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Page 618 out of 625

Cationic polyamines inhibit anthrax lethal factor protease

Goldman ME, Cregar L, Nguyen D, Simo O, O'Malley S, Humphreys T
Product: Anthrax Lethal Factor (LF),Recombinant from B. anthracis

Lethal Factor protease assay:

Lethal Factor (20 nM final concentration) and MAPKK substrate (MAPKKide® 12.5 µM, final concentration) were purchased from List Biological Laboratories, Campbell, CA and used according to the fluorescence resonance energy transfer (FRET) method. ...

Author did not indicate which specific lethal factor was utilized.  List Labs provides Product #172 (Anthrax Lethal Factor (LF), Recombinant from B. anthracis) and Product #169 (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence).

 

PubMed ID: 16762077

Detection of Anthrax Toxin in the Serum of Animals Infected with Bacillus anthracis by Using Engineered Immunoassays

Mabry R, Brasky K, Geiger R, Carrion R Jr, Hubbard GB, Leppla S, Patterson JL, Georgiou G, Iverson BL
Product: Anthrax Protective Antigen, Activated (PA 63) from B. anthracis

Sandwich ELISAs for LF detection:

Recombinant PA83 and PA63 were purchased from List Laboratories (New Jersey). PA63 is a cleavage product that is capable of binding LF (45). For the sandwich ELISA, 50 μl of PA at 63 kDa and 83 kDa (6 μg/ml) was applied to a 96-well plate and blocked with 2% milk-PBS as described above. For initial assays, LF was diluted in PBS or human serum at 5 μg/ml. For assays detecting LF in infected animals, serum was added to the plate initially diluted 1:1 in 2% milk-PBS and then serially diluted across the plate in duplicate. After a 1-h incubation, the plate was washed as described above. Goat anti-LF polyclonal serum (List Labs) was diluted 1:1,000 in 2% milk-PBS and added to the plate for a 1-h incubation in duplicate. The plate was then washed, followed by the addition of goat anti-rabbit IgG-HRP conjugate (Bio-Rad) diluted in 2% milk-PBS for 1 h. ELISA reactions were developed with OPD tablets (Sigma) and quenched by the addition of 50 μl of 4.5 M H2SO4. ...

PubMed ID: 16760326

Anthrax Lethal Toxin Has Direct and Potent Inhibitory Effects on B Cell Proliferation and Immunoglobulin Production

Fang H, Xu L, Chen TY, Cyr JM, Frucht DM
Product: Anthrax PA 63 - FITC Conjugate

Reagents and antibodies:

Recombinant anthrax PA and LF were purchased commercially and were stored in 1:1 glycerol-water at −20°C (List Biological Laboratories) for in vitro studies. Unless otherwise indicated, anthrax LT was administered in excess at concentrations of 2.5 μg/ml PA and 1 μg/ml LF. In selected experiments a proteolytically inactive mutant of LF was used as a negative control (E687C substitution in zinc binding site that eliminates enzymatic activity; List Biological Laboratories). ...

Anthrax PA binding assays:

Purified murine or human B cells were cultured at 4°C for 30 min with FITC-labeled anthrax PA (50 μg/ml; List Biological Laboratories) in the presence or absence of unlabeled anthrax PA (150 μg/ml) to confirm specific binding. Stained cells were then washed with PBS and analyzed by flow cytometry (see below). Unstained cells were analyzed in parallel to establish background levels of autofluorescence.

ELISA:

Primary B cells were cultured in complete RPMI for 4 to 5 h, washed with RPMI 1640, and then stimulated as indicated in the presence or the absence of anthrax LT for 7 days.  ...

Murine in vivo studies:

Mice were treated with varying doses of anthrax LT as indicated, using a fixed ratio of LF/PA of 1:2.5. LF and PA were resuspended in PBS and injected i.p. into mice in a total volume of 1.0 ml of PBS. As a negative control, selected mice were treated with PBS alone. Mice were sacrificed 3 h after treatment, and spleens were harvested for primary B cell isolation as previously described. Primary B cells were then evaluated for proliferation and IgM production.

Author did not indicate which specific lethal factor was utilized.  List Labs provides Product #172 (Anthrax Lethal Factor (LF), Recombinant from B. anthracis) and Product #169 (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence).

PubMed ID: 16670324

Immunization with Staphylococcus aureus Clumping Factor B, a Major Determinant in Nasal Carriage, Reduces Nasal Colonization in a Murine Model

Schaffer AC1, Solinga RM, Cocchiaro J, Portoles M, Kiser KB, Risley A, Randall SM, Valtulina V, Speziale P, Walsh E, Foster T, Lee JC
Product: Cholera Toxin B Subunit (Choleragenoid) from Vibrio cholerae in Low Salt

Intranasal immunization with killed S. aureus:

Mice were immunized with an acapsular mutant of S. aureus Reynolds designated JLO22 (7) that was cultivated in TSB to the logarithmic phase of growth (A650, 0.34). The bacteria were pelleted, suspended in phosphate-buffered saline at a concentration of 108CFU/ml in an open petri dish, and exposed to a UV light source that was 8 cm away for 10 min on a rotator in the dark. The bacteria were concentrated by centrifugation, and 10 μl of the UV-killed bacterial suspension containing 108 CFU S. aureus with or without 5 μg of cholera toxin B (CTB) (List Biological Laboratories, Inc., Campbell, Calif.) was applied to each mouse nose on days 0, 5, and 10. CTB was omitted from the third immunization to reduce nonspecific protection. Two weeks after the third immunization, the mice were inoculated with 108 CFU S. aureus strain Newman cultivated in TSB to the logarithmic phase (A650, 0.34). Colonization was evaluated after 14 days. 

Author did not specify which CTB was utilized.  List Labs has provided Product #103B (Cholera Toxin B Subunit (Choleragenoid) from Vibrio cholerae) and Product #104 (Cholera Toxin B Subunit (Choleragenoid) from Vibrio cholerae in Low Salt).  The former has been discontinued.  The latter may be substituted for the former, if the researcher takes into account the difference in the buffer in which each product is provided and the amount of protein per vial.  When Product #103B was reconstituted with 200 μl of water, it contained 1 mg of protein in 0.05 M Tris, 0.2 M NaCl, 0.001 M Na2EDTA, 0.003 M NaN3, pH 7.5.  When Product #104 is reconstituted with 0.25 ml water, it contains 0.5 mg of Cholera Toxin B Subunit (choleragenoid) in 0.01 M Sodium Phosphate at pH 7.5.

PubMed ID: 16552044

Bovine Peptidoglycan Recognition Protein-S: Antimicrobial Activity, Localization, Secretion, and Binding Properties

Tydell CC, Yuan J, Tran P, Selsted ME
Product: LPS from Salmonella typhimurium

PGRP-S secretion:

Granulocyte-enriched populations of bovine peripheral leukocytes were purified as described above and suspended to 1.2 × 107 cells/ml in HBSS (137 mM NaCl, 5.6 mM glucose, 5 mM KCl, 4 mM NaHCO3 1 mM CaCl2, 0.5 mM MgCl2, 0.4 mM KH2PO4, 0.4 mM Na2HPO4, and 0.4 mM MgSO4 (pH 7.4)). Aliquots of the cell suspension were incubated for 60 min at 37°C in a final volume of 500 μl containing one of the following stimulants: 100 nM PMA, 20 μg/ml lipoteichoic acid (LTA) from Bacillus subtilis, 160 μg/ml muramyl dipeptide (MDP) (Sigma-Aldrich), 100 μg/ml LPS from Staphylococcus typhimurium (List Biological Laboratories), ...

PubMed ID: 16394004