Improvement in the antibody detection sensitivity of the Murex Ab/Ag combination HCV assay

Abstract

Globally, the hepatitis C virus is one of the leading causes of chronic liver diseases, with a high incidence of these diseases developing liver cancer. Hepatitis C infection may be cured, but its effects may only be realised many years after infection. While much has been done to prevent and mitigate the risk of infection, populations in underdeveloped countries remain vulnerable. Thus, in many countries within sub-Saharan Africa, access to high quality healthcare for many transmissible diseases remains limited. It is estimated that only 5% of those infected with hepatitis C virus are diagnosed, while almost none have treatment options. These statistics have alarming implications for the availability and accessibility of safe blood and blood products. In resource-constrained countries, access to traditional screening methods is limited and expensive, requiring burdensome specialised equipment and training. It is, therefore, imperative to find high quality tests that are accurate, simple to use, and require minimal resources. The Murex HCV Ag/Ab Combination assay is a high-quality serological assay that may be used to screen blood, as it is able to simultaneously detect both HCV antigen and anti-HCV antibodies. A positive result may then be confirmed using more expensive molecular techniques. The Murex HCV Ag/Ab Combination assay whilst being able to simultaneously detect both antigen and antibodies to HCV, is more sensitive in its detection of the hepatitis C viral parti-cles. The antibody detection aspect of the assay, although robust in its detection of the anti-bodies to core region 1, 2, and 3 as well as non-structural proteins, has been difficult to prepare. Specifically, detection of antibodies to core region 3 required the addition of large amounts of the detection molecule during preparation to achieve the required assay sensitivity. This not only makes the assay more expensive but also impacts the specificity of the assay by increas-ing the risk of non-specific binding of assay reagents. The aim of this study was to improve the ability of the of the Murex HCV Ag/Ab Combination assay to detect antibodies generated to core region 3 of the HCV genome in blood serum by: optimizing the preparation of the enzyme-peptide conjugate; developing an improved method for the removal of excess/unwanted reagents and optimizing the sensitivity of the core capture molecule coated on the microtitre wells. This experimental study identified variables within the current preparation process for both the capture and detection molecules and evaluated them to determine their effect on the Murex HCV Ab/Ag Combination Assay. A separate capture molecule specific to each region of the core represented in the Murex HCV Ab/Ag Combination Assay was prepared by the linkage of the each of the peptides to a single bovine serum albumin (BSA) molecule via sulfhydryl bonds. This molecule was then coated onto microtitre wells at a concentration of a 0.2 μg/ml. The detection molecule was prepared by: i) increasing the concentration of sulfo-SMCC modified horseradish peroxidase to 1250 μM; ii) providing sulfo-SMCC in 2.5-fold excess to horseradish peroxidase; iii) providing a 500 mM peptide solution for linkage to the modified horseradish peroxidase; iv) cross-linking the core peptide and modified horseradish peroxidase in pH 6.0 buffer and, v) removing excess and unbound reagents using a Sephadex G25 column and eluting with pH 6.0 buffer. The sensitivity, specificity, and stability of the optimised core and detection molecules were evaluated using a quality control containing antibodies specific to core region 3 only, the antibody positive control contained within the Murex HCV Ab/Ag Combination Assay, as well as 90 normal human serum samples known to be negative for hepatitis C antibodies. The incorporation of these steps improved the sensitivity of detection of antibodies to core region 3 by 36%, the specificity of the assay by 13%, and the limit of detection was lowered by 50.9%. In addition, the optimised method did not adversely affect the stability of the Murex HCV Ab/Ag Combination Assay. Incorporation of the suggested steps and reagents may optimize the sensitivity and specificity of the Murex HCV Ab/Ag Combination Assay for the screening of donor blood and blood prod-ucts. This will provide a more reliable, less burdensome, and inexpensive alternative for the screening of blood in countries with resource constraints. Thus, this will allow more people to access treatment options and positively contribute to the World Health Organizations (WHO) target of decreasing mortality by 65% by 2030.