bioelisa
READ HIGHLIGHTED CHANGES
bioelisa HBsAg 3.0
3000-1158 96 tests
3000-1159 480 tests
ELISA test for the detection of hepatitis B surface antigen (HBsAg) in human serum or plasma in clinical
laboratories and as a first-line screening assay in blood centres.
Summary
Hepatitis B is a disease produced by a viral infection during which many serological markers appear. One of these
1
markers is the HBsAg. In 1964 Blumberg and col. were the first to detect an antigen in the blood of an Australian
aborigine which reacted with the serum antibody of a New York haemophilia patient. This antigen was later
identified as hepatitis B surface antigen. The discovery and identification of hepatitis B surface antigen was a great
stride forward in the knowledge and differentiation of viral hepatitis infections. The presence of HBsAg in serum or
plasma constitutes the most important indicator for the diagnosis of a hepatitis B virus (HBV) infection. The enzyme
2 3
immunoassay was first described by Engvall and Perlmann and van Weemen and Schuurs in 1971. The
4,5
technique was subsequently developed by Voller and col. using microplates, that permitted the development of
highly sensitive and accurate diagnostic kits.
Principle
bioelisa HBsAg 3.0 is a direct immunoenzymatic method of the «sandwich» type in which guinea pig anti-HBs
antibodies coated to microplate wells act as the capture antibody and goat anti-HBs antibodies marked with
peroxidase serve as conjugate antibodies. The sample to be analysed is incubated in one of the antibody-coated
wells. If the sample contains HBsAg, the antigen will bind to the antibody on the plate. After washing to eliminate
any unbound material, goat anti-HBs conjugate to peroxidase is added to the well and allowed to react with the
antigen-antibody complex formed in the first incubation. After a second incubation and subsequent washing, an
enzyme substrate containing a chromogen is added. The substrate will develop a blue colour if the sample is
positive for HBsAg. The blue colour changes to yellow after blocking the reaction with sulphuric acid. The intensity
of the colour is proportional to the amount of HBsAg in the test specimens.
Components
1. MCPL MICROPLATE:
12 x 8 wells coated with guinea pig anti-HBs antibodies. Individually separable wells.
2. CONJ 51x CONCENTRATE CONJUGATE:
Goat anti-HBs antibodies conjugated with peroxidase. Contains red dye, protein stabilisers, 0.02% Bronidox
and 0.001% gentamycin sulphate. To be diluted 1/51 with the conjugate diluent before use.
3. DIL CONJ CONJUGATE DILUENT:
Tris buffer containing yellow dye, additives, 0.02% Bronidox and 0.001% gentamycin sulphate. To dilute the
concentrate conjugate.
4. WASH SOLN 10x CONCENTRATE WASHING SOLUTION:
Phosphate buffer concentrate (10x) containing 1% Tween 20 and 0.01% thimerosal. To be diluted 1/10 in
distilled or deionised water before use.
5. SUBS BUF SUBSTRATE BUFFER:
Citrate-acetate buffer containing hydrogen peroxide.
6. SOLN TMB CHROMOGEN:
3,3', 5,5'-Tetramethylbenzidine (TMB) dissolved in dimethylsulphoxide (DMSO). Contains red dye.
7. CONTROL + POSITIVE CONTROL:
Normal human serum diluted in phosphate buffer containing purified and inactivated HBsAg and sodium azide
0.02%. Contains green dye. Ready to use.
8. CONTROL – NEGATIVE CONTROL:
HBsAg negative human serum diluted in phosphate buffer containing sodium azide 0.02%. Contains yellow
dye. Ready to use.
9. H2SO4 1N STOPPING SOLUTION (only in 1 plate kit):
1N sulphuric acid. Ready to use.
BIOKIT, S.A. - Can Malé s/n - 08186 Lliçà d’Amunt - Barcelona - SPAIN 3000-1158 R03 06.2012 eng.doc
0459
bioelisa
10. SEALS ADHESIVE SEALS:
To cover the microplate during incubations.
11. BAG RESEALABLE BAG:
For storage of unused strips.
Precautions
bioelisa HBsAg 3.0 is intended for IN VITRO diagnostic use.
For professional use only.
WARNING: POTENTIALLY BIOHAZARDOUS MATERIAL.
All human source material used in the preparation of this product were found to be negative for the presence of
HIV-1/HIV-2 and HCV antibodies, as well as for the hepatitis B surface antigen, using a commercial licensed
method, except those intended to be positive. The HBsAg contained in the positive control has been inactivated at
60°C for 10 hours. Nevertheless, because no test method can offer complete assurance of the absence of
infectious agents, this product should be handled with caution:
- Avoid contact of reagents with the eyes and skin. If that occurs, wash thoroughly with water.
