Mycobacteria-, Ag85B- and ESAT-6-specific antibodies in serum were measured by indirect enzyme-linked immunosorbent assay using Mtb H37Rv lysate (BEI Resources), recombinant Ag85B (BEI Resources), or recombinant ESAT-6 (BEI Resources) to coat and AP-labeled anti-mouse IgG, IgG1, and IgG2c for detection (SouthernBiotech).
Esat 6
Manufactured by BEI Resources
Sourced in United States, Denmark
ESAT-6 is a laboratory analysis instrument that measures the secretion of ESAT-6 protein. ESAT-6 is a key component of the early secreted antigenic target of Mycobacterium tuberculosis. The ESAT-6 assay is used to detect the presence and quantity of this protein in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Cfp 10, by BEI Resources (6 mentions)
Ag85b, by BEI Resources (4 mentions)
Ag85a, by BEI Resources (2 mentions)
Guava easycyte, by Merck Group (2 mentions)
Culture filtrate protein cfp 10, by BEI Resources (2 mentions)
Lsrfortessa, by BD (2 mentions)
Mtb h37rv lysate, by BEI Resources (1 mentions)
Recombinant ag85b, by BEI Resources (1 mentions)
Fasl pe, by Thermo Fisher Scientific (1 mentions)
Amnis imagestream mark 2, by Merck Group (1 mentions)
Purified lam, by BEI Resources (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Magnetic carboxylated beads, by Bio-Rad (1 mentions)
Psts1, by BEI Resources (1 mentions)
Ag85c, by BEI Resources (1 mentions)
Anti ifn γ apc, by BioLegend (1 mentions)
Anti cd56 fitc, by BioLegend (1 mentions)
Anti cd3 fitc, by BioLegend (1 mentions)
Cd14 antibody, by Miltenyi Biotec (1 mentions)
16 protocols using esat 6
1
Mycobacterial Antigen Antibody Assay
2
Investigating Antigen-Specific T Cell Responses
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Isolated CD4 þ T cells were activated with ESAT-6 (BEI Resources) and retinal crude extract, respectively, in the absence of CD28, for 10 hours under standard cell culture conditions. Then cells were stained for intra/extra cellular proteins, FasL-PE (Invitrogen), LAMP-1 FITC (eBioscience). Samples were acquired and analyzed by using Amnis Image-Stream Mark II (Merck Millipore, Seattle, WA, USA) with appropriate controls.
3
Comparative Assessment of Pathogen Antigens
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Antigens derived from M. tuberculosis , HIV, and various control pathogens were utilized across multiple assays. An HIV-1 clade B/C consensus gp120 antigen was acquired from Immune Technology. PPD was received from the Statens Serum Institute. Purified LAM, ESAT6, CFP10, Ag85A, and Ag85B were all acquired from BEI Resources. Tetanus toxoid was received from Massachusetts Biologics. PPSV23 is the pneumococcal 23-valent vaccine from Merck Sharp & Dohme Corporation. A pool of recombinant influenza hemagglutinin (HA) antigens (HA1-B/Florida/4/2006, HA-B/Malaysia/2506/2004, H1N1-A/Solomon island/3/2006, H3N2-A/Wisconsin/67/X-161/2005, H3N2-A/Brisbane/10/2007, H1N1-A/New Caledonia/20/99, and H1N1-A/Brisbane/59/2007; Immune Technologies) representing dominant strains from the past 10 years were combined to generate the influenza HA control antigen.
4
ESAT-6 Induced NLRP3 Activation in RPE
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ESAT-6 (BEI Resources, USA, catalogue #NR-14868), dissolved in Hanks BSS, as per manufacturer's instructions was used for treating RPE cell cultures. Cells with 80-90% confluence were treated for varying durations with 5µg of ESAT-6 per mL of culture medium in all experiments, except where other doses are mentioned. This dose was chosen based on reported use of 2.5µg dose of ESAT-6 for NLRP3 inflammasome activation in THP-1 macrophages [7] . 10 µg of exponential phase M. tuberculosis RNA, either untreated or following various enzymatic treatments (Table 1) was transfected into human or mouse RPE cells with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. A synthetically produced 300 nt long dsRNA, Alu RNA, that has been shown to cause NLRP3 dependent caspase-1 activation in the RPE (endogenously produced in age-related macular degeneration [14] , was similarly transfected to be used as positive control. Enrichment of ssRNA from total RNA was performed using a ssRNA enrichment kit (Zymo, Irvine, CA).
5
Analyzing T-cell Responses via Intracellular Cytokine Staining
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Cryopreserved PBMCs were thawed, washed and rested in RPMI 1640 media containing 10% heat-inactivated fetal bovine serum (FBS) overnight prior to antigen stimulation with a concentration of 2 x 10 6 cells /mL. PBMCs were counted with Guava easyCyte (Millipore) using Guava ViaCount reagent (Luminex) and GuavaSoft v.2.6 software. Samples with less than 66% viability were discarded. Cells were stimulated with a pool of early secretory Mtb antigen target-6 (ESAT-6) and culture filtrate protein (CFP-10) (BEI Resources). PMA (25ng/mL)/ionomycin (1ug/mL) was used as a positive control and DMSO (0.5%) was used as a negative control. In addition, costimulatory antibody anti-CD28/49d, cytokine secretion inhibitor Brefeldin A and Monensin were added to each stimulation cocktail. Cells were lysed and permeabilized with FACS Lyse and FACS Perm-II buffer and stained on a BD LSRFortessa with an antibody panel developed for analyzing CD4 and CD8 T-cell responses (ICS) including IFN-, TNF, IL-2, and IL-17a [1] (link) (Table S1). [24] Flow cytometry data were analyzed in FlowJo™ v10.7.
