Clinical Chemistry

Product Selection Guide

Sample Preparation, High Purity Water, Quality Assurance

More Than Meets the Eye

Membrane filter devices may look similar, but when you put them to the test, you can see some significant differences. Millipore filter devices are designed to provide the highest levels of quality and performance. They include the latest membrane filter technology and innovative device engineering. Take our Amicon Ultra Centrifugal Devices, for example. Their high recovery Ultracel membrane typically provides >90% protein retention, while the vertical membrane orientation speeds filtration times. No other device provides the same combination of recovery and speed. Of course, you can also count on Millipore to support our products. Our knowledgeable Technical Service Specialists are a phone call or email away. Plus, we offer numerous protocols to assist you in optimizing your separations.

Call your local office for additional information or visit www.millipore.com/techservice.

Selection Guide
Application Recommended Product(s) In-vitro Diagnostic Use CE Mark Page

Concentrating & Desalting Prior to Electrophoresis Bence-Jones proteins Amicon Ultra-4 Centrifugal Device (10K MWCO) Centricon Centrifugal Devices Minicon and Miniplus Concentrators Oligoclonal banding Centricon Centrifugal Devices Minicon and Miniplus Concentrators Deproteinization ELISpot Assay Amicon Ultra-4 Centrifugal Device (10K MWCO) MultiScreen Filter Plates

4 6 7 6 7

4 8 9

Separating Free from Bound Protein in Microsolute Centrifree Micropartition Device PCR Clean-up

HIV genotyping by DNA sequencing Montage PCR Filter Units Montage PCR96 Micro-well Filter Plate Montage SEQ Cleanup Kit Neonatal Screening PKU screening Clinical Analyzer Feedwater Microbiological QC Testing MultiScreen-HV Filter Plates AFS Water Systems Addi-Chek System HPC and Total Count Samplers and Swab Test Kits

11 11 11 13 14 14 14

SELECTION GUIDE 2

Concentrating and Desalting Prior to Electrophoresis

The measurement of specific proteins in urine is important for the diagnosis and management of disease states. In most cases, the content of these proteins in urine or cerebral spinal fluid is too low to be detected and needs to be concentrated. For example, patients with multiple myeloma exhibit a proliferation of one antibody-producing plasma cell that leads to excess production of free immunoglobulin light chains known as Bence-Jones proteins. After sample enrichment in the Amicon Ultra-4 device, immunofixation electrophoresis can be used to identify free light chains (Bence-Jones proteins) by forming a light chain-antibody complex. Also, agarose electrophoresis can be used to quantitate light chains and identify proteins such as albumin, 1 globulins, transferrin and IgG that can be present in renal tubular disorders. Millipore ultrafiltration devices provide a convenient way to rapidly concentrate physiological fluids.

There are three devices to choose from:

Amicon Ultra-4 Centrifugal Device A unique combination of high speed and high recovery (typically > 90%) with convenient direct pipettor access. Centricon Centrifugal Device Very high solute recovery (typically >95%) and a patented deadstop for reliable, reproducible concentrate volumes. Minicon and Miniplus Concentrators No pressure, vacuum, or centrifuge required. Just add the sample. Absorbent pads pull the solvent and microsolute through the ultrafilter to concentrate the sample.

References

1. Christenson, R.H., et al., Clinical Chemistry, 29(6), 1028-1030 (1983). 2. Christenson, R.H. and Russell, M.E., Clinical Chemistry, 31(6), 973 (1985). 3. Gubar, K.T., et al., Clinical Chemistry, 31(6), 973 (1985).

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Amicon Ultra-4 Centrifugal Filters

High speed High recovery High concentration factors In-vitro diagnostic use, CE mark (10K MWCO)

Now you can get fast spin times without sacrificing recovery. Only Amicon Ultra filters give you the ultra-high recoveries you expect from an Amicon device (typically >90%) with ultra-fast processing. The high-recovery Ultracel ultrafiltration membrane has broad chemical compatibility. And its sealed in the device using heatnot adhesives, which can result in downstream extractables. The vertical membrane design allows direct pipette sample access, which eliminates the need for a reverse-spin recovery step. The housing design is transparent for easy viewing of concentrate volumes. And its compatible with most rotor types.

