Description: CD4 Capture ELISA.
Storage: Store Components as specified at 4°C/-75°C.
Stability: Kit should be used within 90 days.
Note: Read instructions and familiarize yourself with kit components before performing experiments with this kit.
A) Plate holder with solid phase capture ligand (gp120) strips (Store at-75°C)
B) Wash Buffer 10 x (Store at 4°C)
C) Blocking/Diluent Buffer (Store at 4°C)
D) CD4 Reference (Store at-75°C)
E) Detector Reagent (Anti-CD4 Peroxidase, Store at 4°C)
F) Substrate/TMB (Store at 4°C)
G) Stop Solution (Store at 4°C)
1. Bring components A, B, and C to room temperature (RT).
2. Dilute wash buffer (Component B) 1 : 10 with dionized water for use.
3. Remove plate holder with strips from shielded bag (Use scissors to cut the sealed end of bag).
4. Add 200µl of wash buffer to each well and leave at RT until ready for the next step.
Section-2 Capture Assay
1. Prepare several dilutions (minimum 200µl) of positive reference CD4(Component B, 1000ng/ml) in diluent buffer (Component C) in Eppendorf or comparable tubes - labeled accordingly: 500ng/ml to 0.5ng/ml in 2-fold serial dilutions.
2. Prepare test samples using diluent buffer (Component C) in 100ng/ml range. Unknown samples may be used in 1:2 to 1:10 dilution range with Diluent Buffer.
3. Continue from Section-1, Step 4: Dump contents of wells and pat dry. Set up strip well holder on a grid.
4. Pipette 100µl of positive reference (Step 1 above) and test samples into wells. Note position on the grid. Tap holder gently against table to dislodge air bubbles in wells. Cover plate and leave at RT for 1 hour.
5. Dump Contents of wells and wash 3 times with 1 x wash buffer (Component B, 300µl/well). Leave last wash until ready to add detector reagent. Pat dry wells.
6. Dilute detector reagent (Component E) 1:100 in diluent buffer (Component C). Add 100µl of detector reagent to each well. Leave plate at RT for 1 hour.
7. Repeat washing as in Step 5 above, leave last wash in wells until ready to add substrate.
8. Dump contents of wells from Step 7 above.
9. Add 100µl of TMB substrate (Component F) to each well. Color (Blue) will develop over 5-30 minutes at RT.
10. Stop color development after10 minutes (Step 9) by adding 50µl of stop solution (Component H) to each well. Color will change to yellow. Read plate at 450nm as soon as possible (no more than 15 minutes).
STRICTLY FOR NON-HUMAN RESEARCH