Intended Use
The Anthrax Protective Antigen IgG ELISA Kit detects and quantifies PA83 IgG in human serum or plasma of vaccinated, immunized and/or non-vaccinated individuals. This immunoassay is suitable for:
Determining immune status relative to non-immune controls;
Assessing efficacy of vaccines, including dosage, adjuvantcy, route of immunization and timing;
Qualifying and standardizing vaccine batches & protocols.
The kit contains no anthrax bacteria (live or killed), and is for research use only (RUO), not for diagnosis, cure or prevention of the disease.
Contents of Kit
1. Wash Solution Concentrate (100×): 10ml
2. Sample Diluent Concentrate (20×): 10ml
3. Anti-Human IgG-HRP Conjugate Concentrate (100×): 150ul
4. PA83 Microwell Strip Plate: 8-well strips (12). Coated with PA83, and post-coated with stabilizers.
5. Anti-PA83 Calibrators: 650ul × 5 (10 U/ml, 20 U/ml, 40 U/ml, 80 U/ml, 160 U/ml). Five (5) vials, each containing anti-PA83 levels in arbitrary activity Units; diluted in buffer with stabilizers antimicrobial.
6. Anti-PA83 Positive Control: 650ul. Antibody reactive with PA83. [Value range is on label]
7. TMB Substrate: 12ml. Chromogenic substrate for HRP containing TMB and peroxide.
8. Stop Solution: 12ml. Dilute sulfuric acid.
Storage
The microtiter well plate and all other reagents, if unopened, are stable at 2-8°C until the expiration date printed on the box label.
Sensitivity
The PA83-coated plate and the anti-Human IgG HRP concentration are optimized to differentiate anti-PA83 IgG from background (non-antibody) signal with human serum samples diluted 1:200.
General Description
Anthrax, a zoonotic disease caused by the spore-forming bacterium Bacillus anthracis, has become a biological warfare agent of concern due to the stability and extreme lethal consequences of human inhalation of spores. Exposure to infected animals or tissue is also a major safety concern. The disease can occur in three forms: cutaneous, gastrointestinal and inhalation. Spores can remain viable and infective in the soil for many years. B. anthracis evades the immune system by producing an anti-phagocytic capsule. In addition, three proteins - protective antigen (PA), lethal factor (LF), and edema factor (EF) – are produced that act in combinations to form two exotoxins known as lethal toxin and edema toxin. Development of improved vaccines for protection of livestock and for human immunization have involved preparations that include combinations of these antigens. Immunoassays that measure titer of host antibody directed against the specific B. anthracis antigens (PA83/LF/EF can be used to study the efficacy of anthrax vaccines and the exposure to the bacterium and/or separate antigens.
Citations
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Bacillus anthracis can cause a severe zoonotic infection known as anthrax. The clinical manifestations and prognosis of human anthrax infection vary depending on the source of infection. Humans can be exposed to the pathogen by dermal, gastrointestinal, or inhalation routes, with inhalation-type symptoms being the most severe. Mortality rates for untreated respiratory, gastrointestinal, and dermal infections have been reported to be 100%, 25%-75%, and 20%, respectively. B. anthracis secretes protective antigen (PA), lethal factor (LF), and edema factor (EF), which are toxic and affect the virulence of B. anthracis. LF and EF enter the cell through a complex formed with a heptamer of PA and cause systemic disease through the binding of their receptors to the membranes of the infected cell. Current anthrax vaccines are made from sterile filtrates of attenuated, non-enveloped strains of the pathogen and consist primarily of PA antigen and very small amounts of LF and EF. According to several studies, protection and vaccine efficacy are achieved through the production of anti-PA-neutralizing antibodies.
Protective antigen (PA), a key component of anthrax toxin, is required for the formation of transmembrane pores, which allow LF and EF to enter the cell cytosol and exert their toxic effects through their enzymatic activities. PA is a single-chain protein consisting of 735 amino acids with a total molecular mass of 83kDa. The monomeric form of PA consists of four predominantly antiparallel β-folded structural domains. Structural domain 1 consists of a pair of calcium ions, a furan cleavage site, and either an LF or an EF-binding site. Structural domain 2 plays a key role in transmembrane pore formation. Structural domain 3 is associated with PA heptamer and product oligomer stabilization. Structural domain 4 contains regions that bind to host cell membrane receptors (capillary morphogenetic protein (CMG2) and tumor endothelial marker 8 (TEM8)). PA83, the 83kDa form of PA, is considered the major anthrax immunogen, and all four structural domains contain neutralizing epitopes that are sufficient to neutralize the toxin.
