Methodology for an Adaptable HCV Immunoassay

With infectious diseases, serology-based diagnosis serves as a primary tool for surveillance and diagnosis. However, there are little to no commercial assays available for certain neglected diseases which mostly impacts low and middle income countries due to prices associated with large scale testing.

A recently published study articulated the development and validation of a multiepitope based on enzyme-linked immunoassay (ELISA) against hepatitis C virus (HCV). The implementation of this assay can be implemented at low cost, according to study investigators.

The methodology has the potential to provide a readily adaptable method that can be utilized for the development of novel ELISA-based diagnostic assays for other infectious pathogens, which could improve diagnosis of neglected diseases.

Developing the MBP-HCV Antigen

A team of investigators including Jannie Pedersen, Département de Microbiologie-Infectiologie et Immunologie, Faculté de Médecine, Université Laval, developed an adaptable enzyme-linked immunoassay (ELISA) using HCV as a proof-of-concept application. They were able to obtain a high concentration of protein suitable for downstream applications after combining the maltose-binding-protein with a multiepitope HCV protein.

“Following optimization, the assay was verified using previously tested human samples from Canada, Denmark and Gabon in parallel with the use of a commercial protein,” investigators wrote. “Sensitivity and specificity were calculated to 98% and 97% respectively, after accounting for non-specific binding and assay optimization.”

The team cloned several HCV specific epitopes, separated by GS-linker, relaying them into the modified pET28a+ vector which were fused with the maltose-binding protein (MBP). The antigen (MBP-HCV) was induced in BL21-DE3 bacterial cells with IPTG and purified by using immobilized metal ion affinity chromatography.

Validating the Serological Assay

Samples were selected from patients with chronic HCV infection and compared with healthy controls for assay optimization. Investigators estimated that the optimal signal-to-noise ratio to be with a 3-hour inactivation at 56 degrees celsius. A modest drop of 17% in optical density was observed under this condition in the positive samples.

At the same time, a 72% reduction was observed in non-specific signal from a mean optical density of 0.11-0.03 in control samples after 3-hour inactivation compared with 30 minutes of inactivation. The best results were achieved by coating each well with 15 ng of MBP-HCV and using a 1:100 dilution of sample material.

“To evaluate the quality of our MBP-HCV protein, the ELISA binding of the same HCV positive and negative samples was analyzed using a commercially available recombinant protein expressing NS3, Core, NS4 and NS5,” investigators wrote.

A similar sensitivity (95%, CI, 91-97%) and specificity (96% CI, 92-98%) were obtained when using this commercial protein. With these provided data, the quality of the team’s in-house produced MBP-HCV protein designed for serological diagnosis of HCV patients was confirmed.

Suggested Implementation

“Here, we document the development of a simple serological test detecting anti-HCV antibodies. This assay can be reproduced and validated in reference laboratories in low and middle income countries, where HCV remains a major health burden,” investigators explained.

If local production of the assay could be implemented by government laboratories or nonprofit organizations, the barriers of high delivery cost, import taxes and manufacturer and distribution profit margins would be lessened or eliminated, according to the study.

“While serological diagnosis cannot be a stand-alone diagnostic tool for HCV infection, the developed ELISA assay is a simple and accurate way to document HCV prevalence in a large population,” investigators stated. “The global fight to decrease hepatitis as one of the major health threats will need broad screening platforms, which could readily test the whole at-risk population rather than solely symptomatic individuals.”

The study, “An Adaptable Platform for in-house Hepatitis C Serology” was published in the Journal of Virological Methods.