnetic microbeads, and an effective dual FMIA was developed by integrating the two single assays. Optimal serum dilution for each assay, correlation with other common swine virus-positive sera, and comparison with ELISA for two PRV antigens were tested for validation. Compared with ELISA, the specificity and sensitivity were 99.26% and 92.3% for gE IgG antibody detection, and 95.74% and 96.3% for the gB IgG antibody detection via dual FMIA. We provide a new method for monitoring PRV protective antibodies in vaccinated pigs and differentiating wild-type PRV infection from vaccinated responses simultaneously.

scholarly journals Help in the Choice of Automated or Semiautomated Immunoassays for Serological Diagnosis of Toxoplasmosis: Evaluation of Nine Immunoassays by the French National Reference Center for Toxoplasmosis

2016 ◽  
Vol 54 (12) ◽  
pp. 3034-3042 ◽  
Author(s):  
O. Villard ◽  
B. Cimon ◽  
C. L'Ollivier ◽  
H. Fricker-Hidalgo ◽  
N. Godineau ◽  
...  
Keyword(s):  
Congenital Toxoplasmosis ◽  
Antibody Detection ◽  
Clinical Settings ◽  
Serum Samples ◽  
Igg Antibody ◽  
Routine Testing ◽  
Content Type ◽  
Specificity And Sensitivity ◽  
National Reference Center ◽  
Serology Testing

Toxoplasmosis, a benign infection, is asymptomatic or paucisymptomatic in over 80% of cases, except in immunocompetent patients suffering from ocular toxoplasmosis or in immunocompromised patients with opportunistic or congenital toxoplasmosis. Diagnosis is based mainly on serology testing. Thus, we compared the performance of the nine most commonly used commercial automated or semiautomated immunoassays for IgG and IgMToxoplasma gondiiantibody detection, that is, the Advia Centaur, Architect, AxSYM, Elecsys, Enzygnost, Liaison, Platelia, VIDAS, and VIDIA assays. The assays were conducted on four panels of serum samples derived during routine testing from patients with an interfering disease and who exhibited a low IgG antibody level in one of two clinical settings, namely, acute or chronic toxoplasmosis. As a result, IgG sensitivities ranged from 97.1% to 100%, and IgG specificities ranged from 99.5% to 100%. For IgG quantification, strong differences in IgG titers (expressed in IU/ml) were noted depending on the assay used. IgM sensitivities ranged from 65% to 97.9%, and IgM specificities ranged from 92.6% to 100%. For defining the best serological strategies to be implemented, it appears crucial to compare the diagnostic performance of the different tests with respect to their specificity and sensitivity in detecting the presence of IgG and IgM antibodies.

scholarly journals Analysis of the Complete Genome Sequence of a Novel, Pseudorabies Virus Strain Isolated in Southeast Europe

2019 ◽  
Vol 2019 ◽  
pp. 1-12
Author(s):  
Zsolt Csabai ◽  
Dóra Tombácz ◽  
Zoltán Deim ◽  
Michael Snyder ◽  
Zsolt Boldogkői
Keyword(s):  
Single Molecule ◽  
Base Composition ◽  
Pseudorabies Virus ◽  
Point Mutations ◽  
Wild Type Virus ◽  
Economic Losses ◽  
Wild Type ◽  
Southeast Europe ◽  
Long Read ◽  
National Programs

Background. Pseudorabies virus (PRV) is the causative agent of Aujeszky’s disease giving rise to significant economic losses worldwide. Many countries have implemented national programs for the eradication of this virus. In this study, long-read sequencing was used to determine the nucleotide sequence of the genome of a novel PRV strain (PRV-MdBio) isolated in Serbia.Results. In this study, a novel PRV strain was isolated and characterized. PRV-MdBio was found to exhibit similar growth properties to those of another wild-type PRV, the strain Kaplan. Single-molecule real-time (SMRT) sequencing has revealed that the new strain differs significantly in base composition even from strain Kaplan, to which it otherwise exhibits the highest similarity. We compared the genetic composition of PRV-MdBio to strain Kaplan and the China reference strain Ea and obtained that radical base replacements were the most common point mutations preceding conservative and silent mutations. We also found that the adaptation of PRV to cell culture does not lead to any tendentious genetic alteration in the viral genome.Conclusion. PRV-MdBio is a wild-type virus, which differs in base composition from other PRV strains to a relatively large extent.

