A novel strategy for interpreting the T-SPOT.TB test results read by an ELISPOT plate imager.

BACKGROUND: The T-SPOT.TB can be read by an ELISPOT plate imager as an alternative to a labor-intensive and time-consuming manual reading, but its accuracy has not been sufficiently discussed to date. METHODS: 1,423 test results obtained from manual reading using a microscope and an ELISPOT plate imager were compared. The agreement of qualitative test results was assessed using Cohen's kappa coefficient. The relationship of spot counts was studied using Bland-Altman analysis. RESULTS: The overall percent agreement of the qualitative test results was 95.43% with a kappa coefficient of 0.91. Positive test results with the maximum net spot count of 8 and borderline test results showed relatively high discordance. The agreement of spot counts in panel A, panel B, and nil control was good, and variability did not increase with higher spot counts. On the basis of study findings, a novel strategy for interpreting the test results by an ELISPOT plate imager was proposed. CONCLUSIONS: To increase diagnostic accuracy, positive test results with the maximum net spot count of 8 and borderline test results should be manually confirmed. Our strategy could be a practical guide for laboratories to build their own strategies for interpreting the test results by an ELISPOT plate imager.

Direct Ex Vivo Functional Analysis of HCV-Specific T Cells.

CD8+ and CD4+ T cells play a critical role in viral clearance during self-limited HCV infection. Moreover, induction of robust and polyfunctional memory T cells is a primary goal in the development of T-cell-based HCV prophylactic vaccines. Direct ex vivo function of HCV-specific T cells without in vitro expansion can be assessed by measuring cytokine secretion or cell proliferation upon in vitro stimulation with HCV antigens using enzyme-linked immunospot (ELISpot) assays, intracellular cytokine staining (ICS) assays, and flow cytometry-based T-cell proliferation assays. In particular, polyfunctionality of T cells can be assessed by ICS assays using multicolor flow cytometry. Here, we provide protocols to measure direct ex vivo functions of HCV-specific T cells.

Towards a Full Automation of the ELISpot Assay for Safe and Parallelized Immunomonitoring.

The ELISpot assay, as a sensitive and specific method, enables the detection of cytokines for immunological purposes and in vaccine development. Here we describe the successful transfer of the manual procedure to a commercially available automated liquid handling platform, based on the work described in Neubauer et al. (Cytotechnology 69:57-73, 2017). Different kinds of technical issues (dead volume reduction, instrumental handling limitations, liquid class improvement) have been solved and biological effects (reagents concentration, selectivity tests, dispensing way, etc.) have been controlled during the implementation process. At the end a maximum of 6% mean delta difference and a lower mean dispersion than the manual assay were reached as well as a turnaround time of four to six times higher than the manual process.

Comparing TSPOT assay results between an Elispot reader and manual counts.

BACKGROUND: The interferon gamma release assay, TSPOT.TB (TSPOT) can be read by several methodologies, including an Elispot reader or manually by technician. We compared the results from these two counting methods. METHODS: Automated and manual TSPOT results among 2481 United States health care workers were compared. Cohen's kappa coefficient was used to determine the inter-rater agreement. Univariate and multiple logistic regression were used to investigate selected variable contributions. RESULTS: No prognostic factors were associated with agreement of TSPOT results between counting methods. Agreement between TSPOT results were 92.3%, 89.5%, 93.0%, and 93.1% at baseline, and at follow-up at 6, 12, and 18 months, respectively. The inter-rater agreement for all test results was good (kappa = 0.71). There was a significant difference between individual technicians kappa coefficients (p < 0.001), but no significant increase in agreement over time for technicians (p = 0.394). CONCLUSION: Commercial Elispot readers and manual counts have good agreement of TSPOT results in a low TB burden setting. Levels of agreement differed between individual technicians and automated reader from moderate to very good, indicating borderline results may be misinterpreted due to inter-rater variability. With no latent tuberculosis infection (LTBI) gold standard, it cannot be determined if one TSPOT reading method is better than another.

ELISPOT Assay for Measurement of Antigen-Specific and Polyclonal Antibody Responses.

The enzyme-linked immunospot (ELISPOT) assay for detection of antigen-specific and polyclonal antibody responses by single antibody-secreting cells has become the method of choice due to its cell-based quantitative value. Antigen stability and specificity and the diversity of antigens that can be used in the assay have contributed to the translational application of ELISPOT as demonstrated by many FDA-approved clinical tests that employ this technique. In addition, the ELISPOT assay can be used to detect two antigenically different secreted antibodies simultaneously by two-color analysis and offers the unique possibility of quantifying the number of antibody molecules secreted per cell.

Immunospot assay based on fluorescent nanoparticles for Dengue fever detection.

Dengue fever is one of the most neglected tropical diseases and of highest international public health importance, with 50 million cases worldwide every year. Early detection can decrease mortality rates from more than 20% to less than 1% and the relevant early diagnosis analyte is the viral non-structural glycoprotein, NS1. Currently, enzyme linked immunosorbent assay (ELISA) is the method of choice to detect NS1. However, this is a time consuming method, requiring 3-5h, and it is the bottleneck for routine of clinical analysis laboratory in epidemic periods, when hundreds of samples should be tested. Here we describe an easy method combining principles of fluorophore linked immunosorbent assay (FLISA) and enzyme linked immunospotting (ELISPOT). For detection, we used mouse anti-NS1 IgG labeled with fluorescent nanoparticles. The presented procedure needs only 4 µL of serum samples and requires 45-60 min. The detection limit, 5.2 ng/mL, is comparable to ELISA tests. The comparison of 83 samples with a commercial ELISA revealed a sensitivity of 81% and specificity of 88%. The use of fluorescent nanoparticles provides a higher sensitivity than an assay using usual fluorescent dye molecules, besides avoiding bleaching effects. Based on the results, the proposed method provides fast, specific and sensitive results, and proves to be a suitable method for Dengue NS1 detection in impoverished regions or epidemic areas.

Dual- and triple-color fluorospot.

Cytokine ELISPOT has become a powerful routine tool for the analysis of disease- as well as vaccine-induced T-cell responses. The method is limited, however, in that only one cytokine at a time is assessed. Fluorospot is based on the principle of ELISPOT, but facilitates the analysis of single cells secreting several cytokines, e.g., polyfunctional T cells, suggested to be of protective importance in various infectious diseases. By detecting each cytokine with a specific fluorophore and analyzing differentially colored spots by fluorophore--specific filter systems, cells producing single or multiple cytokines are identified. Fluorospot maintains the simplicity and sensitivity of the ELISPOT while taking the analysis a step forward toward multiplex analysis.

Overview of membranes and membrane plates used in research and diagnostic ELISPOT assays.

Polyvinylidene fluoride (PVDF) membrane-bottomed, 96-well plates and 8-well strips constitute the formats in which the overwhelming majority of ELISPOT assays used in research and diagnostic applications are performed. PVDF is well suited for ELISPOT because it has a high antibody-binding capacity and because its white color provides an excellent backdrop for ELISPOT enumeration. Nitrocellulose (NC) and PVDF membranes and 96-well plates containing those membranes used in ELISPOT assays were initially commercialized for filtration applications and later optimized for a range of different protein analytical applications. An overview of the development and biotechnology applications of PVDF membrane is provided. Characteristics and attributes of the membrane that are relevant to ELISPOT are summarized. Enhancements in PVDF membrane performance and optimization of devices for automation compatible and diagnostic ELISPOT applications are presented.