IVD Technology Magazine
IVDT Article Index

Originally published January, 1998

Development applications for membrane-bottom microwell plates

Asha A. Oroskar

More than just solid supports, membranes combined with microwell plates have a wide range of applications.

In the 30 years since DNA binding to nitrocellulose membranes was first discovered, membranes have become an integral component of DNA-based assays in both medical diagnostics and forensic laboratory tests. In addition to solid-phase DNA analysis, the Western blots named for the analysis of electrophoretically transferred proteins to nitrocellulose have led to an explosion of membrane-based immunoassays.

Membrane Composition

The manifold varieties of membranes available in the market offer tremendous versatility. Membranes may be used as prefilters, as exemplified by borosilicate glass filters; for general biological sample filtration, as seen with cellulose acetate filters; or for high protein retention and DNA and RNA binding, as seen with nitrocellulose membranes.

Nylon membranes are inherently hydrophilic and work well with aqueous-based samples, but should not be used when maximum protein recovery is the goal. Polysulfone and poly-vinylidene fluoride (PVDF) membranes bind very little pro-tein, exhibit good flow rates, and are chosen depending on the need for solvent resistance. PVDF is highly resistant to most solvents, but polysulfone is generally used for aqueous-based biological samples. Polytetrafluoroethylene (PTFE) membranes are hydrophobic and highly resistant to solvents, acids, alkalides, and propellants. Finally, ultrafiltration membranes (molecular-weight cut-off filters) made up of either cellulose triacetate or polysulfone membranes are generally used for desalting, sample concentration, and deproteinization as well as buffer exchange.^1—4

Membranes offer not only a quick and simple medium for solid-phase fractionation of components but also the ability to easily detect the end products retained on them. These features have enabled the use of membranes as versatile solid supports in cell-based assays, immunoassays, and DNA-based assays, making membrane-based assays one of the fastest- growing classes of diagnostic tests.

In addition to their inherent physicochemical characteristics, membranes are being exploited for their active surface characteristics. The latter may be manipulated either before or after the manufacturing of the membranes.

Realizing the chemical capabilities of membranes, device manufacturers were quick to respond to the demands of assay development by placing them as the bottoms of the traditional plastic 96-well plates. The utility of membrane-bottom plates as versatile formats for assay and process development has in fact been further enhanced by integration with existing systems for liquid handling and detection in the microwell format.

Membrane synthesis and utilization is based on the assumption that a membrane is more than a simple solid support. Indeed, the choice of membrane should be dictated by its final utility to the assay system.

Depth Filters and Screen Filters

Membranes come in a variety of physical and surface chemistries. Simply speaking, they fall into two basic categories: depth filters and screen or microporous filters.^1—4 Photomicrographs of depth filters shown demonstrate their tortuous and complex flow path (see Figure 1).² The structure of the depth filter consists of a matrix of randomly oriented fibers bonded together to form complex channels. This feature provides a high loading capacity.

Figure 1. Photomicrograph showing the complex flow path of a depth filter. Photo Courtesy of Millipore Corp.

The obvious uses of a depth filter are as a prefiltration matrix to extend the life of a membrane filter, and in processes requiring high flow rates and retention of large particulate, such as waste water analysis and receptor assays. The depth filter's disadvantages include the inability to define pore sizes and the retention of large quantities of liquid, a serious impediment to its use with precious fluids.

The screen filter, or microporous membrane filter, as seen in Figure 2, is designed to retain particles on its surface, like a sieve.² The screen filter has a rigid surface with a precisely controlled pore size.

Figure 2. Photomicrograph of particles and bacteria collected on the surface of a microporous membrane (screen filter). Photo Courtesy of Millipore Corp.

The advantages of the screen or microporous membrane filter are low liquid retention and predetermined pore size. It is ideally suited to quantitative retention of particles in such applications as fluid sterilization, fluid clarification, monitoring of air particles, and bioassays. Obviously, low loading capacities lead to rapid clogging of screen filters in high-particle situations. The best functionality of filters is achievable by combining the characteristics of both the depth and screen filter. A depth filter is best used as a prefilter and the screen filter as a final filter.

