Synthesis of a unique highperformance

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Jan 10, 2012 - acids and accomplishment of the process was evaluated with ... nologies have been developed by polymer-based complexes. [1],[4],[7],[8]. ... Dextran T500KD was activated with 37.5 mg/mL of periodate .... added at 37.5ng/mL, respectively. ... formation of constructs with a molecular mass greater than.
Synthesis of a unique high-performance poly-horseradish peroxidase complex to enhance sensitivity of immunodetection systems

Biotechnology and Applied Biochemistry

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  C 2012 International Union of Biochemistry and Molecular Biology, Inc. Volume 59, Number 1, January/February 2012, Pages 45–49 • E-mail: [email protected]

1. Introduction Nowadays, modern diagnostic tests are based on the wellknown 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 important 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 being used plays a major role. Furthermore, many biomolecules exist in picograms and femtograms naturally, causing the detection 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: [email protected] Received 7 June 2011; accepted 9 September 2011 DOI: 10.1002/bab.58 Published online 10 January 2012 in Wiley Online Library (

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.

Keywords: detection technology, dextran–HRP, high sensitivity, immunoassay, immunohistochemistry

Over the past decade, detection enhancement technologies 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 chemiluminescence systems. Basically, the immunological system involved 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 NH3 group of lysine residues of enzymes attacking aldehyde groups of another molecule. The second one is constructed by conversion of the surface carbohydrate’s OH groups to aldehyde groups, and binding them to the NH3 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 provides an elevation in sensitivity of immunodetection systems compared to similar complexes synthesized so far. 45

2. Methods and materials 2.1. Synthesis of the dextran–HRP complex Dextran T500KD was activated with 37.5 mg/mL of periodate in sodium acetate buffer, 0.05 M, pH 5 at 0◦ C for 30 Min. Aldehyde production was checked with 2 mg/mL of dextran– aldehyde and 2,4-dinitrophenylhydrazine (DNPH, 10%) in NaOH (1 M), and formaldehyde was applied as a control. Amination of dextran–aldehyde and l-lysine at the ratio of 1:10 was performed at room temperature for 3 H. Production of dextran– amine was controlled by 200 μg/mL of dextran–amine and 2,4,6-trinitrobenzenesulfonic acid (TNBSA, 1%) and glycine was applied as a control [12]. HRP was activated following a modified procedure used in protein protocols [13]. Peroxidase was anchored onto dextran–amine through its carbohydrate moiety at 2 mg/mL for dextran–amine and 4.8 mg/mL for active HRP for 2 H at room temperature; the process was followed by 2 mg/mL of sodiumborodohydrate at 4◦ C overnight [14–16]. After that, free aldehyde groups were blocked with lysine at excess concentrations (20 mg/mL) by incubation for 1 H at room temperature and were constantly stirred. Removal of additive salts was performed by Sephadex G-25 gel and unbounded HRPs were removed from complexes by Sephacril S-300 (obtained from Pharmacia, New York, NY, USA, SDSPAGE was carried out to confirm the formation of conjugates by using a 10% separating gel and 5% stacking gel and staining with coomassie R-250.

2.2. Biotinylation of dextran–HRP complex Biotin–long-chain N-hydroxysuccinimide (biotin–LC-NHS) in DMF (N,N-dimethylformamide, 78 μg/mL) was added to dextran–HRP (1 mg/mL) in carbonate–bicarbonate buffer (0.1 M, pH 8.5) for 20 Min while stirring at room temperature, followed by dialysis at 4◦ C overnight, a modified procedure of Cho et al. [17]. This biotinylation procedure was tested with a streptavidincoated plate. For this process, microplate wells were coated with 100 μL of streptavidin [1 μg/mL in phosphate-buffered saline (PBS) after washing with wash buffer (PBS), Tween 20 (0.05%)], and the plate was blocked (with bovine serum albumin, 1%), stored, and desiccated at room temperature. In the first strip, biotinylated dextran–HRP complex was added, using twofold serial dilution starting at 120 ng/mL and ending at 700 pg/mL. In the second strip, twofold dilution of biotinylated HRP starting at 120 ng/mL and ending at 700 pg/mL was added as a positive control. In the third strip, dextran–HRP complex was added in the same condition as a negative control. After 1 H incubation at room temperature, each strip was washed. The complexes were detected using 100 μL of maleimide, 3,3 ,5,5 tetramethylbenzidine (TMB) substrate at room temperature for 5 Min, followed by 100 μL of H2 SO4 (2 M) and the optical density at 450 nm was measured applying a 96-well plate ELISA reader apparatus.

2.3. Fabrication of streptavidin on biotin–dextran–HRP complex This stage was performed by trial and error, that is, streptavidin was added to the biotin–dextran–HRP complex. After stirring, 46

the complex formation was checked on both biotin- and streptavidin-coated plates (biotin was coated as biotin–albumin complex). Streptavidin and biotin were coated at 1 μg/mL and biotinylated dextran–HRP complex was added, using twofold serial dilution starting at 120 ng/mL and ending at 700 pg/mL. After using 100 μL of TMB substrate at room temperature for 5 Min and 100 μL of H2 SO4 (2 M), the process was continued until the optical density (OD) at 450 nm was near zero for the complex added to the streptavidin-coated plate and maximum for the complex added to the biotin-coated plate. Removal of unbounded streptavidin from complexes was performed by Sephacril S-300.

