Immunocapture of virion from body fluids

Jeffrey A. Johnson; Sarah Sabour; Jin-fen Li; Jonathan Lipscomb

Jan 09, 2024

Immunocapture of virion from body fluids

PLOS One

Peer-reviewed method

DOI

dx.doi.org/10.17504/protocols.io.e6nvwdom7lmk/v1

Jeffrey A. Johnson¹,
Sarah Sabour¹,
Jin-fen Li¹,
Jonathan Lipscomb¹

¹CDC

PLOS ONE Lab Protocols
Tech. support email: plosone@plos.org

jjohnson

DOI: dx.doi.org/10.17504/protocols.io.e6nvwdom7lmk/v1

External link: https://doi.org/10.1371/journal.pone.0296891

Protocol Citation: Jeffrey A. Johnson, Sarah Sabour, Jin-fen Li, Jonathan Lipscomb 2024. Immunocapture of virion from body fluids. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwdom7lmk/v1

Manuscript citation:

Sabour S, Li Jf, Lipscomb JT, Santos Tino AP, Johnson JA (2024) Immunocapture of cell surface proteins embedded in HIV envelopes uncovers considerable virion genetic diversity associated with different source cell types. PLOS ONE 19(2): e0296891. https://doi.org/10.1371/journal.pone.0296891

License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Protocol status: Working

Working protocol

Created: October 19, 2023

Last Modified: January 09, 2024

Protocol Integer ID: 90186

Disclaimer

The performance of this protocol is claimed by the authors and does not necessarily represent the official view of the CDC.

Abstract

Procedure for immunocapturing HIV virions from blood and seminal plasma, cerebral spinal fluid, and cell culture supernatant by monoclonal antibody-targeting source cell markers in virion envelopes.

Attachments

nwqnb9rdx.pdf

338KB

Guidelines

Workflow Chart:

Definitions:

Materials

Equipment:

Roller-mixer: Stuart SRT9
Microcentrifuge (e.g., Eppendorf 5415D)
Template Tamer/CleanSpot workstation
UV cross-linker
Thermocycler and real-time cycler (e.g., Bio-Rad OPUS)
Genetic sequence analyzer

Reagents and Media:

Monoclonal antibodies: sourced from Santa Cruz Biotechnology (SCBT.com) 
ABCDEFG
S.NoPRODUCT  NAME CAT. # ISOTYPE EPITOPE APPLICATIONS SPECIES
1CD16 (2Q1240)SC-70548mouse  IgG1FL (h)WB,IP,IF,IHC(P),FCM human
2CD14 (61D3)sc-52475mouse  IgG1Extracellular  (h)IP,IF,IFCM human
3PECAM-1/CD31 (158-2B3)sc-65260mouse  IgG1FL (h)WB,IP,IF,FCM human
4CD45RA (4KB5)sc-20057mouse  IgG1FL (h)WB,IP,IF,IHC(P),FCM human
5CD45RO (UCHL1)sc-1183mouse  IgG2aFL (h)WB,IP,IF,IHC(P),FCM human
6HLA-DR/DP (HL-38)sc-51616mouse  IgG2aFL (h)WB,IP,FCM human
7CD27 (H-260)sc-20923 rabbit IgGFL (h)WB,IP,IF,ELISAhuman>mouse, rat
8CD3-ɛ (UCH T1)sc-1179mouse  IgG1FL (h)WB,IP,IF,IHC(P),FCM human
9CD2 (MT910)sc-19638mouse  IgG1FL (h)WB,IP,IF,IHC(P),ELISA human
10CD21 (A3)sc-13135mouse IgG2bAA 21-260 (h)WB,IP,IF,IHC(P),ELISA mouse, human
11Integrin αX/CD11c (B6)sc-46676mouse  IgG1FL (h)WB,IP,IF,IHC(P),ELISA human
12Iba1 (F-4)sc-398406mouse  IgG1FL (h)WB,IP,IF,IHC(P),ELISA human
13CD36 Antibody (SMφ)sc-7309mouse IgM ĸExtracellular  (h)WB, IP, IF, IHC(P), FCMmouse, rat and human
14CD68 (KP1)sc-20060mouse  IgG1Extracellular  (h)WB, IP, IF, IHC(P) and FCMmouse, rat and human
ReagentCD16 Antibody (2Q1240)Santa Cruz BiotechnologyCatalog #sc-70548   
ReagentCD14 Antibody (61D3)Santa Cruz BiotechnologyCatalog #sc-52457  
ReagentCD31/PECAM-1 Antibody (158-2B3)Santa Cruz BiotechnologyCatalog #sc-65260  
ReagentCD45RA Antibody (4KB5)Santa Cruz BiotechnologyCatalog #sc-20057  
ReagentCD45RO Antibody (UCH-L1)Santa Cruz BiotechnologyCatalog #sc-1183  
ReagentHLA-DR/DP Antibody (HL-38)Santa Cruz BiotechnologyCatalog #sc-51616  
ReagentCD3-ε Antibody (UCH-T1)Santa Cruz BiotechnologyCatalog #sc-1179  
ReagentCD2 Antibody (MT910)Santa Cruz BiotechnologyCatalog #sc-19638  
ReagentCD21 Antibody (A-3)Santa Cruz BiotechnologyCatalog #sc-13135  
ReagentIntegrin αX/ITGAX/CD11c Antibody (B-6)Santa Cruz BiotechnologyCatalog #sc-46676  
ReagentIba1 Antibody (F-4)Santa Cruz BiotechnologyCatalog #sc-398406  
ReagentCD36 Antibody (SMφ)Santa Cruz BiotechnologyCatalog #sc-7309  
ReagentCD68 Antibody (KP1)Santa Cruz BiotechnologyCatalog #sc-20060  


