Confirmatory test of HBsAg

KAPG4SAO

DIAsource ImmunoAssays S.A. - Rue du Bosquet, 2 - B-1348 Louvain-la-Neuve - Belgium

: 131114/1
 Confirmatory test of HBsAg
Test kit for the confirmation of the existence of HBsAg in the specimens
KAPG4SAO
en
FOR RESEARCH USE ONLY
DIAsource ImmunoAssays S.A. ­ Rue du Bosquet 2, B­1348 Louvain­la­Neuve, Belgium Tel: +32 10 84 99 11 ­ Fax : +32 10 84 99 90

1) Intended Use
Confirmatory test of HBsAg is a test kit for the confirmation of the existence of HBsAg in the specimens. When
Confirmatory test of HBsAg is used in combination with HBsAg Elisa, it can confirm the presence or absence of HBsAg
in specimens which are positive for HBsAg in screening test.

2) Summary and Test Explanation

Hepatitis B surface antigen (HBsAg) is a serological marker typical of hepatitis B infection. Viral hepatitis of type B is
usually accompanied by the appearance of hepatitis B surface antigen in the blood which can be generally detected days
weeks as early as 2 to 3 weeks before clinical symptoms begin appear.
Its titer reaches a peak at the time when jaundice and changes in the levels of liver­specific enzymes like ALT and AST
are observed and gradually decreases following its elimination. When serum or plasma samples react positive in a
HBsAg screening assay, an in vitro confirmation of the presence of hepatitis B surface antigen (HBsAg) have to be
always carried out.
Assays for HBsAg are routinely used to screen of blood donation in order to reduce hepatitis B transmission by blood
transfusion. Furthermore, these assays are used to diagnose a suspected HBV infection and to monitor the status of
infected individuals i.e. a chronic hepatitis B virus infection.

3) Test description
Confirmatory test of HBsAg is specially formulated from high potency and strong neutralizing antiserum which contains
antibody to HBsAg. Confirmatory test of HBsAg adopts to the basic principles of the neutralization of antibody with
antigen. Even the specimen with very high level of HBsAg still can be accurately confirmed without diluting the
specimen in advance. After the first incubation of the procedure, the plate only absorbed part of the HBsAg content in
the specimen. Then the unbound HBsAg is removed with the serum by washing the plate. Therefore, the anti­HBs
reagent needs to neutralize only that part of HBsAg which has been coupled to the plate.Then only limited quantity of
HBsAg left to react with enzyme labeled antibody that added to the test later on.

4) Description of Materials Provided & Product Code system

● Item 1 ­ 2 in the following reagent table should be refrigerated at 2­8°C. For long period storage, freezing at ­20°C is
recommended.
Qt. per
ITEMS Components Description
96 tests
One bottle (3ml), containing guinea
CONTROL + pig antiserum against human HBsAg
ad and ay subtype and protein
(1) Anti­HBs Positive Control stabilizers. 1 bottle,
Preservatives: 0.003% Gentamycin 3 ml
and 0.01% Thimerosal.

One bottle (3ml), containing normal

CONTROL ­ human serum and protein stabilizers.
(2) Preservatives: 1 bottle,
Anti­HBs Negative Control 0.003% Gentamycin and 0.01% 3 ml
Thimerosal.

Catalogue Nr: KAPG4SAO PI number : 1701342/en Revision Nr : 131114/1

 ● OTHER MATERIALS AND DEVICES NEEDED
ITEMS Components
(1) HBsAg Elisa or equivalent HBsAg screening kits.
(2) Accessories for performing HBsAg test.

4.1) Storage condition and Stability of the kit and components

Kit/components Storage temp. State Stability
Original 12 months
Anti­HBs Positive Control +2～+8°C
Once open 1 month
Original 12 months
Anti­HBs Negative Control +2～+8°C
Once open 1 month

5) Instructions for Use

5.1) Warnings:
5.1.1) This reagent kit is for professional use only.
5.1.2) This reagent kit is for research use only.