Filter Selection

The choice of membrane type should be based on those physical and surface characteristics of the membrane that can directly affect assay performance variables such as sensitivity and reliability. Factors such as retention capabilities, hydrophobic or covalent interactions with the membrane surface, and signal-to-noise ratio of the end point analyzed, are highly dependent on the type of membrane.^1—5

A clear understanding of membrane characteristics therefore is essential to the identification of the right filter for biological assays and process development protocols. The membrane of choice should meet the following criteria:

• Ability to bind the component of choice, leading to high specificity.

• Ability to retain the immobilized molecule in a biologically active state.

• Detection mode compatible with signal-to-noise ratios, leading to high sensitivity.

• High tensile strength to withstand transfer conditions, both physical and chemical.

Biomolecule immobilization optimally occurs under clearly defined conditions of binding, either with hydrophobic or covalent interactions or electrostatic attraction. Depending on pore size, nitrocellulose membranes can bind anywhere from 80 to 150 µg of protein per square centimeter² of membrane.⁴ Variables determined by the mode of biomolecule transfer—such as ionic strength, pH, and high currents used in electrophoretic transfer—affect the degree of sample retention.

High specificity of signal can be achieved only with membranes that allow few if any nonspecific interactions with the active surface. Blocking agents that make the active sites unavailable to further interactions generally increase the signal-to-noise ratio of the analyzed end point.⁵

Detection protocols developed to date include radioisotopic, chromogenic, chemiluminescent, and fluorogenic modes on membrane filters. Nitrocellulose membranes work well with all detection modes except fluorescence.⁴

Nylon membranes such as Nytran (Schleicher & Schuell, Keene, NH) and Biodyne membranes (Pall Gelman Sciences, Port Washington, NY) are generally superior to nitrocellulose membranes in durability and in the nonradioactive (chemiluminescent or fluorescent) detection of DNA, RNA, and proteins.¹ Biodyne nylon membranes are available with four types of chemically integral surfaces, providing specific adsorption parameters. Each of the four types of nylon membranes has distinct chemical groups, and the groups that correspond to differences in surface charge and chemical behavior.

Specific Biodyne membranes perform better in certain detection methods. The Biodyne B membrane is the membrane of choice for radioactive detection. The Biodyne A and the Biodyne Plus membranes provide optimal results with nonradioactive detection systems. The FBI laboratories use the Biodyne A membrane for the restriction fragment length polymorphism (RFLP) profile of human genomic loci from blood stains.⁶ This chemiluminescent detection protocol is in fact the gold standard for forensic laboratories evaluating RFLP profiles.

The Membrane-Bottom Plate

The physicochemical properties, versatility, and proven success of membranes as solid supports with chemically active groups made them obvious candidates for integration into existing high-throughput plastic microwell formats. The integration of membranes into microwell plates yielded membrane-bottom plates.

Membrane integration into the microwell format extended the possible applications of membranes by making possible their use with preexisting accessories designed for microwell formats, such as liquid handling, pipetting operations with existing instrumentation, and detection in commercially available radioisotopic, colorimetric, and chemiluminescence plate readers. Assay development in such microwell formats thus offers a versatile tool for high-throughput sample processing on membranes. Exemption from regulatory approval, or a Class I exempt registration, has been granted in the United States for the membrane-bottom plate used in at least one clinical diagnostic test.

Following is a discussion of the use of currently available 96-well membrane-bound microplates in biological assay and process development systems. Also included is an evaluation of the properties of some of the other available membranes, projecting their potential applications into newer product formats, such as 96-well membrane-bottom microplates.

Solid-phase assay systems have the advantages of target immobilization localized in sufficiently high concentrations, fractionation of components without centrifugation, and ease of handling end products on a manageable matrix. Already representing a sizable proportion of the laboratory and diagnostic test market, immunoassays are also moving increasingly into nonmedical applications, including environmental sample analysis, agricultural assays, and food safety testing. Immunoassays are being employed in drug discovery as well, especially in the elucidation of the possible pathways of biological function. Examples of a few of these assays are explored below.

Cell Proliferation Assays. Silent Monitor plates (previously manufactured and sold by Pall Gelman Sciences but now available from Nalge Nunc International, Rochester, NY) have been shown to support the growth of the eukaryotic Dictyostelium cells.⁷ Membrane-cultured Dictyostelium cells are easily processed for a colony-blot hybridization to detect specific RNA transcripts.

