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)
1 Liter
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)
1 Liter
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
Streamline Your ELISA Liquid Handling with the 8-Port Manifold :
Performing ELISA involves multiple repetitive liquid handling steps such as dispensing coating antibodies, washing buffers, samples, and reagents across many wells. To improve accuracy and save time during these critical steps, the 8-port manifold compatible with the 50ml combi-syringe is an invaluable tool.
This manifold allows you to simultaneously dispense or aspirate liquids across 8 wells perfectly aligned with the standard 9mm microplate spacing. It ensures consistent volume delivery with minimal variation (within 1.7%), reducing manual errors and accelerating your assay workflow.
Whether you’re adding coating antibodies, washing plates, or dispensing substrates, this manifold simplifies your ELISA process saving you time and improving reproducibility.