A New ELISA to Overcome the Pitfalls in Quantification of Recombinant Human Monoclonal Anti-HBs, GC1102, by Commercial Immunoassays

Yong Won Shin,Gi Won Song,Se-Ho Kim,Dong-Hyung Cho

doi:10.1186/s12575-018-0083-8

Abstract

Several methods for the quantification of human anti-HBs, an antibody to hepatitis B surface antigen (HBsAg), have been developed based on enzyme reaction, chemiluminescence, fluorescence, and radioactivity for application to human serum or plasma. Commercial anti-HBs immunoassay kits use a sandwich method in which a bridge is formed by the anti-HBs between a HBsAg immobilized solid matrix and the labeled HBsAg. However, this direct sandwich enzyme-linked immunosorbent assay (ELISA) is insufficient to accurately evaluate the activity of the human monoclonal anti-HBs, GC1102. As an alternative, we developed an indirect anti-HBs ELISA (anti-HBs qELISA_v.1) that improved detection of anti-HBs. In this current study, we further optimized this indirect method to minimize nonspecific binding of human serum, by employing incubation buffers containing animal serum, Tween 20, skim milk, and a low pH washing buffer. This new and improved method, termed anti-HBs qELISA_v.2, showed accurate quantification of plasma-derived hepatitis B immune globulin (HBIG) and was comparable to results obtained with commercial ELISA (r = 0.93) and RIA (r = 0.85) kits. Further, the GC1102 in human serum could be precisely measured using the anti-HBs qELISA_v.2 without limitations of nonspecific binding.

Highlights

Hepatitis B is caused by infection of the liver by the hepatitis B virus (HBV)
According to the World Health Organization (WHO) report in 2015, approximately 257 million people were infected by HBV and 887,000 people succumbed to HBV-induced death, mostly from liver cirrhosis and hepatocellular carcinoma [1, 2]
Quantification of anti-HBs is carried out using a direct sandwich type of enzyme-linked immunosorbent assay (ELISA), which relies on a hepatitis B surface antigen (HBsAg)-coated solid matrix and horseradish peroxidase (HRP) labeled-HBsAg for the detection and estimation of anti-HBs [11]

Summary

Introduction

Hepatitis B is caused by infection of the liver by the hepatitis B virus (HBV). According to the World Health Organization (WHO) report in 2015, approximately 257 million people were infected by HBV and 887,000 people succumbed to HBV-induced death, mostly from liver cirrhosis and hepatocellular carcinoma [1, 2].Hepatitis B immune globulin (HBIG), prepared from hepatitis B vaccine-boosted plasma, is being used effectively for HBV prophylaxis and for preventing hepatitis B recurrence after liver transplantation in patients with hepatitis B-induced liver complications [3,4,5,6]. This indirect method uses a similar HBsAgimmobilized microplate as sandwich ELISA, it makes use of goat anti-human IgG to detect bound antiHBs, instead of HBsAg. This anti-HBs qELISA_v.1 showed accurate quantification of recombinant anti-HBs in cell culture media, PBS buffer and monkey serum, but failed to quantify precise levels of anti-HBs in human serum due to a high nonspecific binding.

Results

Conclusion