BiotinTag Micro Biotinylation kit (BTAG), Sigma BTAG-1KT
 ReagentDimethyl sulfoxideMerck MilliporeSigma (Sigma-Aldrich)Catalog #D5879  
Bicinconinic acid kit, Sigma BCA1-1KT
µMACS Streptavidin MicroBeads, Miltenyi 120-001-017
Equilibration Buffer for nucleic acid applications, Miltenyi 120-001-014
20 µMACS Columns: Miltenyi 120-001-002
PBS 0.01M pH7.4, CDC #4550
ReagentTween 20 100% Nonionic DetergentBio-Rad LaboratoriesCatalog #1706531  
ReagentBovine Serum AlbuminMerck MilliporeSigma (Sigma-Aldrich)Catalog #A9418  
Wash buffer: PBS +1% BSA + 1% Tween 20
Blocking buffer: PBS +1% BSA + 1% Tween 20
Ethanol (96 – 100%)
ReagentDEPC-Treated WaterThermo FisherCatalog #AM9906  
0.1 M Sodium Phosphate Buffer, pH 7.2, Sigma P9693
ReagentQIAamp® Viral RNA Mini QiagenCatalog #52906  
ReagentQIAquick PCR Purification KitQiagenCatalog #28104  
ReagentQubit™ dsDNA HS Assay KitInvitrogen - Thermo FisherCatalog #Q32851  /ReagentQubit® dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32854  
Elution buffer: 0.01 M Tris-Cl in DEPC-treated water
ReagentBigDye XTerminator&trade; Purification KitThermo FisherCatalog #4376486  
SuperScript‱ III RT/ Platinum‱ Taq HiFi: Invitrogen 12574 
ReagentRNase InhibitorThermo FisherCatalog #N8080119  
ReagentPlatinum™ SuperFi II PCR Master MixInvitrogen - Thermo FisherCatalog #12368050  
DNase DNA-free kit, Invitrogen AM1906 (for tissue culture supernatants)



Supplies, Other Materials:
Equipment
Amicon Ultra-0.5 Centrifugal Filter Unit
NAME
Centrifugal Filter Unit
TYPE
Millipore
BRAND
UFC5003BK
SKU
https://www.merckmillipore.com/GB/en/product/Amicon-Ultra-0.5-Centrifugal-Filter-Unit,MM_NF-UFC5003BK
LINK

