Hepatitis B Surface Antigen

Group 10
Bacani, fernando
OCAMPO, TONI JERICO
TORRES, CHELZYLYN
 Hepatitis B Etiology

The Australian antigen, now called Hepatitis B

surface antigen (HBsAg), was discovered in 1966.
HBV is a complex DNA virus that belongs to a new
class called the hepadna viruses. The intact virus is
a double-shelled particle referred to as the “Dane
particle”. It has an outer surface structure, Hepatitis
B surface antigen (HBsAg), and an inner core
component, the hepatitis B core antigen (HBcAg).



 Epidemiology

HBV infection was previously called “serum hepatitis”

or “long incubation hepatitis”.
One of the most frequent clinical infections
transmitted by blood transfusion in the past.


 Persons at risk of exposure to HBV:

 Patients who have received multiple transfusion
 Heterosexual men/women with multiple partners
 Household contacts and sexual partners of HBV carrier
 Infants born to HBV-infected mothers
 Acute hepatitis B

an acute hepatitis B infection occurs when a person

gets infected with the virus. The illness from the
infection lasts six months or less. During the acute
stage , the infected individual can pass the virus to
others. Most healthy adults, about 90 to 95
percent, are able to recover from HBV. They will
develop protective antibodies against future
infections of hepatitis B.

 Chronic hepatitis B

by definition, if a person continues to be infected by



HBV six months after its onset, the infection is no

longer considered acute, but chronic. The persons
become a chronic carrier of hepatitis B for unknown
reasons men are six times more likely than women
to suffer from chronic hepatitis B. Infants and
children are more susceptible.



 Hepatitis B Surface Antigen

The initial detectable marker found in serum during

the incubation period of HBV infection is hepatitis
B surface antigen (HBsAg).
This procedure screens for the presence of the
major coat-protein of the virus (HBsAg) in serum
Is the most reliable serologic marker for HBV
infection.
The presence of HBsAg indicates active HBV
infection, either acute or chronic.



Earliest indicator of HBV infection.
Usually appears in 27-41 days (as early as 14 days)
Peaks as ALT rises and persist during the acute
illness.
Usually disappears 1-13 weeks after onset of
laboratory abnormalities in 90% of cases.
Persistence >6 months defines carrier state. May
also found in chronic infection.
The incubation period is 4 to 26 weeks.

 SEROLOGIC DETECTION OF HBsAg

 The serologic tests used for the detection of HBsAg

include:
 Ouchterlony double diffusion (agar gel diffusion)
 Counterelectrophoresis
 Rheophoresis
 Complement Fixation
 Reversed passive latex agglutination
 Reversed passive hemeagglutination
 Radioimmunoassay
 Enzyme-linked immunosorbent assay (ELISA)

 SEROLOGIC DETECTION OF HBsAg

These tests may be grouped into first generation, second

generation, and third generation, according to sensitivity
—third generation, being the most sensitive and
therefore preferred in most clinical situations.
First generation: Ouchterlony double diffusion (agar gel
diffusion)
Second generation: complement fixation, rheophoresis,
and counterelectrophoresis,
third generation: radioimmunoassay, reversed passive
hemeagglutination, enzyme-linked immunosorbent
assay (ELISA) and reversed passive latex agglutination

Sources of antisera for the detection of HBsAg

Human, including multiple-transfused patients,

patients giving an anamnestic response following
transfusions of blood and blood products,
volunteers stimulated with noninfectious HBsAg-
positive material, and sporadic sources
Animals hyperimmunized with purified HBsAg
Laboratory animals (guinea pigs, rabbits, mice,
monkeys, chimpanzees)
Domestic animals (horses, sheep, goats)

 OUCHTERLONY DOUBLE DIFFUSION
(AGAR GEL DIFFUSION)

Ouchterlony double diffusion (or agar gel diffusion) was

the first method used in establishing the relationship of
Ouchterlony double diffusion (agar gel diffusion) to
type B hepatitis.
 Materials
Buffers—phosphate-buffered saline at pH 7.4.
Gels—agarose.
Plates—3 ¼ x 4 ¼-inch lantern slides. (Microscope
slides or petri dishes work equally well).

