Download biomedica product listHuman Angiopoietin-2 ELISA Kit | BI-ANG2
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Method
Sandwich ELISA, HRP/TMB, 12×8-well detachable strips
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Sample type
Serum, EDTA plasma, citrate plasma, heparın plasma, urine, cell culture supernatant
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Sample volume
20 µl / well
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Assay time
2 h / 1 h / 30 min
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Sensitivity
3.7 pmol/l (= 203 pg/ml)
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Standard range
0 – 400 pmol/l (= 0 – 21 960 pg/ml)
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Conversion factor
1 pmol/l = 54.9 pg/ml (MW: 54.9 kDa)
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Precision
In-between-run (n=9): ≤ 6 % CV
Within-run (n=3): ≤ 8 % CV
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Specificity
According to epitope mapping and sequence analysis the Angiopoietin-2 ELISA should detect all 3 Angiopoietin-2 isoforms.
Endogenous and recombinant human Angiopoietin-2.
-
Cross-reactivity
No cross-reactivity with Angiopoietin-1.
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Use
Research use only
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Validation Data
See validation data tab for: precision, accuracy, dilution linearity, values for healthy donors, etc
Human Ang2 ELISA Product Overview
The Angiopoietin 2 ELISA kit is a 3.5 h, 96-well sandwich ELISA for the quantitative determination of Angiopoietin-2 in human serum, plasma, urine and cell culture supernatant.
The human Angiopoietin-2 ELISA kit uses highly purified, epitope mapped antibodies.
Human Ang2 ELISA Assay Principle
The human Angiopoietin 2 ELISA kit is a sandwich enzyme immunoassay for the quantitative determination of Angiopoietin-2 in human serum, plasma, urine and cell culture supernatant.
The figure below explains the principle of the human Ang-2 sandwich ELISA:
Capture antibody: monoclonal anti-human Angiopoietin-2 antibody
Detection antibody: polyclonal goat anti-human Angiopoietin-2 antibody
Target antigen: human Angiopoietin-2
In a first step, pre-diluted standard/control/sample and biotinylated antibody (goat polyclonal anti-human Angiopoietin-2) are pipetted into the wells of the microtiter strips, which are pre-coated with a monoclonal anti-human angiopoietin-2 antibody. Angiopoietin-2 present in the standard/control/sample binds to the pre-coated antibody in the well and forms a sandwich with the anti-human Angiopoietin-2 antibody. In a washing step all non-specific unbound material is removed. In a second step, the conjugate (Streptavidin-HRP) is pipetted into the wells and reacts with the biotinylated antibody. After another washing step, the substrate (TMB, tetramethylbenzidine) is pipetted into the wells. The enzyme-catalyzed color change of the substrate is directly proportional to the amount of Angiopoietin-2 present in the sample. This color change is detectable with a standard microplate reader. A dose response curve of the absorbance (optical density, OD at 450 nm) using the values obtained from the standards versus the standard concentration is generated. The concentration of Angiopoietin-2 in the sample is determined directly from the dose response curve.
Human Ang2 ELISA Typical Standard Curve
The figure below shows a typical standard curve for the Ang-2 ELISA. The immunoassay is calibrated against recombinant human Angiopoietin-2 protein:
Human Angiopoietin-2 ELISA Kit Components
|
Contents |
Description |
Quantity |
|
PLATE |
Recombinant human monoclonal Angiopoietin-2 antibody pre-coated microtiter strips in stripholder packed in aluminium bag with desiccant |
12 x 8 tests |
|
WASHBUF |
Wash buffer concentrate 20x, natural cap |
1 x 50 ml |
|
STD |
Standards (0; 12.5; 25; 50; 100; 200; 400 pmol/l) recombinant human angiopoietin-2, white caps, lyophilized |
7 vials |
|
CTRL |
Control A and B, yellow cap, lyophilized, exact concentrations see labels |
2 vials |
|
ASYBUF |
Assay buffer, red cap, ready to use |
1 x 22 ml |
|
AB |
Goat polyclonal anti-human Angiopoietin-2 antibody, biotinylated, green cap, ready to use |
1 x 7 ml |
|
CONJ |
Conjugate, (streptavidin-HRPO), amber cap, ready to use |
1 x 13 ml |
|
SUB |
Substrate (TMB solution), blue cap, ready to use |
1 x 13 ml |
|
STOP |
Stop solution, white cap, ready to use |
1 x 7 ml |
Storage instructions: all reagents of the Angiopoietin-2 ELISA kit are stable at 4°C (2-8°C) until the expiry date stated on the label of each reagent.
Serum, EDTA plasma, heparın plasma, citrate plasma, urine and cell culture supernatant are suitable for use in this human Ang2 ELISA kit. Do not change sample type during studies. We recommend duplicate measurements for all samples, standards and controls. The sample collection and storage conditions listed are intended as general guidelines.
Serum & Plasma
Collect venous blood samples in standardized serum separator tubes (SST) or standardized blood collection tubes using EDTA, heparın or citrate as an anticoagulant. For serum samples, allow samples to clot for 30 minutes at room temperature. Perform separation by centrifugation according to the tube manufacturer’s instructions for use. Assay the acquired samples immediately or aliquot and store at -25°C or lower. Lipemic or haemolyzed samples may give erroneous results. Samples are stable for four freeze-thaw cycles. Thawed samples should be assayed as soon as possible.
Urine
Note: the experiments performed to measure Angiopoietin-2 in urine samples did not undergo a full validation according to FDA/ICH/EMEA guidelines. However, our performance check suggests that urine samples can be measured with this ELISA.
