The document describes the development of an improved fluorescent protease assay. The original PDQ protease assay had limitations including a short shelf-life and detection limits between 100 ng to 10 μg. The new fluorescent assay uses a fluorescently labeled protease substrate immobilized on metal-enhanced nanoparticles. This allows for a single-step assay with lower detection limits over 100-fold lower than the original assay. The fluorescent assay is also adaptable to high-throughput systems and has a longer minimum shelf-life of one year, making it more cost-effective to manufacture.
Protecting Protein Stability with a Novel Grade of SucroseMerck Life Sciences
Sucrose is one of the most widely used stabilizers in marketed drug products, ensuring the chemical and physical stability of the therapeutic protein. A challenge with the use of sucrose as an excipient is that nanoparticle impurities (NPI) can originate from the raw materials or be introduced during production.
In this whitepaper, you can find information on NPIs found in commercially available sucrose, their origin and impact on protein stability; and on a novel sucrose purification process designed to minimize the presence of NPIs.
To find more information about this novel grade of sucrose, please visit our website: https://www.sigmaaldrich.com/product/mm/103789
And to learn more about protein stabilization of biomolecules in general, please follow this link: https://www.sigmaaldrich.com/products/pharma-and-biopharma-manufacturing/formulation/protein-stabilizers
In-vitro antioxidant and GC-MS analysis ethanolic extract of poly herbal drugSkyfox Publishing Group
Antioxidants play an important role in inhibiting and scavenging free radicals, thus providing protection to human against
infections and degenerative diseases. Current research is now directed towards natural antioxidants originated from plants due to safe
therapeutics. Poly herbal drugs is used in Indian traditional medicine for a wide range of various ailments. To understand the mechanism
of pharmacological actions, antioxidant properties of the Poly herbal drugs extract were tested using standard in vitro models. The
ethanolic extract of Poly herbal drugs exhibited strong scavenging effect on superoxide, nitric oxide radical and reducing power radical
scavenging assay. The free radical scavenging effect of Poly herbal drugs extract was comparable with that of the reference antioxidants.
The data obtained in the present study suggests that the extract of Poly herbal drugs have potent Invitro antioxidant and Anti Diabetic
activity against free radicals, prevent oxidative damage to major biomolecules and afford significant protection against oxidative damage.
Parvovirus Filtration Best Practices - 25 Years of Hands-On ExperienceMerck Life Sciences
In this webinar, you will learn:
- how to measure filter performance and capacity,
- how to optimize filter virus removal capability,
- and avoid potential pit-falls
Detailed description:
This webinar will cover all aspects of parvovirus filtration best practices: process development/ optimization, pilot scale-up, and validation and explain the important connections between these activities. The rationale for the recommended best practices will be explained by discussing the underlying mechanisms that control filter performance.
— In the present work, impact of UV-B radiation (280-
315nm: 0.4 W m-2) on growth, photosynthetic pigments, protein,
ascorbate, proline and lipid peroxidation have been studied in
two cyanobacteria Nostoc muscorum and Synechocystis PCC
6803. UV-B radiation (2 to 6 hrs) leads to 55% inhibition of
growth in Synechocystis PCC 6803 in comparison to control
where as in Nostoc muscorum growth reduces up to 45%. This
UV-B treatment also significantly decreased the contents of
chlorophyll, carotenoids and phycocyanin. Photosynthetic
pigments decreased with increasing doses of UV-B (2 to 6 hrs)
radiation. However, the inhibitory effect in Synechocystis PCC
6803 was more pronounced than in Nostoc muscorum. With
increasing UV-B exposure period, production of ascorbate (19-
45%), proline (12-29%) and lipid peroxidation was significantly
higher in Synechocystis PCC 6803 as compared to control
sample. It was observed that lipid peroxidation enhanced 33 %
than control sample of Synechocystis PCC 6803. Our result shows
that photosynthetic apparatus is the main target of UV-B
radiation causing degradation of photosynthetic pigments. This
study concluded that Synechocystis PCC 6803 was the susceptible
organism for survival in stress condition than Nostoc muscorum.
