Product Manual

QuickTiter™ Hepatitis C Core Antigen(HCVcAg) ELISA Kit
Catalog Numbers
VPK-151

96 wells

FOR RESEARCH USE ONLY
Not for use in diagnostic procedures

Introduction
Hepatitis C is an infection of the liver caused by the hepatitis C virus. HCV is transmitted by exposure
to infectious blood; forms of transmission include unprotected sexual activity, blood transfusion,mother-to-infant transmission, or occupational exposure to blood. The acute illness causes liver
inflammation, liver fibrosis, vomiting and jaundice, while chronic HCV infection often leads to livercirrhosis and failure.
An estimated 270-300 million people worldwide have been infected with hepatitis C. At least 75% ofpeople infected will develop chronic hepatitis C and have it the rest of their lives.
Diagnosis of chronic hepatitis C virus (HCV) infection has long been based on HCV serology anddetection of HCV antibodies. With the development of therapies for chronic HCV infection, includinginterferon and ribavirin, quantitative detection of HCV has been used increasingly as the mostimportant marker for monitoring HCV titer, disease progression, and assessing antiviral treatment.
Several assays for the quantitative measurement of HCV DNA have been developed, such as PCRbased nucleic acid amplification assays. However, these methods tend to be cumbersome andexpensive.
Cell Biolabs’ QuickTiter™ HCV Core Antigen ELISA Kit is an enzyme immunoassay developed for
detection and quantitation of the HCV core protein. The kit has detection sensitivity limit of 1 ng /mLHCVcAg. Each kit provides sufficient reagents to perform up to 96 assays including standard curveand HCV samples.

Assay PrincipleAn anti-HCVcAg monoclonal coating antibody is adsorbed onto a microtiter plate. HCV core antigen
present in the sample or standard binds to the antibodies adsorbed on the plate; a FITC-conjugatedmouse anti-HCVcAg 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-HCVcAg. 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 HCV 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 HCV core antigen and sample concentration is then determined.

Related Products
1. VPK-150: QuickTiter™ HBV Core Antigen 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

2Kit Components
Box 1 (shipped at room temperature)
1. Anti-HCVcAg Antibody Coated Plate (Part No. 315101): One strip well 96-well plate.
2. FITC-Conjugated Anti-HCVcAg Monoclonal Antibody (Part No. 315102): 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 HCVcAg Standard (Part No. 315103): One 100 µL vial of 10 µg/mL recombinant
HCV Core Antigen in 8 M Urea containing BSA.

Materials Not Supplied
1. HCV 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 HCVcAg Standard at -20ºC and avoid freeze/thaw.
Store all other components at 4ºC.

Safety Considerations
Remember that your samples contain infectious viruses before inactivation; you must follow therecommended 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-HCVcAg Monoclonal Antibody and HRP-Conjugated Anti-FITCMonoclonal 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 HCVcAg Standard in the concentration range of 100
ng/mL to 1 ng/mL by diluting the stock solution in Assay Diluent (Table 1).
StandardTubes
12345678HCVcAg
Standard (µL)
10500 of Tube #1500 of Tube #2500 of Tube #3500 of Tube #4500 of Tube #5500 of Tube #60Assay Diluent (µL)
990
500
500
500
500
500
500
500HCVcAg(ng/mL)
100
5025
12.5
6.25
3.125
1.5625
0Table 1. Preparation of HCVcAg Standard
2. Transfer 225µL of each dilution to a microcentrifuge tube containing 25 µL of Triton X-100Solution. Perform the assay as described in Assay Instructions.

HCV Sample Dilution and Inactivation
1. (Optional) Dilute HCV 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-100Solution, Vortex well.
3. Incubate 30 minutes at 37ºC.
Note: For samples that contain antibodies, release HCVcAg from the virion by incubating samples
at 56ºC for 30 min.

Assay Protocol
1. Prepare and mix all reagents thoroughly before use.
2. Each HCV sample, HCVcAg standard, blank, and control medium should be assayed in duplicate.
3. Add 100 µL of inactivated sample or HCVcAg standard to Anti-HCVcAg 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 Buffer
per 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-HCVcAg 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 HCVcAg ELISA results. One should use the data below forreference only. This data should not be used to interpret actual results.

