Cell Enzyme-linked Immunospot Assay Research Articles

Cellular immunity evaluation of five mycobacterium tuberculosis recombinant proteins and their compositions

Objective: The cellular immunity of 5 Mycobacterium tuberculosis recombinant proteins and their compositions was evaluated. Method: A total of 88 fresh venous blood from peripheral heparin anticoagulant population, 42 of which were from tuberculosis patients treated by The Tuberculosis Prevention and Treatment Center of Changping District, Beijing, and 46 of healthy volunteers were provided by the Infection Diseases of Chinese Center for Disease Control and Prevention. Healthy volunteers without a history of tuberculosis exposure and any clinical signs and symptoms. Using the Mycobacterium tuberculosis standard strain H37Rv DNA as a template, complete genes of the selected 5 recombinant proteins Rv3874, Rv3875, Rv2031c, Rv1411c and Rv3418c by PCR amplified; 5 recombinant proteins were cloned, expressed and purified as stimulants by genetic recombination and protein purification techniques, and the effector T cell enzyme-linked immunospot assay (ELISPOT) was used to detect cellular immunity in the population. Results: The recombinant proteins Rv3874, Rv3875, Rv2031c, Rv1411c and Rv3418c were successfully cloned, expressed and purified; And the sensitivities were 50.00%, 71.43%, 69.04%, 73.81% and 76.19%, and the specificities were 86.96%, 76.09%, 71.74%, 39.13% and 36.96%. In addition, the positive predictive value, negative predictive value, area under the curve and Youden index were 52.46% to 77.78%, 62.96% to 74.47%, 0.511 to 0.754 and 0.129 to 0.475, respectively. Except for Rv1411c and Rv3418c, the number of spot-forming cell (SFC) detected by Rv3874, Rv3875 and Rv2031c in tuberculosis patients was higher than healthy volunteers, and the differences were statistically significant (P<0.001). Among the 26 compositions composed of 5 recombinant proteins, the sensitivity was 80.95% to 95.24%, and the specificity was 68.89% to 24.44%. As the number of recombinant proteins in the composition increases, the sensitivity gradually increased, but the specificity decreased. Conclusion: The recombinant proteins of Mycobacterium tuberculosis Rv3874, Rv3875 and Rv2031c have strong ability to stimulate T cells to produce immune response, and have certain antigenicity. The efficacy of Rv1411c and Rv3418c alone as diagnostic antigens is not ideal, and the composition composed of multi-component antigens has certain application value. This article provides experimental evidence for the immune diagnosis of tuberculosis and the preparation of new anti-tuberculosis vaccines.

Detecting autoreactive B cells in the peripheral blood of people with type 1 diabetes using ELISpot

Type 1 diabetes mellitus (T1D) is an autoimmune disorder where T lymphocytes damage the islet beta cells but B lymphocytes also play an important role. Although changes in peripheral B cell phenotype have been observed, little is known about the B cells that secrete the autoantibodies.We developed a sensitive B cell enzyme-linked immunospot assay (ELISpot assay) to detect individual B cell antibody responses to glutamic acid decarboxylase (GAD) and islet antigen-2 (IA-2).We found that even healthy donors have B cells that secrete antibodies in response to GAD and IA-2 in the ELISpot. There was increased B cell reactivity to autoantigens in the peripheral blood of individuals with newly-diagnosed, but not long-standing, type 1 diabetes. However, no correlation with serum autoantibody levels was found, indicating that additional factors such as antigen affinity or exposure to antigens in vivo are required for antibody secretion, and that even healthy donors have potentially autoreactive B cells.

Significance of T-cell dot test and combined detection of adenosine deaminase in tuberculous pleurisy at different ages

Objective To analyze the clinical value of combined detection of tuberculous T cell enzyme-linked immuno spot assay (T-SPOT.TB) and adenosine deaminase (ADA) in tuberculous pleurisy patients of different ages. Methods From February 2014 to February 2018, three hundred and thirty-six patients with pleural effusion were admitted to Hebei Thoracic Hospital.Among them, two hundred and fifty five cases were diagnosed as tuberculous pleurisy and 81 cases were diagnosed as non-tuberculous pleurisy.The patients were divided into two groups according to their age.The younger group (214 cases) was 16-59 years old and the older group (122 cases) was over 60 years old.The sensitivity and specificity of T-SPOT.TB combined with ADA in the diagnosis of tuberculous pleurisy were compared between the two groups. Results The sensitivity and specificity of T-SPOT.TB were 85.5% (153/179) and 71.4% (25/35) in the young and middle-aged group, 73.7% (56/76) and 58.7% (27/46) in the old group, respectively.The sensitivity of the young and middle-aged group was significantly higher than that of the old group (χ2=4.990, P=0.045). The sensitivity and specificity of T-SPOT.TB combined with ADA were 98.9% (177/179) and 94.3% (33/35) in the young and middle-aged group, 96.1% (73/76) and 89.1% (41/46) in the elderly group, respectively.There was no significant difference in sensitivity and specificity between the two groups (χ2=0.256, P=0.393、χ2=0.655, P=0.218). Conclusion The diagnostic efficacy of T-SPOT.TB combined with ADA in patients with tuberculous pleurisy at different ages has been improved, especially for those who can not tolerate pleural biopsy and elderly patients. Key words: Tuberculous pleurisy; T cell enzyme-linked immuno-spot assay; Adenosine deaminase; Age

