TITLE: Immunocapture of virion from body fluids
AUTHORS: Jeffrey A. Johnson, Sarah Sabour, Jin-fen Li, Jonathan Lipscomb

[DESCRIPTION]
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.


[GUIDELINES]
Workflow Chart:

 
Definitions:

 




[STEPS]
SECTION: Concentrate Antibody
1. Use Amicon Ultra-0.5 Centrifugal Filter Devices.
SECTION: Concentrate Antibody
2. Insert the Amicon device into the microcentrifuge tube.
SECTION: Concentrate Antibody
3. Add up to 500 µL Ab (0.1 2897-0.2 2897) to the filter device and cap it.

SECTION: Concentrate Antibody
4. Insert the capped Amicon Ultra device into a centrifuge tube and place in the centrifuge rotor.
SECTION: Concentrate Antibody
5. Spin the device at 14000 x g, 30 min. 
 
SECTION: Concentrate Antibody
6. 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.
SECTION: Concentrate Antibody
7. Spin the device at 1000 x g, 2 min.
 
SECTION: Concentrate Antibody
8. Add SPB (0.1 Molarity (M) sodium phosphate buffer, pH 7.2) to achieve a final volume 100 µL mAb at a concentration of 1 2897-2 2897.

SECTION: Antibody Biotinylation (Sigma BTAG)
9. Add 30 µL DMSO to the vial of Biotinylation Reagent (BAC-SulfoNHS), and then add 970 µL 0.1 Molarity (M) sodium phosphate buffer.
 

SECTION: Antibody Biotinylation (Sigma BTAG)
10. Immediately add 2 µL of Biotinylation Reagent to the antibody solution with gentle stirring.

SECTION: Antibody Biotinylation (Sigma BTAG)
11. Incubate with gentle stirring for 30 min at Room temperature or 120 min at 2 °C-8 °C.

SECTION: Isolation of Labeled Antibody (Sigma BTAG)
12. Place the column G-50 in a 1.5 ml Eppendorf tube, pre-spin the column for 1 min at 700 x g (3000 rpm).

SECTION: Isolation of Labeled Antibody (Sigma BTAG)
13. Add 200 µL PBS (pH 7.4) to the column, spin the column for 1 min at 700 x g (3000 rpm).
SECTION: Isolation of Labeled Antibody (Sigma BTAG)
14. Repeat two times.
SECTION: Isolation of Labeled Antibody (Sigma BTAG)
15. Label two of 1.5 ml Eppendorf tube.
SECTION: Isolation of Labeled Antibody (Sigma BTAG)
16. Place column in tube 1 and apple the biotinylation reaction mix to the column.
SECTION: Isolation of Labeled Antibody (Sigma BTAG)
17. Centrifuge the column for 2 min at 700 x g and collect flow-through (fraction 1).

SECTION: Isolation of Labeled Antibody (Sigma BTAG)
18. Place column in tube 2 and add 200 up to the column, spin the column for 2 min at 700 x g. collect flow-through (fraction 2).

SECTION: Determine Ab Concentration
19. Use Bicinchoninic Acid Kit, 96 well Immulon II plate assay.
SECTION: Determine Ab Concentration
20. Prepare standard curve dilutions: 
            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  

SECTION: Determine Ab Concentration
21. Prepare BCA Working Reagent: Mix Reagent A(50) and Reagent B(1).
SECTION: Determine Ab Concentration
22. Add 25 µL protein standard solution, PBS, and Ab samples into well of 96 well plate. Duplicate.

SECTION: Determine Ab Concentration
23. Add 200 µL of BCA working to each well (1:8 protein/BCA ratio).

SECTION: Determine Ab Concentration
24. Cover the plate with film and incubate 37 °C for 30 min.

SECTION: Determine Ab Concentration
25. Read the absorbance at 562 1636 (540 1636-590 1636).

SECTION: Determine Ab Concentration
26. Calculate mAb concentration against the standard curve.
SECTION: ELISA to Check Biotinylated Antibody
27. Using Immulon II 96 well plate.
SECTION: ELISA to Check Biotinylated Antibody
28. Coat three wells with a dilution series of mAb beginning with 1 µL mAb in 99 µL PBS (1:100) continuing with two more 10-fold dilutions. Incubate 30 min at 4 °C.
SECTION: ELISA to Check Biotinylated Antibody
29. Wash plate 4 times with PBS+0.05% Tween.
SECTION: ELISA to Check Biotinylated Antibody
30. Add 100 µL blocking buffer to each well, incubate at 37 °C for 60 min.