Aseptically collect the first urine of the day (mid-stream), voided directly into a sterile container. Centrifuge to remove particulate matter, assay immediately or aliquot and store at -25°C or lower. Do not freeze-thaw samples more than four times. Thawed samples should be assayed as soon as possible.
Cell Culture Supernatant
Note: the experiments performed to measure Angiopoietin-2 cell culture supernatant samples did not undergo a full validation according to FDA/ICH/EMEA guidelines. However, our performance check suggests that cell culture supernatant samples can be measured with this ELISA.
Remove particulates by centrifugation and assay immediately or aliquot and store samples at -25°C or lower. Do not freeze-thaw samples more than four times. Thawed samples should be assayed as soon as possible.
Reagent Preparation
Wash Buffer
|
1. |
Bring the WASHBUF concentrate to room temperature. Crystals in the buffer concentrate will dissolve at room temperature. |
|
2. |
Dilute the WASHBUF concentrate 1:20, e.g. 50 ml WASHBUF + 950 ml distilled or deionized water. Only use diluted WASHBUF when performing the assay. |
The diluted WASHBUF is stable up to one month at 4°C (2-8°C).
Standards
|
1. |
Pipette 200 µl of distilled or deionized water into each standard (STD) and control (CTRL) vial. The exact concentration is printed on the label of each vial. |
|
2. |
Leave at room temperature (18-26°C) for 15 min. Vortex gently. |
Reconstituted STDs and CTRLs are stable -25°C or lower until expiry date stated on the label and can be subjected to up to three freeze-thaw cycles.
STD / CTRL must be diluted 1+10 with assay buffer (ASYBUF), e.g. 20 μl STD / CTRL + 200 μl ASYBUF.
Sample Preparation
Bring samples to room temperature and mix samples gently to ensure the samples are homogeneous. We recommend duplicate measurements for all samples.
Samples must be diluted 1+10 with assay buffer (ASYBUF), e.g. 20 μl sample + 200 μl ASYBUF.
Samples with values above STD7 (400 pmol/l) after pre-dilution can be diluted further with ASYBUF (Assay buffer).
Human Ang2 ELISA Assay Protocol
Read the entire protocol before beginning the assay.
|
1. |
Bring samples and reagents to room temperature (18-26°C). |
|
2. |
Mark positions for STD/CTRL/SAMPLE (standard/control/sample) on the protocol sheet. |
|
3. |
Take microtiter strips out of the aluminum bag. Store unused strips with desiccant at 4°C in the aluminum bag. Strips are stable until expiry date stated on the label. |
|
4. |
Pipette 50 μl 1+10 pre-diluted STD/CTRL/SAMPLE (standard/control/sample) in duplicate into respective wells. See reagents and sample preparation for pre-dilution. |
|
5. |
Add 50 μl AB (biotinylated anti angiopoietin-2 antibody) into all wells, swirl gently. |
|
6. |
Cover tightly and incubate for 2 hours at room temperature (18-26°C), in the dark. |
|
7. |
Aspirate and wash wells 5x with 300 μl diluted wash buffer. After final wash, remove remaining wash buffer by strongly tapping plate against paper towel. |
|
8. |
Add 100 μl CONJ (Conjugate, amber cap) into each well, swirl gently. |
|
9. |
Cover tightly and incubate for 1 hour at room temperature (18-26°C), in the dark. |
|
10. |
Aspirate and wash wells 5x with 300 μl diluted wash buffer. After final wash, remove remaining wash buffer by strongly tapping plate against paper towel. |
|
11. |
Add 100 μl SUB (substrate, blue cap) into each well, swirl gently. |
|
12. |
Incubate for 30 min at room temperature (18-26°C), in the dark. |
|
13. |
Add 50 μl STOP (stop solution, white cap) into each well, swirl gently. |
|
14. |
Measure absorbance immediately at 450 nm with reference 630 nm, if available. |
Calculation of Results
Read the optical density (OD) of all wells on a plate reader using 450 nm wavelength (correction wavelength 630 nm). Construct the standard curve from the OD values of the STD. Use commercially available software or graph paper. Obtain sample concentration from this standard curve. The assay was evaluated with 4PL algorithm. Different curve fitting methods need to be evaluated by the user.
If required, the pmol/l concentration can be converted into pg/ml by applying a conversion factor (1 pg/ml = 0.018 pmol/l; MW: 54.9 kDa). Respective dilution factors have to be considered when calculating
the final concentration of the sample.
The quality control protocol supplied with the kit shows the results of the final release QC for each kit at production date. Data for OD obtained by customers may differ due to various influences including the normal decrease of signal intensity throughout shelf life. However, this does not affect validity of results as long as an OD of 1.5 or higher is obtained for the standard with the highest concentration and the control value is in range (target range see label).
Angiopoietin-2 Protein
Angiopoietin-2 (ANG2) is a 56.9 kDa glycosylated growth factor that is specific for endothelial cells (ECs). ANG2 is expressed in embryonic vessels and contributes to the formation of new vasculature. In adults, it is restricted to sites of vascular remodeling (e.g. ovary, uterus, placenta) and wound healing. ANG2 is regulated by the cytokine vascular endothelial growth factor (VEGF). Together with VEGF, ANG2 induces endothelial cell migration, proliferation, and vascular sprouting. During angiogenesis, ANG2 exerts its effects via the angiopoietin-1/TIE2 receptor signaling system on endothelial cells. Disruption of this signaling leads to the loss of endothelial integrity. In consequence, the endothelium responds to various pro-inflammatory cytokines and growth factors. Thus, ANG2 might cause vascular micro-inflammation in patients with chronic kidney disease (CKD). Various studies demonstrated that ANG2 levels increase with CKD stage and are associated with fluid overload and abnormal cardiac structure. Furthermore, ANG2 concentrations correlate with mortality in patients with CKD stages 4–5. Although ANG2 levels recover after successful kidney transplantation, ANG2 continues to be a cardiovascular risk factor in this population.