5Figure 1: HCV Core Antigen ELISA Standard Curve

References
1. Alter, H. J., R. H. Purcell, J. W., Shih, J. C. Melpolder, M. Houghton, Q.-L. Choo, and G. Kuo(1989) N. Engl. J. Med. 321:1494-1500.
2. Choo, Q.-L., G. Kuo, A. J. Weiner, L. R. Overby, D. W. Bradley, and M. Houghton (1989)
Science 244:359-362.
3. Kuo, G., Q.-L. Choo, H. J. Alter, G. I. Gitnick, A. G. Redeker, R. H. Purcell, T. Miyamura, J.
L. Dienstag, M. J. Alter, C. E. Stevens, G. E. Tegtmeier, F. Bonino, M. Colombo, W. S. Lee, C.
Kuo, K. Berger, J. R. Shuster, L. R. Overby, D. W. Bradley, and M. Houghton (1989) Science
244:362-364.
4. Alias, M. B., K. Patel, H. Dahari, S. Beaucourt, P. Larderie, L. Blatt, C. Hezode, G. Picchio, D.
Dhumeaux, A. U. Neumann, J. G. McHutchison, and J. M. Pawlotsky (2002) Hepatology
36:211-218.
5. Aoyagi, K., C. Ohue, K. Iida, T. Kimura, E. Tanaka, K. Kiyosawa, and S. Yagi (1999) J. Clin.
Microbiol. 37:1802-1808.
6. Kurtz, J. B., E. Boxall, N. Qusir, J. Shirley, D. Coleman, and C. Chandler (2001) J. Virol.
Methods 96:127-132.

Recent Product Citations
1. Rani, S.V. et al. (2023). Detection of Hepatitis C Virus Infection and its GenotypicCharacterisation among Hemodialysis Patients by HCV Core Antigen Elisa and RTPCR- A
Hospital- Based Prospective Study in a Tertiary Care Hospital in South India. Int J Acad MedPharm. 5(4): 628-632. doi: 10.47009/jamp.2023.5.4.125.
2. 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.
3. Hammerstad, S.S. et al. (2019). Hepatitis C virus infection of human thyrocytes: metabolic,hormonal, and immunological implications. J Clin Endocrinol Metab. pii: dgz241. doi:
10.1210/clinem/dgz241.
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4. Blackard, J.T. et al. (2019). CCR5 receptor antagonism inhibits hepatitis C virus (HCV) replicationin vitro. PLoS One. 14(10):e0224523. doi: 10.1371/journal.pone.0224523.
5. Plissonnier, M.L. et al. (2019). LARP1 binding to hepatitis C virus particles is correlated withintracellular retention of viral infectivity. Virus Res. doi: 10.1016/j.virusres.2019.197679.
6. Kumar, S. et al. (2019). MARCH8 Ubiquitinates the Hepatitis C Virus Nonstructural 2 Protein andMediates Viral Envelopment. Cell Rep. 26(7):1800-1814.e5. doi: 10.1016/j.celrep.2019.01.075.
7. Hassan, R. et al. (2018). Role of Hepatitis C Virus Core Antigen Assay in Blood Donors Screening
at Zagazig University Hospitals. Afro-Egyptian Journal of Infectious and Endemic Diseases.
8(1):48-54. doi: 10.21608/aeji.2018.8735.
8. Xiao, F. et al. (2018). Interactions between the Hepatitis C Virus Nonstructural 2 Protein and Host
Adaptor Proteins 1 and 4 Orchestrate Virus Release. MBio. 9(2). pii: e02233-17. doi:
10.1128/mBio.02233-17.
9. Blackard, J.T. et al. (2017). A preliminary analysis of hepatitis C virus in pancreatic islet cells.
Virol J. 14(1):237. doi: 10.1186/s12985-017-0905-3.
10. Dvir, R. et al. (2017). Autoimmune hepatitis and occult HCV infection: A prospective single-centre
clinical study. Autoimmun Rev. 16(3):323-325. doi: 10.1016/j.autrev.2017.01.015.
11. Owolabi, O. B. et al. (2015). Hepatitis C virus seroprevalence, antigaenemia and associated risk
factors among pregnant women in Nigeria. Ethiop Med J. 53.
12. Liu, D. et al. (2015). Downregulation of miRNA-30c and miR-203a is associated with Hepatitis Cvirus core protein-induced epithelial-mesenchymal transition in normal hepatocytes andhepatocellular carcinoma cells. Biochem Biophys Res Commun. doi: 10.1016/j.bbrc.2015.07.107.
13. Jittavisutthikul, S. et al. (2015). Humanized-VHH transbodies that inhibit HCV protease andreplication. Viruses. 7:2030-2056.
14. Amet, T. et al. (2014). BST-2 expression in human hepatocytes is inducible by all three types ofinterferons and restricts production of hepatitis C virus. Curr Mol Med. 14:349-360.
15. Kong, L. et al. (2014). HIV infection of hepatocytes results in a modest increase in hepatitis Cvirus expression in vitro. PLoS One. 9:e83728.
16. Carpentier, A. et al. (2014). Engrafted human stem cell–derived hepatocytes establish an infectious
HCV murine model. J Clin Invest. 124:4953-4964.
17. Deng, A. et al. (2014). Human immunodeficiency virus type 1 Vpr increases hepatitis C virus RNA
replication in cell culture. Virus Res. 184:93-102.

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 ORWARRANTY 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.

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Contact Information
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