 Method

1. Pipette 15ml of a 1.1% agarose solution onto a lantern slide,

giving a gel of about 1.5-mm thickness.
2. After the gel has hardened, puch a 7-hole pattern on the
agarose-coated plate with a metal punch. Wells 2 to 3mm in
diameter and from 3 to 4 mm apart give satisfactory
results, although they can be as large as 5 mm in diameter
and 6 to 7 mm apart.
3. The wells are filled with a Pasteur (or capillary) serum sample.
Serum known to contain HBsAg is placed in the upper and
lower wells. Anti-HBs is placed in the central well; this
allows for the observation of lines of identity or nonidentity
between the control precipitins produced by sera under
test, which are placed in the four adjacent wells.
4. Incubate the slide in a moist chamber for 24 to 72 hours.
 Interpretation

Ideally, plates should be read in a darkened room. The

appearance of a white precipitation line (read
against a dark background) indicates the presence of
HBsAg.
 COUNTERELECTROPHORESIS

The technique of counterelectrophoresis (CEP) is

based on the fact that HBsAg and its antibody, anti
HBs, have differing electrophoretic mobilities in an
electrical field.
 Materials
Many variations of this technique exist with respect to
reagents, concentrations, and so on. All of the many
variations have been reported to give satisfactory
results.
Buffer—barbital buffer (0.05M, pH 8.6)
Gels—17ml of agarose (1% concentration) diluted in
buffer (1)
Plates—3 ¼ x 4- inch lantern slides

 Method
1. Pipette 16 ml of 1per cent agarose solution onto 3 ¼ x 4
inch slides to give a gel of uniform thickness.
2.After the agar has cooled and hardened, cut two parallel
rows of 15 wells, 3 mm in diameter and 7 mm apart,
edge in the agar with a metal punch.
3.Fill the wells in one row with the sera under test; fill the
other set of wells with anti-HBs of adequate potency to
form a precipitin reaction in agar gel diffusion.
4.Connect the slides to the barbital buffer in an
electrophoresis cell by wick of chromatographic paper,
with the cell containing anti-HBs proximal to the
anode and wells containing test sera proximal to the
cathode.
5.Electrophoresis is carried out for 2 hours at 15 mA
constant current per slide.
6.
 Interpretation

Slides may be examined for immunoprecipitin

reaction 1 to 24 hours after completion of
electrophoresis. The presence of a white
precipitation line (read against a dark background)
indicates the presence of HBsAg.

 RHEOPHORESIS

In this technique, however, samples containing

HBsAg are forced to migrate towards the anti-HBs
by evaporation.
rheophoresis has sensitivity similar to that of
counterelectrophoresis and complement fixation,
rheophoresis was found to be particularly
convenient for determining HBsAg subtypes with
appropriate subtype-specific antisera.

 Materials
Buffer—0.01 M tris buffer, pH 7.6
Gels—1.25ml 0.8% agarose
Plate—Ouchterlony double diffusion dish (a molded
plastic cup, 3 cm in diameter and approximately 6
mm deep, will serve the purpose)

 Methods

1. Fit a gel diffusion dish with a cylindrical Teflon ring (3

cm outside diameter, and 0.55 cm in length).
2.Pour melted agarose (1.25 ml, 0.8 per cent, pH 7.6)
into the dish, and allow it to solidity.
3.Cut a pattern of stick peripheral wells (5 mm in
diameter) and a central well (3 mm in diameter) in
the agar with a center-to-center distance of 7 mm.
4.Carefully remove the Teflon ring, leaving a circular
moat around the periphery of the dish.
5.Fill the moat with 0.01 M tris buffer, pH 7.6
6.
6. Fill two opposing peripheral wells with serum containing
HBsAg to serve a control. Fill the remaining peripheral
wells with test samples.
7. Fill the central well with anti-HBs.

8. One minute after adding anti-HBs, cover the central well

with a 3-mm plastic cover to prevent evaporation.

9. Place a 3-cm2 plastic cover with a central hole (0.8 cm in

diameter) over the entire place with the center of the

plastic cover located directly over the antibody well.
10. Incubate the plate with at 37° C, and read after 8 to 16

hours’ incubation (16 to 24 hours if incubation is at 28°C

to 32°C.
1.
 Interpretation

The presence of a white precipitate line (read against

a dark background) indicates the presence of
HBsAg.

