Product Specifications: Item#: 106 Description: HIV-1 Reverse Transcriptase (p66) Capture ELISA. Storage: Store Components as specified. Stability: See Kit Label Note: Read instructions and familiarize yourself with kit components before performing experiments with this kit. Some components of this kit are light sensitive. Do not expose to direct sunlight. Component List: A) Plate holder with solid phase capture ligand (Anti-p66 Monoclonal Antibody) strips, store at 4oC B) Wash Buffer 10 x, store at 4oC C) Blocking/Diluent Buffer, store at 4oC D) Reverser Transcriptase (rp66) Reference, store at 4oC E) Detector Reagent, store at 4oC F) Substrate, store at 4oC G) Stop Solution, store at 4oC Section-1 Pre-Test Preparation 1. Bring components A, B, and C to room temperature. 2. Dilute wash buffer (Component B) 1 : 10 with deionized 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 romm temperature until ready for the next step. Section-2 Capture Assay 1. Prepare several dilutions (minimum 200µl) of positive reference rp66 (Component D,1ug/ml ) in diluent buffer (Component C) in Eppendorf or comparable tubes - labeled accordingly: 500ng/ml to 0.5ng/ml in 4-fold serial dilutions. Note: low rp66 detection sensitivity in 100-200pg/ml or 10-20pg/test range. 2. Prepare test samples using diluent buffer (Component C) in 10-100ng/ml range. Unknown samples may be used in 1:2 to 1:10 dilution range with Diluent Buffer (Component C). 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 in dark. 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. Blue color will develop over 10 minutes at RT. 10. Stop color development (Step 9) after 10 minutes by adding 100µl 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 6 and 10 above, plate must be left in a dark place. STRICTLY FOR NON-HUMAN RESEARCH AND DIAGNOSTICS
