The
e antigen of
hepatitis B (
HBeAg) is positively associated with an increased risk of developing
liver cancer and
cirrhosis in
chronic hepatitis B (CHB) patients. Clinical monitoring of
HBeAg provides guidance to the treatment of CHB and the assessment of
disease progression. We describe here an affinity binding assay for
HBeAg, which takes advantage of G-quadruplex aptamers for enhanced binding and stability. We demonstrate a strategy to improve the binding affinity of aptamers by modifying their sequences upon their G-quadruplex and secondary structures. On the basis of predicting a stable G-quadruplex and a secondary structure, we truncated 19
nucleotides (nt) from the primer regions of an 80-nt aptamer, and the resulting 61-nt aptamer enhanced binding affinity by 19 times (Kd = 1.2 nM). We mutated a second aptamer (40 nt) in one loop region and incorporated
pyrrolo-deoxycytidine to replace
deoxycytidine in another loop. The modified 40-nt aptamer, with a stable G-quadruplex and two modified loops, exhibited a 100 times higher binding affinity for
HBeAg (Kd = 0.4 nM) than the unmodified original aptamer. Using the two newly modified aptamers, one serving as the capture and the other as the reporter, we have developed an improved sandwich binding assay for
HBeAg. Analyses of
HBeAg in serum samples (concentration ranging from 0.1 to 60 ng/mL) of 10
hepatitis B patients, showing consistent results with clinical tests, demonstrate a successful application of the aptamer modification strategy and the associated aptamer binding assay.