Bitan:ELONA

This ELONA protocol for Aβ concentration works with soluble oligomers and fibrils of Aβ(40) and Aβ(42). This protocol is nearly identical to Bitan:ELISA with a change in the Primary Antibody.

=Ingredients=

Buffers
PBS PBST 0.1% Tween_20

Coating Buffer
Carbonate/bicarbonate (pH 9.6) 100 ml 	0.293 g		NaHCO3 (anhydrous) 0.159 g		Na2CO3 (anhydrous)
 * Use carbonate/bicarbonate to adjust pH to 9.6*

Blocking Buffer
2% Amersham ECL Advance Blocking Reagent in PBST

Phosphate/Citrate Buffer
50 mM citric acid and 100 mM sodium phosphate 0.74 g dibasic sodium phosphate (anhydrous) 1.3 g citric acid (anhydrous) Dissolve in 50 ml water *Adjust pH to 5.0* *Add 40 μl fresh 30% H2O2 to 100 ml solution before use*

Reagents
Monoclonal 6E10 Biotinylated KM41 (ssDNA aptamer) Streptavidin-HRP o-Phenylene Diamine (OPD 1N Sulfuric Acid =Recipe=

Coating

 * Dilute 6E10 (1mg/ml stock) 1:1000 in Coating Buffer to make the Coating Solution
 * Add 200ul Coating Solution to each well of a 96-well flat-bottom microtiter plate
 * Incubate at room temperature with shaking for 2 hours or overnight in the cold room

Block

 * Add 200ul Blocking Buffer
 * Incubate at room temperature with shaking for at least two hours or overnight in the cold room

Incubation with Aβ preparation

 * Prepare a 9x two-fold dilution series of the Aβ preparation from 500nM using PBS as diluent
 * Add 100ul of each standard to the plate's wells in triplicate or quadruplicate
 * Add 100ul of PBS in triplicate or quadruplicate as a control
 * Incubate at room temperature for 1.5-2h

Primary Antibody

 * Dilute Biotinylated-KM41 (~1ug/ml stock) 1:1000 in Blocking Buffer
 * Add 100ul of the primary antibody solution to each well
 * Incubate at room temperature with shaking for 1-1.5h
 * Wash the plate with four 10min washes with 110ul PBST per well

Secondary Antibody

 * Dilute streptavidin-HRP (1.5mg/ml stock) 1:10000 in Blocking Buffer
 * Add 100ul of the s-HRP solution to each well
 * Incubate at room temperature with shaking for 1-1.5h
 * Wash the plate with four 10min washes with 110ul PBST per well
 * Tap the plate dry on a pad of Kimwipes

Color Development and Detection

 * Dissolve four 1mg OPD tablets in 10ml Phosphate/Citrate Buffer
 * Add 4ul 30% H2O2
 * Add 100ul of the OPD solution to each well
 * Incubate for 10min in the dark
 * Stop the reaction by adding 100ul 1N Sulfuric Acid per well
 * Measure the absorbance at 490nm with the plate reader