t.spot covid
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t.spot covid

HIGHLIGHTS

  • TSPOT.COVID is an ELISpot interferon gamma-release assay for SARS-CoV-2
  • TSPOT.COVID identifies a T cell response to SARS-CoV-2 spike S1 and N peptides
  • 2–8 weeks post SARS-CoV-2 diagnosis TSPOT.COVID detected 98% of infections
  • In comparison, immunoglobulin G (IgG) serology detected 83% of infections in the same period
  • Cellular immune response activated sooner and lasted longer than antibodies

Abstract

Objective

To evaluate the performance of the T-SPOT.COVID test for identifying SARS-CoV-2-responsive T-cells in participants with SARS-CoV-2 infection.

Methods

The T-SPOT.COVID test uses ELISpot interferon-gamma release assay (IGRA) methodology to measure T cell responses to SARS-CoV-2 spike S1 and nucleocapsid peptides. T-SPOT.COVID and anti-N immunoglobulin (Ig) G serology tests were performed on blood from 186 patients with nucleic acid amplification test (NAAT)-confirmed-SARS-CoV-2 infection and 100 control group participants.

Results

In the 2–8 weeks after NAAT-diagnosed SARS-CoV-2 infection, the T-SPOT.COVID test detected 98.4% (63 of 64) of infected participants, while anti-N IgG serology detected 82.8%. In the first 2 weeks after diagnosis, during adaptive immune response activation, there were less reactive T-SPOT.COVID responses (75.7%, 28 of 37 infected participants) and many less seropositive responses (32.4%). Response numbers tapered after 8 weeks; however, T-SPOT.COVID test continued to detect most participants with confirmed infection (83.6%, 56 of 67) and continued to out-perform serology (52.2%). T-SPOT.COVID response due to cross-reactive T cells was ruled out by demonstrating that, of 44 control group participants with T cells responsive to 4 human common cold coronavirus peptides, only 1 was T-SPOT.COVID reactive.

Conclusion

The T-SPOT.COVID test performed well in detecting SARS-CoV-2-sensitized T-cells over many months

Introduction

Long-term protection from infectious agents, such as the SARS-CoV-2 virus, is mediated by T cells and antibody-mediated immunity of the adaptive immune system (

Sette and Crotty, 2021

). The T-SPOT.COVID test was developed to identify the presence of SARS-CoV-2-responsive T cells.

T cells contribute to the understanding of SARS-CoV-2 infections in many ways. T cells can identify past SARS-CoV-2 infections at a time when PCR tests would be negative and antibodies levels may be waning (

Dan et al., 2021

;

Gudbjartsson et al., 2020

;

Poland et al., 2020

). T cells can provide immune memory lasting for months (

Dan et al., 2021

) and perhaps years, as suggested by the discovery of T cells to the SARS-CoV-1 coronavirus 17 years after infection (

Le Bert et al., 2020

). T cells may act independently of antibodies to control a SARS-CoV-2 infection, as shown by the recovery of COVID-19 patients who lack detectable antibodies but have SARS-CoV-2-responsive T cells (

Gallais et al., 2021

;

Sekine et al., 2020

). T cells also show reactivity to numerous SARS-CoV-2 epitopes, so have the potential to protect against many SARS-CoV-2 variants (

Grifoni et al., 2020

;

Tarke et al., 2021

). T cell-based assays can probe the longevity of an immune response following a SARS-CoV-2 infection or vaccination (

Goletti et al., 2021

;

Liu et al., 2021

;

Reynolds et al., 2021

). These various roles suggest that a T cell assay can be a key contributor to SARS-CoV-2 investigations.

The T-SPOT.COVID test, an enzyme-linked immunospot (ELISpot) assay, identifies T cells in peripheral blood that release interferon-gamma (IFN-γ) in response to stimulation with SARS-CoV-2 peptides. The T-SPOT.COVID test builds on the T-SPOT platform (Oxford Immunotec) used worldwide for tuberculosis and cytomegalovirus testing and the research version, the T-SPOT Discovery SARS-CoV-2 test (

Liu et al., 2021

;

t.spot covid
t.spot covid
Wyllie et al., 2021

). The T-SPOT.COVID ELISpot methodology is performed in many laboratories and offers a standardized comparison of T cell immunity among participants. In addition, ELISpot assays normalize the number of peripheral blood mononuclear cells (PBMCs), thus maintaining test effectiveness in participants with lymphopenia, a commonly reported condition in many COVID-19 patients (

Altmann and Boyton, 2020

) and immunosuppressed people.