5.1.3) Bring all kit reagents and samples to room temperature (+20 to +30°C) and mix carefully before use.

5.1.4) Do not use reagent beyond its expiration date.

5.1.5) Do not interchange reagents between different lots.
5.1.6) Do not pipette with the mouth.
5.1.7) Do not smoke or eat in areas where specimens or reagents are handled.
5.1.8) All kit components and specimens should be regarded as potential health hazards. It should be used and
discarded according to your laboratory’s safety procedures. Such safety procedures probably include the
wearing of protective gloves and avoiding the use of aerosols.
5.1.9) Potential infectious specimens and non­acid containing spills or leakages should be wiped up thoroughly with
5% sodium hypochlorite or treated in accordance with your practice for potential bio­hazard control.
5.1.10) Prior to disposing used specimens and kit reagents as general waste; it should be treated in accordance with
the local practice of potential bio­hazardous waste or treated as follows:
Both liquid and solid waste should be autoclaved at +121°C for at least 30 minutes.
Solid waste can also be incinerated.
Non­acidic liquid waste can be treated with sodium hypochlorite diluted to a final concentration of 1%.
Acidic liquid wastes must be neutralized before treatment with sodium hypochlorite as mentioned above and
should stand for 30 minutes to obtain effective disinfection.

5.1.11) 2N Sulfuric Acid is an irritant to skin, eyes, respiratory tract and mucous membranes. Avoid contact of
the 2N sulfuric acid with skin and mucous membranes. In case of contact, flush immediately with abundant
amounts of water. In case of inhalation, find fresh air and seek medical attention in case of pain.

5.1.12) Chromogenic TMB concentrate contains organic solvent, which is flammable.

Chromogenic TMB concentrate contains dimethyl sulfoxide, an irritant to skin and mucous membranes.

5.1.13) Although all human sourced material are tested non reactive for Anti­HCV and Anti­HIV, and
inactivated at +56°C for one hour, the reagent shall be handled as potential infectious material.*2

Catalogue Nr: KAPG4SAO PI number : 1701342/en Revision Nr : 131114/1

5.2) Specimen Collection and Preparation for Analysis
5.2.1) No special preparation of the patient is required prior to blood collection. Blood should be collected by
approved medical techniques.
5.2.2) Either serum or plasma specimens can be used with this test kit. Whole blood specimen should be separated as
soon as possible in order to avoid hemolysis. Any particulates (e.g. fibrin clots, erythrocytes) contained in the
specimen should be removed prior to use.
5.2.3) Specimens must be stored at +2 to +8°C and avoid heat­inactivation to minimize deterioration. For long­term
storage, they should be frozen below ­20°C. Storage in self­defrosting freezer is not recommended.
5.2.4) Frozen specimens must be thoroughly thawed and mixed homogenously before test.
5.2.5) Avoid multiple freeze­thaw procedures.

WARNING
1.The specimen must not contain any compounds of AZIDE, which inhibits the peroxidase activity.
2. Incompletely coagulated sera and microbial­contaminated specimens should not be used.

5.3) Storage conditions and Stability of Reagents

5.3.1) The kit must be stored at +2 to +8°C. Do not freeze.

5.3.2) Return reagents to +2 to +8°C immediately after use.

5.4) Test Procedure

5.4.1) Bring all reagents and specimens to room temperature (+20 to +30°C) before test
5.4.2) Write down the relative numbers of specimens and the wells on the data sheet. For each specimen and control,
four wells are needed and divide the four wells into 2 groups, A group and B group (2 Wells for A and 2 wells
for B) . Reserve one well for blank.
5.4.3) Add 50 µl of specimen or controls into each marked well individually.
5.4.4) Tear off the protective backing of the adhesive slip and press the slip on the plate to seal it.