Protecting Protein Stability with a Novel Grade of SucroseMerck Life Sciences
Sucrose is one of the most widely used stabilizers in marketed drug products, ensuring the chemical and physical stability of the therapeutic protein. A challenge with the use of sucrose as an excipient is that nanoparticle impurities (NPI) can originate from the raw materials or be introduced during production.
In this whitepaper, you can find information on NPIs found in commercially available sucrose, their origin and impact on protein stability; and on a novel sucrose purification process designed to minimize the presence of NPIs.
To find more information about this novel grade of sucrose, please visit our website: https://www.sigmaaldrich.com/product/mm/103789
And to learn more about protein stabilization of biomolecules in general, please follow this link: https://www.sigmaaldrich.com/products/pharma-and-biopharma-manufacturing/formulation/protein-stabilizers
In-vitro antioxidant and GC-MS analysis ethanolic extract of poly herbal drugSkyfox Publishing Group
Antioxidants play an important role in inhibiting and scavenging free radicals, thus providing protection to human against
infections and degenerative diseases. Current research is now directed towards natural antioxidants originated from plants due to safe
therapeutics. Poly herbal drugs is used in Indian traditional medicine for a wide range of various ailments. To understand the mechanism
of pharmacological actions, antioxidant properties of the Poly herbal drugs extract were tested using standard in vitro models. The
ethanolic extract of Poly herbal drugs exhibited strong scavenging effect on superoxide, nitric oxide radical and reducing power radical
scavenging assay. The free radical scavenging effect of Poly herbal drugs extract was comparable with that of the reference antioxidants.
The data obtained in the present study suggests that the extract of Poly herbal drugs have potent Invitro antioxidant and Anti Diabetic
activity against free radicals, prevent oxidative damage to major biomolecules and afford significant protection against oxidative damage.
Parvovirus Filtration Best Practices - 25 Years of Hands-On ExperienceMerck Life Sciences
In this webinar, you will learn:
- how to measure filter performance and capacity,
- how to optimize filter virus removal capability,
- and avoid potential pit-falls
Detailed description:
This webinar will cover all aspects of parvovirus filtration best practices: process development/ optimization, pilot scale-up, and validation and explain the important connections between these activities. The rationale for the recommended best practices will be explained by discussing the underlying mechanisms that control filter performance.
— In the present work, impact of UV-B radiation (280-
315nm: 0.4 W m-2) on growth, photosynthetic pigments, protein,
ascorbate, proline and lipid peroxidation have been studied in
two cyanobacteria Nostoc muscorum and Synechocystis PCC
6803. UV-B radiation (2 to 6 hrs) leads to 55% inhibition of
growth in Synechocystis PCC 6803 in comparison to control
where as in Nostoc muscorum growth reduces up to 45%. This
UV-B treatment also significantly decreased the contents of
chlorophyll, carotenoids and phycocyanin. Photosynthetic
pigments decreased with increasing doses of UV-B (2 to 6 hrs)
radiation. However, the inhibitory effect in Synechocystis PCC
6803 was more pronounced than in Nostoc muscorum. With
increasing UV-B exposure period, production of ascorbate (19-
45%), proline (12-29%) and lipid peroxidation was significantly
higher in Synechocystis PCC 6803 as compared to control
sample. It was observed that lipid peroxidation enhanced 33 %
than control sample of Synechocystis PCC 6803. Our result shows
that photosynthetic apparatus is the main target of UV-B
radiation causing degradation of photosynthetic pigments. This
study concluded that Synechocystis PCC 6803 was the susceptible
organism for survival in stress condition than Nostoc muscorum.