Magnetic Separator: 8 position MACS magnetic stand 007139
Sterile, RNase-free microcentrifuge tubes, 1.5 mL – 2 mL
10 µL, 200µL, 1000µL pipette and tips
RNase-free pipet tips with aerosol barrier
Immulon II flat well 96-well plates, Nunc #96920
Microcentrifuge tube racks
Clear microfilm seals for plates
96-well hard-shell skirted conical bottom PCR plates
96-well non-skirted clear conical bottom sequencing plates
96-well septa mats
Dedicated spaces for reagent preparation, RNA template, PCR/nested PCR, Real-Time PCR, and sequencing. Gloves must be changed as needed to prevent template contamination.

 
Sample Information / Processing (Volume, labeling, handling, storage)

Fresh, non-frozen biologic sample preferred, stored at Temperature4 °C   and used within 48 hours. If frozen, thaw frozen plasma TemperatureOn ice  .
Aliquot desired input plasma volume from Amount200 µL   – Amount400 µL  , equivalent to <= 500,000 virus copies, into a 2 mL microcentrifuge tube.
Note
 If viral load is unknown, determine copies by qPCR or test on a commercial viral load platform.

Concentrate Antibody

32m

Use Amicon Ultra-0.5 Centrifugal Filter Devices.

Insert the Amicon device into the microcentrifuge tube.

Add up to Amount500 µL   Ab (Amount0.1 µL  -Amount0.2 µL  ) to the filter device and cap it.

Insert the capped Amicon Ultra device into a centrifuge tube and place in the centrifuge rotor.

Spin the device at Centrifigation14000 x g, 00:30:00 . 

30m

Remove the device and place it upside down in a clean tube, place in centrifuge, aligning the open cap strap, toward the center of the rotor.

Spin the device at Centrifigation1000 x g, 00:02:00 .

Add SPB (Concentration0.1 Molarity (M)   sodium phosphate buffer, Ph7.2 ) to achieve a final volume Amount100 µL   mAb at a concentration of Amount1 µL  -Amount2 µL  .

Antibody Biotinylation (Sigma BTAG)

2h 30m

Add Amount30 µL   DMSO to the vial of Biotinylation Reagent (BAC-SulfoNHS), and then add Amount970 µL   Concentration0.1 Molarity (M)   sodium phosphate buffer.
Note
The concentration of Biotinylation Reagent is Amount5 µL  .

Immediately add Amount2 µL   of Biotinylation Reagent to the antibody solution with gentle stirring.

Incubate with gentle stirring for Duration00:30:00   at TemperatureRoom temperature   or Duration02:00:00   at Temperature2 °C  -Temperature8 °C  .

2h 30m

Isolation of Labeled Antibody (Sigma BTAG)

Place the column G-50 in a 1.5 ml Eppendorf tube, pre-spin the column for Duration00:01:00   at Centrifigation700 x g  (Centrifigation3000 rpm ).

Add Amount200 µL   PBS (Ph7.4 ) to the column, spin the column for Duration00:01:00   at Centrifigation700 x g  (Centrifigation3000 rpm ).

Repeat two times.

Label two of 1.5 ml Eppendorf tube.

Place column in tube 1 and apple the biotinylation reaction mix to the column.

Centrifuge the column for Duration00:02:00   at Centrifigation700 x g  and collect flow-through (fraction 1).

Place column in tube 2 and add 200 up to the column, spin the column for Duration00:02:00   at Centrifigation700 x g . collect flow-through (fraction 2).

Determine Ab Concentration

30m

Use Bicinchoninic Acid Kit, 96 well Immulon II plate assay.

Prepare standard curve dilutions: 
ABCD
Protein Ci (µg/mL)Protein Input Volume (uL)PBS (µL)Protein Cf (ug/mL)
1000 - -1000
1000400100800
800375125600
600333166400
400250250200
200250250100

Prepare BCA Working Reagent: Mix Reagent A(50) and Reagent B(1).

Add Amount25 µL   protein standard solution, PBS, and Ab samples into well of 96 well plate. Duplicate.

