Bacterial Toxin Research Citations

MATERIALS AND METHODS:

… cholera toxin was obtained from List Biological. …

Stx partitioning assay:

Culture supernatants of Stx1 and Stx2a, purified Stx1 (5 µg ml¹) (BEI Resources) containing an A:B subunit ratio of 1:5 (as demonstrated by staining with Sypro Orange protein dye), and purified cholera toxin (5 µg ml¹) (List Biological) were partitioned via centrifugal filtration (Amicon Ultracel 50-kDa cutoff; Millipore) into two fractions: >50 kDa (corresponding to holotoxin) and <50 kDa (corresponding to unassembled A and B subunits). …

• Product #100B – Cholera Toxin (AZIDE-FREE) from Vibrio cholerae

Reagents: 

 Recombinant full-length protective antigen (PA), lethal factor (LF), edema factor (EF) and 
 fluorescein-conjugated PA were purchased from List Biological Laboratories  (Campbell, CA, 
 U.S.A.).

PA-binding assay:

 To detect PA-binding ability of the cells, cells were washed twice with ice-cold PBS, scraped, and 
 resuspended in ice-cold PBS (4 x 106 cells / ml). Cell suspensions (each 25 l) were reacted with 1
 mg/ml of FITC-PA in 1.5 ml tubes and incubated at 4 C for 60 min in the dark.  The cells were
 then washed twice with 2 ml ice-cold PBS and re-suspended in 0.5 ml ice-cold PBS for
flowcytometric analysis as described above.

RESULTS:

 Sensitivity to ATX-mediated cytotoxicity has been confirmed in several cell types derived from 
 mice and humans.  Murine (Raw264.7 or J774.1) or human (THP1, U937 or K562) cells were 
 treated with 300 ng/ml of LF and with the indicated concentration of PA, and viabilities of the cells 
 were examined 24, 48 or 72 hr after the treatment (Fig. 1A).  In the case of murine cells, ATX
 treatment showed a cytotoxic effect in a PA dose-dependent manner at 24 hr after treatment. 
 Comparing with murine cells, human U937 and K562 cells had exhibited resistance to 
 ATX-mediated cytotoxicity in the presence of a high concentration (500 ng/ml) of PA.

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).

• Product #171 – Anthrax Protective Antigen (PA), Recombinant from B. anthracis 

Author did not specify which List Labs Anthrax Edema Factor was utilized.  List Labs provides Product #178A(Anthrax Edema Factor (EF), Recombinant from B. anthracis) and Product #173B (Anthrax Edema Factor (EF), Recombinant, Mutant S447N from B. anthracis).

…rPA was obtained from List Biological Laboratories, Inc. The assay endpoint is reported as the serum mean concentration of anti-PA specific IgG (g/mL). … viability. rPA and rLF were obtained from List Biological Laboratories, Inc. …

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).

• Product #171 – Anthrax Protective Antigen (PA), Recombinant from B. anthracis

Anthrax LeTx Forming Assay (TFA):

A mouse macrophage cell line (J774A.1 cells, ATCC TIB-67, Manassas, VA) were plated in 96-well flat-bottomed tissue culture plates at 2.5×10⁴ cells/well in 50 L and incubated for 1619 hours at 37C with 5% CO₂. pp-PA83 variants were titrated on the plated cells in the presence or absence of LF (List Biological Laboratories, Campbell, CA).

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).

… Recombinant, nicked anthrax protein PA63 from B. anthracis for the bilayer experiments was obtained from List Biological Laboratories Inc., Campbell, CA. …

• Product #174 – Anthrax Protective Antigen, Activated (PA 63) from B. anthracis

Materials and Reagents:

Anthrax lethal toxins (PA and LF) and Escherichia coli O111:B4 LPS were purchased from the List Biological Laboratories.

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

RESULTS:

HDAC8 inhibition restores the production of pro-IL-1 in response to LPS in LeTx-exposed murine macrophages. LeTx suppresses expression of various inflammatory cytokines through inactivating MEKs (3-5). Consistent with these reports, a sub-lethal dose of LeTx (100 ng/ml PA & LF, a.a. for 4 h) completely cleaved MEK1 and inhibited expression of pro-IL-1 induced by LPS (100 ng/ml) for more than 2 days in RAW264.7 macrophages (Fig. 1A). …

… Alternatively, macrophages were stimulated using E. coli lipopolysaccharide (100 ng ml 1 ; Sigma-Aldrich, St Louis, MO, USA), Anthrax protective antigen and Anthrax lethal factor (List Biological Laboratories, Campbell, CA, USA), …

• Product #171 – Anthrax Protective Antigen (PA),Recombinant from B. anthracis
• Author did not specify which List Labs Lethal Factor was utilized.  List Labs offers Product #172 (Anthrax Lethal Factor (LF), Recombinant from B. anthracis) and Product #169 (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence).

