Ready-to-use ELISpot kit
Ready-to-use ELISpot kit

T-Track® CMV

Code: 11001001

Size: All-in-one IVD ELISpot kit

Price: Available upon request

Delivery time: Approximately 1-3 days

Not available in: Countries outside the EU (in this case please contact us for a research use only version)

 

Get a Quote Ask for Support


T-Track® CMV diagnostic ELISpot kit

The CE-marked in vitro diagnostic test is a highly standardized and ready-to-use ELISpot CMV kit. The test enables highly sensitive detection of CMV-specific effector cells and measurement of CMV-specific CMI in healthy and immunosuppressed individuals.
Possible immune monitoring applications of T-Track® CMV assay are:

  • Measurement of the reactivity of IFN-γ producing effector cells against CMV antigens
  • Assistance in antiviral therapy decision-making (together with CMV virus load determination)

Assessment and follow up of CMV-specific

  • immune reconstitution
  • immunocompetence after immunosuppression
  • immunity under or after antiviral treatment/ CMV-prophylaxis

T-Track® CMV kit components

  • Precoated PVDF microtiter plate (IVD)
  • T-activated® pp65
  • T-activated® IE-1
  • PHA (positive control)
  • Detection mAb
  • All needed dilution and washing buffers
  • Instructions for Use

Specifications

  • CE-marked IVD in Europe
  • Sensitive and standardized ELISpot assay
  • 12 x 8-well microtiter plate including operator control
  • High clinical sensitivity
  • HLA antigen-type independent application
  • Measures the functionality of broad network of clinically-relevant effctor cells (CD4+ and CD8+, NK and NKT-like cells)

Literature

  • Barabas S et al. (2008). Urea-Mediated Cross-Presentation of Soluble Epstein-Barr Virus BZLF1 Protein. PLoS Pathog. 4:e1000198. (Read more)
  • Barabas S et al. (2017). An optimized IFN-γ ELISpot assay for the sensitive and standardized monitoring of CMV protein-reactive effector cells of cell-mediated immunity. BMC Immunol. 18:14. (Read more)
  • Banas B et al. (2017). Validation of T-Track® CMV to assess the functionality of cytomegalovirus-reactive cell-mediated immunity in hemodialysis patients. BMC Immunol. 18:15. (Read more)
  • Reuschel E et al. (2017). Functional impairment of CMV-reactive cellular immunity during pregnancy. J. Med. Virol. 89:324-331. (Read more)

Background information

Since T cells are crucial for the control of CMV infection, adjunctive immune monitoring of CMV-specific T cells might predict individuals at increased risk of CMV disease after transplantation and may be useful in guiding preemptive or prophylactic antiviral therapies.

Interestingly, accumulating evidence suggests that immune monitoring in combination with viral load monitoring may be valuable to overcome major challenges in CMV management and to guide therapy decisions [1]. To meet the needs for improved CMV diagnostic solutions in clinical settings, an assay should be easy-to-use, standardized, reproducible, highly sensitive, and amenable to either widely available platforms or shipping to measurement centers.

Accordingly, we offer the diagnostic ELISpot kit T-Track® CMV as a highly sensitive, reliable and standardized immune monitoring tool, measuring the functionality of clinically relevant CMV-reactive effector cells, including not only CD4+ and CD8+ T cells but also NK and NKT cells [2-5]. 

About CMV-specific CMI  Test principle  T-activation® technologyClinical validation


Testimonial

Thanks to the Lophius T-Track® CMV kit, I am able to accurately monitor and characterize cellular immunity of bone marrow transplant patients in my routine diagnostics.

The easy-to-use ELISpot assay is highly sensitive, enabling the detection of immune reactivity even in strongly immunosuppressed patients. Unlike other ELISpot products, Lophius’ ELISpot generates very low background and a highly homogeneous spot morphology.

Therefore, I recommend Lophius’ ELISpot systems as effective research and diagnostic tools for immunological applications.  