Morphologic Identification of Viral Gastroenteritis Caused by a Human Calicivirus Strain Undetected Initially by Molecular or Immunologic Methods

1997 ◽  
Vol 3 (S2) ◽  
pp. 79-80 ◽  
Author(s):  
C. D. Humphrey ◽  
J. S. Noel ◽  
B. L. Liu ◽  
E. M. Rodriguez ◽  
P. R. Lambden ◽  
...  
Keyword(s):  
High Specificity ◽  
Viral Gastroenteritis ◽  
Norwalk Virus ◽  
Igg Antibody ◽  
Virus Identification ◽  
Specificity And Sensitivity ◽  
Stool Specimens ◽  
Polymerase Chain ◽  
Identification And Characterization ◽  
Virus Strains

Molecular and immunologie assays for virus identification and characterization are well known for their high specificity and sensitivity. High specificity occasionally hampers efforts to detect virus strains distantly related to previously recognized types and may allow infectious agents to be undetected when these techniques are used for identification. We describe in this report such an example with an outbreak of foodborne gastroenteritis.Forty-six cases of gastrointestinal illness were reported in May 1994 among adult staff of a Parkville, Md., school after a catered luncheon. Symptoms included stomach cramps, nausea, diarrhea, and vomiting. Seven stool specimens, negative by routine bacteriological screening, and 14 acute-and convalescent-phase sera from patients involved in the outbreak were tested for viruses. The stool specimens were examined by electron microscopy (EM) and reverse transcription-polymerase chain reaction (RT-PCR) designed to detect small round structured viruses (SRSVs). In addition, serum specimens were tested for IgG antibody to recombinant capsid proteins from Norwalk virus (rNV) and Toronto virus (rTV) using a direct enzyme immunoassay (EIA).

scholarly journals Development and validation of an enzyme immunoassay for detection and quantification of SARS-CoV-2 salivary IgA and IgG

2021 ◽  
Author(s):  
Veronica Costantini ◽  
Kenny Nguyen ◽  
Zoe Lyski ◽  
Shannon Novosad ◽  
Ana C Bardossy ◽  
...  
Keyword(s):  
Immune Response ◽  
Enzyme Immunoassay ◽  
Limit Of Detection ◽  
Natural Infection ◽  
High Specificity ◽  
Cross Reactivity ◽  
Igg Antibodies ◽  
Antibody Isotype ◽  
Salivary Iga ◽  
Specificity And Sensitivity

Oral fluids offer a non-invasive sampling method for the detection of antibodies. Quantification of IgA and IgG antibodies in saliva allows studies of the mucosal and systemic immune response after natural infection or vaccination. We developed and validated an enzyme immunoassay (EIA) to detect and quantify salivary IgA and IgG antibodies against the prefusion-stabilized form of the SARS-CoV-2 spike protein. Normalization against total antibody isotype was performed to account for specimen differences, such as collection time and sample volume. Saliva samples collected from 187 SARS-CoV-2 confirmed cases enrolled in 2 cohorts and 373 pre-pandemic saliva samples were tested. The sensitivity of both EIAs was high (IgA: 95.5%; IgG: 89.7%) without compromising specificity (IgA: 99%; IgG: 97%). No cross reactivity with seasonal coronaviruses was observed. The limit of detection for SARS-CoV-2 salivary IgA and IgG assays were 1.98 ng/mL and 0.30 ng/mL, respectively. Salivary IgA and IgG antibodies were detected earlier in patients with mild COVID-19 symptoms than in severe cases. However, severe cases showed higher salivary antibody titers than those with a mild infection. Salivary IgA titers quickly decreased after 6 weeks in mild cases but remained detectable until at least week 10 in severe cases. Salivary IgG titers remained high for all patients, regardless of disease severity. In conclusion, EIAs for both IgA and IgG had high specificity and sensitivity for the confirmation of current or recent SARS-CoV-2 infections and evaluation of the IgA and IgG immune response.

scholarly journals Meclizine Inhibits Pseudorabies Virus Replication by Interfering With Virus Entry and Release

2021 ◽  
Vol 12 ◽  
Author(s):  
Panrao Liu ◽  
Danhe Hu ◽  
Lili Yuan ◽  
Zhengmin Lian ◽  
Xiaohui Yao ◽  
...  
Keyword(s):  
Viral Entry ◽  
Pseudorabies Virus ◽  
Molecular Mechanisms ◽  
Clinical Symptoms ◽  
Antiviral Effect ◽  
Inhibitory Effect ◽  
Economic Losses ◽  
Swine Industry ◽  
Viral Attachment ◽  
Viral Loads