Figure 1. View of protective antigen protein with colors representing the different domains
(Source: Dahhas MA, et al. 2022)
The Anthrax Protective Antigen IgG ELISA Kit [DEIA04866] provided by Creative Diagnostics can be used to quantitatively detect PA83 IgG in human serum or plasma. Please note that it does not contain live or inactivated anthrax bacteria. The kit can be used to determine the immune status relative to the non-immune control, to evaluate the efficacy of the vaccine (including dose, adjuvant, immune pathway and timing), qualifying and standardizing vaccine batches & protocols.
Alternative Names
Anthrax PA IgG ELISA Kit
References
1. Dahhas MA, et al. Role of site-directed mutagenesis and adjuvants in the stability and potency of anthrax protective antigen. Saudi Pharm J. 2022 May;30(5):595-604.
2. McComb RC, et al. Neutralizing antibody and functional mapping of Bacillus anthracis protective antigen-The first step toward a rationally designed anthrax vaccine. Vaccine. 2016 Jan 2;34(1):13-9.
3. Kondakova OA, et al. Vaccines against anthrax based on recombinant protective antigen: problems and solutions. Expert Rev Vaccines. 2019 Aug;18(8):813-828.
Role of site-directed mutagenesis and adjuvants in the stability and potency of anthrax protective antigen
Saudi Pharm J
Authors: Dahhas MA, Alsenaidy MA.
Abstract
Anthrax is a zoonotic infection caused by the gram-positive, aerobic, spore-forming bacterium Bacillus anthracis. Depending on the origin of the infection, serious health problems or mortality is possible. The virulence of B. anthracis is reliant on three pathogenic factors, which are secreted upon infection: protective antigen (PA), lethal factor (LF), and edema factor (EF). Systemic illness results from LF and EF entering cells through the formation of a complex with the heptameric form of PA, bound to the membrane of infected cells through its receptor. The currently available anthrax vaccines have multiple drawbacks, and recombinant PA is considered a promising second-generation vaccine candidate. However, the inherent chemical instability of PA through Asn deamidation at multiple sites prevents its use after long-term storage owing to loss of potency. Moreover, there is a distinct possibility of B. anthracis being used as a bioweapon; thus, the developed vaccine should remain efficacious and stable over the long-term. Second-generation anthrax vaccines with appropriate adjuvant formulations for enhanced immunogenicity and safety are desired. In this article, using protein engineering approaches, we have reviewed the stabilization of anthrax vaccine candidates that are currently licensed or under preclinical and clinical trials. We have also proposed a formulation to enhance recombinant PA vaccine potency via adjuvant formulation.
Anthrax toxin component, Protective Antigen, protects insects from bacterial infections
PLoS Pathog
Authors: Alameh S, Bartolo G, O'Brien S, Henderson EA, Gonzalez LO, Hartmann S, Klimko CP, Shoe JL, Cote CK, Grill LK, Levitin A, Martchenko Shilman M.
Abstract
Anthrax is a major zoonotic disease of wildlife, and in places like West Africa, it can be caused by Bacillus anthracis in arid nonsylvatic savannahs, and by B. cereus biovar anthracis (Bcbva) in sylvatic rainforests. Bcbva-caused anthrax has been implicated in as much as 38% of mortality in rainforest ecosystems, where insects can enhance the transmission of anthrax-causing bacteria. While anthrax is well-characterized in mammals, its transmission by insects points to an unidentified anthrax-resistance mechanism in its vectors. In mammals, a secreted anthrax toxin component, 83 kDa Protective Antigen (PA83), binds to cell-surface receptors and is cleaved by furin into an evolutionary-conserved PA20 and a pore-forming PA63 subunits. We show that PA20 increases the resistance of Drosophila flies and Culex mosquitoes to bacterial challenges, without directly affecting the bacterial growth. We further show that the PA83 loop known to be cleaved by furin to release PA20 from PA63 is, in part, responsible for the PA20-mediated protection. We found that PA20 binds directly to the Toll activating peptidoglycan-recognition protein-SA (PGRP-SA) and that the Toll/NF-κB pathway is necessary for the PA20-mediated protection of infected flies. This effect of PA20 on innate immunity may also exist in mammals: we show that PA20 binds to human PGRP-SA ortholog. Moreover, the constitutive activity of Imd/NF-κB pathway in MAPKK Dsor1 mutant flies is sufficient to confer the protection from bacterial infections in a manner that is independent of PA20 treatment. Lastly, Clostridium septicum alpha toxin protects flies from anthrax-causing bacteria, showing that other pathogens may help insects resist anthrax. The mechanism of anthrax resistance in insects has direct implications on insect-mediated anthrax transmission for wildlife management, and with potential for applications, such as reducing the sensitivity of pollinating insects to bacterial pathogens.
COA
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