TITLE: LEGEND MAX™ Human α-Synuclein ELISA Kit Protocol
AUTHORS: Sam Li

[STEPS]
?. [I. Preparation of 1X Wash Buffer]
Label a 1L bottle as “1X Wash Buffer”.
?. [I. Preparation of 1X Wash Buffer]
Dilute 5X Wash Buffer 1:5 using lab grade water* and mix well.*Note: Lab grade filtered water such as injection grade, cell culture grade, Reverse Osmosis De-Ionization (RODI).
?. [II. Preparation of 1X Reagent Diluent]
Label an appropriate sized bottle as “1X Reagent Diluent”.
?. [II. Preparation of 1X Reagent Diluent]
Dilute 2X Reagent Diluent to 1X by adding 30mL of 2X Reagent Diluent to 30mL of lab grade water in the bottle labeled “1X Reagent Diluent”.
?. [II. Preparation of 1X Reagent Diluent]
Mix well by vortex.
?. [III. Preparation of Standard Intermediates (Refer to Table 1)]
Reconstitute lyophilized standard with 50µL of lab grade water and mix well by vortex. The concentration after reconstitution will be 500µg/mL.
?. [III. Preparation of Standard Intermediates (Refer to Table 1)]
Label (2) 1.5mL microcentrifuge tubes as intermediate #1 & intermediate #2.
?. [III. Preparation of Standard Intermediates (Refer to Table 1)]
Aliquot 990µL of 1X Reagent Diluent into intermediate #1 & intermediate #2 tubes.
?. [III. Preparation of Standard Intermediates (Refer to Table 1)]
Remove 10µL from the vial of reconstituted standard and add to 990µL of 1X Reagent Diluent in intermediate tube #1. Mix well by vortex.
?. [III. Preparation of Standard Intermediates (Refer to Table 1)]
Remove 10µL from intermediate #1 and add to 990µL of 1X Reagent Diluent in intermediate #2 tube. Mix well by vortex.
?. [III. Preparation of Standard Intermediates (Refer to Table 1)]
The final concentration of intermediate tube #2 will be 50ng/mL.
?. [IV. Preparation of Standard Curve (Refer to Table 2)]
Label (8) 1.5mL microcentrifuge tubes as #1-8.
?. [IV. Preparation of Standard Curve (Refer to Table 2)]
Add 1280µL of 1X Reagent Diluent to tube #1 and 825µL of 1X Reagent Diluent to tubes #2-8.
?. [IV. Preparation of Standard Curve (Refer to Table 2)]
Remove 40µL from intermediate #2 and add to 1280µL of 1X Reagent Diluent in tube #1.
?. [IV. Preparation of Standard Curve (Refer to Table 2)]
Mix well by vortex (this will be the top point of the standard curve, 1500pg/mL).
?. [IV. Preparation of Standard Curve (Refer to Table 2)]
Remove 550µL from tube #1 and add to 825µL of 1X Reagent Diluent in tube #2.
?. [IV. Preparation of Standard Curve (Refer to Table 2)]
Mix well by vortex.
?. [IV. Preparation of Standard Curve (Refer to Table 2)]
Continue making 2.5 fold serial dilutions by adding 550µL ofthe previous dilution to 825µL of 1X Reagent Diluent in tubes #3-7. Mix well by vortex between each dilution. Note: Tube #8 will be the zero or blank sample and should only contain 1X Reagent Diluent.
?. [VI. Running the Assay - Day 1]
Remove the plate from the foil pouch.
?. [VI. Running the Assay - Day 1]
Add 300µL/well of 1X Wash Buffer.
?. [VI. Running the Assay - Day 1]
Dump out wash buffer and pat dry.
?. [VI. Running the Assay - Day 1]
Repeat 3 more times for a total of 4 washes.
?. [VI. Running the Assay - Day 1]
Add 200µL of each standard to the plate in duplicate or triplicate. Follow the plate layout below.Note: The volumes in Table 2 are sufficient for running the standard curve in triplicate.
?. [VI. Running the Assay - Day 1]
Add 200µL of each sample dilution to the plate.
?. [VI. Running the Assay - Day 1]
Cover the plate with the plate sealer provided.
?. [VI. Running the Assay - Day 1]
Incubate overnight at 2-8° C, while shaking.
?. [VII. Running the Assay - Day 2]
Notes: Allow kit components to be brought to room temperature before use.
