Se-Hui Jung

Label-free and quantitative analysis of C-reactive protein in human sera by tagged-internal standard assay on antibody arrays.

We have developed a new, high-throughput, competition-based tagged-internal standard (TIS) assay to measure the levels of blood proteins in human serum. In this assay, target proteins in the sample serum compete with tagged-internal standard proteins for binding to an antibody array. Antibody arrays are fabricated by immobilizing a target protein-specific antibody on the carboxylate-modified latex bead surface of well-type arrays. A solution of Alexa 546-conjugated target protein is added to a sample of human serum and applied to the well-type antibody array. The array is then analyzed with a fluorescence scanner and the level of unlabeled target protein in the human sera is inferred from the amount of tagged protein bound to the array. We successfully applied this assay to measure the level of C-reactive protein (CRP) in 92 unlabeled human sera. The TIS assay was found to be specific and reproducible for the quantitative analysis of CRP. The antibody array data from the TIS assay correlate well with clinical laboratory data obtained using the commercialized latex-enhanced turbidimetry immunoassay (n=3, r=0.967, CV=0.32%). Thus, the antibody array-based TIS assay system is high-throughput, quantitative, and label-free and may be useful in the rapid serodiagnosis of human disease.

Rapid analysis of matrix metalloproteinase-3 activity by gelatin arrays using a spectral surface plasmon resonance biosensor.

We developed a novel assay system using an array-based spectral surface plasmon resonance (SPR) biosensor for a high-throughput analysis of matrix metalloproteinase (MMP)-3 activity. Gelatin arrays were fabricated by immobilizing gelatin, a MMP-3 substrate, on amine-modified gold arrays. MMP-3 activity was determined by monitoring the shift of SPR wavelength caused by gelatin proteolysis. The gelatinolytic activity of MMP-3, which caused a decrease of the SPR wavelength, was verified by SPR spectroscopy, atomic force microscopy, and fluorescence-based protein arrays. MMP-3 activity increased by three non-ionic detergents in a dose-dependent manner, and Brij-35 was most effective. The array-based SPR biosensor was successfully applied to the rapid analysis of dose-dependent MMP-3 activity and its inhibition with tissue inhibitors of metalloproteinase 1 and GM6001, MMP inhibitors. Therefore, this new assay system using a spectral SPR biosensor is simple, label-free, and high-throughput, and is likely to have a strong potential for inhibitor screening.

High-throughput analysis of mumps virus and the virus-specific monoclonal antibody on the arrays of a cationic polyelectrolyte with a spectral SPR biosensor

We investigated the potential use of a spectral surface plasmon resonance (SPR) biosensor in a high‐throughput analysis of mumps virus and a mumps virus‐specific mAb on the arrays of a cationic polyelectrolyte, poly(diallyldimethylammonium chloride) (PDDA). The PDDA surface was constructed by electrostatic adsorption of the polyelectrolyte onto a monolayer of 11‐mercaptoundecanoic acid (MUA). Poly‐L‐lysine was also adsorbed onto the MUA monolayer and compared with the PDDA surface in the capacity of mumps virus immobilization. The PDDA surface showed a higher adsorption of mumps virus than the poly‐L‐lysine surface. The SPR signal caused by the virus binding onto the PDDA surface was proportional to the concentration of mumps virus from 0.5 × 105 to 14 × 105 pfu/mL. The surface structure of the virus arrays was visualized by atomic force microscopy. Then, a dose‐dependent increase in the SPR signal was observed when various concentrations of the antimumps virus antibody in buffer or human serum were applied to the virus arrays, and their interaction was specific. Thus, it is likely that the spectral SPR biosensor based on the cationic polyelectrolyte surface may provide an efficient system for a high‐throughput analysis of intact virus and serodiagnosis of infectious diseases.

Rapid determination of blood coagulation factor XIII activity using protein arrays for serodiagnosis of human plasma.

