COMPETITION ELISA
Cameron Haase-Pettingell

Last updated: 6/21/01

Day 1

• Coat wells of at least four (4) 96-well plates with 100 µl of 1 µg/ml native tailspike in PBS; leave Column 1 and Rows A and H blank
• Incubate plates @ RT for 3 hr, or store at 4°C for up to two months (wrap to prevent dehydration) with the plate tape and in a zip lock bag.
• Dilute 1-50 µl of sample to 250 µl with PBS+TWEEN; make it larger then take out the 250 µl.
• Antibodies Dilution:
-Low Kd 10^-11 mix with 250 µl of .08 µg/ml 1° Ab
-Hi Kd 10^-8 about 0.02 µg/ml
-aB124 0.04 µg/ml
• Native tspk dilution series for standard curve 0.625 - 0.0391 µg/ml tailspike, 1:1 dilution
• Mix sample with 1°Ab. 250 µl/tube. Do 1°Ab positive controls (2 per plate), mixed with 250 µl PBS+TWEEN. 6.6 ml/plate. For all, seal, wrap and store overnight @ 4° C with the plate tape
-For conversion of % Recognition to [Native Tspk Ag], it is really necessary to use % Rec. values below 70% (below 60% is ideal).

Day 2

• Wash plate wells with PBS+TWEEN, 3x3 min dump out and slam dry, add with the filler
• Sample Aliquot - 100 µl of mixtures into four wells of one column: three coated wells and one uncoated control (for example, Column 2 Row A-D); repeat for all samples, standards and blanks. DO NOT put sample in Column 1; save as blank.
NOTE: Use timer to regulate rate of sample addition, starting a new sample every 20 sec.
• Incubate samples 30 min @ RT; aspirate out samples at same rate as sample addition (starting new sample every 20 sec); when not aspirating, refill emptied wells with 200 µl PBS+TWEEN, using distriman washes
• Wash 3 x 3 min each
• 2°Ab Aliquot 100 µl (1:2000 dilution of goat anti-mouse alkaline phosphatase) into each sample well (do not fill Column 1)
• Incubate 2°Ab 30 min @ RT
• wash wells 3x
• Aliquot 100 µl PNPP substrate solution into each well; incubate until bright yellow (~60 min; check color development with plate reader) 2.5 ml/tablet 10 m./plate
• Read A₄₀₅ of each plate

REAGENTS

• 10x PBS: 80g NaCl, 2 g KH₂PO₄, 11.1 g Na₂HPO₄ (anhydrous), 2 g KCl, to 1 L w/ H2O; dilute 1/10 to use
• PBS+TWEEN: PBS plus 0.05% Tween-20 (from a 10% stock solution)
• PNPP substrate solution: 1 M ethanolamine, 1 mM MgSO₄, to pH 9.8 with HCl; use 2.5 ml of this to dissolve each 5 mg tablet of PNPP