Insulin ELISA
Remove pancreas into tared eppendorf tube
Weigh pancreas and note weight in mg
Add 20ul of cold Acid Ethanol (75ml ETOH + 25ml H2O + 1.5ml Conc HCL) per mg wet weight of pancreas
Rock overnight at 4oC
Next morning spin 20,000 X g for hr at 4o
Remove supe into clean 1.5ml tube
Neutralize pH with 1M Tris Base (200ul per ml extract)– check pH with pH paper. Note that if higher dilutions
(1/5000, 1:10,000) are used for the assay, this dilution step is not necessary. The neutralize Acid Ethanol Extract must be diluted a minimum of 1/100 so that the Ethanol doesn’t interfere with the ELISA
Dilute 1/5000-1/20000 in Crystal Chem Kit Diluent 1. Use 50ul per well as described in the Crystal Chem kit protocol for assaying insulin for samples that are not serum or plasma.
Follow the Crystal Chem Rat Insulin ELISA kit instructions for samples that are not serum or plasma. Run mouse insulin standards in duplicate. If possible, run unknown samples in duplicate as well. Vortex samples very well prior to dispensing into the wells of the microtiter plate.
Read abs 490nm and 650nm
Subtract abs 490-650nm
Plot Standard Curve and calculate the insulin concentration based on equation.
Sample calculation:
Sample 1350A: Ave conc for duplicate samples is 17116ng insulin/ml or 17.1ug insulin/ml
For each well 50ul (of a 1/10,000 dilution) is used
Because all pancreas organs are weighed prior to extraction in 20ul Acid Ethanol per mg wet weight, each sample is .05mg/ul wet weight pancreas.
.05mg/ul wet weight X 50ul = 2.5mg wet weight pancreas used
17.1ug insulin/ml/2.5mg wet weight pancreas = 6.84ug insulin/ml/mg wet weight pancreas
Reference :Rastogi, G.K, et. al (1970) “Immunoreactive insulin content of 203 pancreases from Foetuses of healthy mothers” Diabetologia 6: 445-446