In cancer, targeting the TIE2-Angiopoietin pathway has shown promising results in some pre-clinical and clinical trials, including studies on recurrent or metastatic breast and renal cell carcinomas.
In COVID-19 patients, ANG2 was recently reported to be a relevant factor to predict transfer to the ICU as it was associated with poor lung compliance. Thus, showing that endothelial activation reinforces the hypothesis of a COVID-19-associated microvascular dysfunction. In this context, another study demonstrated that ANG2 levels in critically ill COVID-19 patients correlate with disease severity, hypercoagulation, and mortality. The researchers also provided novel in vivo evidence for a direct role for ANG2 in coagulation through binding to and inhibition of thrombomodulin-mediated anticoagulation. The scientists suggest that inhibition of ANG2 might be beneficial for treating critically ill COVID-19 patients, as well as other patients with hypercoagulation.
|
Molecular weight |
56.9 kDa |
|
Cellular localization |
Extracellular, plasma membrane |
|
Post-translational modifications |
Glycosylation, lipidation (GPI-anchor) |
|
Alternative names |
ANG2, ANGPT2, Angiopoietin 2, Angiopoietin-2, ANG-2, Angiopoietin-2B, Angiopoietin-2a, Tie2-Ligand |
|
Entrez/NCBI ID |
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Genecards |
|
|
OMIM |
|
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Protein Atlas |
|
|
Uniprot ID |
Areas of interest:
- Ischemic pathologies (PAD, CAD)
- Inflammation (Bowel disease, Chron’s disease, cirrhosis, sepsis)
- Autoimmune disease (rheumatoid arthritis, psoriasis)
- Artherosclerosis
- Chronic kidney disease
- Diabetic retinopathy
- Cancer
- COVID-19
- Biomedical significance of endothelial cell specific growth factor, angiopoietin.
Koh, G.Y., Kim, I., Kwak, H.J., Yun, M.J., Leem J.C., 2002. Exp. Mol. Med., 34(1):1–11.
PMID:11989972 - Control of vascular morphogenesis and homeostasis through the angiopoietin–Tie system.
Augustin, H.G., Koh, G.Y., Thurston G., Alitalo, K., 2009. Nat. Rev. Mol. Cell. Biol., 10(3): 165–177.
PMID:19234476 - Crystal structures of the Tie2 receptor ectodomain and the angiopoietin-2–Tie2 complex.
Barton, W.A., Tzvetkova-Robev, D., Miranda, E.P., Kolev, M.V., Rajashankar, K.R., Himanen, J.P., Nikolov, D.B., 2006; Nat. Struct. Mol. Biol., 13(6): 524–532.
PMID:16732286 - Characterization and Expression of a Novel Alternatively Spliced Human Angiopoietin-2.
Kim, I., Kim, J.H., Ryu, Y.S., Jung, S.H., Nah, J.J., Koh, G.Y., 2000. J. Biol. Chem., 275(24): 18550–18556.
PMID:10766762 - Angiopoietin-1 and Angiopoietin-2 Inhibitors: Clinical Development.
Gillen, J., Richardson, D., Moore, K., 2019. Curr. Oncol. Rep., 21(3): 22.
PMID:30806847 - Targeting the Angiopoietin-2/Tie-2 axis in conjunction with VEGF signal interference.
Biel, N.M., Siemann D.W., 2016. Cancer Lett., 380(2): 525–533.
PMID:25312939 - Angiopoietin-2: a multifaceted cytokine that functions in both angiogenesis and inflammation: Angiopoietin-2 and inflammation.
Scholz, A., Plate, K.H., Reiss, Y. 2015. Ann. N. Y. Acad. Sci., 1347(1): 45–51.
PMID:25773744 - Circulating Angiopoietin-2 levels predict mortality in kidney transplant recipients: a 4-year prospective case-cohort study.
Molnar, M.Z., Kümpers, P., Kielstein, J.T., Schiffer, M., Czira, M.E., Ujszaszi, A., Kovesdy, C.P., Mucsi, I., 2014; Transpl. Int., 27(6): 541–552.
PMID:24628855 - Circulating angiopoietin-2 levels increase with progress of chronic kidney disease.
David, S., Kümpers, P., Lukasz, A., Fliser, D., Martens-Lobenhoffer, J., Bode-Böger, S.M., Kliem, V., Haller, H., Kielstein, J.T., 2010. Nephrol Dial Transplant, 25: 2571-2579.
PMID:20179005 - The interaction between fluid status and angiopoietin-2 in adverse renal outcomes of chronic kidney disease.
Tsai, Y.C., Chiu, Y.W., Kuo, H.T., Lee J.J., Lee, S.C., Chen, T.H., Lin, M.Y., Hwang, S.J., Kuo, M.C., Hsu, Y.L., Chen, H.C., 2017. PLoS One, 12 (3): e173906.
