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Multiplexed chemiluminescence imaging assay of protein biomarkers using DNA microarray with proximity binding-induced hybridization chain reaction amplification.

A multiplexed chemiluminescence (CL) imaging assay was designed for sensitive screen detection of a panel of protein biomarkers by integrating DNA microarray with proximity binding-induced hybridization chain reaction (HCR) amplification. The DNA microarray was manufactured by printing hairpin DNAs for different analytes detection in an array format onto the sensing cells arranged on an aldehyde-functionalized glass chip. The existence of target proteins induced the formation of sandwich immunocomplexes along with the hybridization of DNA strands labeled on the pair of antibodies via proximity effect, which subsequently unfolded their corresponding spotting hairpin DNAs to expose prelocked primer and trigger HCR assembly. Through the streptavidin-biotin conjugation, numerous horseradish peroxidase (HRP) was coupled on the HCR assemblies to catalyze HO-luminol reaction and produce amplified CL signals for sensitive CL imaging assay of protein targets. As a proof of concept, the DNA microarray was used to CL imaging assay of carcinoembryonic antigen, α-fetoprotein and carcinoma antigen 125 in ranges of 0.05-500 ng mL, 0.06-600 ng mL and 0.02-200 U mL, respectively. The CL microarray imaging assay could also be extended to screen detect other biomarker groups by printing different hairpin DNAs on the microarray and using corresponding antibody-DNA pairs. The proposed CL microarray imaging assay showed advantages of small sample consumption, good cost effectiveness, and multiplex detection performance, exhibiting good applicability in clinical diagnosis.

2478 related Products with: Multiplexed chemiluminescence imaging assay of protein biomarkers using DNA microarray with proximity binding-induced hybridization chain reaction amplification.

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Investigating Colorimetric Protein Array Assay Schemes for Detection of Recurrence of Bladder Cancer.

A colorimetric microarray for the multiplexed detection of recurrence of bladder cancer including protein markers interleukin-8 (IL8), decorin (DCN), and vascular endothelial growth factor (VEGF) was established to enable easy and cheap read-out by a simple office scanner paving the way for quick therapy monitoring at doctors' offices. The chip is based on the principle of a sandwich immunoassay and was optimized prior to multiplexing using IL8 as a model marker. Six different colorimetric assay formats were evaluated using a detection antibody (dAB) labeled with (I) gold (Au) nanoparticles (NPs), (II) carbon NPs, (III) oxidized carbon NPs, and a biotinylated dAB in combination with (IV) neutravidin-carbon, (V) streptavidin (strp)-gold, and (VI) strp-horseradish peroxidase (HRP). Assay Format (III) worked best for NP-based detection and showed a low background while the enzymatic approach, using 3,3',5,5'-tetramethylbenzidine (TMB) substrate, led to the most intense signals with good reproducibility. Both assay formats showed consistent spot morphology as well as detection limits lower than 15 ng/L IL8 and were thus applied for the multiplexed detection of IL8, DCN, and VEGF in synthetic urine. Colorimetric detection in urine (1:3) yields reaction signals and measurement ranges well comparable with detection in the assay buffer, as well as excellent data reproducibility as indicated by the coefficient of variation (CV 5-9%).

2783 related Products with: Investigating Colorimetric Protein Array Assay Schemes for Detection of Recurrence of Bladder Cancer.

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Orthogonal enzyme arrays on a DNA origami scaffold bearing size-tunable wells.

A new waffle-like DNA origami assembly (DNA waffle) with nine nanometer-scale wells in a 3 × 3 matrix pattern has been successfully constructed and used as a scaffold for selective nano-patterning of individual protein molecules. The folding pattern of the scaffold was specially designed so that the dimensions of each well could be independently tuned according to the dimensions of the guest nanoparticles. We demonstrated that two distinct proteins, streptavidin (SA) tetramer (d = 5 nm) and anti-fluorescein antibody (IgG) (inter-paratope distance ∼ 14.0 nm), could be selectively captured in size-variable wells of dimensions 6.8 × 12 × 2.0 nm for SA and 6.8 × 12 × 2.0 nm or 10.2 × 12 × 2.0 nm for IgG, respectively, through the attachment of two biotins or two fluoresceins at the two edges of each well. This allowed the formation of a heterogeneous protein nanoarray of individual molecules. The position of SA or IgG capture can be fully controlled by placement of biotins or fluoresceins in the nanoarray well. Moreover, a hetero-nanoarray consisting of two kinds of enzyme: horseradish peroxidase-labeled streptavidin (HRP-SA) and alkaline phosphatase-labeled anti-FITC antibody (AP-IgG) was successfully constructed through selective attachment of biotin or fluorescein in any desired wells. Successful enzyme-heteroarray formation was confirmed by enzymatic activity analyses after purification of mixtures of enzymes and DNA waffles.

2624 related Products with: Orthogonal enzyme arrays on a DNA origami scaffold bearing size-tunable wells.

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Use of lectin microarray to differentiate gastric cancer from gastric ulcer.

To investigate the feasibility of lectin microarray for differentiating gastric cancer from gastric ulcer.

1674 related Products with: Use of lectin microarray to differentiate gastric cancer from gastric ulcer.

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Quantitative assessment of Tn antigen in breast tissue micro-arrays using CdSe aqueous quantum dots.

In this study, we examined the use of CdSe aqueous quantum dots (AQDs) each conjugated to three streptavidin as a fluorescent label to image Tn antigen expression in various breast tissues via a sandwich staining procedure where the primary monoclonal anti-Tn antibody was bound to the Tn antigen on the tissue, a biotin-labeled secondary antibody was bound to the primary anti-Tn antibody, and finally the streptavidin-conjugated AQDs were bound to the biotin on the secondary antibody. We evaluated the AQD staining of Tn antigen on tissue microarrays consisting of 395 cores from 115 cases including three tumor cores and one normal-tissue core from each breast cancer case and three tumor cores from each benign case. The results indicated AQD-Tn staining was positive in more than 90% of the cells in the cancer cores but not the cells in the normal-tissue cores and the benign tumor cores. As a result, AQD-Tn staining exhibited 95% sensitivity and 90% specificity in differentiating breast cancer against normal breast tissues and benign breast conditions. These results were better than the 90% sensitivity and 80% specificity exhibited by the corresponding horse radish peroxidase (HRP) staining using the same antibodies on the same tissues and those of previous studies that used different fluorescent labels to image Tn antigen. In addition to sensitivity and specificity, the current AQD-Tn staining with a definitive threshold was quantitative.

1101 related Products with: Quantitative assessment of Tn antigen in breast tissue micro-arrays using CdSe aqueous quantum dots.

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Development and application of antibody microarray for lymphocystis disease virus detection in fish.

Lymphocystis disease virus (LCDV) is the causative agent of lymphocystis disease affecting marine and freshwater fish worldwide. Here an antibody microarray was developed and employed to detect LCDV in fish. Rabbit anti-LCDV serum was arrayed on agarose gel-modified slides as capture antibody, and Cy3-conjugated anti-LCDV monoclonal antibody (MAbs) was added as detection antibody. The signals were imaged with a laser chip scanner and analyzed by corresponding software. To improve the sensitivity, different substrate binders (poly-L-lysine, MPTS, aldehyde, APES and agarose gel modified slides, and commercially available amino-modified slides), markers (fluorescein isothiocyanate, Cy3, horseradish peroxidase, biotin or colloidal gold) conjugated to anti-LCDV Mabs, and storage time of the antibody were assessed. The results showed that the antibody microarrays based on agarose gel-modified slides gave a lower detection limit of 0.55μg/ml of LCDV when Cy3 and HRP conjugated anti-LCDV MAbs were used as detection antibody; and the lowest detectable LCDV protein concentration was 0.0686 μg/ml when streptavidin-biotin conjugated to anti-LCDV MAbs served as detection antibody. The developed antibody microarray proved to have a high specificity for LCDV detection and a shelf-life of more than 8 months at -20°C. Furthermore, the LCDV detection results of the microarray in fish gills or fins (n=50) presented a concordance rate of 100% with enzyme-linked immunosorbent assay (ELISA) and 98% with immunofluorescence assay technique (IFAT). These results revealed that the developed antibody microarray could serve as an effective tool for diagnostic and epidemiological studies of LCDV in fish.

1443 related Products with: Development and application of antibody microarray for lymphocystis disease virus detection in fish.

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Single-wall carbon nanotube forest arrays for immunoelectrochemical measurement of four protein biomarkers for prostate cancer.

Protein arrays that measure multiple protein cancer biomarkers in clinical samples hold great promise for reliable early cancer detection. Herein, we report a prototype 4-unit electrochemical immunoarray based on single-wall carbon nanotube forests for the simultaneous detection of multiple protein biomarkers for prostate cancer. Immunoarray procedures were designed to measure prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), platelet factor-4 (PF-4), and interleukin-6 (IL-6) simultaneously in a single serum sample. All of these proteins are elevated in serum of patients with prostate cancer, but they have widely different relative levels of serum concentration. Horseradish peroxidase (HRP) was used as label on detection (secondary) antibodies in a sandwich immunoassay scheme. Biotinylated secondary antibodies (Ab(2)) that bind specifically to streptavidin-HRP conjugates provided 14-16 labels per antibody and gave the necessary higher sensitivity required for PF-4 and IL-6 detection at physiological levels. Conventional singly labeled Ab(2)-HRP conjugates were sufficient for PSA and PSMA detection. Immunoarrays were used to measure four biomarkers in clinical human serum samples of prostate cancer patients and controls with excellent correlation to referee enzyme-linked immunosorbent (ELISA) assays.

2008 related Products with: Single-wall carbon nanotube forest arrays for immunoelectrochemical measurement of four protein biomarkers for prostate cancer.

Bone Morphogenetic Protei COOH Modified Single Wall Protein A (Liquid form) Protein A (Liquid form) Protein A (Liquid form) Protein A (Liquid form) NATIVE HUMAN PROLACTIN, P Native Human Lactoferrin, NATIVE HUMAN PROLACTIN, P Anti-daf-2(Abnormal dauer Anti daf 2(Abnormal dauer Anti-BMP-1 (Bone Morphoge

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Biotin-free systems provide stronger immunohistochemical signal in oestrogen receptor evaluation of breast cancer.

Biotin-free polymeric visualisation systems (BFPS) were compared with streptavidin-biotin systems (SABS) in the evaluation of immunoreactivity for oestrogen receptor (ER) in breast carcinomas.

1469 related Products with: Biotin-free systems provide stronger immunohistochemical signal in oestrogen receptor evaluation of breast cancer.

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A quantitative analysis of N-myc downstream regulated gene 2 (NDRG 2) in human tissues and cell lysates by reverse-phase protein microarray.

N-myc downstream regulated gene 2 (NDRG2) belongs to the NDRG family, which is comprised of 4 members, NDRG1-4. Recently, NDRG2 was reported as a new candidate for a tumor suppressor gene. We developed a reverse-phase protein microarray assay to access NDRG2 levels in human tissue specimens and cell lines.

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Rabbit Anti-Cell death in Rabbit Anti-Cell death in Rabbit Anti-APIP Apaf1 In Rabbit Anti-APIP Apaf1 In Proteins and Antibodies H Astra Blue 6GLL, Stain fo Anti C Reactive Protein A Human Macrophage Inflamma Human Macrophage Inflamma cell cycle progression 2 Recombinant Human Inhibin Recombinant Human Inhibin

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Cytokine protein arrays.

Cytokines play important roles in many aspects of cell physiology and pathology. The simultaneous determination of multiple cytokine-expression levels is receiving much attention in the research community. Multiple cytokine-expression levels can be simultaneously determined using enzyme-linked immunosorbent assay (ELISA)-based protein array technology. In this approach, target proteins are captured by the arrayed capture antibody and then detected in a sandwich ELISA format using a cocktail of biotinylated detection antibodies. The signals are visualized by either horseradish peroxidase (HRP)-conjugated streptavidin and enhanced chemiluminescence, or cy3-conjugated streptavidin and laser scanner. Several key factors and steps are described, including selection of solid supports, selection of suitable antibodies, determination of specificity and sensitivity of cytokine protein arrays, array design, sample preparation, and detailed experimental procedures for macroarray and microarray formats. An account of the successful development and application of cytokine protein arrays is presented.

1741 related Products with: Cytokine protein arrays.

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