Ag85a

Manufactured by BEI Resources

Ag85A is a recombinant protein produced by BEI Resources. It is a component of the tuberculosis (TB) bacterium Mycobacterium tuberculosis. Ag85A is a key antigen involved in the host immune response to TB infection.

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Lab products found in correlation

Ag85b, by BEI Resources (4 mentions) Cfp 10, by BEI Resources (3 mentions) Esat 6, by BEI Resources (2 mentions) Ag85c, by BEI Resources (2 mentions) Purified lam, by BEI Resources (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) Anti cd3 clone ucht1, by Thermo Fisher Scientific (1 mentions) Fitc anti cd8 clone 53 6, by BioLegend (1 mentions) γ irradiated m tb h37rv, by BEI Resources (1 mentions)

5 protocols using ag85a

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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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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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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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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Comprehensive Immune Cell Profiling

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FITC anti-CD4 (clone GK1.5, Biolegend), FITC anti-CD8 (Clone 53–6.7, Biolegend), FITC anti-CD14 (Clone 61D3, eBiosciences), qdot605 anti-CD14 (clone Tük4, Invitrogen), V450 anti-CD2 (clone RPA-2.10, eBioscience), anti-CD3 (clone UCHT1, eBioscience), anti-CD19 (clone HIB19, eBioscience), anti-CD20 (clone 2H7, eBioscience), anti-CD56 (clone B159, eBioscience), PE-Cy7 anti-CD16 (clone 3G8, Biolegend), APC-Cy7 anti-HLA-DR (clone L243, BD biosciences), PE anti-CD142/TF (clone HTF-1,eBioscience) and PerCP-Cy5.5 anti-TNF-α (clone MAb11, eBioscience) were used for flow cytometry, γ-irradiated M. tb H37Rv and Ag85a was obtained from BEI Resources.

Venkatasubramanian S., Tripathi D., Tucker T., Paidipally P., Cheekatla S., Welch E., Raghunath A., Jeffers A., Tvinnereim A.R., Schechter M.E., Andrade B.B., Mackman N., Idell S, & Vankayalapati R. (2015). TISSUE FACTOR EXPRESSION BY MYELOID CELLS CONTRIBUTES TO PROTECTIVE IMMUNE RESPONSE AGAINST Mycobacterium tuberculosis INFECTION. European journal of immunology, 46(2), 464-479.

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