Ficus sycomorus latex: An efficient alternative Egyptian source for horseradish peroxidase in labeling with antibodies for immunodiagnostic kits

Research (Published online: 01-10-2018)

1. Ficus sycomorus latex: An efficient alternative Egyptian source for horseradish peroxidase in labeling with antibodies for immunodiagnostic kits

Azza M. Abdel-Aty, Mohamed Belal Hamed, Abdul Aziz M. Gad, Amr E. El-Hakim and Saleh A. Mohamed

Veterinary World, 11(10): 1364-1370

ABSTRACT

Aim: In view of various peroxidase applications, the searching for new sources of unique peroxidase properties is highly desirable. The present study aims to evaluate the efficiency of the peroxidase of locally grown sycamore latex (POL) for conjugation with antibodies and to study the conjugate optimal conditions, storage stability, and affinity toward different substrates as compared with commercial horseradish peroxidase (HRP).

Materials and Methods: Anti-mouse antibodies were prepared in rabbits and purified by protein A sepharose affinity column chromatography. The POL and HRP conjugates were prepared by one-step glutaraldehyde coupling method. The reactivity of the prepared conjugates was examined using the enzyme-linked immunosorbent assay (ELISA). The optimal enzymatic conditions, storage stability, and affinity toward substrates were also determined for both the conjugates.

Results: The POL showed higher percent recovery (98%) than HRP (78%) over the initial activity after conjugation process. The POL and HRP conjugates showed ELISA titers of 1:120 and 1:80, respectively, demonstrating high binding affinity of POL-conjugate. The POL-conjugate showed high thermal stability up to 70°C compared with HRP-conjugate up to 40°C. After conjugation, POL had wide pH stability (5.0-8.0) compared with HPR (4.5-6.0). Both of the prepared conjugates had a high affinity toward the substrates used in immunoassays with lower Km values. The POL-conjugate showed high storage stability for its enzymatic activity and ELISA titer compared with HRP-conjugate after 1 year at −20°C.

Conclusion: The POL of Ficus sycomorus latex is an efficient source for labeling antibodies and could be utilized in immunodiagnostic kits.

Keywords: conjugation, Ficus sycomorus, horseradish peroxidase, immunodiagnostic assays, latex.