PMID: 28333979 - Angiopoietin-2, Angiopoietin-1 and subclinical cardiovascular disease in Chronic Kidney Disease.
Tsai, Y.C., Lee, C.S., Chiu, Y.W., Kuo, H.T., Lee, S.C., Hwang, S.J., Kuo, M.C., Chen, H.C., 2016. Scientific Reports, 6: 39400.
PMID:27991547 - Angiopoietin-1 and Angiopoietin-2 Inhibitors: Clinical Developments.
Gillen, J., Richardson, D., Moore, K., 2019. Current Oncology Reports, 21: 22.
PMID:30806847 - Angiopoietin-2 as a marker of endothelial activation is a good predictor factor for intensive care unit admission of COVID-19 patients.
Smadja, DM., Guerin, CL., Chocron, R., Yatim, N., Boussier, J., Gendron, N., Khider, L., Hadjadj, J., Goudot, G., Debuc, B., Juvin, P., Hauw-Berlemont, C., Augy, JL., Peron, N., .. Diehl, JL. 2020. Angiogenesis, 2020;1-10.
PMID:32458111 - Elevated Angiopoietin-2 inhibits thrombomodulin-mediated anticoagulation in critically ill COVID-19 patients.
Hultstrom M., Fromell, K., Larsson, A., Quaggin, SE., Betsholz, C, Frithiof, R., Lipcsey, M. 2021. MedRxiv preprint server, 2021.
PMID:11989972All Biomedica ELISAs are validated according to international FDA/ICH/EMEA guidelines. For more information about our validation guidelines, please refer to our quality page and published validation guidelines and literature.
- ICH Q2(R1) Validation of Analytical Procedures: Text and Methodology.
- EMEA/CHMP/EWP/192217/2009 Guideline on bioanalytical method validation.
- Bioanalytical Method Validation, Guidance for Industry, FDA, May 2018
Calibration
The Angiopoietin-2 immunoassay is calibrated against recombinant human soluble Angiopoietin-2 protein (O15123 (Uniprot ID)).
Human Ang2 ELISA Detection Limit & Sensitivity
To determine the sensitivity of the Angiopoietin-2 ELISA, experiments measuring the lower limit of detection (LOD) and the lower limit of quantification (LLOQ) were conducted.
The LOD, also called the detection limit, is the lowest point at which a signal can be distinguished above the background signal, i.e. the signal that is measured in the absence of Angiopoietin-2, with a confidence level of 99%. It is defined as the mean back calculated concentration of standard 1 (0 pmol/l of Angiopoietin-2, five independent measurements) plus three times the standard deviation of the measurements.
The LLOQ, or sensitivity of an assay, is the lowest concentration at which an analyte can be accurately quantified. The criteria for accurate quantification at the LLOQ are an analyte recovery between 75 and 125% and a coefficient of variation (CV) of less than 25%. To determine the LLOQ, standard 2, i.e. the lowest standard containing Angiopoietin-2, is diluted, measured five times and its concentration is back calculated. The lowest dilution, which meets both criteria, is reported as the LLOQ.
The following values were determined for the Angiopoietin-2 ELISA:
|
LOD |
3.7 pmol/l |
|
LLOQ |
6.3 pmol/l |
Human Ang2 ELISA Precision
The precision of an ELISA is defined as its ability to measure the same concentration consistently within the same experiments carried out by one operator (within-run precision or repeatability) and across several experiments using the same samples but conducted by several operators using different ELISA lots (in-between-run precision or reproducibility).
Within-Run Precision
Within-run (intra-assay) precision was assessed by measuring two samples of known concentrations three times within one Angiopoietin-2 ELISA kit lot by one operator.
|
ID |
n |
Mean Angiopoietin-2 [pmol/l] |
SD [pmol/l] |
CV (%) |
|
Sample 1 |
3 |
25 |
2.1 |
8 |
|
Sample 2 |
3 |
201 |
3.0 |
1 |
In-Between-Run Precision
In-between-run (inter-assay) precision was assessed by measuring two samples of known concentrations nine times within three FGF23 (intact) ELISA lots by two operators.
|
ID |
n |
Mean Angiopoietin-2 [pg/ml] |
SD [pg/ml] |
CV (%) |
|
Sample 1 |
9 |
26 |
1.6 |
6 |
|
Sample 2 |
9 |
201 |
5.1 |
3 |
Human Ang2 ELISA Accuracy
The accuracy of an ELISA is defined as the precision with which it can recover samples of known concentrations.
The recovery of the Angiopoietin-2 human ELISA was measured by adding recombinant Angiopoietin-2 to human samples containing a known concentration endogenous Angiopoietin-2. The % recovery of the spiked concentration was calculated as the percentage of measured compared over the expected value.
