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should be avoided.

Specimen with extensive particulate should be clarified by

HBsAg ELISA Kit 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
Enzyme-linked immunosorbent assay for the detection of HBsAg in serum or
plasma negative and positive controls each should be run in every assay.

INTRODUCTION PRECAUTIONS
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 1. Caution: Some components of this kit contain human serum. No known test
screening of blood and blood products to be used for transfusion and an aid for method can offer complete assurance that products derived from human
the diagnosis of existing or previous hepatitis B infection. blood will not transmit infectious agents. Therefore, all blood derivatives
should be considered potentially infectious. It is recommended that these
INTRODUCTION reagents and human specimens be handled using established good laboratory
working practices.
HBsAg is one of the earliest markers that appear in the blood following infection 2. Wear disposable gloves while handling kit reagents and specimens and
with Hepatitis B virus (HBV). This infection of the liver is transmitted by thoroughly wash hands afterwards.
homosexual or heterosexual activity, blood borne exposure, mother - infant, close 3. Dispose off all specimens and materials used to perform the test as if they
personal contact and by intake of contaminated water and food products. In the contained infectious agents.
HBV infected people, the virus persists for the rest of their lives and can be passed 4. Do not mix reagents from kits with different lot numbers.
on to others. Therefore Hepatitis B has become a global public health problem. 5. Cross contamination between reagents will invalidate the test results.
6. All reagents and components except the conjugate must be equiliberated at
Infection with HBV results in the appearance of a number of serological markers room temperature prior to use.
and one of the first of such markers is Hepatitis B surface antigen (HBsAg). The STORAGE OF TEST KITS AND INSTRUMENTATION
HBV infection causes a wide variety of liver damages such as acute self-limiting Unopened test kits should be stored at 2-8C upon receipt. Micro titer plate, once
infection, fulminating hepatitis, chronic hepatitis with progression to cirrhosis and opened, should be kept in a sealed bag with desiccants to minimize exposure to
liver failure, and a symptomatic chronic carrier state. 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.
Hepatitis B surface antigen (HBsAg) appears 1-7 weeks before biochemical This is very important because absorbed atmospheric moisture by cold plates
evidence of liver disease or jaundice. Three weeks after the onset of acute significantly reduces their shelf life. Opened test kits will remain stable until the
hepatitis almost half of the patients will still be positive for HBsAg. In the chronic expiration date shown in 4C, provided it is stored as described above. A micro
carrier state, the HBsAg persists for long periods (6-12 months) with no titer plate reader with a bandwidth of 10 nm or less and an optical density range of
seroconversion to the corresponding antibodies. Therefore, screening for HBsAg 0-2 OD or greater at 450 nm wavelength is acceptable for use in absorbance
is highly desirable for all donors, pregnant women and people in high-risk groups. measurement.

PRINCIPLE OF THE TEST WORKING REAGENT PREPARATION, STORAGE AND STABILITY

The HBsAg EIA is a solid-phase simultaneous sandwich immunoassay, which No reagent preparation is required except for wash buffer, which is supplied as a
employs monoclonal antibodies and polyclonal antibodies specific for HBsAg. 20 X concentrate.
Microtiter well are coated with monoclonal antibodies specific for HBsAg. A
serum specimen is added to the antibody coated Microtiter wells together with WORKING WASH BUFFER
enzyme conjugated polyclonal antibodies. HBsAg, if present, will form an
antibody-HBsAg-antibody-enzyme complex. The plate is then washed to remove Dilute the 20X wash buffer concentrate with deionized or distilled water 1:20. For
unbound material. Finally, a solution of substrate is added to the wells and example, 5 ml of wash buffer concentrate should be diluted to a total volume of 100
incubated. A blue color will develop in proportion to the amount of HBsAg mL with deionized or distilled water.
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 STABILITY OF OPENED KIT COMPONENTS AND DILUTED
wavelength of 450 nm. REAGENTS

