Product Manual

QuickTiter™ Hepatitis B Core Antigen(HBVcAg) ELISA Kit
Catalog Numbers
VPK-150

96 assays

VPK-150-5

5 x 96 assays

FOR RESEARCH USE ONLY
Not for use in diagnostic procedures

Introduction
Hepatitis B is an infection of the liver caused by the hepatitis B virus. HBV is transmitted by exposure
to infectious blood or body fluids (e.g. saliva, semen, urine); forms of transmission include unprotected
sexual activity, blood transfusion, mother-to-infant transmission, or consuming contaminatedfood/water. The acute illness causes liver inflammation, vomiting and jaundice, while chronic HBV
infection often leads to liver cirrhosis and cancer.
Roughly one third of the world’s population have been infected with hepatitis B virus. 5-10% of adults
and 90% of babies who have been infected will have the virus for the rest of their lives. The infection
is preventable by vaccination.
Diagnosis of chronic hepatitis B virus (HBV) infection has long been based on HBV serology andmeasurement of hepatocytic enzymes. With the development of therapies for chronic HBV infection,
including interferon and lamivudine, quantitative detection of HBV has been used increasingly as themost important marker for monitoring HBV replication activity, disease progression, and assessing
antiviral treatment. Several assays for the quantitative measurement of HBV DNA have beendeveloped, such as PCR-based nucleic acid amplification assays. However, these methods tend to becumbersome and expensive.
Cell Biolabs’ QuickTiter™ HBV Core Antigen ELISA Kit is an enzyme immunoassay developed fordetection and quantitation of the HBV core protein. The kit has detection sensitivity limit of 1 ng /mL
HBcAg. Each kit provides sufficient reagents to perform up to 96 assays including standard curve andHBV samples.

Assay Principle
An anti-HBVcAg monoclonal coating antibody is adsorbed onto a microtiter plate. HBV core antigen
present in the sample or standard binds to the antibodies adsorbed on the plate; a FITC-conjugatedmouse anti-HBVcAg antibody is added and binds to the antigen captured by the first antibody.
Following incubation and wash steps, a HRP-conjugated mouse anti-FITC antibody is added and binds
to the FITC conjugated anti-HBVcAg. Unbound HRP-conjugated mouse anti-FITC antibody isremoved during a wash step, and substrate solution reactive with HRP is added to the wells.
A colored product is formed in proportion to the amount of HBV core antigen present in the sample.
The reaction is terminated by addition of acid and absorbance is measured at 450 nm. A standard curve
is prepared from recombinant HBV core antigen and sample concentration is then determined.

Related Products
1. VPK-151: QuickTiter™ Hepatitis C Core Antigen (HCcAg) ELISA Kit
2. VPK-108-H: QuickTiter™ Lentivirus Quantitation Kit (HIV-1 p24 ELISA)
3. VPK-107: QuickTiter™ Lentivirus Titer Kit (Lentivirus-Associated HIV p24)
4. VPK-112: QuickTiter™ Lentivirus Quantitation Kit
5. LTV-200: ViraDuctin™ Lentivirus Transduction Kit

2Kit Components
Box 1 (shipped at room temperature)
1. Anti-HBVcAg Antibody Coated Plate (Part No. 315001): One strip well 96-well plate.
2. FITC-Conjugated Anti-HBVcAg Monoclonal Antibody (Part No. 315002): One 20 µL vial.
3. HRP-Conjugated Anti-FITC Monoclonal Antibody (Part No. 310811): One 20 µL vial.
4. Assay Diluent (Part No. 310804): One 50 mL bottle.
5. Triton X-100 Solution (Part No. 310805): One 15 mL bottle containing 5% Triton X-100 inTBS.
6. 10X Wash Buffer (Part No. 310806): One 100 mL bottle.
7. Substrate Solution (Part No. 310807): One 12 mL amber bottle.
8. Stop Solution (Part. No. 310808): One 12 mL bottle.
Box 2 (shipped on blue ice packs)
1. Recombinant HBVcAg Standard (Part No. 315003): One 100 µL vial of 10 µg/mL recombinant
HBV Core Antigen in PBS containing BSA.

