Download biomedica product listHuman Endostatin ELISA | BI-20742
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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
10 µl / well
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Assay time
3 h / 1 h / 30 min
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Sensitivity
4 pmol/l (= 80 pg/ml)
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Standard range
0 – 800 pmol/l (= 0 – 16,000 pg/ml)
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Conversion factor
1 pmol/l = 20 pg/ml (MW: 20 kDa)
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Specificity
Endogenous and recombinant human endostatin.
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Precision
In-between-run (n=16): ≤ 5 % CV
Within-run (n=5): ≤ 6 % CV
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Cross-reactivity
Human endostatin only. No cross-reactivity with COL15A1.
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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
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Synonyms
COL18
Collagen 18
Collagen XVIII
Endostar
Human Endostatin ELISA Product Overview
The Endostatin ELISA is a 3.5 hour, 96-well sandwich ELISA for the quantitative determination of Endostatin in human serum, plasma and urine.
Human Endostatin ELISA Assay Principle
The Human Endostatin ELISA kit is a sandwich enzyme immunoassay for the quantitative determination of Endostatin in human serum, plasma and urine samples.
The figure below explains the principle of the Endostatin ELISA:
Capture antibody: polyclonal goat anti-human Endostatin antibody
Detection antibody: polyclonal rabbit anti-human Endostatin antibody, biotin labeled
Target antigen: human Endostatin
In a first step, standard/control/sample and detection antibody (biotinylated polyclonal rabbit anti-human Endostatin) are pipetted into the wells of the microtiter strips, which are pre-coated with polyclonal goat anti-human Endostatin antibody. Endostatin present in the standard/control/sample binds to the pre-coated antibody in the well and forms a sandwich with the detection antibody. After a washing step, which removes non-specifically unbound material, the conjugate (streptavidin-HRP) is pipetted into the wells and reacts with the detection 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 Endostatin present in the sample. This color change is detectable with a standard microtiter plate reader. The concentration of Endostatin in the sample is determined directly from the dose response curve.
Human Endostatin ELISA Typical Standard Curve
The figure below shows a typical standard curve for the human Endostatin ELISA. The immunoassay is calibrated against recombinant human Endostatin:
Human Endostatin ELISA Kit Components
|
Contents |
Description |
Quantity |
|
PLATE |
Polyclonal goat anti-human Endostatin antibody pre-coated microtiter strips in a strip holder, packed in an aluminum bag with desiccant |
12 x 8 tests |
|
WASHBUF |
Wash buffer concentrate 20x, natural cap |
1 x 50 ml |
|
STD |
Standards 1-7, (0; 25; 50; 100; 200; 400; 800 pmol/l), recombinant human Endostatin in assay buffer, white caps, ready to use |
7 x 400 µl |
|
CTRL |
Control A and B, yellow cap, ready to use, exact concentrations see labels |
2 x 400 µl |
|
ASYBUF |
Assay buffer, red cap, ready to use |
1 x 100 ml |
|
AB |
Polyclonal rabbit anti-human Endostatin antibody – biotin labeled, green cap, green dye, ready to use |
1 x 7 ml |
|
CONJ |
Conjugate, (streptavidin-HRP), amber bottle, amber cap, ready to use |
1 x 22 ml |
|
SUB |
Substrate (TMB solution), amber bottle, blue cap, ready to use |
1 x 22 ml |
| STOP | Stop solution, white cap, ready to use | 1 x 7 ml |
Storage instructions: All reagents of the Endostatin ELISA kit are stable at 4°C (2-8°C) until the expiry date stated on the label of each reagent.
Sample Collection & Storage
Serum, EDTA plasma, heparın plasma, citrate plasma and urine are suitable for use in this Endostatin ELISA. 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. Do not freeze-thaw samples more than four times.
Urine
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.
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).
Sample Preparation
Bring samples to room temperature and mix samples gently to ensure the samples are homogenous. We recommend duplicate measurements for all samples.
