TITLE: MSD Gold Streptavidin Antibody Preparation and Plate Run Protocol
AUTHORS: Kamaljot Gill, Maria  Sckaff, Claire D Clelland

[DESCRIPTION]
This protocol describes how to conjugate antibodies and run the Meso Scale Discovery (MSD) Sandwich enzyme-linked immunosorbent assay (ELISA) on MSD GOLD 96-well Small Spot Streptavidin SECTOR Plates. This protocol is adapted from MSD GOLD™ Streptavidin Plate and Avidin Plates Quick Guide 1 and MSD GOLD™ SULFO-TAG NHS-Ester Conjugation Quick Guide 2 and optimized for C9orf72 dipeptide repeat detection from human iPSC derived neurons.

[GUIDELINES]
References
1MSD GOLD™ SULFO-TAG NHS-Ester Conjugation Quick Guide
https://www.mesoscale.com/~/media/files/handout/msd%20gold%20sulfo-tag%20conjugation%20quick%20guide.pdf

2MSD® Biotin Conjugation Quick Guide
https://www.mesoscale.com/~/media/files/handout/biotin%20conjugation%20quick%20guide%20v2.pdf

3MSD® GOLD Streptavidin and Avidin Plates Quick Guide
https://www.mesoscale.com/~/media/files/handout/msd%20gold%20strep_avidin%20plates%20quick%20guide.pdf

[STEPS]
SECTION: Buffer Exchange the Antibodies
2. Chill PBS (or MSD Conjugation Buffer) and ultrapure water on ice.

SECTION: Buffer Exchange the Antibodies
3. Equilibrate Zeba Spin Desalting Columns, MSD Storage Buffer, and Sulfo-NHS-LC-Biotin at Room temperature.

SECTION: Buffer Exchange the Antibodies
3.1. Use one Zeba column per 70 µL of antibody.

SECTION: Buffer Exchange the Antibodies
3.2. In order to both biotinylate and sulfo-tag the antibody, you need at least 140 µL of antibody at an optimal concentration of 1.0 mg/mL.
SECTION: Biotinylate the Antibody
14. 
 

Calculate how much Sulfo-NHS-LC-Biotin you need per antibody, using the following formula:
1,000 × ([Concentration of antibody in mg/mL]/ 150,000 Da) × 20 × 70 µL of antibody = nmol of Biotin needed
This nmol of Biotin needed divided by 0.5 nmol/μL Biotin reagent = μL of Sulfo-NHS-LC Biotin needed
See attached examples at the end of this document
SECTION: Buffer Exchange the Antibodies
3.3. It still possible to move forward with a less concentrated sample.
SECTION: Buffer Exchange the Antibodies
3.4. Dilute the antibodies with ice-cold PBS if necessary.
SECTION: Buffer Exchange the Antibodies
4. Remove the Zeba columns' bottom closure and loosen the cap.
 

SECTION: Buffer Exchange the Antibodies
5. Place the column in a collection tube to remove the storage buffer.
SECTION: Buffer Exchange the Antibodies
6. Spin at 1500 x g, 1 min. Empty collection tube.

SECTION: Buffer Exchange the Antibodies
7. Wash 1: Add 300 µL of PBS (or MSD Conjugation Buffer) to the column. Spin at 1500 x g, 1 min. Empty collection tube.

SECTION: Buffer Exchange the Antibodies
8. Wash 2: Add 300 µL of PBS (or MSD Conjugation Buffer) to the column. Spin at 1500 x g, 1 min. Empty collection tube.

SECTION: Buffer Exchange the Antibodies
9. Wash 3: Add 300 µL of PBS (or MSD Conjugation Buffer) to the column. Spin at 1500 x g, 3 min. Empty collection tube.

SECTION: Buffer Exchange the Antibodies
10. Change collection tube to a clean Eppendorf tube for sample recovery. Label one Eppendorf tube/sample for each sample to be biotinylated or sulfo-tagged.
SECTION: Buffer Exchange the Antibodies
11. Pipette 70 µL of the antibody to the spin column.

SECTION: Buffer Exchange the Antibodies
12. Spin at 1500 x g for 3-4 minutes.
SECTION: Buffer Exchange the Antibodies
13. Save the eluent on ice. 
 


SECTION: Biotinylate the Antibody
15. Add 180 µL of ultrapure H2O to the 1 mg vial to Sulfo-NHS-LC-Biotin.

SECTION: Biotinylate the Antibody
16. Dilute the Sulfo-NHS-LC-Biotin by adding 10 µL of the stock to 190 µL of cold water. Once formed, this is highly unstable and should be used immediately.

SECTION: Biotinylate the Antibody
17. Add the calculated volume of diluted reconstituted Sulfo-NHS-LC-Biotin to each antibody.
SECTION: Biotinylate the Antibody
18. Let the antibody and biotin incubate at Room temperature for 120 min in the dark.
SECTION: Sulfo-tag the Antibody
19. 
 

