Description: HIV-1 Nef Capture ELISA.
Storage: Store components as specified.
Stability: See Kit Label
Note: Read instructions carefully and familiarize yourself with kit components before performing experiments with this kit. Perform assays away from direct light, HRP step must be performed in dark.
Some components of this kit are light sensitive. Do not expose to direct light.
A) Plate holder with solid phase capture ligand (Anti-nef Monoclonal Antibody) strips, store at 4oC
B) Wash Buffer 10 x, store at 4oC
C) Blocking/Diluent Buffer, store at 4oC
D) Recombinant Nef Reference, Lyophilized.
E) Detector Reagent, store at 4oC
F) Substrate, store at 4oC
G) Stop Solution, store at 4oC
1. Bring components A, B, C and D to room temperature.
2. Dilute wash buffer (Component B) 1: 10 with de-ionized 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 room temperature until ready for the next step.
1. Lyophilized nef reference (component D): Solubilize vial contents in 50ul MQ water (cold). Vortex gently to solubilize protein. This nef reference may be frozen at -20C for further use- 4 freeze/thaw cycles, maximum.
2. Remove 5ul of the solubilized nef reference (Section 2,1 above) and quantitatively transfer to 1ml of the diluent buffer ( component C). Label nef reference dilution 1:1. This is positive nef reference for your assays and is stable for up to one week at 4C.
3. Serially dilute nef reference (Section 2, 2 above) in 4X dilution steps, 50ul nef reference 1:1 and 150ul diluent buffer to a final dilution of approximately 1:4000.
4. Prepare plasma/serum test samples using diluent buffer (Component C) in 1:2 to1:10 dilution range.
3. Continue from Section-2, Step 4 above: Pipette 100µl of each nef positive reference (Step 2 above) to wells 1A-1G, well IH as blank, and test samples (section2, step 4 above) into wells 2-12A/2-12H. Note position of reference and test samples on the grid. Tap plate holder gently against table to dislodge air bubbles in wells. Cover plate and leave at RT for 1 hour.
4. Dump Contents of wells and wash wells 3 times with 1 x wash buffer (Component B, 300µl/well). Leave last wash until ready to add detector reagent. Using a stack of paper towels, pat dry wells for the next step.
5. Dilute detector reagent (Component E) 1:100 in diluent buffer (Component C). Add 100µl of detector reagent to each well. Cover plate and leave at RT for 1 hour in dark.
6. Repeat washing as in Step 4 above, leave last wash in wells until ready to add substrate.
7. Dump contents of wells from Step 6 above. Pat dry wells for the next step.
8. Add 100µl of TMB substrate (Component F) to each well. Blue color will develop over a period of 10 minutes at RT.
9. Stop color development (Step 8) after 10 minutes by adding 50µl of Stop Solution (Component G) to each well. Color will change to yellow. Read plate at 450nm as soon as possible (no more than 10 minutes).
Caution: Steps 4 and 10 above, plate must be left in a dark place.
STRICTLY FOR NON-HUMAN RESEARCH AND DIAGNOSTICS
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