Enzyme-Linked
Immunosorbent Assay (ELISA) Protocol
1.
Prepare
plate:
a)
Add
100 µl of coating antibody to each
well, wrap plate in Saran wrap and store at 4˚C overnight. Plates can be stored for up to a week at 4oC.
b)
Wash
plate 3 times with 300 µl washing buffer
per well.
All residual
washing buffer must be removed.
c)
Add 100 µl of anti-insulin antibody to each well, wrap
plate in Saran wrap and store at 4˚C overnight. Plates can be stored for up to one week at 4oC.
2.
Remove
antibody and wash plate 2 times with 300 µl washing buffer each well.
3.
Add
300 µl sample buffer (NaFAM) per well, incubate at 37˚C
for 30 minutes.
4.
Remove
sample buffer and wash plate 3 times with 300 µl washing buffer per well.
5.
Add
100 µl insulin standards or samples to appropriate wells, incubate
at 37˚C for 1 hour.
Standard
curve (run in triplicate):
Tube Number |
NaFam (µl) |
Insulin Std. (µl) (100 ng/ml) |
Std. Conc. (ng/ml) |
1 |
399.5 |
0.5 |
0.125 |
2 |
399 |
1 |
0.25 |
3 |
398 |
2 |
0.5 |
4 |
396 |
4 |
1 |
5 |
390 |
10 |
2.5 |
6 |
380 |
20 |
5 |
7 |
360 |
40 |
10 |
8 |
340 |
60 |
15 |
9 |
320 |
80 |
20 |
10 |
300 |
100 |
25 |
6.
Add
100 peroxidase-labeled insulin to
each well, incubate at 37˚C for 1 hour.
DO NOT REMOVE INSULIN STANDARDS OR SAMPLES BEFORE ADDING
PEROXIDASE-LABELED INSULIN.
7.
Wash
plates 3 times with 200 µl washing
buffer each well.
8. Add 100 µl ATBS to each
well, wrap to shield from light, incubate at room temperature. Read the absorbance at OD415 at
20, 30, and 40 minutes.
Buffers for
Insulin ELISA:
1. Coating Buffer for coating antibody (0.5 M
carbonate bicarbonate buffer, pH 9.6)
1 Liter
Na2CO3 1.59 g
NaHCO3 2.93
g
NaN3 0.2 g
dH2O to 1
liter
2. Incubation buffer for anti-insulin antibody
(0.05 M phosphate buffer, pH 7.4)
Na2HPO4 4.6 g
NaH2PO4 1.05 g
BSA 1.0 g
Sodium merthiolate 0.24 g
dH2O to 1 liter
3. NaFam Sample Buffer (0.05 M phosphate buffer,
pH 7.4)
NaHPO4 4.6
g
NaH2PO4 1.05
g
BSA (RIA grade) 60.0 g
NaCl 6.0 g
Sodium merthiolate 0.24 g
dH2O to 1 liter
4. Washing Buffer [0.15 M Phosphate Buffered
Saline (PBS), pH 7.2]
NaCl 8.0 g
KCl 0.2 g
Na2HPO4 1.15 g
Tween 20 0.5
ml (0.05%)
dH2O 1 liter
5. Citrate Buffer (0.1 M)
Citric acid monohydrate 4.8 g/250 ml dH2O
Dissolve 4.8 g of citric acid in 200 ml of water and
adjust pH with 1M NaOH to 4.0 (about 30 ml needed) and bring up the volume to
250 ml with dH2O. Stable at 4˚C for 2-3 month.
6. Substrate solution for peroxidase
ABTS (chromagen) 1
ml (4 mg/ml stock)
H2O2 (30%,
substrate) 10 µl
Citrate buffer 11 ml
Materials
and Reagents for Insulin ELISA
1. Coating Antibody: Rabbit anti-guinea pig
EY Laboratory, San Manteo, CA
Catalog # AT-2358-2
Initial Concentration: 10 mg/2 ml (5
mg/ml)
Add 500 µl of 5 mg/ml stock to 250 ml coating
antibody buffer pH 9.6. This gives a 10
mg/l final concentration.
Aliquot 10 ml volumes and store at
-20˚C.
2. Anti-insulin antibody: Guinea pig anti-rat
insulin
Linco Research Products
Catalog #1013
1000 tube quantity/bottle
Add 100 ml of incubation buffer pH 7.4
to bottle
Aliquot and store at –20˚C
3. Insulin standard
Linco Research Products #8013
The vial contains 100 µg of insulin
and 1 mg of human albumin
Add 1 ml of NaFam pH 7.4 to get 100
ng/ml stock
Stock solutions are stable for at
least one year at -20˚C
4.
Horse Radish Peroxidase (HRP)-labeled insulin (light-sensitive)
Sigma, catalog # I-2133
0.5 mg/bottle
Dissolve 0.5 mg in bottle in 2.5 ml of fresh NaFam
(sample buffer) to make a 0.2 mg /ml stock.
Aliquot 50 µl of HRP-labeled insulin stock to glass
tubes and
store at –20˚C.
Before
use add 10 ml of NaFam to the 50 µl HRP-labeled insulin stock. This is the working stock for the assay.
5. ABTS: 2,2'-Azino-bis
(3-ethylbenzythiazoline-6-sulfonic acid)
Sigma catalog # A1888
4 mg/ml in dH2O
Aliquot into Eppendorf tubes and wrap
with aluminum foil
Store at –20˚C