Diagnosis of tuberculous pleurisy with combination of adenosine deaminase and interferon-γ immunospot assay in a tuberculosis-endemic population: A prospective cohort study.

The aim of this study was to identify the optimal cut-off value of T cell enzyme-linked immunospot assay for tuberculosis (T-SPOT.TB) and evaluate its diagnostic performance alone (in the peripheral blood) or in combination with the adenosine deaminase (ADA) activity test (in peripheral blood and the pleural fluid) in patients with tuberculous pleurisy.Adult patients presenting with pleural effusion were included in this prospective cohort study. Tuberculous pleurisy was diagnosed by T-SPOT.TB in peripheral blood and a combination of T-SPOT.TB and ADA activity test in pleural fluid and peripheral blood. Receiver operating characteristic (ROC) curve in combination with multivariate logistic regression was used to evaluate the diagnostic performance of the assays.Among a total of 189 patients with suspected tuberculous pleurisy who were prospectively enrolled in this study, 177 patients were validated for inclusion in the final analysis. ROC analysis revealed that the area under the ROC curve (AUC) for T-SPOT.TB in pleural fluid and peripheral blood was 0.918 and 0.881, respectively, and for the ADA activity test in pleural fluid was 0.944. In addition, 95.5 spot-forming cells (SFCs)/2.5 × 105 cells were determined as the optimal cut-off value for T-SPOT.TB in pleural fluid. Parallel combination of T-SPOT.TB and ADA activity test in pleural fluid showed increased sensitivity (96.9%) and specificity (87.5%), whereas serial combination showed increased specificity (97.5%). The combination of 3 assays had the highest sensitivity at 97.9%, with an AUC value of 0.964.T-SPOT.TB in pleural fluid performed better than that in peripheral blood and the ADA activity test in pleural fluid for tuberculous pleurisy diagnosis. The optimal cut-off value of T-SPOT.TB in pleural fluid was 95.5 SFCs/2.5 × 105 cells. Combination of 3 assays might be a promising approach for tuberculous pleurisy diagnosis.

Diagnostic performance of T-SPOT.TB on peripheral blood in combination with adenosine deaminase on pleural fluid for the diagnosis of tuberculous pleurisy within different age group

Objective: To evaluate the performance of T cell enzyme-linked immuno-spot assay (T-SPOT) on peripheral blood in combination with adenosine deaminase (ADA) on pleural fluid for diagnosis of tuberculous (TB) pleurisy within different age groups. Methods: The data of patients with pleural effusion from the Department of Pulmonary and Critical Care Medicine of the First Affiliated Hospital of Wenzhou Medical University from April 2012 to November 2016 were retrospectively analyzed, and the diagnoses of these patients were histopathologically confirmed through medical thoracoscopy. The cases who had confirmed diagnosis, in the same time, received peripheral blood T-SPOT.TB were enrolled. The performance of peripheral blood T-SPOT.TB in combination with pleural fluid ADA on diagnosing TB pleurisy in the younger patients (16-59 years old) and elderly patients (≥60 years old) were analyzed respectively. The sensitivity, specificity and the receiver operating characteristic (ROC) curve were adopted for statistical analysis. Results: A total of 448 cases were finally enrolled, 341(76.1%) confirmed with TB pleurisy, 224 males, 117 females, (47±19) years old; and 107 (23.9%) classified as non-TB pleurisy, 65 males, 42 females, (61±14) years old. There were 285 cases who were classified as younger group, and the other 163 cases were classified as elderly group. The sensitivity and specificity of peripheral blood T-SPOT.TB were 85.4% (204/239) and 71.7% (33/46) in the younger patients, 76.5% (78/102) and 59.0% (36/61) respectively in the elderly patients. The sensitivity of peripheral blood T-SPOT.TB in the younger patients was significantly higher than that in the elderly patients (P=0.047). The sensitivity and specificity were 99.2% and 95.7% in combination with peripheral blood T-SPOT.TB and pleural fluid ADA respectively in the younger patients. The area under ROC curve (AUC) of T-SPOT.TB in the younger patients was 0.833, AUC of T-SPOT.TB combined with ADA was 0.911. The combination test of 2 tests had the sensitivity of 96.1% and the specificity of 90.2% respectively in the elderly patients. The AUC of T-SPOT.TB in the elderly patients was 0.747, AUC of T-SPOT.TB combined with ADA was 0.911. Conclusion: Peripheral blood T-SPOT.TB combined with pleural fluid ADA can improve the diagnostic performance for TB pleurisy with different ages, especially for elderly patients who can't tolerate pleural biopsy.