5.4.5) Incubate the plate at room temperature ( 20 to 30°C ) for 20±4 hours.
5.4.6) At the end of incubation, wash the plate by following the plate washing procedure of HBsAg Elisa.
5.4.7) Add 50µl Anti HBs positive control to A group and equal volume of Anti HBs negative control to B group.
5.4.8) Cover the plate with the adhesive slip and incubate at 40±1°C for 1 hour.
5.4.9) Remove the slip and add 50µl of Anti­HBs · HRPO conjugate solution into each well (except the blank) Seal the
plate with a new adhesive slip.
5.4.10) Incubate the plate at 40±1°C for 1 hour.
5.4.11) Repeat the step 5.4.6) to wash the plate.
5.4.12) Add 100 µl pre­mixed chromogenic TMB concentrate and substrate buffer solution (1:1) into each well.
Avoided­light incubation at room temperature for half an hour.
5.4.13) Stop the reaction by adding 100μl of 2N H2SO and detect the OD 450/620­690nm (450nm reading wavelength
with 620 – 690 nm reference wavelength)*1.

Catalogue Nr: KAPG4SAO PI number : 1701342/en Revision Nr : 131114/1

5.5) Calculations of Results
Following the calculation methods of HBsAg Elisa to obtain the result:
5.5.1) Calculation the Negative Control mean (NCx). All values of Negative Control should meet the following criteria,
otherwise those tests will be invalided.

HBsAg Elisa
NC≦0.1 (Absorbance)

5.5.2) Calculation the average test value of group A (Ax) and B (Bx) of each specimen and control individually. The
mean of B group values should meet the following criteria, otherwise it is impossible to confirm this specimen.

HBsAg Elisa : Bx ≧ NCx + 0.05

5.5.3) Percentage of neutralization can be calculated by the following formula:

Bx – Ax

­­­­­­­­­­­­­­­­× 100 % = Neutralization Percentage

Bx – NCx

Note: 1. Not to calculate the Neutralization Percentage with negative samples.

2. A weak­positive sample in the HBsAg Elisa may be negative tested in the confirmation test.

5.6) Interpretation of Results

5.6.1) A specimen is confirmed positive for HBsAg if the mean of B group values is equal to or greater than cutoff
value (NCx + 0.05) and Neutralizing Percentage is equal to or greater than 50 %.
Note: The Positive control must be confirmed HBsAg POSITIVE at first, or the whole experiment will be
considered invalid and should be repeated again.
5.6.2) In cases of samples with high HBsAg concentrations (e.q. saturated OD without neutralization antibodies) and
neutralization percentages lower than 50 %, the test should be repeated after 1:100 dilution of the sample.

5.7) Troubleshooting
If the result cannot be reproduced, perform a preliminary troubleshooting by checking the possibilities listed below:
5.7.1) Improper washing procedure.
5.7.2) Contamination with positive specimen.
5.7.3) Wrong volume of sample, conjugate or substrates.
5.7.4) Contamination of the well rim with conjugate.
5.7.5) Improper specimen, such as hemolyzed serum or plasma, specimen containing sediments and specimen not
thoroughly mixed before use.
5.7.6) Wrong incubation time or temperature.
5.7.7) Obstructed or partial obstructed washer aspirate/dispense head and needles.
5.7.8) Insufficient aspiration.

Catalogue Nr: KAPG4SAO PI number : 1701342/en Revision Nr : 131114/1

5.8) Limitations and Interferences
5.8.1) This reagent kit is to be used for un­pooled human serum or plasma only.
5.8.2) The reagent kit has not been validated for use with cadaveric samples.
5.8.3) A negative HBsAg result without other evidence should not be used to exclude an HBV infection.
5.8.4) Interfering Substances:
The following results were obtained in respective experiments:
1. No interferences with different anticoagulants such as lithium heparin, EDTA, citrate have been observed.
2. Samples containing potential interfering substances [e.g. triglycerides (lipemia), hemoglobin (hemolysis),
bilirubin (icterus), monoclonal immunoglobulin components, elevated levels of autoimmune antibodies
(rheumatoid factor­RF, antinuclear antibodies­ANA, or antimitochondrial antibodies­ANA)] and samples from
pregnant women did not interfere with the Confirmatory test of HBsAg assay.