Serum & Plasma
Serum and plasma samples must be diluted 1+100 with assay buffer (ASYBUF), e.g. 10 µl sample + 1000 µl ASYBUF. Diluted samples are stable at 4°C (2-8°C) overnight. Therefore, dilutions can be prepared one day prior to analysis.
Urine
Use urine samples undiluted. Urine samples for which the OD value exceeds the highest point of the standard range can be diluted 1+3 with assay buffer (ASYBUF).
Human Endostatin ELISA Assay Protocol
Read the entire protocol before beginning the assay.
|
1. |
Bring samples and reagents to room temperature (18-24°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 100 µl ASYBUF (assay buffer, red cap) into each well. |
|
5. |
Add 20 µl STD/SAMPLE/CTRL into the respective wells. |
|
6. |
Add 50 µl AB (biotinylated anti-Endostatin antibody, green cap) into each well. Swirl gently. |
|
7. |
Cover the plate tightly, and incubate for 3 hours at room temperature (18-24°C). |
|
8. |
Aspirate and wash wells 5 x with 300 µl diluted WASHBUF (wash buffer). After the final wash, remove the remaining WASHBUF by strongly tapping plate against a paper towel. |
|
9. |
Add 200 µl CONJ (conjugate, amber cap) into each well. |
|
10. |
Cover tightly and incubate for 1 hour at room temperature. |
|
11. |
Aspirate and wash wells 5 x with 300 µl diluted WASHBUF. After the final wash, remove remaining WASHBUF by strongly tapping plate against a paper towel. |
|
12. |
Add 200 µl SUB (substrate, blue cap) into each well. |
|
13. |
Incubate for 30 min at room temperature in the dark. |
|
14. |
Add 50 µl STOP (stop solution, white cap) into each well. |
|
15. |
Measure absorbance immediately at 450 nm with reference 630 nm, if available. |
Calculation of Results
Construct a standard curve from the absorbance read-outs of the standards using commercially available software capable of generating a four-parameter logistic (4-PL) fit. Alternatively, plot the standards’ concentration on the x-axis against the mean absorbance for each standard on the y-axis and draw a best fit curve through the points on the graph. Curve fitting algorithms other than 4-PL have not been validated and will need to be evaluated by the user.
Obtain sample concentrations from the standard curve. If required, pmol/l can be converted into pg/ml by applying a conversion factor (1 pg/ml =0.005 pmol/l (MW: 20 kDa)). Respective dilution factors, e.g. 1:101 for serum and plasma samples, have to be considered when calculating the final concentration of the sample.
The quality control (QC) 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 1.50 or more is obtained for the STD with the highest concentration and the value of the CTRL is within the target range (see label).
Endostatin Protein
Endostatin, a 20 kDa C-terminal proteolytic fragment of collagen XVIII, is an endogenous angiogenesis inhibitor localized in the vascular basement membrane in various organs (http://www.uniprot.org/uniprot/P39060). The biological functions of the Endostatin network involve SPARC, thrombospondin-1, glycosaminoglycans, collagens and integrins.
|
Molecular weight |
20 kDa |
|
Cellular localisation |
Extracellular |
|
Post-translational modifications |
Proteolytic processing, glycosylation, hydroxylation |
|
Sequence similarities |
Member of the collagen familiy |
|
Alternative names |
Endostar (Jiangsu Simcere Pharmaceutical) |
|
Pubchem ID |
187888 link: https://pubchem.ncbi.nlm.nih.gov/compound/Endostatin |
|
Genecards |
COL18A1 link: https://www.genecards.org/cgi-bin/carddisp.pl?gene=COL18A1 |
|
OMIM |
120328 link: https://www.omim.org/entry/120328 |
|
PDB |
1KOE link: http://www.rcsb.org/structure/1KOE |
|
Pfam |
PF06482 link: http://pfam.xfam.org/family/endostatin |
|
Protein Atlas |
COL18A1 link: https://www.proteinatlas.org/ENSG00000182871-COL18A1/tissue |
|
Uniprot ID |
P39060 (COIA1_HUMAN) link: https://www.uniprot.org/uniprot/P39060 |
Endostatin Function
Endostatin is expressed during the progression of renal fibrosis in tubular cells of injured tissue. In renal micro-vascular disease, observed in late stages of patients with chronic kidney disease, increased Endostatin levels are possibly the consequence of enhanced extracellular matrix degradation. Thus Endostatin may become an important marker for progressive microvascular renal disease in patients with chronic kidney disease (Chen et al., 2012; Futrakul et al., 2008; Iqbal et al., 2011; Moskowitz-Kassai et al., 2012). Endostatin levels in blood are also likely to increase in patients with other microvascular tissue injuries, including atherosclerosis, myocardial- and brain ischemia. In ischemic stroke patients, high Endostatin plasma levels predict a worse long-term clinical outcome (Navarro-Sobrino et al., 2011; Sodha et al., 2009).