Pseudorabies virus (PRV) is a pathogen that causes substantial economic losses to the swine industry. With the emergence and widespread of PRV variants since 2011 in China, current commercial vaccines cannot provide complete protection against PRV infection. Therefore, antiviral drugs may work as an alternative way to control and prevent PRV. In this study, the inhibitory effects and underlying molecular mechanisms of meclizine against PRV were studied. Meclizine displayed a significant inhibitory effect against PRV when it was added before, simultaneously with, or after virus infection. The inhibitory effect of meclizine occurred during viral entry and cell-to-cell spreading but not at viral attachment into PK-15 cells. Meclizine also inhibited viral particle release at the late stage of infection. The antiviral effect of meclizine was tested in mice, and the results showed that meclizine reduced the severity of clinical symptoms and the viral loads in tissues, and delayed the death, after PRV challenge. The above results indicated that meclizine had an inhibitory effect on PRV. Our findings will contribute to the development of potential therapeutic drugs against PRV infection.

scholarly journals Recombinant cold shock domain containing protein is a potential antigen to detect specific antibody during early and late infections of Haemonchus contortus in goat

2020 ◽  
Author(s):  
Muhammad Ali-ul-Husnain Naqvi ◽  
KaLiBiXiaTi Aimulajiang ◽  
Muhammad Ali Memon ◽  
Muhammad Waqqas Hasan ◽  
Sana Zahra Naqvi ◽  
...  
Keyword(s):  
Haemonchus Contortus ◽  
Cold Shock ◽  
Fasciola Hepatica ◽  
Trichinella Spiralis ◽  
Early Stage ◽  
Cross Reactivity ◽  
Antibody Detection ◽  
Economic Losses ◽  
Diagnostic Potential ◽  
Specificity And Sensitivity

Abstract Background : Haemonchus contortus ( H. contortus ) is one of the most important parasites that cause huge economic losses to small ruminant industry worldwide. Effective prognosis and treatment depend upon the early diagnosis of H. contortus infection. To date, no widely-approved methods for the identification of prepatent H. contortus infection are available to identify prepatent H. contortus infection. The aim of this study was to evaluate the diagnostic potential of recombinant cold shock H. contortus protein (rHc-CS) during early and late infections of H. contortus in goat. Results : Purified rHc-CS exhibited a clear band, with a molecular weight about 38 kDa. H. contortus eggs were not detected by fecal egg count technique from feces collected at 0 to 14 days post infection (D.P.I). However, eggs were detected at 21, 28 and 35 D.P.I. Hence, results of immunoblotting assay showed specific anti rHc-CS antibody detection in all goat sera collected at early stage (14 D.P.I) and late stage (21-103 D.P.I) of H. contortus infection. Furthermore, no cross reactivity was observed against Trichinella spiralis , Fasciola hepatica and Toxoplasma gondii or uninfected goats. Among several evaluated rHc-CS indirect-ELISA format variables, favorable antigen coating concentration was found 0.28μg/well at 37℃ 1h and overnight at 4°C. Moreover, optimum dilution ratio of serum and rabbit anti-goat IgG was recorded as 1:100 and 1:4000, respectively. The best blocking buffer was 5% Bovine Serum Albumin (BSA) while the best time for blocking, serum incubation and TMB reaction were recorded as 60, 120 and 10 min, respectively. The cut-off value for positive and negative interpretation was determined as 0.352 (OD 450 ). The diagnostic specificity and sensitivity of the rHc-CS, both were recorded as 100%. Conclusion : These results validated that rHc-CS is a potential immunodiagnostic antigen to detect the specific antibodies during early and late H. contortus infections in goat.

scholarly journals Highlighted Prospects of an IgM/IgG Antibodies Test in Identifying Individuals With Asymptomatic Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection

2020 ◽  
Vol 145 (1) ◽  
pp. 39-45
Author(s):  
Yaqing Li ◽  
Qiang He ◽  
Rizhen Yu ◽  
Hui Jiang ◽  
Weizhong Wang ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Severe Acute Respiratory Syndrome ◽  
Ct Scanning ◽  
Immunoglobulin M ◽  
Antibody Detection ◽  
Igg Antibodies ◽  
Igg Antibody ◽  
Imaging Results ◽  
Close Contacts ◽  
Nucleic Acid Tests