MultiScreen HV plates (Millipore Corp., Bedford, MA) also support eukaryotic cell growth. In experiments measuring tritiated thymidine uptake in the MultiScreen HV plates, HB-124 cells (a murine hybridoma cell line, ATCC, Rockville, MD) were easily cultured and processed for subsequent liquid scintillation counting. Figure 3 shows a typical increase in DNA synthesis measured by thymidine uptake.⁸ The micro-porous membranes thus effectively allow nutrients to be available for cell growth on the solid support.

Figure 3. Cell culture and processing with the MultiScreen system. (A) Thymidine uptake versus time in culture for glass-fiber disk and MultiScreen membrane production of cells.. HB-124 cells, in standard culture media containing tritiated thymidine, were added to individual wells (1 x 10⁵ cells/well) of sterile MultiScreen HV plates, and the plates were incubated at 37°C in a 5% carbon dioxide atmosphere. Nonspecific binding controls were prepared by adding tritium-labeled thymidine to wells containing media without cells. The total volume per well was 0.2 ml; 0.2 ml of ultrapure Milli-Q system water was added to unused wells of the plate. At indicated times, the MultiScreen plates were removed from the incubator and processed. The results show a typical increase in DNA synthesis with time measured by thymidine uptake. (B) Thymidine uptake effect of stimulation by serum. HB-124 cells were added to individual wells (5 x 10⁴ cells/well) of a sterile MultiScreen-HV plate and incubated at 37°C in a 5% carbon dioxide atmosphere for four days to deplete the medium of nutrients. On day 4, 50 µl of 10 different dilutions (0.01—8%) of bovine calf serum were added, and the incubation continued overnight. At the end of serum stimulation, tritiated thymidine (concentration: 0.05 µCi/well) was added and incubated for two hours. The plates were read following processing. The results demonstrate that following nutrient depletion of HB-124, cells are stimulated by as little as 0.1—0.5% serum in a dose-response fashion. Courtesy of Millipore Corp.

Cell-Based Receptor-Binding Assays. Receptor-binding assays require extensive incubation, washing, and quantitative collection of filtrates and filters. The hydrophilic microporous Durapore PVDF membranes in MultiScreen formats are used in several cell-based receptor-binding assays. In this application, the PVDF filters allow for the separation of bound labeled ligand from free labeled ligand.

Figure 4 shows the inhibition of 125I-angiotensin II binding with a prototypical experimental drug in adrenal gland preparations.⁹ 125I-labeled angiotensin was added to adrenal membranes in MultiScreen-HV 0.45-µm plates and incubated for 90 minutes at room temperature. Nonspecific binding was measured in the presence of 125I-angiotensin II, where antagonists were also present. This rapid screening system has been standardized for routine determinations of the binding affinities of newly developed chemical compounds.⁹

Figure 4. Cell-based receptor-binding assay. 125-I angiotensin II was added to adrenal membranes in MultiScreen HV (0.45 µm) plates and incubated 90 minutes at room temperature. Each 250-µl incubate consisted of the following (final concentration): 50 mM Tris, 120 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol, 0.05% bovine serum albumin, 0.1 nM 125I-angiotensin II and 8—15 µg adrenal membrane protein. Antagonists were added in concentrations from 10 nM to 100 µM. Nonspecific binding was measured in the presence of 0.1 µM SAR1, Ile8-angiotensin II. Binding was terminated by applying vacuum to filter plates. Receptor-ligand complex trapped on filters was washed three times with 300 µl ice-cold wash solution (50 mM Tris, 120 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol). Filter disks were dried, punched out, and counted in gamma counter (52% efficiency). Courtesy of Millipore Corp.

Enzyme Assays. Membrane-bottom microwell plates facilitate exploitation of novel matrices, like beads, in traditional immunoassays. The surface area for ligand binding is greater with beads than with simple coating of the wells of a traditional microwell plate. The coated immunosorbent beads may provide less steric hindrance and therefore improved sensitivity of the immobilized antibody.

This principle is evident in a report published by Orthner and colleagues of the American Red Cross (Rockville, MD).¹⁰ Their assay was designed to measure enzymatically active activated protein C (APC) in plasma samples using MultiScreen Durapore type DV membranes. APC is a serine protease and a naturally occurring antithrombotic enzyme. It functions as an anticoagulant by proteolytically inactivating coagulation factors VIIIa and Va. APC bound to a monoclonal antibody on immunosorbent beads was analyzed following its elution through Durapore MultiScreen plates. The immunosorbent bead protocol proved to yield a much more sensitive assay than did the monoclonal antibody-coated microwell system.