The First Device to Combine High Speed and High Recovery

92% 80% 65%
Spin Conditions: 10K membrane with 0.25 mg/mL Cytochrome c. Starting Volumes: 4 mL Ultra and Brand V; 3.5 mL Brand P.

35 Mins.

Percent Retentate Recovery Spin Time

15 Mins.

10 Mins.

Brand V

Brand P

Amicon Ultra-4

C O N C E N T R AT I N G A N D D E SA LT I N G P R I O R T O E L E C T R O P H O R E S I S 4

Precision Analysis of CSF Sample

Amicon Ultra-4 Centrifugal Filters

Specifications
Amicon Ultra-4

(continued)

Amicon Ultra-15

Maximum Initial Sample Volume Swinging Bucket Fixed Angle 4 mL 4.0 mL (45 and 35 rotor angles) 3.5 mL (23 rotor angle) Typical Final Concentrate Volume Final Deadstop Volume
Analysis of 20 aliquots of a CSF sample that were identically diluted and then concentrated using an Amicon Ultra-4 device showed a mean albumin percentage and standard deviation of 68.8+1.3 and a coefficient of variance of 1.9%. Analysis of two samples that were not concentrated or diluted showed no statistical difference. (Data courtesy of Helena Laboratories.)

15 mL 12 mL (35 rotor angle)

50 L 20 L

200 L 50 L

Maximum Relative Centrifugal Force (x g) Swinging Bucket Fixed Angle 4000 7500* 4000 (3500 for concentrated or viscous solutions)* 5000

*Validated at 25 mg/mL concentrated solute.

Ordering Information
MWCO 8/pk 24/pk 96/pk

Precision Analysis of Urine Samples

Amicon Ultra-4 Centrifugal Devices 5K 10K* 30K 50K 100K UFC8 005 08 UFC8 010 08 UFC8 030 08 UFC8 050 08 UFC8 100 08 UFC8 005 24 UFC8 010 24 UFC8 030 24 UFC8 050 24 UFC8 100 24 UFC8 005 96 UFC8 010 96 UFC8 030 96 UFC8 050 96 UFC8 100 96

U39
1 2 3 4 1

U41
2 3 4

U42
1 2

Amicon Ultra-15 Centrifugal Devices 5K

U44
1 2 3 4 1

UFC9 005 08 UFC9 010 08 UFC9 030 08 UFC9 050 08 UFC9 100 08

UFC9 005 24 UFC9 010 24 UFC9 030 24 UFC9 050 24 UFC9 100 24

UFC9 005 96 UFC9 010 96 UFC9 030 96 UFC9 050 96 UFC9 100 96

U45
2 3 4

10K 30K 50K 100K

*In-vitro diagnostic use. Analysis of five groups of urine samples that were identically diluted and then concentrated using an Amicon Ultra-4 device showed a mean albumin percentage and standard deviation of 43.04+1.54. (Data courtesy of Helena Laboratories.)

For Further Information Urine Concentration, Protocol Note, PC1050EN00 Desalting and Buffer Exchange, Application Note, AN1050EN00

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Centricon Centrifugal Filters

Recommended for samples 2 mL High recoveries Patented deadstop volume

Centricon filters let you reproducibly concentrate and desalt small volumes (2 mL) of urine, CSF, and other clinical samples usually in less than 60 minutes. The devices feature the low adsorptive Amicon YM membrane for consistent recoveries (typically >95%). Fluid and low molecular weight materials pass into the filtrate cup while proteins are retained and concentrated in the sample reservoir. The driving force for filtration is provided by centrifugation in a fixed angle rotor at 1000 to 5000 x g. Recommended for Detection of Bence-Jones Proteins in CSF Centricon YM-10 devices are recommended for detection of Bence-Jones proteins in CSF, which is an indicator of multiple myeloma. After sample enrichment in the Centricon YM-10 device, immunofixation electrophoresis is used to identify free light chains (Bence Jones proteins) by forming a light chain-antibody complex. Agarose electrophoresis is then used to quantify the light chains and to identify proteins that can be present in tubular diseases such as albumin, 1 globulins, transferrin, and IgG.

Specifications Maximum Initial Sample Volume Typical Final Concentrate Volume Max. Centrifugal Force (YM-10 and 30) 2 mL 2550 L dependent on rotor angle 5,000 x g

Ordering Information
Description* NMWL Qty/Pk Catalogue No.

