anti-HBc Safety Notes

Do not swallow the reagents. Avoid contact with eyes, skin and mucous
ELISA Test for the Detection of Anti-HBc
Antibodies in Human Serum or Plasma
Package Size
[REF] 51260 96 Tests Complete Test Kit
[IVD]
Intended Use
Viral hepatitis is an infectious disease most often caused by hepatitis B
virus (HBV), which affects about 5% of the world population with some
regional variations. The disease may manifest as asymptomatic, acute
(with fulminant and mortal cases), or chronic hepatitis with possible
degeneration into cirrhosis and/or hepato-cellular carcinoma and death.
The disease is usually transmitted through the exchange of body fluids
between healthy and infected individuals. The path of transmission is
through either parenteral (infected serum, blood products, blood
transfusions, etc.) or non-parenteral (saliva, tears, sweat, urine, semen;
skin wounds etc.) route.
The progress of HBV infection can be monitored by the determination of
anti-HBc. Anti-HBc appears in serum of a HBV infected individual shortly
after HBsAg is found but may be weeks or even months before anti-HBs
appears. In acute HBV infection, anti-HBc will persist after the
disappearance of HBsAg and before the appearance of anti-HBs. During
this window period, anti-HBc may be the only serological marker for
recent HBV infection and potentially infectious status.
Principle
The ANTI-HBc test is based on the principle of competitive binding
between anti-HBc in a test specimen and anti-HBc-peroxidase conjugate
(human) for a limited number of binding sites on the HBcAg coated well.
Thus the amount of anti-HBc-peroxidase conjugate bound to the well is
inversely proportional to the concentration of anti-HBc in the specimen.
After incubation of specimen and anti-HBc-peroxidase unbound enzyme
conjugate is washed off. Substrate solution is added and during further
incubation a blue colour develops. The intensity of this colour, which
changes to yellow after stopping the reaction with acidic solution, is
inversely proportional to the amount of anti-HBc in the specimen.
Within certain limits, the optical density at 450 nm (OD450) inversely
reflects the level of HBV core antibody in the specimen. An OD450 reading
less than the cut-off value is considered reactive for anti-HBc. An OD450
reading equal to or greater than the cut-off value is considered negative
for anti-HBc test. Specimen yielding repeatedly reactive readings by this
assay may be positive for the presence of anti-HBc.
Reagents and Contents
[MIC] 12 Microtiter Strips
breakable 8-well strips coated with HBcAg
(recombinant)
membranes. All patient specimens and controls should be handled as
potentially infectious. The positive control has been inactivated by heat-
ing. The negative control has been found negative for Hepatitis B mark-
ers. Both controls have been found negative for HCV and HIV-1/2 anti-
bodies. Wear protective clothing and disposable gloves according to Good
Laboratory Practices.
All materials contaminated with patient specimens or controls should be
inactivated by validated procedures (autoclaving or chemical treatment)
in accordance with applicable regulations.
[STOP], [NC], [SA] and [SB] irritates eyes, skin and mucous membranes.
Upon contact, rinse thoroughly with copious amounts of water and
consult a doctor.
Stability
The reagents are stable up to the stated expiry dates on the individual
labels when stored at 2...8°C.
After opening reagents have to be stored at 2...8°C and used within one
month (see also “Note”).
[WS] (20x) should be stored at room temperature to avoid crystallisation.
[MIC]
- Sealed in an plastic bag with a desiccant
- Must be at room temperature before opening!
- Unused: return to the zip-lock bag and store in this way at 2...8°C for 3
months.
- Do not touch the upper rim or the bottom of the wells with fingers.
Reagent Preparation
Bring all reagents to room temperature (15...25°C) before use.
Reagents not in use should always be stored at 2...8°C.
Working wash solution [WASH]
- Crystals in [WS] have to be dissolved by heating at 35...37°C
- Dilute [WS] 1 + 19 with fresh, germ free, distilled or deionised water,
e.g. 50 ml [WS] + 950 ml = 1000 ml.
- About 3 ml [WASH] required for each well
- Prepare only the quantity required for the current run.
Stability : 1 week at 2...8°C.
Working Substrate Solution [SUB] (see Alternative procedures A 2)
- Prepare required volume by mixing equal volumes of [SA] and [SB] in a
clean plastic container, rinsed with deionised water, prior to use.
- [SA] should be colourless to light blue, otherwise it should be discarded.
- Handle [SUB] carefully and avoid contamination ! Do not use, if it
appears blue!

