Detecting Neurodegenerative Disease-Associated Protein α-Synuclein Using ELISA

Abstract

Source: Bétemps, D., et al., Detection of Disease-associated α-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated α-synuclein. J. Vis. Exp. (2015).

This video demonstrates the procedure to detect neurodegenerative disease-associated protein α-synuclein in brain tissue from transgenic mice using ELISA. The steps include tissue homogenization, centrifugation, and ELISA detection of α-synuclein protein to quantify its disease-associated forms.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. α-Synuclein (αS) Extraction from Dissected Brain Regions

1. Dissect a whole brain in a 35 mm plastic petri dish on ice with a low power magnifier (8X magnification) using two forcipes whose ends are kept together except when dissecting the hippocampus. Do not exceed 10 min to preserve brain integrity. Place the brain right side up and retrieve the following brain regions in this order:
   1. Separate one of the two olfactory bulbs using forceps placed just behind it. Detach it from the brain by moving it downward. Repeat this operation for the second bulb.
   2. Gently wedge the forceps in between the two cortices and move it forward to facilitate dissociation of the two cortices. Keeping the brain in place with one forceps, use another to separate the cortex from the hippocampus.
   3. Position the forceps 2 mm below the cortex. Maintain a gentle pressure on the forceps until the top of the hippocampus is visible. Peel off the first part of the cortex, and repeat with the second part. Use the forceps to separate the two cortices starting at the hippocampus and moving towards the front of the brain.
   4. Position the open forceps around one of the hippocampi. Close the forceps at the bottom of the hippocampus then gently remove it, recovering as much as possible. Repeat the procedure for the second hippocampus.
   5. Position the open forceps below one of the striata and gently separate it from the brain. Use the forceps to remove any remaining cortex from the striatum. Repeat this procedure for the second striatum.
   6. Use the forceps to gently depress the contour of the cerebellum by 2 mm to facilitate its separation from the brain. Place the forceps just behind the cerebellum and remove them by moving the forceps forward.
   7. Use the wide part of the forceps to raise the mesencephalon in order to clearly see where it joins the brain stem. Make a vertical incision at the junction then remove the brain stem.
   8. Position the forceps behind the mesencephalon, which is composed of four rounded structures. Incise vertically until the mesencephalon has been completely separated from the remaining brain.
2. Prepare homogenates of variable percentage (weight/volume) in high salt (HS) buffer, depending on the number of available tissues: 5% homogenate for a weight between 10 and 30 mg, 10% for a weight between 30 and 80 mg, and 20% for a weight above 80 mg.
   1. Add an adequate volume of HS buffer to the dissected brain regions to obtain the expected % of homogenate.
   2. Vortex and check that the tissues are fully immersed in the HS buffer.
   3. Homogenize samples prepared from the dissected brain regions or the cervical spinal cord with a tissue grinder composed of a borosilicate glass tube and two pestles, A and B.
   4. Pour each brain region to be crushed directly into the tube. Insert pestle A into the tube and retract it. Repeat this movement about ten times to dissociate the tissue. Then use pestle B to continue grinding the tissue with a further 20 movements. Transfer the homogenates into a 1.5 ml tube with a 1 ml transfer pipette.
3. Centrifuge the samples at 1,000 x g for 5 min at 4 °C to eliminate any unground brain fragments. Retrieve the supernatants, divide them up into 200 µl aliquots, and keep them at -80 °C for subsequent analysis.

