Bioanalytical Development Tools

Nearly every process conducted in a biotechnology company requires analytical methods to back it up. Since BioPharm's last guide published in December 2001,¹scientists have developed exciting, new tools for conducting research. Listed here is a sampling of new technological developments unveiled in 2005.

Amino Acid Analysis

AAA is hydrolysis of a protein or peptide into its individual components and is often used for compositional analysis of naturally occurring amino acids. Advancing work concentrates on automation and handling multiple samples simultaneously.

Nanogen, Inc. has two new US patents: "Amino Acid Sequence Pattern Matching" and "Enzymatic modification of a nucleic acid-synthetic binding unit conjugate."² ParAllele BioScience, Inc. offers an extremely large assay panel with a capacity of 20,000 non-synonymous single nucleotide polymorphism (SNPs) to be used in human genotypifg studies.³ Sigma-Aldrich launched its Panorama Human p53 Protein Functional Microarray. Developed by Procognia and distributed exclusively by Sigma-Aldrich, the p53 protein microarray is based on a novel, proprietary technology that ensures the protein is correctly folded and confers full functionality.⁴

Biocalorimetry

Biocalorimetry studies molecular interactions and conformational energetics by direct measurement of the change in enthalpy for a biological process. Technical societies are the best source of information. On the current calendar is the 2005 North American Thermal Analysis Society (NATAS) Conference in Universal City CA, September 18-21, 2005. Prior to the central meeting, a short course is offered by NATAS September 17-18, 2005, at Sheraton Universal Hotel, Universal City, CA.⁵

The International Society for Biological Calorimetry is a technical society devoted to this topic. The society will be holding its 14th conference (founded in 1973) in Gdansk Poland June 2-8, 2006.⁶ Major topics scheduled for discussion include:

• Instrumentation and Theoretical Approaches

• Biological Materials

• Biochemical and Pharmaceutical Aspects

• Microorganisms and Tissue Cultures

• Plants and Photocalorimetry

• Insects and Social Communities

• Aquatic Animals

• Medical Aspects

The Society also has expanded its reach to South America. The second Campinas Symposium on Calorimetry and Chemical Thermodynamics will be held at the University of Campinas, Campinas, Brazil April 9-13 2006. For more information, refer to http://www.biocalorimetry.org.

Capillary Electrophoresis

Capillary electrophoresis (CE) is a miniaturized instrumental version of traditional electrophoresis. CE has been applied to separations historically performed by gel electrophoresis. The most notable example is the adaptation of CE technology in multi-channel DNA sequencers, which enabled the sequencing of the human genome in less than two years. In a sister publication (LCGC North America), Ted Weir related the use of CE to replace SDS-PAGE.⁷

"The CE-SDS technique has two unique advantages that eliminate the problems encountered in other CE techniques such as capillary zone electrophoresis. First, the high viscosity of the sieving polymer reduces EOF (electroosmotic flow), to a very low level. This eliminates the major source of poor reproducibility of migration times and peak areas in CE. Second, the high negative charge density of SDS-protein complexes eliminates the problem of protein adsorption to the capillary wall.

"Recently, chip-based instruments for analysis of biopolymers have been introduced by three companies. These employ photolithographic technologies to microfabricate devices with channels etched into substrates such as glass and plastics for electrically driven separations. All three commercial systems provide chips and application kits for the CE-SDS chemistry."

Circular Dichroism

Circular Dichroism (CD) is an excellent method for analyzing protein and nucleic acid secondary structure in solution. It can be used to track changes in folding as a function of temperature, and is also useful for measuring protein-ligand and nucleic acid-ligand interactions.

CD measures the difference in absorption between left- and right-circularly polarized light as a function of wavelength. When light passes through an optically active chromophore, differential absorption results in light with an electronic vector that is elliptical rather than circular.

