Development of a Simple Horseradish Peroxidase-linked Hybrid Conjugates System for Sensitive Detection of Alpha-fetoprotein

Abstract

Alpha-fetoprotein (AFP) is frequently used for the detection and diagnosis of various cancers, including liver, endodermal, ovarian, stomach, and other malignant tumors. Additionally, it is utilized to identify conditions such as miscarriage, Down syndrome, and neural tube defects during pregnancy. Therefore, the present study focuses on the development/design of a highly sensitive hybrid conjugate system for the diagnosis of AFP. In this regard, the hybrid conjugate was synthesized using horse radish peroxidase, specific antibodies of AFP and copper (II) ion as inorganic component. The conjugate synthesized with high encapsulation efficiency (97%) was characterized using SEM, EDX, FTIR, and XRD analyses. The hybrid conjugate analyses were conducted in direct and sandwich ELISA test systems, and it was found that the direct method was ineffective. Optimal working conditions were determined in the sandwich ELISA test system. In this method, a linear relationship was plotted between A450, and the AFP concentration in the range of 15.6 to 500 ng/mL(R2 = 0.990), and the cut-off value for this test was calculated as 0.465. The detection range of the conjugate was found to be close to commercial ELISA test kits. The findings indicate that functionalized hybrid conjugate systems have the potential to offer a more economical application in the diagnostic field. Protein-inorganic hybrid material technology shows promise for developing ELISA kits in the diagnostic industry, pending further research on sensitivity, repeatability, and stability. The presented biomolecular engineering approach is promising for the development of optional hybrid systems.