5.9) Performance Characteristics

5.9.1) Analytical Specificity
Spiking experiment with HBsAg material were performed with paired non­reactive serum and plasma with the three
anticoagulants in order to show equivalence in the test results between serum and different types of plasma in the
Confirmatory test of HBsAg.
The lipemic, hemolytic, icteric samples and samples with high monoclonal and elevated levels of autoimmune
antibodies do not interfere with the test result. Pregnancy is not influencing the test result HBsAg. No false positive
and false negative results are observed with samples with these characteristics.
5.9.2) Analytical Sensitivity

The neutralization capacity was determined using the HBsAg ad and ay reference preparations of the Paul Ehrlich
Institute, Langen/Germany with 95% Confidence Interval.

The confirmatory activity of Ad subtype is 0.019 PEI U/ml.

The confirmatory activity of Ay subtype is 0.075 PEI U/ml.

The Confidence Interval has to be 95%.

5.9.3) Diagnostic Specificity

20 false positive samples which were reactive with the HBsAg Elisa assay were tested with the Confirmatory test of
HBsAg assay to evaluate the diagnostic specificity.
None of these samples could be confirmed with the Confirmatory test of HBsAg assay. The diagnostic
specificity was 100%.

5.9.4) Diagnostic Sensitivity

5.9.4.1) HBV infected individuals

All more than 300 positive specimens could be confirmed with both the Confirmatory test of HBsAg and the Dade
Behring Enzygnost HBsAg confirmatory assay

5.9.4.2) Commercial seroconversion panels

1. The diagnostic sensitivity determined in the 15 commercial seroconversion panels to following results:
Sample No. of sample Reactive Sensitivity
HBsAg positive serum and plasma 15 15 100％
Diagnostic sensitivity = 15/15 = 100％

Catalogue Nr: KAPG4SAO PI number : 1701342/en Revision Nr : 131114/1

 2. The results of the testing of 15 seroconversion panels with Confirmatory test of HBsAg assay as following tables:
Reference Assay vs.
HBsAg Confirmed Results from Initial Draw Date Confirmatory test of
HBsAg Assay
Panel ID Reference Conf. Assay Confirmatory test of
Difference in Bleed #s*
(# Days) HBsAg Assay (# Days)
PHM 903 6 10 ­1
PHM 904 7 18 ­1
PHM 906 137 150 ­1
PHM 909 9 9 0
PHM 910 35 42 ­1
PHM 918 7 12 ­1
PHM 923 15 21 ­1
PHM 924 29 29 0
PHM 925 8 14 ­1
PHM 926 13 15 ­1
PHM 927 4 7 ­1
PHM 928 9 14 ­1
PHM 929 14 18 ­1
PHM 931 19 19 0
PHM 932 61 61 0
Over all ­­­­ ­­­­ Sum= ­ 11 bleed #s
­­­­ Average ­11/15 = ­ 0.73 bleed #s
Summary of the evaluation of all tested serocoversion panel:
The results have shown that the Confirmatory test of HBsAg is nearly equivalent to the reference assay. On
average the Confirmatory test of HBsAg test is only 0.73 bleed #s later in the 15 tested seroconversion panels in
comparison with the reference assay.

5.9.5) Precision

5.9.5.1) Intra-assay reproducibility

Intra­assay precision was determined using the HBsAg Elisa positive control sample provided and two patient
serum samples of different HBsAg titer were confirmed with the Confirmatory test of HBsAg in replicates of 10
in a single run over 3 days.