Materials Not Supplied
1. HBV Sample: purified virus or unpurified viral supernatant
2. Cell Culture Centrifuge
3.
4.
5.
6.
7.

0.45 µm filter
10 µL to 1000 µL adjustable single channel micropipettes with disposable tips
50 µL to 300 µL adjustable multichannel micropipette with disposable tips
Multichannel micropipette reservoir
Microplate reader capable of reading at 450 nm (620 nm as optional reference wave length)

StorageUpon receiving, aliquot and store recombinant HBVcAg Standard at -20ºC and avoid freeze/thaw.
Store all other components at 4ºC.

Safety Considerations
Remember that your lentiviral samples contain infectious viruses before inactivation; you must follow
the recommended NIH guidelines for all materials containing infectious organisms.

Preparation of Reagents•1X Wash Buffer: Dilute the 10X Wash Buffer Concentrate to 1X with deionized water. Stir tohomogeneity.
3•FITC-Conjugated Anti-HBVcAg Monoclonal Antibody and HRP-Conjugated Anti-FITC
Monoclonal Antibody: Immediately before use dilute the FITC-conjugated antibody 1:1000 andHRP-conjugated antibody 1:1000 with Assay Diluent. Do not store diluted solutions.

Preparation of Standard Curve
1. Prepare a dilution series of Recombinant HBVcAg Standard in the concentration range of 100
ng/mL – 1 ng/mL by diluting the stock solution in Assay Diluent (Table 1).
StandardTubes
12
34
5
678

HBVcAgStandard (µL)
10500 of Tube #1
500 of Tube #2
500 of Tube #3
500 of Tube #4
500 of Tube #5500 of Tube #60

Assay Diluent
(µL)
990
500
500
500
500
500
500
500

HBVcAg(ng/mL)
1005025
12.5
6.25
3.125
1.56250

Table 1. Preparation of HBVcAg Standard
2. Transfer 225µL of each dilution to a microcentrifuge tube containing 25 µL of Triton X-100
Solution. Perform the assay as described in Assay Instructions.

HBV Sample Dilution and Inactivation
1. (Optional) Dilute HBV sample in culture medium. Include culture medium as a negative control.
2. Transfer 225 µL of each sample to a microcentrifuge tube containing 25 µL of Triton X-100
Solution, Vortex well.
3. Incubate 30 minutes at 37ºC.
Note: For samples that contain antibodies, release HBVcAg from the virions by incubating samplesat 56ºC for 30 min.

Assay Protocol
1. Prepare and mix all reagents thoroughly before use.
2. Each HBV sample, HBVcAg standard, blank, and control medium should be assayed in duplicate.
3. Add 100 µL of inactivated sample or HBVcAg standard to Anti-HBVcAg Antibody Coated Plate.
4. Cover with a Plate Cover and incubate at 37ºC for 2 hours.

45. Remove plate cover and empty wells. Wash microwell strips 5 times with 250 µL 1X Wash Bufferper well with thorough aspiration between each wash. After the last wash, empty wells and tap
microwell strips on absorbent pad or paper towel to remove excess 1X Wash Buffer.
6. Add 100 µL of the diluted FITC-Conjugated Anti-HBVcAg Monoclonal Antibody to each well.
7. Cover with a plate cover and incubate at room temperature for 1 hour on an orbital shaker.
8. Remove plate cover and empty wells. Wash the strip wells 5 times according to step 5 above.
9. Add 100 µL of the diluted HRP-Conjugated Anti-FITC Monoclonal Antibody to all wells.
10. Cover with a plate cover and incubate at room temperature for 1 hour on an orbital shaker.
11. Remove plate cover and empty wells. Wash microwell strips 5 times according to step 5 above.
Proceed immediately to the next step.
12. Warm Substrate Solution to room temperature. Add 100 L of Substrate Solution to each well,including the blank wells. Incubate at room temperature on an orbital shaker. Actual incubation
time may vary from 5-20 minutes.
Note: Watch plate carefully; if color changes rapidly, the reaction may need to be stopped sooner
to prevent saturation.
13. Stop the enzyme reaction by adding 100 µL of Stop Solution into each well, including the blankwells. Results should be read immediately (color will fade over time).
14. Read absorbance of each microwell on a spectrophotometer using 450 nm as the primary wave
length.

