Application Note IGF006: IGF-I ELISA Procedure

IGF-I ELISA FOR THE QUANTITATIVE DETERMINATION OF INSULIN-LIKE GROWTH FACTOR-I (IGF-I)

Introduction:

Insulin-like Growth Factor-I (IGF-I)  is a polypeptide of 70 amino acids. It has a high sequence homology to IGF-II and Insulin. IGF-I is a growth factor for a wide range of cell types. In serum and other body fluids, IGF-I is bound with high affinity to Insulin-like Growth Factor Binding Proteins (IGFBP´s). Until today six of the high affinity IGFBP´s have been found. In serum the predominant binding protein is IGFBP-3 and more than 95 % of the IGF-I in serum is bound to IGFBP-3. This complex associates with an additional protein, the Acid Labile Subunit, ALS. 

 

Specificity:

The antibody used for coating reacts specifically with human, porcine, bovine and guinea pig IGF-I and R3-IGF-I, an analog of human IGF-I with an Arg substitution at position 3. The relative binding potency to chicken IGF-I is 70 %, rat IGF-I 25 %, Long R3 - IGF-I 20 %. The cross-reaction to human IGF-II is less than 1%. The test is supplied with standards of human IGF-I, but may be used for other species using standards prepared from our recombinant IGF-I from other species.

Principle of the test:

IGF-I in samples competes with biotinylated IGF-I for binding to the antiserum bound to the microtiter plate. After washing to remove excess reagents, a Streptavidin-Peroxidase complex  is added, which binds to biotinylated IGF-I. After a washing step, the substrate is added and colour develops. The intensity of the colour is inversely related to the quantity of IGF-I in the sample. The reaction is stopped by addition of stopping solution and the extinction is read at 450 nm.

 

Contents of the kit:

1. Microtiterplate: coated with anti-IGF-I antibody, 12 strips with 8 wells each

2. Standards: 0 - 5 - 10 - 50 - 150 - 300 – 600 ng/ml, ready for use, 1 ml

3. 1M HCl, 1vila, 3 ml, ready to use.

4. Neutralisation buffer, 1 vial, 3 ml, ready to use.

5. Enzyme-Conjugate: Biotinylated IGF-I, ready for use, 14 ml

6. Enzyme Complex: Streptavidin-Peroxidase, ready for use, 20 ml

7. Substrate: one bottle TMB, ready for use, 14 ml

8. Stopping Solution: 0.5 M H2SO4, ready for use, 14 ml, Caution: Caustic!

9. Washing Solution: 40 x concentrated, 30 ml

10. Working instructions

 

Storage:

The kit should be stored at 2- 8 °C in the dark

 

1. Test preparation:

1.1 Bring all reagents and microtiter plate to room temperature before use.

1.2  Sample and Standards preparation:

Pipette 200 µl Sample and Standard in 1.5 ml Reaction cups (e.g. Eppendorf-Cups).

Please note: The standards should be acidified and neutralized too, according to the procedure described below.

Add 20 µ, 1M HCl.

Mix and incubate for 15 minutes.

For Neutralization add 20 µl Neutralization Buffer to all cups and mix the solution

Immediately continue with step 2.1 of the procedure.

Caution: Samples, as e.g. serum of unknown origin may contain infectious agents, so all samples should be treated as potentially infectious. The components of the kit do not contain proteins of human origin.

1.3 Fix the required amount of microtiter wells in the frame. The wells, that are not used immediately, must be stored in the resealed bag with the desiccant.

1.4 Dilute the required amount of 1 volume of washing solution with 29 volumes of demineralized or distilled water, i.e. 30 ml plus 1170 ml of demineralized or distilled water.

 

 

2. ELISA procedure:

2.1 Add 50 µl of the acidified and neutralized samples and standards in duplicates to the wells of the microtiter plate.

2.2 Add 100 µl of the biotinylated IGF (enzyme-conjugate) to each well

2.3 Incubate at room temperature for two hours.

2.4 Discard the solution in the wells.

2.5 Wash the wells with 300 µl  of the diluted washing solution per well three times for 20 seconds per wash.

2.6 Add 150 µl of the enzyme complex into each well.

2.7 Incubate one hour at room temperature.

2.8 Wash the wells with 300 µl  of the diluted washing solution per well three times for 20 seconds per wash.

2.9 Add 100 µl of substrate to each well and incubate for 15 min.

2.10 The reaction is stopped by the addition of 100 µl of stopping solution.

2.11 The extinction is read with a microtiterplate reader at 450 nm. As reference wave length 620 nm is recommended.

It is recommended, that the substrate and the stopping solution should be added to the wells in timed intervals to improve the coefficient of variantion.

 

3. Calculation of the results:

Results are calculated best using 4-parameter logistics. Alternatively, linear regression and a semilogarithmic standard curve may be used. Draw the standard curve with extinction values on the linear Y-axis and the concentration on the logarithmic X-axis.