Prof. Dr. med. Monika Lindemann
Head of the Research Group
AG Lindemann - Essen University Hospital


Downloads and Information


T-Track® CMV Ordering Information

Code: 11001001

Size: All-in-one IVD ELISpot kit

Price: Available upon request

Delivery time: Approximately 1-3 working days

Not available in: Countries outside the EU (in this case please contact us for a research use only version)

 

Get a QuoteAsk for Support


CMV-specific CMI

Immunological protection against CMV involves cell-mediated immunity and the participation of a broad network of cells from both adaptive and innate immunity, in particular CD4+ and CD8+ T cells, natural killer (NK) and natural killer T (NKT) cells [6]. A virus-specific T cell response develops within 6 weeks after primary antigen exposure. CMV-specific T cell response is dynamic. Primary infection is dominated by a CD8+ T cell response targeting mainly the CMV immediate early-1 (IE-1) antigen while CD4+ T helper cells are associated with long-term recovery, predominantly targeting the CMV lower matrix phosphoprotein 65 (pp65). In addition to the interaction of antigen-presenting cells (APC) with antigen-specific T cells, co-stimulatory receptors on the surface of both T cell and APC are required to assure efficient immune protection. Furthermore, innate lymphocytes such as natural killer (NK) and natural killer T (NKT) cells, contribute to the protection against CMV reactivation by bystander activation and cross-talk with APC. Upon activation, CMV-specific reactive effector cells secrete cytokines, in particular IFN-γ, which can be measured as a surrogate marker for functional CMV-specific cell-mediated immunity.

Upregulation of inhibitory receptors by T cells, such as programmed cell death protein 1 (PD-1), can on the other hand lead to persistent virus replication. This causes a loss of function (including a reduction or loss in IFN-γ production) and is commonly known as T-cell exhaustion [7].

In children and adults, primary CMV infection and reactivation are typically asymptomatic [1]. As a result, many people are unaware that they have been infected. The highly variable frequency and functionality of CMV-specific CD8+ and CD4+ T cells correlates with varying levels of protection and can be quantified by various methods. To improve immune monitoring for effective CMV management in infected individuals and transplant patients, correlates of protection from CMV disease need to be validated.

 

Need for improved CMV diagnostic solutions

Through its direct and indirect effects, CMV is associated with significant clinical illness, allograft loss, and mortality after transplantation as well as the leading cause of congenital infections worldwide [1,8].

Beyond the direct clinical manifestations of CMV syndrome or tissue-invasive disease, the virus indirectly increases predisposition to allograft rejection and opportunistic infections. Reported allograft nephropathy or even allograft loss illustrate the challenge of delayed-onset primary CMV disease and its impact on transplantation outcome despite antiviral prophylaxis. CMV is also the most common non-genetic cause of childhood hearing loss and an important cause of neurodevelopmental delay, driven by non-primary maternal infection [8].

Antiviral CMV treatment is costly, has serious side effects and decision to treat is still solely based on viral load detection. Since the strongest risk factor for CMV disease is a lack of CMV-specific immunity, immunodiagnostic CMV assays should assess a potential way to improve individualized CMV management strategies [1].


Test principle and interpretation

Peripheral blood mononuclear cells (PBMC) are isolated from Li-heparinized whole blood by density gradient centrifugation, adjusted to the required cell density and seeded on an ELISpot membrane coated with IFN-γ-specific antibodies. After stimulation for 17-21 hours with two CMV-specific antigens (T-activated® immediate-early IE-1 protein and phosphoprotein pp65) and phytohemagglutinin (PHA) as a positive control, cells are removed and secreted IFN-γ that was captured by IFN-γ-specific antibodies is detected by another enzyme-conjugated IFN-γ-specific detection antibody.

Following addition of a soluble substrate, an enzymatic reaction produces an insoluble colored precipitate and spots are revealed. Thereby one spot represents the footprint of a single antigen-reactive IFN-γ-producing effector cell (Figure 1).

