Citations

Materials:

Anthrax rPA and recombinant LF rLF were obtained from List Biological Laboratories, Inc. (Campbell, California). …

Macrophage Toxicity Assay

Mouse macrophages are sensitive to LeTx(the combination of PA and LF )-induced necrosis.. Here, rLF was added to the reconstituted powder formulation and the activity/stability of rPA was evaluated by measuring the PA dependent toxicity of rLF using mouse macrophage J774A.1 cells …

Anthrax lethal toxin neutralization assay:

Mouse macrophages are sensitive to LeTx(the combination of PA and LF )-induced necrosis. An in vitro lethal toxin (LeTx) neutralization assay using J774A.1 mouse macrophage cells was performed to determine the functional titer of the anti-PA antibody present in rabbit serum. The details of the assay were reported previously ³⁵. Briefly, rabbit sera were incubated with PA for 1 hr at 37C to allow neutralization to occur, followed by incubation with added LF at room temperature for 1 hr. The serum-PA/LF mixture was then added to the plate containing macrophage cells. The starting serum dilution on the final plate is 1:128 or 1:2⁷ with a final concentration of 0.1875 g of LeTx (rPA+rLF) per ml. Percent neutralization was calculated based on (sample OD value LeTx standard OD value)/(cells only OD value-LeTx standard OD value) 100. The percent neutralization was plotted vs serum log₂ dilutions and fitted with a second order polynomial curve. The serum dilution able to neutralize 50% of LeTx (NT50) was calculated. …

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

Immunization and infection of mice:

Mice were immunized by subcutaneous (s.c.) injection with 10 g of recombinant PA (List Biological Laboratories Inc.) and 0.3% aluminum hydroxide gel (Sigma-Aldrich) in 100 l of vaccine 20 days before infection, with a booster given 10 days before infection. …

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

… Each well of a high binding microtiter plate (Thermo Fisher Scientific, Waltham, MA) was coated with 0.25 µg of cholera toxin (List Biological Laboratories, Campbell, CA) diluted in coating buffer over night at 4°C …

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

-Allergic response – Oral sensitization and immune challenge:

Cv, Gn and Gf mice (2730 per group) were divided into two subgroups of c. 15 mice each. Oral sensitizations were performed by intragastric infusion. One subgroup received whey proteins (WP, Lacprodan 80; Arla, Lyon, France; 15 mg per mouse) and cholera toxin (CT) as an adjuvant (List Biological, Campbell, California; 10 µg per mouse) in 0.9% NaCl (sensitized group). The other subgroup received CT alone in 0.9% NaCl (control group). The sensitizations were performed five times, at weekly intervals, from day 16 to day 44. …

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

Mice and immunizations with the YopE69-77 peptide:

Female C57BL/6J mice (Jackson Laboratory) were used for all experiments. Animals that were 9 to 10 weeks old were anesthetized with a mixture of ketamine (60 mg/kg of body weight) and xylazine (10 mg/kg) delivered by intraperitoneal (i.p.) injection. A mixture of 1 µg of cholera toxin (CT; List Biological Laboratories Inc., Campbell, CA) … Control mice received CT in 15 µl of PBS only. …

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

… The mice were orally sensitized twice per week for 4 weeks with 1 mg of Ovm (19) adjuvanted with 10 µg of cholera toxin (CT; List Biologicals, Campbell, CA, USA) (8), as outlined in Fig. 1. The negative control group received CT alone (n = 10). …

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

… These mononuclear cells were stimulated by 100 ng/ml LPS (Salmonella minnesota R595 ultra pure LPS; List Biological Laboratories, Campbell, CA) and sampled at 0 (before stimulation), 2, 4, 8, and 24hr. …

• Product #434 – ULTRA PURE LPS from Salmonella minnesota R595 (Re)

Reagents:

… An ultrapurified form of E. coli 0111:B4 LPS was purchased from List Biological Lab. Inc. (Campbell, CA). …

• Product #421 – ULTRA PURE LPS from Escherichia coli O111:B4

… Ultra pure LPS from Escherichia coli 0111;B4 was obtained from List Biological Laboratories (CA). …

• Product #421 – ULTRA PURE LPS from Escherichia coli O111:B4

Treatment of reproductive tract cells with PAMP:

To determine whether PAMP affected antigen presentation by uterine and vaginal cells, PAMP were added to individual wells in antigen presentation assays at the start of each experiment. To measure the effect of PAMP on co-stimulatory molecule expression, PAMP were added to epithelial cells, stromal cells and vaginal cells (5 10⁵ cells/100 ml) at the start of each 24 hr incubation period as previously described.^28,44 The TLR agonists used in these studies included TLR1/TLR2: Pam₃Cys-Ser-(Lys)4 (P3C, EMC Microcollections, Tbingen, Germany) at a final concentration of 10 g/ml; TLR2/TLR6: Peptidoglycan from Staphylococcus aureus (InvivoGen), 10 g/ml; TLR3: Poly(I:C) (InvivoGen), 25 g/ml; TLR4: ultra-pure lipopolysaccharide from Salmonella minnesota (LPS; List Biological Laboratories, Inc., Campell, CA), … Wells containing media alone were included in all experiments as controls. The TLR agonists were tested for LPS contamination using the Limulus assay …

• Product #304 – LPS from Salmonella minnesota R595 (Re)