Development and validation of an ultra-sensitive ELISA using APTES-based chemical crosslinking method for detection of anti-HDV in human serum.

BACKGROUND

Hepatitis delta virus (HDV) is a defective RNA virus that causes severe liver diseases. Despite its endemicity in a few countries, the worldwide prevalence of HDV is unequal. Therefore, the development of sophisticated diagnostic assays for detecting HDV infection is less attractive and the diversity of commercially available kits is limited.

METHODS

Recombinant 23 kDa His-tagged sHDAg protein was produced in E. coli and purified by Ni-NTA chromatography. An ELISA plate was assembled using a 3-Aminopropyl triethoxysilane (APTES) linker to create highly capacitive and organized binding. For analysis, prequalified 460 samples (Positive group: HBsAg and anti-HDV/HDV-RNA positive, n = 220; Negative group: HBsAg positive and anti-HDV/HDV-RNA negative, n = 40, anti-HCV and HBsAg negative, n = 200) were used. Based on the optimized protocol, cut-off values were identified using different methods.

RESULTS

We found that the ELISA assay is ultra-sensitive and can detect up to 10^9 times diluted samples. Three different cut-off values (0.8201, 0.7232, and 0.7285) were obtained using the ROC curve, distribution curve, and standard deviation-based methods. The assay demonstrated stable results, including a specificity of 96.25 %-97.08 %, a sensitivity of 96.36 % and an area under the curve (AUC) was 0.98843 (95 %CI=0.97897-0.99789, SE=0.0048, p < 0.001).

CONCLUSION

ELISA developed using a chemical crosslinking method can detect HDV infection in highly sensitive and rapid ways and should be introduced in clinical practice.