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TNGP100GlykoPrep® Guidebook

TNGP101UPLC® Rapid Methods

TNGP102GlykoPrep® Microfuge Method - Rapid N-Glycan Preparation with 2-AB

TNGP103GlykoPrep® Microfuge Method - Rapid N-Glycan Preparation with InstantAB™

TNGP104GlykoPrep® Microfuge Method - Rapid N-Glycan Preparation with APTS

TNFC300FACE®N-Linked Glycan Sequencing

TNGK200Glyko Enzyme Guide

Please contact us for consultation

TNGK200.1Comparing PNGase F Activity

Genzyme's patent for recombinant PNGase F in Europe (EP Patent No. 0472651 B1, Endo F-Free PNGase F) was upheld May 4, 2006. Users, who wish to purchase from a supplier licensed under the patent, have inquired about the conversion of units from Roche Applied Science (Roche) to ProZyme's N-Glycanase®.

TNGL100Principles of Glycobiology

TNGL101Biosynthesis of N- and O- Glycans

TNGL102GPI Membrane Anchors

TNGL103Unusual Modifications

TNGL104Biological Roles of Oligosaccharides

TNGL105Pharmacological Effects of Glycosylation

TNGL106The Role of Glycosylation in Disease

TNGL107Selectins and Glycosylation in Inflammation

TNGL108Plant Lectins

TNGL109Animal and Bacterial Lectins

TNGL110Lectins as Indicators of Disease

TNGS300.1An Enzyme-based Sialic Acid Quantitation Assay for Rapid Screening of Therapeutic Glycoproteins During Process Development: A Potential Process Analytical Technology

Sialic acid serves a critical role in mediating the effectiveness of recombinant therapeutic proteins, especially those intended for intra-vascular administration. The presence or absence of this 9-carbon carbohydrate can dramatically effect the pharmacokinetics of the protein, as well as its immunogenicity; most recently, sialic acid has been directly implicated in the function and effectiveness of therapeutic immunoglobulins (Scallon et al., 2006; Kaneko et al., 2006). It has clearly been demonstrated that cell culture conditions; the cell type used for expression host; and cell culture media components can alter sialic acid content and the distribution of sialic acid species. Given these potential variations in sialic acid levels during process optimization and for biosynthesis of antigenic forms of this carbohydrate, continual monitoring of both sialic acid content and its various molecular species is a mandatory requirement of any process development effort.

In order to meet the need for a rapid, high-throughput means for screening a large number of samples, we have developed a fluorometric (or colorimetric), enzyme-coupled method for sialic acid screening, which utilizes low levels (10 - 50 µg) of protein in a 96-well plate format. Detection as low as 200 pmols of sialic acid can be made with an inter-assay relative error of about 5%. Sialic acid on recombinant proteins with very low levels of sialic acid, as found on monoclonal antibodies, can be readily quantitated. Intra-assay variations are about 5 - 7%. Assays on ~90 samples can be made in about 70 minutes. Sialic acid content was determined for a wide variety of sialylated glycoproteins, including immunoglobulins. The results are consistent with previously reported values for sialic acid content using well-established, but significantly more complex and time consuming assays.

Presented at WCBP 2007: 11th Symposium of Regulatory and Analytical Sciences for Biotechnology Health Products January 29 - 31, 2007, Washington, DC, USA

TNGS300.2Qualification of a Process Analytical Technology for Quantifying Sialic Acid On Therapeutic Proteins Using Two Instrument Platforms

Sialic acid serves a critical role in mediating the effectiveness of recombinant therapeutic proteins. It has been well established that cell culture conditions, host cell type and media components can alter sialic acid content and the distribution of sialic acid species. These considerations underscore the importance of monitoring both sialic acid content and its various molecular species over the course of any therapeutic protein process development effort.

