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Background: T cell immunity plays a pivotal role in mitigating the severity of coronavirus disease 2019 (COVID-19). Therefore, reliable functional T cell assays are required to evaluate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cell immunity in specific patient populations., Methods: We recruited a cohort of 23 healthcare workers who received their bivalent Omicron BA.1 / ancestral mRNA booster vaccination or were infected with the Omicron variant at a median of 144 days and 227 days before blood collection, respectively. In this cohort, we compared the performances of two widely utilized commercial SARS-CoV-2 interferon-gamma release assays (IGRAs), i.e., QuantiFERON SARS-CoV-2 and T-SPOT.COVID, and an in-house designed Omicron enzyme-linked immunospot (ELISpot)., Results: The QuantiFERON SARS-CoV-2 and T-SPOT.COVID assays detected SARS-CoV-2 spike-specific T cells in 34.8 % and 21.7 % of participants, respectively. Moreover, our in-house designed ELISpot that included Omicron BA.4 and BA.5 full-spike peptides detected T cell responses in 47.8 % of participants and was strongly associated with the T-SPOT.COVID., Conclusion: The evaluation of SARS-CoV-2 T cell immunity using commercially accessible assays may yield disparate outcomes as results from different assays are not directly comparable. A specific Omicron ELISpot should be considered to assess Omicron-specific T cell immunity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Coxsackievirus A16 (CA16) is one of the major pathogens responsible for hand, foot and mouth disease (HFMD). The assessment of the humoral immunity response is indispensable in the development of vaccines against enteroviruses. The neutralization test based on the inhibition of cytopathic effects (Nt-CPE) is a common method for measuring neutralizing antibodies against CA16. However, an efficient neutralization test needs to be developed for seroepidemiological surveys and clinical trials of CA16 vaccines because Nt-CPE is time-consuming and labor-intensive. In this study, a high-throughput neutralization test for CA16 based on the enzyme-linked immunospot assay (Nt-ELISPOT) was developed. The monoclonal antibody 7D10, which reacted with the viral protein VP1, was used to detect the cells infected with CA16. The neutralizing titers of sera were proven to be unchanged over an infectious dose range from 10 to 10,000TCID50 per well. The Nt-ELISPOT results correlated well with the Nt-CPE results (R(2) = 0.9250), and the detection period was shortened from five days to approximately 30h. Overall, the Nt-ELISPOT is a reliable and efficient method for measuring neutralizing antibodies against CA16., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Conventional rotavirus infectivity assays are time consuming, labor intensive, and with low sample throughput. To overcome these problems, a 96-well microplate enzyme-linked immunospot assay (Elispot) was developed for the measurement of rotavirus infectious titers. The infected MA104 cells were stained with a horseradish peroxidase-conjugated anti-VP6 monoclonal antibody followed by detection with an ELISPOT analyzer. A linear relationship was found between spot number and input of rotavirus dose in SA11 and 10 rotavirus isolates of different genotypes. The propagation of rotavirus SA11 in MA104 cells was monitored, and the neutralizing activity of serum samples and monoclonal antibodies was determined. The 50% neutralizing titer (NT50) of serum and 50% inhibitory concentration (IC50) of monoclonal antibodies were correlated well with the results determined by ELISA-based neutralization assay. In conclusion, a rapid and semi-automated procedure to determine rotavirus infectivity was developed, which will be useful to study the infectivity and the neutralizing epitopes of rotavirus., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Plaque-reduction assays have been used to detect varicella-zoster virus (VZV)-neutralizing antibodies in sera for many decades. The current study characterized the mouse monoclonal antibody (MAb) 18A10, specific for VZV envelope glycoprotein K (gK), and applied this antibody to a new type of neutralization assay in the VZV field. The procedure is called the neutralization enzyme-linked immunosorbent spot (N-ELISPOT) assay and evolved from the VZV immunoperoxidase focus assay. Optimization of the assay involved defining the optimum combination of virus plaque-forming units (PFU) and antibody dilution, which were found to be 0-100 PFU and 