Evaluation of interferon-gamma release assay (T-SPOT.TB(™) ) for diagnosis of tuberculosis infection in rheumatic disease patients.

Patients with rheumatic diseases are at higher risk of developing tuberculosis (TB). The objective of this prospective study was to evaluate the T-SPOT.TB assay (T cell enzyme-linked immuno-spot assay), for the diagnosis of tuberculosis infection in rheumatic disease patients in China. Prospectively enrolled patients came from the Department of Rheumatology, Renji Hospital, Shanghai Jiao Tong University School of Medicine between July 2008 and Aug 2012 for TB screening. Subjects' histories of TB infection, previous TB contact or bacille Calmette-Guérin vaccination and concurrent immunosuppressive therapy, were reviewed carefully and recorded in detail. TST (tuberculin skin test) and TSPOT.TB assay were performed on the subjects. A prospective evaluation by Chi-square test was used for sensitivity and specificity of both TST and T-SPOT assay. A total of 311 subjects included 114 (36.66%) male and 197 (63.34%) female subjects, with a median age of 37.7 ± 12.6 years (range 17-72). Thirty-two patients (10.29%) had a history of TB infection or previous TB contact; 256 patients (82.32%) were using glucocorticoids or immunosuppressants; 28 patients (9.0%) were clinically diagnosed as having TB infection. The sensitivity and specificity of TST for TB screening in rheumatic disease patients was 81.82% (9/11) and 67% (67/100), respectively. However, the sensitivity and specificity of T-SPOT assay was statistically higher at 92.86% (26/28) and 93.64% (265/283), respectively (P < 0.05). As a new immunoassay for TB diagnosis, the sensitivity and specificity of T-SPOT is higher than TST. It is of great importance in the diagnosis of active or latent TB infection in rheumatic disease patients.

Gamma Interferon Immunospot Assay of Pleural Effusion Mononuclear Cells for Diagnosis of Tuberculous Pleurisy

Diagnosis of tuberculous pleurisy remains a challenge in the clinic. In this study, we evaluated the usefulness of a previously developed Mycobacterium tuberculosis antigen-specific gamma interferon enzyme-linked immunospot (ELISPOT) assay in the diagnosis of tuberculous pleurisy by testing a cohort of 352 patients with pleural effusion. We found that M. tuberculosis antigen-specific gamma interferon-producing cells were enriched four to five times in pleural fluid compared with their levels in peripheral blood from patients with tuberuclous pleurisy assayed in parallel. The sensitivity, specificity, positive predictive value, and negative predictive value of the pleural fluid mononuclear cell ELISPOT assay for the diagnosis of tuberculous pleurisy were 95.7%, 100%, 100%, and 81.0%, respectively. In comparison, the sensitivity and specificity of the ELISPOT assay using peripheral blood mononuclear cells were 78.3% and 86.3%, respectively. The sensitivity and specificity of the pleural fluid adenosine deaminase activity test were 55.5% and 86.3%, respectively. These results demonstrate that the M. tuberculosis antigen-specific ELISPOT assay performed on pleural fluid mononuclear cells provides an accurate, rapid diagnosis of tuberculous pleurisy.

B cell memory is directed toward conformational epitopes of parvovirus B19 capsid proteins and the unique region of VP1.

Loss of antibody reactivity against linear epitopes of parvovirus B19 (B19) capsid proteins VP1 and VP2 occurs after infection; however, it is unclear whether B cell memory is established against linear epitopes. B cell enzyme-linked immunospot assay was used to evaluate B19-specific B cell memory in volunteer donors (n=22). B cell memory is maintained against conformational epitopes of VP2 and is absent against linear epitopes of VP2. Individuals seronegative for IgG against the unique region of VP1 have detectable B cell memory, with the potential to mount a humoral response on reexposure to B19. Conversely, in mice immunized with VP2, long-lasting IgG against linear epitopes of VP2 and a strong B cell-memory response are observed. B cell memory is established and maintained against conformational epitopes of VP2 and against linear epitopes of VP1 but not against linear epitopes of VP2. These findings further our understanding of the immune response to B19 and suggest that analysis of B19-specific B cell memory merits consideration for future B19-vaccine studies.