1. Detection of αS by ELISA

1. Dilute the coating antibodies to 0.01 ng/ml. Use either anti-αS rabbit polyclonal or monoclonal clone 42 antibody in 50 mM Na₂CO₃/NaHCO₃ buffer (pH 9.6).
2. Coat the 96-well microplates with 100 µl per well of this coating solution, and leave at 4 °C O/N. Use the anti-αS rabbit polyclonal antibody in the coating solution for ELISAs using detection antibodies syn514, clone 42, LB509, AS11, 4D6, or 8A5. Use the anti-αS monoclonal antibody clone 42 as a coating solution in combination with the anti-pSer129 αS detection antibody.
   NOTE: If necessary, the plates may be kept at 4 °C for one week before the ELISA is performed.
3. Use a plate washer to wash the plates five times with 300 µl of phosphate-buffered saline with 0.05% Tween 20 (PBST) per well. From this step onward, incubation is at RT.
4. Add 200 µl of T20 PBS (phosphate-buffered saline) blocking buffer per well. Shake for 1 hr at 150 rpm. Wash the plates five times with PBST.
5. Dilute the brain homogenates (dilution 1:100 of the 20% homogenates, 1:50 of the 10% homogenates, and 1:25 of the 5% homogenates in PBST BSA 1%), and add 100 µl to each well. Then incubate for 2 hr while shaking at 150 rpm. Wash the plates five times with PBST.
6. Add the different αS detection antibodies in PBST with bovine serum albumin (BSA) 1% at the dilutions mentioned in the Materials List. Incubate for 1 hr at 150 rpm. Wash the plates five times with PBST.
7. Add either anti-mouse or anti-rabbit IgG HRP conjugates diluted 1:8,000 in PBST supplemented with BSA 1% for 1 hr while shaking at 150 rpm. Wash the plates five times with PBST.
8. Add 100 µl of 3,3’,5,5’-tetramethylbenzidine (TMB) solution to each well and incubate for 15 min in the dark while shaking at 150 rpm.
9. Stop the reaction by adding 100 µl of 1 N HCl per well then measure the absorbance at 450 nm with the microplate reader.
10. For data analysis, subtract the OD value obtained in a well with all the reagents except any mouse brain samples (blank well) from the OD values measured for each of the analyzed samples.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
LB509		Abcam	ab27766	Detection antibody 1/2000
AS11		Produced at Anses		Detection antibody 1/1000
4D6		Abcam	ab1903	Detection antibody 1/2000
PSer129		Abcam	ab59264	Detection antibody 1/3000
PSer129 EP1536Y		Abcam	ab51253	Detection antibody 1/1000
Syn514		Abcam	ab24717	Detection antibody 1/500
Clone 42		BD Biosciences	610787	Coating and detection antibody (1/2000)
8A5		Provided by Dr. Anderson		Detection antibody 1/2000
Polyclonal anti-alpha syn antibody		Millipore	AB5038P	Coating antibody
Anti-mouse IgG HRP conjugate		Southern Biotech	1010-05
Anti-rabbit IgG HRP conjugate		Southern Biotech	4010-05
Goat anti-mouse IgG HRP conjugate		Dianova	115-035-164
HS buffer	Tris-HCl 50 mM	Euromedex	26-128-3094-B	Adjust at pH 7.5 and keep at 4°C
NaCl 750 mM	Euromedex	1112-A
EDTA 5 mM	Euromedex	EU0007-B
DTT 1 mM	Sigma	43815
PBS	Disodium hydrogen phosphate1 mM	Euromedex	1309	Adjust at pH 7.5
Monopotassium phosphate 1.5 mM	Euromedex	2018
NaCl 137 mM	Euromedex	1112-A
KCl 2.7 mM	Euromedex	P017
Tween 20		Euromedex	2001-C
BSA		Sigma	A7906
Microplate MaxiSorpTM		Thermo Scientific	442404
Tampon carbonate 50 mM pH 9.6	Sodium carbonate decahydrate	Sigma	71360	2.86 g/L
Sodium bicarbonate	Merk	6329	3.36 g/L, pH9.6
Superblock T20 PBS blocking buffer		Pierce	E6423H	10 X concentrated
TMB		Sigma	T0440	Used for ELISA
TMB		Analytik Jena AG	847-0104200302	Used for epitope mapping
HCl 1N		Chimie plus	40030
Ribolyser		Thermo	Fast prep FP120	Keep on ice at this step
Grinding tubes		Biorad	355-1197
Plate washer		Tecan	Columbus Pro
Plate reader		Biorad	Model 680
Low power magnifier		VWR	630-1062	X8 magnification
Forceps Dumont#7		WPI	14097	For dissection steps
Transfer pipette 1ml Samso		Samso	43231
1.5 ml tubes		Dutscher	33290