- Wear gloves.
- Do not pipette by mouth.
- Do not smoke.
- Dispose all used materials in a suitable biohazardous waste container. Remains of samples, controls,
aspirated reagents and pipette tips should be collected in a container for this purpose and autoclaved 1-hour
at 121°C or treated with 10% sodium hypochlorite (final concentration) for 30 min before disposal. (Remains
containing acid must be neutralised prior addition of sodium hypochlorite).
- Certain reagents in this kit contain sodium azide as preservative. Sodium azide may react with lead or copper
pipes and plumbing creating highly explosive metal azides. Flush drains with water thoroughly after disposing
of the remains of reagents.
Handling instructions:
- Adjust washer to the plate used (flat bottom) in order to wash properly.
- Do not mix reagents from different lots.
- Do not use reagents after expiration date.
- Do not use the reagent if you observed any change in appearance of components included in the kit.
- Extreme care should be taken to avoid microbial contamination and cross contamination of reagents.
- Use a new pipette tip for each specimen and each reagent.
- It is very important to prepare the substrate-TMB solution just 5-10 minutes before use. Keep it in a
well-sealed container and avoid light exposure.
- Soaps and/or oxidising agents remaining in containers used for preparation of substrate-TMB solution can
interfere with the reaction. If glass containers are used to prepare the solution, they should be washed with 1N
sulphuric or hydrochloric acid, rinsed well with distilled water and dried before use. We recommend using
disposable plastic containers.
Storage and stability
The components will remain stable through the expiration date shown on the label if stored between 2-8°C. The
bag containing the microplate should be brought to room temperature before opening to avoid condensation in the
wells. Once opened the bag, microplate strips are stable for 3 months at 2-8°C in the plastic bag tightly sealed, with
the silicagel. Once diluted, the washing solution is stable for two weeks if stored between 2-8°C. Once diluted, the
conjugate is stable for 7 days at 2-8°C. Store the chromogen in the dark. As the substrate-TMB solution is not
stable once prepared, instructions for its use should be closely followed.
Available packaging
- 1 plate kit (96 tests), REF 3000-1158.
Contains: 1 plate, 1 x 0.4 ml concentrate conjugate, 1 x 16 ml conjugate diluent, 2 x 50 ml concentrate washing
solution, 1 x 14 ml substrate buffer, 1 x 1.5 ml chromogen, 1 x 1.7 ml positive control, 1 x 5 ml negative control,
1 x 12 ml stopping solution, 1 resealable bag and adhesive seals.
- 5 plates kit (5 x 96 tests), REF 3000-1159.
Contains: 5 plates, 1 x 1.3 ml concentrate conjugate, 2 x 30 ml conjugate diluent, 3 x 100 ml concentrate
washing solution, 5 x 14 ml substrate buffer, 1 x 1.5 ml chromogen, 1 x 1.7 ml positive control, 1 x 5 ml negative
control, 1 resealable bag and adhesive seals.
BIOKIT, S.A. - Can Malé s/n - 08186 Lliçà d’Amunt - Barcelona - SPAIN 3000-1158 R03 06.2012 eng.doc
0459
bioelisa
Material required not provided
- Distilled or deionised water.
- Multichannel pipettes and micropipettes (100 µl) and disposable tips.
- Dry or wet incubator at 37°C 1°C.
- Timer.
- Microplate reader with a 450 nm filter. Reference filter of 620 or 630 nm is advisable.
- Manual or automated wash system.
- Stopping solution (5-plates kit): 1N sulphuric acid. 2N or 4N sulphuric acid could also be used.
Sample collection
Use fresh serum or plasma (citrate/EDTA). Other anticoagulants should be evaluated before use. Samples can be
stored at 2-8°C for 3 days. For longer periods, samples should be frozen (-20°C). Avoid repeated freezing and
thawing. Specimens showing visible particulate matter should be clarified by centrifugation. Serum or plasma
samples should not be heat inactivated, since that may cause incorrect results.
Automatic processing
Automated or semi-automated assay may be used with different instruments. It is very important to validate any
automated system to demonstrate that results obtained for samples are equivalent to the ones obtained using
manual assay. It is recommended that the user validate periodically the instrument. If there is any difficulty in the
setting of Biokit automatic processors, please contact your distributor.
