IGFBP-3 Direct Proteolysis Assay Kit

Instructions for Use of the Direct IGFBP-3 Proteolysis Assay Kit:

1.             Introduction

Proteolysis is one of the posttranslational modifications of the six IGFBP´s. It results in fragments with no or reduced affinity to IGF-I and IGF-II and results in increased availability to target cells (2). The fragments obtained by proteolysis may also have IGF-independent functions (1).

 

2.         Applications

IGFBP-3 proteolytic activities have initially been found in pregnancy serum. Later fragments have been detected in other body fluids (1). Increased proteolysis has been observed in malignancies, as e.g. breast cancer (3) and leukemia (7), in malnourished elderly people (4), in insulin dependent diabetes mellitus (5) and after extensive exercise (6).

The assay results are not affected by intrinsic IGFBP-3 or free IGF´s in the samples. Though in our experiments with Plasmin we found, that the biotinylated IGFBP-3 is degraded somewhat slower than the non-biotinylated form (see Application Note IGF018) we could show, that the fragment pattern obtained is equivalent to the fragment pattern obtained with non-biotinylated IGFBP-3. Therfore the IGFBP-3 Direct Proteolysis Assay Kit is recommended for use as a  rapid and very sensitive screening test in samples which may contain intrinsic IGFBP-3 and for comparison of protease activities e.g. in normal and disease state samples.

 

3.         Principle of the Test

The test uses biotinylated IGFBP-3 as a substrate for proteases. The proteolysed Biotin-IGFBP-3 is bound to the wells of a Streptavidin coated microtiterplate. Unproteolysed IGFBP-3 is detected by a monoclonal antibody to human IGFBP-3 followed by a goat-anti-mouse-peroxidase-conjugate.

 

4.         Kit Contents and Storage

1.Testplate: 12 strips with 8 wells each, coated with Streptavidin

2. Biotin-IGFBP-3: biotinylated human recombinant IGFBP-3, 2.5 µg, lyophilized

3. Antibody: 50 µg anti-human IGFBP-3, mouse monoclonal, lyophilized

4. Conjugate: Goat-anti mouse IgG-Peroxidase conjugate, 50 ml dilute 1:2000

5. Assay Buffer, buffered solution for dilution of proteolyzed samples and Conjugate. 50 ml. Ready for-use.

6. Washing Buffer, buffered solution, with detergent, 10- fold concentrated. 25 ml

7. Substrate, TMB, 11 ml. Ready for use.

8. Stop Solution, 0,2 M sulfuric acid11 ml. Ready for use. Caution: caustic.

9. Instructions for use.

The kit should be stored at 2 - 8  °C. The unopened kit is stable until the expiry date printed on the kit label. Openened components are stable until the expiry date, except the reagents and working solutions mentioned in 7.1.

 

5.         Materials required, but not supplied

-Distilled or deionized water

-Disposable test tubes for sample preparation

-Pipettes

-Vortex mixer

-ELISA Reader

 

6.         Precautions for Users

The ibt direct IGFBP-3 proteolysis assay kit is for in-vitro use only!

This product must be used strictly in accordance with the instructions for use. ibt - immunological & biochemical testsystems GmbH will not be held responsible for any loss or damage howsoever caused, arising out of non-compliance with the instructions provided.

The assay buffer contains BSA. Though serum is not considered as a high risk material for BSE, the assay buffer should be treated as potentially infectious.

The stop solution contain acids that is caustic. Avoid contact with skin, eyes and mucous membranes.

TMB Substrate is a suspected carcinogen. Avoid contact with skin, eyes and mucous membranes.

The reagents contain a preservative in very low concentration. Avoid contact with skin, eyes and mucous membranes.

 

7.         Test Procedure

 

7.1       Test Preparation:

7.1.1    Bring all reagents and microtiter plate to room temperature.

Remove the required quantity of strips from the microtiter plate. Strips not to be used immediately must be stored in the sealed bag with the desiccant at 4 °C.

 

7.1.2    Dissolve BiotinIGFBP-3 with 100 µl distilled or demineralized water to yield a 25 µg/ml solution. Mix carefully and allow keep at room temperature for at least 30 minutes to ensure complete dissolution. The Biotin-IGFBP-3 is stable for one week at 4 °C. Reconstituted Biotin-IGFBP-3 may be frozen in aliquots and is stable for at least one month.

 

7.1.3    Reconstitute the Antibody with  50 µl of distilled or demineralized water to yield a 1 mg/ml stock solution. The stock solution is stable for at least one month at 4°C. Prepare the required quantity of working solution by dilution of 1 volume of the stock solution with 2000 volumes of Assay Buffer, e.g. 1 µl antibody to 2 ml Assay Buffer.