?. [VII. Running the Assay - Day 2]
Label a 15mL centrifuge tube as “Biotinylated Primary Antibody”.
?. [VII. Running the Assay - Day 2]
Dilute the biotinylated primary antibody by adding 6µL* of the Biotinylated Primary antibody stock to 6mL of 1X Reagent Diluent in the 15mL tube labeled “Biotinylated Primary Antibody”*Note: A quick spin in a centrifuge is suggested prior to pipetting to ensure liquid is at the bottom of the vessel.
?. [VII. Running the Assay - Day 2]
Mix well by vortex.
?. [VII. Running the Assay - Day 2]
Remove plate from refrigerator and dump contents.
?. [VII. Running the Assay - Day 2]
Add 300µL/well of 1X Wash Buffer.
?. [VII. Running the Assay - Day 2]
Dump out wash buffer and pat dry.
?. [VII. Running the Assay - Day 2]
Repeat 3 more times for a total of 4 washes.
?. [VII. Running the Assay - Day 2]
Add 50µL/well of the Biotinylated Primary Antibody to the plate (prepared above).
?. [VII. Running the Assay - Day 2]
Cover and incubate for 2 hours at room temperature.
?. [VII. Running the Assay - Day 2]
Preparation of Streptavidin HRP (Refer to Table 4)
?. [VII. Running the Assay - Day 2]
Label a 1.5mL microcentrifuge tube as “HRP Intermediate”.
?. [VII. Running the Assay - Day 2]
Label a 50mL tube as “Diluted Streptavidin HRP”.
?. [VII. Running the Assay - Day 2]
Make the “HRP Intermediate” by adding 10µL* of Streptavidin-HRP stock to 990µL of 1X Reagent Diluent in the 1.5mL microcentrifuge tube labeled “HRP Intermediate”.*Note: A quick spin in a centrifuge is suggested prior to pipetting to ensure liquid is at the bottom of the vessel.
?. [VII. Running the Assay - Day 2]
Mix well by vortex.
?. [VII. Running the Assay - Day 2]
Remove 150µL from the HRP Intermediate tube and add to 22.35mL of 1X Reagent Diluent in the 50mL tube labeled “Diluted Streptavidin HRP”.
?. [VII. Running the Assay - Day 2]
Mix well by vortex.
?. [VII. Running the Assay - Day 2]
Remove plate from incubation and dump contents.
?. [VII. Running the Assay - Day 2]
Add 300µL/well of 1X Wash Buffer.
?. [VII. Running the Assay - Day 2]
Dump out wash buffer and pat dry
?. [VII. Running the Assay - Day 2]
Repeat 3 more times for a total of 4 washes.
?. [VII. Running the Assay - Day 2]
Add 200µL/well of Diluted Streptavidin HRP to the plate (prepared above).
?. [VII. Running the Assay - Day 2]
Cover and incubate for 1 hour at room temperature.
?. [VII. Running the Assay - Day 2]
Remove plate from incubation and dump contents.
?. [VII. Running the Assay - Day 2]
Add 300µL/well of 1X Wash Buffer.
?. [VII. Running the Assay - Day 2]
Dump out wash buffer and pat dry.
?. [VII. Running the Assay - Day 2]
Repeat 3 more times for a total of 4 washes.
?. [VII. Running the Assay - Day 2]
Mix chemiluminescent substrates for use:
?. [VII. Running the Assay - Day 2]
Add 5.5mL ofsubstrate A to a 15mL centrifuge tube.
?. [VII. Running the Assay - Day 2]
Add 5.5mL of substrate B to the same 15mL centrifuge tube.
?. [VII. Running the Assay - Day 2]
Mix well by vortex.
?. [VII. Running the Assay - Day 2]
Add 100µL of mixed substrate per well.Note: If reading multiple plates add substrate one plate at a time; do not add substrate to all plates at the same time. Add substrate to each plate immediately before reading.
?. [VII. Running the Assay - Day 2]
Shake plate on either a plate shaker or using the shaking mechanism within the plate reader for 10-15 seconds.
?. [VII. Running the Assay - Day 2]
Read plate immediately.Note: The recommended luminometer settings are to read at a mid-range sensitivity level for 1 second per well. These settings will vary between plate reader manufacturers, please consult your owner’s manual prior to performing this assay.