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Angiogenesis in Cancer
Bladder cancer (Cheng et al., 2012; Szarvas et al., 2012)
Gastric cancer (Koç et al., 2006; Wang et al., 2015)
Breast cancer (Guo et al., 2016; Kuroi et al., 2001)
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Cardiovascular Disease
Cardiovasuclar disease (Ruge et al., 2017)
Stroke (Xue et al., 2017)
Coronary artery disease (Sponder et al., 2016)
Diabetes mellitus (Sponder et al., 2014; Wuopio et al., 2018)
Hypertension (Carlsson et al., 2013; Damico et al., 2015; Kim and Kim, 2014)
Heart failure (Barroso et al., 2017; Gouya et al., 2014; Ruge et al., 2018)
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Kidney Disease
Chronic kidney disease (Carlsson et al., 2016; Chauhan et al., 2018; Futrakul et al., 2008; Kanbay et al., 2016; Kato et al., 2018)
Renal fibrosis (Lin et al., 2016, 2014)
Literature
Plasma endostatin predicts kidney outcomes in patients with type 2 diabetes.
Chauhan, K., Verghese, D.A., Rao, V., Chan, L., Parikh, C.R., Coca, S.G., Nadkarni, G.N., 2018. Kidney Int.
PMID: 30591223
Association of the Serum Endostatin Level, Renal Function, and Carotid Atherosclerosis of Healthy Residents of Japan: Results from the Kyushu and Okinawa Population Study (KOPS).
Kato, Y., Furusyo, N., Tanaka, Y., Yamasaki, S., Ueyama, T., Takayama, K., Mitsumoto-Kaseida, F., Murata, M., Ikezaki, H., Hayashi, J., 2018. J. Atheroscler. Thromb. 25, 829–835.
PMID: 29398676; PMCID: PMC6143776
Serum Endostatin Levels in Oral Squamous Cell Carcinoma.
Mardani, M., Andishe Tadbir, A., Ranjbar, M.A., Khademi, B., Fattahi, M.J., Rahbar, A., 2018. Iran. J. Otorhinolaryngol. 30, 125–130
PMID: 29876326; PMCID: PMC5985613
Circulating endostatin and the incidence of heart failure.
Ruge, T., Carlsson, A.C., Ingelsson, E., Risérus, U., Sundström, J., Larsson, A., Lind, L., Ärnlöv, J., 2018. Scand. Cardiovasc. J. SCJ 1–6.
PMID: 29893146
The association between circulating endostatin and a disturbed circadian blood pressure pattern in patients with type 2 diabetes.
Wuopio, J., Östgren, C.J., Länne, T., Lind, L., Ruge, T., Carlsson, A.C., Larsson, A., Nyström, F.H., Ärnlöv, J., 2018. Blood Press. 27, 215–221.
PMID: 29488402
Endostatin a Potential Biomarker for Heart Failure with Preserved Ejection Fraction.
Barroso, M.C., Boehme, P., Kramer, F., Mondritzki, T., Koehler, T., Gülker, J.-E., Karoff, M., Dinh, W., 2017. Arq. Bras. Cardiol. 109, 448–456.
PMID: 28977054; PMCID: PMC5729781
Endostatin: a promising biomarker in the cardiovascular continuum?