PROCEDURE (See procedural flow chart)
Previous operations
Allow all the reagents to reach room temperature (20-25°C) before running the assay.
Gently mix all liquid reagents before use.
Dilute the concentrate washing solution 1/10 with distilled or deionised water. For one plate, mix 50 ml of the
concentrate solution with 450 ml of water. If less than a whole plate is used, prepare the proportional volume of
solution.
Dilute the concentrate conjugate 1/51 with the conjugate diluent. For one plate, add 240 µl of concentrate
conjugate (red colour) to 12 ml of conjugate diluent (yellow colour). Mix gently. For less than one plate follow
table 1. THE WORKING CONJUGATE SOLUTION HAS AN ORANGE COLOUR.
TABLE 1
Strips required 1 2 4 6 8 10 12
Conjugate diluent ml 1.0 2.0 4.0 6.0 8.0 10.0 12.0
Concentrate conjugate µl 20 40 80 120 160 200 240
Sample and reagent addition monitoring
The fact that the sample doesn’t need predilution and that all the reagents and controls are coloured allows
spectrophotometric monitoring of sample and reagent addition to the microplate. For this purpose, after each
addition to the microplate, it should be read at 450 nm (without reference filter). The expected absorbance values
are the following:
SERUM / PLASMA / CONTROLS: 0.100
WORKING CONJUGATE (orange colour): 0.500
WORKING SUBSTRATE (pink colour): 0.050
In case that a well doesn’t perform according to these specifications it indicates that some problem in the
dispensing may have occurred, and should be investigated further.
Assay procedure
1. Transfer 100 µl of each control, positive and negative, to the assigned wells. Use at least three wells for the
negative control, 1 well for the positive control and one well for the substrate blank, in all microplates or
fractions used in each run, regardless of the number of samples to be tested. Leave the blank well empty
(it is recommended that well A1 be used for blank).
2. Transfer 100 µl of each sample to be analysed to the corresponding well.
3. Cover the plate with an adhesive seal and incubate for 60 5 minutes at 37°C.
BIOKIT, S.A. - Can Malé s/n - 08186 Lliçà d’Amunt - Barcelona - SPAIN 3000-1158 R03 06.2012 eng.doc
0459
bioelisa
4. Remove and discard the adhesive seal. Aspirate the contents of the wells and fill them completely
(approximately 350 µl) with the diluted washing solution. Repeat the process of aspiration and washing 3 more
times. Ensure that each column of wells soak for at least 15 seconds before the next aspiration cycle. After the
last washing blot the microplate on absorbent tissue to remove any excess liquid from the wells.
5. Transfer 100 µl of diluted conjugate into each well of the microplate, except the blank well.
6. Cover the plate with an adhesive seal and incubate for 30 2 minutes at 37°C.
7. During the last 5-10 minutes of this incubation prepare the substrate-chromogen solution. If the entire plate is
used add 280 µl of chromogen (TMB) to the bottle containing the substrate buffer (14 ml) and mix well.
THE WORKING SUBSTRATE SOLUTION HAS A PINK COLOUR; discard if it becomes blue. If the entire
plate is not used, follow table 2.
TABLE 2
Strips required 1 2 4 6 8 10 12
Substrate buffer ml 1.0 2.0 4.0 6.0 8.0 10.0 12.0
Chromogen (TMB) µl 20 40 80 120 160 200 240
NOTE: The TMB is dissolved in DMSO. As the melting point of the DMSO is 18°C, the chromogen solution should
be allowed to reach a temperature of 20-25°C and be well mixed before use.
8. Remove and discard the adhesive seal. Aspirate and wash the plate as in step 4.
9. Add 100 µl of substrate-TMB solution to each well, including the blank.
10. Incubate for 30 2 minutes at room temperature (20-25°C).
11. Stop the reaction by adding 100 µl of stopping solution in the same sequence and time intervals as for the
substrate-TMB.
12. Blank the reader at 450 nm with the blank well and read the absorbance of each well, within 30 minutes. It is
recommended to read in bichromatic mode using a 620 - 630 nm reference filter.
Quality control
Results of an assay are valid if the following criteria are accomplished:
1. Substrate blank.
Absorbance value must be less than or equal to 0.100.
2. Negative control mean (NCx).
Calculate the mean absorbance value of the negative control. Each of the individual values obtained should be
equal to or greater than 0.5 times NCx and equal to or less than 1.5 times NCx. Any value outside this range should
be discarded, and the mean recalculated. If two values are outside the range, the assay should be repeated.
