Bitan:ELISA

This ELISA protocol for Aβ concentration works with soluble oligomers and fibrils of Aβ(40) and Aβ(42).

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 4G8
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-4G8 (1mg/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