Ruge, T., Carlsson, A.C., Larsson, A., Ärnlöv, J., 2017. Biomark. Med. 11, 905–916.
PMID: 28976780
Changes in serum vascular endothelial growth factor and endostatin concentrations associated with circulating endothelial progenitor cells after acute ischemic stroke.
Xue, L., Chen, H., Zhang, T., Chen, J., Geng, Z., Zhao, Y., 2017. Metab. Brain Dis. 32, 641–648.
PMID: 28093678
Diagnostic Values of sVEGFR-1 and Endostatin in Malignant Pleural Effusions in Patients with Lung Cancer.
Zhu, Y.-Y., Wu, H.-M., Liu, R.-Y., 2017. Clin. Lab. 63, 1371–1378.
PMID: 28879702
The association between endostatin and kidney disease and mortality in patients with type 2 diabetes.
Carlsson, A.C., Östgren, C.J., Länne, T., Larsson, A., Nystrom, F.H., Ärnlöv, J., 2016. Diabetes Metab. 42, 351–357.
PMID: 27080454
Endostatin inhibits the growth and migration of 4T1 mouse breast cancer cells by skewing macrophage polarity toward the M1 phenotype.
Guo, H., Liu, Y., Gu, J., Wang, Y., Liu, L., Zhang, P., Li, Y., 2016. Cancer Immunol. Immunother. CII 65, 677–688.
PMID: 27034233
Endostatin in chronic kidney disease: Associations with inflammation, vascular abnormalities, cardiovascular events and survival.
Kanbay, M., Afsar, B., Siriopol, D., Unal, H.U., Karaman, M., Saglam, M., Gezer, M., Taş, A., Eyileten, T., Guler, A.K., Aydin, İ., Oguz, Y., Tarim, K., Covic, A., Yilmaz, M.I., 2016. Eur. J. Intern. Med. 33, 81–87.
PMID: 27394925
Endostatin and transglutaminase 2 are involved in fibrosis of the aging kidney.
Lin, C.H.S., Chen, J., Zhang, Z., Johnson, G.V.W., Cooper, A.J.L., Feola, J., Bank, A., Shein, J., Ruotsalainen, H.J., Pihlajaniemi, T.A., Goligorsky, M.S., 2016. Kidney Int. 89, 1281–1292.
PMID: 27165830; PMCID: PMC4868664
Physical inactivity increases endostatin and osteopontin in patients with coronary artery disease.
Sponder, M., Fritzer-Szekeres, M., Marculescu, R., Litschauer, B., Strametz-Juranek, J., 2016. Heart Vessels 31, 1603–1608.
PMID: 26661073
Serum endostatin is a genetically determined predictor of survival in pulmonary arterial hypertension.
Damico, R., Kolb, T.M., Valera, L., Wang, L., Housten, T., Tedford, R.J., Kass, D.A., Rafaels, N., Gao, L., Barnes, K.C., Benza, R.L., Rand, J.L., Hamid, R., Loyd, J.E., Robbins, I.M., Hemnes, A.R., Chung, W.K., Austin, E.D., Drummond, M.B., Mathai, S.C., Hassoun, P.M., 2015. Am. J. Respir. Crit. Care Med. 191, 208–218.
PMID: 25489667; PMCID: PMC4347439
Effects of changes in serum endostatin and fibroblast growth factor 19 on the chemotherapeutic sensitivity in acute myeloid leukemia patients.
Su, Y.Z., Wang, C.B., Zhou, Y., Sun, N.T., 2015. Genet. Mol. Res. GMR 14, 5181–5187.
PMID: 26125711
Correlation of serum levels of endostatin with tumor stage in gastric cancer: a systematic review and meta-analysis.
Wang, Z.-H., Zhu, Z.-T., Xiao, X.-Y., Sun, J., 2015. BioMed Res. Int. 2015, 623939.
PMID: 25685799; PMCID: PMC4313525
Association of endostatin with mortality in patients with chronic heart failure.