Context.— Covert severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections could be seeding new outbreaks. How to identify asymptomatic SARS-CoV-2 infections early has become a global focus. Objective.— To explore the roles of immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies detection, nucleic acid tests, and computed tomography (CT) scanning to identify asymptomatic SARS-CoV-2 infection. Design.— The clinical data of 389 individuals with close contacts, including in general characteristics, SARS-CoV-2 etiology, serum-specific IgM and IgG antibody detection and CT imaging results, were systematically analyzed. Results.— The present study showed that only 89 of 389 individuals with close contacts were positive after the first nucleic acid test, while 300 individuals were still negative after 2 nucleic acid tests. Among the 300 individuals, 75 did not have pneumonia, and the other 225 individuals had pulmonary imaging changes. A total of 143 individuals were eventually diagnosed as having asymptomatic infection through IgM antibody and IgG antibody detection. The sensitivity, specificity, and false-negative rate of IgM and IgG antibody detection were approximately 97.1% (347 of 357), 95.3% (204 of 214), and 4.67% (10 of 214), respectively. It also indicated that during approximately 2 weeks, most individuals were both IgM positive and IgG positive, accounting for 68.57% (72 of 105). During approximately 3 weeks, the proportion of IgM-positive and IgG-positive individuals decreased to 8.57% (9 of 105), and the proportion of IgM-negative and IgG-positive individuals increased to 76.19% (80 of 105). Conclusions.— There are highlighted prospects of IgM/IgG antibody detection as a preferred method in identifying the individuals with asymptomatic SARS-CoV-2 infection, especially combined with nucleic acid tests and pulmonary CT scanning.

scholarly journals Optimize Clinical Laboratory Diagnosis of COVID-19 from Suspect Cases by Likelihood Ratio of SARS-CoV-2 IgM and IgG antibody

2020 ◽  
Author(s):  
Feng Yangchun
Keyword(s):  
Nucleic Acid ◽  
Likelihood Ratio ◽  
Posterior Probability ◽  
Clinical Laboratory ◽  
Laboratory Diagnosis ◽  
Antibody Detection ◽  
Nucleic Acid Detection ◽  
Igg Antibodies ◽  
Igg Antibody ◽  
Antibody Tests

ObjectiveTo optimize clinical laboratory diagnosis of COVID-19 from suspect cases by Likelihood Ratio of SARS-CoV-2 IgM and IgG antibody.MethodsBy reinterpreting the data in the article “Diagnostic Value of Combined Detection of Serum 2019 novel coronavirus IgM and IgG Antibodies in novel coronavirusin Infection”, the positive likelihood ratio of IgM and IgG antibody in diagnosis of COVID-19 (nucleic acid positive patients) was calculated, and the posterior probability of IgM and IgG antibodies and their tandem detection to diagnose was finally calculated.ResultsThe positive likelihood ratios of single IgM and IgG antibody were 18.50 and 12.65 respectively, and the posterior probabilities were 90.18% and 86.26% respectively. However, the posterior probability of the two antibodies tandem detection is 99.15%, which can give clinicians quantitative confidence in the diagnosis of COVID-19 from suspected cases. According to the results of this study, combining the advantages and disadvantages of nucleic acid detection and antibody detection, the clinical pathway for clinicians to diagnose COVID-19 is found.ConclusionFor suspected cases, IgM and IgG antibody tests should be firstly done at the same time. If the antibody tests are all positive, COVID-19 can be confirmed. If not, nucleic acid detection (one or more times) is performed, and in extreme cases, high-throughput viral genome sequencing is performed.

scholarly journals A survey of coxsackie A16 virus antibodies in human sera

1984 ◽  
Vol 93 (2) ◽  
pp. 205-212 ◽  
Author(s):  
G. E. D. Urquhart
Keyword(s):  
Spontaneous Abortion ◽  
Adult Female ◽  
Foot And Mouth Disease ◽  
Antibody Detection ◽  
Igg Antibodies ◽  
Igg Antibody ◽  
Detection Rates ◽  
Common Cause ◽  
Human Sera ◽  
Antibody Tests

SummaryA comparison of neutralizing and immunofluorescent (IF) IgG antibody tests in 18 sera from 10 cases of hand, foot and mouth disease (HFMD) showed a variety of responses but all sera taken more than one week after infection had both neutralizing and IF IgG antibodies.A survey of IF IgG antibody in 80 paediatric and 80 adult non-HFMD case sera gave antibody detection rates of 47·5% and 11·3% respectively. This difference could be attributed to a decline in IF IgG antibody with time after infection. Three point nine per cent of 2G sera from possible adult carditis cases had IF antibody suggesting that coxsackie A16 virus was not a common cause of adult carditis. Forty-eight point three per cent of 29 sera from cases of spontaneous abortion had detectable IF antibody, a rate similar to the paediatric sera and significantly greater than that in adult male (7·9%) and other adult female (13%) sera tested. This interesting observation requires further study.

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