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Detection of hepatitis C virus core protein circulating within different virus particle populations
O V Masalova1, S N Atanadze, E I Samokhvalov
1D.I. Ivanovsky Institute of Virology, Moscow, Russia.
Insights
Researchers developed a new method to detect hepatitis C virus (HCV) core protein in patient plasma, overcoming challenges of low concentration and antibody interference. This improved detection aids in understanding HCV pathogenesis and diagnosis.
Area of Science:
- Virology
- Immunology
- Biochemistry
Background:
- Hepatitis C virus (HCV) diagnosis and pathogenesis studies are hindered by low viral protein concentrations and antibody interference.
- Accurate detection of HCV proteins is crucial for reliable diagnosis and understanding disease mechanisms.
Purpose of the Study:
- To develop a sensitive method for detecting hepatitis C virus (HCV) nucleocapsid (core) protein in plasma using monoclonal antibodies (MABs).
- To overcome challenges in HCV protein detection, including low viral load and antibody binding.
Main Methods:
- Plasma samples from HCV-infected individuals were analyzed using enzyme immunoassay (EIA) with four MABs targeting different determinants of the core protein.
- Pellets from ultracentrifuged plasma were treated with Tween 80 and KBr to dissociate immune complexes and expose viral proteins.
- The assay's sensitivity was determined for both recombinant and natural core proteins.
Main Results:
- A monoclonal sandwich assay detected HCV core protein with a sensitivity of 1 ng/ml in the pellet, or 5 pg/ml normalized to plasma volume.
- Optimal detection of natural core protein required four MABs, suggesting conformational differences from recombinant analogs.
- Core protein was detected in 19 out of 21 HCV RNA-positive plasma samples using the developed method.
Conclusions:
- The developed method effectively detects hepatitis C virus (HCV) core protein in patient plasma, even in the presence of antibodies.
- This technique enhances the reliability of HCV diagnosis and provides a valuable tool for studying viral pathogenesis.
- The findings highlight the utility of MABs in overcoming detection challenges for viral proteins in complex biological samples.
Abstract:
Progress in studying pathogenesis and increasing the reliability of hepatitis C diagnosis can be achieved by analysis of different forms of virus particles circulating in blood of both patients and infected persons. Detection of hepatitis C virus (HCV) proteins faces two basic difficulties: low concentration of HCV proteins, and their blocking by antibodies. The aim of this work was to develop a method for the detection of nucleocapsid (core) protein in the plasma of HCV-infected persons using monoclonal antibodies (MABs). Twenty-seven anti-HCV-positive donor plasmas were studied of which 21 contained HCV RNA and 6 were negative. The plasmas were centrifuged for 3 hr at 143,000 g and the antigenic activity of core-protein was studied in the pellets by EIA using four MABs able to recognize four nonoverlapping determinants, two at N-terminus and two at C-terminus of recombinant core (1-150 aa). The determinants detected were present in the natural core protein of at least two genotypes (1b and 3a). Maximal efficiency of recombinant protein detection was achieved with 2 MABs, whereas a combination of 4 MABs was necessary for optimal detection of natural core protein. This is indicative of different conformational structures of natural protein and its gene-engineered analog. The sensitivity of core detection by monoclonal sandwich assay was 1 ng/ml in the pellet or 5 pg/ml after normalization to the initial plasma volume. To dissociate immune complexes, the pellet was treated with 2.5 M KBr after first treating the pellet with the nonionic detergent Tween 80 to remove the virus lipid envelope. Using this treatment protocol, core protein was found in 19 of 21 RNA positive plasmas.