Add Amount200 µL   of BCA working to each well (1:8 protein/BCA ratio).

Cover the plate with film and incubate Temperature37 °C   for Duration00:30:00  .

30m

Read the absorbance at Amount562 µL   (Amount540 µL  -Amount590 µL  ).

Calculate mAb concentration against the standard curve.

ELISA to Check Biotinylated Antibody

2h 45m

Using Immulon II 96 well plate.

Coat three wells with a dilution series of mAb beginning with Amount1 µL   mAb in Amount99 µL   PBS (1:100) continuing with two more 10-fold dilutions. Incubate DurationOvernight   at Temperature4 °C  .

30m

Wash plate 4 times with PBS+0.05% Tween.

Add Amount100 µL   blocking buffer to each well, incubate at Temperature37 °C   for Duration01:00:00  .

Wash plate 4 times with PBS+0.05% Tween.

Add Amount100 µL   of 1:5000 ExtrAvidin_Peroxidase diluted with blocking buffer to each well. Cover plate and incubate at Temperature37 °C   for Duration01:00:00  .

Wash plate 4 times with PBS+0.05% Tween.

Add Amount100 µL   TMB substrate to each well.

Develop plate at TemperatureRoom temperature   in the dark for Duration00:15:00  .

15m

Add Amount100 µL   of stop solution to each well.

Read the absorbance of each well at Amount450 µL   and Amount550 µL  . OD values of the 1:100 dilution (first well) of ≥0.6 indicates adequate biotin labelling of antibody.

Streptavidin coated beads_ Biotinylated Ab + HIV → bead-Ab_HIV complex

1h 18m

Dilute Biotinylated Ab to Amount0.4 µL   with Concentration0.1 Molarity (M)   sodium phosphate buffer.

Incubate Amount100 µL   of Streptavidin coated beads with Amount5 µL   PBS (negative Ab control) or Amount2 µg   (Amount5 µL   of Amount0.4 µL  ) biotinylated Ab for Duration00:10:00   at TemperatureRoom temperature   on a roller platform.

10m

Centrifuge bead-Ab complex at Centrifigation8000 rpm, 00:10:00 .

10m

Remove supernatant and wash pellet with Amount100 µL   PBS+1% BSA +1% Tween 20, centrifuge beads Ab complex at Centrifigation8000 rpm, 00:10:00 . Wash 3 times.

10m

Add Amount100 µL   Blocking buffer (PBS+1% BSA +1% Tween 20) to the tube and incubate at Temperature4 °C   DurationOvernight  .

10m

Centrifuge bead-Ab complex at Centrifigation8000 rpm, 00:08:00  and then remove supernatant.

If working with tissue culture supernatants first DNase treat and inactivate.

Add Amount200 µL   HIV-positive material (plasma, CSF, Semen, Culture or flow-through) to the designated bead-Ab complex and incubate for Duration00:30:00   at TemperatureRoom temperature  . Mixing gently on a roller-mixer.

30m

Prepare µMACs column

Attach µMACs column to the magnetic multistand.

Add Amount100 µL   equilibration buffer for nucleic acid applications to the column.

Rinse column with Amount100 µL   wash buffer (PBS+1% BSA +1% Tween 20), twice.

Binding HIV-bead-Ab complex to the column and collecting the flow-through

30m

Apply HIV-bead-Ab complex onto the top of column, collecting the flowthrough in a clean microfuge tube or eluting directly into the next tube of biotinylated mAb-bead complex. Let reaction pass through the column completely, captured virus will be retained on the column and flow-through will contain non-target virus (see figure below).

Add Amount30 µL   wash buffer to the column and collect the flow.
Note
This accounts for the column void volume and maintains a Amount200 µL   sample volume.

Incubate the flowthrough with next mAb-bead complex for Duration00:30:00   on the roller-mixer at
TemperatureRoom temperature  .

30m

To the just-eluted column, rinse the column 3 times with Amount400 µL   of wash buffer to remove nonspecifically bound material, allowing the column drain completely. Discard the wash.