Gouya, G., Siller-Matula, J.M., Fritzer-Szekeres, M., Neuhold, S., Storka, A., Neuhofer, L.M., Clodi, M., Hülsmann, M., Pacher, R., Wolzt, M., 2014. Eur. J. Clin. Invest. 44, 125–135.PMID: 24188329
Serial changes of serum endostatin and angiopoietin-1 levels in preterm infants with severe bronchopulmonary dysplasia and subsequent pulmonary artery hypertension.
Kim, D.-H., Kim, H.-S., 2014. Neonatology 106, 55–61.
PMID: 24818792
Endostatin and kidney fibrosis in aging: a case for antagonistic pleiotropy?
Lin, C.H.S., Chen, J., Ziman, B., Marshall, S., Maizel, J., Goligorsky, M.S., 2014. Am. J. Physiol.-Heart Circ. Physiol. 306, H1692–H1699.
PMID: 24727495
Exercise increases serum endostatin levels in female and male patients with diabetes and controls.
Sponder, M., Dangl, D., Kampf, S., Fritzer-Szekeres, M., Strametz-Juranek, J., 2014. Cardiovasc. Diabetol. 13, 6.
PMID: 24393402; PMCID: PMC3913788
Association between circulating endostatin, hypertension duration, and hypertensive target-organ damage.
Carlsson, A.C., Ruge, T., Sundström, J., Ingelsson, E., Larsson, A., Lind, L., Arnlöv, J., 2013. Hypertens. Dallas Tex 1979 62, 1146–1151.
PMID: 24082055
Elevated serum endostatin levels are associated with poor survival in patients with advanced-stage nasopharyngeal carcinoma.
Mo, H.-Y., Luo, D.-H., Qiu, H.-Z., Liu, H., Chen, Q.-Y., Tang, L.-Q., Zhong, Z.-L., Huang, P.-Y., Zhao, Z.-J., Zhang, C.-Q., Zhang, Y., Mai, H.-Q., 2013. Clin. Oncol. R. Coll. Radiol. G. B. 25, 308–317.
PMID: 23290342
Elevated plasma levels of endostatin are associated with chronic kidney disease.
Chen, J., Hamm, L.L., Kleinpeter, M.A., Husserl, F., Khan, I.E., Chen, C.-S., Liu, Y., Mills, K.T., He, C., Rifai, N., Simon, E.E., He, J., 2012. Am. J. Nephrol. 35, 335–340.
PMID: 22456114; PMCID: PMC3362190
Clinical value of vascular endothelial growth factor and endostatin in urine for diagnosis of bladder cancer.
Cheng, D., Liang, B., Li, Y., 2012. Tumori 98, 762–767.
PMID: 23389364
Excretion of anti-angiogenic proteins in patients with chronic allograft dysfunction.
Moskowitz-Kassai, E., Mackelaite, L., Chen, J., Patel, K., Dadhania, D.M., Gross, S.S., Chander, P., Delaney, V., Deng, L., Chen, L., Cui, X., Suthanthiran, M., Goligorsky, M.S., 2012. Nephrol. Dial. Transplant. 27, 494–497.
PMID: 22253069; PMCID: PMC3275786
Serum endostatin levels correlate with enhanced extracellular matrix degradation and poor patients’ prognosis in bladder cancer.
Szarvas, T., László, V., Vom Dorp, F., Reis, H., Szendröi, A., Romics, I., Tilki, D., Rübben, H., Ergün, S., 2012. Int. J. Cancer 130, 2922–2929.
PMID: 21815140
Early-onset coronary artery disease after pediatric kidney transplantation: implicating the angiogenesis inhibitor, endostatin.
Iqbal, C.W., Dean, P.G., Ishitani, M.B., 2011. Am. Surg. 77, 731–735
PMID: 21679642
Recombinant human endostatin, Endostar, enhances the effects of chemo-radiotherapy in a mouse cervical cancer xenograft model.