Filtration Strategies for Optimal Development and Purification of a FMD Virus...Merck Life Sciences
This poster presentation outlines the different filtration strategies and performances in the upstream and downstream process to develop a scalable, cost-efficient and GMP-compliant Foot and Mouth Disease (FMD) vaccine production:
• Introduction to foot and mouth disease (FMD) and background on FMD vaccines
• Review of cell culture, clarification, and concentration/diafiltration steps for the production and purification of the FMD vaccine
• Suggested further actions based on data outlined in this poster
To learn more about this topic or collaborate with our technical experts, schedule an in-person or remote visit at our M Lab™ Collaboration Centers: www.merckmillipore.com/mlab
Cell Culture Media Filtration – Filter Selection and SizingMilliporeSigma
The purpose of this application note is to provide estimated filtration areas for different sterilizing-grade filters with a panel of media used for Chinese Hamster Ovary (CHO) cell culture processes.
High-Capacity Capture of a Recombinant Growth Factor Directly From Refold Sol...Merck Life Sciences
In this work, the authors evaluate a salt tolerant tentacle resin targeting the purification of biomolecules from feeds at high salt concentration:
• Reviews process challenges presented by recombinant growth factors which are commonly expressed in E. coli as inclusion bodies
• Demonstrates how a salt tolerant resin facilitates easy process development with straightforward binding and elution conditions
• Presents cost analysis that compares salt tolerant with conventional cation exchanger for the growth factor purification
To learn more about this topic or collaborate with our technical experts, schedule an in-person or remote visit at our M Lab™ Collaboration Centers: www.merckmillipore.com/mlab
Nanoparticulate impurities in excipients - a threat to protein stabilityMerck Life Sciences
Watch the presentation of this webinar here: https://bit.ly/36cAQWT
Pharmaceutical-grade sucrose – an important stabilizer for biopharmaceuticals – contains nanoparticulate impurities (NPIs) that are a potential threat to protein stability. The scientists behind the discovery explain their impact on drug development and introduce a new sucrose with low NPI content.
A main reason for using excipients is to stabilize the active pharmaceutical ingredient and by that, ensuring an effective and safe therapy. Pharmaceutical grade sucrose, an important and commonly used stabilizer, contains nanoparticulate impurities (NPIs) of 100-200 nm size originating from the starting raw material. In this webinar, the scientists behind the discovery discuss the characteristics of these NPIs, explain their impact on aggregate/particle analytics and show, on the example of antibodies, that NPIs are a threat to protein stability. Their research has triggered the development of improved sucrose grades, that are low in NPIs to reduce the risk of unwanted instability.
In this webinar, you will learn:
• The role of sucrose as a stabilizer in pharmaceuticals
• Presence and characteristics of nanoparticulate impurities (NPIs) in pharmaceutical-grade sucrose
• Impact of NPIs on protein stability
• Succrose with low NPI content
Austin Biomolecules: open access is a peer reviewed, scholarly journal dedicated to publish articles covering all areas of Biomolecules.
The journal aims to promote latest information and provide a forum for doctors, researchers, physicians, and healthcare professionals to find most recent advances in the areas of Biomolecules. Austin Biomolecules: open access accepts research articles, reviews, mini reviews, case reports and rapid communications covering all aspects of Biomolecules.
Austin Biomolecules: open access strongly supports the scientific up gradation and fortification in related scientific research community by enhancing access to peer reviewed scientific literary works. Austin Publishing Group also brings universally peer reviewed journals under one roof thereby promoting knowledge sharing, mutual promotion of multidisciplinary science.
Lateral Flow: Making Magnetic Particles A Viable And Easier To Use Replacemen...Anteo Technologies
This application note titled Lateral Flow: Making Magnetic Particles A Viable And Easier To Use Replacement To Gold Nanoparticles outlines the preparation of 200nm magnetic particles conjugates for use in an anti-hepatitis B surface antigen based lateral flow assay.
This presentation reviews the results of a study in which the authors investigated the effects of poly-diallydimethylammonium chloride (pDADMAC) flocculation and clarification on the performance and longevity of protein A resin.