Centricon YM-10

10K

8 24 100 500 8 24 100 500

4241 4205 4206 4207 4242AM 4208 4209 4210

Centricon YM-30

30K

*Other cut-offs available. Contact Millipore for more information.

For Further Information Contact Millipore or visit www.millipore.com/publications Centricon Devices, Data Sheet, PF461EN00 CSF and Urine Concentration, Application Note, AN1000EN00 Desalting with Centricon, Application Note, AN001EN00

C O N C E N T R AT I N G A N D D E SA LT I N G P R I O R T O E L E C T R O P H O R E S I S 6

Minicon and Miniplus Concentrators

Concentrate up to 10 mL from 20x to 200x No other equipment required No cross-contamination between cells Graduation lines indicate concentration level

Static concentrators allow labs that dont have access to a centrifuge to concentrate clinical samples for analysis by electrophoresis or immunoelectrophoresis. Simply add sample to the cells, wait for the sample to be concentrated, and remove the enriched material. Absorbent pads pull the solvent and microsolute through the ultrafilter, concentrating the sample. A treated area near the bottom of each Minicon cell prevents filtration to dryness. The concentrators can be used to concentrate Bence-Jones and other proteins in urine, CSF, or other bodily fluids. They can also be used to enrich serum or plasma or to concentrate enzymes or isoenzymes. Typical performance is 100x concentration in 80 to 120 minutes (Minicon) and 200x concentration in 3 to 4 hours (Miniplus).
Specifications
Minicon B15 Minicon CS15 Minicon A25 Miniplus

NMWL Cell Capacity Cells/Unit Max Concentration Factor Min Recovery Volume Dimensions (mm) WxLxH

15K 5 mL 8 100x 50 L 45 x150 x100

15K 2.5 mL 10 80x 30 L 30 x140 x 70

25K 0.75 mL 8 20x 40 L 40 x110 x 50

10K 10 mL 4 200x 50 L 37x100 x120

Ordering Information
Description Tests/Pk Catalogue No.

Minicon B15 Minicon CS15 Minicon A25 Miniplus

Minicon Concentrators (left) have 8 or 10 cells. Miniplus Concentrators (right) have 4 cells.

(8 x 5) 40 (10 x 5) 50 (8 x 5) 40 (4 x 5) 20

9031 9051 9011 9061

For Further Information Contact Millipore or visit www.millipore.com/publications Minicon/Miniplus, Data Sheet, PF1234EN00

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ELISpot Assays
MultiScreen Assay Plates
High protein binding capacity with low background staining Reliable sensitivity from lot-to-lot. Tight CVs from plate-to-plate. Available with white plate material, which reduces optical cross-talk when used with imaging systems
1.

Millipore offers three MultiScreen plates designed for ELISpot assays. Each of these sterile, 96-well, filter-bottomed plates provides excellent spot recovery and spot definition. Choose from either styrene or acrylic plate materials. The acrylic is available in clear or white plastic, which reduces optical cross-talk when used with imaging systems. Protocol
2. 3. 4. 5.

1. 2. 3. 4. 5.

Coat membrane with antibodies. Add immune and target cells and incubate Responding cells produce cytokines Wash to remove cells. Add biotinylated antibodies Add avidin-enzyme conjugate Each responding cell produces one spot

Ordering Information
Description Membrane Plate Material (color) Sterility Qty/Pk Catalogue No.

MultiScreen-HA MultiScreen-HV

MF (MCE)

Styrene (clear)

Sterile Sterile Sterile

10 10 10

MAHA S45 10 MAIP S45 10 S2EM 004 M99

Immobilon Acrylic (PVDF) (clear) Acrylic (white)

Accessories Single-well membrane punch

MELI PUN CH

For Further Information Contact Millipore or visit www.millipore.com/publications IFN- ELISpot Assays, Technical Note, TN1075EN00

E L I S P O T A S S AY S 8

Separating Bound vs. Free Microsolute

Centrifree Micropartition Devices
Produces 100 L ultrafiltrate from 500 L serum in minutes Retains >99.9% serum protein 10 L or less hold-up volume Recommended for nearly all serum-protein-bound ligands