 

7.1.4 Prepare the required amount of washing buffer by dilution of 1 volume of the concentrated washing buffer with 9 volumes of distilled or demineralized water. Use within one week.

7.1.5. Prepare the required amount of conjugate working solution by dilution of 1 volume of the concentrated Conjugate with 2000 volumes of assay buffer (e.g. 2 µl Conjugate plus 2 ml assay buffer.

7.2       Preparation of Samples and Standards:  

7.2.1    Samples

Proteolyse suitable aliquots of biotinylated IGFBP-3. After proteolysis stop reaction with suitable protease inhibitors. Dilute samples to fit into the standard curve.

Please note: The proteolysis of IGFBP-3 may beaffected by IGF´s. There are differences in proteolysis of glycosylated and non-glycosylated IGFBP-3 as it is used commonly in the assays with I125-IGFBP-3 (10).

The Assay Buffer contains BSA and may affect proteolysis.

7.2.2. Examples for applications

7.2.2.1 1 Digestion of biotinylated IGFBP-3 by Plasmin:

As an example we have tested degradation of biotinylated IGFBP-3 with plasmin using the conditions described in a paper by Vorwerk et al. (13). For proteolysis, we incubated 0.5 µg of biotinylated IGFBP-3 with 10 µg, 5 µg, 1.25 µg 0.25 mg and 0 mg (as control) in a total volume of 40 µl Tris-HCl pH 7.5 for 30 min at 37 °C. The reaction was stopped adding 10 µl of a stock solution of a protease inhibitor cocktail (1 Complete Mini tablet, Roche Applied Science, in 1.5 ml water). An aliquot was diluted to  10 ng/ml and the concentrations of the nondigested IGFBP-3 was measured.

The concentrations of the plasmin digested samples are found in the range of 4.54 ng/ml to 7.71 ng/ml, corresponding to a degradation of biotinylated IGFBP-3 from 54.6 to 22.9 %. The concentration in the control was calculated as 10.29 ng/ml, corresponding to a recovery rate of 102.9 %.

7.2.2.2 IGFBP-3 Proteolysis in normal and cancer serum

 

In another experiment we measured IGFP-3 in different cancer sera compared to a normal serum as a control. 50 µl of a 1 µg/ml solution of biotinylated IGFBP-3 in assay buffer were mixed with 50 µl of normal serum or cancer serum and incubated over night (18 h) at 37 °C. The reaction was stopped with 25 µl of a stock solution of a protease inhibitor cocktail (1 Complete Mini tablet, Roche Applied Science, in 1.5 ml water). The concentrations of undigested biotinylated IGFBP-3 have been measured in a diluted sample.

 

In some of the cancer samples we observed strongly increased IGFBP-3 proteolysis. As the samples of cancer serum have been form a commercial source, no detailed clincial information on disease stage etc. was available and the number of samples was limited. Therefore no interpretation of the significance of increased IGFBP-3 proteolysis for the disease is possible. However the usefulness of the Direct IGFBP-3 Proteolysis Assay kit as a screening assay in complex samples of biological origin has been demonstrated.

Sample

% biotinylated IGFBP-3 digested

Normal Serum

6.5

Colon Cancer

44.7

Uterine Cancer

17.7

Testicular Cancer

17.7

Langerhans Islet Cancer

4.9

Acute Lymphoblastic Leukemia

24.7

Bronchial Cancer

76.2

Breast Cancer

14.6

Acute Myelogenous Leukemia

11.2

Melanoma

24.4

Prostate Cancer

25.9

7.2.3 Standard Curve:

 

Prepare a standard curve in the range of 8 ng to 0.4 ng by dilution with Assaybuffer. First prepare a standard stock solution: 2 µl Biotin IGFBP-3 plus 498 µl Assaybuffer (=100 ng/ml). Prepare Biotin IGFBP-3 Standards according to the following schedule:

Standard

Standard stock solution

Assaybuffer

Concentration (ng/ml)

1

20 µl

230 µl

8

2

10 µl

240 µl

4

3

5 µl

245 µl

2

4

2 µl

248 µl

0.8

5

1 µl

249 µl

0.4

7.3       Assay Procedure:

7.3.1    Dispense 100 µl of assay buffer per well as blank value. Dispense 100 µl per well of each standard or sample in duplicates into the wells of the microtiter plate.

Note: these should be dispensed within a period of 10 minutes to minimise drift.

Incubate for 30 minutes to 1 hour at room temperature (18-25° C).

7.3.2    Wash all wells three times with working wash solution:

Automatic plate wash: Set plate washer to dispense 250 µl of washing buffer per well and a minimum of 20 seconds per washing step. Fill and aspirate for 3 cycles.

