Jan 08, 2024

Public workspaceImmunocapture of virion from body fluids

PLOS One
CheckPeer-reviewed method
DOI
dx.doi.org/10.17504/protocols.io.e6nvwdom7lmk/v1
  • Jeffrey A. Johnson1,
  • Sarah Sabour1,
  • Jin-fen Li1,
  • Jonathan Lipscomb1
  • 1CDC
jjohnson
Open access
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 08, 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
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™ 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
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 undetermined -Amount0.2 undetermined ) to the filter device and cap it.

Pipetting
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
Centrifigation
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 .

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

Pipetting
Antibody Biotinylation (Sigma BTAG)
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 undetermined .


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

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

2h 30m
Incubation
Isolation of Labeled Antibody (Sigma BTAG)
Isolation of Labeled Antibody (Sigma BTAG)
6m
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 ).

1m
Centrifigation
Add Amount200 µL PBS (Ph7.4 ) to the column, spin the column for Duration00:01:00 at Centrifigation700 x g (Centrifigation3000 rpm ).
1m
Centrifigation
Pipetting
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).

2m
Centrifigation
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).

2m
Centrifigation
Determine Ab Concentration
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.

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

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

30m
Incubation
Read the absorbance at Amount562 undetermined (Amount540 undetermined -Amount590 undetermined ).

Calculate mAb concentration against the standard curve.
ELISA to Check Biotinylated Antibody
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
Incubation
Overnight
Wash plate 4 times with PBS+0.05% Tween.
Wash
Add Amount100 µL blocking buffer to each well, incubate at Temperature37 °C for Duration01:00:00 .

1h
Incubation
Pipetting
Wash plate 4 times with PBS+0.05% Tween.
Wash
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 .

1h
Incubation
Pipetting
Wash plate 4 times with PBS+0.05% Tween.
Add Amount100 µL TMB substrate to each well.

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

15m
Add Amount100 µL of stop solution to each well.

Pipetting
Read the absorbance of each well at Amount450 undetermined and Amount550 undetermined . 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
Streptavidin coated beads_ Biotinylated Ab + HIV → bead-Ab_HIV complex
1h 18m
Dilute Biotinylated Ab to Amount0.4 undetermined 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 undetermined ) biotinylated Ab for Duration00:10:00 at TemperatureRoom temperature on a roller platform.

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

10m
Centrifigation
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
Centrifigation
Wash
Add Amount100 µL Blocking buffer (PBS+1% BSA +1% Tween 20) to the tube and incubate at Temperature4 °C DurationOvernight .

10m
Incubation
Pipetting
Centrifuge bead-Ab complex at Centrifigation8000 rpm, 00:08:00 and then remove supernatant.

8m
Centrifigation
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
Incubation
Pipetting
Mix
Prepare µMACs column
Prepare µMACs column
Attach µMACs column to the magnetic multistand.
Add Amount100 µL equilibration buffer for nucleic acid applications to the column.

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

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


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

30m
Incubation
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.

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)
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.

Pipetting
Add another Amount150 µL AVL lysis buffer to the column and pass through the column completely.
Pipetting
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
Incubation
Pipetting
RNA extraction: QIAamp Viral RNA Mini Kit
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
Centrifigation
Pipetting
Mix
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.

1m
Centrifigation
Pipetting
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.

1m
Centrifigation
Pipetting
Add Amount500 µL Buffer AW2. Close the cap and centrifuge at full speed (Centrifigation20000 x g ; Centrifigation14000 rpm ) for Duration00:03:00 .
3m
Centrifigation
Pipetting
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 .

1m
Centrifigation
Pipetting
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 .

2m
Incubation
Centrifigation
RT PCR: SuperScript™ III One-Step RT-PCR System with Platinum™ Taq High Fidelity DNA Polymerase
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.
Centrifigation
Mix
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 .
Pipetting
Gently mix and make sure that all the components are at the bottom of the amplification tube.
Mix
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.

PCR
Nested PCR (nPCR): Platinum™ SuperFi II PCR Master Mix
Nested PCR (nPCR): Platinum™ SuperFi II PCR Master Mix
Thaw, vortex briefly to mix and centrifuge each component before use.
Centrifigation
Mix
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.

Pipetting
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):

PCR
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
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
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.