Separate free from protein-bound microsolute with a single centrifugal spin. Simply pipette 0.15 to 1 mL of serum, plasma, or other biological sample into the device, place it in the fixed angle rotor of your centrifuge, and spin at 1000 to 2000 x g for 5 to 10 minutes. Protein-bound microsolute and other macromolecules are retained by the 30,000 MWCO membrane. Free microsolute collects in a filtrate cup, ready for analysis or storage. No dilutions or specialized equipment are required. There are no changes in pH, ion composition, or unbound microsolute concentration.13 The devices have been shown to improve accuracy when measuring free ligand concentration, binding capacity, and affinity constants.35 Centrifree is ideal for therapeutic drug monitoring, as well as drug metabolism, free hormone, serotonin, amino acid, free ion and other analyses. In addition, you can conveniently process multiple samples under identical conditions with maximum efficiency. Fast Separation of Free from Bound Microsolute
300

200 Starting Volume 100

10

20

30

Centrifugation Time (minutes)

Typical ultrafiltration rates using serum spun in a 33 fixed-angle rotor at 1000 xg at 25 C.

9 S E PA R AT I N G B O U N D V S . F R E E M I C R O S O L U T E

Specifications
Maximum sample volume Minimum sample volume Hold-up volume Maximum centrifugal force Autoclave Rotor compatibility

1.0 mL 0.15 mL 0.01 mL 2000 x g The devices are NOT autoclavable Any fixed-angle rotor that accommodates 17100 mm tubes and is capable of 1000 2000 x g

Ordering Information
Description Qty/Pk Catalogue No.

Centrifree Micropartition Device

50

4104

For Further Information Contact Millipore or visit www.millipore.com/publications Centrifree Devices, Data Sheet, PF002EN00 Assay of Free Anticonvulsant Drugs, Application Note, AN1002EN00 Overview of Clinical Guidelines for Monitoring Free Drug Concentration, Application Note, AN1003EN00 Preparing Physiological Samples for Amino Acid Analysis, Application Note, AN100EN00 Centrifree On-line Bibliography, www.millipore.com/publications

References

1. Gilbert, G.A., and Jenkins, R.C., Proc. Roy. Soc. A., 253, 42037 (1959) 2. Paulus, H., Anal. Biochem. 32, 91100 (1969) 3. Sophianopoulos, J.A., et al., Arch. Biochem. Biophys., 187, 1329 (1978) 4. Geiseler, D. and Ritter, M., Clin. Chem., 30, 2832 (1984) 5. Siiteri, P., Science, 223, 191193 (1984)

S E P A R A T I N G B O U N D V S . F R E E M I C R O S O L U T E 10

HIV Genotyping
PCR and Sequencing Cleanup

No alcohol. No bind-wash-elute. No gels. Fast processing times >99% primer removal

11 H I V G E N O T Y P I N G

Millipore's unique size-exclusion technology eliminates the use of alcohol, gels, and bind-wash-elute steps. The result: purer DNA, longer read lengths, and higher pass rates. Single-sample PCR Cleanup Montage PCR Filter Units allow you to remove primers and unincorporated dNTPs with a single centrifugal spin. High Throughput PCR Cleanup The Montage PCR96 Micro-well Filter Plate is ideal for high throughput genotyping labs. The micro-well format allows recovery in small volumes, and studies show that recovery of PCR products is 2X greater than other size-exclusion filter plates.* Sequencing Reaction Cleanup Our unique, size-exclusion technology eliminates the need for costly gel filtration resins and bind-wash-elute columns. Montage SEQ cleanup kits increase the amount of useable data from each sequencing reaction by eliminating dye blobs and increasing sequencing read lengths. In addition, theyre optimized for BigDye Terminator and ET Terminator sequencing chemistries.

*Results from internal study. Visit www.millipore.com/montage to view results.

High Purity DNA for Genotyping

Sample A2 Genotype: G/G

Sample B1 Genotype: T/T

Sample D1 Genotype: G/T

A 301 bp product specific for the human triglyceride lipase gene (Accession Number M29189) was amplified and the resultant PCR products were purified using the Montage PCR96 plate. Sequencing reactions were cleaned-up using the Montage SEQ kit. Both heterozygote (D1) and homozygote (A2 and B1) examples are shown.

Ordering Information Other 96-well plates and kits are also available. Contact Millipore for additional information.
Description Qty/Pk Catalogue No.

