should be avoided.
Specimen with extensive particulate should be clarified by
HBsAg ELISA Kit
Enzyme-linked immunosorbent assay for the detection of HBsAg in serum or
plasma
INTRODUCTION
HBsAg ELISA is used for the qualitative determination of Hepatitis B surface
antigen (HBsAg) in human serum or plasma. This test is indicated for the
screening of blood and blood products to be used for transfusion and an aid for
the diagnosis of existing or previous hepatitis B infection.
INTRODUCTION
HBsAg is one of the earliest markers that appear in the blood following infection
with Hepatitis B virus (HBV). This infection of the liver is transmitted by
homosexual or heterosexual activity, blood borne exposure, mother - infant, close
personal contact and by intake of contaminated water and food products. In the
HBV infected people, the virus persists for the rest of their lives and can be passed
on to others. Therefore Hepatitis B has become a global public health problem.
Infection with HBV results in the appearance of a number of serological markers
and one of the first of such markers is Hepatitis B surface antigen (HBsAg). The
HBV infection causes a wide variety of liver damages such as acute self-limiting
infection, fulminating hepatitis, chronic hepatitis with progression to cirrhosis and
liver failure, and a symptomatic chronic carrier state.
Hepatitis B surface antigen (HBsAg) appears 1-7 weeks before biochemical
evidence of liver disease or jaundice. Three weeks after the onset of acute
hepatitis almost half of the patients will still be positive for HBsAg. In the chronic
carrier state, the HBsAg persists for long periods (6-12 months) with no
seroconversion to the corresponding antibodies. Therefore, screening for HBsAg
is highly desirable for all donors, pregnant women and people in high-risk groups.
PRINCIPLE OF THE TEST
The HBsAg EIA is a solid-phase simultaneous sandwich immunoassay, which
employs monoclonal antibodies and polyclonal antibodies specific for HBsAg.
Microtiter well are coated with monoclonal antibodies specific for HBsAg. A
serum specimen is added to the antibody coated Microtiter wells together with
enzyme conjugated polyclonal antibodies. HBsAg, if present, will form an
antibody-HBsAg-antibody-enzyme complex. The plate is then washed to remove
unbound material. Finally, a solution of substrate is added to the wells and
incubated. A blue color will develop in proportion to the amount of HBsAg
present in the specimen. The enzyme-substrate reaction can be stopped and the
result is visualized by naked eye or read by EIA plate reader for absorbance at the
wavelength of 450 nm.
REAGENTS
Materials provided with the kits:
1. Microtiter Well: 8x12 or 12x8, coated with monoclonal anti-HBs antibody
2. Negative Control: 0.5ml HBsAg negative serum.
3. Positive Control: 0.5ml HBsAg positive serum.
4. Enzyme Conjugate: 6 ml, Goat anti-HBsAg-HRP
5. Wash Buffer Concentrate (20x): 25 ml, The buffer should be diluted 20
times with distilled water before use.
6. Substrate Solution A: 6 ml Urea Peroxide
7. Substrate Solution B: 6 ml TMB solution
8. Stop Solution: 6 ml 2N Sulfuric Acid
Materials required but not provided:
1. Precision pipettes: 0.02, 0.05, 0.10, 0.15, 0.20, and 1.0 ml.
2. Disposable pipette tips.
3. Distilled water.
4. Humidified Box capable of maintaining 37C
5. Absorbent paper or paper towel.
6. Microtiter plate or strip-well washer
7. Microtiter plate reader.
SPECIMEN COLLECTION AND PREPARATION
No special preparation of the patient is required prior to blood collection. Blood
should be collected by approved medical techniques. Remove serum or plasma
from the clot or blood cells as soon as possible to avoid hemolysis. Grossly
hemolytic, lipidic or turbid samples should not be used. Plasma samples
containing EDTA, heparin or oxalate may interfere with test procedures and
centrifugation prior to use. Covered specimens may be stored for up to 48 hours at
2-8C prior to assaying. Specimens held for a longer time can be frozen at -20C
for mix prior to testing. Avoid repeated freeze thaw. At least, two wells of
negative and positive controls each should be run in every assay.
PRECAUTIONS
1. Caution: Some components of this kit contain human serum. No known test
method can offer complete assurance that products derived from human
blood will not transmit infectious agents. Therefore, all blood derivatives
should be considered potentially infectious. It is recommended that these
reagents and human specimens be handled using established good laboratory
working practices.
2. Wear disposable gloves while handling kit reagents and specimens and
thoroughly wash hands afterwards.
3. Dispose off all specimens and materials used to perform the test as if they
contained infectious agents.
4. Do not mix reagents from kits with different lot numbers.
5. Cross contamination between reagents will invalidate the test results.
6. All reagents and components except the conjugate must be equiliberated at
room temperature prior to use.
STORAGE OF TEST KITS AND INSTRUMENTATION
Unopened test kits should be stored at 2-8C upon receipt. Micro titer plate, once
opened, should be kept in a sealed bag with desiccants to minimize exposure to
damp air. To remove the required number of strips from the micro titer plates,
bring the sealed pouches to room temperature first and then open the pouches.
This is very important because absorbed atmospheric moisture by cold plates
significantly reduces their shelf life. Opened test kits will remain stable until the
expiration date shown in 4C, provided it is stored as described above. A micro
titer plate reader with a bandwidth of 10 nm or less and an optical density range of
0-2 OD or greater at 450 nm wavelength is acceptable for use in absorbance
measurement.
WORKING REAGENT PREPARATION, STORAGE AND STABILITY
No reagent preparation is required except for wash buffer, which is supplied as a
20 X concentrate.
WORKING WASH BUFFER
Dilute the 20X wash buffer concentrate with deionized or distilled water 1:20. For
example, 5 ml of wash buffer concentrate should be diluted to a total volume of 100
mL with deionized or distilled water.
STABILITY OF OPENED KIT COMPONENTS AND DILUTED
REAGENTS
The diluted wash buffer is stable for at least one week when stored at room
temperature. Substrate is stable for the expiration date of the kit. The micro titer
plates should be opened after they have been kept at room temperature for 20-30
minutes. After removing the required number of strips, the plates should be resealed
in the foil pouch bags along with the desiccant and stored at 2-8C. Exposure of
HBsAg plates to humidity drastically reduces the shelf life.
ASSAY PROCEDURE:
It is strongly advised to analyze each specimen and controls in duplicate. All the
reagents should equilibrate to room temperature before use.
1. Dispense one drop (50 ul) of Positive Control as well as Negative Control in
duplicate into respective wells. Set one blank well as background control,
and 50ul of serum or plasma samples into respective test wells
2. Add one drop (50 ul) of Enzyme Conjugate to each well. Mix it gently by
swirling the microtiter plate on flat bench for 1 minutes. Do not add Enzyme
Conjugate to the blank well.
3. Place the microtiter plates into a humidified box, and incubate at 37C for
30 minutes.
4. Wash each well 4 times by filling each well with diluted wash buffer, then
inverting the plate vigorously to get all water out and blocking the rim of
wells on absorbent paper for a few seconds.
5. Add one drop (50 ul) of Substrate Solution A (HRP-substrate) to each well,
then add one drop (50 ul) of Substrate Solution B (TMB) to each well. Mix
gently and incubate at 37C for 15 minutes. .
6. Add 1 drop (50 ul) of Stop Solution to each well to stop the color reaction.
Read O.D. at 450 nm with an EIA plate reader.
ASSAY VALIDITY
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hbSaG
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EIA Plate Reader at 450 nm (using the OD value of the blank well to correct all the
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OD reading from all wells, OD value of Positive Control should be between 1.5-
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2.5).