Each sample was compensated and gated manually.
Each sample was compensated and gated manually.
6
Recombinant Mtb Antigen Protein Preparation
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Recombinant Mtb proteins Ag85B and ESAT-6 were obtained from BEI Resources (Manassas, VA, USA), and dissolved in 1X PBS at a concentration of 5 mg/mL. The alpha-galactosylceramide (α-GalCer) was procured from Diagnocine LLC (Hackensack, NJ, USA) and dissolved in dimethyl sulfoxide, (Sigma, St. Louis, MO, USA) at a concentration of 1 mg/mL.
7
Multiplex Assay for Tuberculosis Antibodies
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A customised TB multiplex assay was designed to assess for Ab specific for Ag85B, MPT64, TX114 proteins, TB peptidoglycan, Ag85 complex and ESAT‐6 (BEI resources, Manassas, USA). ATBI‐positive serum from a different cohort was used as a control to verify Mtb antigen binding. H3 influenza haemagglutinin (Sino Biological A/Switzerland/9715293/2013, HA; Sino Biological, Beijing, China) was also included in the array as a positive antigen control. Briefly, magnetic carboxylated beads (Bio‐Rad, California, USA) were covalently coupled to Mtb and Flu antigens by carbodiimide reaction as previously described.47 The isotypes and subclasses (IgG, IgA1, IgA2, IgG1‐4) of antigen‐specific Abs were assessed as previously described.48
8
Preparation of Mycobacterium tuberculosis Antigens
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9
Detailed Multiparametric Immunophenotyping Protocol
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For flow cytometry, we used anti-CD3 FITC (BioLegend, USA, Cat#300440) and PerCP (BD Biosciences, USA, Cat#347344), anti-CD56 FITC (BioLegend, USA, Cat#318304) and APC (BD Biosciences, USA, Cat#555518), anti-CD16 PE (BioLegend, USA, Cat#302008); for staining memory cells anti-KLRG1 FITC (BioLegend, USA, Cat#138410), anti-CD27 PE (BD Biosciences, USA, Cat#555441); for intracellular staining anti-IL-32α (R&D Systems, USA, Cat#IC30402A) and anti-IFN-γ APC (BioLegend, USA, Cat#502512) antibodies were used. Magnetic beads conjugated to anti -CD56 (Miltenyi Biotec, Germany, Cat# 120-000-307) and -CD14 antibodies (Miltenyi Biotec, Germany, Cat# 120-000-305) were used for positive selection of NK cells and monocytes respectively.
For in vitro stimulation assays, we used ESAT-6 (BEI Resources, USA, Cat#NR-50711) and CFP-10 (BEI Resources, USA, Cat#NR-50712) peptide pools consisting of 21 and 22 individual peptides belonging to 6-kDa ESAT-6 and 10-kDa CFP-10 respectively. γ-irradiated Mtb H37Rv whole cells were used for some experiments (BEI resources, USA, Cat#49098).
For in vitro stimulation assays, we used ESAT-6 (BEI Resources, USA, Cat#NR-50711) and CFP-10 (BEI Resources, USA, Cat#NR-50712) peptide pools consisting of 21 and 22 individual peptides belonging to 6-kDa ESAT-6 and 10-kDa CFP-10 respectively. γ-irradiated Mtb H37Rv whole cells were used for some experiments (BEI resources, USA, Cat#49098).
10
Cryopreserved PBMC Stimulation and ICS
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Cryopreserved PBMCs were thawed, washed and rested in RPMI 1640 media containing 10% heat-inactivated fetal bovine serum (FBS) overnight prior to antigen stimulation with a concentration of 2 x 106 cells /mL. PBMCs were counted with Guava easyCyte (Millipore) using Guava ViaCount reagent (Luminex) and GuavaSoft v.2.6 software. Samples with less than 66% viability were discarded. Cells were stimulated with a pool of early secretory Mtb antigen target-6 (ESAT-6) and culture filtrate protein (CFP-10) (BEI Resources). PMA (25ng/mL)/ionomycin (1ug/mL) was used as a positive control and DMSO (0.5%) was used as a negative control. In addition, costimulatory antibody anti-CD28/49d, cytokine secretion inhibitor Brefeldin A and Monensin were added to each stimulation cocktail. Cells were lysed and permeabilized with FACS Lyse and FACS Perm-II buffer and stained on a BD LSRFortessa with an antibody panel developed for analyzing CD4 and CD8 T-cell responses (ICS) including IFN-ɣ, TNF, IL-2, and IL-17a (Table A in S1 Appendix ) [24 (link)]. Flow cytometry data were analyzed in FlowJo™ v10.7. Each sample was compensated and gated manually.
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