[NC] 2.0 ml Anti-HBc Negative Control (yellow cap) - Store protected from bright light.
ready for use, human serum free of Hepatitis B markers Stability: 30 minutes at room temperature (15...25°C)
[PC] 1.5 ml Anti-HBc Positive Control (red cap)
Specimen
ready for use, human serum containing anti-HBc
Serum or plasma (EDTA, Heparin, Oxalate)
in buffer solution
Samples may be stored for 7 days at 2...8°C, for 30 days at -20°C. Freeze
[CON] 8.0 ml anti-HBc Enzyme Conjugate (black cap) pH 7.0 0.1
and thaw once only. Thawed specimen must be homogenised. Eliminate
Anti-HBc horseradish peroxidase conjugate (human),
particulate matter by centrifugation or filtration.
coloured yellow 0.2 µg/ml
Tris / HCl buffer 0.1 mol/l Procedure
[WS] 58 ml Wash Solution (20x) (black cap) pH 6.5 1.0 Follow the procedure exactly as described.
Concentrate for ca. 1000 ml
Phosphate buffered saline 1 mol/l Procedural Notes
Tween-20 0.2 % P1: Do not mix or use components with different lot numbers. Do not
[SA] 12 ml Substrate Reagent A (white cap) pH 5.1 0.2 mix caps of vials (risk of contamination). Do not use reagents after
Hydrogen peroxide 0.03 % their expiration date.
Citric acid buffer 0.06 mol/l P2: Do not use reagents that could be contaminated or look or smell
[SB] 12 ml Substrate Reagent B (black cap) different than usual.
3,3', 5,5'-tetramethylbenzidin (TMB) 0.6 mg/ml P3: Record specimens and controls carefully on the spread sheet
DMSO 0.7 mol/l supplied with the kit.
[STOP] 12 ml Stop Solution (white cap) P4: [MIC] - select the required number of Microtiter Strips.
Sulphuric acid, ready for use 1.0 mol/l
P5: Run duplicates or triplicates for positive resp. negative control.
(R36/38), Xi irritant
Pipette controls and specimen on the bottom in the microwells.
1 Strip Holder
P6: Always add reagents in the same order and timing to minimise
Adhesive Strips reaction time differences between wells. This is important for
The following preservatives are contained in the reagents: reproducible results. Pipetting of specimens should not exceed 5
minutes. Otherwise pipette the controls in the indicated positions at
0.01 % Thimerosal in [CON], [NC], [PC]
half way time of the series. If more than 1 plate is used, repeat the
0.003 % Gentamycin in [CON], [NC], [PC]
controls for each plate.
P7: Avoid/remove air bubbles prior to incubations and reading of Interpretation of Results
absorbance. Result Interpretation
P8: [SUB] – incubate in the dark. [SUB] initiates a kinetic reaction, which
A450 (specimen) 1.1 x COV negative for anti-HBc
is terminated by [STOP].
non-reactive
Wash Procedure A450 (specimen) 0.9 x COV positive for anti-HBc
The wash procedure is critical. Insufficient washing will result in poor initially reactive: retest
precision or falsely high absorbances. Retest in duplicate: Anti-HBc
W1: Remove Adhesive Strips, aspirate off the contents into 5% sodium
A450 (specimen) COV repeatedly reactive: perform
hypochlorite solution and add [WASH] to each well, aspirate off after
confirmatory test
30 sec. soak time and repeat washing 5 times.
W2: In case of automatic washers fill and prime with [WASH]. Subse- Limitations of the Procedures
quently wash strips 6 times. Ensure the washer fills all wells 1. The ANTI-HBc ELISA test is limited to the detection and semi-
completely and aspirates off efficiently after 30 sec. (remaining quantitation of anti-HBc in serum, plasma, or recalcified plasma.
liquid: <15 µl).
2. As with all diagnostic tests, a definitive clinical diagnosis should not be
W3: After washing, remove remaining liquid by tapping the plate upside based on the results of a single test, but should only be made by the
down on tissue paper. physician after all clinical and laboratory findings have been evaluated.