INTERPRETATION OF RESULTS
Positive: P/N value is equal to or greater than 2.1

Negative: P/N value is less than 2.1

P/N value = OD value of specimen .

Average OD value of Negative Control

If the OD value of the negative control is less than 0.05, it should be reported as
0.05. If it is more than 0.05, it should be reported as the actual OD value measured.

LIMITATIONS OF THE PROCEDURE

1. HBsAg kit is used for the detection of HBsAg in human serum of plasma.
Based on a single reactive test result, a sample should not be considered
HBsAg positive. Further testing, including confirmatory testing, should be
performed before a specimen is considered positive for HBsAg. A non-
reactive test result does not exclude the possibility of exposure to hepatitis B
virus. Levels of HBsAg may be undetected both in early infection and late
after infection. Specimens containing precipitate may give inconsistent test
results.
2. As the other sensitive immunoassays, there is the possibility that non-
repeatable reaction may occur due to inadequate washing. So do aspirate the
well or get rid of entire content of wells completely before adding the
washing solution.
3. The positive control in the test kit is not to be used to quantify assay
sensitivity. The positive control is used to verify that the test kit components
are capable of detecting a reactive specimen provided the procedure is
followed as defined in the kit and the storage conditions have been strictly
adhered to.

REFERENCES

1. Rubin. E. Acute and chronic viral hepatitis. Federation Proceedings. 38 (13):

2665-2673 (1979).
2. Ling C. M. Radioimmunoassay for hepatitis B virus. Manual of clinical
immunology. Rose N. and Firiedman H. editors.
3. Wolters G. et al., Enzyme linked immunoasorbent essay for hepatitis B
surface antigen. J. Infect. Dis. 136:311 (1977)
4. Pderson, D.L.. Nath N and Gravilanes E Structure of hepatitis B surface
antigen. The J d Bid.Chern. 257 (17):10414-10410 (1982).

Manufacturer:
Biocare Diagnostics Ltd.
6F, Building B, 108 Xinghua Road
Xiangzhou, Zhuhai, China 519000
Tel: +86-756-8238560
E-mail: info@ivdbiocare.com
Website: www.ivdbiocare.com

April 10, 2008 Revision: 03

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elisa kits
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fsh
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h pylori
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