The tables shows the summary of the recovery experiments in the human Angiopoietin-2 ELISA in human samples:
|
|
% Recovery |
||||
|
+36 pmol/l |
+180 pmol/l |
||||
|
Sample matrix |
n |
Mean |
Range |
Mean |
Range |
|
Serum |
6 |
81 |
72-94 |
93 |
77-100 |
|
Citrate plasma |
1 |
100 |
- |
95 |
- |
|
|
% Recovery |
||||
|
+40 pmol/l |
+200 pmol/l |
||||
|
Sample matrix |
n |
Mean |
Range |
Mean |
Range |
|
EDTA plasma |
6 |
85 |
73-94 |
78 |
71-85 |
|
Heparın plasma |
1 |
89 |
- |
79 |
- |
Data showing % recovery of recombinant Angiopoietin-2 in human serum samples:
|
Angiopoietin-2 [pmol/l] |
% Recovery |
|||||
|
Sample matrix |
ID |
Reference |
+ 36 pmol/l |
+ 180 pmol/l |
+ 36 pmol/l |
+ 180 pmol/l |
|
Serum |
s1 |
18 |
51 |
181 |
88 |
96 |
|
Serum |
s2 |
20 |
52 |
185 |
94 |
97 |
|
Serum |
s3 |
63 |
84 |
202 |
76 |
95 |
|
Serum |
s4 |
15 |
39 |
147 |
72 |
77 |
|
Serum |
s5 |
30 |
55 |
195 |
78 |
100 |
|
Serum |
s6 |
33 |
58 |
187 |
78 |
95 |
|
Mean |
81 | 93 | ||||
|
Min |
72 | 77 | ||||
|
Max |
94 | 100 | ||||
Data showing % recovery of recombinant Angiopoietin-2 in human citrate sample:
|
Angiopoietin-2 [pmol/l] |
% Recovery |
|||||
|
Sample matrix |
ID |
Reference |
+ 36 pmol/l |
+ 180 pmol/l |
+ 36 pmol/l |
+ 180 pmol/l |
|
Citrate plasma |
c1 |
16 |
51 |
180 |
100 |
95 |
Data showing % recovery of recombinant Angiopoietin-2 in human serum samples:
|
Angiopoietin-2 [pmol/l] |
% Recovery |
|||||
|
Sample matrix |
ID |
Reference |
+ 40 pmol/l |
+ 200 pmol/l |
+ 40 pmol/l |
+ 200 pmol/l |
|
EDTA plasma |
e1 |
13 |
45 |
164 |
81 |
79 |
|
EDTA plasma |
e2 |
16 |
50 |
168 |
87 |
80 |
|
EDTA plasma |
e3 |
48 |
78 |
166 |
87 |
71 |
|
EDTA plasma |
e4 |
11 |
47 |
160 |
94 |
77 |
|
EDTA plasma |
e5 |
19 |
53 |
180 |
89 |
85 |
|
EDTA plasma |
e6 |
30 |
56 |
164 |
73 |
74 |
|
Mean |
85 |
78 |
||||
|
Min |
73 |
71 |
||||
|
Max |
94 |
85 |
||||
Data showing % recovery of recombinant Angiopoietin-2 in human citrate sample:
|
Angiopoietin-2 [pmol/l] |
% Recovery |
|||||
|
Sample matrix |
ID |
Reference |
+ 40 pmol/l |
+ 200 pmol/l |
+ 40 pmol/l |
+ 200 pmol/l |
|
Heparın plasma |
h1 |
14 |
49 |
165 |
89 |
79 |
Human Ang2 ELISA Dilution Linearity & Parallelism
Tests of dilution linearity and parallelism ensure that both recombinant and endogenous samples containing Angiopoietin-2 (ANG2) behave in a dose dependent manner and are not affected by matrix effects. Dilution linearity assesses the accuracy of measurements in diluted human samples spiked with known concentrations of recombinant analyte. By contrast, parallelism refers to dilution linearity in human samples and provides evidence that the endogenous analyte behaves in the same way as the recombinant one. Dilution linearity and parallelism are assessed for each sample type and should be within 20% of the expected concentration.
Dilution Linearity
Dilution linearity was assessed by serially diluting human samples spiked with recombinant Angiopoietin-2 with assay buffer.
The table below shows the mean recovery and range of serially diluted recombinant Angiopoietin-2 :
|
|
|
% Recovery of recombinant Angiopoietin-2 in diluted samples |
|||
|
|
1+1 |
1+3 |
|||
|
Sample matrix |
n |
Mean |
Range |
Mean |
Range |
|
Serum |
6 |
120 |
112-141 |
124 |
117-140 |
|
EDTA plasma |
6 |
106 |
103-110 |
122 |
114-131 |
|
Citrate plasma |
1 |
118 |
- |
117 |
- |
|
Heparın plasma |
1 |
97 |
- |
111 |
- |
Data showing dilution linearity of human serum samples containing recombinant Angiopoietin-2:
|
Angiopoietin-2 [pmol/l] |
Recovery (%) |
|||||
|
Sample matrix |
ID |
Reference |
1+1 |
1+3 |
1+1 |
1+3 |
|
Serum |
s1 |
181 |
104 |
54 |
116 |
119 |
|
Serum |
s2 |
185 |
108 |
56 |
117 |
122 |
|
Serum |
s3 |
202 |
114 |
60 |
112 |
118 |
|
Serum |
s4 |
147 |
103 |
51 |
141 |
140 |
|
Serum |
s5 |
195 |
112 |
57 |
115 |
117 |
|
Serum |
s6 |
187 |
114 |
60 |
122 |
127 |
|
|
|
|
|
Mean |
120 |
124 |
|
|
|
|
|
Min |
112 |
117 |
|
|
|
|
|
Max |
141 |
140 |
Data showing dilution linearity of human EDTA plasma samples containing recombinant Angiopoietin-2:
|
Angiopoietin-2 [pmol/l] |
Recovery (%) |
|||||
|
Sample matrix |
ID |
Reference |
1+1 |
1+3 |
1+1 |
1+3 |
|
EDTA plasma |
e1 |
164 |
86 |
49 |
105 |
119 |
|
EDTA plasma |
e2 |
168 |
93 |
51 |
110 |
121 |
|
EDTA plasma |
e3 |
166 |
86 |
54 |
103 |
130 |
|
EDTA plasma |
e4 |
160 |
87 |
46 |
109 |
116 |
|
EDTA plasma |
e5 |
180 |
93 |
51 |
104 |
114 |
|
EDTA plasma |
e6 |
164 |
87 |
53 |
106 |
131 |
|
|
|
|
|
Mean |
106 |
122 |
|
|
|
|
|
Min |
103 |
114 |
|
|
|
|
|
Max |
110 |
131 |
Data showing dilution linearity of human citrate plasma samples containing recombinant Angiopoietin-2:
|
Angiopoietin-2 [pmol/l] |
Recovery (%) |
|||||
|
Sample matrix |
ID |
Reference |
1+1 |
1+3 |
1+1 |
1+3 |
|
Citrate plasma |
c1 |
173 |
102 |
51 |
118 |
117 |
Data showing dilution linearity of human heparın plasma samples containing recombinant Angiopoietin-2:
|
Angiopoietin-2 [pmol/l] |
Recovery (%) |
|||||
|
Sample matrix |
ID |
Reference |
1+1 |
1+3 |
1+1 |
1+3 |
|
Heparın plasma |
h1 |
165 |
80 |
46 |
97 |
111 |
Parallelism
Parallelism was assessed by serially diluting serum samples containing endogenous Angiopoietin-2 with assay buffer.
