Enzyme-Linked Immunosorbent Assay (ELISA) Protocol

 

1.     Prepare plate:

a)     Add 100 l 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 l washing buffer per well.

All residual washing buffer must be removed.

 

c)     Add 100 l 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 l washing buffer each well.

 

3.     Add 300 l sample buffer (NaFAM) per well, incubate at 37˚C for 30 minutes.

 

4.     Remove sample buffer and wash plate 3 times with 300 l washing buffer per well.

 

5.     Add 100 l insulin standards or samples to appropriate wells, incubate at 37˚C for 1 hour.

Standard curve (run in triplicate):

Tube

Number

NaFam (l)

Insulin Std. (l)

(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 l washing buffer each well.

 

8. Add 100 l 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 l

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 l 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 g 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 l of HRP-labeled insulin stock to glass tubes and

store at 20˚C.

Before use add 10 ml of NaFam to the 50 l 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