Microplate Method
Predilution of samples
The predilution is performed to prevent interference from inhibitors, lipemic samples, icteric or hemolysed samples.
| Standards, plasma samples, and controls | 50 ml |
| Buffer | 1000 ml |
Kinetic Method
| Step | Amount / Time |
| Add pre-diluted Sample | 50 ml |
| Incubate at 37°C | 3 – 4 min. |
| Add chromogenic substrate | 50 ml |
| Mix well and add within | 30 sec |
| RVV + CaCl2 (20 – 25°C) | 50 ml |
| Mix well |
Transfer the microplate to a microplate reader immediately after addition of the RVV + CaCl2 and measure the absorbance change in a microplate reader at 405 nm. The microplate reader must be pre-incubated at 37°C.
If performing the acid-stopped method, mix and incubate at 37°C for 3 minutes after addition of RVV + CaCl2, then add 50 ml of 20% Acetic acid. Read the absorbance of the samples at 405 nm. The color is stable for at least 4 hours.