Application Note IGF020: Instructions for use ibt human IGFBP-4 ELISA

1.         Introduction

Insulin-like growth factor binding-protein-4 (IGFBP-4) is a member of a famuily of six high-affinity IGF-binding proteins (IGFBP-1 to -6). IGFBP-4, the smallest member of the family is found in all biological fluids with the highest levels in amniotica and follicular fluid. It exists in both non-glycosylated and N-glycosylated forms. It acts as a transport protein for IGF-I and IGF-II and inhibits IGF-induced cellular growth both in vitro and in vivo. The levels of IGFBP-4 are regulated by proteolyis. Pregnancy-associated plasma protein-A has been identified as the major IGFBP-4 protease.

The serum levels of IGFP-4 do not change significantly with age. They are increased in ovarian cancer and chronic renal failure. Increased levels of IGFBP-4 have been observed in pregnancies with fetal growth restriction.

 

2.         Principle of the test

The biotinylated antibody binds to the streptavidin coated microtiterplate. The IGFBP-4 of the standards/samples binds to this antibody. The biotinylated antibody and the standards/ samples are incubated on the microtiter plate at the same time. After a washing step the bound IGFBP-4, is then detected by a horseradish peroxidase-conjugated anti-IGFBP-4 antibody. After a second washing step the conjugate is added and incubated. The reaction with the substrate is stopped by addition of acid and the blue colour turns to yellow. The measured absorbance is directly proportional to the quantity of IGFBP-4.

 

3.         Kit contents and storage

1.       1.Microtiter Plate: 96 wells (12 x 8 well strips in a foil pouch with desiccant), coated

with streptavidin.

2.      Biotinylated Antibody: monoclonal antibody to human IGFBP-4 (1 mg/ml) 25 µl.

3.      Standard, humanIGFBP-4, 1 µg, lyophilized.

4.      Conjugate: horseradish-peroxidase conjugated monoclonal antibody to IGFBP-4 (100 ug/ml, 50 µl.

5.      Assaybuffer: 50 ml, ready for use.

6.      Washing buffer: 20 ml, 10-fold concentrated

7.      Substrate: TMB, 12.5 ml, ready for use

8.       Stop solution: 0,2 M sulfuric acid, 12.5 ml, ready for use (Caution: caustic)

9.      Instructions for use

Store the kit at 2-8°C.

 

4.         Materials required, but not supplied

- Distilled or deionized water

- Disposable test tubes for sample preparation

- Pipettes

- Vortex mixer

- ELISA Reader

 

5.         Precautions for Users

The ibt IGFBP-4 ELISA is for in-vitro research 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 bovine serum albumin. Therefore it should be treated as infectious materials and precautions should be taken as e.g. adequate clothing, wearing gloves etc. All waste should be disposed following the regulations for infective materials.

The stop solution contains acid that is harmful. 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.

 

6.         Test procedure

 

6.1      Preparation of reagents and working solutions

6.1.1 Bring all reagents and the 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.

6.1.2 Reconstitute the standard with 100 µl of the Assay Buffer. The reconstituted standard is stable for at least 2 weeks at 4 °C or for 1 month at -20 °C. Avoid repeated freeze-thaw cycles.

6.1.3. Preparation of the Biotinylated Antibody working solution:Mix 3 µl of the Biotinylated Antibody with 6 ml of Assay Buffer. Use the same day

6.1.4 Conjugate: Biotinylated Antibody working solution:Mix 30 µl of the Biotinylated Antibody with 12 ml of Assay Buffer. Use the same day

6.1.5 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.

6.1.6 Sera: In normal sera a dilution of 1 + 9 is recommended.

 

 

6.2      Preparation of standards

Standard 1 (40 ng/ml): Mix 4 µl Reconstituted Standard with 996 µl Assaybuffer

Standard 2 (20 ng/ml): Mix 500 µl Standard 1 with 500 µl Assaybuffer

Standard 3 (10 ng/ml): Mix 500 µl Standard 2 with 500 µl Assaybuffer

Standard 4 (5 ng/ml): Mix 500 µl Standard 3 with 500 µl Assaybuffer

Standard 5 (2.5 ng/ml): Mix 500 µl Standard 4 with 500 µl Assaybuffer

Standard 6 (1.25 ng/ml): Mix 500 µl Standard  5 with 500 µl Assaybuffer

Standard 7 (0.625  ng/ml): Mix 500 µl Standard 6 with 500 µl Assaybuffer.

 

6.3      Assay Procedure

6.3.1 Dispense 100 µl of Assaybuffer in Well A1 and A2 (Blank) of the microtiter plate (Blank).

Dispense 50 µl of the standards and samples in duplicates per well.

Dispense 50 µl of Biotinylated antibody working solution in each well

Incubate for one hour at room temperature (22-25 °C).

Note: a constant temperature is important for a low coefficient of variation. Avoid draughts. Incubation is best done in a closed incubator with constant temperature.

 

6.3.2 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. When washing is done manually, tap the inverted plate firmly on absorbent tissue to ensure complete removal of washing buffer before proceeding to the next step.

 

6.3.3 Dispense 100 µl conjugate working solution per well and incubate for one hour at room temperature.

 

6.3.4 Wash all wells three times as described in 6.3.2

 

6.3.5 Dispense 100 µl substrate solution per well. To minimize imprecision, this should be done by a multichannel pipette or in timed intervals. Incubate for 15 minutes at room temperature.

 

6.3.6 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 6.3.5. The blue colour will turn yellow upon addition of the stop solution.

 

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

 

7.         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.

 

 

 

8.         Typical Test Results

 

Typical standard curve (not to be used for calculation of actual results):

References

References:

1.) Van Doom et al.: Plasma levels of insulin-like growth factor binding protein-4 (IGFBP-4) under normal and pathological conditions. Clin Endocrinol (Oxf). 2001 May;54(5):655-64.

2.)Zhou et al.: IGF-binding protein-4: biochemical characteristics and functional consequences. Journal of Endocrinology (2003) 178, 177–193.

http://intl-joe.endocrinology-journals.org/content/178/2/177.full.pdf

3.)Mazerbourg et al.: Up date on IGFBP-4: regulation of IGFBP-4 levels and functions, in vitro and in vivo. Growth Horm IGF Res. 2004 Apr;14(2):71-84.

 

4.) Mosig et al.: IGFBP-4 tumor and serum levels are increased across all stages of epithelial ovarian cancer. J Ovarian Res. 2012; 5: 3. www.ncbi.nlm.nih.gov/pmc/articles/PMC3271973/

5.) Qiu et al. : Significance of IGFBP-4 in the development of fetal growth restriction. J Clin Endocrinol Metab. 2012 Aug;97(8):E1429-39