Wallace has speculated on the future of this method. "Synchrotron radiation sources produce much brighter light than can be obtained from Xenon arc lamps now used as the light sources in commercial CD instruments. As a result, a Synchrotron Radiation Circular Dichroism (SRCD) instrument will enable more-accurate spectra to be measured to lower wavelengths. One of two places in the world where such an instrument now exists is at the Centre for Protein and Membrane Structure and Dynamics located at the Daresbury Lab in Cheshire UK."⁸

Electron Microscopy

Electrons have a shorter wavelength than light allowing magnifications up to many thousand times. The theme in this older technology is automation, resolution, and speed. FEI Company has published a useful, amusing instructional booklet on this topic.⁹

On April 18, 2005 FEI reported that the Titan 80-300, its newest scanning electron microscope, achieved atomic scale imaging with a resolution below 0.7 Angstrom.10 FEI claims this as a new record for a commercial tool. On August 1, 2005 this firm released new software and hardware for the Tecnai G2 transmission electron microscope.¹¹ Technai 3.0 software runs under Windows XP and takes full digital control of the microscope and detectors.

Electrophoresis

The separation of proteins, DNA, RNA, and peptides in an electric field is based on their relative electric charge. Greater charges migrate faster. BioPharm International published an application article in April 2005; which discussed a case study of developing analytical methods for an API.¹² One that was improved is SDS-PAGE. The excerpt from the article follows:

"GEL ELECTROPHORESIS Formulation development could not progress until an initial method to evaluate product quality was available. Although not required as a release method, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was the first technique established because of the simplicity of method development. SDS-PAGE separates proteins based on size and can be used to detect impurities, truncation, or aggregation. All materials used for SDS-PAGE were purchased from Invitrogen.

SDS-PAGE was used to analyze all early formulation development samples and was invaluable in identifying formulations prone to aggregation. In particular, a significant dimer band was observed in samples freeze-dried with only mannitol and stored at 37°C for two weeks. Dimer was not observed in frozen controls or frozen solutions of the same formulation. Also, dimer was not observed in samples freeze-dried with only sucrose, or with mixtures of sucrose and mannitol.

SDS-PAGE required little development time, but it was not an ideal product release method. The technique was useful for comparing the purity of samples within one analysis, but intermediate precision was generally poor. An analysis of the purity results by densitometry was subjective because streaks, bubbles, and background must be excluded from the purity calculation. In addition, the limit of quantitation (LOQ) was high. While not experimentally determined, the LOQ can be estimated from linearity data to be approximately 60 μg/mL or 10 percent aggregates (proteins stuck together but still soluble). Aggregates are undesirable because there are concerns that they can cause immunogenic reactions in patients.

SDS-PAGE was optimized during initial method development. We loaded samples at 0.5, 1, 2, 3, 4, and 5 μg to determine optimum loading conditions. The gel was scanned with a desktop scanner and analyzed by gel analysis software. We then plotted the amount of protein loaded against optical density to determine the linear range of staining, which was from 0.5 to 3μg with a 0.999 coefficient of determination."

Fluorescence in situ Hybridization (FISH)

The FISH technology platform is a system in which genes and chromosomes are probed by fluorescent-labeled DNA and then illuminated to allow for clearer identification. It has been shown that by identifying abnormalities in chromosomal DNA, clinicians can effect better treatments.

Cell nuclei from urine appear red (chromosome 3), green (chromosome 7), yellow (chromosome 9), and blue (chromosome 17) after hybridization with FISH assay probes. (Photograph courtesy of Kerstin Juniker, Ph.D.)

Research work at the University of Jena first noted that FISH would be useful in detecting bladder cancer. Guttman noted that the sensitivity of the FISH assay was superior in all stages of disease compared with cytology, and it correctly identified all invasive tumors (pT2-pT4).¹³

The FISH assay is a multi-color kit that uses four probes to detect aneuploidy for chromosomes 3, 7, and 17 and loss of the 9p21 locus in cells from voided urine samples. To perform the test, cells are fixed onto glass slides by centrifugation or pipetting for identification of the 25 nuclei that are most abnormal morphologically according to DAPI counterstaining. Each of the selected cells are classified into one of three groups based on enumeration of signals for the four chromosome targets, and the specimen is determined to be negative or positive for tumor according to the classification results.

