Anthrax Protective Antigen
IgG ELISA
Catalog No. 130BQ 015G
(96 tests)
NAME AND INTENDED USE
The Anthrax protective antigen (PA) IgG ELISA is
intended for use in the evaluation of patient’s immune status or exposure to
Anthrax.
SUMMARY AND EXPLANATION OF THE TEST
Bacillus anthracis, the etiologic agent of anthrax, is a large, gram-positive,
nonmotile, spore-forming bacterial rod. The three virulence factors of B.
anthracis are edema toxin, lethal toxin and a capsular antigen. Human anthrax
has three major clinical forms: cutaneous, inhalation, and gastrointestinal.
Cutaneous anthrax is a result of introduction of the spore through the skin;
inhalation anthrax, through the respiratory tract; and gastrointestinal anthrax,
by ingestion. In the United States, incidence of naturally acquired anthrax is
extremely low. Gastrointestinal anthrax is rare but may occur as explosive
outbreaks associated with ingestion of infected animals. Worldwide, the
incidence is unknown, though B. anthracis is present in most of the world. If
untreated, anthrax in all forms can lead to septicemia and death. Early
treatment of cutaneous anthrax is usually curative, and early treatment of all
forms is important for recovery. Patients with gastrointestinal anthrax have
reported case- fatality rates ranging from 25% to 75%. Case-fatality rates for
inhalational anthrax are thought to approach 90 to 100%. Because B. anthracis
has a high probability for use as an agent in biologic terrorism, many centers
are involved in studying the epidemiological and laboratory diagnostic of this
bacterium. ELISA test for the detection of IgG antibody to Anthrax Protective
Antigen (PA) can be used to study the efficacy of experimental anthrax vaccine
and the exposure to this antigen.
PRINCIPLE OF THE TEST
Diluted patient serum is added to wells coated with purified antigen. IgG
specific antibody, if present, binds to the antigen. All unbound materials are
washed away and the enzyme conjugate is added to bind to the antibodyantigen
complex, if present. Excess enzyme conjugate is washed off and
substrate is added. The plate is incubated to allow the hydrolysis of the
substrate by the enzyme. The intensity of the color generated is proportional to
the amount of IgG specific antibody in the sample.
MATERIALS PROVIDED
1. Microwell Strips: PA recombinant antigen coated wells (12 x 8 x 1 wells)
2. Sample Diluent: 1 bottle (22 mL). Ready to use.
3. Calibrator: Yellow Cap. (1.50 mL/vial). Ready to use.
4. Positive Control: Red Cap. (1.50 mL/vial). Ready to use.
5. Negative Control: Blue Cap. (1.50 mL/vial). Ready to use.
6. Enzyme Conjugate: 1 bottle (12 mL). Ready to use.
7. TMB Substrate: 1 bottle (12 mL). Ready to use.
8. Stop Solution: 1N H2SO4; 1 bottle (12 mL). Ready to use.
9. Wash Concentrate: 1 bottle (50 mL), 20X concentrate.
STORAGE AND STABILITY
1. Store the kit at 2-8° C.
2. Keep microwells sealed in a dry bag with desiccants.
3. The reagents are stable until expiration of the kit.
4. Do not expose test reagents to heat, sun or strong light during storage or
usage.
WARNINGS AND PRECAUTIONS
1. Potential biohazardous materials:
The calibrator and controls contain human source components, which
have been tested and found non-reactive for hepatitis B surface antigen
as well as HIV antibody with FDA licensed reagents. However, there is
no test method that can offer complete assurance that HIV, Hepatitis B
virus or other infectious agents are absent. These reagents should be
handled at the Biosafety Level 2, as recommended in the Centers for
Disease Control/National Institutes of Health manual, "Biosafety in
Microbiological and Biomedical Laboratories." 1984.
2. Optimal results will be obtained by strict adherence to the test protocol.
Precise pipetting as well as following the exact time and temperature
requirements is essential.
3. Do not pipette by mouth. Do not smoke, eat, or drink in the areas in which
specimens or kit reagents are handled.
4. The components in this kit are intended for use as an integral unit. The
components of different lots should not be mixed.
5. Control sera and sample diluent contain preserved with sodium azide.
Sodium azide may react with lead and copper plumbing to form explosive
metal azide. On disposal, flush with a large volume of water.
SPECIMEN COLLECTION AND HANDLING
1. Collect blood specimens and separate the serum.
2. Specimens may be refrigerated at 2–8° C for up to seven days or frozen
for up to six months. Avoid repetitive freezing and thawing of samples.
PREPARATION FOR ASSAY
1. Bring all specimens and kit reagents to room temperature (20-25° C) and
gently mix.
2. Prepare washing buffer by adding the contents of the bottle (50 mL, 20X
Wash concentrate) to 950 mL of distilled or deionized water in one-liter
container. Store at room temperature.
ASSAY PROCEDURE
1. Place the desired number of coated strips into the holder.
2. Negative control, positive control, and calibrator are ready to use.
Prepare 1:41 dilution of test samples, by adding 5 μL of the sample to
200 μL of sample diluent. Mix well.
3. Dispense 100 μL of diluted sera, calibrator and controls into the
appropriate wells. For the reagent blank, dispense 100μL sample diluent
in 1A well position. Tap the holder to remove air bubbles from the liquid
and mix well. Incubate for 30 minutes at room temperature.
4. Remove liquid from all wells. Repeat washing three times with wash
buffer.
5. Dispense 100 μL of enzyme conjugate to each well and incubate for 30
minutes at room temperature.
6. Remove enzyme conjugate from all wells. Repeat washing three times
with wash buffer.
7. Dispense 100 μL of TMB substrate solution and incubate for 10 minutes
at room temperature.
8. Add 100 μL of 1N H2SO4 to stop reaction.
9. Read O.D. within 30 min at 450 nm using microwell reader.