To learn more about this topic or collaborate with our technical experts, schedule an in-person or remote visit at our M Lab™ Collaboration Centers: http://www.emdmillipore.com/mlab
Biosynthesis and characterization of silver nanoparticles using ficus benghal...eSAT Publishing House
IJRET : International Journal of Research in Engineering and Technology is an international peer reviewed, online journal published by eSAT Publishing House for the enhancement of research in various disciplines of Engineering and Technology. The aim and scope of the journal is to provide an academic medium and an important reference for the advancement and dissemination of research results that support high-level learning, teaching and research in the fields of Engineering and Technology. We bring together Scientists, Academician, Field Engineers, Scholars and Students of related fields of Engineering and Technology
HIV Vaccines Process Development & Manufacturing - Pitfalls & PossibilitiesKBI Biopharma
Originally presented at the HIV Vaccine Manufacturing Workshop –July 19th& 20th, 2017 by Abhinav A. Shukla, Ph.D.Senior Vice PresidentDevelopment & ManufacturingKBI Biopharma, Durham NC
Filtration Strategies for Optimal Development and Purification of a FMD Virus...Merck Life Sciences
This poster presentation outlines the different filtration strategies and performances in the upstream and downstream process to develop a scalable, cost-efficient and GMP-compliant Foot and Mouth Disease (FMD) vaccine production:
• Introduction to foot and mouth disease (FMD) and background on FMD vaccines
• Review of cell culture, clarification, and concentration/diafiltration steps for the production and purification of the FMD vaccine
• Suggested further actions based on data outlined in this poster
To learn more about this topic or collaborate with our technical experts, schedule an in-person or remote visit at our M Lab™ Collaboration Centers: www.merckmillipore.com/mlab
Cell Culture Media Filtration – Filter Selection and SizingMilliporeSigma
The purpose of this application note is to provide estimated filtration areas for different sterilizing-grade filters with a panel of media used for Chinese Hamster Ovary (CHO) cell culture processes.
High-Capacity Capture of a Recombinant Growth Factor Directly From Refold Sol...Merck Life Sciences
In this work, the authors evaluate a salt tolerant tentacle resin targeting the purification of biomolecules from feeds at high salt concentration:
• Reviews process challenges presented by recombinant growth factors which are commonly expressed in E. coli as inclusion bodies
• Demonstrates how a salt tolerant resin facilitates easy process development with straightforward binding and elution conditions
• Presents cost analysis that compares salt tolerant with conventional cation exchanger for the growth factor purification
To learn more about this topic or collaborate with our technical experts, schedule an in-person or remote visit at our M Lab™ Collaboration Centers: www.merckmillipore.com/mlab
Nanoparticulate impurities in excipients - a threat to protein stabilityMerck Life Sciences
Watch the presentation of this webinar here: https://bit.ly/36cAQWT
Pharmaceutical-grade sucrose – an important stabilizer for biopharmaceuticals – contains nanoparticulate impurities (NPIs) that are a potential threat to protein stability. The scientists behind the discovery explain their impact on drug development and introduce a new sucrose with low NPI content.
A main reason for using excipients is to stabilize the active pharmaceutical ingredient and by that, ensuring an effective and safe therapy. Pharmaceutical grade sucrose, an important and commonly used stabilizer, contains nanoparticulate impurities (NPIs) of 100-200 nm size originating from the starting raw material. In this webinar, the scientists behind the discovery discuss the characteristics of these NPIs, explain their impact on aggregate/particle analytics and show, on the example of antibodies, that NPIs are a threat to protein stability. Their research has triggered the development of improved sucrose grades, that are low in NPIs to reduce the risk of unwanted instability.
In this webinar, you will learn:
• The role of sucrose as a stabilizer in pharmaceuticals
• Presence and characteristics of nanoparticulate impurities (NPIs) in pharmaceutical-grade sucrose
• Impact of NPIs on protein stability
• Succrose with low NPI content
Austin Biomolecules: open access is a peer reviewed, scholarly journal dedicated to publish articles covering all areas of Biomolecules.
