新工作流程:浓缩和浓度检测说明书

New Workflow for Lentivirus

Purification, Concentration, and

Immunodetection

Janet Smith

1

, Sara Gutierrez

1

, Charles Neville

1

, Mikhail Kozlov, PhD

2

, and Kathleen Ongena, PhD

1

Millipore Corporation, Billerica, MA USA

Presented at the 2008 Gene Therapy Conference

May, 2008-Boston, MA USA

Millipore is a registered trademark of Millipore Corporation. Amicon, Steriflip, Ultrafree, and Immobilonare

registered trademarks of Millipore Corporation. Fast-Trap and SNAP id are trademarks of Millipore Corporation.

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Quick Titer is a trademark of Cell Biolabs. Syprois a trademark of Molecular Probes, Inc.

Transblotis a trademark of BioRadLaboratortiesInc. Tweenis a trademark of ICI Americas Inc

Lit. No. PS2076ENUS Printed in U,S.A. 2/08

©Millipore Corporation, Billerica, MA 01821 USA All rights reserved.

New Workflow for Lentivirus

Purification, Concentration, and Immunodetection

Janet Smith

1

, Sara Gutierrez

1

, Charles Neville

1

, Mikhail Kozlov, PhD

2

and Kathleen Ongena, PhD

1

1

Millipore Corporation, BioScience Division, Danvers, MA, United States, 01923 and

2

Millipore Corporation, Bedford, MA, United States, 01730

Abstract

Lentivirus is a negatively charged, enveloped, single stranded

RNA virus from the Retroviridaefamily that is often used as a

vector to transport genetic material into cells. These viral vectors

can be used for genetic modification, RNAi, gene therapy, and

vaccine production. Before viral preparations/propagations can be

used for any of these applications, researchers need to purify their

virus sample.

Traditionally, time-consuming density gradient centrifugation

separation and/or chromatographic techniques have been used.

The lab-scale chromatographic devices are commonly syringe or

column-based. To process the virus sample, these devices

require hand pressure or gravity; this may lead to messy and to

potentially unsafe handling conditions during assembly and

disassembly.

A new workflow has been developed to clarify, purify, and

concentrate/buffer exchange a crude lentivirus sample. For added

safety and improved handling, the clarification and purification

steps are performed in a closed vacuum-based device. This

purification produces high recovery of virus particles in about one

hour with similar or improved results as compared to traditional

methods. Purity was visualized by gel electrophoresis and

confirmed by western blotting using an innovative vacuum-based

immunodetection system that allows detection of the protein of

interest in less than forty minutes.

Here we show the results of the purification and the

immunodetection of a Lentivirus-VSVG pseudotype that encodes

green fluorescent protein (GFP). We demonstrate the viral titer,

the percent recovery of infectious particles, and the purity of the

virus sample.

Lentivirus Fast-Trap

™

Virus Purification & Concentration Kit combined with SNAP i.d.

™

Immunodetection enables results in less than three hours.

New Workflow: Virus Purification & Concentration

Fast-Trap Virus Purification

and Concentration Kit

Initial

Harvest

Benzonase

Treatment

(Optional)ClarifyPurifyConcentrate

Immunodetection Workflow With the SNAP i.d. Protein Detection System

SNAP i.d.

Immunodetection

Electrophoresis

45 min. -1 hr.

Membrane

TransferBlocking

20 sec.

Antibody

Additions &

Washes

20 min.

Detection

Result: Purified Virus

in 1 to 2.5 hrs.

Steps:

1 2

30 min.

3A

30 sec.

3B

0.5 -2 hr.

3C

20 min.

Steps: 456A 6B7

Result: Detection of

Protein in < 40 min.

0.5 -2.5 hrs.

Transfer to:

Immobilon-P

Membrane

5 -15 min.

•Genetic modification

•RNAi

•Gene therapy

•Vaccine production

Detect with:

Immobilon

Western HRP

Substrate

Purification Method:

¾Step 1:

¾Step 2:

¾Step 3:

Step 3A:

Step 3B:

Step 3C:

Initial harvest of lentivirus

(Optional) Benzonase treatment 30 minutes at 37

°

C

Purification with Fast-Trap Lentivirus Purification & Concentration Kit (Millipore #FTLV00003):

Clarify using provided 0.45 µm HV Steriflip

®

filter unit by vacuum filtration

Purify virus using Fast-Trap purification device & buffers

Concentrate/buffer exchange of eluted lentivirus with supplied 100 kDa Amicon

®

Ultra device.

