Glyko TechNotes
PhycoLink TechNotes

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The TNGL TechNotes were transferred to ProZyme from Glyko.  We offer them here at the request of our customers. 

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 

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 

FRET Specific TechNotes
General TechNotes

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.
 

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

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.
 

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.
 

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).
 

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.
 

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). 
 

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.
 

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.

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.
 

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

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

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.
 

Methods for calculating concentrations, molarities and molecular weights of fluorescent molecules and conjugates.

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 

Poster displayed at Luminex Planet xMAP 2007Symposium

Abstract 
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 2007Symposium

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. 
 

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