2.3. Glucose assay

1. To make the glucose standard curve, prepare eight clean glass test tubes (5 ml) and label them S1 through S8.

2. Aliquot 0.5, 1, 5, 10, 30, 50 and 100 µl glucose standard solution (1 mg/ml) into tubes S2-S8.

Do not add any standard to tube S1 as this tube will be the blank.

3. Label appropriate number of clean glass test tubes (5 ml) for samples.

4. Keep the tubes on ice.

5. Take the haemolymph samples from the -80 ºC freezer and keep them on ice.

6. Add 1 ml enzyme mix solution to each standard tube and sample tube.

7. Invert tubes 4-6 times.

8. Centrifuge tubes for 30 s to spin down contents.

9. Equilibrate reaction mix at room temperature for 15 min.

10. Transfer 200 µl aliquots of each standard (S1-S8) and sample into cuvettes or into a 96-well microplate for spectrophotometry readings.

11. Set spectrophotometer (for cuvettes) or ELISA reader (for microplates) for reading absorbance at 340 nm.

Standards and samples should be read in replicates or triplicates.

12. To calculate glucose concentrations, plot absorbance at 340 nm as a function of glucose concentration of the standard curve samples.

The concentrations of glucose standards (0.5, 1, 5, 10, 30, 50 and 100 µg/ml) are plotted on the X-axis; respective absorbance is on the Y-axis.

13. Determine the equation of the line by linear regression.

14. Each glucose concentration is calculated as:

Glucose concentration

15. If a sample had to be diluted during preparation, the result must be multiplied by the dilution factor, F.