The objective of this study was to evaluate the ability of the T-SPOT.COVID test to detect T cell responses in participants with or without a history of SARS-CoV-2 infection and to compare the T-SPOT.COVID test results with anti-N immunoglobulin (Ig)G serology results in the first several months after infection.

Materials and Methods

2.1 Participant recruitment

Participants for this single-center, cross-sectional study were recruited from patients who had attended the outpatient Primacare medical center in Fall River, Massachusetts, USA, between November 30, 2020, and March 24, 2021, a time of high demand for COVID-19 testing. Among other healthcare services, Primacare provided COVID-19 testing to anyone wanting or required to be tested. The New England Center for Clinical Research (NECCR) invited participants to join the study if they had received a positive SARS-CoV-2 nucleic acid amplification test (NAAT) at Primacare or if NECCR deemed them to be at low risk of SARS-CoV-2 infection. As this study was run independently from the participants’ healthcare providers, clinical data such as chest x-rays and hospitalizations records were not obtained. Informed consent and study approval were obtained from the Advarra institutional review board by NECCR at Primacare.
Confirmed-infection group: A NAAT, which detects the presence of the SARS-CoV-2 virus, was used to identify people infected with SARS-CoV-2 at the time of testing (

Rai et al., 2021

). Participants in the confirmed-infection group were recruited from asymptomatic and symptomatic patients who had had a positive SARS-CoV-2 NAAT result within the past 9 months. The date of the first positive NAAT result was considered the date of diagnosis of SARS-CoV-2 infection. Blood was drawn for Abbott SARS-CoV-2 chemiluminescent microparticle immunoassay (CMIA) anti-N IgG serology and T-SPOT.COVID tests between 0 to 249 days after diagnosis.

The analysis of responses was divided into 3 time periods: 0 to 2 weeks after diagnosis (0 to 14 days); 2+ to 8 weeks after diagnosis (15 to 56 days); and 8+ weeks after diagnosis (57+ days).
Control group: Many SARS-CoV-2 studies use frozen pre-pandemic blood for control samples; however, the T-SPOT platform requires fresh blood to ensure consistent results. Therefore fresh blood was obtained from control group participants prospectively recruited from individuals with low risk of prior SARS-CoV-2 infection. Requirements for enrollment included no current or prior signs or symptoms of COVID-19, no known contact with a confirmed SARS-CoV-2-infected individual, no prior history of a positive SARS-CoV-2 NAAT, no SARS-CoV-2 vaccination, and no prior diagnosis with SARS-CoV-1 or Middle Eastern Respiratory Syndrome (MERS). In addition, the BIOHIT HealthCare SARS-CoV-2 lateral flow anti-N IgM/IgG serology test was performed at enrollment, and the 1 person with a positive BIOHIT result was not enrolled. Blood was drawn at enrollment for testing with T-SPOT.COVID and the Abbott CMIA anti-N IgG serology test and anyone with a positive serology result was excluded from the control group.

2.2 T-SPOT.COVID test

The T-SPOT.COVID test includes over 250 SARS-CoV-2 peptides (15-mer peptides overlapping by 11 amino acids) in 2 antigen peptide pools; one pool contains peptides from the spike S1 protein, including the receptor-binding domain, and the other contains peptides from the nucleocapsid protein.
Blood samples for the T-SPOT.COVID test were processed and analyzed according to the manufacturer’s instructions. Briefly, blood samples were drawn into lithium heparin tubes which were shipped overnight to Oxford Immunotec (Abingdon, UK) in temperature-controlled shipping boxes. Next, the T-Cell Xtend reagent (Oxford Immunotec) was added to the samples, and PBMCs were isolated by density gradient centrifugation, washed, counted, and 250 000 cells/well were plated into 4 wells of a 96-well plate.

COVID-19 variant pack

SB072 0.5
EUR 2325.81
Description: SARS-CoV-2 Spike (S) glycoprotein:Spike (S) glycoprotein corresponds to one of the leading targets for COVID-19 disease. Present on the surface of Sars-CoV-2 virus, Spike S protein in a class I fusion protein that allows the virus to enter host cells.Variants package available:Some proteins of Sars-CoV-2 are identified as leading targets for COVID-19 therapies. SB-PEPTIDE offers a special pack pre-made peptide libraries of Spike protein including peptides from COVID-19 variants: UK COVID-19 variant B.1.1.7, South Africa COVID-19 variant B.1.351, Brazil COVID-19 variant B.1.1.248. SB-PEPTIDE offers additional plate containing only peptides with Spike S protein mutation of COVID-19 B.1.1.7, B.1.3.5.1 and B.1.1.248 (cited below).Variants package and Spike (S) glycoprotein peptide library can be used for T-cell assays, immune monitoring, antigen specific T-cell stimulation, T-cell expansion and cellular immune response.