1:200, respectively. Furthermore, the N-ELISPOT assay produced results consistent with that obtained for the plaque-reduction neutralization assay. Considering that the plaque-reduction neutralization assay is time-consuming and labor-intensive, the VZV N-ELISPOT assay offers several advantages including reproducibility and applicability for high-throughput analysis of humoral immune responses to VZV., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Assessing the CMV specific CMI in transplant subjects represents a promising strategy to determine the risk of infection on individual basis. In this study 61 adult CMV IgG seropositive solid organ transplant recipients were examined in order to improve the efficacy of CMI detection. For this purpose, pair-wise comparisons were conducted comparing positive control stimuli PWM and PMA/iono and CMV stimuli, pp65 peptide pool and whole CMV particle. Rosette pre-depletion of blood was also investigated for detecting CD4+ or CD8+ T-cell responses using the IFN-g ELISPOT assay. In the time-points 30-180 days after transplantation, PMA/iono produced statistically significant higher responses compared to PWM, probably because PMA/iono activation pathway is independent from the effect of immunosuppressive drugs. The data showed that 11% of transplant patients displayed very low or undetectable responses to pp65 peptide pool antigen while having sustained high responses to whole CMV particle. In addition, in all the subjects analyzed, CMI responses to CMV particle produced a statistically significant higher number of spots compared to pp65 peptide pool antigen. Rosette pre-depletion of whole blood proved to be effective in detecting CD4+ or CD8+ T-cell responses similarly to flow cytometry. Taken together, the following recommendations are suggested to optimize the CMV-ELISPOT for transplantation settings: (1) use PMA/iono as positive control; (2) whole virus particle should be used to avoid peptide-related false negative responses; (3) a rosette pre-depletion step may be useful to detect CD4+ or CD8+ T-cell responses., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The baculovirus expression vector system (BEVS) is one of the most powerful methods for production of recombinant proteins for research or commercial purposes. Titration of viable virus in insect cell culture is often required when BEVS is used for basic research or bioprocessing. An enzyme-linked immunosorbent spot (ELISPOT) assay using monoclonal antibodies against the major capsid protein VP39 of both Autographa californica nuclear polyhedrosis virus (AcMNPV) and Bombyx mori nuclear polyhedrosis virus (BmNPV) was developed for baculovirus quantitation at 48h post-infection. The titer was determined by visualizing infected insect cells as blue spots and automated spot counting was achieved with ELISPOT hardware and software. Log-scale comparison of the results between the ELISPOT assay and a conventional end point dilution assay using a fluorescent marker showed a good correlation for both AcMNPV (R(2)=0.9980, p<0.05) and BmNPV (R(2)=0.9834, p<0.05). In conclusion, a novel, rapid and semi-automated procedure for titrating baculovirus was developed based on the specific immunostaining of infected cells followed by automated spot counting., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Dengue envelope (E) protein is a dominant immune inducer and E protein-based vaccines elicited partial to complete protection in non-human primates. To study the immunogenicity of these vaccines in humans, an enzyme linked immunospot (ELISPOT) assay for measuring interferon gamma (IFN-γ) production was developed. Cells from two subject groups, based on dengue-exposure, were selected for assay development. The unique feature of the IFN-γ ELISPOT assay is the utilization of dendritic cells pulsed with E proteins as antigen presenting cells. IFN-γ production, ranging from 53-513 spot forming units per million peripheral blood mononuclear cells (PBMCs), was observed in dengue-exposed subjects as compared to 0-45 IFN-γ spot forming units in dengue-unexposed subjects. Further, both CD4(+) and CD8(+) T cells, and cells bearing CD45RO memory marker, were the major sources of IFN-γ production. The assay allowed quantification of E-specific IFN-γ-secreting memory T cells in subjects 9 years after exposure to a live-attenuated virus vaccine and live-virus challenge. Results suggested that the dendritic cell-based IFN-γ assay is a useful tool for assessing immunological memory for clinical research., (Copyright © 2011 Elsevier B.V. All rights reserved.)