Jia, Y., Liu, M., Cao, L., Zhao, X., Wu, J., Lu, F., Li, Y., He, Y., Ren, S., Ju, Y., Wang, Y., Li, Z., 2011. Eur. J. Gynaecol. Oncol. 32, 316–324
PMID: 21797125
Prognostic significance of the angiogenic factors angiogenin, endoglin and endostatin in cervical cancer.
Landt, S., Mordelt, K., Schwidde, I., Barinoff, J., Korlach, S., Stöblen, F., Lichtenegger, W., Sehouli, J., Kümmel, S., 2011. Anticancer Res. 31, 2651–2655
PMID: 21778318
A large screening of angiogenesis biomarkers and their association with neurological outcome after ischemic stroke.
Navarro-Sobrino, M., Rosell, A., Hernández-Guillamon, M., Penalba, A., Boada, C., Domingues-Montanari, S., Ribó, M., Alvarez-Sabín, J., Montaner, J., 2011. Atherosclerosis 216, 205–211.
PMID: 21324462
Endostatin and angiostatin are increased in diabetic patients with coronary artery disease and associated with impaired coronary collateral formation.
Sodha, N.R., Clements, R.T., Boodhwani, M., Xu, S.-H., Laham, R.J., Bianchi, C., Sellke, F.W., 2009. Am. J. Physiol. Heart Circ. Physiol. 296, H428-434.
PMID: 19074676; PMCID: PMC2643884
Elevated serum endostatin levels are associated with favorable outcome in acute myeloid leukemia.
Aref, S., El-Sherbiny, M., Azmy, E., Goda, T., Selim, T., El-Refaie, M., Twafik, E., 2008. Hematol. Amst. Neth. 13, 95–100.
PMID: 18616876
A defective angiogenesis in chronic kidney disease.
Futrakul, N., Butthep, P., Laohareungpanya, N., Chaisuriya, P., Ratanabanangkoon, K., 2008. Ren. Fail. 30, 215–217.
PMID: 18300124
Serum endostatin levels in gastric cancer patients: correlation with clinicopathological parameters.
Koç, M., Göçmen, E., Kiliç, M., Ozbay, M., Oktem, M., Tez, M., 2006. Hepatogastroenterology. 53, 616–618
PMID: 16995474
Circulating levels of endostatin in cancer patients.
Kuroi, K., Tanaka, C., Toi, M., 2001. Oncol. Rep. 8, 405–409
PMID: 11182064
We validate all Biomedica ELISAs according to FDA/ICH/EMEA guidelines. For more
information about our validation guidelines, please refer to our quality (->link) page and published validation guidelines and literature.
CPMP/ICH/381/95
ICH Topic Q2 (R1) „Validation of Analytical Procedures: Text and Methodology“
including:
ICH Q2A “Text on Validation of Analytical Procedures”
ICH Q2B “Validation of Analytical Procedures: Methodology”
EMEA/CHMP/EWP/192217/2009
“Guideline on Validation of Bioanalytical Methods”
Calibration
The Endostatin immunoassay is calibrated against recombinant human Endostatin protein (processed Uniprot protein P39060).
Human Endostatin ELISA Detection Limit & Sensitivity
To determine the sensitivity of the Endostatin 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 from the background signal, i.e., the signal that is measured in the absence of Endostatin, with a confidence level of 99%. It is defined as the mean back-calculated concentration of Endostatin-free sample plus three times the standard deviation of three independent 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%. The lowest concentration of Endostatin, which meets both criteria, is reported as the LLOQ.
The following values were determined for the Endostatin ELISA:
|
LOD |
4 pmol/l |
| LLOQ | 3 pmol/l |
Human Endostatin 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 at different locations using different ELISA lots (in-between-run precision or reproducibility).