SECTION: ELISA to Check Biotinylated Antibody
31. Wash plate 4 times with PBS+0.05% Tween.
SECTION: ELISA to Check Biotinylated Antibody
32. Add 100 µL of 1:5000 ExtrAvidin_Peroxidase diluted with blocking buffer to each well. Cover plate and incubate at 37 °C for 60 min.

SECTION: ELISA to Check Biotinylated Antibody
33. Wash plate 4 times with PBS+0.05% Tween.
SECTION: ELISA to Check Biotinylated Antibody
34. Add 100 µL TMB substrate to each well.

SECTION: ELISA to Check Biotinylated Antibody
35. Develop plate at Room temperature in the dark for 15 min.

SECTION: ELISA to Check Biotinylated Antibody
36. Add 100 µL of stop solution to each well.

SECTION: ELISA to Check Biotinylated Antibody
37. Read the absorbance of each well at 450 1636 and 550 1636. OD values of the 1:100 dilution (first well) of ≥0.6 indicates adequate biotin labelling of antibody.
SECTION: Streptavidin coated beads_ Biotinylated Ab + HIV → bead-Ab_HIV complex
38. Dilute Biotinylated Ab to 0.4 2896 with 0.1 Molarity (M) sodium phosphate buffer.

SECTION: Streptavidin coated beads_ Biotinylated Ab + HIV → bead-Ab_HIV complex
39. Incubate 100 µL of Streptavidin coated beads with 5 µL PBS (negative Ab control) or 2 µg (5 µL of 0.4 2896) biotinylated Ab for 10 min at Room temperature on a roller platform.

SECTION: Streptavidin coated beads_ Biotinylated Ab + HIV → bead-Ab_HIV complex
40. Centrifuge bead-Ab complex at 8000 rpm, 10 min.

SECTION: Streptavidin coated beads_ Biotinylated Ab + HIV → bead-Ab_HIV complex
41. Remove supernatant and wash pellet with 100 µL PBS+1% BSA +1% Tween 20, centrifuge beads Ab complex at 8000 rpm, 10 min. Wash 3 times.

SECTION: Streptavidin coated beads_ Biotinylated Ab + HIV → bead-Ab_HIV complex
42. Add 100 µL Blocking buffer (PBS+1% BSA +1% Tween 20) to the tube and incubate at 4 °C 10 min.

SECTION: Streptavidin coated beads_ Biotinylated Ab + HIV → bead-Ab_HIV complex
43. Centrifuge bead-Ab complex at 8000 rpm, 8 min and then remove supernatant.

SECTION: Streptavidin coated beads_ Biotinylated Ab + HIV → bead-Ab_HIV complex
44. If working with tissue culture supernatants first DNase treat and inactivate.
SECTION: Streptavidin coated beads_ Biotinylated Ab + HIV → bead-Ab_HIV complex
45. Add 200 µL HIV-positive material (plasma, CSF, Semen, Culture or flow-through) to the designated bead-Ab complex and incubate for 30 min at Room temperature. Mixing gently on a roller-mixer.
SECTION: Prepare µMACs column
46. Attach µMACs column to the magnetic multistand.
SECTION: Prepare µMACs column
47. Add 100 µL equilibration buffer for nucleic acid applications to the column.

SECTION: Prepare µMACs column
48. Rinse column with 100 µL wash buffer (PBS+1% BSA +1% Tween 20), twice.

SECTION: Binding HIV-bead-Ab complex to the column and collecting the flow-through
49. 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).
 
SECTION: Binding HIV-bead-Ab complex to the column and collecting the flow-through
50. Add 30 µL wash buffer to the column and collect the flow.
 

SECTION: Binding HIV-bead-Ab complex to the column and collecting the flow-through
51. Incubate the flowthrough with next mAb-bead complex for 30 min on the roller-mixer at
Room temperature.

SECTION: Binding HIV-bead-Ab complex to the column and collecting the flow-through
52. To the just-eluted column, rinse the column 3 times with 400 µL of wash buffer to remove nonspecifically bound material, allowing the column drain completely. Discard the wash.

SECTION: Binding HIV-bead-Ab complex to the column and collecting the flow-through
53. Repeat this process until all mAb-bead columns in the series are completed.
SECTION: Elute target virion RNA from the column (using the QIAamp Viral RNA Mini kit)
54. After washing the column, place the column of bound virion in a new 1.5 mL Eppendorf tube.
SECTION: Elute target virion RNA from the column (using the QIAamp Viral RNA Mini kit)
55. Add 50 µL AVL lysis buffer to the column and pass through the column completely.