The detection of reactive CMV-specific effector cells is considered positive when the geometric mean of the quadruplicate spot count frequency (spot-forming cells of SFC per 200,000 PBMC) for at least one stimulation (IE-1 and/or pp65) is ≥ 10 AND when the ratio of the geometric mean of stimulated to unstimulated conditions is ≥ 2.5. A negative test result or a low or decreasing CMV-CMI might indicate an increased risk potential for treatment-requiring CMV complication.

Note: the described method allows a semi-quantitative assessment of CMV-specific immunocompetence in CMV-seropositive patients, but is not suitable for the detection of a CMV infection.

Figure 1 - T-Track® CMV results showing exemplary findings from allogenic hematopoietic stem cell transplantation (HSCT) patients: in each case only 1 exemplary well per measurement is shown; Numbers on the upper right side of each well indicate the number of detected reactive CMV-specific effector cells. The operator control verifies proper assay performance.

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Model for evaluating the risk of CMV disease

 T-Track® CMV test results should only be interpreted in the context of the overall clinical picture. It is advisable to carry out the T-Track® CMV ELISpot in parallel to other CMV-specific diagnostic tests (such as CMV DNAemia PCR or pp65 antigenemia) and to evaluate the results in consideration of existing symptoms.        

To eventually improve assessment of the risk for CMV disease, the following model (Figure 2) illustrates a possible risk stratification of clinically-relevant CMV reactivation post-transplantation based on CMV-specific CMI.  T cell-based control of CMV replication could be affected after immunosuppressive treatment and/or T cell-depleting therapy post-transplantation. In this model, a high and stable CMV-specific CMI indicates a reduced risk for clinical complications following CMV reactivation, whereas a low and/or decreasing CMV-specific CMI could imply an increased risk for post-transplantational CMV disease.

 

 

Figure 2 - Model for risk stratification of CMV-related clinical complications following solid-organ transplantation based on CMV-CMI

Recommendations for risk stratification based on viral load detection together with T-Track® CMV assay could be drawn as a matrix (Figure 3) from low and intermediate risk to a high risk of CMV-related clinical complication.

Observation of a decreasing or low viral load with an increasing or high CMV-specific CMI might indicate a low risk for CMV complication (Figure 3, upper left quadrant). Antiviral therapy might not be necessary or might be discontinued. Adjustment of immunosuppressive treatment might not be necessary. In this case, occasional viral load monitoring in parallel to T-Track® CMV measurement would be recommended.

Observation of an increasing viral load and simultaneous low or decreasing CMV-specific CMI might indicate a high risk for CMV reactivation and related clinical complications (Figure 3, lower right quadrant). Frequent monitoring of viral load in parallel to T-Track® CMV would be recommended. Immunosuppressive treatment might be adjusted. Decision to start or continue antiviral therapy is per clinician’s assessment based on test results (viral load and T-Track® CMV) and the patient’s overall clinical picture.

In case of intermediate or borderline viral load but stable CMV-CMI, frequent monitoring in parallel to T-Track® CMV might help stratify the risk for future CMV complications and guide the clinician in his/her decision to initiate, delay or discontinue antiviral therapy.

Figure 3 – Matrix for risk stratification of clinically-relevant CMV reactivation post-transplantation based on CMV-CMI and viral load

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Immunological monitoring for CMV

Current immune monitoring assays for CMV are based on MHC multimer staining (by FACS), intracellular staining (ICS) of cytokines (by FACS), and interferon-gamma (IFN-γ) release assays (IGRAs) combined with an ELISA or ELISpot read-out.

Each of these assays present limitations in terms of sensitivity (ELISA), ability to measure cell functionality (Multimer staining) and standardization capability (ICS-FACS), as well as in their ability to predict CMV disease. T cell-based IFN-γ ELISpot assays rely on the detection of IFN-γ-secreting CMV-reactive effector cells after stimulation of whole blood or peripheral blood mononuclear cells (PBMC) with CMV-specific antigens or peptides.

