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Updated: Jan 14, 2026
Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-α
Published on: June 14, 2018
Development and validation of an ultra-sensitive ELISA using APTES-based chemical crosslinking method for detection
Nomin Ariungerel1, Badmaarag Munkhjin2, Ulziigerel Erdembileg1
1Department of Chemical and Biological Engineering, National University of Mongolia, Ulaanbaatar 14201, Mongolia.
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.
