Component List:

A) Plate holder with solid phase hCEM-15 ligand strips (96 well plate). Store at 4°C.

B) Wash Buffer 10 X, (dilute 1:10 with deion-ized water) before use.

C) Sample Dilution Buffer (Store at 4oC)

D) HIV-1 rvif Standard (2ug/ml, Store at 4°C).

E) Detector Reagent: Murine anti-vif mAb peroxidase (dilute 1:100 before use).

F) Peroxidase Substrate (Store at 4°C)

G) Stop Solution (Store at 4°C)

Bring Diluent Buffer (Component C) to room temperature before use.

Section-1         

vif Capture

1. vif detection range: 200ng/test to 2ng/test.Transfer 100ul of Dilution Buffer (Component C) to wells B-H. Using a 200ul pipettor, transfer 100ul of vif reference (Component D) to wells A and B each.

2. Using a new pipette  tip mix well B contents by drawing the liquid from the well into the pipette tip and gently returing the liquid to the well 10 times without  letting any air bubbles into the well.  Remove 100ul of mix from well B and transfer to well C. Using the same tip repeat the mixing procedure as in 2 above from wells C to G (2-fold serial dilution ). Leave well H as a blank.

3. Gently tap the plate holder againt a solid object to dislodge any air bubbles in the wells and leave plate at room temperature for 1 hr.

4. Dump contents of wells and wash wells with 1 x wash buffer (Component B:  1:10 diluted with De-ionized H2O) 4 times.  Leave last wash in wells until ready for the next step.

Section-2 

vif Detection

1. Dilute detector reagent (Component E) 1:100 in Diluent Buffer (Component C).  Dump contents of wells (step 4 above) and pipette 100µl of diluted detector reagent (Component E) into each well.  Cover plate and leave in a dark place at room temperature for 1 hour.

2. Dump contents of wells, and wash wells 4 times with 1 x wash buffer (Component B).  Dump last wash before continuing to the next step.

3. Add 100µl TMB substrate (Component F) to each well.

Note: The TMB Substrate is light sensitive.  Do not expose to sustained, direct light.

4. Blue color will develop in Positive Reference wells over 10 minutes at room temperature.

5. Stop color development after10 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).

Section-3      

Inhibitor Assay

Before adding vif  reference to the wells Section 1 above, , add 10 ul of the inhibitor test compound to the wells so that after the addition of 100ul of  vif reference to the well, the concentration of the inhibitor in the desired range ( pM,  nM, uM and mM) is achieved. Inhibitor test studies may be performed in duplicate or triplicate with appropriate amounts of vif  (Section 1 above) to produce the  color signal measured at 450nm for readings in 1.5-1.8 OD range. This corresponds to a vif concentration of approximately 100ng/well.