We developed a novel on-chip assay using protein arrays for quantitative and rapid analysis of blood coagulation factor XIII (FXIII) activity in human plasma. FXIII is activated by concerted action of thrombin and Ca(2+) and plays essential roles in hemostasis, angiogenesis, and wound healing. We fabricated protein arrays by immobilizing fibrinogen onto the 3-aminopropyltrimethoxysilane layer of well-type arrays and determined FXIII activity by analyzing biotinylated fibrinogen with Cy3-conjugated streptavidin. We determined optimal concentrations of Ca(2+), thrombin, and 5-(biotinamido)pentylamine (BAPA) for the on-chip activity assay, and the detection limit was 0.01 Lowey U/mL (9.9 pM). Using the on-chip activity assay, hepatocellular carcinoma patients (n = 24), but not hepatitis (n = 24) or liver cirrhosis patients (n = 41), had significantly lower FXIII activities (p < 0.001) than normal individuals (n = 41), indicating that FXIII activity is a possible diagnostic marker for hepatocellular carcinoma. In addition, we have successfully used this activity assay to reveal individual variations (37-57%, n = 65) in the inhibition rate of FXIII activity by isoniazid, the first-line antituberculosis agent. Thus, our optimized on-chip FXIII activity assay provides a quantitative and high-throughput approach to investigating the role(s) of FXIII in human diseases. Moreover, it has a strong potential to be applied toward FXIII-related personalized medicines.

Quantitative and rapid analysis of transglutaminase activity using protein arrays in mammalian cells

We developed a novel on-chip activity assay using protein arrays for quantitative and rapid analysis of transglutami-nase activity in mammalian cells. Transglutaminases are a family of Ca2+-dependent enzymes involved in cell regulation as well as human diseases such as neurodegenerative disorders, inflammatory diseases and tumor progression. We fabricated the protein arrays by immobilizing N,N′-dimethylcasein (a substrate) on the amine surface of the arrays. We initiated transamidating reaction on the protein arrays and determined the transglutaminase activity by analyzing the fluorescence intensity of biotinylated casein. The on-chip transglutaminase activity assay was proved to be much more sensitive than the [3H]putrescine-incorporation assay. We successfully applied the on-chip assay to a rapid and quantitative analysis of the transgluta-minase activity in all-trans retinoic acid-treated NIH 3T3 and SH-SY5Y cells. In addition, the on-chip transglutaminase activity assay was sufficiently sensitive to determine the transglutaminase activity in eleven mammalian cell lines. Thus, this novel on-chip transglutaminase activity assay was confirmed to be a sensitive and high-throughput approach to investigating the roles of transglutaminase in cellular signaling, and, moreover, it is likely to have a strong potential for monitoring human diseases.

Sensitivity enhancement of spectral surface plasmon resonance biosensors for the analysis of protein arrays

A novel method for sensitivity enhancement of spectral surface plasmon resonance (SPR) biosensors was presented by reducing the refractive index of the sensing prism in the analysis of protein arrays. Sensitivity of spectral SPR biosensors with two different prisms (BK-7, fused silica) was analyzed by net shifts of resonance wavelength for specific interactions of GST–GTPase binding domain of p21-activated kinase-1 and anti-GST on a mixed thiol surface. Sensitivity was modulated by the refractive index of the sensing prism of the spectral SPR biosensors with the same incidence angle. The sensitivity of a spectral SPR biosensor with a fused silica prism was 1.6 times higher than that with a BK-7 prism at the same incidence angle of 46.2°. This result was interpreted by increment of the penetration depth correlated with evanescent field intensity at the metal/dielectric interface. Therefore, it is suggested that sensitivity enhancement is readily achieved by reducing the refractive index of the sensing prism of spectral SPR biosensors to be operated at long wavelength ranges for the analysis of protein arrays.