Immunosorbent resins may be used in the creation of microcolumns in 96-well membrane-bottom microwell plates. With the increase in surface area, the beads very likely favored an increased diffusion of reactants, such as APC to antibody-coated porous beads, resulting in improved assay sensitivity. Similarly, minicolumns have also been used with Alumina to quantify c-AMP phosphodiesterases, and with Sephadex G-50 for the purification of DNA.^11,12

Nylon membranes in the Silent Screen plates available from Nalge Nunc International (and previously in the Silent Monitor plates) have proved to be excellent supports for immunoassays in the rapid diagnosis of influenza A infection.¹³

Efficient Filtration and Quantitative Filtrate Collection. Federal law mandates that neonates be tested for phenylketonuria and galactosemia, disorders that result in the infant's inability to produce enzymes necessary in the breakdown of certain food products. The bacterial inhibition assay and a fluorometric assay are currently available to test for these enzyme deficiencies.¹⁴ Infant blood samples collected on a Guthrie card (Schleicher & Schuell) are punched out and tested for the levels of phenylalanine by the bacterial inhibition assay described by Guthrie and Susi.¹⁴ This test provides only a semiquantitative reading, is subject to interference by antibiotics, and lacks automatability. Several fluorometric tests have now been developed to replace it.

Hoffman and associates pioneered the use of fluorometric detection of the enzyme digestion end point in the 1980s.¹⁵ Both the Shield Diagnostics Quantase system (available in the United States and Canada from Wallac, Inc., Gaithersburg, MD) and the AP1300 System from Astoria Pacific Corp. (formerly Alpkem, Inc., Clackamas, OR) were developed for automated fluorometric assays for phenylalanine, galactose, and other mandated tests. The fluorometric assay system, however, is very sensitive to blood debris and contaminating fibers from the Guthrie cards. Filtration through membrane-bottom plates clears the reactants of filter fibers or blood debris, enabling a clear filtrate to be read fluorometrically.¹⁶ The Millipore Durapore plate made with PVDF filters is used with both commercially available fluorometric systems. Quantase system kits include MultiScreen DV plates, which are registered in the United States as Class I exempt devices specifically for PKU and galactose screening.

Purification of PCR Products. Purification or fractionation of large-molecular-weight DNA, such as PCR products, from oligonucleotides and unincorporated nucleotides is easily accomplished by column chromatography through resins such as Sephacryl-500HR (Pharmacia LKB, Uppsala, Sweden). Such a procedure may be easily adapted into high-throughput 96-mini-spin-columns by packing the prewetted Sephacryl-500HR filtration matrix into either the Silent Monitor (Nalge Nunc International) or the MultiScreen filtration plate (Millipore Corp.).¹⁷

Packing of the column in each well is achieved through centrifugation of the resin-loaded membrane-bottom plate to achieve a column height of about three-quarters of the depth of the well. PCR products are applied to the center of each column with a multichannel pipette, then centrifuged using a microplate rotor. Both the flow-through material and the material eluted with buffer contain pure PCR products. In the 96-well membrane-bottom plate, therefore, column chromatography of single samples evolved into spin-column chromatography of 96 samples in parallel.

High-Throughput Preparation of Yeast Artificial Chromosome DNA. The yeast artificial chromosome (YAC) is an important vector for cloning large pieces of DNA. With DNA fragments as long as 250 to 300 kilobases, YAC grown in a microwell plate system does not reach optimal density levels. DNA preparation from YAC cells is laborious and time consuming.

The use of MultiScreen plates allows high cell densities and simplifies DNA extraction from YAC cells.^18,19 As illustrated in Figure 5, the MultiScreen 96-well filter-bottom plate is first embedded in solid growth media poured into the lid of a 96-tip yellow-tip rack. With additional medium available through dialysis of nutrients by the membrane, YAC cell densities of much greater than 10⁷ cells per well are achieved. YAC DNA extraction is accomplished through the zymolase treatment of the YAC cells embedded in agarose plugs in the MultiScreen plate followed by dialysis of the DNA through the micro-porous membrane of the MultiScreen plate.