The journal aims to promote latest information and provide a forum for doctors, researchers, physicians, and healthcare professionals to find most recent advances in the areas of Biomolecules. Austin Biomolecules: open access accepts research articles, reviews, mini reviews, case reports and rapid communications covering all aspects of Biomolecules.
Austin Biomolecules: open access strongly supports the scientific up gradation and fortification in related scientific research community by enhancing access to peer reviewed scientific literary works. Austin Publishing Group also brings universally peer reviewed journals under one roof thereby promoting knowledge sharing, mutual promotion of multidisciplinary science.
Lateral Flow: Making Magnetic Particles A Viable And Easier To Use Replacemen...Anteo Technologies
This application note titled Lateral Flow: Making Magnetic Particles A Viable And Easier To Use Replacement To Gold Nanoparticles outlines the preparation of 200nm magnetic particles conjugates for use in an anti-hepatitis B surface antigen based lateral flow assay.
This presentation reviews the results of a study in which the authors investigated the effects of poly-diallydimethylammonium chloride (pDADMAC) flocculation and clarification on the performance and longevity of protein A resin.
To learn more about this topic or collaborate with our technical experts, schedule an in-person or remote visit at our M Lab™ Collaboration Centers: http://www.emdmillipore.com/mlab
Biosynthesis and characterization of silver nanoparticles using ficus benghal...eSAT Publishing House
IJRET : International Journal of Research in Engineering and Technology is an international peer reviewed, online journal published by eSAT Publishing House for the enhancement of research in various disciplines of Engineering and Technology. The aim and scope of the journal is to provide an academic medium and an important reference for the advancement and dissemination of research results that support high-level learning, teaching and research in the fields of Engineering and Technology. We bring together Scientists, Academician, Field Engineers, Scholars and Students of related fields of Engineering and Technology
HIV Vaccines Process Development & Manufacturing - Pitfalls & PossibilitiesKBI Biopharma
Originally presented at the HIV Vaccine Manufacturing Workshop –July 19th& 20th, 2017 by Abhinav A. Shukla, Ph.D.Senior Vice PresidentDevelopment & ManufacturingKBI Biopharma, Durham NC
Supercritical fluid (CO2) chromatography for quantitative determination of se...Ratnakaram Venkata Nadh
In the present study, two cancer therapeutic drugs (docetaxel and bortezomib) were separated from their
potential impurities on a chromatographic platform by utilizing CO2 gas (supercritical state) and quantified.
The chromatographic separations were achieved on two short columns BEH-2EP (100mm 3mm, 1.7 mm)
and CHIRALPAK AD-3 (100 mm 4.6 mm, 3 mm) for docetaxel and bortezomib, respectively. The present
work describes the role of organic modifiers in the separation of polar compounds by supercritical fluid
chromatography. The two new methods were fully validated in accordance with the current ICH
(International Council for Harmonization of technical requirements for pharmaceuticals for human use)
guidelines. The stability indicating power of the methods was demonstrated from the stress studies
conducted on the injection formulations of the two compounds. The methods are precise with % RSD of
0.4, linear with the correlation coefficient of r2 $ 0.999 and accurate in the range of 50–150% of the
target assay concentration. The two methods can be equally employed for the assay determination of
docetaxel and bortezomib APIs as well.