On January 25, 2005 FDA approved Abbott Laboratories' UroVysion DNA probe-assay for use as an aid in the initial diagnosis of bladder cancer in patients with hematuria (blood in urine) and suspected of having bladder cancer.¹⁴ With this approval, UroVysion represents the first gene-based test available for both diagnosis and monitoring of bladder cancer recurrence. The test is designed to detect genetic changes in bladder cells in urine specimens FISH. Names get confusing because the FISH products are researched and marketed by a subsidiary named Vysis and not directly by Abbot. In addition to UroVysion, FISH is utilized in an FDA-approved device, PathVysion, to quickly identify which patients with late-stage breast cancer are suitable candidates for Herceptin therapy. Abbot scientists are also exploring FISH's diagnostic capabilities for cervical, esophageal, melanoma, and hematological cancers.

Teamed up with Vysis to solve cancer problems is Applied Imaging Corp., which has received 510(k) clearance from FDA to market its Ariol Her-2/neu FISH application.15 This is designed to detect Her-2/neu gene amplification in breast cancer biopsy samples via FISH. The application complements and completes the breast cancer panel on the Ariol system, which now includes 510(k) cleared assays for Her-2/neu Immunohistochemistry (IHC), Her-2/neu FISH, Estrogen Receptor, and Progesterone Receptor. The Ariol Her-2/neu FISH application assists in the analysis of a complex test that is an important factor in the evaluation and selection of certain breast cancer patients for Genentech's Herceptin (trastuzumab) therapy.

HPLC

High-performance liquid chromatography is similar to larger-scale liquid chromatography but is set up to be more powerful. The resin particles in the stationary phase are smaller to increase the number of plates and the loading is quite low and there are many choices of the mobile phases.

R&D Magazine selected the Corona CAD (Charged Aerosol Detector) for its prestigious 2005 R&D 100 Award.¹⁶ The Corona is a breakthrough, universal detector technology for HPLC (High Performance Liquid Chromatography). The Corona CAD is a breakthrough device because it is the only available technology to simultaneously offer the following benefits in one, rugged, cost-effective, easy-to-use instrument:

• Excellent sensitivity

• Wide dynamic range

• Superior reproducibility

• More consistent response

• Broad applicability

• Intuitive operation

Immunoassay

Antibodies detect the presence of certain molecules in a sample. Monoclonal antibodies (Mabs) bind very specifically to their targets at low concentrations. These Mabs must be tagged in some way to permit measurements. Sporadic news is available about this topic as many companies make products in this wide-ranging field. Enclosed are three from 2005.

BioArray Solutions, Ltd. announced FDA 510(k) clearance of a novel immunoassay for the simultaneous detection of six antibodies to different extractable nuclear antigens (ENA). The ENA IgG BeadChip Test System, for use on the company's Array Imaging System (AIS 400), utilizes BioArray Solutions' proprietary BeadChip format to simultaneously detect multiple analytes of interest on a tiny silicon chip holding a planar array of color encoded microparticles. The AIS 400 fully automates image acquisition and integrated analysis to rapidly generate assay results.¹⁷

Bayer HealthCare Diagnostics Division received approval from FDA for its automated HBsAg (hepatitis B surface antigen) and HBsAg Confirmatory assays.18 The fully automated tests, which are used to aid in the diagnosis and confirmation of acute or chronic hepatitis B infections, are available on the ADVIA Centaur Immunoassay System. The ADVIA Centaur HBsAg Assay is an in vitro diagnostic immunoassay for the qualitative detection of hepatitis B surface antigen (HBsAg) in human serum and plasma using the ADVIA Centaur system. The ADVIA Centaur HBsAg Confirmatory Assay is an in vitro diagnostic immunoassay for the qualitative confirmation of the presence of hepatitis B surface antigen (HBsAg) in human serum and plasma using the ADVIA Centaur system.

