Immune Netw.  2017 Dec;17(6):451-459. 10.4110/in.2017.17.6.451.

ELISA for Quantitative Determination of Hepatitis B Virus Surface Antigen

• Kim SH

• KMID: 2400630
• DOI: http://doi.org/10.4110/in.2017.17.6.451

Abstract

Several studies have reported a good correlation between levels of serum hepatitis B virus surface antigen (HBsAg) and covalently closed circular DNA (cccDNA) before and after antiviral therapy. As a result, the quantification of HBsAg levels has attracted much attention in recent years as an important approach to evaluate viral activity. In this study, mAbs against HBsAg were generated and 9 mAbs (H17, H30, H31, H67, H73, H97, H101, H118, and H128) were investigated for optimization of HBsAg quantitation ELISA. Determination of the best combinations of mAbs for sandwich ELISA identified H17 and H31 mAbs as the ideal capture and horseradish peroxidase (HRP) conjugate mAbs, respectively. A standard curve for the current assay system exhibited linearity up to 40 ng/ml of HBsAg while a detection limit of approximately 1 ng/ml of HBsAg was also estimated, which was comparable to that of the other commercial ELISA kits. The ELISA system established in this study is particularly differentiated from other commercial kits in using mAbs for both capture and HRP conjugate, which provides a solution to inconsistency of quality and ethical issues in polyclonal antibodies production using laboratory animals.

Keyword

ELISA; Hepatitis B virus surface antigen; Monoclonal antibody; Quantification

MeSH Terms

Animals, Laboratory
Antibodies
Antigens, Surface
DNA, Circular
Enzyme-Linked Immunosorbent Assay*
Ethics
Hepatitis B Surface Antigens
Hepatitis B virus*
Hepatitis B*
Hepatitis*
Horseradish Peroxidase
Limit of Detection
Antibodies
Antigens, Surface
DNA, Circular
Hepatitis B Surface Antigens
Horseradish Peroxidase

Figure

• 

  Figure 1 The result of primary screening of mAb pairs to screen out the candidates for further evaluation for quantitative HBsAg ELISA. ELISA signals (A) with respect to the immobilized mAbs and (B) with respect to the HRP-conjugated mAbs. ELISA signals were measured for each combination of the 9 immobilized mAbs (H17, H30, H31, H67, H73, H97, H101, H118, and H128) vs. the 7 mAb-HRP conjugates (H17, H31, H67, H73, H97, H101, and H128). Ten ng/ml of HBsAg in 1% BSA-PBS was used as a positive control. Each assay was performed in duplicate. Mean values are shown in the graph.

• 

  Figure 2 The result of confirmatory screening of mAb pairs to decide the candidates for development of quantitative HBsAg ELISA kit. ELISA signals (A) with respect to the immobilized mAbs and (B) with respect to the HRP-conjugated mAbs. ELISA signals were measured for each combination of the 5 immobilized mAbs (H17, H67, H101, H118, and H128) vs. the 3 mAb-HRP conjugates (H31, H67, and H101). Ten ng/ml of HBsAg in 1% BSA-PBS was used as a positive control and 1% BSA-PBS was used as a background control. Each assay was performed in duplicate. Mean values are shown in the graph.

• 

  Figure 3 Standard curve for the current HBsAg ELISA system exhibiting linearity within the range of the standards (0–40 ng/ml in 1% BSA-PBS). Each assay was performed in duplicate. Mean values are shown in the graph.

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