ELISPOT:

Peripheral blood mononuclear cells (PBMCs) were prepared and washed in RPMI medium supplemented with 10% fetal calf serum, penicillin, streptomycin, and L-glutamine. …The antigen, recombinant Protective Antigen (rPA), LF or EF (List Biological Laboratories Campbell, Calif.) was added at a final concentration of 12.5 g/ml for PA or 25 g/ml for either LF or EF. The plates were incubated overnight at 37C in a 5% CO₂, humidified atmosphere. …

Quantitative anti-PA IgG ELISA:

Wells of 96-well microtiter plates (Immulon 2HB; Dynex Technologies, Chantilly, Va.) were coated by adding of 100 l rPA (List Biological Laboratories Campbell, Calif.) diluted to 1 g/ml in PBS. Plates were incubated overnight at 4C. …

Lethal toxin neutralization assay (TNA):

…Lethal toxin (LeTx; 100 ng of rPA/ml and 50 ng of LF/ml, final concentrations) was prepared in the same medium and pre-incubated with the sample dilutions in a humidified incubator set at 37C, 5% CO2 for 1 h. LeTx was prepared using PA obtained from List Biological Laboratories …

End-point ELISA for LF and EF:

Wells of 96-well microtitre plates (Immulon 2HB) were coated with either LF or EF (List Biological Laboratories) at 1 g/ mL in PBS (100 L per well). …

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).

• Product #171 – Anthrax Protective Antigen (PA), Recombinant from B. anthracis

 Author did not specify which List Labs Anthrax Edema Factor was utilized.  List Labs provides Product #178A(Anthrax Edema Factor (EF), Recombinant from B. anthracis) and Product #173B (Anthrax Edema Factor (EF), Recombinant, Mutant S447N from B. anthracis).

Chemicals and Reagents:

… Recombinant anthrax lethal factor (LF) from B. anthracis was from List Biological Laboratories (Campbell, California).

Characterisation of the thresholds for LF and EF detection:

In a first step, we characterised the threshold for the enzymatic quantification of LF and EF by using plasma and ear tissue samples derived from mice infected…

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).

Bacterial Strains and Reagents:

… Activated protective antigen was procured form List Biological Laboratories (USA). …

2.6. Binding to LF and Native-PAGE

For the confirmation of functional activity of the expressed PA, 2g of trypsin digested PA was incubated with 2g of LF at room temperature for 30 minutes. The samples were run on 4% native polyacrylamide gel at 4C and visualized by staining the gel with coomassie brilliant blue.

2.7. Animal Immunization

Six female Balb/c mice were procured from Institutional Animal Care Center, DRDE, and were provided with food and water ad libitum. Each mouse was immunized subcutaneously with 10g PA adsorbed on aluminium hydroxide adjuvant on days 0, 14, and 28. Mice receiving only PBS were taken as control. All mice were bled prior to first immunization on day 0 and on 35th day. Serum was separated and stored at 20C until further use.

2.8. Enzyme Linked Immunosorbent Assay

To test the reactivity of PA immunized sera, indirect ELISA was performed by coating 1gmL¹ PA in ELISA plate overnight at 4C. After blocking with 5% skim milk for one hour, serial twofold dilution of PA immunized sera starting from 1:1000 was added and incubated for one hour at room temperature. …

2.9. Cell Culture

Mouse macrophage cell line J774.1 was procured from National centre for cell sciences, Pune, India and was maintained in high glucose Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS) and 100UmL¹ penicillin and streptomycin as antibiotics. One day before the experiment 4 10⁴ cells were seeded in a 96 well plate and incubated at 37C in presence of 5% humidified CO₂. The next day spent media was replaced by a fresh medium containing gradient concentration of soluble PA and native PA of B. anthracis from List Biologicals (0.005gmL¹ to 5gmL¹) and was incubated at 37C along with 0.125gmL¹ LF. After 4 hours, 20L of 5mgmL¹ MTT (3-(4,5 dimethylthiazol-2 yl)-2,5 diphenyl tetrazolium bromide) was added to each well and further incubated for 30 minutes.

• Product #171 – Anthrax Protective Antigen (PA), Recombinant from B. anthracis
• Product #174 – Anthrax Protective Antigen, Activated (PA 63) from B. anthracis