Calculate how much SULFO-TAG NHS-Ester you need per antibody, using the following formula:
1,000 × ([Concentration of antibody in mg/mL]/ 150,000 Da) × 20 × 70 µL of antibody = nmol of Sulfo-Tag reagent needed
This nmol of Sulfo-Tag needed divided by 3.0 nmol/µL  = µL of sulfo-tag ester solution needed
See attached examples at the end of this document

SECTION: Sulfo-tag the Antibody
20. Add 50 µL of ice-cold ultrapure H2O to the 150 nmol SULFO-TAG NHS-Ester Vial to make a 3 nmol/μL solution. Once formed, this is highly unstable and should be disposed.

SECTION: Sulfo-tag the Antibody
21. Vortex the solution lightly.
SECTION: Sulfo-tag the Antibody
22. Add the calculated volume of diluted reconstituted SULFO-TAG NHS-Ester reagent to each antibody.
SECTION: Sulfo-tag the Antibody
23. Let the antibody and SULFO-TAG NHS-Ester incubate at Room temperature for 120 min in the dark.

SECTION: Clean-up the Biotinylated Antibody
24. Equilibrate Zeba Spin Desalting Columns, MSD Storage Buffer at Room temperature.

SECTION: Clean-up the Biotinylated Antibody
25. Place the column in a collection tube to remove the storage buffer.
SECTION: Clean-up the Biotinylated Antibody
26. Remove the columns' bottom closure and loosen the cap
 

SECTION: Clean-up the Biotinylated Antibody
27. Spin at 1500 x g, 1 min. Empty collection tube.
SECTION: Clean-up the Biotinylated Antibody
28. Wash 1: Add 300 µL of MSD Conjugate Storage Buffer to the column. Spin at 1500 x g, 1 min. Empty collection tube.

SECTION: Clean-up the Biotinylated Antibody
29. Wash 2: Add 300 µL of MSD Conjugate Storage Buffer to the column. Spin at 1500 x g, 1 min. Empty collection tube.

SECTION: Clean-up the Biotinylated Antibody
30. Wash 3: Add 300 µL of MSD Conjugate Storage Buffer to the column. Spin at 1500 x g, 3 min. Empty collection tube.

SECTION: Clean-up the Biotinylated Antibody
31. Change collection tube for sample recovery.
SECTION: Clean-up the Biotinylated Antibody
32. Pipette 70 µL of the unpurified biotinylated antibody to the spin column.

SECTION: Clean-up the Biotinylated Antibody
33. Spin at 1500 x g for 3-4 minutes.

SECTION: Clean-up the Biotinylated Antibody
34. Save the eluent on ice. 
 


SECTION: Cleanup the Sulfo-Tagged Antibody
35. Equilibrate Zeba Spin Desalting Columns, MSD Storage Buffer at 4 Room temperature.

SECTION: Cleanup the Sulfo-Tagged Antibody
36. Remove the columns' bottom closure and loosen the cap.
 

SECTION: Cleanup the Sulfo-Tagged Antibody
37. Place the column in a collection tube to remove the storage buffer.
SECTION: Cleanup the Sulfo-Tagged Antibody
38. Spin at 1500 x g, 1 min. Empty collection tube.
SECTION: Cleanup the Sulfo-Tagged Antibody
39. Wash 1: Add 300 µL of MSD Conjugate Storage Buffer to the column. Spin at 1500 x g, 1 min. Empty collection tube.

SECTION: Cleanup the Sulfo-Tagged Antibody
40. Wash 2: Add 300 µL of MSD Conjugate Storage Buffer to the column. Spin at 1500 x g, 1 min. Empty collection tube.

SECTION: Cleanup the Sulfo-Tagged Antibody
41. Wash 3: Add 300 µL of MSD Conjugate Storage Buffer to the column. Spin at 1500 x g, 3 min. Empty collection tube.

SECTION: Cleanup the Sulfo-Tagged Antibody
42. Change collection tube for sample recovery.
SECTION: Cleanup the Sulfo-Tagged Antibody
43. Pipette 70 µLof the unpurified SULFO-TAG NHS-Ester tagged antibody to the spin column.

SECTION: Cleanup the Sulfo-Tagged Antibody
44. Spin at 1500 x g for 3-4 minutes.

SECTION: Cleanup the Sulfo-Tagged Antibody
45. Save the eluent on ice. 
 


SECTION: Day 1 of MSD: Coating the Plate with Capture Antibody
46. Dilute biotinylated capture antibodies in 1x DPBS to your desired concentration.
SECTION: Day 1 of MSD: Coating the Plate with Capture Antibody
47. Add 30 µL of the diluted antibody to the corner of each well of the MSD 96 Streptavidin Small. Spot Plate according to the plate format. 
 