Exploring the Versatility of Micro-flow Technology – From Peptide Biomarkers ...Waters Corporation
Presenter: Corey D. Broeckling, Ph.D., Associate Director, Proteomics and Metabolomics Facility, Joint Assistant Professor, Colorado State University
Microfluidic technology offers multiple advantages including ease of use, robustness and sensitivity. Coupled with a tandem quadrupole mass spectrometer (such as the Xevo TQ-S) we can create an optimal and versatile “middle ground” platform in which these advantages can be exploited for both small molecule and peptide quantitative applications. For example, most small molecule applications are performed using standard flow chromatography (in the range of 600-100 L/min) consuming a high level of both solvent and sample which increases the cost (both fiscally and environmentally). The use of microfluidic technology for these small molecule applications can reduce solvent consumption by upwards of 150-fold and can significantly increase on-column sensitivity, thus reducing sample consumption. Conversely, quantitative peptide assays are almost exclusively performed using nanoscale chromatography (~400 nL/min) to achieve the required sensitivity for detection of these low abundance molecules within a complex matrix (e.g. serum, urine, etc.). We have found that the use of microfluidic technology for peptide quantitation yields the same or better sensitivity when compared to a nanoscale platform and has the additional, very significant advantages of ease of use, robustness, and improved chromatographic resolution (e.g. peak capacity). Thus, with a single analytical platform we can perform quantitative analysis for a wide range of compounds spanning from lipids/metabolites to peptides. One application in which the technology has struggled is the analysis of compounds in negative ionization mode. This limitation has been overcome in the development of a next generation microfluidic device that incorporates post-column addition of isopropanol to improve ionization and spray stability in negative mode applications. With this new capability we can now perform quantitative experiments in negative mode or with polarity switching.
This presentation was given at the 11th International Conference of the Metabolomics Society (Metabolomics 2015, #metsoc2015 on Twitter), June 29, 2015, in San Francisco.
Antibody Coupling - Single Coupling Approach To Bind Antibodies Diverse Speci...Anteo Technologies
Traditional covalent chemistries use harsh chemicals and require expertise in the techniques of diverse covalent coupling methodologies. The Antibody Coupling Kit was made to address issues such as: difficulties in binding certain antibodies with traditional covalent chemistries, antibody wastage, and incorrect antibody orientation. Anteo’s technology offers scientists the flexibility to bind any antibody onto a solid support surface through the use of polymeric metal complexes. The polymeric metal nature of the technology allows multi-valent binding of the target antibody through chelation to the electron donating groups located in the Fc region of the antibody. Anteo’s kit promotes gentle monolayer binding, meaning proteins assemble in the correct orientation while reducing the amount of damaged proteins, leading to increased functionality of antibodies and less antibodies used for the experiment.
This application note demonstrates the ability of the Antibody Coupling Kit to bind Mouse IgG, Mouse IgM, Rabbit IgG, Human IgG and Human IgM antibodies onto 200 nm magnetic particles using a particle-based fluorescent antibody loading assay.
Improved coverage of the proteome using gel eluted liquidExpedeon
It has long been understood that sample fractionation is critically important to generating quality, comprehensive proteomics data. In spite of the continual improvements in speed and sensitivity of mass spectrometers, these instruments are still unable to adequately overcome the enormous challenge
of most biological samples without multiple dimensions of separation prior to mass analysis.
Lignocellulose Biomass- Hydrolysis & Fermentation Lab Protocols
YOU AGREE TO INDEMNIFY BiorefineryEPCTM , AND ITS AFFILIATES, OFFICERS, AGENTS, AND EMPLOYEES AGAINST ANY CLAIM OR DEMAND, INCLUDING REASONABLE ATTORNEYS' FEES, RELATED TO YOUR USE, RELIANCE, OR ADOPTION OF THE DATA FOR ANY PURPOSE WHATSOEVER. THE DATA ARE PROVIDED BY BiorefineryEPCTM "AS IS" AND ANY EXPRESS OR IMPLIED WARRANTIES, INCLUDING BUT NOT LIMITED TO THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE ARE EXPRESSLY DISCLAIMED. IN NO EVENT SHALL BiorefineryEPCTM BE LIABLE FOR ANY SPECIAL, INDIRECT OR CONSEQUENTIAL DAMAGES OR ANY DAMAGES WHATSOEVER, INCLUDING BUT NOT LIMITED TO CLAIMS ASSOCIATED WITH THE LOSS OF DATA OR PROFITS, WHICH MAY RESULT FROM ANY ACTION IN CONTRACT, NEGLIGENCE OR OTHER TORTIOUS CLAIM THAT ARISES OUT OF OR IN CONNECTION WITH THE USE OR PERFORMANCE OF THE DATA.