Montage PCR Filter Units Montage PCR96 Filter Plates Montage SEQ Cleanup Kit

50 units 250 units 10 plates 50 plates 1 plate 4 plates 24 plates 1

UFC7 PCR 50 UFC7 PC2 50 LSKM PCR 10 LSKM PCR 50 LSKS 096 01 LSKS 096 04 LSKS 096 24 SAVM 384 01

Montage Vacuum Manifold*

*Manifold for use with Montage PCR96 and SEQ products. Montage PCR Filter Units are centrifugal filters.

For Further Information Visit www.millipore.com/montage and click on Genomics

H I V G E N O T Y P I N G 12

Neonatal Screening
MultiScreen-HV Plate for PKU Screening

High throughput 96-well filter plate format Underdrain prevents leaking during incubation No cross-contamination

The MultiScreen-HV filter plate has an individual Durapore membrane sealed at the bottom of each well to prevent cross-contamination. It also has an underdrain to prevent leakage during incubation. After incubation, eluates are quickly and easily transferred to a microtiter plate using vacuum filtration, which eliminates time-consuming and labor-intensive pipetting. In a published study, the MultiScreen-HV plate was shown to improve the sensitivity of a phenylalanine assay by allowing a larger part of the eluate of the bloodspots to be available to the enzyme reaction (78% as opposed to 40%).1 In addition, the authors note the elimination of a pipetting step, which diminishes the chance of cross-contamination, as well as the removal of fibers from the blotting paper that could interfere in the assay.

Ordering Information
Plate Material (color)

Description

Membrane

Sterility

Qty/Pk

Catalogue

MultiScreen-HV

Durapore (PVDF)

Styrene (clear)

Non-sterile

50

MAHV N45 50

See the back cover for information about syringe filter units for HPLC sample preparation.

References

1 Elvers, L.H., et al, "Phenylketonuria Screening Using the Quantase Phenylalanine Kit in Combination with a Microfilter System and the Dye Tartrazine," Screening 3 (1995) 209223.

13 N E O N A T A L S C R E E N I N G

Clinical Analyzer Feedwater

AFS Feedwater Systems
Water is a clinical reagent. The quality of the water used to feed clinical analyzers directly affects results and impacts the productivity of the clinical lab. AFS Feedwater Systems from Millipore are specifically designed to produce NCCLS Type I reagent water for clinical analyzers. The water is used in a variety of on-board applications, including reagent reconstitution, probe rinsing, and cuvette washing, as well as general laboratory applications, such as constanttemperature water baths. Unlike water systems that rely on a single water purification technology, Millipore AFS feedwater systems employ a combination of purification technologies and water quality monitoring techniques. This multi-technology approach guarantees high water quality and consistency. For Further Information Visit www.millipore.com/afs

Microbiological Testing
Admixture Preparations, Water, and Surfaces
The Addi-Chek II Test Kit is designed for use in a statistically valid Quality Assurance (QA) program for microbial analysis of prepared admixtures. The test canister captures and cultures microorganisms that may have been introduced into an admixture from environmental contaminants. Everything needed for quality control testing is included: disposable test canister with bacterially retentive filter, connecting tubes and spikes, and double-peel label.

HPC and Total Count Samplers/Swab Test Kits are self-contained devices that are ideal for monitoring the bacterial quality of surfaces and the water used in dialysate. Theres no media to mix, no agar plates to prepare, nothing to sterilize or clean.

14

HPLC Sample Preparation

Millex Filter Units

Millex filter units are available in a variety of diameters with a choice of membranes, including PVDF, nylon, and hydrophilic or hydrophobic PTFE with low-extractable high density polyethylene (HDPE) housings. Clinical applications for these products include removing particulates and red blood cells for PKU analysis (13 mm Millex units with 0.22 m Durapore PVDF membrane, cat. no. SLGV013NL).

For Further Information Millipore Quick Reference Guide, PB1951EN00 Sample Filtration Prior to Chromatography, TB025EN00

Internet: www.millipore.com Technical Service: www.millipore.com/techservice

Millipore, Addi-Chek, Amicon, AFS, Centricon, Centrifree, Durapore, Immobilon, MF-Millipore, Microcon, Millex, Millipore Express, Minicon, Miniplus, Montage, MultiScreen, and Ultracel are trademarks of Millipore Corporation. BigDye is a trademark of Applera Corporation. Lit. No. PB3441ENOO Printed in U.S.A. 10/03 Rev. 03-260 2003 Millipore Corporation, Billerica, MA. All rights reserved.