The table below shows the mean recovery and range of serially diluted endogenous Angiopoietin-2 in human samples:
| % Recovery of endogenous Angiopoietin-2 in diluted samples | |||||
|
1+1 |
1+3 |
||||
|
Sample matrix |
n |
Mean |
Range |
Mean |
Range |
|
Serum |
6 |
105 |
98-115 |
105 |
98-120 |
|
EDTA plasma |
6 |
110 |
100-128 |
122 |
110-139 |
|
Citrate plasma |
1 |
104 |
- |
115 |
- |
|
Heparın plasma |
1 |
107 |
- |
109 |
- |
Data showing dilution linearity of human serum samples containing endogenous Angiopoietin-2:
|
Angiopoietin-2 [pmol/l] |
Recovery (%) |
|||||
|
Sample matrix |
ID |
Reference |
1+1 |
1+3 |
1+1 |
1+3 |
|
Serum |
s1 |
73 |
42 |
22 |
115 |
120 |
|
Serum |
s2 |
81 |
42 |
21 |
104 |
101 |
|
Serum |
s3 |
87 |
42 |
21 |
98 |
99 |
|
Serum |
s4 |
80 |
43 |
22 |
106 |
109 |
|
Serum |
s5 |
83 |
43 |
20 |
104 |
98 |
|
Serum |
s6 |
79 |
41 |
20 |
104 |
101 |
|
|
|
|
|
Mean |
105 |
105 |
|
|
|
|
|
Min |
98 |
98 |
|
|
|
|
|
Max |
115 |
120 |
Data showing dilution linearity of human EDTA plasma samples containing endogenous Angiopoietin-2:
|
Angiopoietin-2 [pmol/l] |
Recovery (%) |
|||||
|
Sample matrix |
ID |
Reference |
1+1 |
1+3 |
1+1 |
1+3 |
|
EDTA plasma |
e1 |
76 |
48 |
25 |
128 |
133 |
|
EDTA plasma |
e2 |
81 |
45 |
24 |
111 |
120 |
|
EDTA plasma |
e3 |
72 |
36 |
20 |
101 |
110 |
|
EDTA plasma |
e4 |
67 |
35 |
20 |
105 |
118 |
|
EDTA plasma |
e5 |
67 |
34 |
19 |
100 |
115 |
|
EDTA plasma |
e6 |
52 |
29 |
18 |
112 |
139 |
|
|
|
|
|
Mean |
110 |
122 |
|
|
|
|
|
Min |
100 |
110 |
|
|
|
|
|
Max |
128 |
139 |
Data showing dilution linearity of human citrate plasma samples containing endogenous Angiopoietin-2:
|
Angiopoietin-2 [pmol/l] |
Recovery (%) |
|||||
|
Sample matrix |
ID |
Reference |
1+1 |
1+3 |
1+1 |
1+3 |
|
Citrate plasma |
c1 |
117 |
61 |
33 |
104 |
115 |
Data showing dilution linearity of human heparın samples containing endogenous Angiopoietin-2:
|
Angiopoietin-2 [pmol/l] |
Recovery (%) |
|||||
|
Sample matrix |
ID |
Reference |
1+1 |
1+3 |
1+1 |
1+3 |
|
Heparın plasma |
h1 |
160 |
85 |
44 |
107 |
109 |
Human Ang2 ELISA Specificity
This assay recognizes endogenous and recombinant human Angiopoietin-2. According to epiptope mapping and sequence analysis Angiopoietin-2 ELISA should detect all 3 isoforms. Angiopoietin-1 is not detected in this assay.
The specificity of an ELISA is defined as its ability to exclusively recognize the analyte of interest. The specificity of the human Angiopoietin-2 ELISA was shown by characterizing both the capture and the detection antibody through epitope mapping. Both antibodies used in the human Angiopoietin-2 ELISA bind to Angiopoietin-2 with high affinity. Moreover, the specificity of the ELISA was established through competition experiments, which measure the ability of the antibodies to exclusively bind to Angiopoietin-2.