Procyon Biopharma Inc. entered into a licensing and distribution agreement with Medicorp Inc. granting the latter the exclusive worldwide rights to develop, manufacture and commercialize PSP94-based test kits for research purposes as well as the rights to sub-license for clinical diagnostic applications.¹⁹ PSP94 (Prostate Secretory Protein of 94 amino acids) is one of the three major proteins secreted in the seminal fluid, together with Prostate Specific Antigen (PSA) and Prostatic Acid Phosphatase (PAP). PSP94-based test kits measure the amount of free PSP94, bound PSP94 and PSP94 binding protein present in the blood, the relative ratios of which are believed to have utility in prostate cancer prognosis, diagnosis and monitoring. Recent studies to be presented at major conferences this year also indicate that PSP94-based test kits were able to predict patients suffering from a more aggressive disease. PSP94 was found to be a strong predictor of relapse post-radiotherapy as well as following radical prostatectomy.

Isoelectric Focusing

Isoelectric focusing (IEF) is a high-resolution, stand-alone technique that can be used as an analytical method or tool for protein purification. The only current book on the market, the Handbook of Isoelectric Focusing and Proteomics is a one-stop source for germane information in this discipline.²⁰

BioPharm International ran an application article in April 2005; which discussed a case study of developing analytical methods for an API.¹² One that was improved is IEF. The excerpt from this article follows:

"The first step we took in analytical support of the formulation development was to determine the protein's isoelectric point (pI) by gel IEF. The pI is the solution pH at which a protein carries no net charge. In solutions with moderate salt concentration, the solubility of most proteins is, at a minimum, near their pI. An electric field is applied to a gel matrix containing a pH gradient to separate proteins on the basis of their charge. IEF can be one of the most powerful separation methods for evaluating charge differences and can resolve a single charge difference between large protein molecules.

The first IEF test used materials purchased from Invitrogen because these were available in the laboratory. An API sample was desalted using a 5K molecular-weight-cut-off spin filter. The sample was diluted in pH 3-10 IEF sample buffer and then samples and pI markers were loaded on a pH 3-10 IEF gel. Three major, poorly resolved bands migrated between the pH 5.3 and 6.0 markers.

Because multiple bands were observed, a method for further characterization was required to ensure that formulation and process conditions were not affecting the pattern of charge variants. The IEF bands' sharpness and resolution were improved by using a larger gel on a flat-bed system. A recirculating chiller assured temperature control. Flat-bed IEF was performed using equipment and materials purchased from Amersham Biosciences. An Ampholine PAGEplate pH 3.5-9.5 gel was used, with 1M phosphoric acid as the anode buffer and 1M NaOH as the cathode buffer. After staining with Coomassie blue, the gels were placed on a white-light transilluminator and photographed with the Kodak EDAS 290 system.

Resolution was better. Six bands, which migrated between the pH 5.2 and 5.85 markers, were resolved. Because the protein was not glycosolated and had no disulfide bonds, the charge variants observed by IEF were the major forms of molecular heterogeneity requiring characterization. "

LC/MS/MS

Liquid chromatography with tandem mass spectrometry detection has the abbreviation LC/MS/MS when the detector is an atmospheric pressure ionization triple-quadropole mass spectrometer. This combination of well-known technologies has a long history. Advances are incremental.

The June 5-9, 2005 meeting of the American Society for Mass Spectrometry in San Antonio accepted three useful applications of this technology. Enclosed is a list of the titles and authors taken from the abstract, available at the ASMS web site. [http://www.asms.org/Default.aspx?tabid=43].

Zhang YL, Christians U, Simultaneous quantitation of Naysol and Bakuchiol, Two multiple biological activities compounds isolated from Chinese herbal medicine by semi-automated LC-MS/MS. University of Colorado Health Sciences Center, Denver CO.