Identification and ultrastructural imaging of photodynamic therapy-induced microfilaments by atomic force microscopy

Atomic force microscopy (AFM) is an emerging technique for imaging biological samples at subnanometer resolution; however, the method is not widely used for cell imaging because it is limited to analysis of surface topology. In this study, we demonstrate identification and ultrastructural imaging of microfilaments using new approaches based on AFM. Photodynamic therapy (PDT) with a new chlorin-based photosensitizer DH-II-24 induced cell shrinkage, membrane blebbing, and reorganization of cytoskeletons in bladder cancer J82 cells. We investigated cytoskeletal changes using confocal microscopy and atomic force microscopy. Extracellular filaments formed by PDT were analyzed with a tandem imaging approach based on confocal microscopy and atomic force microscopy. Ultrathin filaments that were not visible by confocal microscopy were identified as microfilaments by on-stage labeling/imaging using atomic force microscopy. Furthermore, ultrastructural imaging revealed that these microfilaments had a stranded helical structure. Thus, these new approaches were useful for ultrastructural imaging of microfilaments at the molecular level, and, moreover, they may help to overcome the current limitations of fluorescence-based microscopy and atomic force microscopy in cell imaging.

Simultaneous Activity Assay of Two Transglutaminase Isozymes, Blood Coagulation Factor XIII and Transglutaminase 2, by Use of Fibrinogen Arrays

We developed an on-chip activity assay system to simultaneously determine the transamidating activities of blood coagulation factor XIII (FXIII) and transglutaminase 2 (TG2) by use of fibrinogen arrays. FXIII and TG2 are transglutaminase family members that are involved in various physiological functions, including vascular pathophysiology, bone development, and cancer progression. However, investigation of their differential functions is limited by the lack of high-throughput and isozyme-specific activity assays. For the on-chip activity assay, we fabricated protein arrays by immobilizing fibrinogen onto the 3-aminopropyltrimethoxysilane surface of well-type arrays, and we determined transamidating activity by probing biotinylated fibrinogen with Cy3-conjugated streptavidin on arrays. We optimized assay conditions, such as buffer pH, concentrations of dithiothreitol and 5-(biotinamido)pentylamine, and incubation time, and we created equations to determine specific FXIII and TG2 activities in samples. We successfully applied this assay system to monitor changes in FXIII and TG2 activities in THP-1 monocytic cells differentiated with phorbol 12-myristate13-acetate and interleukin-4. This activity assay is sensitive and suitable for high-throughput determination of FXIII and TG2 activities and thus has a strong potential for investigating the differential functions of these isozymes in cell signaling and cardiovascular pathophysiology research.

A novel array-based assay of in situ tissue transglutaminase activity in human umbilical vein endothelial cells

Transglutaminases (TGs), a family of calcium-dependent transamidating enzymes, are involved in functions such as apoptosis andinflammation and play a role in autoimmune diseases and neurodegenerative disorders. In this study, we describe a novel array-based approach to rapidly determine in situ TG activity in human umbilical vein endothelial cells and J82 human bladder carcinoma cells. Amine arrays were fabricated by immobilizing 3-aminopropyltrimethoxysilane on glass slides. The assay was specific and highly reproducible. The average coefficient of variation between spots was 2.6% (n=3 arrays), and the average correlation coefficients between arrays and between arrays/reactions were 0.998 and 0.976, respectively (n=3 arrays). The assay was successfully applied to detect changes in TG activity induced by maitotoxin and to analyze inhibition of the TG activation with cystamine and monodansyl cadaverine. In addition, the assay demonstrated that intracellular reactive oxygen species regulate the maitotoxin-induced activation of TG. Thus, the array-based in situ TG activity assay constitutes a rapid and high-throughput approach to investigating the roles of TGs in cell signaling.

Integrative Proteomic Profiling of Protein Activity and Interactions Using Protein Arrays

Proteomic studies based on abundance, activity, or interactions have been used to investigate protein functions in normal and pathological processes, but their combinatory approach has not been attempted. We present an integrative proteomic profiling method to measure protein activity and interaction using fluorescence-based protein arrays. We used an on-chip assay to simultaneously monitor the transamidating activity and binding affinity of transglutaminase 2 (TG2) for 16 TG2-related proteins. The results of this assay were compared with confidential scores provided by the STRING database to analyze the functional interactions of TG2 with these proteins. We further created a quantitative activity-interaction map of TG2 with these 16 proteins, categorizing them into seven groups based upon TG2 activity and interaction. This integrative proteomic profiling method can be applied to quantitative validation of previously known protein interactions, and in understanding the functions and regulation of target proteins in biological processes of interest.