Jul 29, 2025

Sandwich ELISA for the quantification of alpha-synuclein

DOI
https://dx.doi.org/10.17504/protocols.io.q26g748jqgwz/v1
  • 1Ottawa Hospital Research Institute;
  • 2Paracelsus-Elena-Klinik, Kassel, Germany
Julianna Tomlinson
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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.
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Materials
Materials:

Coating buffer:
AB
NaHCO3 200 mM
Sodium azide (pH9.6)0.02%
Blocking buffer:
AB
PBS
Gelatin2.50%
Tween0.03%
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 200 millimolar (mM) NaHCO3 with 0.02% sodium azide (9.6 ).

1 0 antibody most used is syn1 at 1:500 dilution (BD 610787).
Add 25 µL per well (avoiding outer wells).

Incubate on rocker at 4 °C Overnight .

Blocking - Day 2
Make blocking buffer (PBS+2.5% gelatin, 0.025% tween).
Wash plate 3X using 1xPBS-T in plate washer.
Blot on paper towel.
Add 80 µL per well of blocking buffer.

Incubate at 37 °C for 01:00:00 .

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

Samples- Day 2
Standards – use full length recombinant alpha-synuclein. Stock aliquoted upon opening.
Make serial dilutions in blocking buffer, starting from 10 Mass Percent stock.

Dilute samples in blocking buffer.
Wash plate 3X, blot on paper towel.
Add 25 µL per well of standards and samples (in duplicate).

Incubate at Room temperature for 02:00:00 .

2h
Detection antibody - Day 2
Bring blocking buffer to Room 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.
Add 25 µL per well of detection antibody.

Incubate at 37 °C for 01:00:00 .

1h
Avidin-alkaline phosphatase - Day 2
Wash plate 5X, blot on paper towel.
Use avidin-alkaline phosphatase at 0.6 Mass Percent of blocking buffer.

Add 25 µL per well of AAP.

Incubate at 37 °C for 00:30:00 .

30m
Substrate - Day 2
1h 2m 30s
Make pNPP - 1 of each tablet for 5 mL ddH2O (cover tube in aluminum, rock to dissolve).

Wash plate 5X, blot on paper towel.
Add 25 µL /well of pNPP, read plate immediately.

Read absorbance at 405nm, every 2.5 minutes for 01:00:00 .

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