Immunoassay analysis of poly(GP)

POLY(GP) IMMUNOASSAY

The purified mouse monoclonal poly(GP) antibody (TALS 828.179) used for the poly(GP) assay was acquired from the Target ALS Foundation. Please see the following link for instructions on how to request this antibody: https://www.targetals.org/research/resources-for-scientists/resource-antibody-core/

Prior to commencing the assay, biotinylate a portion of the antibody according to the manufacturer’s protocol so it can be used as the capture antibody (Fisher, EZ-Link™ Sulfo-NHS-LC-Biotin, No-Weigh™ Format 8 x 1mg, Catalogue #21327). 

Using the remaining antibody portion, prepare Sulfo-tagged TALS 828.179 to be used as the detection antibody following the manufacturer’s protocol (Meso Scale Discovery, MSD Gold Sulfo-tag NHS Ester Conjugation Pack, Catalogue #R31AA-1).

1. On the first day of the assay, prepare the capture antibody solution to coat wells of MSD GOLD 96-well Streptavidin SECTOR plate(s) (Meso Scale Discovery, Catalog # L15SA-2). Taking into account the number of samples to be tested, the number of wells you will use for each sample (e.g., testing samples in singlet or duplicate), and the fact that 30 ul of capture antibody solution is used per well, calculate the total volume of capture antibody solution needed making sure to prepare 10% more in case there is loss due to pipetting. To prepare your capture antibody solution, dilute biotinylated TALS 828.179 to 2 ug/ml in Tris-buffered saline (TBS). Note: the antibody concentration can be adjusted as needed.
2. Add 30 ul of capture antibody per well. Shake the plate(s) at 1500 rpm for 15 seconds on a plate shaker. Make sure that bottoms of wells are evenly coated. Continue to shake at 600 rpm for 15 min.              
3.  Seal plate(s) tightly and incubate at 4°C overnight.           
4. Make Tris-buffered saline + 0.2% Tween (TBST; 2 ml of Tween in 1 L of TBS)
5. Make blocking buffer. To 100 ml of TBST add 3 g of BSA (Meso Scale Discovery, MSD Blocker A Kit, Catalog # R93AA-1). Mix well. Store at 4°C.
6. On the second day of the assay, flick off antibody solution and wash wells once with TBST using 150 ul/well.
7.  Flick off TBST, tap plate upside down on clean paper towel to remove any remaining buffer, and add blocking buffer using 150 ul/well. Seal plate(s) and incubate for 1 hour while shaking at 600 rpm at room temperature.
8. Near the end of the incubation period, prepare samples to be tested by diluting them in TBS to the desired concentration keeping in mind that no more than 50 ul of sample should be added per well, and that all samples should be tested at the same concentration and volume, ideally in duplicate or triplicate.
9. At end of the incubation period, remove blocking buffer and wash plate(s) 3 times with TBST (150 ul/well).
10. Add samples to respective wells. Seal Plate. Incubate plate for 1.5 h while shaking at 600 rpm at room temperature. 
11. Determine how much detection antibody solution will be needed such that 25 ul is added to each well. Dilute the Sulfo-tagged detection antibody in Blocking Buffer at a concentration of 4 ug/ml (the concentration may be adjusted as needed).
12.  Flick the plate(s) over a proper waste receptacle to remove samples, and wash plate(s) 3 times with TBST (150 ul/well).
13.   Remove last wash, tap plate(s) upside down on clean paper towel to remove any remaining buffer, and add 25 ul of detection antibody solution to each well. Seal Plate. Incubate plate for 1 hour while shaking at 600 rpm at room temperature.
14. Once incubation is complete, flick off detection antibody solution and wash plate(s) 3 times with TBST (150 ul/well). Remove last wash, tap plate upside down on clean paper towel to remove any remaining buffer. Add 150 ul of MSD GOLD 1X Read Buffer (Catalogue # R92TG). 
15. Read plate(s) on the MSD QUICKPLEX SQ120 platform (or equivalent MSD platform) using small spot mode.