COVID-19 Spike Antigen

30-2018 1 mg
EUR 400
Description: COVID-19 Spike recombinant antigen

COVID-19 Spike Antigen

30-2019 1 mg
EUR 400
Description: COVID-19 Spike recombinant antigen

Moderna COVID-19 Vaccine

H7N7-319 1 vial
EUR 798.4
Description: Protein

SARS & COVID-19 coronavirus

3862 each
EUR 330
Description: nucleoprotein

SARS & COVID-19 coronavirus

3863 each
EUR 330
Description: nucleoprotein

SARS & COVID-19 coronavirus

3864 each
EUR 330
Description: nucleoprotein

COVID-19 Negative Control

MBS412771-05mL 0.5mL
EUR 100

COVID-19 Negative Control

MBS412771-5x05mL 5x0.5mL
EUR 305

Coronavirus COVID-19 Spike (COVID-19, SARS-CoV-2, 2019-nCoV) ELISA Kit

MBS7612502-10x96StripWells 10x96-Strip-Wells
EUR 3900

Coronavirus COVID-19 Spike (COVID-19, SARS-CoV-2, 2019-nCoV) ELISA Kit

MBS7612502-48StripWells 48-Strip-Wells
EUR 340

Coronavirus COVID-19 Spike (COVID-19, SARS-CoV-2, 2019-nCoV) ELISA Kit

MBS7612502-5x96StripWells 5x96-Strip-Wells
EUR 2045

Coronavirus COVID-19 Spike (COVID-19, SARS-CoV-2, 2019-nCoV) ELISA Kit

MBS7612502-96StripWells 96-Strip-Wells
EUR 455

COVID-19 S Protein / (GFP)– (6His) VLP COVID-19 S Protein / (GFP)– (6His) VLP

VLP001 1x108 VP/ml x 200ul
EUR 455
Description: COVID-19 Spike protein (S) Virus Like Particle, packaged with GFP genomic material. Particles were concentrated and provided in PBS solution

COVID-19 Nucleocapsid protein

30-2005 1 mg
EUR 700
Description: COVID-19 Nucleocapsid protein recombinant antigen

COVID-19 Nucleocapsid protein

30-2006 1 mg
EUR 700
Description: COVID-19 Nucleocapsid protein recombinant Antigen

COVID-19 Nucleocapsid antigen

30-2020 1 mg
EUR 400
Description: COVID-19 Nucleocapsid recombinant protein

COVID-19 Nucleocapsid antigen

30-2021 1 mg
EUR 400
Description: COVID-19 Nucleocapsid recombinant protein

Coronavirus (COVID-19) Antibody

MBS569951-1mg 1mg
EUR 655

Coronavirus (COVID-19) Antibody

MBS569951-5x1mg 5x1mg
EUR 2760

COVID-19 Nucleocapsid Protein

MBS669369-005mg 0.05mg
EUR 500

COVID-19 Nucleocapsid Protein

MBS669369-025mg 0.25mg
EUR 1010

COVID-19 Nucleocapsid Protein

MBS669369-5x025mg 5x0.25mg
EUR 4525

COVID-19 Nucleoprotein antibody

10-2890 1 mg
EUR 420
Description: Purified COVID-19 Nucleoprotein Antibody

COVID-19 Nucleoprotein antibody

10-2891 1 mg
EUR 420
Description: Purified COVID-19 Nucleoprotein Antibody

COVID-19 Nucleoprotein antibody

10-2894 1 mg
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Description: Purified COVID-19 Nucleoprotein Antibody

COVID-19 Nucleoprotein antibody

MBS5316694-1mg 1mg
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COVID-19 Nucleoprotein antibody

MBS5316694-5x1mg 5x1mg
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COVID-19 Nucleoprotein antibody

MBS5316695-1mg 1mg
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COVID-19 Nucleoprotein antibody

MBS5316695-5x1mg 5x1mg
EUR 1480

STAT-NAT COVID-19 B

BSV-qPCR-12 24 tests
EUR 316
Description: STAT-NAT COVID-19 B

STAT-NAT COVID-19 HK

BSV-qPCR-13 24 tests
EUR 316
Description: STAT-NAT COVID-19 HK

COVID-19 Spike ELISA Kit

MBS7608267-10x96StripWells 10x96-Strip-Wells
EUR 3900

COVID-19 Spike ELISA Kit

MBS7608267-48StripWells 48-Strip-Wells
EUR 340

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