Within-Run Precision
Within-run precision was tested by measuring the same samples three times within one Endostatin ELISA lot. One operator conducted the experiment.
|
ID |
n |
Mean Endostatin [pmol/l] |
SD [pmol/l] |
CV [%] |
|
Sample 1 |
5 |
49 |
3.5 |
7 |
|
Sample 2 |
5 |
392 |
22.6 |
6 |
In-Between-Run Precision
In-between-run precision was assessed by measuring the same samples 16 times within multiple Endostatin ELISA lots. The measurements were carried out by several operators.
|
ID |
n |
Mean Endostatin [pmol/l] |
SD [pmol/l] |
CV [%] |
|
Sample 1 |
16 |
50 |
2.1 |
4 |
|
Sample 2 |
16 |
405 |
21.6 |
5 |
Human Endostatin 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 Endostatin ELISA was measured by adding recombinant Endostatin to human samples containing a known concentration of endogenous Endostatin. The % recovery of the spiked concentration was calculated as the percentage of measured over the expected value.
This table shows the summary of the recovery experiments in the Endostatin ELISA in different sample matrices:
|
% Recovery |
|||||
|
Sample matrix |
n |
+100 pmol/l |
+400 pmol/l |
||
|
Mean |
Range |
Mean |
Range |
||
|
Serum |
5 |
98 |
86 - 109 |
121 |
107 - 131 |
|
EDTA plasma |
5 |
100 |
90 - 110 |
95 |
80 - 111 |
|
Citrate plasma |
5 |
88 |
76-113 |
96 |
81-118 |
|
Heparın plasma |
4 |
96 |
92 - 99 |
94 |
80 - 109 |
|
Urine |
4 |
74 |
65 - 78 |
||
|
|
Endostatin [pmol/l] |
% Recovery |
||||
|
Sample matrix |
ID |
Reference |
+ 100 pmol/l |
+ 400 pmol/l |
+ 100 pmol/l |
+ 400 pmol/l |
|
Serum |
S1 |
45.9 |
147.1 |
568.3 |
101 |
131 |
|
Serum |
S2 |
39.4 |
148.5 |
563.8 |
109 |
131 |
|
Serum |
S3 |
40.2 |
129.6 |
489.3 |
89 |
112 |
|
Serum |
S4 |
53.5 |
157.7 |
548.9 |
104 |
124 |
|
Serum |
S5 |
49.1 |
134.9 |
475.3 |
86 |
107 |
|
Mean |
98 |
121 |
||||
|
|
Min |
86 |
107 |
|||
|
Max |
109 |
131 |
||||
Data showing recovery of recombinant Endostatin in human EDTA plasma samples:
|
|
|
Endostatin [pmol/l] |
% Recovery |
|||
|
Sample matrix |
ID |
Reference |
+ 100 pmol/l |
+ 400 pmol/l |
+ 100 pmol/l |
+ 400 pmol/l |
|
EDTA plasma |
E1 |
51.8 |
157.4 |
390.9 |
106 |
85 |
|
EDTA plasma |
E2 |
43.1 |
132.7 |
487.2 |
90 |
111 |
|
EDTA plasma |
E3 |
43.2 |
153.4 |
445.1 |
110 |
100 |
|
EDTA plasma |
E4 |
43.6 |
136.8 |
435.8 |
93 |
98 |
|
EDTA plasma |
E5 |
42.6 |
144.6 |
361.3 |
102 |
80 |
|
Mean |
100 |
95 |
||||
|
|
|
|
|
Min |
90 |
80 |
|
|
|
|
|
Max |
110 |
111 |
Data showing recovery of recombinant Endostatin in human citrate plasma samples:
|
|
|
Endostatin [pmol/l] |
% Recovery |
|||
|
Sample matrix |
ID |
Reference |
+ 100 pmol/l |
+ 400 pmol/l |
+ 100 pmol/l |
+ 400 pmol/l |
|
Citrate plasma |
C1 |
71.8 |
157.5 |
477.5 |
86 |
101 |
|
Citrate plasma |
C2 |
47.3 |
133.2 |
439.8 |
86 |
98 |
|
Citrate plasma |
C3 |
53.0 |
166.1 |
523.6 |
113 |
118 |
|
Citrate plasma |
C4 |
40.4 |
117.5 |
363.8 |
77 |
81 |
|