Recombinant Factor C (rFC) is a recombinantly manufactured protein used for the detection of bacterial endotoxins. They can be cloned and expressed in yeast, Escherichia coli and mammalian cells. It is an alternative to the Limulus amebocytelysate (LAL) which is widely used for the detection of lipopolysaccharides (LPS). However, rFC is a better alternative as it is animal free, simpler, more specific and sensitive as it strongly binds to the LPS and detect their presence.
1. PDQ™ Protease Assay
Dave Samaroo, Rosalind Ramsey, and Sheldon E. Broedel, Jr.
Athena Environmental Sciences, Inc. 1450 South Rolling Road, Baltimore, MD 21227
Introduction
Importance:
Proteases are well known commercial enzymes that are exploited in the
detergent, food, pharmaceutical, diagnostics, and fine chemical industries.
Their use as detergent additives is the largest application of industrial enzymes
in terms of volume and value. Interest in proteases has increased with the
realization that they play a critical role in a variety of diseases. Modifications in
proteolytic systems may trigger multiple pathological conditions such as
cancer, neurodegenerative disorders, and cardiovascular diseases. In industrial
settings, the formation of biofilms is an economic problem and proteases are
often used to remove and clean these biofilms from surfaces. Further,
researchers and manufacturers of high valued proteins are concerned with
undesirable degradation by contaminating proteases. All of these applications
require the use of a quantitative measure of protease activity.
Background of PDQ™ Protease Assay:
The PDQ™ Protease Assay is a colorimetric assay that was designed to
simplify the measurement of protease activity. The assay format eliminates the
separation steps often used in historic protease assays thereby allowing for a
single step assay. The assay is a vial-based system consisting of cross-linked
sol-gel composed of albumin, azoalbumin, and gelatin. This substrate matrix is
susceptible to a wide range of enzymes commonly used in industry and
research. However, the manufacture of the assay is labor intensive and the
product suffers from a short shelf-life. Further, the detection limit is between
100 ng and 10 µg depending on the enzyme.
Objective of new format:
To develop an assay that has a lower detection limit, is adaptable to high
throughput systems and is more cost effective to manufacture, we evaluated the
use of fluorescence tagged protease substrate. The new format consists of a 96-
well plate using metal-enhanced nano-particles to increase the fluorescent
signal. The fluorescently labeled substrate is immobilized on the surface of the
metal nano-particles and protease activity is quantified by measuring the
amount of residual fluorescence remaining in the well or the amount of
fluorescence released into the buffer. This allows for a one step procedure with
reaction conditions that are matrix-independent including the use of opaque
samples.
References:
Kirk, O., Borchert, T., & Fuglsang, C. (2002). Industrial enzyme applications. Current Opinion
In Biotechnology, 13(4), 345-351. doi:10.1016/s0958-1669(02)00328-2.
Lopez-Otin, C., & Bond, J. (2008). Proteases: Multifunctional Enzymes in Life and
Disease.Journal Of Biological Chemistry, 283(45), 30433-30437. doi:10.1074/jbc.r800035200.
Selan, L., Berlutti, F., Passariello, C., Comodi-Ballanti, M., & Thaller, M. (1993). Proteolytic
enzymes: a new treatment strategy for prosthetic infections?. Antimicrobial Agents And
Chemotherapy, 37(12), 2618-2621. doi:10.1128/aac.37.12.2618
Royer, G. P. and Broedel, Jr., S. E. (1996). One Step Protease Assay Technical Brief. Retrieved 8
January 2015, from http://www.athenaes.com/tech_brief_protease.php (accessed 8 Jan 2015).
Aslan, K., Gryczynski, I., Malicka, J., Matveeva, E., Lakowicz, J., & Geddes, C. (2005). Metal-
enhanced fluorescence: an emerging tool in biotechnology. Current Opinion In
Biotechnology, 16(1), 55-62. doi:10.1016/j.copbio.2005.01.001.