Example:
Negative control Absorbance
1 0.045
2 0.050
3 0.049
Total 0.144
0.144
NCx = = 0.048
3
0.5 x 0.048 = 0.024
1.5 x 0.048 = 0.072
BIOKIT, S.A. - Can Malé s/n - 08186 Lliçà d’Amunt - Barcelona - SPAIN 3000-1158 R03 06.2012 eng.doc
0459
bioelisa
None of the values in this example has to be discarded.
The mean absorbance of the negative controls must be less than 0.120, after subtracting the blank.
NCx 0.120
3. Positive control (PC).
The absorbance value obtained for the positive control should be equal to or higher than 0.700, after
subtracting the blank.
PC 0.700
If any of the criteria above is not accomplished, the assay is not valid and should be repeated.
Results
The presence or absence of HBsAg in the samples analysed is determined by relating the absorbance value of
each sample to the cut-off value.
1. Calculate the cut-off value by adding 0.040 to the mean absorbance of the negative control.
Cut-off = NCx + 0.040
2. Divide the sample absorbance by the cut-off value.
Positive: ratio absorbance/cut-off 1.0
Negative: ratio absorbance/cut-off 0.9
Equivocal: ratio absorbance/cut-off 0.9 1.0
Interpretation of results
A repeatedly positive result for HBsAg is indicative of a hepatitis B virus infection. To determine whether it is acute or
chronic, other serological markers of hepatitis B should be analysed taking into account the clinical picture of the patient.
Limitations of the procedure
Samples with positive or equivocal result must be reanalysed in duplicate. If the result is repeatedly positive or
equivocal, the sample should be analysed by another similar test.
Although this test is qualified as a third generation method for HBsAg detection, it is recognised that currently
existing methods for HBsAg detection are not sufficiently sensitive to detect all possible cases of hepatitis B.
Expected results
The prevalence of HBsAg chronic carriers varies widely in the world, from high (> 8%, e.g., Africa, Asia and
Western Pacific) to intermediate (2-7%, e.g., Southern and Eastern Europe) and low (< 2%, e.g., Western Europe,
North America and parts of South America). In Western Europe the HBsAg carrier rate is between 0.1 and 0.5%
13
and in Southern Europe the prevalence of carriers varies between 1 and 5%.
Performance characteristics
Analytical sensitivity
The sensitivity obtained is, at least, 0.100 HBsAg units/ml for ad and ay subtypes, using HBsAg standards
traceable to the standards from Paul Ehrlich Institute (PEI-Germany, ref. 87). For the Second International HBsAg
standard subtype adw2, genotype A from the World Health Organisation (NIBSC code: 00/588) the sensitivity is of
at least 0.125 IU/ml.
Evaluations
- In a study to evaluate the performance of the kit, 416 samples presumptively positive for HBsAg were tested
and compared to a reference test. A sensitivity of 100% was obtained in this study.
- 200 samples from hospitalized patients have been tested and compared to a reference test. 196 samples were
found negative by both tests, 3 were confirmed positive and 1 was false positive. A specificity of 99.5% was
obtained in this study.
- 440 serum samples from a blood bank were tested with three lots of reagent and compared to the routine method
used for screening of HBsAg. A specificity of 100% was obtained with two lots and 99.5% with the third lot.
BIOKIT, S.A. - Can Malé s/n - 08186 Lliçà d’Amunt - Barcelona - SPAIN 3000-1158 R03 06.2012 eng.doc
0459
bioelisa
- In a blood bank (EFS Centre Atlantique, France), 4871 samples from unselected donors were analysed in
parallel with the routine method used for screening of HBsAg. 11 samples were initially reactive (0.23%), 3 of
which were repeatedly reactive (0.06%). It was obtained a specificity of 99.94%.
Precision
Intra-assay reproducibility:
The coefficient of variation obtained for the absorbance values of a positive sample assayed in 72 replicates were
2.2%, 2.5% and 2.6% in three lots studied.
Inter-assay reproducibility:
The positive control was tested in 5 different days with 3 different lots of bioelisa HBsAg 3.0. The coefficients of
variation obtained for the ratios absorbance/cut-off of the control for the three lots were 1.2%, 1.0% and 2.4%
respectively.
Interferences
Interference by addition
No interference has been found for haemoglobin (2 mg/ml), bilirubin (0.2 mg/ml) and triglycerides (13 mg/ml).
Cross-reactivity
In a study of possible interferences, 144 samples with potential risk of producing false positive results were tested.
