1.Plates: Nunc-Immuno 96-Well Plates (Maxisorp Surface Treatment) (Fischer Cat. No. 12-565-136)
Primary: Monoclonal mAb211, 1:1000 dilution for coating, store at -20°C
Secondary: Polyclonal FL140, 1:10000 dilution for incubation, store at 2-8°C
3. 30% Hydrogen Peroxide (H2O2) (Fischer Cat. No. H325-100; $62.44 for 100ml bottle)
4. o-Phenylenediamine dihydrochloride (OPD) (1mg tablets) (Sigma Product No. P6662-50TAB; $150.50 for 50 tablets)
5. 96-well microtiter plate (Fischer Cat. No. 12-565-136; $183.25 for 60 plates)
6. 1N Sulfuric acid (Fischer Cat. No. 83004; $8.89 for 120ml bottle)
B.Buffers and Solutions
1.Coating Buffer: Carbonate/bicarbonate (pH 9.6) 100ml
**Use carbonate/bicarbonate to adjust pH to 9.6**
2.PBS-Tween (PBST): 0.05% (v/v) Tween-20 in PBS (50μl Tween-20 in 100ml PBS)
3.PBST-BSA: PBS w/ 0.05% (v/v) Tween-20 and 0.5% (w/v) BSA (250mg BSA in 50ml PBST)
4.Phosphate-Citrate: 50mM citric acid and 100mM sodium phosphate
0.74g Dibasic sodium phosphate and 1.3g Citric acid
Dissolve in 50ml water
Adjust pH to 5.0
Add 40ul fresh 30% H2O2 to 100ml solution before use
5.Blocking: 0.5% (w/v) BSA in PBS 500mg BSA 100ml PBS
6.HRP substrate: (OPD) in phosphate-citrate buffer
Dissolve 1mg OPD tablet in 2.5ml buffer
Vortex to mix
Do not touch tablet with fingers or metallic forceps
7.Stopper: 1N Sulfuric Acid
8-well multichannel pipette
Airtight sandwich box
1 Dilute mAB211 1:1000 in Coating Buffer to approximately 2.5 μg/ml.
2 Add 100ul of mAB211 dilution from step 1 to each well of microtiter plate. Incubate overnight (o/n) @ 4°C sealed plate sealer.
3 Wash plates 3x w/ PBST. Aspirate and blot dry with Kimwipe.
4 Store individually in plastic bags @ -20°C. Plates may be stored for
1. Thaw plates before opening bags to prevent condensation in the wells.
2. Add 200ul Blocking Buffer to each well and incubate 1 hour @ room temperature (RT).
3. Wash 3x w/ PBST. Aspirate and blot dry.
4. Prepare standards and samples in PBS.
5. Add 100ul of dilutions to wells.
6. Incubate plate 2-3h @ RT.
7. Incubate o/n @ 4C.
8. Wash 3x w/ PBST. Aspirate and blot dry.
9. Dilute secondary antibody FL140 in PBST + 0.5% Bovine Serum Albumin (BSA).
10. Add 100ul diluted Ab to each well. Incubate 2h @ RT.
11. Wash 3x w/ PBST. Aspirate and blot dry.
12. Prepare Peroxidase Substrate immediately before use.
13. Add 100ul Peroxidase Substrate to each well and incubate 10min in the dark @ RT.
14. Add 100ul Stopper Solution to wells and read plate at 490nm.