Latex Hev b 6.02 ELISA Assay

Additional Information

Assay Principle

FITkit Hev b 6.02 test is based on the immunoenzymetric assay technique. Microtiter wells are coated with Hev b 6.02-specific monoclonal capture antibody that during the first incubation binds Hev b 6.02 molecules from the sample extract. After incubation, unbound material is removed by washing the wells. During the second incubation, horseradish peroxidase (HRP) labelled Hev b 6.02-specific monoclonal detection antibody binds to a different epitope of Hev b 6.02 molecules. As such, the two antibodies are a matched pair that forms a “sandwich” around the target Hev b 6.02 allergen. After washing, HRP substrate is added and the intensity of the colour produced is directly proportional to the Hev b 6.02 concentration of the sample.

Assay Procedure

1. Dispense 100 μL of Assay Buffer into each well.
2. Pipette 25 μL of calibrator, control and sample into appropriate wells in duplicate.
3. Cover the microwell plate. Incubate the microwell plate for 60 minutes at room temperature on a microwell plate shaker (100-200 rpm).
4. Aspirate and wash the wells 4 times with 300 μL of washing solution.
5. Dispense 100 μL of Enzyme Conjugate into the wells in duplicate.
6. Cover the microwell plate. Incubate the microwell plate for 30 minutes at room temperature on a microwell plate shaker (100-200 rpm).
7. Aspirate and wash the wells 4 times with 300 μL of washing solution.
8. Add 100 μL of HRP Substrate Solution at fixed time points into each well.
9. Cover the microwell plate. Incubate the microwell plate for 15 minutes at room temperature on a microwell plate shaker (100-200 rpm).
10. Stop the reaction by adding 100 μL of Stopping Solution into each well at the same fixed time points as in Step 8 so that exactly the same substrate reaction time is achieved. Shake the microwell plate for 1-2 minutes to mix the solutions.
11. Measure the absorbance at 414 nm using a microwell plate or strip reader, preferably immediately but no more than 60 minutes after stopping the reaction. If the microwell plate is not read immediately, protect the microwell plate from light.

Typical Standard Curve