Presented at Glycobiology 2007, November, 2007, Boston, MA, USA &
Post Translational Modifications, November, 2007, Prague, Czech Republic

TNGS300.3Improved Sialic Acid Quantitation Assay Suitable For Robotics And Process Analytical Technology Applications

We previously developed a sensitive, high-throughput, enzyme-based sialic acid assay suitable for application in a 96-well plate format. This method relies on the detection of hydrogen peroxide, generated through a coupled enzyme system in which sialic acid molecules are converted to mannosamine and pyruvate via the neuraminyl aldolase followed by a subsequent conversion of pyruvate into hydrogen peroxide and acetylphosphate with pyruvate oxidase. Although this method has been proven to be useful for routine monitoring of the sialic acid content of therapeutic proteins, the execution is moderately complex and time consuming, involving multiple steps and reagents. For example, three sets of reagents along with distinct buffers or organic solvent (DMSO) are added at different stages.

We have now simplified the method so that only one reagent mixture is required to carry out the two-step enzyme process with all enzymes acting simultaneously. These modifications significantly reduce the reagent preparation time, eliminate the need for the DMSO solvent, and streamline the method by consolidating multiple pipetting operations into a single step. Furthermore, this new simplified method has greater precision, higher signal-to-background ratio compared to the previous method, and is much easier to adapt to robotics. For several well-known glycoproteins, relative assay errors were reduced by half (2 — 3 % for replicate analysis, compared with about 5 — 7 % with the previous method) and signal-to-background ratios were tripled (>30:1 at 1000 pmol levels, compared with 10:1 with the previous assay). Detection with fluorescence can be made at 50 — 1000 pmol of sialic acid and absorbance detection can be made at 1000 — 5000 pmol.

Presented at Glycobiology 2008, November, 2008, Fort Worth, TX, USA &
Post Translational Modifications, November, 2008, Munich, Germany

TNGS300.4Screening Sialic Acid Content to Optimize Pharmacokinetics and Pharmacodynamics of Glycoprotein Therapeutics

Sialic acid serves a critical role in mediating the effectiveness of recombinant therapeutic proteins, especially those intended for intravascular administration. The presence or absence of this terminal sugar on the glycan side chains can dramatically alter the residence time in the plasma (pharmacokinetics) and can also affect the pharmacodynamics of the protein. The absence of sialic acid is a useful indicator that other carbohydrate residues, such as mannose, galactose or N-acetylgalactosamine, occupy terminal positions on the glycan, which can interact with cell surface receptors and drive clearance of the therapeutic from circulation (Hara et al. 1989; Hudson et al. 1973). In addition, the level of sialic acid and type of linkage to the penultimate galactose residue on the Fc-glycan chains of therapeutic antibodies elicit either an inflammatory or an anti-inflammatory response (Kaneko et al. 2006; Scallon et al. 2007).

Presented at WCBP 2010, January 2010, Washington, DC, USA; the Society for Glycobiology, November, 2009, San Diego, CA, USA; Post Translational Modifications, September, 2009, Prague, Czech Republic & the Society for Biomolecular Sciences 15th Annual Conference & Exhibition, April, 2009, Lille, France

TNGS300.5A New Platform for High Throughput Micro Chromatography Analysis

The advent of Quality by Design (QbD) has dramatically increased the experimentation required to develop a robust biotherapeutic manufacturing process. Even relatively simple assays such as antibody concentration have become a significant bottleneck for large designed experiments. Highly complex assays, such as glycan profile analysis, are equally important for process design space, but are well beyond current throughput capabilities.

The AssayMAP™ platform was designed to enable microliter-scale analytical bind/elute chromatography and enzymatic digestion to be performed in a high-throughput, parallel format compatible with microplate liquid handling.

Presented at WCBP 2010, January 2010, Washington, DC, USA & LabAutomation2010, January 2010, Palm Springs, CA, USA

AssayMAP™ is a trademark of BioSystem Development, LLC, Madison, WI, USA


TNPB100Phycobiliprotein Overview

Overview of Phycobiliproteins, including nomenclature, structure, properties, and detailed descriptions.

