Jul 29, 2025

Public workspaceSandwich ELISA for the quantification of alpha-synuclein

DOI
https://dx.doi.org/10.17504/protocols.io.q26g748jqgwz/v1
  • Nathalie Lengacher1,
  • Brit Mollenhauer2,
  • Michael G. Schlossmacher1
  • 1Ottawa Hospital Research Institute;
  • 2Paracelsus-Elena-Klinik, Kassel, Germany
Julianna Tomlinson
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.q26g748jqgwz/v1
Protocol Citation: Nathalie Lengacher, Brit Mollenhauer, Michael G. Schlossmacher 2025. Sandwich ELISA for the quantification of alpha-synuclein. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g748jqgwz/v1
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
We use this protocol and it's working.
Created: January 26, 2022
Last Modified: July 29, 2025
Protocol Integer ID: 57479
Keywords: Sandwich ELISA , alpha-synuclein, synuclein from tissue, synuclein this protocol, synuclein, biological fluids by elisa, sandwich elisa for the quantification, sandwich elisa, biological fluid
Abstract
This protocol details the procedure to quantify total alpha-synuclein from tissue and biological fluids by ELISA.
Attachments
Icon for gegmbqbhp.pdf
gegmbqbhp.pdf
123KB
Materials
Materials:

Coating buffer:
AB
NaHCO3 200 mM
Sodium azide (pH9.6)0.02%
Blocking buffer:
AB
PBS
Gelatin2.50%
Tween0.03%
Troubleshooting
Sandwich ELISA for alpha-synuclein

Note
Note: Antibody pairing determined based on sample type and should be optimized.

Coating - Day 1
Coat 384-well plate with primary antibody.
Make Coating buffer Concentration200 millimolar (mM) NaHCO3 with 0.02% sodium azide (Ph9.6 ).

1 0 antibody most used is syn1 at 1:500 dilution (BD 610787).
Add Amount25 µL per well (avoiding outer wells).

Pipetting
Incubate on rocker at Temperature4 °C DurationOvernight .

Incubation
Overnight
Blocking - Day 2
Make blocking buffer (PBS+2.5% gelatin, 0.025% tween).
Wash plate 3X using 1xPBS-T in plate washer.
Wash
Blot on paper towel.
Add Amount80 µL per well of blocking buffer.

Pipetting
Incubate at Temperature37 °C for Duration01:00:00 .

1h
Incubation
During incubation, prepare standards and samples TemperatureOn ice , dilute in blocking buffer (BB).

Incubation
Samples- Day 2
Standards – use full length recombinant alpha-synuclein. Stock aliquoted upon opening.
Make serial dilutions in blocking buffer, starting from Concentration10 Mass Percent stock.

Dilute samples in blocking buffer.
Wash plate 3X, blot on paper towel.
Wash
Add Amount25 µL per well of standards and samples (in duplicate).

Pipetting
Incubate at TemperatureRoom temperature for Duration02:00:00 .

2h
Incubation
Detection antibody - Day 2
Bring blocking buffer to TemperatureRoom temperature before use.

Dilute detection antibody in blocking buffer.
  • 2 o antibody most used is biotinylated-hSA4, at 1:200 dilution (commercially MJFR1 ab138501; biotinylated in-lab).
Wash plate 3X, blot on paper towel.
Wash
Add Amount25 µL per well of detection antibody.

Pipetting
Incubate at Temperature37 °C for Duration01:00:00 .

1h
Incubation
Avidin-alkaline phosphatase - Day 2
Wash plate 5X, blot on paper towel.
Wash
Use avidin-alkaline phosphatase at Concentration0.6 Mass Percent of blocking buffer.

Add Amount25 µL per well of AAP.

Pipetting
Incubate at Temperature37 °C for Duration00:30:00 .

30m
Incubation
Substrate - Day 2
1h 2m 30s
Make pNPP - 1 of each tablet for Amount5 mL ddH2O (cover tube in aluminum, rock to dissolve).

Pipetting
Wash plate 5X, blot on paper towel.
Wash
Add Amount25 µL /well of pNPP, read plate immediately.

Pipetting
Read absorbance at 405nm, every 2.5 minutes for Duration01:00:00 .

1h
Use data from a read when the standards are linear.