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 4^oC.

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 4^oC.

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

�� NaH₂PO_{4��}�� 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