TNPJ100Techniques for Optimizing the Sensitivity of Fret Assays

Homogeneous FRET assays have become popular for the detection of molecular interactions, driven both by the inherent robustness of fluorescence assays and by the logistic simplicity of their implementation. Perceived limits to their sensitivity, however, have mitigated against their use with lower affinity molecular interactions. Through a systematic reexamination of FRET assay design and signal detection, opportunities for significantly enhanced assay sensitivity can be identified.

TNPJ100.01bMultiple-Lot Comparison of PhycoLink PJ25S Streptavidin-Allophycocyanin conjugates in a Performance Assay

ProZyme's performance testing of streptavidin-APC demonstrates the historical consistency of the product and assures the consistency of new lots.

TNPJ100.02Precision of FRET Assays: S/N vs. S/B

The sensitivity of an assay is indicated by its signal-to-noise ratio (S/N). S/N should not be confused with the signal-to-background ratio (S/B), which can provide misleading indications when improperly interpreted as equivalent to S/N.

TNPJ100.02aMeasuring the Precision of FRET Assays: S/N and Z'

S/N and Z' are useful indices of assay precision for FRET assays, and incorporate the same assay response parameters. The choice between them should be based on the needs of the investigator: Z' is particularly sensitive in discriminating between assays with poor precision; S/N provides clearer distinctions between higher precision assays.

TNPJ100.03Background Correction and Spectral Overlap Compensation in FRET Assays

When proximity between two fluorescent molecules leads to FRET, the total fluorescence emission spectrum of the mixture is different from the spectrum of the same molecules mixed randomly in solution. The spectral differences reflect changes in the magnitudes of the donor and acceptor emission spectra, added together and superimposed on background fluorescence from various sources. These components of the complex emission spectra are identified and discussed to illustrate the principals behind the various methods of calculating FRET results (TNPJ100.04 FRET Calculations).

TNPJ100.04FRET Calculations

The calculation of FRET results requires both correction for blanks and compensation for spectral overlap between channels. Moreover, the final results of FRET assays may be expressed in several different ways, either in terms of FRET counts or as ratios. Equations are provided for these various output parameters.

TNPJ100.05Dissecting FRET Data: Quench-FRET Analysis

Quench-FRET analysis goes beyond standard FRET parameters (such as A/B ratio and Net FRET) by examining donor Quench, FRET and their ratio (Q/F). It is useful for detecting false positives and other artifacts produced by interference from absorbent/fluorescent sample compounds. Appropriate for both TR-FRET and PB-FRET assays, it is particularly suited to the latter because of the strong donor Quench and low noise in PB-FRET assays.


Phycobiliprotein-FRET (PB-FRET) can achieve signal-to-noise ratios significantly higher than those achieved with time-resolved FRET (TR-FRET).

TNPJ100.14Selection of Donor and Acceptor Reagents in a TR-FRET Assay

TR-FRET assays in which the more costly lanthanide fluor, rather than the less expensive phycobiliprotein fluor, is conjugated to streptavidin provide similar performance at a lower overall cost.

TNPJ100.16Microplate Color Comparison in a TRF Assay

Time-resolved FRET (TRF) assays were originally developed to overcome problems with sample background autofluorescence in proximity assays. Through the use of long lifetime fluorescence donors, detection is delayed until fluorescence from short-lived sources subsides, thereby eliminating most background.

TNPJ100.19Microplate Color Comparison in a TRF Assay

PB-FRET results that may be compromised due to candidate absorbance or fluorescence are readily identified by Quench-FRET analysis. The cost of the additional controls is minimal compared to the improved discrimination capability.

TNPJ100.20Self-quenching in FRET Assays [06/13/01]

At high reagent concentrations, fluorescent reagents can reabsorb their own emitted fluorescence, leading to unexpected nonlinearity in reagent concentration effects on fluorescence.

TNPJ100.23PB-FRET™: Ilumination and Detection Windows for Filter-based Instruments

By examining excitation and emission spectra for donor and acceptor fluors, optimum detection windows for FRET assays can be established.