Sample-ID Day 1 Day 2 Day 3

Neutralization Neutralization Neutralization
Percentage [%] Percentage [%] Percentage [%]
1 82,44 69,40 78,15
1 80,31 67,53 74,61
1 82,22 73,42 74,84
1 76,84 71,54 73,06
1 82,51 74,33 80,27
1 86,37 59,82 78,39
1 84,19 72,48 79,55
1 80,86 61,65 81,28
1 81,42 66,55 77,83
1 79,83 65,61 84,36
M 81,71 68,23 77,90
SD 2,77 4,93 3,41
CV 3,39 7,23 4,37
Table 1: within run precision of patient sample 1.

Catalogue Nr: KAPG4SAO PI number : 1701342/en Revision Nr : 131114/1

 Sample-ID Day 1 Day 2 Day 3
Neutralization Neutralization Neutralization
Percentage [%] Percentage [%] Percentage [%]
2 88,63 78,79 67,89
2 82,15 69,58 66,05
2 81,94 77,06 66,80
2 76,80 68,50 72,90
2 81,41 73,03, 61,63
2 76,69 67,29 64,75
2 87,26 75,71 70,50
2 79,44 67,01 70,64
2 81,49 75,97 70,29
2 80,68 72,21 64,90
M 82,01 72,51 67,63
SD 3,67 4,28 3,45
CV 4,48 5,90 5,10
Table 2: Within run precision of patient sample 2.

Sample-ID Day 1 Day 2 Day 3

Neutralization Neutralization Neutralization
Percentage [%] Percentage [%] Percentage [%]
PC 97,00 96,39 96,01
PC 96,40 95,05 96,02
PC 97,71 94,10 95,60
PC 96,42 96,16 94,28
PC 96,22 95,77 95,30
PC 96,55 94,73 94,16
PC 97,25 96,16 95,92
PC 96,67 95,35 96,03
PC 97,06 95,25 96,05
PC 96,64 96,60 95,01
M 96,79 95,56 95,44
SD 0.46 0.80 0.73
CV 0.47 0.83 0.77
Table 3: Within run precision of positive control.

The calculated CV’s ranged between 0.47% and 7.23%. (=acceptable value for an immunoassay in
microtiter plate format).

Catalogue Nr: KAPG4SAO PI number : 1701342/en Revision Nr : 131114/1

5.10) Flow chart of the test procedure
Test Procedure When test with HBsAg Elisa
Write down the relative numbers of specimens and the wells
on the data sheet. For each specimen, four wells are needed
and other four wells for positive control. Thus, four pairs are
formed. Two of the pairs are marked as AA and the others are
BB groups. Three additional wells are needed for negative
control into each marked well individually.
↓
Add 50 µl of specimen or Controls into each marked well,
individually.
↓
Incubate the plate at Room Temperature (15 ­30°C) for
20±4 hours.
↓
Wash the plate.
↓
Add 50 µl Anti HBs positive control to AA group and the
same amount of Anti HBs negative control to BB group and
the wells with negative control.
↓
Incubate in a 40°C water bath or incubator for 1 hour.
↓
Add 50µl of Anti-HBs · HRPO conjugate solution into each
wells (including the Negative Control wells).
↓
Incubate the plate in the 40°C water bath or incubator for 1
hour.
↓
Wash the plate
(Choice one of the following two methods for enzymatic
reaction with color development

↙ ↘
Mix equal volumes of Add 50µl of chromogenic
chromogenic TMB TMB concentrate to wells
concentrate and substrate and then add 50µl of
buffer. Add 100µl of the substrate buffer. Mix
mixed solution to well, well, gently.
↘ ↙
Incubate at R.T. for 30 minutes.
↓
Add 100 µl of 2N sulfuric acid into each well.
↓
Determine absorbance using 450 nm as reading
wavelength with 620­690nm reference
wavelength*1