2.4. Analyzing the capability of synthesized complex in detecting trace amount of antigens ELISA was used as a method to compare the potential of synthesized streptavidin–HRP complex with the available golden standard streptavidin–poly-HRP in detecting nanograms or picograms of tissue plasminogen activator (t-PA) as an antigen. To achieve this goal, microtiter plates were coated with t-PA (obtained from Boehringer Ingelheim, Ingelheim am Rhein, Germany, 500 ng/mL at a twofold dilution serially up to 300 pg/mL. After blocking, biotinylated anti-t-PA antibody was added to the wells at 1 μg/mL and incubated for 1 H. Synthesized streptavidin– dextran–HRP and golden standard streptavidin–HRP were added at 37.5 ng/mL, respectively. Finally, OD450nm was read in the ELISA reader.

2.5. Application of the synthesized complex in immunohistochemistry This experiment was performed in the following two steps: a) Preparation of slides for immunohistochemistry: Briefly, formalin-fixed, paraffin-embedded human blood vessel tissue was deparaffinized using a Xylene substitute. Threemicrometer sections were prepared and rehydrated by soaking in different ethanol concentrations for 10 Min starting from 99.6%, 80%, 40%, and 20% and ending in deionized water. Slides were placed in silan 2% dissolved in acetone for 20 Min, and then tissue sections were placed on the slides. Antigen retrieval was performed by placing the slides in sodium citrate buffer (10 mM, 0.05% Tween 20, pH 6). b) Detection of cell surface markers of angiosarcoma in human blood vessel tissue: Endogenous peroxidase activity was blocked by incubating samples in H2 O2 (0.1%) for 30 Min. After washing with PBS, tissue sections on slides were blocked with fetal calf serum (FCS, 2%) for 30 Min. To block endogenous biotin molecules, streptavidin was added in excess (100 μg/mL) for 15 Min. To confirm the occupancy of all the streptavidin-binding sites, biotin was also added at 30 μg/mL for 15 Min to the tissue sections to avoid attachment of our biotinylated antibody to streptavidinfree sites. After washing with PBS, biotinylated antibody Biotechnology and Applied Biochemistry

vascular endothelial growth factor receptor 2 (55B11) rabbit monoclonal antibody (mAb; supplied from Dako, Glostrup, Denmark, was added to the tissue sections at 20 μg/mL and incubated for 1 H. Slides were washed with PBS and synthesized streptavidin–dextran–HRP and golden standard streptavidin–HRP were added to the slides and incubated for 1 H. Washing with PBS was performed and diaminobenzidine (0.5 mg; Sigma, St. Louis, MO, USA,, 30% H2 O2 (0.15 μL), and PBS (1 mL) were applied and images were visualized using a light microscope. All of the materials were purchased from Sigma (www unless otherwise mentioned above.

3. Results and discussion 3.1. Synthesis of the dextran–HRP complex After activation of dextran with periodate, production of aldehyde groups on dextran had been checked with 2 mg/mL of dextran–aldehyde and DNPH 10% (data not shown). In the next step, dextran–aldehyde was aminated using lysine and the production of dextran–amine was controlled by 1% TNBSA because the TNBSA reagent assesses primary chemical adduction with the amino group [18]. On the basis of the fact that about three amino acids having free amino groups are available at the surface of the folded structure of HRP, the efficiency of the protocol that conjugated dextran–aldehyde to amino groups on HRP to prepare dextran–HRP complex is low, so we have applied the method that conjugated dextran–amine to activate HRP (containing functional aldehyde groups) for synthesizing the dextran–HRP complex. Finally, SDS-PAGE was carried out to confirm the formation of conjugates. The results showed the appearance of polydisperse bands in the separating gel that suggested the formation of constructs with a molecular mass greater than that of HRP (Fig. 1). Active HRP was used as a control in the set-up process of synthesizing dextran–HRP conjugates; thus, the observable band was related to free HRP molecules (data not shown). According to this finding, polydispersed bands in dextran–HRP samples were related to conjugation of HRPs with dextrans. This method had been previously used to improve conjugation of a kind of protein with a chemical molecule in production of albumin–dextran complexes [19],[20]. On the basis of the fact that proteins larger than 1,000 kDa are not capable of entering the stacking polyacrylamide gel, the bands at the top of the stacking gel demonstrated the formation of dextran–HRP conjugates with a molecular weight more than 1,000 kDa. Here, we should stress a very important point; after conjugating aminated dextran to activated HRPs, there were still many free aldehyde groups on both HRP and dextran molecules because of strict hindrances. Blocking these free aldehyde groups with lysine amino acid caused an increase in the amount of free NH3 groups, which were platforms of biotin binding, so that the amount of biotin molecules on the complex increased, as could be seen in the results of ELISA for biotinylation of

Synthesis of a unique high-performance poly-HRP complex

Fig. 1. SDS-PAGE of dextran–HRP conjugates. From left to right: lane 1, molecular weight standard marker (β-galactosidase, Escherichia coli, 116.0 kDa; bovine serum albumin, bovine plasma, 66.2 kDa; ovalbumin, chicken egg white, 45.0 kDa; lactate dehydrogenase, porcine muscle, 35.0 kDa; RE Bsp98I, E. coli, 25.0 kDa; β-lactoglobulin, bovine milk, 18.4 kDa); Lane 2, free HRP; Lane 3, dextran–HRP complex.

dextran–HRP complex in the next step. This process is a reason for the significant increase in the efficiency of our synthesized complex as compared with similar ones prepared by Marquette et al. [1].

