Human Anti-Cholera Toxin B IgG ELISA kit

Sample

serum, plasma

Species Reactivity

Human

Detection Method

iELISA

Intended Use

The Human Anti-Cholera Toxin B Subunit IgG ELISA Kit detects and quantifies anti-Cholera Toxin B IgG in human serum or plasma of exposed or immunized individuals. This immunoassay is suitable for:
o Determining immune status relative to non-immune controls;
o Assessing efficacy of vaccines, including dosage, adjuvantcy, route of immunization and timing;
o Qualifying and/or standardizing vaccine batches and protocols.
This kit is for research use only (RUO), not for diagnosis or therapeutic purposes.

Contents of Kit

1. Wash Solution Concentrate (100×): 10ml, Dilute the entire volume 10 ml + 990 ml with distilled or deionized water into a clean stock bottle. Label as Working Wash Solution and store at 4°C for long term and RT for short term.
2. Sample Diluent Concentrate (20×): 10ml, Dilute the entire volume, 10 ml + 190 ml with distilled or deionized water into a clean stock bottle. Label as Working Sample/Conjugate Diluent and store at 2-8°C until the kit lot expires or is used up.
3. Anti-Human IgG HRP Conjugate Concentrate (100×): 0.15ml. Peroxidase conjugated anti-human IgG in buffer with detergents and antimicrobial. Dilute fresh as needed; 10ul of concentrate to 1ml of Working Sample Conjugate Diluent is sufficient for 1 8-well strip. Use within the working day and discard. Return 100× to 2-8°C storage.
Ready For Use
4. Cholera Toxin B Subunit Coated Strip Plate: Coated with Cholera Toxin B proteins; postcoated with stabilizers.
5. Anti- Cholera Toxin B Calibrators: 1 U/ml, 2.5 U/ml, 5 U/ml and /10 U/ml. 0.65ml/each. Four (4) vials, each containing antiCholera Toxin B antibodies; in buffer with antimicrobial.
6. Anti-Cholera Toxin B Positive Control: 0.65 ml. Anti- Cholera Toxin B diluted in buffer with protein, detergents and antimicrobial. [Value range on label].
7. Low NSB Sample Diluent: 30 ml. TBTm. Not for HRP Conjugate dilution. Buffer with protein, detergents and antimicrobial. Use as is for sample dilution.
8. TMB Substrate: 12 ml, Chromogenic substrate for HRP containing TMB and peroxide.
9. Stop Solution: 12 ml. Dilute sulfuric acid.

Storage

The microtiter well plate and all other reagents, if unopened, are stable at 2-8 º C until the expiration date printed on the box label. Stabilities of the working solutions are indicated under Reagent Preparation.

Performance Characteristics

Specificity
Purified cholera toxin B subunit from Vibrio Cholerae is used to coat the microwells; thus the assay is specific for antibodies directed to cholera toxin B. The Anti-Human IgG-HRP conjugate reacts specifically with human IgG class antibodies; IgA, IgM and IgE antibody would not be measured above background signals.
Assay Sensitivity
The Cholera Toxin B coating level, HRP conjugate concentration and Low NSB Sample Diluent are optimized to differentiate antiCholera Toxin B IgG from background (non-antibody) signal with human serum samples diluted 1:100.

General Description

Cholera toxin (sometimes abbreviated to CTX, Ctx, or CT) is a protein complex secreted by the bacterium Vibrio cholerae. CTX is responsible for the harmful effects of cholera infection. The cholera toxin is an oligomeric complex made up of six protein subunits: a single copy of the A subunit and five copies of the B subunit. The two parts are connected by a disulfide bond. The five B subunits, each weighing 12 kDa, form a five-membered ring. The A subunit has two important segments. The A1 portion of the chain (CTA1) is a globular enzyme payload that ADPribosylates G proteins, while the A2 chain (CTA2) forms an extendedm alpha helix which seats snugly in the central pore of the B subunit ring. Once secreted, the B subunit ring of CTX will bind to GM1 gangliosides on the surface of the host's cells. After binding takes place, the entire CTX complex is internalized by the cell and the CTA1 chain is released. CTA1 catalyzes ADP ribosylation leading to the activation of adenylate cyclase. Increased adenylate cyclase activity increases cAMP synthesis causing massive fluid and electrolyte efflux resulting in diarrhea. Neutralizing antibodies against subunit B can prevent the binding of subunit B to epithelial cells preventing the release of Sub A. Currently, recombinantly produced CTB and killed V. cholerae (Serotypes O1 and 0139) are used as a component of internationally licensed oral cholera vaccines (Dukoral® SBL Vaccin AB and Shanchol® Shantha Biotechnics Limited), as the protein induces potent humoral immunity that can neutralize CT in the gut. Additionally, recent studies have revealed that CTB administration leads to the induction of antiinflammatory mechanisms in vivo.