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
Materials provided with the kits: plates should be opened after they have been kept at room temperature for 20-30
1. Microtiter Well: 8x12 or 12x8, coated with monoclonal anti-HBs antibody minutes. After removing the required number of strips, the plates should be resealed
2. Negative Control: 0.5ml HBsAg negative serum. in the foil pouch bags along with the desiccant and stored at 2-8C. Exposure of
3. Positive Control: 0.5ml HBsAg positive serum. HBsAg plates to humidity drastically reduces the shelf life.
4. Enzyme Conjugate: 6 ml, Goat anti-HBsAg-HRP
5. Wash Buffer Concentrate (20x): 25 ml, The buffer should be diluted 20 ASSAY PROCEDURE:
times with distilled water before use.
6. Substrate Solution A: 6 ml Urea Peroxide It is strongly advised to analyze each specimen and controls in duplicate. All the
7. Substrate Solution B: 6 ml TMB solution reagents should equilibrate to room temperature before use.
8. Stop Solution: 6 ml 2N Sulfuric Acid 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,
Materials required but not provided: and 50ul of serum or plasma samples into respective test wells
1. Precision pipettes: 0.02, 0.05, 0.10, 0.15, 0.20, and 1.0 ml. 2. Add one drop (50 ul) of Enzyme Conjugate to each well. Mix it gently by
2. Disposable pipette tips. swirling the microtiter plate on flat bench for 1 minutes. Do not add Enzyme
3. Distilled water. Conjugate to the blank well.
4. Humidified Box capable of maintaining 37C 3. Place the microtiter plates into a humidified box, and incubate at 37C for
5. Absorbent paper or paper towel. 30 minutes.
6. Microtiter plate or strip-well washer 4. Wash each well 4 times by filling each well with diluted wash buffer, then
7. Microtiter plate reader. inverting the plate vigorously to get all water out and blocking the rim of
wells on absorbent paper for a few seconds.
SPECIMEN COLLECTION AND PREPARATION 5. Add one drop (50 ul) of Substrate Solution A (HRP-substrate) to each well,
No special preparation of the patient is required prior to blood collection. Blood then add one drop (50 ul) of Substrate Solution B (TMB) to each well. Mix
should be collected by approved medical techniques. Remove serum or plasma gently and incubate at 37C for 15 minutes. .
from the clot or blood cells as soon as possible to avoid hemolysis. Grossly 6. Add 1 drop (50 ul) of Stop Solution to each well to stop the color reaction.
hemolytic, lipidic or turbid samples should not be used. Plasma samples Read O.D. at 450 nm with an EIA plate reader.
containing EDTA, heparin or oxalate may interfere with test procedures and
ASSAY VALIDITY
www.ct.gov/dph/lib/dph/infectious_diseases/ctepinews/vol22no... Yahoo! Search What exactly is HBsAg and HBsAb? - Yahoo... Feb 28, 2010 ... What exactly is the difference between HBsAg and HBsAb? ... Well said, "Kelle". Ag means antigen. An antigen is a protein substance that doesn't ... answers.yahoo.com/question/index?qid=20100228202123AA1xxu1 Google Hepatitis B Foundation: Blood Test FAQ What is the hepatitis B surface antibody (HBsAb)? ... Its appearance with the protective surface antibody (positive HBsAb or anti-HBs) indicates prior ...
www.hepb.org/patients/hepatitis_b_blood_tests.htm Google Blood -- How I treat and monitor viral h... Another 6 transplant recipients in the study were initially HBsAb-positive. ... These donors are usually both HbcAb and HBsAb positive.97,99 Some donors may have anti ... bloodjournal.hematologylibrary.org/cgi/content/full/113/14/3... Yahoo! Search Wyoming Department of Health HBsAb(positive) results indicate that the patient has antibody to hepatitis B virus. ... HBsAb(negative) results indicate that the patient has no antibody to hepatitis B ... wdh.state.wy.us/phsd/lab/hephiv.html Yahoo! Search
HBsAb definition from the online medical... Definition of
hbSaG
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www.HepatitisABCSolution.com/ Sponsored: Ads by Google HBsAg - Wikipedia, the free encyclopedia The genome organisation of HBV. The genes overlap. (ORF S, in green, encodes HBsAg). HBsAg is the surface antigen of the Hepatitis-B-Virus (HBV). ... en.wikipedia.org/wiki/HBsAg Google, Yahoo! Search Management of the HBsAg Positive Patient The marker for the surface is HBsAg (hepatitis B surface antigen) and the markers for the core are HBeAg (hepatitis B "e" antigen) and HBV-DNA. ... www.comeunity.com/adoption/health/hepatitis/smith.html Google, Yahoo! Search Hepatitis B:
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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:
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Website: www.ivdbiocare.com

April 10, 2008 Revision: 03

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colloidal gold
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elisa kit
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elisa kits
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fsh
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by Google Fsh levels Relax. Take a deep breath. We have the answers you seek. www.RightHealth.com/ Sponsored: Ads by Google Follicle-stimulating hormone - Wikipedia... Follicle-stimulating hormone (FSH) is a hormone found in humans and other animals. It is synthesized and secreted by gonadotropes of the anterior pituitary ... en.wikipedia.org/wiki/Follicle-stimulating_hormone Google, Yahoo! Search FSH: The Test Describes when the FSH test is ordered, how the FSH test is used, and what the results of an FSH test might mean. www.labtestsonline.org/understanding/analytes/fsh/test.html
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h pylori
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pylori -Peptic Ulce
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