Biotechnology and Applied Biochemistry Synthesis of a unique high-performance poly-horseradish peroxidase complex to enhance sensitivity of immunodetection systems Fahimeh Charbgoo, Manouchehr Mirshahi, ∗ Sina Sarikhani, and Mahboobeh Saifi Abolhassan Department of Biochemistry, Faculty of Biological Science, Tarbiat Modares University, Tehran, Iran Abstract. Because early detection is the first step in successful therapy, increasing the sensitivity of detection systems has always been considered as one of the major trends in development of these technologies. Therefore, we have fabricated a high-performance poly-horseradish peroxidase (HRP) complex and analyzed it in different formats of immunodetection systems. To construct this complex, dextran–aldehyde was prepared by oxidation of dextran in the presence of sodium periodate. Activated polymer was then coupled to lysine amino acids and accomplishment of the process was evaluated with trinitrobenzenesulfonic acid. Following conjugation of HRP to free amino groups of lysine, the stage’s accuracy and the rate of conjugation were demonstrated by SDS-PAGE. Then, conjugation of poly-HRP complex to streptavidin by biotin was performed. The results of a series of experiments confirmed the complete synthesis of streptavidin–poly-HRP complex by this procedure. Finally, we compared our harvested complex with the golden standard complex available for ELISA and immunohistochemistry (IHC). The results showed the high efficiency of the synthesized complex. Consequently, this complex can be applicable in highly sensitive detection technologies. Conjugating this complex to any antibody by using biotin–streptavidin bridging and preparing poly-HRP-labeled antibodies will be a valuable multifold approach to increase the sensitivity of detection systems, which can be applicable in ELISA, immunocytochemistry, and IHC methods. C  2012 International Union of Biochemistry and Molecular Biology, Inc. Volume 59, Number 1, January/February 2012, Pages 45–49 • E-mail: mirshahi@modares.ac.ir Keywords: detection technology, dextran–HRP, high sensitivity, immunoassay, immunohistochemistry 1. Introduction Nowadays, modern diagnostic tests are based on the well- known enzyme immunoassay formats. Such assays have been found to be first and foremost safer, easier, and more precise than the early radioimmunoassays. Detection is the most im- portant step in all therapeutic processes. Many attempts have been made to increase the sensitivity of immunoassays that deal with detection technology [1–6]. For detection of many infectious agents and cell surface antigens, sensitivity of the method be- ing used plays a major role. Furthermore, many biomolecules exist in picograms and femtograms naturally, causing the de- tection process to be almost impossible unless using highly sensitive systems. Abbreviations: HRP, horseradish peroxidase; biotin–LC-NHS, biotin–long-chain N-hydroxysuccinimide; t-PA, tissue plasminogen activator. ∗ Address for correspondence: Dr. Manouchehr Mirshahi, Department of Biochemistry, Faculty of Biological Science, Tarbiat Modares University, Tehran, Iran. Tel.: + 98 21 82884408; Fax: + 98 21 82884484; e-mail: mirshahi@modares.ac.ir. Received 7 June 2011; accepted 9 September 2011 DOI: 10.1002/bab.58 Published online 10 January 2012 in Wiley Online Library (wileyonlinelibrary.com) Over the past decade, detection enhancement tech- nologies have been developed by polymer-based complexes [1],[4],[7],[8]. Marquette et al. [1] provided a kind of dextran– horseradish peroxidase (HRP) complex, which increased the sensitivity of detection; however, it was just applicable in chemi- luminescence systems. Basically, the immunological system in- volved in ELISA and the enzyme detection system applied in this assay ensure accuracy of the test [9]. HRP, which is widely used for immunodetection, is a glycosylated enzyme in which carbohydrate builds 30% of its total molecular weight [10]. Because of the existence of carbohydrate moieties, conjugation of HRP to different biomolecules will be possible by generation of Schiff base bonds. Furthermore, there are two methods of constructing a Schiff base. The first one is triggered by the NH 3 group of lysine residues of enzymes attacking aldehyde groups of an- other molecule. The second one is constructed by conversion of the surface carbohydrate’s OH groups to aldehyde groups, and binding them to the NH 3 group of the second molecule. Almost always the second way of conjugating HRPs to other molecules is used [11]. In this research, a novel process for synthesis of the streptavidin–HRP complex has been introduced, which pro- vides an elevation in sensitivity of immunodetection systems compared to similar complexes synthesized so far. 45