Protein Detection Method:

¾Step 4:Electrophoresis-Samples denatured & reduced before SDS-PAGE

¾Step 5:Transfer to Immobilon

®

-P membrane (Millipore #IPVH07850)

¾Step 6: Immunodetection with SNAP i.d. Protein Detection System (Millipore #WBAVDBASE)

Step 6A: Apply blocking reagent containing Tween

®

20 surfactant and vacuum filter

Step 6B:Apply antibodies (primary, secondary) and wash between antibody additions

¾Step 7:Add Immobilon

®

Western Chemiluminescent HRP substrate (Millipore #WBKLS0500) for

chemiluminescence detection of protein of interest and visualizeimmunoreactive proteins

Performance of the Fast-Trap Lentivirus Purification & Concentration Kit and SNAP i.d. Protein Detection Workflow

Methods

Fast-Trap Purification

Virus: Crude lentivirus (University of North Carolina [UNC] Vector Core) was

benzonase treated, clarified, purified, and concentrated using the new Fast-

Trap kit workflow

Recovery of Infectious Particles with the Fast-Trap

Purification and Concentration Kit

R

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i

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P

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)

Figure 1

Sample Purity & Capsid Protein P24 Detection

Figure 2

Ratio of Infectious to Non-Infectious Viral Particles

Table 1Figure 3

SDS-PAGE

Samples: Virus samples normalized for protein concentration, denatured and reduced

Gel: NuPAGE

4 –12% Bis-Tris gel (Invitrogen) 200 V, 35 minutes

Gel Markers:M =Mark12

unstained standard or MagicMarkXP Western standard

(Invitrogen)

Stain:SYPRO

Ruby stain (Invitrogen) overnight, destain, and visualize, or

membrane transfer for immunodetection

100%

75%

50%

25%

0%

Average

Concentration by ELISA

(vp/mL x 10

10

)

Unpurified

Lentivirus

3.6

2.8

1.3

Purified

Lentivirus

22.5

12.9

1.7

BSA

Infective particle Input

P24

Clarification

HV Steriflip Device

Purification

Fast-Trap Kit using

Steriflip Virus

Device

Concentration

Amicon Ultra Filter

Unit

Workflow Total

Membrane Transfer

Transfer:SD Semi-dry Transfer cell) to

Immobilon-P membrane at 10 V, 35 minutes with Tris-Glycine buffer

Semi-dry transfer method (BioRad Trans-Blot

(Ivp x 10

6

)

Ratio

(vp/Ivp x 10

4

)

SNAP i.d. Immunodetection

Blocking: Add blocking buffer (0.5% non-fat dry milk in Tris Buffered Saline with 0.1%

Tween-20 surfactant, TBST) and vacuum

1°Antibody: Probe with Mouse anti-HIV, p24 (Millipore #MAB8790) diluted 1:13,000 in

blocking buffer, and incubate for 10 minutes

Wash:Wash with TBST buffer using constant vacuum

2°Antibody:Add Goat anti-Mouse IgG, HRP-conjugate (Millipore #AP124P) diluted

1:10,000 in blocking buffer, and incubate for 10 minutes

Wash:Wash with TBST buffer using constant vacuum

Detect:Incubate 5 minutes with Immobilon Western Chemiluminescent HRP

substrate, and visualize by exposing to x-ray film

Figure 1:Human Embryonic Kidney (HEK 293A, Invitrogen) cells were transduced with serial dilutions of

crude, clarified, purified, and concentrated viral fractions. After three days, fluorescence of virus-transduced cells

expressing GFP were quantified by image analysis and/or manual counting. Viral titer and percent recovery of

infectious particles (Ivp) were determined. All of the lentivirus was recovered during clarification step (input ~ 1 x

10Ivp). Clarified lentivirus (~ 3.9 x 10Ivp) was purified using the Fast-Trap purification device, and efficient

recovery observed (avg. 72%). The eluted virus (~ 2 x 10Ivp) was concentrated/buffer exchanged and high

recovery achieved (~2 x 10, avg. 94%). The total lentivirus recovery for this workflow was ~ 67%.

Figure 2:SYPRO Ruby Stain of SDS-

PAGE. Using equal protein load (3 µg per

lane), the presence ofserum protein

bovine serum albumin (BSA) was

observed in the input (IN), flow through

(FT) and wash (W) fractions. A reduction

of BSA was observed in both the eluted

(E) and concentrated (C) fractions.

