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 to 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. The binding of IGF-I to IGFBP-3 and other IGFBP´s prevents binding to antibodies. Therefore, for ELISA

IGF-I has to be extracted before measurement. Another alternative is, that samples are acidified to a pH of 2.3. Under this conditions, the IGFBP-3/IGF-I/ALS complex dissociates. If the sample is neutralised in the presence of an excess of IGF-II, the IGFBP´s are blocked, and IGF-I remains unbound and is accessible for antibodies.

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 - 1 - 5 - 10 - 50 - 150 - 300 ng/ml, ready for use, 0.5 ml
3. Conjugate: Biotinylated IGF-I, ready for use, 5.5 ml
4. Enzyme Complex: Streptavidin-Peroxidase, ready for use, 11 ml
5. Substrate: one bottle TMB, ready for use, 11 ml
6. Stopping Solution: 0.5 M H2SO4, ready for use, 6 ml, Caution: Caustic!
7. Washing Solution: 10 x concentrated, 100 ml
8. Working instructions

Storage:

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

1. Test preparation:

1.1 Sample preparation:

In samples containing IGFBP`s, IGF-I has to be extracted using one of the standard methods, that are described in the scientific literature, e.g. by chromatography under acidic conditions, chromatography on C18 columns, centrifugation with Bio-Spin columns, alcohol precipitation etc. Some references for extractions methods of serum, milk, urine and other body fluids are listed below.

Alternatively, the samples can be acidifed to a pH of 2.3 and neutralised in the presence of an excess of IGF-II (we recommend a 25-fold excess of IGF-II) to block IGF-I binding to IGFBP´s.

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.

NOTE: The standards are ready for use, DO NOT EXTRACT THE STANDARDS!

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

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 9 volumes of demineralized or distilled water.

2. ELISA procedure:

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

2.2 Add 50 µl of the biotinylated IGF (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 100 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 50 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.

4. Ordering information:

1 kit Product Code: IGF-I ELISA     400.- €

5. References:

5.1 Acidic chromatography of serum:

Juul, A. et al.: Serum Insulin-like Growth Factor-I in 1030 Healthy Children, Adolescents and Adults: Relation to Age, Sex, Stage of Puberty, Testicular Size, and Body Mass Index. J Clin Endocrinol Metab 78: 744-752, 1994.

5.2 Comparison of different methods (centrifugation, C18 chromatography, acid-ethanol extraction, gel chromatography under acidic conditions):

Mohan, S. and Baylink, D.J. : Development of a Simple Valid Method for the Complete Removal of Insulin-Like Growth Factor (IGF)-Bindin Proteins from IGFs in Human Serum and other body fluids:Comparison with Acid-Ethanol Treatment and C18 Sep-Pak Separation. J Clin Endocrinol Metab 80: 637-647, 1995.

5.3 Gel filtration of urine and serum samples:

Ratcliffe, S.G. et al: Urinary Insulin-like Growth Factor I in Normal Children: Relationship to Age, Pubertal Status and Urinary Growth Hormone. Growth Regulation 5, 53-59, 1995.

5.4 Acidic chromatography of milk and serum samples:

Donovan, S.M., Hintz, R. L. and Rosenfeld, R.G.: Investigation into the potential pyhsiological sources of rat milk IGF-I and IGF-binding proteins. J Endocrinol 145, 569-578, 1995.

5.5 Ultrafiltration of acidified samples:

Raynaud-Simon et al.: IGF-I, IGF-I-binding proteins and GH-binding protein in malnourished elderly patients with inflammation receiving refeeding therapy. European Journal of Endocrinology (2002) 146 657–665. Link to article: eje-online.org/cgi/reprint/146/5/657

http://eje-online.org/cgi/reprint/146/5/657