Pipetting Scheme 3. The detection of anti-HBc should not be the only indicator for the
diagnosis of HBV infection. In very early HBV infection the anti-HBc
Follow the procedure exactly as described. Pay particular attention to
titer may be below the detection limit of the method.
the washing procedure!
4. In old infection the presence of anti-HBc does not necessarily indicate
Reagents and specimens should be at room temperature before use. the existence of active virus replication.
Step 1 Well [µl] 5. As in other sensitive microwell tests, inconsistent readings may be
A1 B1-D1 E1/F1 G1... obtained due to inadequate washing. Therefore it is important to
Blank [NC] [PC] Sample aspirate wells completely before adding the Washing Solution or liquid
reagents.
[NC] in triplicate -- 50 -- --
[PC] in duplicate -- -- 50 -- Performance Characteristics
Samples -- -- -- 50 Typical performance data can be found in the Verification Report,
accessible via:
[CON] -- 50 50 50
www.human.de/data/gb/vr/el-a-hbc.pdf
Mix carefully
www.human-de.com/data/gb/vr/el-a-hbc.pdf
[MIC] cover with Adhesive Strips
Note
Incubate 60 min. at 37°C
The handling should always be in compliance with common GLP re-
Wash 6 times as described (see W1 - W3) quirements (*)! The validation criteria must be met!
[WASH] 400 400 400 400 (*This includes: Proper caps being replaced on the vials and firmly tightened / Remove only
Step 2 reagents required for a run from stock solutions if they could come into contact with other
contaminating solutions like patient specimens etc. / Stock solutions always returned to
[SUB] 100 100 100 100 2...8°C when not in use.)
Mix carefully
References
Incubate 30 min. at 15...25°C (see P8)
1. Polesky H. F., Olson C., The incidence and significance of antibody to
[STOP] 100 100 100 100 Australia antigen in blood donors, Am. J. Clin. Pathol. 56, 129 (1971)
Mix carefully 2. Blumberg B.S. et al., Australia antigen and hepatitis, New Eng. J. Med.
Measure the absorbance at 450 nm as soon as possible or within 30 283, 349-354 (1970)
min. after terminating the reaction, using a reference wavelength of 3. Hoofnagle J. H. et al., Antibody to Hepatitis-B-Virus Core in Man. Lancet
630 – 690 nm (if available). ii: 869-873, 1973

Alternative procedures intended for automated ELISA analyzers:
A 2:
(Step 2) dispense 50 µl [SA] first, then 50 µl [SB] into each well
including A1; mix well and proceed as described
4. Szmuness W. et al., Antibody Against the Hepatitis Type B Core
Antigen, Am. J. Epidemiol. 104, 256-262 (1976)
5. Katchaki J. N. et al., Detection and Significance of Anti-HBc in the blood
Bank: Preliminary Results of a Controlled Prospective Study, J. Virol
Methods 2, 199-125 (1980)

Calculation of Control Values, Cut-off and Cut-off index

EL-A-HBC INF 5126001 GB 10-2010-13
Mean absorbance values of [NC] in wells B1, C1 and D1 (MNC) and [PC] in
wells E1 and F1 (MPC) are calculated according to:
A450 (B1) + A450 (C1) + A450 (D1) A450 (E1) + A450 (F1)
MNC = MPC =
3 2
Cut-off value COV = MNC + 0.025
The test run may be considered valid provided that the following criteria
are met:
1. Colour in A1: colourless or light yellow, otherwise the test is invalid and
should be repeated.
2. MNC 0.400
3. MPC 0.100
4. MPC – MNC 0.300.

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