Cohen M, Zhang YL, Galinkin J, Zuk J, Christians U. Quantitative determination of ketorolac in human plasma by HPLC/MS/MS. University of Colorado Health Sciences Center, Denver, CO; and The Children's Hospital of Denver, Denver, CO;

Christianson CD, Needham SR. Development of a novel HILIC HPLC/MS/MS bioanalytical method for the quantitative analysis of carboplatin from the plasma of mouse. Alturas Analytics, Inc., Moscow ID.

Limulus Amebocyte Lysate (LAL)

NASA does not want to send endotoxins into space. NASA uses a variety of methods to measure, control and reduce spacecraft microbial contamination for planetary protection purposes.²¹ Assembly of spacecraft hardware is carefully controlled and often takes place in clean-room facilities using, aseptic techniques in order to meet planetary protection requirements. Dry heat microbial reduction techniques first used on the Viking spacecraft are still used today. Measurement techniques are cultivation-based microbial assays using well-characterized biological methods.

The Limulus Amebocyte Lysate (LAL) assay method currently under development tests for the presence of microbial cell wall materials. The method is based upon an enzyme cascade, isolated from the blood cells, or amebocytes, of the primitive horseshoe crab, Limulus polyphemus, which is part of its anti-microbial defense mechanisms. The method detects lipopolysaccharide (endotoxins) and beta glucan from Gram-negative bacteria, yeast and mold, which is a general indicator of the bioburden present on space-bound hardware.

The LAL method was originally developed for use in the pharmaceutical industry, where the presence of endotoxin in injectable drugs and medical devices is known to cause fever in patients. Its application under development for planetary protection has been adapted to the use of ultra-clean polyester swabs to sample spacecraft surfaces. Microbial material is extracted from the swab with water and quantified using either a portable instrument or laboratory-based microplate reader. The method has several strong points; it is exquisitely sensitive (10-12 g lipopolysaccharide), rapid (15-30 min), and reactive with both live organisms as well as organic material from dead or non-cultivable organisms. Research is continuing that would further increase the breadth of microbes reactive to the assay. This would further improve its usefulness to assess microbial bioburden and organic cleanliness of spacecraft.

Microbial Testing

Electronic gadgetry speeds up microbial testing. While bioburden still has to be grown in culture media, colorimetric methods make analysis easier. The BacT/ALERT 3D is a system patented by bioMérieux that demonstrates excellent recovery of a wide range of organisms with greater than 95 percent recovery within 24 hours and greater than 98 percent within 72 hours. It must use a matching culture media to detect aerobic microorganisms and high-acid-producing organisms such as lactobacillus, yeasts, and molds.²²

BacT/Alert 3D´s patented colorimetric sensor-and-detection technology

BacT/Alert 3D's patented colorimetric sensor-and-detection technology detects microorganism growth by tracking CO₂ production. Notification of positives is immediate and results are dependable.

Here's how it works.

1. Microorganisms multiply in the media, generating CO₂. As CO₂ increases, the sensor in the bottle turns yellow.

2. Measuring reflected light, the BacT/ALERT 3D monitors and detects color changes in the sensor.

3. Algorithms analyze the data to determine positivity, and the laboratory is notified immediately with visual and audible alarms.

4. Changes in the sensor are permanent and visible to the unaided eye, unlike any other method.

Near-Infrared Spectroscopy

The near-infrared region of the electromagnetic spectrum extends from 700 to 2,500 nm. This is an old technology and so the news from an Advanstar publication is market-oriented.²³

"Infrared and near-infrared (NIR) instruments represent about 27 percent of the molecular spectroscopy market, and both are experiencing moderate growth in the 5-6 percent range. Pharmaceutical and biotech applications represent growth opportunities, but food analysis, especially milk and grains, forensics, plastic recycling, and cellular analysis are the most important application areas. Color measurement instrument demand improved in 2004 and should grow at around 5 percent in 2005. Major industrial users such as paints, textiles, and printing have rebounded, while prospects in Asia, especially China, are burgeoning. "

NMR Spectroscopy

In a magnetic field, atomic nuclei with magnetic movements acquire a magnetization parallel to the field. A burst of energy at the right frequency, in the FM radio frequency range, can perturb that magnetization. This creates a small voltage than can be detected.

