Application Note CHEM002: Instructions for use for the human SDF-1 alpha ELISA Kit

Application Note CHEM002:Instructions for use for the human CXCL12/SDF-1  ELISA Kit.

 

1.         Introduction

 

SDF-1α (CXCL12) is a CXC chemokine [1]. The small basic protein (ca. 8 kDa) has no glycosylation site in its sequence [2].

The homology properties of SDF-1α are unique among the chemokines. It has a high degree of conservation within the mammalians, with 99 % identity between the human and the murine SDF-1α sequences. But it shows a weak homology to the other CXC chemokines and the SDF-1 gene is on chromosome 10, whereas the other chemokine genes are on chromosome 4 and 17 [3].

Except for SDF-1α there is another isoform of this chemokine, SDF-1β. SDF-1α and SDF-1β result from alternative splicing [1].

SDF-1α binds to and activates the receptors CXCR4 and CXCR7 [1], whereupon SDF-1α is the only known ligand for CXCR4 [2]. SDF-1α binds also to fibronectin-1 [4], several glycosaminoglycans and heparin [5].

SDF-1α is constitutively expressed by bone marrow stromal cells [1] and many other tissues (for example heart, liver, brain, muscle, kidney) [3].

The function of SDF-1α is multifaceted. It plays a role in the prenatal development of the neural, hematopoietic and cardiovascular systems and is involved in the postnatal migration of CXCR4- and CXCR7-positive stem cells [1]. Furthermore, it has a chemoattractant effect on CXCR4-positive lymphocytes [1] and plays a role in the growth and the metastasis of CXCR4- and CXCR7-positive tumors [6]. Except for the chemoattractant effects SDF-1α has an inhibiting effect on HIV-1 in vitro [7].

 

2.         Principle of the test

 

The biotinylated antibody binds to the streptavidin coated microtiterplate. The SDF-1 α of the standards/samples binds to this antibody and is detected by the primary antibody. The biotinylated antibody, the primary antibody and the standards/samples are incubated on the microtiterplate altogether at the same time. The primary antibody, which is bound to SDF-1α, is then detected by a secondary peroxidase-conjugated antibody. 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 SDF-1α.

 

3.         Kit contents and storage

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

2. Biotinylated Antibody: antibody to human SDF-1α, 6 µg, lyophilized, reconstitute in 60 µl Assaybuffer, dilute 1:100.

3. Detection Antibody solution: Monoclonal Antibody: antibody to human SDF-1, 360 µl (100 µg/ml)

4. Standard, human SDF-1 alpha, 1 µg, lyophilized.

5. Conjugate: Goat-anti mouse IgG-Peroxidase conjugate, 50 µl, dilute 1:2000

6. Assaybuffer: 40 ml, ready for use.

7. Washing buffer: 20 ml, 10-fold concentrate.

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

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

10. nstructions 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 SDF-1 alpha-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 250 µl of the Assay Buffer .

6.1.3. Reconstitute the biotinylated antibody with 60 µl Assaybuffer (stock solution).

Mix carefully and keep at room temperature for at least 30 minutes to ensure complete dissolution. The dissolved antibody is stable for one month at 4 °C.

6.1.4  Preparation of the Antibody working solution:

The antibody working solution should be prepared as follows

Spin down the contents of the vials with the reconstituted Capture Antibody and the Detection Antibody solution by centrifugation and transfers contents into a vial with 6 ml Assaybuffer. Use the same day.

For a half plate: Mix 3 ml of assaybuffer with 30 µl Capture antibody and 180 µl Detection antibody solution.

6.1.5 Conjugate: Dilute 1 + 2000 with Assaybuffer, e.g. 6 µl plus 12 ml Assaybuffer for 1 plate. Use the working solution the same day

6.1.6 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.

 The kit should be stored at 2-8°C.