TNPJ100.25Suitability of Fluorescent Molecules in Fret Assays

The cyanine dye Cy5 has similar spectral characteristics to APC but is a weaker fluorescence acceptor. APC-streptavidin gave six times the FRET counts and twice the signal:noise as Cy5-streptavidin in a tyrosine kinase TR-FRET assay.

TNPJ200PhycoLink® Conjugate Evaluations

Describes techniques to evaluate PhycoLink conjugates via absorbance, affinity chromatography, and HPLC/gel filtration. Methods for calculating concentrations, molarities and molecular weights of fluorescent molecules and conjugates.

TNPJ200.1Lot-to-Lot Comparison of Streptavidin-PE Conjugates in Two Commercial Assays

The properties of the conjugate incorporating the reporter dye (streptavidin-PE) impact assay performance to a great degree. The optimal conjugate is bright, exhibits minimal non-specific binding and demonstrates consistency lot to lot. Three lots of ProZyme's PhycoLink PJ31S were compared in two commercial assays.

Poster displayed at Luminex Planet xMAP 2006 Europe Symposium

TNPJ200.2Choice of Streptavidin-Phycoerythrin Conjugate is a Critical Element for Success in Assay Development

Poster displayed at Luminex Planet xMAP 2007 Symposium

TNPJ200.3Pushing the Limit of Detection: Signal Amplification and Screening for the Optimal Reporter Achieves Sub-attomole Sensitivity

Maximizing signal-to-background ratios is crucial in developing highly sensitive assays. During assay development for the detection of specific nucleic acid sequences, significant effort is spent identifying optimal sequences for high specificity while minimizing hybridization artifacts from secondary and tertiary structures. However, in general, very little effort is given to optimizing the signal generation or reporter portion of the assay. Here we demonstrate that the use of a signal can increase assay sensitivity by greater than one order of magnitude as measured by assessing the limit of detcection in a standard nucleic acid hybridization assay developed for the Luminex® xMAP® technology.

Poster displayed at Luminex Planet xMAP 2007 Symposium

TNPJ200.4New SA-PE Conjugates Reduce Capture-antibody-specific Background in Sandwich Immunoassays

Achieving high signal levels with low background is a principle objective of Luminex xMAP assay development. The conjugated reporter dye, streptavidin-phycoerythrin (SA-PE), can greatly influence both the signal strength and the background in an assay. We present as an example a panel of antibody-coupled microspheres which showed significant variation in background binding of SA-PE in the absence of both antigen and detection antibody. Several different types of SA-PE were tested against this panel for background binding. These same SA-PEs were tested in commercial assays for Human IL-4 and IFN-y to compare relative signal strengths.

Three new SA-PEs showed dramatic reduction of high backgrounds observed with some of the antibodies. These conjugates also produced higher signal strength. These improved properties can make a major contribution to assay optimization, and are illustrative of the continuous improvement we are making in this area.

Poster displayed at Luminex Planet xMAP 2008 Symposium

TNPJ200.5New SA-PE Conjugates Improve Critical Assay Parameters in Sandwich Immunoassays Performed using Luminex® xMAP® Technology

In Luminex® xMAP® assay development, the conjugated reporter dye streptavidin-phycoerythrin (SA-PE) can greatly influence such critical assay parameters as the signal strength, limit of detection, background and reproducibility of an assay. We present as an example a pair of antibody-coupled microspheres which showed significant differences in these parameters. Several different types of SA-PE were evaluated and compared.