3.2. Biotinylation of dextran–HRP complex After biotinylation of dextran–HRP conjugate with biotin– LC-NHS dissolved in DMF, the process was checked with streptavidin-coated microplate. The biotin–dextran–HRP conjugate, dextran–HRP complex, and biotin–HRP at 120 ng/mL in dilution buffer were used to perform the process. The results demonstrated that biotinylation of dextran–HRP complex was performed (Fig. 2). The biotin molecules in dextran–HRP complex are capable of attaching to free amine groups on both dextran and HRP molecules, which caused an increase in the biotinylation efficiency. The consequence of this will be an increase in the number of dextran–HRP complexes fabricated on streptavidin. As a rule of thumb, the more HRP molecules attached to the structure of mAb, the better the signals will be. Because the number of biotin molecules bonded to dextran– HRP complex was unknown, the amount of biotin in each batch








Fig. 2. Biotinylated dextran–HRP conjugate at 120 ng/mL on streptavidin-coated plate ( ); biotinylated HRP at ); 120 ng/mL on streptavidin-coated plate ( dextran–HRP complex at 120 ng/mL on streptavidin-coated ). plate (

Fig. 3. Detection of t-PA (160 pg/mL–500 ng/mL) in direct ) and ELISA format with synthesized streptavidin–HRP ( ) at 37.5 ng/mL. golden standard one (

of the sample was not determined either. Thus, the amount of streptavidin required for blocking biotins on the complex was different for each batch of the biotin–dextran–HRP complex. Because of this challenge, performance of the process was just possible by trial-and-error experiments.

research demonstrated a sevenfold increase in signals from ELISA through the use of a supermacromolecular complex in comparison to the golden standard streptavidin–HRP complex. In assaying many infectious agents, sensitivity is the most important parameter that ultimately determines the informative value of the test. The fact that validates this research is the low concentration of many components in biological fluids, which is detectable only by these sensitive systems.

3.3. Analyzing the capability of synthesized complex in detecting trace amount of antigens

3.4. Application of the synthesized complex in immunohistochemistry

With the intention of developing a highly sensitive ELISA, enabling measurements of low antigen levels, t-PA was selected as an appropriate candidate and then was coated in nanogram to pictogram range. t-PA is a suitable antigen because its blood concentration is trace and is approximately 5 ng/mL. As demonstrated in Fig. 3, the synthesized streptavidin–HRP complex was capable of detecting 320 pg/mL of t-PA, which yields the OD of 0.7. In comparison, the golden standard sample did not have a significant OD in the same condition. It is possible to measure t-PA at approximately 320 pg/mL with an OD of about 0.7. This

After performing tissue preparation steps and blocking the tissue sections, human blood vessel–mounted tissues were incubated for 1 H with biotinylated antibody at 20 μg/mL, followed by 1 H incubation in synthesized streptavidin–HRP complex and golden standard streptavidin–HRP complex, respectively. Fig. 4 illustrates that immunohistochemistry (IHC) staining with synthesized streptavidin–HRP was as strong as immunostaining with the golden standard complex. This experiment demonstrates that synthesized streptavidin–HRP is applicable in both ELISA and IHC assays.

Fig. 4. Immunostaining of blood vessel biopsies 50 × with (A) golden standard streptavidin–HRP complex; (B) and (C) synthesized streptavidin–HRP that was diagnosed as angiosarcoma positive (the difference between images B and C is just in the location of sections prepared from biopsy).


Biotechnology and Applied Biochemistry

4. Conclusion Researchers have studied many different methods to increase the sensitivity of detection systems. Applying methods that gather several molecules of HRP together on each antibody will lead to a high sensitivity in diagnostic tests. The present study proposed a new approach for enhancing ELISA and IHC signals through the synthesis of a macromolecular complex composed of a dextran–lysine structure carrying a large number of grafted HRP that are conjugated to streptavidin. The enhancement was obtained maximally (seven times) at a low complex concentration, demonstrating steric hindrance of a massive macromolecular complex. Nevertheless, significant enhancement in detection sensitivity was observed both in ELISA for t-PA and IHC for angiosarcoma markers. Consequently, poly-HRP conjugates are useful in a range of immunoassays with different formats, where HRP is a suitable reporter [1,2].

Acknowledgements The authors would like to thank Dr. Jahnzad, Cancer institute, Imam Khomeini Hospital, for his help with IHC.

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Synthesis of a unique high-performance poly-HRP complex

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