It is as yet the most sensitive assay, measure CMV-specific cell functionality at a single-cell level and present the potentiality for standardization. One outstanding advantage of the CE-marked in vitro diagnostic IFN-γ ELISpot test T-Track® CMV is to measure the functionality of a broad range of CMV-specific effector cells, including CD8+ T cells (cytotoxic T cells or CTL), CD4+ T cells (T helper or Th), natural killer (NK) cells and NKT cells.

The patented T-activation® technology enhances the immune stimulatory capacity of CMV immediate early-1 protein (IE-1) and of phosphoprotein 65 (pp65) proteins, reflecting more closely the uptake, processing and presentation of natural antigens, and thus resulting in a high assay sensitivity.

Because of the recall of a wide CMV-specific T cell repertoire (CD8+ and CD4+), and of the bystander activation of CMV-reactive cells of the innate immunity (NK, NKT or NKT-like), a positive test result is expected in most CMV-seropositive individuals, independently of their HLA haplotype.

Following an easy, standardized and reproducible procedure, results are available within 24 hours and can be analysed by specialized laboratories and diagnostic departments.

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Lophius' patented T-activation® Technology platform

The T-Track® CMV ELISpot kit is based on the in vitro stimulation of mononuclear cells of the peripheral blood with two immunogenic CMV-specific proteins:

  • T-activated® IE-1
  • T-activated® pp65

In contrast to peptides and unmodified proteins, T-activated® proteins (formulated with Lophius patented T-activation® buffer) are processed and presented via both the exogenous (MHC-II) pathway and endogenous (MHC-I) pathway (cross-presentation) by functional antigen presenting cells (APC), thus mimicking a natural infection (Figure 1).

Therefore, T-activated® proteins result in a more efficient and HLA type-independent stimulation of a broad spectrum of clinically relevant subpopulations of antigen-reactive effector cells (CD4+ and CD8+ T cells and bystander activation of NK and NKT-like cells), as outlined in Figure 2.

Cross-presentation of T-activated proteins
Figure 1 - Presentation of T-activated® antigens by APC via the exogenous (MHC-II) pathway and the endogenous (MHC-I) pathway (cross-presentation). The specific interaction of a peptide / MHC complex with a T cell receptor (TCR) on the surface of a T helper (Th) or cytotoxic T cell (CTL) results in T cell activation and secretion of IFN-γ.
Network of antigen-reactive effector cells
Figure 2 - Network of antigen-reactive effector cells activated following stimulation with T-activated® proteins

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References

  1. Kotton CN et al. (2013). Updated international consensus guidelines on the management of cytomegalovirus in solid-organ transplantation. Transplantation 96:333-360. (Read more)

  2. Barabas S et al. (2008). Urea-Mediated Cross-Presentation of Soluble Epstein-Barr Virus BZLF1 Protein. PLoS Pathog. 4:e1000198. (Read more)

  3. Barabas S et al. (2017). An optimized IFN-γ ELISpot assay for the sensitive and standardized monitoring of CMV protein-reactive effector cells of cell-mediated immunity. BMC Immunol. 18:14. (Read more)

  4. Banas B et al. (2017). Validation of T-Track® CMV to assess the functionality of cytomegalovirus-reactive cell-mediated immunity in hemodialysis patients. BMC Immunol. 18:15. (Read more)

  5. Reuschel E et al. (2017). Functional impairment of CMV-reactive cellular immunity during pregnancy. J. Med. Virol. 89:324-331. (Read more)

  6. Hanley PJ, Bollard CM (2014). Controlling cytomegalovirus: helping the immune system take the lead. Viruses 6:2242-2258. (Read more)

  7. Sester U et al. (2008). PD-1 Expression and IL-2 Loss of Cytomegalovirus-Specific T Cells Correlates with Viremia and Reversible Functional Anergy. Am. J. Transplant. 8:1486-1497. (Read more)

  8. Leung AKC et al. (2003). Congenital cytomegalovirus infection. J. Natl. Med Assoc. 95:213-218. (Read more)

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