 

4.         Materials required, but not supplied

 

6.2      Preparation of standards

Standard 1 (40 ng/ml): Mix 10 µl Reconstituted Standard with 990 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

If overnight incubation is used:

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

Dispense 50 µl of the assay buffer into the other wells of the microtiter plate.

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

Dispense 50 µl of antibody working solution in each well

Incubate for one hour or overnight 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 30 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 tandard curve (not to be used for calculation of actual results):

 

 

Validation and references

9.         Validation of the assay

 

Expected SDF-1 alpha levels in Normal and Disease State Sera:

 

 

Mean sdf-1 alpha Concentration (ng/ml)

Range

Normal sera (n = 10)

2.86

0.31 – 15.1

Tumor sera (n = 13)

20.31

0.82 - 75.62

Diabetes Sera (n = 10)

11.63

1.17 – 39.23

Rheuma Sera (n = 10)

18.65

2.90 – 60.37

 

Serum-Plasma-Pairs:

10 serum plasma pairs (EDTA-plasma) have been tested. The correlation between the absorbance values of plasma and serum was 0.9957.

 

Interfering Substances:

No interference has been observed in spiked samples with up to 1mg/ml bilirubin, 5 mg hemoglobin and 4 mg/ml triglycerides. Anyway, we do not recommend to use highly lipemic or grossly hemolyzed samples.

 

Intra-assay Precision:                                                            

The intra-assay precision was determined by testing plasma samples 9 times on one plate. The CV was 0.80 % (sample 1) and 2.24 % (sample 2).

 

Inter–assay Precision:

The intra-assay precision was determined by testing plasma samples (4-fold determinations) on five days. The Inter-assay precision was 8.37 %.

 

Sensitivity:

The minimum detectable dose was determined by adding two standard deviations to the mean optical density value of the zero-standard and calculating the corresponding concentration. The minimum detectable dose was 0.31 ng/ml.

 

Linearity:

To assess the linearity of the assay, a sample containing an elevated concentration of SDF-1 alpha was diluted with assay buffer to produce samples with values within the range of the standard curve:

 

Sample dilution

Measured concentration,

Recalculated, ng/ml

1:2

33

1:2,5

29.96

1:5

33,6

1:10

30,65

Mean Value/CV

31,8/ 5,77

Intra-assay Precision:                                                            

The intra-assay precision was determined by testing plasma samples 9 times on one plate. The CV was 0.80 % (sample 1) and 2.24 % (sample 2).

 

Inter–assay Precision:

The intra-assay precision was determined by testing plasma samples (4-fold determinations) on five days. The Inter-assay precision was 8.37 %.

 

Sensitivity:

The minimum detectable dose was determined by adding two standard deviations to the mean optical density value of the zero-standard and calculating the corresponding concentration. The minimum detectable dose was 0.31 ng/ml.

 

10.      References

 

[1] Takahashi M (2010) "Role of the SDF-1/CXCR4 System in Myocardial Infarction." Circulation Journal 74 (3): 418-423

[2] Rollins BJ (1997) "Chemokines." Blood 90: 909–928

[3] Bleul CC et al (1996) "A Highly Efficacious Lymphocyte Chemoattractant, Stromal Cell-derived Factor 1 (SDF-1)." Journal of Experimental Medicine 184: 1101-1109

[4] Pelletier AJ et al. (2000) "Presentation of chemokine SDF-1a by fibronectin mediates directed migration of T cells." Blood 96: 2682-2690

[5] Mbemba E et al (2000) "Glycan and glucosaminoglycan binding properties of stromal cell-derived factor (SDF)-1α." Glycobiology 10 (1): 21-29

[6] Majka M et al (2006) "SDF-1 alone and in co-operation with HGF regulates biology of human cervical carcinoma cells." Folia Histochemika et Cytobiologica 44 (3): 155-164

[7] Maréchal V (1999) "Opposite Effects of SDF-1 on Human Immunodeficiency Virus Type 1 Replication." Journal of Virology 73 (5): 3608-3615.