Example of Results
The following figures demonstrate typical HBVcAg ELISA results. One should use the data below forreference only. This data should not be used to interpret actual results.

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Figure 1: HBV Core Antigen ELISA Standard Curve

References
1. Bottcher, B., S. A. Wynne, and R. A. Crowther (1997) Nature 386:88-91. 4
2. Bredehorst, R., H. von Wulffen, and C. Granato (1985) J. Clin. Microbiol. 21:593-598. 5
3. Kimura, T., A. Rokuhara, Y. Sakamoto, S. Yagi, E. Tanaka, K. Kiyosawa, and N. Maki (2002)
J. Clin. Microbiol. 40:439-445. 15
4. Usuda, S., H. Okamoto, F. Tsuda, T. Tanaka, Y. Miyakawa, and M. Mayumi (1998) J. Virol.
Methods 72:95-103. 30

Recent Product Citations
1. Miyakawa, K. et al. (2022). Galectin-9 restricts hepatitis B virus replication via p62/SQSTM1mediated selective autophagy of viral core proteins. Nat Commun. 13(1):531. doi: 10.1038/s41467022-28171-5.
2. Domfeh, S.A. et al. (2021). Cryptolepine and Nibima inhibit hepatitis B virus replication. Sci Afr.
doi: 10.1016/j.sciaf.2021.e00942.
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3. Kong, L. et al. (2021). The synthetic opioid fentanyl enhances viral replication in vitro. PLoS One.
16(4):e0249581. doi: 10.1371/journal.pone.0249581.
4. Akahori, Y. et al. (2020). Establishment of a novel hepatitis B virus culture system using
immortalized human hepatocytes. Sci Rep. 10(1):21718. doi: 10.1038/s41598-020-78655-x.
5. Peiffer, K.H. et al. (2020). Quadruple mutation GCAC1809-1812TTCT acts as a biomarker inhealthy European HBV carriers. JCI Insight. doi: 10.1172/jci.insight.135833.
6. Jiang, B. et al. (2020). The N-Terminus Makes the Difference: Impact of Genotype-Specific
Disparities in the N-Terminal Part of The Hepatitis B Virus Large Surface Protein on
Morphogenesis of Viral and Subviral Particles. Cells. 9(8):E1898. doi: 10.3390/cells9081898.
7. Colant, N. et al. (2020). A rational approach to improving titer in E. coli-based cell-free protein
synthesis reactions. Biotechnol Prog. doi: 10.1002/btpr.3062.
8. Jiang, B. et al. (2019). Formation of semi-enveloped particles as a unique feature of a hepatitis B
virus PreS1 deletion mutant. Aliment Pharmacol Ther. doi: 10.1111/apt.15381.
9. Biswas, B. et al. (2017). A G-quadruplex motif in an envelope gene promoter regulates
transcription and virion secretion in HBV genotype B. Nucleic Acids Res. 45(19):11268-11280.
doi: 10.1093/nar/gkx823.

Warranty
These products are warranted to perform as described in their labeling and in Cell Biolabs literature when used inaccordance with their instructions. THERE ARE NO WARRANTIES THAT EXTEND BEYOND THIS EXPRESSEDWARRANTY AND CELL BIOLABS DISCLAIMS ANY IMPLIED WARRANTY OF MERCHANTABILITY OR
WARRANTY OF FITNESS FOR PARTICULAR PURPOSE. CELL BIOLABS’s sole obligation and purchaser’s
exclusive remedy for breach of this warranty shall be, at the option of CELL BIOLABS, to repair or replace the products.
In no event shall CELL BIOLABS be liable for any proximate, incidental or consequential damages in connection with theproducts.

Contact Information
Cell Biolabs, Inc.
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San Diego, CA 92111
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E-mail: tech@cellbiolabs.com
www.cellbiolabs.com©2010-2024: Cell Biolabs, Inc. - All rights reserved. No part of these works may be reproduced in any form without
permissions in writing.

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