Using enzyme-linked aptamer assay to detect hepatitis B surface antigen in comparison to enzyme-linked immunosorbent assay

Abstract

Background: Clinical diagnostic kits for detecting hepatitis B are based on antibodies, and have inefficient sensitivity, stability, and cost. Therefore, researches about aptamers and using them instead of antibodies may make these defects up. In this study, a biotinylated anti-hepatitis B virus surface antigen (anti-HBsAg) aptamer sequence was used to evaluate the possibility of specific detection of HBsAg via enzyme-linked aptamer assay (ELAA) method using biotin-streptavidin system. Methods: HBsAg was immobilized in Maxibinding plate (SPL, Korea) by using carbonate-bicarbonate buffer (pH: 9.4), and by considering different variables. Immobilization of HBsAg was evaluated by commercial kit. Aptamer sequence was cloned and imbalance polymerase chain reaction (PCR) was improved and performed using plasmid as DNA template. Then, biotinylated aptamer was utilized in enzyme-linked aptamer assay method via taking advantage of streptavidin-biotin signal amplification system. Findings: Antigen immobilization was set up using conjugates number 1 and 2 of commercial kit. Colony polymerase chain reaction confirmed aptamer cloning in Escherichia coli (E. coli) Top10 host. The best gradient polymerase chain reaction result was achieved at 64° C annealing temperature. Control group in assays showed accuracy of immobilization; but no repetitive specific signal was obtained that could prove joining of aptamer to HBsAg, and detection of that. Conclusion: Three-dimensional structure of an immobilized antigen on a solid surface may vary from that which exists on the surface of viruses or cells. Negative results might be due to the ability of aptamers to attach into antigens with totally intact shape. Conjugation of aptamer with biotin may interfere in aptamer conformation; so, a connective arm between aptamer and biotin could be a suggestion, which needs more assessments. © 2018, Isfahan University of Medical Sciences(IUMS). All rights reserved.