Synthesis of a Unique Dextran Polymer-Conjugated Antibody and Horseradish  Peroxidase Complex

Bahareh Zamani; Niloofar Agharezaee; Farshid Moosavi; Saeideh Zamani  Koukhaloo; Parisa  Yousefi; Jafar   Mahmoudian

doi:10.18502/ajmb.v17i3.19276

Bahareh Zamani Monoclonal Antibody Research Center, Avicenna Research Institute (ARI), Academic Center for Education, Culture and Research (ACECR), Tehran, Iran
Niloofar Agharezaee Monoclonal Antibody Research Center, Avicenna Research Institute (ARI), Academic Center for Education, Culture and Research (ACECR), Tehran, Iran
Farshid Moosavi Monoclonal Antibody Research Center, Avicenna Research Institute (ARI), Academic Center for Education, Culture and Research (ACECR), Tehran, Iran
Saeideh Zamani Koukhaloo Monoclonal Antibody Research Center, Avicenna Research Institute (ARI), Academic Center for Education, Culture and Research (ACECR), Tehran, Iran
Parisa Yousefi Monoclonal Antibody Research Center, Avicenna Research Institute (ARI), Academic Center for Education, Culture and Research (ACECR), Tehran, Iran
Jafar Mahmoudian Monoclonal Antibody Research Center, Avicenna Research Institute (ARI), Academic Center for Education, Culture and Research (ACECR), Tehran, Iran

DOI: https://doi.org/10.18502/ajmb.v17i3.19276

Keywords: Antibodies, Dextrans, Horseradish peroxidase, Immunohistochemistry

Abstract

Background: Immunohistochemistry (IHC) is a practical technique that utilizes the specific binding between an antigen and antibody to detect and localize specific anti-gens in tissue and cells. The optimal sensitivity in IHC is of utmost importance to achieve reliable results even when antigens are present at low abundance on the sam-ples. Here, a dextran polymer labeled with Horseradish Peroxidase (HRP) and an anti-body to improve the sensitivity of the IHC technique was synthesized.

Methods: To this end, free thiol groups were introduced on sodium periodate-activated 30 kDa dextran using cystamine, followed by attachment of sulfo-MBS-activated goat anti-mouse antibody and sulfo-MBS-activated HRP to the activated dextran.

Results: The production of Poly-HRP Antibody (PHA) was confirmed by the appear-ance of a new protein band exceeding 150 kDa on Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). Additionally, Enzyme-Linked Im-munosorbent Assay (ELISA) and IHC techniques were employed to characterize PHA’s functionality. The data demonstrated that PHA effectively detected target antigens in these assays.

Conclusion: The newly synthesized PHA has the potential to provide a more sensitive platform for early detection of biomarkers in IHC. Further research is needed to evalu-ate its cost-effectiveness.