Figure 5. High-throughput preparation of yeast artificial chromosome (YAC) DNA using MultiScreen filtration plates. (A). Growing YACs to high density (growth plate). The MultiScreen filter-bottom plates are embedded in solid culture media so that the yeast, while isolated in wells, is able to take advantage of more medium by dialysis of nutrients through membrane. This technique produces 10 times the cells from each clone, for about 3 µg of DNA. (B). Extracting DNA (dialysis plate). This method is a modification of a standard protocol using agarose blocks for DNA preparation for pulsed-field gel electrophoresis. The blocks are formed within the wells of the MultiScreen filtration plates. Dialysis through the membrane bottom allows DNA preparation without manipulation of the blocks. For high throughput, several plates may be si-multaneously dialyzed in a simple "flooding" chamber. Courtesy of Millipore Corp.

High-Throughput Synthesis of Drugs. Membrane-bottom plates are now being used in the process development of 96-well automated synthesis of small-molecule drug libraries. Figure 6 demonstrates the setup of the Protogene polypropyl-ene filter-bottom plate with a polypropylene filter (Protogene Laboratories, Inc., Palo Alto, CA) used in the synthesis of 96 different chemical libraries for drug discovery.²⁰ The polypro-pylene filter-bottom microwell plate may be substituted for assays where resin usage is combined with the filtration mode, as for nucleic acid purification, desalting, or ion-exchange chromatography. The option of substituting a variety of filters allows a more versatile adaptation of the Protogene filter plate to both assay and process development. Indeed, other commercially available and user-fabricated membrane-bottom plates are also being used as reaction vessels in nucleic acid extraction and purification, as well as in the parallel synthesis of 96 different chemical libraries per unit.

Figure 6. The Protogene Laboratories, Inc., filter-bottom plate, a V-bottom polypropylene plate. (A) Each well ends in a capillary exit designed to prevent any cross-talk. The format allows use of the plate as a filter, a spin-column support, or an active membrane support in a 96-well format. (B) The plate is a reaction chamber in the 96-well parallel automated synthesis of small-molecule drug-library by combinatorial chemistry protocols.

Future Developments

In addition to the membrane types currently available in microwell plates, functionally specific membranes developed for future products may be useful for high-throughput sample preparation for DNA diagnostics. Huang and associates have published a report of DNA preparation in a high-throughput format in the Silent Monitor plates (now available as Silent Screen plates).²¹ In plasmid and cosmid DNA preparation in 96-well deep-well plates, the cell debris from lysed bacterial cells is mixed with a polyelectrolyte protein-precipitating reagent (Affinity Technologies, Inc., Fairfield, NJ) and processed for DNA purification by filtration through the Biodyne membranes of Silent Screen plates. Furthermore, there is an acute need for DNA sample preparation before blood is accepted for genotyping and before screening tests are carried out for infectious agents in donated blood.

Leukosorb medium, a fibrous material that selectively binds leukocytes (see Figure 7), is used in Pall leukocyte reduction filters (Pall Gelman Sciences). The leukocyte reduction filters were developed with the Leukosorb matrix for the depletion of leukocytes from transfusion products. The Leukosorb membrane in a 96-well membrane-bottom format is suitable for use in sample preparation or diagnostic tests on leukocytes. Such tests may include genotyping of the leukocytic DNA and identification of intracellular viruses harbored in the leukocytes retained by the Leukosorb medium.

Figure 7. Leukosorb medium is a white, highly wettable, fibrous matrix designed for use in procedures requiring separation of leukocytes from clinical fluids such as blood. Photo Courtesy of Pall Gelman Sciences.

The Hemasep V membrane (Pall Gelman Sciences) provides a noncentrifugal method of sample preparation.²² In this method, plasma is vertically separated from small quantities of whole blood. Thus extracellular viruses, such as hepatitis viruses and human immunodeficiency viruses, could theoretically be identified from the plasma of the blood separated on the membrane.

The Immunodyne ABC membrane (Pall Gelman Sciences) also has potential applications in 96- or higher multiple-well formats. A chemically activated nylon 6,6 membrane, it covalently binds proteins or amino-terminated oligonucleotides. Covalently bound molecules are especially useful in biosensor applications.

Conclusion

Although membrane-bottom plates have been around for just over a decade, they are now used as much more than merely solid supports for reactants. Membranes offer a higher surface area than traditional plastic surfaces. Their porosity increases their versatility. Surface-modification capabilities for reactive groups and charges on membranes allow ionic, hydrophobic, or covalent binding with target molecules. Membranes that are chemically resistant to strong organic solvents enable high-throughput organic synthesis of chemicals.