Manual wash: Decant the contents of the wells by inverting sharply. Dispense 250 µl of diluted wash buffer to all wells. Decant and repeat twice. Tap the inverted plate firmly on absorbent tissue to ensure complete removal of washing buffer before proceeding to the next step.

7.3.3    Add 100 µl of the diluted antibody solution to each well and incubate for 1 hour at room temperature.

7.3.4    Wash all wells three times with working wash solution as described in 7.3.2.

7.3.5    Add 100 µl of the conjugate working solution to each well and incubate for 1 hour at room temperature.

7.3.6    Wash all wells three times with working wash solution as described in 7.3.2.

7.3.7    Add 100 µl of substrate to each well of the microtiter plate. To minimize imprecision, this should be done by a multichannel pipette or in timed intervals. Incubate for 30 minutes at room temperature.

7.3.8    Stop reaction by adding 100 µl of stop solution to each well. To minimize imprecision, this should be done by a multichannel pipette or in timed intervals as in step 5. The blue colour will turn yellow upon addition of the stop solution.

7.3.9    Measure the absorbance of each well at 450 nm (reference 650 nm).

7.4       Trouble shooting

Dissolution of lyophilized reagents – it is essential to dissolve the lyophilized reagents completely!

Washing – Insufficient washing (especially incomplete removal of reagents and washing buffer) can result in high coefficients of variationhigh background, and poor results.

Use of a shaker - may increase sensitivity and decrease variation of duplicates.

8.         Calculation of the Results

The results are calculated by linear regression. The standard curve is plotted by drawing the regression curve with the absorbance on the y-axis and the concentration on x-axis. Calculate the results of the samples and multiply by the dilution factor.

9          Typical Test Results

Standard curve of the example (not to be used for calculation of actual results):

 

 

 

 

 

10. Control Experiments for Detection of biotinylated IGFBP-3 Protease Activity by Immunoblotting

You can use the biotinylated IGFBP-3 as well for control experiments  by immunoblotting (see Application Note IGF007). The monoclonal antibody of this kit may be used at a dilution of 1: 500 to 1:1000, and the conjugate may be used at a dilution of 1:1000. For an example of an immunoblot see

Application Note IGF018.

11. References:

1.) The IGF System: Molecular Biology, Physiology, and Clinical Applications

By Charles T. Roberts, Ron G. Rosenfeld. Published by Humana Press, 1999,

ISBN 0896036928, 9780896036925.

Link to book preview:

http://books.google.com/books?id=9CVyOzQk95EC&pg=PA360&lpg=PA360&dq=IGFBP-1+Proteolysis&source=web&ots=zgI8oi_8MY&sig=lCdkM-kB5cpAvAyLiNnjrfrb4BQ&hl=en&sa=X&oi=book_result&resnum=1&ct=result

 

2.) In vivo proteolysis of serum insulin-like growth factor (IGF) binding protein-3 results in increased availability of IGF to target cells. Blat, et al. J. Clin. Invest. Volume 93, May 1994, 2286-2290.

Link to article: http://www.jci.org/articles/view/117229/pdf

 

3.) Plasma insulin-like growth factor binding protein-3 proteolysis is increased in primary breast cancer. Helle1 et al. British Journal of Cancer (2001) 85(1), 74–77.

Link to article: http://www.nature.com/bjc/journal/v85/n1/pdf/6691860a.pdf

 

4.) IGF-I, IGF-I-binding proteins and GH-binding protein in malnourished elderly patients with inflammation receiving refeeding therapy. Raynaud-Simon et al. European Journal of Endocrinology (2002) 146 657–665.

Link to article: http://www.eje-online.org/cgi/reprint/146/5/657

5.) Macrosomia Associated With Maternal Serum Insulin-Like Growth Factor-I and -II in Diabetic Pregnancy. Lauszus et al. Obstet Gynecol 2001;97:734–41

Link to article: http://www.greenjournal.org/cgi/reprint/97/5/734

 

6.) Effect of Intense Exercise on Inflammatory Cytokines and Growth Mediators in Adolescent Boys.  Nemetet al. Pediatrics 2002;110;681-689.

Link to article: http://pediatrics.aappublications.org/cgi/reprint/110/4/681.pdf

 

7) Insulin-like Growth Factor Binding Protein (IGFBP)-3 Protease Activity Secreted by MCF-7 Breast Cancer Cells: Inhibition by IGFs Does Not Require IGF-IGFBP Interaction. Salahifar et al. Endocrinology Vol. 138, No. 4 1683-1690.
Link to article: http://endo.endojournals.org/cgi/reprint/138/4/1683