Poster displayed at SBS 2008 14th Annual Conference

TNPJ200.6Optimizing Nucleic Acid Detection by Altering Detection Chemistry

Standard amplified nucleic acid hybridization assays require significant in silico and experimental testing to determine the optimal primers and probes for the detection of specific nucleic acid sequences. However, very little analysis or effort is spent optimizing the detection molecules used for a particular hybridization assay. Previously, we have demonstrated that optimization of the specific streptavidin-phycoerythrin conjugate (SA-PE) can increase sensitivity greatly in a nucleic acid hybridization assay using Luminex xMAP® technology. In the present study, we have examined the effects of changing spacer chemistry between the xMAP® microsphere surface and the hybridizing probe, and the primer sequence and the 5'-biotin moiety used to capture the SA-PE conjugate, and varying the SA-PE conjugate structure. The combined effects of these changes demonstrate that varying spacer chemistry and detection conjugate structure significantly enhances the sensitivity of nucleic acid hybridization assays performed xMAP® technology.

Poster displayed at Luminex Planet xMAP 2008 Symposium

TNPJ200.7Evaluation of Streptavidin-Phycoerythrin Reporter Conjugates in a Model TSH Immunoassay

As Luminex® xMAP® technology expands into new application areas requiring greater dynamic range, increased emphasis and resources are being channeled into assay development, with the aim of generating assays with the highest possible sensitivity. The streptavidin-phycoerythrin reporter conjugate (SA-PE) has historically been regarded as a generic assay component and has not typically been the focus of assay optimization efforts. However, relative to other assay components, screening for the optimal SA-PE can be a less complicated and more economical approach to enhancing assay performance. Utilizing a well-characterized sandwich immunoassay for human thyroid stimulating hormone (TSH), we demonstrate that SA-PEs designed specifically for the Luminex platform can improve overall assay performance through superior brightness and/or low background, and are therefore not simply generic components but rather key factors in achieving assay optimization.

Poster displayed at Luminex Planet xMAP 2009 Symposium

TNPJ200.8Room Temperature Stability Study of Streptavidin-Phycoerythrin Reporter Conjugates Using a Model TSH Immunoassay

Luminex® xMAP® technology has proven to be suitable for a wide variety of applications. Increasingly, this includes field applications where storage conditions are not as ideal as in laboratory settings. Reagent stability is always a concern for assay manufacturers, but in these cases there is a special need to demonstrate reagent stability. Utilizing a well-characterized sandwich immunoassay for human thyroid stimulating hormone (TSH), we demonstrate that SA-PEs designed specifically for the Luminex platform can be stored at room temperature for extended periods with no loss of performance.

Poster displayed at Luminex Planet xMAP 2009 Europe Symposium

TNPJ200.9Evaluation of anti-Human IgG Phycoerythrin Reporter Conjugate in a Model Human IgG Immunoassay

The most common embodiment of Luminex® xMAP® technology involves the use of biotinylated detection agents (antibodies or oligonucleotides) coupled with a streptavidin-RPE reporter conjugate. Different configurations are potentially useful in certain circumstances. In this study we examine the use of a different reporter conjugate, one that consists of polyclonal α-Human IgG conjugated directly to RPE (α-Human IgG-RPE). We show how this conjugate can be used to detect human IgG in a capture sandwich immunoassay.

Poster displayed at Luminex Planet xMAP 2010 Symposium

TNPJ210Conjugate Brightness

TNPJ220How to Make the Best Darn Conjugates (~10MB)

Researchers increasingly conjugate their own antibodies because they want direct conjugates; have only limited quantities to work with; can't find the right color on the desired marker; want to reserve the brightest tags for their dimmest antigens; or are driven by the need for more cost-effective reagents. Others may just want to do it themselves or understand the basic principles. We've got the answers for all of you. Please join us for a tutorial on conjugating PE, APC, PerCP and other phycoproteins (with subsequent conjugate purification) using ProZyme's fast and easy kits. We will also focus on those factors that make conjugates bright, reducing the scale (50 ug or less), evaluating conjugates for consistency lot-to-lot, scaling up and troubleshooting. Get the benefit of years of experience in one short hour from the people who know phycobiliproteins.

TNPJ220mSame tutorial as above at medium resolution (~2.5MB)

TNPJ300Alternative Conjugation Protocols

Protocols for Iminothiolane and SPDP conjugations when the standard protocol doesn't produce an acceptable conjugate.

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