Improvements are rolling out from leading manufacturers of the instruments. There is steady improvement in the software and the magnets. Some machines now have superconducting magnets. One manufacturer claims a range of 4.7 to 14.1 Tesla is now commercial.²⁴

Probes get hot working in salty biological samples and damage the experiments. University of Illinois Urbana-Champlain and a partner company have teamed up to develop a scroll coil that runs cooler in salty samples than a solenoid coil.²⁵ A patent has been applied for. This company has also developed a cryogenically cooled probe that also solves the local overheating problem.²⁶

NMR has been applied to many research projects. Springer Netherlands has a specialty publication, Journal of Biomolecular NMR, edited by Nobel Medalist (Chemistry 2002) Kurt W_rtrich. Access it at www.springeronline.com.

Peptide Mapping

Proteins are selectively cleaved into fragments that are subsequently separated to create a characteristic pattern. As you can imagine, it takes a number of tools and computer programs to be successful. BioPharm International located a recent paper combining mass spec and powerful computer programs to discern the peptide sequence and quotes parts of it with permission.²⁷

Background

"Protein-protein, protein-DNA and protein-RNA interactions are of central importance in biological systems. Quadrapole Time-of-flight (Q-TOF) mass spectrometry is a sensitive, promising tool for studying these interactions. By combining this technique with chemical crosslinking, it is possible to identify the sites of interactions within these complexes. Due to the complexities of the mass spectrometric data of crosslinked proteins, new software is required to analyze the resulting products of these studies.

Result

We designed a Cross-Linked Peptide Mapping (CLPM) algorithm that takes advantage of all of the information available in the experiment including the amino acid sequence from each protein, the identity of the crosslinker, the identity of the digesting enzyme, the level of missed cleavage, and possible chemical modifications. The algorithm does in silico digestion and crosslinking, calculates all possible mass values and matches the theoretical data to the actual experimental data provided by the mass spectrometry analysis to identify the crosslinked peptides.

Conclusion

Identifying peptides by their masses can be an efficient starting point for direct sequence confirmation. The CLPM algorithm provides a powerful tool in identifying these potential interaction sites in combination with chemical crosslinking and mass spectrometry. Through this cost-effective approach, subsequent efforts can quickly focus attention on investigating these specific interaction sites."

Polymerase Chain Reaction

Polymerase chain reaction exponentially amplifies a specific region of DNA, making it easier to study by other analytical techniques. It is turning out to be a very good test method for disease. Two related articles have appeared in Advanstar publications.

A PCR-based assay of antenatal maternal serum can determine fetal Rh status in over 99 percent of cases with 100 percent accuracy, according to a recently published French study involving 285 pregnant women. Researchers were able to determine fetal RhD status in all but two cases. (In those two cases, the RhD-negative phenotype of the mother was not the result of a complete RHD gene deletion.) No false-positive or false-negative results occurred; all sera from women carrying an RhD-positive fetus had positive results for RHD gene detection, and all sera from women carrying an RhD-negative fetus had negative results.²⁸

Methylation-specific polymerase chain reaction (M-PCR) is a sensitive, specific, fast, and relatively inexpensive method of diagnosing Klinefelter syndrome, according to data presented by Cornell University researchers. M-PCR may serve as an adjunct to karyotype and Y chromosome microdeletion assay, both of which are widely recommended forms of screening for men with low sperm production.

Of equal importance, the M-PCR test using the FMR1 and XIST genes was found to be 100 percent sensitive and 100 percent specific for the diagnosis of Klinefelter syndrome. The investigators could detect a minimum of one percent of XX/XY mosaicism using methylated-to-unmethylated FMR1 PCR product ratio. Total material cost per test, excluding labor and overhead, was an inexpensive $5.49, with a turnaround time of 48 hours.²⁹

Surface Plasmon Resonance

SPR arises from propagation of electromagentic waves (plasmons) along the surface of a thin metal layer. Optical sensors pick up the reflection of these waves. Correct matching of the medium next to the metal and the angle of the incoming light goes into resonance and there is no reflection. That data is used for analysis.