THE MICROTITER COMPLEMENT FIXATION
TECHNIQUE

more sensitive than double diffusion or

counterelectrophoresis for measuring HBsAg or
anti-HBs but are less sensitive than
hemagglutination or radioimmunoassay techniques
for measuring anti-HBs.
Both quantitative and qualitative complement-
fixation test can be completed within 2hours;
involves simple, easily obtainable equipment; they are
easy to perform with standardized reagents; and
they can be automated.
 Method

1. Heat serum for testing to 56°C for 30 minutes.

2.Prepare a series of twofold dilution in veronal buffered
saline supplemented with calcium and magnesium.
Dilution should start at 1.5, using microtiter plates.
3.Add 0.025 ml of complement, 1:7 to 2 µ, and 0.025 ml of
antibody, 2 to 4 µ to each dilution, and allow to
incubate for 16 to 20 hours at 4°C
4.Note: in practice, overnight fixation at 4°C is commonly
used, but short fixation at 37°C for 1 hour allow for
complement of the test within 2 hours. Tests by
Schmidt and Lennette (1971) revealed that although
the shorter test tended to give slightly lower antigen
titers, its sensitivity was comparable to that of the
overnight test.

7. Add 0.025 ml of 1 per cent suspension of hemolysin-
sensitized sheep red blood cells.
8. Incubate for 30 minutes at 35°C, shaking the plates

to keep the cells in suspension.

9. Read microscopically.


 Interpretation

The endpoint is the highest serum dilution at

which 3-to 4-plus fixation of complement
occurs.

 REVERSED PASSIVE LATEX
AGGLUTINATION

The reversed passive latex agglutination test is the

most rapid and the most simple first-generation test
for HBsAg.
The test is, in principle, the agglutination of latex
participles coated with anti-HBs by HBsAg in test
samples. Because false positive results are
frequently seen, confirmation of this reaction (and
all other agglutination reactions) by another method
of equal or greater sensitivity is essential.

 Method

Using a small rod, mix one drop of serum under test

with one drop of latex suspension on a black plastic
or glass slide.
Set up a control in parallel, using serum known to
possess HBsAg.
Tilt the slide by hand or with an appropriate
mechanical shaker for 5 minutes at room
temperature.

 Interpretation

Agglutination of the particles becomes apparent not

later than 5 minutes after mixing when HBsAg is
present in the serum under test.

REVERSED PASSIVE HEMAGGLUTINATION

Agglutination of red cells coated with anti-HBsAg

(reversed passive hemagglutination) provides a
rapid, sensitive, and simple method for detecting
HBsAg.
The same caution given for reversed passive latex
agglutination applies here; because false-positive
results are frequently seen, another method is
essential before results are finally interpreted.

 Materials

Test cells- 1 per cent suspension of formalinized tanned

turkey erythrocytes with purified horse anti-HBs
dispersed in phosphate-buffered saline (pH 7.2, 0.15 m
containing 5 per cent sucrose, 1.5 per cent sodium
azide). This preparation is available from commercial
sources.
Control cells- formalinized, tanned turkey erythrocytes
coated with normal horse globulin.
Buffer- sterile 0.15 m phosphate-buffered saline, pH 7.2,
containing normal horse serum, normal human serum,
and 0.1 per cent sodium azide.
Titration plates- U-bottom, disposable.

 Method

1. Prepare two dilutions of each test serum by placing

three drops of diluent in the first row of wells of a
U-bottom, disposable titration plate and one drop
in a second row of wells, using a disposable 0.025-
ml dropper. Using a 0.025 ml microtiter loop, take
up 0.025 ml of the patient’s serum and mix with
the diluent in the first well, giving a 1:4 dilution.
Transfer the diluter to the second well, giving a 1:8
dilution. Rinse the diluter in strong bleach (Javex),
and follow with two distilled water rinses. Repeat
for each test serum.

2.Set up positive and negative controls using 1 ml of
heat-inactivated, diluted HBsAg-positive human
serum as a positive control and 1 ml of normal
human serum as negative control.
3.To each of the 1:8 dilutions, add one drop (0.025-ml
dropper) of the test cells.
4.Mix the contents of the wells by gently shaking the
plates, cover with a plastic sealer, and allow to
settle at room temperature.
5.Read after 30 to 60 minutes.
6.
 Interpretation

Positive reaction
 Cells may be partially or completely
agglutinated to form a carpet at the bottom of the
well (Fig. 7-10)
Weak Positive Reaction

 Incomplete agglutination with a central carpet

of agglutinated cells surrounded by a definite ring
pattern is seen. The ring is of larger diameter than
that of a negative pattern and can usually be
distinguished by its slightly crenelated edge (Fig. 7-
10).

