scholarly journals Validation of a commercially available indirect assay for SARS-CoV-2 neutralising antibodies using a pseudotyped virus assay

2021 ◽  
Vol 82 (5) ◽  
pp. 170-177
Author(s):  
Matthew J. Murray ◽  
Megan McIntosh ◽  
Claire Atkinson ◽  
Tabitha Mahungu ◽  
Edward Wright ◽  
...  
Keyword(s):  
Pseudotyped Virus ◽  
Neutralising Antibodies ◽  
Virus Assay ◽  
Indirect Assay

scholarly journals Validation of a commercially available indirect assay for SARS-CoV-2 neutralising antibodies using a pseudotyped virus assay

2021 ◽  
Author(s):  
Matthew J Murray ◽  
Megan McIntosh ◽  
Claire Atkinson ◽  
Tabitha Mahungu ◽  
Edward Wright ◽  
...  
Keyword(s):  
Immune Responses ◽  
Significant Positive Correlation ◽  
Pseudotyped Virus ◽  
Serum Samples ◽  
Neutralising Antibodies ◽  
Virus Assay ◽  
Human Sera ◽  
Surrogate Assay ◽  
Indirect Assay ◽  
Virus Assays

Abstract Objectives To assess whether a commercially available CE-IVD, ELISA-based surrogate neutralisation assay (cPass, Genscript) provides a genuine measure of SARS-CoV-2 neutralisation by human sera, and further to establish whether measuring responses against the RBD of S was a diagnostically useful proxy for responses against the whole S protein.Methods Serum samples from 30 patients were assayed for anti-NP responses, for ‘neutralisation’ by the surrogate neutralisation assay and for neutralisation by SARS-CoV-2 S pseudotyped virus assays utilising two target cell lines. Correlation between assays was measured using linear regression.Results The responses observed within the surrogate neutralisation assay demonstrated an extremely strong, highly significant positive correlation with those observed in both pseudotyped virus assays.Conclusions The tested ELISA-based surrogate assay provides an immunologically useful measure of functional immune responses in a much quicker and highly automatable fashion. It also reinforces that detection of anti-RBD neutralising antibodies alone is a powerful measure of the capacity to neutralise viral infection.

scholarly journals Validation of a commercially available indirect assay for SARS-CoV-2 neutralising antibodies using a pseudotyped virus assay

2021 ◽  
Author(s):  
Matthew J Murray ◽  
Megan McIntosh ◽  
Claire Atkinson ◽  
Tabitha Mahungu ◽  
Edward Wright ◽  
...  
Keyword(s):  
Immune Responses ◽  
Significant Positive Correlation ◽  
Pseudotyped Virus ◽  
Serum Samples ◽  
Neutralising Antibodies ◽  
Virus Assay ◽  
Human Sera ◽  
Surrogate Assay ◽  
Indirect Assay ◽  
Virus Assays

Abstract Objectives To assess whether a commercially available CE-IVD, ELISA-based surrogate neutralisation assay (cPass, Genscript) provides a genuine measure of SARS-CoV-2 neutralisation by human sera, and further to establish whether measuring responses against the RBD of S was a diagnostically useful proxy for responses against the whole S protein.Methods Serum samples from 30 patients were assayed for anti-NP responses, for ‘neutralisation’ by the surrogate neutralisation assay and for neutralisation by SARS-CoV-2 S pseudotyped virus assays utilising two target cell lines. Correlation between assays was measured using linear regression.Results The responses observed within the surrogate neutralisation assay demonstrated an extremely strong, highly significant positive correlation with those observed in both pseudotyped virus assays.Conclusions The tested ELISA-based surrogate assay provides an immunologically useful measure of functional immune responses in a much quicker and highly automatable fashion. It also reinforces that detection of anti-RBD neutralising antibodies alone is a powerful measure of the capacity to neutralise viral infection.

scholarly journals Validation of a commercially available indirect assay for SARS-CoV-2 neutralising antibodies using a pseudotyped virus assay

2021 ◽  
Author(s):  
Matthew J Murray ◽  
Megan McIntosh ◽  
Claire Atkinson ◽  
Tabitha Mahungu ◽  
Edward Wright ◽  
...  
Keyword(s):  
Immune Responses ◽  
Significant Positive Correlation ◽  
Pseudotyped Virus ◽  
Serum Samples ◽  
Neutralising Antibodies ◽  
Virus Assay ◽  
Human Sera ◽  
Surrogate Assay ◽  
Indirect Assay ◽  
Virus Assays

Abstract Objectives To assess whether a commercially available CE-IVD, ELISA-based surrogate neutralisation assay (cPass, Genscript) provides a genuine measure of SARS-CoV-2 neutralisation by human sera, and further to establish whether measuring responses against the RBD of S was a diagnostically useful proxy for responses against the whole S protein.Methods Serum samples from 30 patients were assayed for anti-NP responses, for ‘neutralisation’ by the surrogate neutralisation assay and for neutralisation by SARS-CoV-2 S pseudotyped virus assays utilising two target cell lines. Correlation between assays was measured using linear regression.Results The responses observed within the surrogate neutralisation assay demonstrated an extremely strong, highly significant positive correlation with those observed in both pseudotyped virus assays.Conclusions The tested ELISA-based surrogate assay provides an immunologically useful measure of functional immune responses in a much quicker and highly automatable fashion. It also reinforces that detection of anti-RBD neutralising antibodies alone is a powerful measure of the capacity to neutralise viral infection.

