T-Track® CMV diagnostic ELISpot kit
The CE-marked in vitro diagnostic test is a highly standardized and ready-to-use IFN-γ ELISpot CMV kit. It enables highly sensitive detection of CMV-specific effector cells and measurement of CMV-specific cell-mediated immunity (CMV-CMI), based on CMV-antigen-induced IFN-γ production, in healthy and immunosuppressed individuals.
The recently launched next generation T-Track® CMV requires 50% less patient blood sample and allows an extended sample storage time of 24 hours. For fast and easy result evaluation the kit is delivered together with the CE-marked T-Track® CMV Calculator software.
Possible immune monitoring applications of T-Track® CMV
- Assessment of CMV-specific immune reconstitution or immunocompetence after immunosuppression
- Assessment of CMV-specific immunity under or after antiviral treatment/CMV-prophylaxis for assistance in antiviral therapy decision-making (together with CMV viral load determination)
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
- Sensitive and standardized ELISpot assay
- 12 x 8-well microtiter plate including operator control for 12 tests
- High clinical sensitivity
- HLA-type-independent application
- Measures the functionality of a broad network of clinically relevant effector cells (CD4+ and CD8+, NK and NKT-like cells)
- Low amount of patient blood sample required
- 24 hour sample storage time
- Delivered with the T-Track® CMV Calculator software
- CE-marked IVD in Europe
Principle and evaluation of T-Track® CMV at a glance
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 T-activated® 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 an 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 shows exemplary T-Track® CMV results from allogenic hematopoietic stem cell transplantation (HSCT) patients. In each case only one 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.
We recommend using the provided T-Track® CMV Calculator software (freeware) for an accurate validation and qualitative evaluation of the results (please refer to the corresponding T-Track® CMV Calculator Instructions for Use). In short, the respective duplicate values of SFC (per 2 x 105 cells) from stimulated and unstimulated conditions are square-root-transformed (sqrt). The test is positive if at least one of the stimulated conditions (T-activated® IE-1 and/or T-activated® pp65) fulfills the two following criteria:
A, mean of sqrt(stimulated) for 2 x 105 cells ≥ 3.16
B, mean of sqrt(stimulated) - mean of sqrt(unstimulated) ≥ 1.0496.
The test is confirmed negative if neither of the stimulated conditions fulfill these two criteria AND the positive control (PHA) concurrently shows ≥ 10 spots per 2 x 105 cells. Please refer to the T-Track® CMV Instructions for Use for complete and comprehensive instructions.
Cytomegalovirus is associated with significant clinical illness and mortality after transplantation. Beyond the direct clinical manifestations of CMV, the virus indirectly increases predisposition to allograft rejection, especially in the case of delayed-onset primary CMV disease, and opportunistic infections. It is also the leading cause of congenital infections worldwide, responsible for serious birth defects, including hearing loss and neurodevelopmental delay [1, 2].
Antiviral CMV treatment is costly, has serious side effects and decision to treat transplant recipients 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 might offer a way to improve individualized CMV management strategies [1, 3]. Immunological protection against CMV involves cell-mediated immunity (CMI) which requires 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-like) cells . As T cells are crucial for the control of CMV infection, adjunctive immune monitoring of CMV-specific T cell response may identify individuals at increased risk of CMV-related complications after transplantation. Thus, in combination with viral load measurement, monitoring CMV-specific CMI may be valuable to overcome major challenges in CMV management and to guide therapy decisions.
According to the third international consensus guidelines on CMV, “an assay should ideally be simple to perform, inexpensive, highly reproducible, and amenable to either widely available platforms or shipping to specialized reference laboratories” . Meeting those needs, the diagnostic ELISpot kit T-Track® CMV is 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-like cells [5 - 10].
Convincing T-Track® CMV ELISpot technology
The IFN-γ ELISpot assay is as yet the most sensitive method to measure CMV-specific immune cell functionality at a single-cell level. The ELISpot technology overcomes limitations of other available assay systems, as it is more sensitive than ELISA, easily standardized compared to FACS and able to address cell functionality in contrast to MHC multimer staining.
The patented T-activation technology enhances the immune stimulatory capacity of CMV immediate early-1 protein (IE-1) and of phosphoprotein 65 (pp65) proteins. Besides, the use of proteins instead of peptides reflects more closely the uptake, processing and presentation of natural antigens. Consequently, a positive test result is expected in most CMV-seropositive individuals, independently of their HLA haplotype, due to the recall of a wide CMV-specific T cell repertoire (CD8+ and CD4+) and the bystander activation of CMV-reactive cells of the innate immunity (NK and NKT-like). Following an easy, standardized and reproducible procedure, results are available within 24 hours and can be analyzed by specialized laboratories and diagnostic departments.
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 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.
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 complications. Decision to start or discontinue antiviral therapy is per clinician’s assessment based on test results (viral load and T-Track® CMV) and the patient’s overall clinical picture. 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. 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 the decision to initiate, postpone or discontinue antiviral therapy.
Clinical evidence for the risk evaluation model
Our recent clinical study in renal transplant recipients supports the risk stratification model described above. In the CMValue study, T-Track® CMV was able to monitor a functional impairment in CMV-CMI induced by immunosuppressive agents, as CMV-specific T cell response was globally reduced by approximately 60% (IE-1) and 40% (pp65) after treatment with high-dose steroid (≥ 1mg/kg body weight)  .
Moreover, decreased CMV-CMI was associated with occurrence of opportunistic infections, while increased CMV-CMI correlated with future occurrence of graft rejection. Therefore, T-Track® CMV might identify patients with negative T-Track® CMV results as potentially over-immunosuppressed and at risk for opportunistic infections, while strong CMV-specific cellular immunity might indicate under-immunosuppression and a potential risk for graft rejection .
"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
- Kotton CN et al. (2018). The Third International Consensus Guidelines on the Management of Cytomegalovirus in Solid-organ Transplantation. Transplantation 102: 900–931. (Read more)
- Leung AKC et al. (2003). Congenital cytomegalovirus infection. J. Natl. Med Assoc. 95:213-218. (Read more)
- Ljungman P et al. (2019). Guidelines for the management of cytomegalovirus infection in patients with haematological malignancies and after stem cell transplantation from the 2017 European Conference on Infections in Leukaemia (ECIL 7). Lancet Infect Dis. 2019 May 29, [Epub ahead of print]. (Read more)
- Hanley PJ, Bollard CM (2014). Controlling cytomegalovirus: helping the immune system take the lead. Viruses 6:2242-2258. (Read more)
- 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)
- 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)
- Banas B et al. (2018). Clinical validation of a novel ELISpot assay-based in vitro diagnostic assay to monitor cytomegalovirus-specific cell-mediated immunity in kidney transplant recipients: a multicenter, longitudinal, prospective, observational study. Transpl. Int. 31(4):436-450. (Read more)