Enzym immunoassay voor de quantitatieve bepaling van chlooramfenicol acetyltransferase (CAT).

Protocol

Preparation of solutions:

1. Solution II - wash buffer:
   Mix 1 part 10X wash buffer (bottle with red cap) with 9 parts distiled water
   For 10 samples (each analysed twice), approximately 25 ml wash buffer is required for each wash step
2. Solution III - sample buffer: with blue cap
3. Solution VII - CAT enzyme :
   Dilute 20 µl CAT enzyme (stored at -20°C) with sample buffer (sol. III) up to 2 ml (final concentration 1 ng/ml)
   For the calibration curve, following dillutions are prepared (each in double, with 200 µl per reaction) :
   -----------------------------------------------
   concentration CAT enzyme Sample buffer
   (pg/200µl) (sol. VII) (sol. III)
   -----------------------------------------------
   0 - 1 ml
   25 0.125 ml 0.875 ml
   50 0.25 ml 0.75 ml
   100 0.5 ml 0.5 ml
   200 1.0 ml -
   -----------------------------------------------
4. Solution IV - anti-CAT-DIG :
   Dilute 100 µl anti-CAT-DIG (stored at -20°C) with 9,9 ml sample buffer (sol. III) to a final concentration of 2 µg/ml
5. Solution V - anti-DIG-POD :
   Dilute 60 µl anti-DIG-POD (bottle with orange cap) with 9,94 ml sample buffer (sol. III) to 150 mU/ml
6. Solution VI - POD substrate: Bottle with green cap

1. Make dillutions of the CAT enzyme for the calibration curve (see above)
   Dilute samples with sample buffer (sol. III) - about ± 50 µg protein/well in a volume of 2 X 200 µl.
2. Open het required number of MTP-modules (24 wells per pack - 2 X 6 required for calibration curve and blanks)
   Place them in the holder
3. Rehydrate the MTP modules by adding 300 µl sample buffer to each of the wells
   Incubate for 5 min at room temperature
4. Discard the solution
   Tap MTP module on dry tissue
   Transfer 2 x 200 µl of each sample into wells
   Cover MTP with plastic
   Incubate for 1 hour at 37°C
5. Discard solution
   Wash 4 X with 300 µl wash buffer per well
6. Add 200 µl anti-CAT-DIG
   Cover MTP with plastic
   Incubate for 1 hour at 37°C
7. Discard solution
   Wash 4 X with 300 µl wash buffer per well
8. Add 200 µl anti-DIG-POD
   Cover MTP with plastic
   Incubate for 1 hour at 37°C
9. Discard solution
   Wash 4 X with 300 µl wash buffer per well
10. Add 200 µl POD-substrate
    Cover MTP with plastic
    Incubate for 0,5 to 1 hour at room temperature with constant shaking
11. Measure extinction of the samples with an elisa reader at 405 nm.