scholarly journals Addition of a single N-glycan to street rabies virus glycoprotein enhances virus production

2013 ◽  
Vol 94 (2) ◽  
pp. 270-275 ◽  
Author(s):  
Kentaro Yamada ◽  
Kazuko Noguchi ◽  
Daichi Nonaka ◽  
Muneshin Morita ◽  
Aiko Yasuda ◽  
...  
Keyword(s):  
G Proteins ◽  
Rabies Virus ◽  
Virus Production ◽  
Functional Expression ◽  
Pseudotyped Virus ◽  
Enhanced Production ◽  
Glycosylation Sites ◽  
Virus Assay ◽  
Rabies Virus Strain ◽  
Correct Structure

Most street rabies virus G proteins have two N-glycosylation sites, i.e. Asn37 and Asn319, whereas additional sites are found in fixed (laboratory adapted) viruses. In this study, we performed a pseudotyped virus assay using G-deficient rabies virus and demonstrated that single-N-glycan additions to the G protein of street rabies virus strain 1088, which are found in adapted strains, enhanced virus production in neural and non-neural cell lines, while additions to Asn194 or Asn247 enhanced production greatly. Moreover, we found that N-glycan additions at Asn194 or Asn247 facilitated the production of cell-associated virus. In contrast, deletion of the sequon at Asn37 reduced viral production, while a deletion at Asn319 resulted in extensive loss of production. Furthermore, G proteins lacking an N-glycan at Asn319 failed to fold into their correct structure and lost their fusion activity, indicating that Asn319 N-glycosylation is important for the functional expression of street virus G proteins.

scholarly journals Seroprevalence of Neutralizing Antibodies against Japanese Encephalitis Virus among Adolescents and Adults in Korea: A Prospective Multicenter Study

Vaccines ◽  
2020 ◽  
Vol 8 (2) ◽  
pp. 328
Author(s):  
Byung Ok Kwak ◽  
Young Se Kwon ◽  
Young Jin Hong ◽  
Chung Hyun Nahm ◽  
Woori Jang ◽  
...  
Keyword(s):  
Japanese Encephalitis ◽  
Neutralizing Antibodies ◽  
Age Groups ◽  
Neutralizing Antibody ◽  
Korean Population ◽  
Pseudotyped Virus ◽  
Virus Assay ◽  
Primary Series ◽  
Adolescents And Adults ◽  
Adult Immunization

The immunization schedule for the Japanese encephalitis (JE) vaccine in Korea is a two-dose primary series at 12–24 months of age, followed by booster doses 12 months after the second dose and at the ages of 6 and 12 years. Although the number of JE cases has markedly decreased after the universal vaccination program, JE predominantly occurs in adults. The aim of this study was to assess the age-specific prevalence of the JE-neutralizing antibody (NTAb) among adolescents and adults in Korea. A total of 1603 specimens were collected from a healthy Korean population above 15 years old in five provinces. The JE-NTAb titers were measured with the pseudotyped virus assay and considered to be positive at ≥ 1:50. The seropositivity of JE-NTAb was the highest in the 15–29 years category (>95%) and gradually began to decrease in the age group of 30–44 years (89.42%). The lowest and second lowest JE-NTAb seropositive rates were observed among those aged 70 years or older (59.77%) and those aged 55–59 years (75.24%), respectively. Subjects from Seoul exhibited the highest JE-NTAb titer in all age groups compared to other provinces. In conclusion, the JE-NTAb seropositive rates and titers have maintained appropriate levels in the general Korean population. We propose that adult immunization and boosters at 12 years of age against JE are not strongly recommended in Korea.

scholarly journals Evaluation of a Pseudotyped Virus Neutralisation Test for the Measurement of Equine Influenza Virus-Neutralising Antibody Responses Induced by Vaccination and Infection

Vaccines ◽  
2020 ◽  
Vol 8 (3) ◽  
pp. 466
Author(s):  
Rebecca Kinsley ◽  
Stéphane Pronost ◽  
Manuelle De Bock ◽  
Nigel Temperton ◽  
Janet M. Daly ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Haemagglutination Inhibition ◽  
Antibody Responses ◽  
Pseudotyped Virus ◽  
Equine Influenza Virus ◽  
Serum Samples ◽  
Neutralising Antibodies ◽  
Protective Antibody ◽  
Equine Influenza ◽  
Antibody Assays

