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Ag85b

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Ag85B is a recombinant protein that serves as a diagnostic antigen. It is produced using Escherichia coli as the expression system.

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Lab products found in correlation

Esat 6, by BEI Resources (4 mentions) Ag85a, by BEI Resources (4 mentions) Cfp 10, by BEI Resources (3 mentions) Ag85c, by BEI Resources (2 mentions) Mtb h37rv lysate, by BEI Resources (1 mentions) Recombinant ag85b, by BEI Resources (1 mentions) Purified lam, by BEI Resources (1 mentions) Dimethyl sulfoxide (dmso), by Merck Group (1 mentions) Psts1, by BEI Resources (1 mentions) Purified protein derivative, by Statens Serum Institut (1 mentions) 3 3 5 5 tetramethylbenzidine (tmb), by BD (1 mentions)

7 protocols using ag85b

1

Mycobacterial Antigen Antibody Assay

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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).
Platteel A.C., Nieuwenhuizen N.E., Domaszewska T., Schürer S., Zedler U., Brinkmann V., Sijts A.J, & Kaufmann S.H. (2017). Efficacy Testing of H56 cDNA Tattoo Immunization against Tuberculosis in a Mouse Model. Frontiers in Immunology, 8, 1744.
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2

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.
van Woudenbergh E., Irvine E.B., Davies L., de Kock M., Hanekom W.A., Day C.L., Fortune S, & Alter G. (2020). HIV Is Associated with Modified Humoral Immune Responses in the Setting of HIV/TB Coinfection. mSphere, 5(3), e00104-20.
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3

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.
Khan A., Singh S., Galvan G., Jagannath C, & Sastry K.J. (2017). Prophylactic Sublingual Immunization with Mycobacterium tuberculosis Subunit Vaccine Incorporating the Natural Killer T Cell Agonist Alpha-Galactosylceramide Enhances Protective Immunity to Limit Pulmonary and Extra-Pulmonary Bacterial Burden in Mice. Vaccines, 5(4), 47.
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4

Preparation of Mycobacterium tuberculosis Antigens

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Mtb fractions and recombinant Mtb proteins (CFP10, PstS1, Ag85A, Ag85B, Ag85C, MPT32 and ESAT6) were obtained through BEI Resources (Manassas, Virginia). Mtb fractions and antigens were dissolved in either DMSO or PBS and stored as aliquots at -20°C.
Shang S., Siddiqui S., Bian Y., Zhao J, & Wang C.R. (2016). Nonclassical MHC Ib-restricted CD8+ T Cells Recognize Mycobacterium tuberculosis-Derived Protein Antigens and Contribute to Protection Against Infection. PLoS Pathogens, 12(6), e1005688.
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5

T Cell and Antibody Assays for Tuberculosis

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For T cell assays, overlapping peptide pools targeting ESAT-6 (BEI Resources Cat#NR-50711), CFP-10 (BEI Resources Cat#NR-50712), Ag85A (BEI Resources Cat#NR-34827), Ag85B (BEI Resources Cat#NR-34828), and TB10.4 (BEI Resources Cat#NR-34826) as well as H37Rv M.tb whole cell lysate (BEI Resources Cat#NR-14822) were used. Staphylococcal Enterotoxin Type B (SEB) (List Biological Laboratories Cat#122) was a positive assay control, and dimethyl sulfoxide (DMSO) (Sigma-Aldrich Cat#D8418) was a negative assay control.
For antibody assays, M.tb antigens tested were: purified protein derivative (PPD) (Statens Serum Institute), Ag85A and B in a 1:1 ratio (BEI Resources Cat#NR-49427 and #NR-53526), recombinant ESAT-6 (BEI Resources Cat#NR-49424) and CFP-10 (BEI Resources Cat#NR-49425) in a 1:1 ratio, HspX (BEI Resources Cat#NR-49428), 1-tuberculosyladenosine (1-TbAd) (provided by Dr. Branch Moody), and lipoarabinomannan (LAM) (BEI Resources Cat#NR-14848). An equal mixture of influenza antigens from HA1(B/Brisbane/60/2008) and HA1(H1N1) (A/New Caledonia/20/99) (Immune Technology Corp ITIT-003-001p and IT-003-B3p) was used as a positive assay control.
Davies L.R., Smith M.T., Cizmeci D., Fischinger S., Shih-Lu Lee J., Lu L.L., Layton E.D., Grant A.D., Fielding K., Stein C.M., Boom W.H., Hawn T.R., Fortune S.M., Wallis R.S., Churchyard G.J., Alter G, & Seshadri C. (2023). IFN-γ independent markers of Mycobacterium tuberculosis exposure among male South African gold miners. eBioMedicine, 93, 104678.
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6

Intradermal Antigen Response in Guinea Pigs

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Nine weeks after the last immunization, guinea pig flanks were shaved and injected intradermally with 10 μg of either Ag85B (BEI Resources) or 2 μg PPD (Statens Serum Institute) in 100 μl of PBS. After 24 h, the diameter of erythema and induration was measured.
Gupta T., LaGatta M., Helms S., Pavlicek R.L., Owino S.O., Sakamoto K., Nagy T., Harvey S.B., Papania M., Ledden S., Schultz K.T., McCombs C., Quinn F.D, & Karls R.K. (2018). Evaluation of a temperature-restricted, mucosal tuberculosis vaccine in guinea pigs. Tuberculosis (Edinburgh, Scotland), 113, 179-188.
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7

Antibody Binding Assay for Mycobacterial Antigens

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Ag85A (catalog number NR-14871; BEI Resources), Ag85B (our purified recombinant), or Ag85C (catalog number NR-14858; BEI Resources) was used to coat wells at 0.5 µg/well in phosphate-buffered saline (PBS) and incubated overnight at 4°C. The plates were washed 3 times with PBS (pH 7.4) with 0.05% Tween 20 and blocked with PBS containing 1.0% BSA for 1 h. A starting concentration of 5 µg/ml of mAb 710, 711, or 712 was serially diluted, and 200 µl/well was incubated at room temperature for 2 h. Plates were washed 5 times and incubated with goat anti-mouse IgG-HRP (MP Biomedicals) for 1 h at room temperature. Plates were washed 7 times and developed with the 3,3′,5,5′-tetramethylbenzidine (TMB) substrate according to the manufacturer’s instructions (BD). The reaction was stopped with 2 M sulfuric acid, and the absorbance was read at 450 nm with a Synergy H1 microplate reader (BioTek).
Ernst J.D., Cornelius A, & Bolz M. (2019). Dynamics of Mycobacterium tuberculosis Ag85B Revealed by a Sensitive Enzyme-Linked Immunosorbent Assay. mBio, 10(2), e00611-19.
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