20 of these samples were sourced from RPR positive sera, 15 were sourced from mononucleosis positive sera,
18 were positive for anti-nuclear antibodies (ANA), 9 were positive for rheumatoid factor (RF), 32 were from
pregnant women, 20 were positive for HCV antibodies, 20 were positive for HIV antibodies and 15 were positive for
HAV antibodies. Two samples were found positive and were confirmed as positive by a reference test. All other
samples were negative by the bioelisa HBsAg 3.0.
BIOKIT, S.A. - Can Malé s/n - 08186 Lliçà d’Amunt - Barcelona - SPAIN 3000-1158 R03 06.2012 eng.doc
0459
bioelisa
bioelisa: Troubleshooting guide
Problem Possible causes Solution
1. Controls out of 1a. Incorrect temperature, Check procedure. Repeat
validation. incubation or pipetting. assay.
1b. Improper preparation of Check procedure. Repeat
reagents, error of dilution, assay.
reagents not well mixed.
1c. Cross-contamination of Pipette carefully. Do not
controls. interchange caps. Repeat
assay.
1d. Incorrect reading filter. Check that the wavelength
of the filter used is 450 nm.
If no reference filter of
620 - 630 nm is used,
absorbance increases
approximately 0.050.
1e. Interference in the optical Check the reader. Clean or
pathway. dry the bottom of wells. Check
for air bubbles. Repeat
reading.
1f. Used components from Do not use components from
different lots. different lots as they are
adjusted for each batch
released.
1g. Expired reagents. Check the kit expire date. Use
a non- expired kit and
reagents.
2. No colour or only a light 2a. One or more reagents not Check procedure. Repeat
colour developed at the added or added in wrong assay.
end of the assay. sequence.
2b. Inactive conjugate: wrong Check for contamination.
dilution, improper Check procedure. Repeat
conservation. assay.
2c. Inactive microplate: Always keep unused strips in
improper conservation. the bag very well closed, with
the desiccant inside. Repeat
assay.
2d. Inactive substrate: Always use a freshly prepared
improper conservation or dilution of TMB in substrate
dilution, the container used buffer. Use disposable
affects substrate stability, containers or wash with acid
cross-contamination with or ethanol and rinse with
the stopping solution. deionised water before
re-use. Check procedure.
Repeat assay.
BIOKIT, S.A. - Can Malé s/n - 08186 Lliçà d’Amunt - Barcelona - SPAIN 3000-1158 R03 06.2012 eng.doc
0459
bioelisa
bioelisa: Troubleshooting guide
Problem Possible causes Solution
3. Too much colour in all 3a. Contaminated, oxidised Check that working substrate
microplate wells. or improperly prepared is pink, discard if blue. Make
substrate. sure that TMB is completely
liquid before using. Make sure
that TMB is well mixed in the
substrate buffer. Use
disposable containers or
washed with acid or ethanol.
Repeat assay.
3b. Contaminated or Check for contamination
improperly prepared (turbid aspect). Check
reagents. dilutions. Repeat assay.
3c. Contaminated washing Check the quality of distilled
solution (1x). or deionised water used for
dilution. Repeat assay.
3d. Insufficient washing or Check the washer. Fill wells
washing not consistent: with washing solution close to
filling volume and/or the top, aspirate completely.
aspiration insufficient or Increase the number of
not uniform. Insufficient wash cycles and soak time.
number of washing After washing, blot the
cycles, contaminated inverted microplate on tissue
device. paper.
3e. Using of a washing Use only biokit washing
solution from other solution.
manufacturer.
4. Poor reproducibility 4a. Washing problems. See 3c, 3d, 3e.
or high number of 4b. Uncalibrated pipettes or Use only calibrated pipettes,
non-repeatable reactive tips not well fitted. with well-fitted tips and pipette
samples. Improper pipetting. carefully, without bubbles and
splashing. Repeat assay.
4c. Reagents and sera not at Equilibrate reagents and sera
room temperature or not to room temperature and mix
well mixed before using. thoroughly before using.
4d. Air currents over the Keep the microplate protected
microplate during from air currents.
incubations.
4e. Too long time for addition Develop consistent and
of samples and/or uniform technique.
reagents. Inconsistency in
time intervals. Air
bubbles.
4f. Interference in the optical See 1e.
pathway.
BIOKIT, S.A. - Can Malé s/n - 08186 Lliçà d’Amunt - Barcelona - SPAIN 3000-1158 R03 06.2012 eng.doc
0459