SECTION: Day 1 of MSD: Coating the Plate with Capture Antibody
48. Tap the plate on its edges to ensure the antibody is spread evenly across the well.
SECTION: Day 1 of MSD: Coating the Plate with Capture Antibody
50. Store in the plate at 4 °C to incubate 120 min without shaking.

SECTION: Day 2 of MSD
51. Tap out the plate to dispose of the capture antibody.
SECTION: Day 2 of MSD
52. Add 150 µL/well of Blocking Solution (3% Blocker A (or BSA) + PBS) per well.
 
      3% Blocker A in 1x PBS (Store at 4°C)    For 100 mL    Blocker A  3 g    1X PBS  100 mL    (Stir or   shake overnight at 4°C to make sure it is dissolved completely.)  
 
SECTION: Day 2 of MSD
53. Seal the plate and incubate at 4 Room temperature with shaking at  .

SECTION: Day 2 of MSD
54. Prepare the lysate samples according to plate layout, by diluting the protein samples in the lysate buffer to the desired lysate concentrations.
SECTION: Day 2 of MSD
55. Tap out the plate.
SECTION: Day 2 of MSD
56. Wash 1x with 150 µL/well of PBS - T (0.05% Tween).

SECTION: Day 2 of MSD
57. Discard the wash solution, without letting the plate dry.
SECTION: Day 2 of MSD
58. Add 25 µL/well of the lysate to the target wells. 
 


SECTION: Day 2 of MSD
59. Seal the plate and incubate at 4 Room temperature with shaking at   for 1-2 hours.

SECTION: Day 2 of MSD
60. While the lysates incubate, prepare the detection antibodies (your sulfo-tagged antibodies) in 1% MSD Blocker A in 1x DPBS.
SECTION: Day 2 of MSD
61. Discard the lysate solution. 
 

SECTION: Day 2 of MSD
62. Wash with PBS - T (0.05% Tween).
SECTION: Day 2 of MSD
63. Discard the wash solution, but do not let the plate dry.
SECTION: Day 2 of MSD
64. Add 25 µL/well of sulfo-tag antibodies (in 1% Blocker A in DPBS) to the plate layout.

SECTION: Day 2 of MSD
65. Seal the plate and incubate at 4 Room temperature with shaking at  .

SECTION: Day 2 of MSD
66. Tap out the plate.
SECTION: Day 2 of MSD
67. Wash with PBS - T (0.05% Tween).
SECTION: Day 2 of MSD
68. Tap out the plate.
SECTION: Day 2 of MSD
69. Add 150 µL/well of Read Buffer A using reverse pipetting to avoid making bubbles. 
 

SECTION: Day 2 of MSD
70. Read the plate immediately.
SECTION: Day 2 of MSD
62.1. Wash with 150 µL/well of PBS - T (0.05% Tween). (1/3)

SECTION: Day 2 of MSD
62.2. Wash with 150 µL/well of PBS - T (0.05% Tween). (2/3)
SECTION: Day 2 of MSD
62.3. Wash with 150 µL/well of PBS - T (0.05% Tween). (3/3)
SECTION: Day 2 of MSD
67.1. Wash with 150 µL/well of PBS - T (0.05% Tween). (1/3)
SECTION: Day 2 of MSD
67.2. Wash with 150 µL/well of PBS - T (0.05% Tween). (2/3)
SECTION: Day 2 of MSD
67.3. Wash with 150 µL/well of PBS - T (0.05% Tween). (3/3)
SECTION: Sample calculations
71. 
 

SECTION: Sample Biotinylation Calculations
72. Poly-GR MABN778 and Poly-PR ABN1354
  
4.67 nmol of sulfo-NHS-LC Biotin required
SECTION: Sample Biotinylation Calculations
73. 
  
9.34 µL of sulfo-NHS-LC Biotin stock solution .

SECTION: Sample Biotinylation Calculations
74. Poly-GA MABN889
  
3.08 nmol of sulfo-NHS-LC Biotin required

SECTION: Sample Biotinylation Calculations
75.   
6.16 µL of sulfo-NHS-LC Biotin stock solution.

SECTION: Sample Sulfo-tag Calculations
76. Poly-GR MABN778 and Poly-PR ABN1354
  
4.67 nmol of sulfo-tag reagent required.

SECTION: Sample Sulfo-tag Calculations
77.   
1.56 µL of sulfo-tag ester solution .

SECTION: Sample Sulfo-tag Calculations
78. Poly-GA MABN889
 
3.08 nmol of sulfo-tag reagent required.

SECTION: Sample Sulfo-tag Calculations
79.   
1.03 µL of sulfo-tag ester solution.

SECTION: Day 1 of MSD: Coating the Plate with Capture Antibody
49. Seal the plate with parafilm to avoid loss of antibody due to evaporation.
SECTION: Buffer Exchange the Antibodies
1.