Figure 3:Detection of Capsid Protein

P24 by SNAP i.d. Immunodetection.

Using equal protein load (3 µg per lane), the

presence of lentiviral Capsid Protein P24

was confirmed in the eluted (E) and

concentrated (C) fractions.

Table 1:The ratio for unpurified and purified lentivirus (using the Fast-Trap Purification and

Concentration Kit) was determined byEnzyme-Linked ImmunoSorbent Assay (ELISA) Quick Titer™

Lentivirus Quantitation kit (Cell Biolabs). This ratio is maintained when using Fast-Trap Lentivirus

Purification and Concentration kit.

Lentivirus Purification: Comparison of the Fast-Trap Kit to Other Formats/Methods (Syringe, Column, Precipitation, & Traditional Sucrose Gradient)

Processing Time & Virus Recovery

Table 2

Recovery of

Processing Processing Infectious Viral

Purification FormatKey

Volume (ml)Time (min.)Particles, Ivp

(%)

Vacuum, closed systemFast: Fast-Trap 9657%

Lentivirus

Gravity syringe S1 9119%

BioVintage,

91021%Gravity syringe S2 Cell

Biolabs, ViraBind™

Gravity syringe S3 91129%

Sartorius Vivapure

Column Col 930038%

BioMiga, Viratrap

Precipitation/Centrifugation Ppt 1012 Hr. + 3557%

System

Table 2:Crude lentivirus (Input 1.4 x 10Ivp) was purified with the Fast-Trap Virus Purification and

Concentration Kit, three types of gravity syringe chromatographic columns (S1, S2 & S3), one column

(Col), and one precipitation (Ppt) method (following manufacturer’s instructions). The table compares

overall performance and processing time (for bind/wash/elute, orprecipitation/centrifugation steps only).

The Fast-Trap purification method has the highest recovery and the shortest processing time. The

closed, vacuum-based Fast-Trap’s purification device was easy to handle without the messy

assembly/disassembly process or flow regulation that some formats require.

Conclusions

¾Results can be generated in less than three hours using

the Fast-Trap Virus Purification and Concentration kit

followed by SNAP i.d. immunodetection

¾Fast-Trap Virus Purification and Concentration Kit:

¾Enables efficient recovery of high titer lentivirus

¾Maintains ratio of non-infective to infective viral particles

¾Outperforms other chromatographic and precipitation

formats for processing time, percent recovery of

infectious particles, purity, and handling

¾Provides an alternative to precipitation, column or

chromatography-based, and traditional gradient

purification methods

¾Vacuum-based SNAP i.d. Protein Detection System:

¾Shortens immunodetection process to ~ 30 minutes

¾Offers a faster alternative to traditional methods without

consumption of additional reagents or loss of sensitivity

Acknowledgements

¾UNC Vector Core & Dr. R. Jude Samulski for providing lentivirus samples

Copyright 2008

Millipore Corporation

Sample Purity & Capsid Protein P24 Detection

Figure 4Figure 5

Fast-Trap Virus Purification and

Concentration Kit versus Traditional Sucrose

Gradient Method

Figure 6Figure 7

Figure 4:SYPRO Ruby Stain of SDS-PAGE gel.

Purity of input (IN) and elution fractions were

visualized on the SDS-PAGE gel loading equal protein

(5 µg per lane). The gel demonstrates reduction of

BSA in the Fast-Trap Virus method (Fast) and other

formats. These include three syringe-based (S1, S2,

& S3), one column (C), and one precipitation (Ppt).

Contaminating protein (BSA) is observed in the

column (Col) and precipitation (Ppt) methods.

Figure 5:Detection of Capsid Protein P24 by

SNAP i.d. Immunodetection. Using 5 µg of protein

per lane, the presence of the lentivirus Capsid Protein

P24 was confirmed in the Fast-Trap Virus (Fast),

Syringe S2, and precipitation (Ppt) methods.

Figure 6:SYPRO Ruby Stain of Figure 7:Detection of Capsid Protein P24

SDS-PAGE gel. Using equal protein by SNAP i.d. Immunodetection. Using

load ( 3 µg per lane), purity of the input equal protein load (3 µg per lane), the

(In), the Fast-Trap kit’s eluted (E) and presenceof lentivirus Capsid Protein P24

concentrated (C) fractions are was confirmed in the eluted (E), concentrated

compared to a traditional sucrose (S) (C), and sucrose gradient method (S,

gradient method. Removal of BSA is provided by UNC Vector Core).

observed in the purified samples.