The inclusion of membranes in 96-well membrane-bottom formats has combined all the advantages mentioned above with all the capabilities of 96-well formats. The latter include use in robotic workstations, with modularization and automated liquid handling and assay end point reading. Future developments include a move beyond 96-well formats into 384- or higher multiple-well formats for high-throughput manipulation.

References

1. "Applications Guide for Pall Membranes," Port Washington, NY, Pall Gelman BioSupport Div., 1996.

2. "Millipore Corporation Catalog," Bedford, MA, Millipore, Corp., 1997.

3. "Alltech Bioscience Lab Resources," 1st ed, Deerfield, IL, Alltech Bioscience, 1997.

4. "Products for Life Science Research," Keene, NH, Schleicher & Schuell, 1995—1996.

5. Dubitsky A, "Blocking Strategies for Nylon Membranes Used in Enzyme-Linked Immunosorbent Assays," IVD Technol, 3(4):53—59, 1997.

6. Guisti AM, and Budowle B, "A Chemiluminescent-Based Detection System for Human DNA Quantitation and Restriction Length Polymorphism (RFLP) Analysis,"Appl Theoret Electrophoresis, 58:89—98, 1995.

7. Maniak M, Saur U, and Nellen W, "A Colony-Blot Technique for the Detection of Specific Transcripts in Eukaryotes," Anal Biochem, 176:78—81, 1989.

8. "MultiScreen Methods: Thymidine Uptake Assays Using the MultiScreen System," lit #TB038, Bedford, MA, Millipore Corp., 1994.

9. "MultiScreen Methods: Cell-Based Receptor Binding Assays Performed with the MultiScreen Assay System," lit #MM011, Bedford, MA, Millipore Corp., 1996.

10. Orthner CL, Kolen B, and Drohan WN, "A Sensitive and Facile Assay for the Measurement of Activated Protein C Activity Levels in Vivo," Thromb Haemost, 69(5):441—447, 1993.

11. Daniels DV, and Alvarez R, "A Semiautomated Method for the Assay of Cyclic Adenosine 5'-Monophosphate Phosphodiesterase," Anal Biochem, 236:367—369, 1996.

12. "MultiScreen Methods," Bedford, MA, Millipore Corp., in press.

13. Duverlie G, et al., "A Nylon Membrane Enzyme Immunoassay for Rapid Diagnosis of Influenza A Infection," J Virol Methods, 40:77—84, 1992.

14. Guthrie R, and Susi A, "A Simple Phenylalanine Method for Detecting Phenylketonuria in Large Populations of Newborn Infants," Pediatrics, 32:328, 1963.

15. Hoffman GL, Laessig RH, Hassemer DJ, et al., "Dual-Channel Flow System for Determination of Phenylalanine and Galactose Application to Newborn Screening," Clin Chem, 30(2):287—290, 1984.

16. Elvers LH, Diependaal GAM, Blonk HJ, et al., "Phenylketonuria Screening Using the Quantase Phenylalanine Kit Combination with a Microfilter System and the Dye Tartrazine," Screening, 3(4):209—223, 1995.

17. Wang K, Gan L, Boysen C, et al., "A Microtiter Plate-Based High-Throughput DNA Purification Method," Anal Biochem, 226:85—90, 1995.

18. MacMurray AJ, Weaver A, Shin H-S, et al., "An Automated Method for DNA Preparation from Thousands of YAC Clones," Nucleic Acids Res, 19(2):385—390, 1991.

19. "MultiScreen Filtration System: High-Throughput Preparation of YAC DNA Using MultiScreen Filtration Plates," lit #TB061, Bedford, MA, Millipore Corp., 1991.

20. Molinari RJ, "Solid-Phase Synthesis of Small Molecule Drug Libraries Using Second-Generation 96-Well Array Synthesizer," presented at the Second Annual Solid-Phase Synthesis Meeting, Coronado, CA, February 6—7, 1997.

21. Huang GM, Wang K, Kuo C, et al., "A High Throughput Plasmid DNA Preparation Method," Anal Biochem, 223:35—38, 1994.

22. Alter J, "Single-Step Vertical Plasma Separation of Whole Blood for Test and Sample Prep," Genet Engineer N, November 15, 1996.

Asha A. Oroskar, PhD, is president of Oros Technologies, Inc. (Oak Brook, IL), and head of industrial research at the Center for Biotechnology, Northwestern University (Evanston, IL).