SECTION: Elute target virion RNA from the column (using the QIAamp Viral RNA Mini kit)
56. Add another 150 µL AVL lysis buffer to the column and pass through the column completely.
SECTION: Elute target virion RNA from the column (using the QIAamp Viral RNA Mini kit)
57. Add 360 µL AVL lysis buffer to the tube of eluted lysate and incubate tube at Room temperature for 10 min. Continue with the extraction kit instructions as follows.
 


SECTION: RNA extraction: QIAamp Viral RNA Mini Kit
58. Add 560 µL ethanol (96–100%) to the sample and mix by pulse-vortexing for 15 s. After mixing, briefly centrifuge the tube to remove drops from inside the lid.
SECTION: RNA extraction: QIAamp Viral RNA Mini Kit
59. Carefully apply 630 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 6000 x g (8000 rpm) for 1 min. Place the QIAamp Mini column into a clean 2 ml collection tube and discard the tube containing the filtrate.

SECTION: RNA extraction: QIAamp Viral RNA Mini Kit
60. Repeat this step until all of the lysate has been loaded onto the spin column.
SECTION: RNA extraction: QIAamp Viral RNA Mini Kit
61. Add 500 µL Buffer AW1. Close the cap, and centrifuge at 6000 x g (8000 rpm) for 1 min. Place the QIAamp Mini column in a clean 2 ml collection tube.

SECTION: RNA extraction: QIAamp Viral RNA Mini Kit
62. Add 500 µL Buffer AW2. Close the cap and centrifuge at full speed (20000 x g; 14000 rpm) for 3 min.
SECTION: RNA extraction: QIAamp Viral RNA Mini Kit
63. Place the QIAamp Mini column in a new 2 mL collection tube and discard the old collection tube with the filtrate. Centrifuge at 20000 x g (full speed) for 1 min.

SECTION: RNA extraction: QIAamp Viral RNA Mini Kit
64. 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 60 µL Buffer AVE equilibrated to Room temperature.

SECTION: RNA extraction: QIAamp Viral RNA Mini Kit
65. Close the cap and incubate at Room temperature for 1 min. Then centrifuge at 6000 x g (8000 rpm) for 1 min.

SECTION: RT PCR: SuperScript™ III One-Step RT-PCR System with Platinum™ Taq High Fidelity DNA Polymerase
66. Thaw, vortex briefly to mix and centrifuge each component before use.
SECTION: RT PCR: SuperScript™ III One-Step RT-PCR System with Platinum™ Taq High Fidelity DNA Polymerase
67. Prepare 45 µL reaction mast mix in a PCR workstation. 
        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  

SECTION: RT PCR: SuperScript™ III One-Step RT-PCR System with Platinum™ Taq High Fidelity DNA Polymerase
68. Add 5 µL of template RNA. Final reaction volume is 50 µL.
SECTION: RT PCR: SuperScript™ III One-Step RT-PCR System with Platinum™ Taq High Fidelity DNA Polymerase
69. Gently mix and make sure that all the components are at the bottom of the amplification tube.
SECTION: RT PCR: SuperScript™ III One-Step RT-PCR System with Platinum™ Taq High Fidelity DNA Polymerase
70. Place the reaction in the preheated thermal cycler programmed as described above. Collect the data and analyze the results.
SECTION: RT PCR: SuperScript™ III One-Step RT-PCR System with Platinum™ Taq High Fidelity DNA Polymerase
71. Program the thermal cycler to amplify with the following conditions:
 
 

SECTION: Nested PCR (nPCR): Platinum™ SuperFi II PCR Master Mix
72. Thaw, vortex briefly to mix and centrifuge each component before use.
SECTION: Nested PCR (nPCR): Platinum™ SuperFi II PCR Master Mix
73. For each sample, prepare 48 µL reaction master mix in a PCR workstation as follows: 
        Component  Volume (uL)    Platinum‱ SuperFi II PCR Master Mix  25    F primer (10 μM)  1    R primer (10 μM)  1    Water  21    Total  48  

SECTION: Nested PCR (nPCR): Platinum™ SuperFi II PCR Master Mix
74. Transfer new reaction microfuge tubes and RT-PCR samples to Nested PCR room. Add 2 µL of each RT-PCR sample per tube.

SECTION: Nested PCR (nPCR): Platinum™ SuperFi II PCR Master Mix
75. 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):
 
SECTION: Nested PCR (nPCR): Platinum™ SuperFi II PCR Master Mix
76. 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.
SECTION: Nested PCR (nPCR): Platinum™ SuperFi II PCR Master Mix
77. Identify samples with clean amplicon bands for further analysis.
SECTION: Nested PCR (nPCR): Platinum™ SuperFi II PCR Master Mix
78. Perform Sanger sequencing with available platform.
SECTION: Sequence analysis
79. 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)
SECTION: Sample Retention and Storage
80.