Epitope Mapping
Antibody binding sites were determined by epitope mapping using microarray analysis (Pepperprint GmbH). The capture antibody shows no linear epitope, but has a structural epitope which binds to receptor binding site of human ANG2. This receptor binds to TEL/TIE2 protein. The detection antibody binds to several epitopes distributed over the entire Angiopoietin-2 molecule.
CAB Coating Antibody: recombinant monoclonal mouse anti-human Angiopoietin-2
DAB Detection Antibody: polyclonal goat anti-human Angiopoietin-2
Epitope mapping of detection antibody
Microarray of polyclonal detection antibody reveals seven linear epitopes on human ANG-2 sequence. Fluorescent signals (green) illustrate where the antibody binds to peptide sequences on a microarray chip. These peptides were spotted as 15mers with an 13 amino acid overlap in duplicates on the chip and represent the whole human Angiopoietin-2 sequence. Red fluorescent signals mark the position of control peptides
High affinity antibodies
Biolayer interferometry binding kinetics demonstrate low dissociation of both coating (blue) and detection antibody (orange) towards human Angiopoietin-2 protein (kdis<1.0E-07 s-1) Association and dissociation were determined for 600s (x-axis).
Antibody epitopes on human ANG-2 molecule
SWISS model of human Angiopoietin-2 monomer (122-493 aa, O15113/ 3ghg.1.B) with antibody epitopes. The monoclonal coating antibody binds to the bioactive receptor-binding site within the C-terminus (structural epitope, blue). Linear epitopes (purple) of the detection antibody range over the whole protein. The N-terminus and three additional epitopes are not shown here.
Competition of Signal
Competition experiments were carried out by pre-incubating human samples containing endogenous levels of Angiopoietin-2 with an excess of capture antibody (CAB). The concentration measured in this mixture was then compared to a reference value, which was obtained from the same sample but without the pre-incubation step. Mean competition was 87%, and is within the range of acceptance of international guidelines.
|
Angiopoietin-2 [pmol/l] |
|
||
|
ID |
Reference |
Reference + capture AB |
% Competition |
|
s1 |
295 |
17 |
94 |
|
s2 |
36 |
7 |
81 |
|
s3 |
66 |
14 |
79 |
|
s4 |
103 |
16 |
84 |
|
s5 |
141 |
24 |
83 |
|
s6 |
398 |
3 |
99 |
| s7 |
157 |
19 |
88 |
|
|
Mean |
87 |
|
Isoforms
According to epiptope mapping and sequence analysis Angiopoietin-2 ELISA should detect all 3 isoforms. Angiopoietin-1 is not detected in this assay.
Sample Stability
Serum, EDTA plasma, heparın plasma, and citrate plasma are suitable for use in this assay. Do not change sample type during studies. We recommend duplicate measurements for all samples, standards and controls. The sample collection and storage conditions listed are intended as general guidelines.
Freeze-thaw Stability
The stability of endogenous Angiopoietin-2 (ANG2) was tested by comparing four measurements in samples that had undergone four freeze-thaw cycles (F/T).
For freeze-thaw experiments, samples were collected according to the supplier’s instruction using blood collection devices and stored at -80°C. Reference samples were freeze-thawed once. The mean recovery of sample concentration after four freeze-thaw cycles is 94%.
Angiopoietin-2 concentrations of samples after freeze-thaw (F/T) cycles:
|
|
|
Angiopoietin-2 [pmol/l] |
Recovery (%) 4x F/T vs Reference |
||||
|
Sample matrix |
ID |
Reference |
1x |
2x |
3x |
4x |
|
|
Serum |
s1 |
46 |
43 |
42 |
43 |
47 |
102 |
|
EDTA plasma |
e1 |
48 |
45 |
39 |
38 |
40 |
84 |
|
Citrate plasma |
c1 |
37 |
35 |
33 |
34 |
33 |
91 |
|
Heparın plasma |
h1 |
42 |
41 |
43 |
47 |
41 |
98 |
|
Mean |
96 |
||||||
All samples should undergo a maximum of four freeze-thaw cycles.
Benchtop Stability
The benchtop stability of endogenous Angiopoietin-2 was tested by comparing Angiopoietin-2 measurements in human samples that had been stored at different temperatures.
For the assessment of the benchtop stability, a set of undiluted human samples was aliquoted and stored at at room temperature or at 4°C. Samples can be stored for at least three hours at room temperature as well as overnight at 4°C. The mean recovery of sample concentrations after overnight storage at 4°C is 93%.
Angiopoietin-2 concentrations of samples stored at -25°C (reference), at room temperature (RT) or overnight (oN) at 4°C:
|
|
|
Angiopoietin-2 [pmol/l] |
Recovery (%) oN 4°C vs Reference |
|||
|
Sample matrix |
ID |
Reference |
1 h RT |
3 h RT |
oN 4°C |
|
|
Serum |
s1 |
65 |
75 |
68 |
63 |
98 |
|
EDTA plasma |
e1 |
65 |
63 |
57 |
58 |
90 |
|
Citrate plasma |
c1 |
66 |
56 |
56 |
53 |
81 |
|
Heparın plasma |
h1 |
66 |
74 |
68 |
68 |
104 |
|
Mean |
93 |
|||||
Sample Values
Angiopoietin-2 Values in Apparently Healthy Individuals
To provide values for circulating Angiopoietin-2 (ANG2), a panel of samples from apparently healthy donors was tested. Each individual donated blood for all tested sample matrices.
