Learning activity # 8 Proper

performance of the serologic test for

the determination of hepatitis B
Surface Antigen (HBsAg)
HBsAg:

Found in the outer envelope of the

virus.
HBsAg is found in non-infectious
andtubular particles that lack viral
DNA and circulate freely in the
blood.
Measured by enzyme
immunoassay (EIA),
Radioimmunoassay or reversed
passive hemagglutinantion (RPHA)
o EIA and radioimmunoassay
are widely used because of
good sensitivity.
o RPHA method is rapid and
simple to perform, but it
lacks sensitivity and the
results are difficult to
interpret.
Useful in establishing the initial
diagnosis of hepatitis B and in
monitoring the course of infection.
The HBsAg is the first maker to
appear becoming detectable in 210 weeks after exposure to HBV.
And its levels peaks during acute
infection.
In patients with Acute Hepatitis B,
Serum HBsAg usually becomes
undetectable by 4 6 months after
onset of symptoms.
However, in Px. With Chronic HBV
infection, HBsAg remains elevated
for 6 months or more.

Specimen: Serum or plasma

Principle: the test is an
immunochromatographic assay for the
qualitative detection of hepatitis B surface
antigen (HBsAg). It is a double antibody
sandwich immunoassay colloidal cold
conjugate anti-HBsAg antibody complexes
are dry-immobilized in the test device.

When the sample is added, it migrates by

capillary diffusion through the strip
rehydrating the gold conjugate complexes.
If present, HBsAg will react with the gold
conjugate complexes forming particles.
These particles will continue to migrate
along the strip until the Test Zone (T)
where they are captured by anti-HBsAg
Antibodies immobilized there and visible
red line appears.
If there is no HBsAg in the sample, no red
line will appear in the Test Zone (T).
The gold conjugate complexes will
continue to migrate until they are
captured in the Control Zone (C) by
immobilized goat anti-mouse IgG anitbody
aggregating a red line, which indicates the
validity of the test.
Preparation of specimens
- Cloudy, hyperlipidemic specimens,
specimens by heavy hemolysis and
heat treatment may give
inconsistent test result.
- Specimen should be refrigerated at
2-8C with 1% Sodium azide
(NaN3) for uo to three days
following collection. if not tested
immediately. But if three days is
not possible, specimens should be
frozen at -20C
- Expiry date: 24 months from the
manufacturing date.
Assay procedure
1. Dispense 1oo l of sample into the
well of the devise
2. Read the result for 30 mins.
Interpretation of results
1. Negative: A pink or red band
appears in the control region.
2. Positive: Two pink or red bands
appear in the control and test
region.
3. Retest: No color band appears in
both regions.
*In case strong color development
In the test region only, retest after
diluting the specimen 10-fold with
buffered saline.