Human Anti HIV 1&2 Elisa Test Kit For Human Immunodeficiency Virus

3. ASSAY PROCEDURE

• Allow all components to reach room temperature before use. Dilute concentrated wash buffer 1:20 with ddH2O.
• Set two positive and three negative controls. Dispense 100μl of Positive Control(HIV-1 and HIV-2) as well as Negative Control in duplicate into respective wells without sample diluent. Set one blank well as background control without adding any liquid. Add 50μl of sample diluent into each test well, add 50 μl test serum or plasma into test wells mix thoroughly.
• Place the microtiter plate into a humidified box and incubate at 37°C for 30 min.
• Discard reaction solution and add 350μl diluted wash buffer to each well. Discard the wash buffer after soaking for 15-30seconds. Repeat the washing procedure 5 times and then inverting the plate vigorously to get all water out and blocking the rim of wells on absorbent paper for a few seconds.
• Add 100μl of Enzyme Conjugate to each well. Mix it gently by swirling the microtiter plate on flat bench for 1 min. Do not add Enzyme Conjugate to the blank well..
• Place the microtiter plate into a humidified box and incubate at 37°C for 30 min.
• Wash each well 5 times by filling each well with diluted 1X wash buffer, then inverting the plate vigorously to get all water out and blocking the rim of wells on absorbent paper for a few seconds.
• Add 50μl of Substrate Solution A (HRP substrate) to each well, then add 50μl of Substrate Solution B (TMB) to each well. Mix gently and incubate at 37°C for15 min.
• Add one drop (50μl) of Stop Solution to each well to stop the color reaction.
• Read the OD value at 450 nm/630 nm with dual filter plate reader. It is option to read the OD value at 450 nm with single filter plate reader. (using the OD value of the blank well to correct all the OD reading from all wells