A summary of the results is shown below:
|
|
|
Angiopoietin-2 [pmol/l] | ||
|
Sample matrix |
n |
Mean |
Range |
Median |
|
Serum |
11 |
30 | 15-63 | 28 |
|
EDTA plasma |
11 |
26 | 11-48 | 24 |
|
Citrate plasma |
11 |
23 | 8-43 | 23 |
|
Heparın plasma |
11 |
27 | 10-57 | 25 |
It is recommended to establish the normal range for each laboratory.
Angiopoietin-2 Values in Disease Panels
In addition to samples from apparently healthy donors, panels of samples from chronic kidney disease (CKD) patients, CKD patients on dialysis, CKD patients with a kidney transplant (NTX), as well as an unselected hospital panel were tested.
Summary of the results obtained with several disease panels:
|
|
|
Angiopoietin-2 [pmol/l] | ||
|
Sample matrix |
n |
Mean |
Range |
Median |
|
Serum apparently healthy |
11 |
30 | 15-63 | 28 |
|
Serum Nephro 2 |
11 |
118 | 55-204 | 132 |
|
Serum Nephro 1 |
8 |
135 | 66-313 | 114 |
|
Serum NTX |
29 |
71 | 60-87 | 69 |
|
EDTA plasma CKD |
9 |
34 | 18-52 | 34 |
Kidney disease patients (mixed)
Chronic kidney disease patients
Transplant patients
Matrix Comparison
To assess whether all tested matrices behave the same way in the Angiopoietin-2 (ANG2) ELISA, concentrations of ANG2 were measured in serum, EDTA, heparın, and citrate plasma samples prepared from 11 apparently healthy donors. Each individual donated blood in all tested sample matrices.
Data and graph for apparently healthy donors are shown below:
|
|
Angiopoietin-2 [pmol/l] |
|||
|
Donor ID |
Serum |
EDTA plasma |
Citrate plasma |
Heparın plasma |
|
#1 |
46 |
48 |
37 |
42 |
|
#2 |
18 |
13 |
14 |
14 |
|
#3 |
20 |
16 |
16 |
14 |
|
#4 |
63 |
48 |
43 |
57 |
|
#5 |
15 |
11 |
8 |
10 |
|
#6 |
30 |
19 |
20 |
22 |
|
#7 |
33 |
30 |
27 |
30 |
|
#8 |
22 |
19 |
18 |
20 |
|
#9 |
28 |
26 |
26 |
29 |
|
#10 |
33 |
29 |
24 |
30 |
|
#11 |
25 |
24 |
23 |
25 |
Comparison with other human Angiopoietin-2 ELISA assays
Assay Characteristics of different human Angiopoietin-2 ELISA assays
|
|
Biomedica |
Another Manufacturer |
|
Method |
Sandwich ELISA, |
Sandwich ELISA |
|
Sample type |
Plasma, serum, cell culture supernatant, urine |
Plasma, serum, cell culture supernatant, urine, saliva |
|
Sample volume |
20 µl |
20 µl |
|
Assay time |
3.5 h |
4.5 h |
|
Assay range |
686 -21,960 pg/ml (assay range optimized for clinical samples- no additional testing required) |
46.9 - 3,000 pg/ml
|
|
Specificity |
Endogenous and recombinant human ANG2. The ANG2 ELISA should detect all 3 ANG-2 isoforms |
Natural and recombinant human ANG2 |
|
Antibodies |
Recombinant human monoclonal ANG-2, polyclonal anti-human ANG2 (epitope-mapped antibodies) |
monoclonal anti-human ANG2, monoclonal anti-human ANG2 |
|
Standard Matrix |
Human serum matrix containting recombinant human ANG2 7 ready to use standards |
Protein buffered matrix containing recombinant human ANG2 1 stock standard vial |
|
Values of apparently healthy samples |
Plasma mean value (n=11): 1317 pg/ml (604-2635 pg/l) |
Plasma mean value (n=35): 1964 pg/ml (1071-4389 pg/ml) |
|
Values of clinical samples |
CKD Serum mean value/range (n=32): 7105 pg/ml (2641-19,311 pg/ml)
|
not indicated |
|
Controls |
2 vials (high, low), included |
not included |
|
Validation |
According to FDA/ICH/EMEA guidelines |
not indicated |
|
Use |
RUO |
RUO |
Correlation of human samples measured with two different Angiopoietin-2 ELISA assay kits
The Biomedica human Angiopoietin-2 ELISA was compared with another human Angiopoietin- ELISA assay using serum and plasma samples from various cohorts (n=77). The correlation between the Biomedica and the competitor assay is R2 = 0.9157.
Sample panel:
- Cardio panel (n=16) – plasma
- Nephro panel (n= 32) - serum
- Control panel (apparently healthy) (n=77) - serum, plasma
Comparison of sample values [pg/ml] from a CKD-cohort measured with the Biomedica Angiopoietin-2 ELISA and an ELISA from another manufacturer
Nephrology CKD panel (n=32) -serum :
Biomedica mean values: 7105 +/- 635 pg/ml
Competitor mean values: 1532 +/-185 pg/ml
- Angiopoietin-2 predicts all-cause mortality in male but not female end-stage kidney disease patients on hemodialysis. Chu C, Chen X, Hasan AA, Szakallova A, Krämer BK, Tepel M, Hocher B. Nephrol Dial Transplant. 2021 Nov 18:gfab332. doi: 10.1093/ndt/gfab332. Epub ahead of print. PMID: 34792167.
- ERA-EDTA Poster 2019 Human Angiopoietin-2