 RADIOIMMUNOASSAY

Radioimmunoassays are the most sensitive methods

for the detection of HBsAg and anti-HBs. The two
most widely used techniques are the solid phase
radioimmunoassay and the double-antibody or
radioimmunoprecipitation (RIP) tests.

 Materials

The Kit Contains



Beads- coated with HBsAg (guinea pig)

Vials (10 ml each) of anti-HBs 125l (human)-
approximately 7 µCi per vial; preservative-0.1 per cent
sodium azide
Vial (5 ml) of negative control (non reactive for HBsAg);
preservative-0.1 per cent sodium azide
Vial (3 ml) of positive control (positive for HBsAg);
preservative-0.1 per cent sodium azide
Four reaction trays-(20 wells each), 8 sealers, and 80
tubes identification inserts.
Counting tubes (8 cartons, 20 tubes each) properly
positioned for transfer of beads from reaction trays

 Procedure

1. Adjust the temperature of the water bath to 45°C

2. Remove the cap from the clear plastic tube that contains the
antibody-coated beads. Hold the bead dispenser directly
over the top of the reaction tray incubation well, and push
down with the index finger to release one bead into the well
for each sample to be tested.
3. Using precision pipettes, add 0.2 ml of serum and positive and
negative controls to the bottom of their respective wells.
Ensure that the antibody-coated bead is completely
surrounded by serum. Tap the reaction tray to release any
air bubbles that may be trapped in the serum sample.
4. Apply a cover sealer to each tray, and incubate the trays in the
45°c water bath for 2 hors.
5.
6.
5. At the end of two hours, remove the trays from the water bath.
Remove the cover sealer and discard. Using a semiautomated
aspiration and rinsing system, aspirate the serum; rinse each well
and bead with a total of 5 ml of distilled or deionized water.
Repeat this wash procedure one additional time. Note: a manual
system of washing the wells and beads may also be used. Using
disposable pipette or cannulas and a cornwall syringe delivery
system, or equivalent, and a vacuum source, rinse each well and
bead with extreme care not to overflow the reaction well but
ensuring that the bead is totally immersed throughout the wash
procedure. Place the pipette or cannula, attached to the vacuum
source, into the bottom of the well next to the bead, and
simultaneously add slowly a Cornwall syringe, 5ml of distilled or
deionized water.
6. With precision pipettes, add 0.2 ml of 1251-labelled anti-HBs
(Human) to the bottom of each reaction well. Ensure that the
antibody-coated bead is surrounded by the labeled antibody
solution. Tap the tray to release any air bubbles that may be
trapped in the solution.
7.
7. Apply a new cover sealer to each tray, and incubate the trays in 45 degree
Celsius water bath for 1 hour.
8. At the end of 1 hour, remove the trays from the water bath. Remove the cover
sealer, aspirate the antibody solution from each well, rinse the well and
antibody-coated bead it contains with a total of 5ml of distilled or
deionized water, as in step 5.
9. Transfer the beads from the reaction wells to properly identified counting
tubes; align the inverted rack of oriented counting tubes over the
reaction tray, press the tubes tightly over wells, and then invert the tray
and tubes together so that the beads fall into the properly labeled tubes.
10.Place the counting tubes in a suitable well-type gamma scintillation counter,
and determine the count rate. The position of the bead at the bottom of
the counting tubes is not important. Although it is not critical that the
counting be done immediately, it should be performed as soon as
practicable. All control samples and unknown must be counted together.
11.
 Interpretation

The presence or absence of HBsAg is determined by

relating net counts per minute of the unknown
sample to net counts per minute of the negative
control mean times the factor 2.1. Unknown samples
whose net count rate is higher than the mean cutoff
value established with the negative control are to be
considered positive with respect to HBsAg.
The mean value of the positive control samples should
be at least five times the negative control mean. If
not, the technique may be suspect, and the run
should be repeated.

 Development of Hepatitis B Vaccine

Article taken from The Journal of the American

Medical Association, Published July 15, 2009
The authors stated that the source of the vaccine was
concentrated, highly purified, formalin-treated,
Hepatitis B virus coat (HBsAg) from 4 overtly
healthy human hepatitis carrier plasma donors.

 References

Neville Bryant, Laboratory Immunology and Serology,

1992
Mary Louise Turgeon, Immunology and Serology
James Chow, The Encyclopedia of Hepatitis and
Other Liver Disease
Jacques Wallach, Interpretation of Diagnostic Test