To increase the sensitivity and specificity of the binding assays, particularly in the case of antibody binding, users can take advantage of an extension of SPR when surface plasmon fluorescence spectroscopy (SPFS) is applied simultaneously with SPR.³⁰

The electromagnetic field intensity at the gold surface is strongly related to the reflectivity of the system. Detecting the fluorescence intensity of the labeled molecule in addition to the SPS reflectivity, improves the sensitivity of our device by at least one order of magnitude. Adsorption of dye-labeled molecules on the metal film enhances the fluorescence emission.³⁰

Surface plasmons on gold substrates cannot be efficiently excited using a wavelength lower than 500nm due to the increasing absorption of the gold related to interband transitions. To obtain a well-defined surface plasmon mode with the corresponding electro-magnetic field enhancement an excitation wavelength above 600 nm should be chosen. This fact limits the number of suitable dyes to the long wavelength absorbing molecules.³⁰

According to our HeNe laser providing an excitation wavelength of 632.8nm, we had to choose an adequate label for the use dye. The CY5 dye, possessing an absorption maximum at 650nm and an emission maximum at 670nm, match in this configuration. This improved detection device allows for the simultaneous monitoring of the surface plasmon reflectivity and fluorescence emission.³⁰

Total Organic Carbon Analysis

The TOC analyzer is so elemental an instrument that it defies radical change. TOC of water is measured by oxidizing organic carbon to produce CO₂, which is measured. New developments are incremental and depend on the specifications for the water. A search of listings in www.globalspec.com showed 32 companies competing. Attacking it differently as a word search pulled up 1,455 entries; but only 14 were directly related.

Ultracentrifugation

An analytical centrifuge is essentially s preparative centrifuge with an optical system for measuring the distribution of molecules inside while the rotor is spinning. Computer programs that interpret the data are the newest development. Peter Schuck of the National Institutes of Health is the webmaster of a homepage for SEDFIT, software for the analysis of analytical ultracentrifugation and other hydrodynamic data, which was written at NIH and is distributed without charge for research use. The program can be downloaded from the home page, www.analyticalultracentrifugation.com/default.htm. (It is also available directly from NIH.) SEDFIT is closely related to SEDPHAT, which provides global modeling capability for both sedimentation equilibrium and sedimentation velocity data. It also can serve as a platform for isotherm analyses.

A general introduction to the study of protein interactions by analytical ultracentrifugation can be found at the website of the PBR/DBEPS at NIH. This includes an introduction to the general principles of AUC and detailed experimental protocols. A general introduction of analytical ultracentrifugation can be found in Protein Science 2002; 11:2067-2079. A workshop on hydrodynamic and thermodynamic sedimentation analysis with SEDFIT and SEDPHAT at NIH in Bethesda is scheduled to take place September 20-22, 2005.

UV-VIS Spectroscopy

A research-grade scanning spectrometer measures the intensity of transmitted light in a narrow bandpass, and scans the wavelength over time in order to collect a spectrum. Because absorption is a ratiometric measurement, these instruments generally require the user to measure two spectra, one sample and one blank. The blank should be identical to the sample in every way except that the absorbing species of interest is not present. This can be done either consecutively with a single-beam instrument followed by a ratio calculation, or simultaneously with a dual-beam instrument. The dual-beam method is faster and has the added advantage that lamp-drift and other slow-intensity fluctuations are properly accounted for in the ratio calculation. Collecting spectra with scanning spectrometers is slow, but the instruments often have very high resolving power owing to the use of photo multiplier tube detectors, which can be used with very narrow slit widths.³¹