Repeat this process until all mAb-bead columns in the series are completed.

Elute target virion RNA from the column (using the QIAamp Viral RNA Mini kit)

10m

After washing the column, place the column of bound virion in a new 1.5 mL Eppendorf tube.

Add Amount50 µL   AVL lysis buffer to the column and pass through the column completely.

Add another Amount150 µL   AVL lysis buffer to the column and pass through the column completely.

Add Amount360 µL   AVL lysis buffer to the tube of eluted lysate and incubate tube at TemperatureRoom temperature   for Duration00:10:00  . Continue with the extraction kit instructions as follows.
Note
Take Amount140 µL   of final flow through after all columns are completed and add Amount560 µL    of lysis buffer.  Continue with lysis kit steps.

10m

RNA extraction: QIAamp Viral RNA Mini Kit

8m 15s

Add Amount560 µL   ethanol (96–100%) to the sample and mix by pulse-vortexing for Duration00:00:15  . After mixing, briefly centrifuge the tube to remove drops from inside the lid.

15s

Carefully apply Amount630 µL   of the sample solution to the QIAamp Mini column (in a 2 ml collection tube) without wetting the rim. Close the cap, and centrifuge at Centrifigation6000 x g  (Centrifigation8000 rpm ) for Duration00:01:00  . Place the QIAamp Mini column into a clean 2 ml collection tube and discard the tube containing the filtrate.

Repeat this step until all of the lysate has been loaded onto the spin column.

Add Amount500 µL   Buffer AW1. Close the cap, and centrifuge at Centrifigation6000 x g  (Centrifigation8000 rpm ) for Duration00:01:00  . Place the QIAamp Mini column in a clean 2 ml collection tube.

Add Amount500 µL   Buffer AW2. Close the cap and centrifuge at full speed (Centrifigation20000 x g ; Centrifigation14000 rpm ) for Duration00:03:00  .

Place the QIAamp Mini column in a new 2 mL collection tube and discard the old collection tube with the filtrate. Centrifuge at Centrifigation20000 x g  (full speed) for Duration00:01:00  .

Place the QIAamp Mini column in a clean 1.5 ml microcentrifuge tube. Discard the old collection tube containing the filtrate. Carefully open the QIAamp Mini column and add Amount60 µL   Buffer AVE equilibrated to TemperatureRoom temperature  .

Close the cap and incubate at TemperatureRoom temperature   for Duration00:01:00  . Then centrifuge at Centrifigation6000 x g  (Centrifigation8000 rpm ) for Duration00:01:00  .

RT PCR: SuperScript™ III One-Step RT-PCR System with Platinum™ Taq High Fidelity DNA Polymerase

Thaw, vortex briefly to mix and centrifuge each component before use.

Prepare Amount45 µL   reaction mast mix in a PCR workstation. 
AB
ComponentVolume (uL)
2x Reaction Mix25
F primer (10 μM)1
R primer (10 μM)1
SuperScript III RT/Platinum Taq High Fidelity Enzyme Mix2
RNA Inhibitor (40 U/µL)1
Water15
Total45

Add Amount5 µL   of template RNA. Final reaction volume is Amount50 µL  .

Gently mix and make sure that all the components are at the bottom of the amplification tube.

Place the reaction in the preheated thermal cycler programmed as described above. Collect the data and analyze the results.

Program the thermal cycler to amplify with the following conditions:

Note
You may check for primary PCR product by gel electrophoresis or real-time detection.  Due to the potential for low copy numbers perform nested reactions.

Nested PCR (nPCR): Platinum™ SuperFi II PCR Master Mix

Thaw, vortex briefly to mix and centrifuge each component before use.

For each sample, prepare Amount48 µL   reaction master mix in a PCR workstation as follows: 
AB
ComponentVolume (uL)
Platinum SuperFi II PCR Master Mix25
F primer (10 μM)1
R primer (10 μM)1
Water21
Total48

Transfer new reaction microfuge tubes and RT-PCR samples to Nested PCR room. Add Amount2 µL   of each RT-PCR sample per tube.

