Ninety-six-well microtiter plates (Corning, Lowell, MA) were coated with 1 μg/well of recombinant PA (List Biological Laboratories, Campbell, CA) or 0.02 μg/well recombinant HBs (Cell Sciences, Canton, MA). Diluted plasma was added, followed by anti-human IgG conjugate (Jackson ImmunoResearch, West Grove, PA) and para-nitrophenylphosphate substrate (Sigma-Aldrich, St. Louis, MO), with washing between steps. The optical density (OD) was detected using a Dynex MRX II microplate reader (Dynex Technologies, Chantilly, VA). Concentration of PA and HBs antibodies were calculated from standard curves of the reference sera AVR801 (BEI Resources, Manassas, VA) [27 (link)] and WHO international standard 07/164 (NIBSC, Potters Down, UK), respectively. Greater than 10 IU/L of anti-HBs is defined as positive or protected, and a good response is defined as 100 IU/L [8 (link), 28 (link)]. Non-human primate studies estimate the level of anti-PA IgG that predicts 80% survival in the year following a 3 dose priming series varies from 5.4–97.3 μg/mL depending on the time since last vaccination [29 (link)]. Therefore, individuals with anti-PA IgG < 5.4 μg/mL, 5.4–97.3 μg/mL, and >97.3 μg/mL are considered negative, equivocal, and positive, respectively.
Recombinant pa
Manufactured by List Biological Laboratories
Sourced in United States
Recombinant PA is a recombinant protein produced by List Biological Laboratories. It is the protective antigen component of the anthrax toxin.
Automatically generated - may contain errors
5 protocols using recombinant pa
1
Quantitative Antibody Assay for PA and HBs
2
Protease-based Anthrax Detection Assay
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All chemicals, including Protease Type XIII from Aspergillus saitoi (≥0.6 unit/mg) and deuterium oxide (≥99.6 atom % D), were purchased from Sigma-Aldrich (Milwaukee, WI, USA) unless noted otherwise. An ACE® Excel® SuperC18™ column (100 mm × 2.1 mm, 1.7 μm, 90 Å) was purchased from Advanced Chromatography Technologies Ltd. (Aberdeen, Scotland). Recombinant PA was purchased from List Biological Laboratories, Inc. (Campbell, CA, USA). Fully human monoclonal antibodies p6C01, p1C03, p1A06, and p6C04 were produced in HEK293 cells as previously described [9 (link),19 (link)].
3
Efferocytosis Assessment in M2 Macrophages
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Efferocytosis was assessed by flow cytometry [68 (link)]. Briefly, 5 × 105/well monocyte-derived human M2(IL-4+IL-10+Dex) macrophages or M2(Dex) macrophages were preincubated with 10 nM recombinant PA (List Biological Laboratories, Campbell, CA, USA), 10 nM recombinant EF (List Biological Laboratories), or various concentrations of PA + EF (ET) for 4 h at 37 °C in 5% CO2 atmosphere. Fluorescently-labeled apoptotic human PMN were preincubated with 10% serum, Protein S, or MFGE8 for 1 h unless otherwise noted and then added to the macrophages, resulting in a macrophage:PMN ratio of 1:5. After 1 h at 37 °C in 5% CO2 atmosphere, the percentage macrophages containing intracellular (eFluor 670+) but not surface-bound (surface CD66b−) PMN was assessed by flow cytometry.
4
CD4+ T Cell Activation and Toxin Treatment
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CD4 + T cells were isolated using the above procedure for two human subjects. After equilibration in RPMI-1640 with 10% FBS, cells were stimulated with 25 ng/mL PMA and 1 mM Ionomycin (P/I or π) or vehicle control (2.5 µL EtOH and 1.66 µL DMSO in 10 mL of culture media). Thirty minutes after activation or addition of the vehicle control, cells were treated with 2.5 µg/mL Recombinant PA (List Biological Laboratories 171D) and 500 ng/mL Recombinant LF (List Biological Laboratories 172A) or vehicle control. Twenty-four hours later, media from non-activated, non-activated with toxin treatment, activated, and activated with toxin treatment wells was collected for ELISA.
5
Quantitative Serum Anthrax Toxin Assay
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PA was measured
in serum using PA-ECL screening assay kit MesoScale Discovery (L15CA-1),
which utilizes the anti-PA antibody. To quantitate the levels of PA
in each serum sample, a standard curve (0–100 ng/mL) was analyzed
in parallel on each assay day by using recombinant PA (List Biological
Laboratories, Inc., 171E). Test samples were assayed in duplicate.
Anti-PA IgGs were measured in serum via ECL similar to the PA-ECL
screening assay. Biotinylated recombinant PA63 were bound
to streptavidin-coated plates (MSD) and used as the capture antigen.
Detection was accomplished using SULFO-TAG labeled antirabbit antibody
and read buffer (MSD).
in serum using PA-ECL screening assay kit MesoScale Discovery (L15CA-1),
which utilizes the anti-PA antibody. To quantitate the levels of PA
in each serum sample, a standard curve (0–100 ng/mL) was analyzed
in parallel on each assay day by using recombinant PA (List Biological
Laboratories, Inc., 171E). Test samples were assayed in duplicate.
Anti-PA IgGs were measured in serum via ECL similar to the PA-ECL
screening assay. Biotinylated recombinant PA63 were bound
to streptavidin-coated plates (MSD) and used as the capture antigen.
Detection was accomplished using SULFO-TAG labeled antirabbit antibody
and read buffer (MSD).
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