A photodiode spectrometer houses a one- or two-dimensional stack of individual photodiode detectors, each of which makes an independent measurement of the incident light intensity at its particular location. Typically, 2-D arrays are used for electronic imaging applications while the one-dimensional arrays are used in spectroscopic instrumentation. If the array is placed at the focal plane of a monochromator, then the position of each photodiode will be associated with a specific bandwidth of light. ³¹

A popular model (Agilent 8453) has 1,024 diodes in the linear array associated with a 1 nm band of light spanning from 900 nm to 1,000 nm. The individual diodes are separated by about 25 microns and the physical slit width matches this spacing. Thus the maximum resolution of the instrument is about 1 nm. This is not as good as the typical resolution of a scanning instrument (resolutions of about 0.2 nm are not uncommon with scanning instruments), but for many applications (especially molecular solution spectroscopy where adsorption bands are very broad) 1 nm is adequate. The real advantage of he instrument is its speed. A single spectrum can be taken in about 0.1 sec.³¹

Peter Silverberg is an associate editor with Biopharm International.

References

1. Montgomery SA, ed.The BioPharm Guide to Bioanalytical Methods. Supplement to BioPharm 2001 December.

2. Nanogen, Inc. Press release. Nanogen issued patents for biomarker discovery and nucleic acid manipulation Technologies. San Diego CA. 24 May 2005. Available at www.nanogen.com

3. Parallele BioSciences, Inc. Press release. ParAllele Announces Largest Ever Assay Panel of Non-Synonymous SNPs - Promises a High Pay Off When Used in Disease Association Studies. South San Francisco CA. 19 April 2005. Available at www.parallelebio.com.

4. Sigma-Aldrich. Press release. Sigma-Aldrich Launches Novel Functional p53 Protein Microarray. St. Louis MO. 21 Feb. 2005. Available at www.sigma-aldrich.com/p53

5. NATAS web site: http://www.wku.edu/natas/Natas2005.htm.

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11. FEI Company. FEI introduces hardware and software upgrades for Technai G2 TEM. Press release 1 August 2005. Hillsboro OR. Available at: www.feicompany.com.

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13. Guttman C. FISH assay may be useful monitor of TCC recurrence. Urology Times 2002 Dec.

14. Abbott Laboratories. FDA approves first gene-based test, UroVysion, for diagnosis of patients suspected of having bladder cancer. Press release 25 January 2005. Abbott Park IL. Available at: www.vysis.com.

15. Applied Imaging Corp. Applied Imaging announces FDA clearance to market breast cancer assessment test. Press release. 10 May 2005. San Jose CA. Available at: www.aicorp.com.

16. ESA Biosciences Inc., ESA Corona CAD wins 2005 R&D award. Press release. 5 July 2005. Chelmsford MA.

17. BioArray Solutions, Ltd. New bead array technology test system from BioArray Solutions receives FDA market clearance. Press release. 15 June 2005. Warren NJ. Available at: www.bioarrays.com/.

18. Bayer HealthCare, Diagnostics Division. Bayer Diagnostics receives FDA approval for automated hepatitis B assays; completes acute hepatitis panel on the Advia Centaur immunoassay system. Press release. 6 June 2005. Tarrytown NY. Available at: www.bayerdiag.com/.

19. Procyon Biopharma Inc. Procyon and Medicorp enter into a licensing and distribution agreement for the commercialization of PSP94-based test kits for prostate cancer. 2 March 2005. Montreal, Que. Available at: www.procyonbiopharma.com/.

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21. National Administration for Space and Aeronautics. planetaryprotection.nasa.gov/pp/methods/

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24. Varian, Inc., Varian unveils new magnetic resonance imaging system. Press release. 9 May 2005. Palo Alto CA. Available at: www.varianinc.com.

25. Varian, Inc. Varian, Inc. announces unique probe technology breakthrough for biological solids NMR 6 April 2005. Palo Alto CA. Available at www.varianinc.com.

26. Varian, Inc., Varian, Inc. introduces the world's first salt toleranet cold probe for biological NMR and metabolics studies. 12 April 2005. Palo Alto CA. Available at www.varianinc.com.

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