Bacterial Toxin Research Citations

Diphtheria-Tetanus-Acellular Pertussis Multiplex Immunoassay

Total IgG concentrations against vaccine antigens pertussis toxin (PT), pertactin (PRN), filamentous hemagglutinin (FHA), fimbriae 2/3 (FIM 2/3), tetanus toxin (TT), and diphtheria toxoid (DT) were measured using an in-house multiplex bead-based immunoassay. PT, PRN, and FHA were kindly provided by GlaxoSmithKline (Belgium). TT was purchased from Sigma-Aldrich while DT and FIM 2/3 was sourced from List biological laboratories (USA). A standard curve was generated using a 10-step 3-fold serial dilution of an in-house reference sera previously quantified against National Institute for Biological Standards and Control reference sera: PT (06/140), TT (TE-3), and DT (10/262). The concentration of FIM 2/3 IgG was previously assigned to 06/140 by an international collaborative study (22). Blanks and two QC samples were included on every plate to calculate% critical variance across all assays, which fell between 6 and 12.7%. Assay specificity was determined using inhibition and interference assays. No cross reactivity was detected (data not shown).

• Product #150 – Diphtheria Toxin, Unnicked, from Corynebacterium diphtheriae
• Product #186 – Fimbriae 2/3 from B. pertussis

Reagents

Allergens

… Cholera toxin (lots 10165A1 and 10167A2; List Biological Laboratories, …

Immunization and LN collection (see note 1)

1. Immunize mice with allergen (eg, intranasal A alternata/NP-OVA, intragastric peanut/cholera toxin).9
a. Intranasal immunization: For each mouse, prepare 10 μg A alternata extract and 25 μg NP-OVA in 50 μL PBS. Anesthetize mice with methoxyflurane using the open-drop method. Once the mice are under anesthesia and their breathing is slow and steady, administer 50 μL of antigen diluted in PBS through the nares using a pipette.
b. Intragastric immunization: For each mouse, prepare 5 mg ground peanut and 10 μg cholera toxin in 200 μL 0.2 mol/L sodium bicarbonate. Anesthetize mice with 30% isoflurane using the open-drop method. Once the mice are under anesthesia, gently gavage 200 μL of antigen diluted in 0.2 mol/L sodium bicarbonate using a feeding tube connected to a 1-mL syringe.

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

Protective Antigen Binding Assays

For all PA binding assays, activated PA (63 kDa, List Biological Laboratories, Inc.; Campbell, CA, USA) was labeled using an Alexa Fluor® 647 Microscale Protein Labeling Kit (Molecular Probes; Waltham, MA, USA) following the manufacturer’s instructions. RAW 264.7, human BAL cells, and human leukocytes were processed as described for anthrax surface receptor expression through the point of Fc receptor blocking. At this point, cells were either left untreated, or incubated with 3000 or 10,000 ng/mL unlabeled PA as indicated for 30 min on ice. Cells were then exposed to AF647-labeled PA at 10, 100, or 1000 ng/mL for an additional 30 min on ice. In the case of lung and blood cells, antibodies used for cell subset-specific identification were also added at the same time as AF647-PA. All samples were centrifuged at 300× g for 5 min and washed twice with 1X PBS-2. Samples were fixed with 2% paraformaldehyde prior to flow cytometry.

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

Reagents

… pertussis toxin was obtained from List Biological Laboratories (Campbell, CA, USA). …

EAE induction and treatment studies

… The mice were intraperitoneally injected with 200 ng pertussis toxin on days 0 and 2 post immunization. …

Author did not specify which List Labs Pertussis Toxin was utilized. List Labs provides the following Pertussis Toxin products:
• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer
• Product #181 – Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free)
• Product #179A – Pertussis Toxin from B. pertussis (in Glycerol)

EAE induction and behavioral assessment

… followed by inoculations with 300 ng of pertussis toxin, Bordetella pertussis, (List Biological Labs) on the day of induction and 48 hours later. …

Author did not specify which List Labs Pertussis Toxin was utilized. List Labs provides the following Pertussis Toxin products:
• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer
• Product #181 – Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free)
• Product #179A – Pertussis Toxin from B. pertussis (in Glycerol)

Animals and induction of disease

… Experimental autoimmune encephalomyelitis was induced with 50 μg myelin oligodendrocyte glycoprotein (MOG35-55; Synpeptide, Shanghai, China), 1 mg/ml complete Freund’s adjuvant (CFA; Sigma Aldrich, Oakville, Ontario, Canada) and 100–600 ng of pertussis toxin (PTX) for behavioral work (lot #180235A1A, CHO cell assay 0.03 ng/ml; List Biological Laboratories, Campbell, CA). A second lot of PTX was also used for molecular biology work, with PTX doses titrated to ensure similar clinical scores (80–180 ng, lot #180237A1, CHO cell assay 0.003 ng/ml), representing outcomes similar to “100–200 ng” from lot #180235A1A. …

• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer

… To deplete inflammatory monocytes, CCR2-DTR mice or their cohoused control littermates were injected intraperitoneally with 200 ng (10 ng/g body weight) of DT (List Biological Laboratories) as noted for a total of four doses …

• Product #150 – Diphtheria Toxin, Unnicked, from Corynebacterium diphtheriae

Protective Antigen (PA) List Biological Laboratories, Inc., Campbell, USA

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

… Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free) List Biological Laboratories Cat# 181 …

• Product #181 – Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free)

Chemicals, Peptides, and Recombinant Proteins

Pertussis toxin List Biological Laboratories Cat# 180

EAE

Mice were immunized subcutaneously in the flanks with an emulsion containing MOG35–55 (80 μg/mouse) and M. tuberculosis H37Ra extract (3 mg/ml, Difco Laboratories) in CFA (100 μl/mouse). Pertussis toxin (100 ng/mouse, List Biological Laboratories) was administered intraperitoneally on days 0 and 2. Mice were monitored and assigned grades for clinical signs of EAE as previously described (Xiao et al., 2015). To evaluate the suppressive ability of Tim-1+ B cells, purified total B cells or Tim-1+ B cells were transferred i.v. into WT recipients. Hosts were then immunized to induce EAE.

• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer