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Search results for: Alkaline Phosphatase HRP

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#31720524   2019/10/22 To Up

Protein-Stabilizing Effect of Amphiphilic Block Copolymers with a Tertiary Sulfonium-Containing Zwitterionic Segment.

Tertiary sulfonium-containing zwitterionic block copolymers consisting of -acryloyl-l-methionine methyl sulfonium salt (A-Met(S)-OH) and -butyl acrylate (BA) were newly synthesized to develop a novel protein stabilizer. The zwitterionic block copolymers were prepared by reversible addition-fragmentation chain-transfer (RAFT) polymerization of BA using a hydrophilic macro-chain-transfer agent (CTA) obtained from -acryloyl-l-methionine (A-Met-OH) and subsequent postmodification. RAFT polymerization of A-Met-OH using poly(BA) macro-CTA, followed by postmodification, also afforded the target poly(A-Met(S)-OH)--poly(BA). The block copolymers stabilized horseradish peroxidase (HRP) during storage at 37 °C for 5 days, and the protein-stabilizing effect was enhanced with increase in the A-Met(S)-OH content. In particular, the block copolymer with ∼85% A-Met(S)-OH content showed a significant protein-stabilizing effect at a temperature (37 °C) higher than the room temperature, which is highly desirable for practical and industrial applications. The addition of sucrose into the block copolymer-protein solution led to a considerable increase in the HRP activity under the same conditions. Excellent alkaline phosphatase stabilization at 37 °C for 12 days was also achieved using the block copolymers. The zwitterionic block copolymers with the optimal hydrophilic/hydrophobic balance were found to serve as efficient protein-stabilizing agents, in comparison with the corresponding homopolymer and random copolymers. Dynamic light scattering, zeta potential, transmission electron microscopy, and circular dichroism measurements revealed that the zwitterionic block copolymer stabilizes an enzyme by wrapping with a slight change in the size, whereas the secondary and ordered structures of the enzyme are maintained.
Ryutaro Imamura, Hideharu Mori

2508 related Products with: Protein-Stabilizing Effect of Amphiphilic Block Copolymers with a Tertiary Sulfonium-Containing Zwitterionic Segment.

1 mL100ug500 100ug Lyophilized100ug Lyophilized1 Set1 Set50 ug1 Set100ug1 Set1 Set

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#31540182   2019/09/15 To Up

Cord-Based Microfluidic Chips as A Platform for ELISA and Glucose Assays.

This paper describes the development and application of microfluidic cord-based analytical devices (µCADs) in two enzyme-linked immunosorbent assays (ELISAs) and glucose assay. In this study, biotinylated goat anti-mouse immunoglobulin (IgG) antibody, rabbit IgG antibody, and glucose are quantitatively detected. In the ELISA systems, the antibody is spotted on the cord at the detection site and a series of washes, followed by streptavidin-alkaline phosphatase (Strep-ALP) or alkaline phosphatase (ALP)-conjugated secondary antibody and colorimetric substrate, completing the experiment. The devices are subsequently scanned and analyzed yielding a correlation between inverse yellow or inverse blue intensity and antibody concentration. For the first ELISA, a linear range of detection was observed at lower concentrations (2.50 × 10-1.75 × 10 mg/mL) of Strep-ALP with saturation of the enzyme achieved at higher concentrations (>2.50 × 10). For the second ELISA, the L was demonstrated to be 167.6 fmol/zone. The glucose assay consisted of spotting increasing concentrations of glucose on the analysis sites and transporting, via capillary action, a solution containing glucose oxidase (GOx), horseradish peroxidase (HRP), and potassium iodide (KI) to the detection sites realizing a yellow-brown color indicating oxidation of iodide to iodine. The device was then dried, scanned, and analyzed to show the correlation between yellow inverse intensity and glucose. Glucose in artificial urine showed good correlation using the devices.
Jenny Elomaa, Laura Gallegos, Frank A Gomez

1543 related Products with: Cord-Based Microfluidic Chips as A Platform for ELISA and Glucose Assays.

96 assays1 x 96 assays100 assays100 assays1,000 tests100 assays100 assays1 L100 assays96 assays100 assays

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#31371474   2019/08/01 To Up

Detection of an Antigen on an Immunoblot.

Detection of the antigen on an immunoblot can be achieved by either enzyme-based detection systems or using detection reagents labeled with fluorochromes. Two major types of enzyme-based detection systems are used in immunoblotting based on either horseradish peroxidase (HRP)-or alkaline phosphatase (AP)-coupled antibodies, and a range of soluble substrates that yield insoluble colored products (chromogenic detection) or generate light (chemiluminescent detection). This protocol describes chromogenic detection with AP as well as both chromogenic and chemiluminescent methods of detection with HRP. Before the detection step, the nitrocellulose or polyvinylidene fluoride (PVDF) membrane is incubated with the antigen using enzyme-conjugated primary antibodies or secondary detection reagents (such as species-specific anti-immunoglobulin antibodies, streptavidin, or Protein A) and washed. An appropriate substrate solution is applied to the membrane, and the signal is detected in the course of the enzymatic reaction, resulting either in development of insoluble colored products (chromogenic substrates) or in generation of light (chemiluminescent substrates). This protocol also describes use of detection reagents labeled with fluorochromes. The nitrocellulose or PVDF membrane incubated with fluorochrome-labeled detection reagents is rinsed with PBS and processed for image capturing with appropriate imaging equipment.
Larisa Litovchick

1687 related Products with: Detection of an Antigen on an Immunoblot.

96 tests96 tests0.251mg100 ug1 mg50ul (2mg/ml)0.1ml1000 Units100 µg0.1mg0.1ml

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#31287664   2019/07/09 To Up

Symmetric-Key Encryption Based on Bioaffinity Interactions.

The research presented here shows a bridge between biochemistry and cryptography. Enzyme-based assays were used in a new methodology linked to ciphers and cipher systems. Three separate enzyme assays, alkaline phosphatase (ALP) (E.C. 3.1.3.1), lysozyme (E.C. 3.2.1.17), and horseradish peroxidase (HRP) (E.C. 1.11.1.7), were used to create a cipher key in order to encrypt a message. By choosing certain parameters for one's experiment that are performed in the same way as a person receiving the message, correct encryption and decryption keys would be produced, resulting in a correct encryption and decryption of a message. It is imperative that both parties perform the same experiment under the same conditions in order to correctly interpret the message. Bioaffinity-based assays, in particular enzymatic assays, provide a specific, yet flexible mechanism to use for the encryption of messages. Because of the nature of this process there are a multitude of sets of parameters that may be chosen, each of which would result in a different key being produced, heightening the security and the robustness of the method. This paper shows that by using this concept of forming encryption keys using a bioaffinity-based approach, one is able to properly encrypt and decrypt a message, which could be viable for other biochemically based techniques.
Leif K McGoldrick, Elizabeth A Weiss, Jan Halámek

2599 related Products with: Symmetric-Key Encryption Based on Bioaffinity Interactions.

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#31281909   // To Up

Simultaneous detection of carcinoembryonic antigen and neuron-specific enolase in human serum based on time-resolved chemiluminescence immunoassay.

In the clinical diagnosis of tumor, the immunological detection of single tumor markers may lead to errors and missed inspection. Therefore, it is necessary to establish an accurate and effective method for the simultaneous detection of multiple tumor markers. Thus, we developed a time-resolved chemiluminescence immunoassay (TRCLIA) to simultaneously detect carcinoembryonic antigen (CEA) and neuron-specific enolase (NSE) in human serum. Horseradish peroxidase (HRP) and alkaline phosphatase (ALP) were used as the detection probes to label the monoclonal antibodies of CEA and NSE by strain-promoted azide-alkyne cycloaddition (SPAAC), respectively. Based on a sandwich immunoassay, the targets in the samples were captured by antibodies immobilized on the surface of carboxylate-modified polystyrene microspheres (CPSMS) and sandwiched by other antibodies labeled with HRP and ALP. Since HRP and ALP had different dynamic characteristics, the CEA and NSE signals were recorded at 0.5 s and 20 min, respectively, and cross-interference could be avoided effectively. The whole signal detection processes could be completed in 20 min. The linear ranges of CEA and NSE were 0.1-64 ng mL-1 and 0.05-64 ng mL-1 and the limits of detection were 0.085 ng mL-1 and 0.044 ng mL-1 (S/N = 2), respectively. Also, 45 human serum samples obtained from patients having lung disease were tested by TRCLIA and commercial chemiluminescence enzyme-linked immunoassay (CLEIA) kits with good correlation. The correlation coefficients of CEA and NSE were 0.985 and 0.970, respectively. The results demonstrated a novel, effective, reliable and convenient TRCLIA method for the clinical diagnosis of CEA and NSE. The TRCLIA method has the potential to be an effective clinical tool for the early screening of lung cancer and can be applied in clinical diagnosis.
Yanhua Mao, Nana Wang, Fei Yu, Songcheng Yu, Lie Liu, Yongmei Tian, Jia Wang, Yilin Wang, Leiliang He, Yongjun Wu

2254 related Products with: Simultaneous detection of carcinoembryonic antigen and neuron-specific enolase in human serum based on time-resolved chemiluminescence immunoassay.

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#31201790   2019/06/13 To Up

Comparison of soybean peroxidase with horseradish peroxidase and alkaline phosphatase used in immunoassays.

Enzyme labeling of an antigen or an antibody helps to visualize and amplify the signal and is an important reagent used in immunoassays for the detection of a target of interest. In this research, soybean peroxidase (SBP), a less commonly used enzyme reporter, was compared in immunoassays with the two most commonly used reagents, horseradish peroxidase (HRP) and alkaline phosphatase (ALP). The enzyme-antibody conjugates were evaluated by their performance in an indirect competitive enzyme-linked immunosorbent assay (icELISA) and in an indirect competitive chemiluminescent enzyme immunoassay (icCLEIA) for ractopamine (RAC). The results revealed that the more affordable SBP offers a long-lasting chemiluminescent signal, which outperformed ALP and HRP. SBP-antibody conjugate (SBP-Ab) based immunoassays produced lower limits of detection (LODs) and better accuracy in the detection of RAC in animal urine samples. Additionally, SBP-Ab has advantages in being more resistant to heat, acid and organic solvents. These results suggest that SBP could be a potentially excellent alternative to HRP and ALP for the development of immunoassay in food safety field.
Huijuan Yang, Candace S Bever, Huiyan Zhang, Ghulam Mujtaba Mari, Hongfang Li, Xiya Zhang, Liuchuan Guo, Ziwen Wang, Pengjie Luo, Zhanhui Wang

2012 related Products with: Comparison of soybean peroxidase with horseradish peroxidase and alkaline phosphatase used in immunoassays.

500 ml 100ul100 μg 125 ml 100ul 125 ml 100ul100 μg 15 ml 1 g 15 ml 8 ml

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#30902335   2019/02/02 To Up

Chemiluminescent dual-enzyme immunoassays capable of simultaneously quantifying carbohydrate antigen 19-9 and carcinoma embryonic antigen in a sample.

Using the internal chemiluminescence resonance energy transfer (Inter-CRET) of luminescent dye and the high-energy intermediate formed in 1,1'-oxalyldiimidazole chemiluminescence (ODI-CL) reaction, we developed for the first time dual-enzyme immunoassays operated with alkaline phosphatase (ALP) and horseradish peroxidase (HRP) for the simultaneous quantifications of carbohydrate antigen 19-9 (CA19-9) and carcinoma embryonic antigen (CEA) in a sample. Fluorescein formed from the ALP enzyme immunoassay emits green light in ODI-CL reaction, while resorufin formed from the HRP enzyme immunoassay emits red light. Green and red CL lights emitted in a detection cell were measured individually with two photomultiplier tubes with an optical filter capable of sensing green or red emission without interferences. The limits of detection for CA 19-9 (0.09 U/ml) and CEA (0.11 ng/ml) determined using the accurate and reproducible dual-enzyme immunoassays were as low as those calculated using the conventional single-enzyme immunoassays capable of sensing CA 19-9 or CEA in a sample. In conclusion, we confirmed that the highly selective dual enzyme immunoassays can be applied as a new analytical method capable of early diagnosing and monitoring colon and pancreatic cancers with the simultaneous analyses of CA 19-9 and CEA.
Yujung Lee, Seung Sun Kim, Ji Hoon Lee

1957 related Products with: Chemiluminescent dual-enzyme immunoassays capable of simultaneously quantifying carbohydrate antigen 19-9 and carcinoma embryonic antigen in a sample.

96tests96tests0.1ml96tests0.1ml500 tests50ul0.1ml0.1ml100 0.1ml (1mg/ml)1 mg

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#30851843   2019/02/10 To Up

Enzymatic in situ generation of covalently conjugated electron acceptor of PbSe quantum dots for high throughput and versatile photoelectrochemical bioanalysis.

Most of the photoelectrochemical (PEC) bioassays need to immobilize biomolecules on electrodes, which lead to tedious modification processes, damaged biomolecules, as well as crippled sensitivity/accuracy and low throughput of the performances. To overcome these drawbacks, we now introduce an exquisitely split-mode (which separates the bioreaction (performed in microplates) from the PEC detection (conducted in PEC cell)) cathodic photoelectrochemistry for probing versatile biocatalytic events with high throughput. Specifically, the enzymatically in situ generated 1,2-bezoquinone was covalently attached onto the PbSe quantum dots (QDs) modified indium tin oxide (ITO) (ITO/PbSe) photocathode through the connector of chitosan (CS). And the attached 1,2-bezoquinone acted as an efficient electron acceptor to promote the cathodic photocurrent of the ITO/PbSe electrode, enabling us to probe quinones-generating oxidoreductase (by taking horseradish peroxidase (HRP) as a model) coupled biocatalytic cascades including the alkaline phosphatase (ALP)/HRP and the glucose oxidase (GOx)/HRP cascades. Quantitative probing for ALP activity in a wide linear range of 5.0 × 10 to 10 U/L with the detection limit of 1.2 × 10 U/L was realized. While a wide linear range of 5.0 × 10 to 1.0 × 10 moL/L with a quite low detection limit of 1.0 × 10 moL/L was obtained for the glucose assay. In addition, this testing protocol was also extended to an immunoassay (taking carcinoembryonic antigen (CEA) as an example) using HRP as a catalytic tracer. The developed bioassays show high sensitivity and good selectivity for CEA detection in the linear range from 0.1 pg/mL to 100 ng/mL with a detection limit of 0.02 pg/mL. Moreover, the proposed detection has distinctive merits because it not only avoids the adverse effects of the surface confined biomolecules for crippling the signal transduction, but also it has enhanced throughput.
Hong Wang, Fang Yuan, Xiuming Wu, Yuming Dong, Guang-Li Wang

2523 related Products with: Enzymatic in situ generation of covalently conjugated electron acceptor of PbSe quantum dots for high throughput and versatile photoelectrochemical bioanalysis.

2 Pieces/Box100ug Lyophilized500 MG100ug Lyophilized100ug100ug Lyophilized100ug100ug100ug Lyophilized4 Arrays/Slide100ug Lyophilized

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#30566330   2019/01/24 To Up

Synthesis of Zwitterionic Polymers Containing a Tertiary Sulfonium Group for Protein Stabilization.

The present study demonstrates the controlled synthesis and biological potential of poly( N-acryloyl-l-methionine methyl sulfonium salt)s (poly(A-Met(S)-OH)s), which mimic dimethylsulfoniopropionate (DMSP), a compound produced by marine algae to protect their proteins. The novel sulfonium-containing zwitterionic polymers were synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization of the amino acid-based monomer N-acryloyl-l-methionine (A-Met-OH) followed by a postmodification process in which the sulfide groups were reacted with iodomethane. The DMSP-mimic zwitterionic macromolecules were shown for the first time to exhibit low cytotoxicity and the ability to stabilize proteins. By adding the resulting poly(A-Met(S)-OH)s to horse radish peroxidase (HRP) solution, the activity of HRP was maintained even after storage at 4 °C for several days. In addition, the protein activities were tested using peroxidase-labeled antibody to mouse immunoglobulin G (IgG-HRP) and alkaline phosphatase (ALP) after storage and for HRP after freeze-thaw cycles. Amphiphilic random copolymers, poly(A-Met(S)-OH- co-BA)s, also exhibited excellent properties for protein stabilization.
Ryutaro Imamura, Hideharu Mori

2002 related Products with: Synthesis of Zwitterionic Polymers Containing a Tertiary Sulfonium Group for Protein Stabilization.

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#30516449   2018/12/05 To Up

Generation by phage display and characterization of drug-target complex-specific antibodies for pharmacokinetic analysis of biotherapeutics.

Anti-idiotypic antibodies play an important role in pre-clinical and clinical development of therapeutic antibodies, where they are used for pharmacokinetic studies and for the development of immunogenicity assays. By using an antibody phage display library in combination with guided in vitro selection against various marketed drugs, we generated antibodies that recognize the drug only when bound to its target. We have named such specificities Type 3, to distinguish them from the anti-idiotypic antibodies that specifically detect free antibody drug or total drug. We describe the generation and characterization of such reagents for the development of ligand binding assays for drug quantification. We also show how these Type 3 antibodies can be used to develop very specific and sensitive assays that avoid the bridging format. Abbreviations: BAP: bacterial alkaline phosphatase; CDR: complementarity-determining regions in VH or VL; Fab: antigen-binding fragment of an antibody; HRP: horseradish peroxidase; HuCAL®: Human Combinatorial Antibody Libraries; IgG: immunoglobulin G; LBA: ligand binding assay; LOQ: limit of quantitation; NHS: normal human serum; PK: pharmacokinetics; VH: variable region of the heavy chain of an antibody; VL: variable region of the light chain of an antibody.
Stefan Harth, Andre Ten Haaf, Christian Loew, Christian Frisch, Achim Knappik

1289 related Products with: Generation by phage display and characterization of drug-target complex-specific antibodies for pharmacokinetic analysis of biotherapeutics.

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