Equine influenza is a major respiratory disease of horses that is largely controlled by vaccination in some equine populations. Virus-neutralising antibodies, the mainstay of the protective immune response, are problematic in assaying for equine influenza virus, as most strains do not replicate efficiently in cell culture. Surrogate measures of protective antibody responses include the haemagglutination inhibition (HI) test and single radial haemolysis (SRH) assay. For this study, a pseudotyped virus, bearing an envelope containing the haemagglutinin (HA) from the Florida clade 2 equine influenza virus strain A/equine/Richmond/1/07 (H3N8), was generated to measure HA-specific neutralising antibodies in serum samples (n = 134) from vaccinated or experimentally-infected ponies using a pseudotyped virus neutralization test (PVNT). Overall, the results of PVNT were in good agreement with results from the SRH assay (100% sensitivity, 68.53% specificity) and HI test (99.2% sensitivity, 49.03% specificity). The PVNT was apparently more sensitive than either the SRH assay or the HI test, which could be advantageous for studying the antibody kinetics, particularly when antibody levels are low. Nevertheless, further studies are required to determine whether a protective antibody level can be defined for the SRH assay and to ascertain the inter-laboratory reproducibility. In conclusion, the PVNT efficiently measures neutralising antibodies after immunization and/or experimental infection in the natural host, and may complement existing antibody assays.

scholarly journals Selenium and Dogs: A Systematic Review

Animals ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 418
Author(s):  
Viola Zentrichová ◽  
Alena Pechová ◽  
Simona Kovaříková
Keyword(s):  
Male Fertility ◽  
Clinical Signs ◽  
Complex Data ◽  
Selenium Status ◽  
Dog Food ◽  
Balanced Diet ◽  
Naturally Occurring ◽  
Correct Function ◽  
Indirect Assay

The intent of this review is to summarize the knowledge about selenium and its function in a dog’s body. For this purpose, systematic literature search was conducted. For mammals, including dogs, a balanced diet and sufficient intake of selenium are important for correct function of metabolism. As for selenium poisoning, there are no naturally occurring cases known. Nowadays, we do not encounter clinical signs of its deficiency either, but it can be subclinical. For now, the most reliable method of assessing selenium status of a dog is measuring serum or plasma levels. Levels in full blood can be measured too, but there are no reference values. The use of glutathione peroxidase as an indirect assay is questionable in canines. Commercial dog food manufactures follow recommendations for minimal and maximal selenium levels and so dogs fed commercial diets should have balanced intake of selenium. For dogs fed home-made diets, complex data are missing. However, subclinical deficiency seems to affect, for example, male fertility or recovery from parasitical diseases. Very interesting is the role of selenium in prevention and treatment of cancer.

scholarly journals Human Immunodeficiency Viruses Pseudotyped with SARS-CoV-2 Spike Proteins Infect a Broad Spectrum of Human Cell Lines through Multiple Entry Mechanisms

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 953
Author(s):  
Chuan Xu ◽  
Annie Wang ◽  
Ke Geng ◽  
William Honnen ◽  
Xuening Wang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Viral Entry ◽  
Broad Spectrum ◽  
A549 Cells ◽  
Cell Types ◽  
Pseudotyped Virus ◽  
Angiotensin Converting Enzyme 2 ◽  
Human Immunodeficiency Viruses ◽  
Tyrosine Protein Kinase ◽  
Overexpressing Cell

Severe acute respiratory syndrome-related coronavirus (SARS-CoV-2), the causative agent of coronavirus disease 19 (COVID-19), enters cells through attachment to the human angiotensin converting enzyme 2 (hACE2) via the receptor-binding domain (RBD) in the surface/spike (S) protein. Several pseudotyped viruses expressing SARS-CoV-2 S proteins are available, but many of these can only infect hACE2-overexpressing cell lines. Here, we report the use of a simple, two-plasmid, pseudotyped virus system comprising a SARS-CoV-2 spike-expressing plasmid and an HIV vector with or without vpr to investigate the SARS-CoV-2 entry event in various cell lines. When an HIV vector without vpr was used, pseudotyped SARS-CoV-2 viruses produced in the presence of fetal bovine serum (FBS) were able to infect only engineered hACE2-overexpressing cell lines, whereas viruses produced under serum-free conditions were able to infect a broader range of cells, including cells without hACE2 overexpression. When an HIV vector containing vpr was used, pseudotyped viruses were able to infect a broad spectrum of cell types regardless of whether viruses were produced in the presence or absence of FBS. Infection sensitivities of various cell types did not correlate with mRNA abundance of hACE2, TMPRSS2, or TMPRSS4. Pseudotyped SARS-CoV-2 viruses and replication-competent SARS-CoV-2 virus were equally sensitive to neutralization by an anti-spike RBD antibody in cells with high abundance of hACE2. However, the anti-spike RBD antibody did not block pseudotyped viral entry into cell lines with low abundance of hACE2. We further found that CD147 was involved in viral entry in A549 cells with low abundance of hACE2. Thus, our assay is useful for drug and antibody screening as well as for investigating cellular receptors, including hACE2, CD147, and tyrosine-protein kinase receptor UFO (AXL), for the SARS-CoV-2 entry event in various cell lines.

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