Catalogue Nr: KAPG4SAO PI number : 1701342/en Revision Nr : 131114/1

6) Bibliography
1. Blumberg BS, Alter HJ, Visnich S. A „new“ antigen in leukemia sera. JAMA, 1965;191:101­ 106.
2. Dane DS, Cameron CH, Briggs M. Virus­like particles in serum of patients with Australia­antigen­associated
hepatitis. Lancet 1970; 1: 695 ­ 698.
3. Aach RD, Grisham JW, Paker CW. Detection of Australia antigen by radioimmunoassay. Proc Natl Acad Sci. USA
1971;68:1056­1060.
4. Kim CY, Tikes JG. Purification and biophysical characterization of hepatitis antigen. J Clin Invest. 1973; 52:1176­
1186.
5. Wolters G. Cuijpers LP, Kacaki J, Schuurs AH, Enzyme linked immunosorbent assay for hepatitis B surface antigen.
J Infect. Dis 1977;136:Suppl 311­377.
6. Shih JW, Cote PJ Jr, Dapolito GM, Gerin JL. Production of monoclonal antibodies against hepatitis B surface antigen
(HBsAg)by somatic cell hybrids. J Virol Methods. 1980;1:257­273.
7. Hoofnagle JH, Di Bisceglie AM. Serologic diagnosis of acute and chronic viral hepatitis. Semin Liver Dis.
1991;11:73­83

7) Notes
*1 The reference wavelength of the photometer to be used can be 620 nm to 690 nm. However, the user should validate
the photometer in combination with HBsAg Elisa before use.
*2 Incomplete inactivation of hepatitis B virus after heat treatment at +60°C for 10 hours, J. Infect. Dis. 138:242­244.

Revision date : 2013-11-14

Catalogue Nr: KAPG4SAO PI number : 1701342/en Revision Nr : 131114/1

P.I. Number : 1701000 Revision nr 130513
Used symbols
Consult instructions for use
Storage temperature
Use by
Batch code
Catalogue number
Control
In vitro diagnostic medical device
Manufacturer
Contains sufficient for <n> tests
WASH SOLN CONC Wash solution concentrated
CAL 0 Zero calibrator
CAL N Calibrator #
CONTROL N
Control #
Ag 125I Tracer
Ab 125I Tracer
Ag 125I CONC Tracer concentrated
Ab 125I CONC Tracer concentrated
Tubes
INC BUF Incubation buffer
ACETONITRILE Acetonitrile
SERUM Serum
DIL SPE Specimen diluent
DIL BUF Dilution buffer
ANTISERUM Antiserum
IMMUNOADSORBENT Immunoadsorbent
DIL CAL Calibrator diluent
REC SOLN Reconstitution solution
PEG Polyethylene glycol
EXTR SOLN Extraction solution
ELU SOLN Elution solution
GEL Bond Elut Silica cartridges
PRE SOLN Pre-treatment solution
NEUTR SOLN Neutralization solution
TRACEUR BUF Tracer buffer
Microtiterplate
Ab HRP HRP Conjugate
Ag HRP HRP Conjugate
Ab HRP CONC HRP Conjugate concentrate
Ag HRP CONC HRP Conjugate concentrate
CONJ BUF Conjugate buffer
CHROM TMB CONC Chromogenic TMB concentrate
CHROM TMB Chromogenic TMB solution
SUB BUF Substrate buffer
STOP SOLN Stop solution
INC SER Incubation serum
BUF Buffer
Ab AP AP Conjugate
SUB PNPP Substrate PNPP
BIOT CONJ CONC Biotin conjugate concentrate
AVID HRP CONC Avidine HRP concentrate
ASS BUF Assay buffer
Ab BIOT Biotin conjugate
Ab Specific Antibody
SAV HRP CONC Streptavidin HRP concentrate
NSB Non-specific binding
2nd Ab 2nd Antibody
ACID BUF Acidification Buffer
DIST Distributor
TRAY Incubation trays
PMSF PMSF solution
Protect from light
STRIP Dot Strip
SUB Substrate
EXTR SOLN CONC Extraction Buffer Concentrate
CART Cartridge
SAV HRP Streptavidin HRP
PIPETTE Pipette
WASH SOLN Wash buffer