Use a designated 2nd round PCR thermocycler – vortex and quick spin samples before inserting into thermocycler. Amplify with the following conditions (specific for primers used):

DNA is quantified and PCR amplicon size is verified via the Agilent 2200 Tapestation after nested PCR is performed for sequencing. Alternatively, bands can be checked by agarose gel.

Identify samples with clean amplicon bands for further analysis.

Perform Sanger sequencing with available platform.

Sequence analysis

Compare relatedness of HIV sequences in alignment software (e.g., Geneious) and MEGA to generate neighbor-joining trees and perform genetic distance analysis. Perform best model fit (typically Tamura 92 is the best fit)

Sample Retention and Storage

Note
Frozen plasma specimens should be stored at Temperature-80 °C   until ready for testing.
Extracted genetic material should be stored at Temperature-80 °C   for long-term storage.
Amplified RT-PCR can be stored for two weeks at Temperature4 °C   but should be stored at Temperature-80 °C   for longer storage.
RT-PCR amplicons should not be stored with clinical samples.

A	B	C	D	E	F	G
S.No	PRODUCT NAME	CAT. #	ISOTYPE	EPITOPE	APPLICATIONS	SPECIES
1	CD16 (2Q1240)	SC-70548	mouse IgG1	FL (h)	WB,IP,IF,IHC(P),FCM	human
2	CD14 (61D3)	sc-52475	mouse IgG1	Extracellular (h)	IP,IF,IFCM	human
3	PECAM-1/CD31 (158-2B3)	sc-65260	mouse IgG1	FL (h)	WB,IP,IF,FCM	human
4	CD45RA (4KB5)	sc-20057	mouse IgG1	FL (h)	WB,IP,IF,IHC(P),FCM	human
5	CD45RO (UCHL1)	sc-1183	mouse IgG2a	FL (h)	WB,IP,IF,IHC(P),FCM	human
6	HLA-DR/DP (HL-38)	sc-51616	mouse IgG2a	FL (h)	WB,IP,FCM	human
7	CD27 (H-260)	sc-20923	rabbit IgG	FL (h)	WB,IP,IF,ELISA	human>mouse, rat
8	CD3-ɛ (UCH T1)	sc-1179	mouse IgG1	FL (h)	WB,IP,IF,IHC(P),FCM	human
9	CD2 (MT910)	sc-19638	mouse IgG1	FL (h)	WB,IP,IF,IHC(P),ELISA	human
10	CD21 (A3)	sc-13135	mouse IgG2b	AA 21-260 (h)	WB,IP,IF,IHC(P),ELISA	mouse, human
11	Integrin αX/CD11c (B6)	sc-46676	mouse IgG1	FL (h)	WB,IP,IF,IHC(P),ELISA	human
12	Iba1 (F-4)	sc-398406	mouse IgG1	FL (h)	WB,IP,IF,IHC(P),ELISA	human
13	CD36 Antibody (SMφ)	sc-7309	mouse IgM ĸ	Extracellular (h)	WB, IP, IF, IHC(P), FCM	mouse, rat and human
14	CD68 (KP1)	sc-20060	mouse IgG1	Extracellular (h)	WB, IP, IF, IHC(P) and FCM	mouse, rat and human

A	B	C	D
Protein Ci (µg/mL)	Protein Input Volume (uL)	PBS (µL)	Protein Cf (ug/mL)
1000	-	-	1000
1000	400	100	800
800	375	125	600
600	333	166	400
400	250	250	200
200	250	250	100

	A	B
	Component	Volume (uL)
	2x Reaction Mix	25
	F primer (10 μM)	1
	R primer (10 μM)	1
	SuperScript III RT/Platinum Taq High Fidelity Enzyme Mix	2
	RNA Inhibitor (40 U/µL)	1
	Water	15
	Total	45

	A	B
	Component	Volume (uL)
	Platinum SuperFi II PCR Master Mix	25
	F primer (10 μM)	1
	R primer (10 μM)	1
	Water	21
	Total	48

Public workspaceImmunocapture of virion from body fluids

Immunocapture of virion from body fluids