Methods: QuantaWell™ strip wells were coated as in Fig. 3 and stored at 50ºC. At
the times indicated, a duplicate set of strips were reacted with 0.1 ml trypsin and the
amount of residual fluorescence measured as in Fig. 3.
Methods: QuantaWells™ were coated as in Fig. 3 and duplicate wells incubated at
37ºC with 0.1 ml of seven different proteases at concentrations ranging from 100
µg/ml to 10 pg/ml. After 1 hour, the amount of residual fluorescence was determined.
Summary and Conclusions:
The Fluorescent PDQ™ Protease Assay using metal nano-particle coated
QuantaWell™ microtiter plates exhibits:
• Lower detection limits by two or more orders of magnitude compared to
the original assay.
• Ability to measure a broad spectrum of proteases.
• Lower manufacturing costs.
• Increased product shelf life.
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
0.0 200.0 400.0 600.0 800.0 1000.0
RFU
Coating Concentration (µg/ml)
Released Substrate using 40 BAEE/ml Trypsin
Abstract
The colorimetric PDQ™ Protease Assay was first introduced in 1996. It
has since become a routinely used assay for measuring protease activity in
process samples or for certifying reagents as protease-free. However, while
the sol-gel substrate matrix allows for a one-step assay with nanogram
detection limits, the substrate matrix only has a three month shelf-life.
Alternative matrix formats, including the use of fluorescent substrates, have
not yielded a more robust assay. Here we describe development of a protease
assay using a fluorescent protease substrate in combination with metal nano-
particle coated microtiter wells. The metal-enhanced fluorescent signal
permitted the development of an assay with a detection limit more than 100-
fold lower than the original assay with detection limits below 10 pg for
certain proteases. An accelerate stability study suggested that the shelf-life
of the substrate is at least one year at 4ºC. Further, the assay is adaptable to
high-throughput formats.
Figure 1. Metal nano-particle coated plates increase the fluorescence
signal yielding a more sensitive assay.
Method: The wells of a Maxisorb™ (Nunc) and QuantaWell™ (Athena) plate were
coated with 100 µg of fluorescently labeled BSA. Duplicate wells were incubated with
0.1 ml of trypsin at different concentrations ranging from 50 to 0.05 BAEE Units/ml
and the residual level of fluorescence measured after 1 hour incubation at 37ºC.
Figure 2. The signal is proportional to the amount of substrate coated on
the well.
Methods: A QuantaWell™ plate was coated with 50 µg fluorescently labeled BSA.
Duplicate wells were incubated with 0.1 ml of 100 to 0.001 BAEE units/ml trypsin and
the residual fluorescence measured after 1 hour at 37ºC. The resulting calibrator
curve (right panel) was compared to the calibrator curve generated by the colorimetric
assay (left panel).
Figure 3. Enhanced fluorescence assay exhibited a 100-fold lower
detection limit compared to the standard colorimetric assay.
Method: A QuantaWell™ plate was coated with different concentrations of
fluorescently labeled BSA. Duplicate wells were incubated with 0.1 ml of 40 BAEE
units/ml trypsin for 1 hour at 37ºC and the amount of residual (not shown) and
released fluorescence measured. The selected coating concentration for further
development was 500 µg/ml (50 µg per well).
Released Residual
Figure 4. Measuring the residual fluorescence permitted detection of
lower amounts of protease activity.
Method: QuantaWells™ (Athena) were coated as in Fig. 3. Duplicate wells were
incubated with 0.1ml of trypsin at concentrations ranging from 102 to 10-3 BAEE
Units/ml and the amount of fluorescence released (left panel) and residual (right
panel) measured after 1 hour incubation at 37ºC.
LOD ~ 0.01 Unit/mlLOD ~ 1 Unit/ml
Trypsin Conc. (Units/ml)
Figure 6. The activity of a range of different type of proteases can be
measured using the fluorescent PDQ™ Protease Assay.
Figure 5. The substrate matrix coated on QuantaWells™ was stable at
elevated temperature for 48 days. This indicates a product shelf-life of 1
year at 4ºC.