Citrate plasma |
C5 |
48.9 |
124.6 |
377.2 |
76 |
82 |
|
|
Mean |
88 |
96 |
|||
|
Min |
76 |
81 |
||||
|
Max |
113 |
118 |
||||
Data showing recovery of recombinant Endostatin in human heparın plasma samples:
|
|
|
Endostatin [pmol/l] | % Recovery | |||
|
Sample matrix |
ID |
Reference |
+ 100 pmol/l |
+ 400 pmol/l |
+ 100 pmol/l |
+ 400 pmol/l |
|
Heparın plasma |
H1 |
4.8 |
148.5 |
443.8 |
99 |
99 |
|
Heparın plasma |
H2 |
45.8 |
144.2 |
398.3 |
98 |
88 |
|
Heparın plasma |
H3 |
50.1 |
146.5 |
372.0 |
96 |
80 |
|
Heparın plasma |
H4 |
47.5 |
139.1 |
483.8 |
92 |
109 |
|
Mean |
96 |
94 |
||||
|
|
Min |
92 |
80 |
|||
|
|
Max |
99 |
109 |
|||
Data showing recovery of recombinant Endostatin in human urine samples:
|
|
|
Endostatin [pmol/l] |
% Recovery |
|
|
Sample matrix |
ID |
Reference |
+ 100 pmol/l |
+ 100 pmol/l |
|
Urine |
U1 |
74 |
139 |
65 |
|
Urine |
U2 |
66 |
144 |
78 |
|
Urine |
U3 |
22 |
100 |
78 |
|
Urine |
U4 |
19 |
94 |
75 |
|
Mean |
71 |
|||
|
Min |
65 |
|||
|
Max |
78 |
|||
Human Endostatin ELISA Dilution Linearity & Parallelism
Tests of dilution linearity and parallelism ensure that both, endogenous and recombinant samples containing Endostatin, 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 to the recombinant one. Dilution linearity and parallelism are assessed for each sample type and should be within 20% of the expected concentration.
The table below shows the mean recovery and range of serially diluted recombinant Endostatin in assay buffer:
|
% Recovery of recombinant Endostatin in diluted samples |
|||||||||||
|
Sample |
n |
1+16 |
1+32 |
1+64 |
1+128 |
1+256 |
|||||
|
Mean |
Range |
Mean |
Range |
Mean |
Range |
Mean |
Range |
Mean |
Range |
||
|
Sample 1 |
5 |
100 |
96 - 106 |
100 |
92 - 112 |
101 |
96 - 112 |
98 |
80 - 112 |
101 |
96 - 104 |
Parallelism was assessed by serially diluting human samples containing endogenous Endostatin with assay buffer.
The figure and table below show the mean recovery and range of serially diluted endogenous Endostatin in several sample matrices:
|
% Recovery of endogenous Endostatin in diluted samples |
|||||||||
|
Sample matrix |
n |
1+2 |
1+29 |
1+59 |
1+119 |
||||
|
Mean |
Range |
Mean |
Range |
Mean |
Range |
Mean |
Range |
||
|
Serum |
3 |
119 |
107 - 130 |
120 |
99 - 129 |
116 |
101 - 127 |
||
|
EDTA plasma |
3 |
127 |
117 – 132 |
138 |
120 - 160 |
141 |
120 – 163 |
||
|
Citrate plasma |
3 |
108 |
102 -114 |
100 |
91 – 114 |
95 |
90 - 104 |
||
|
Heparın plasma |
3 |
116 |
112 - 123 |
114 |
111 - 120 |
120 |
119 – 121 |
||
|
Urine |
5 |
120 |
97 - 138 |
||||||
|
|
|
Endostatin [pmol/l] |
% Recovery |
|||||
|
Sample matrix |
ID |
Reference (1+15) |
1+29 |
1+59 |
1+119 |
1+29 |
1+59 |
1+119 |
|
Serum |
S1 |
356 |
190 |
88 |
45 |
107 |
99 |
101 |
|
Serum |
S2 |
240 |
145 |
79 |
36 |
121 |
132 |
121 |
|
Serum |
S3 |
311 |
202 |
100 |
50 |
130 |
129 |
127 |
|
|
Mean |
119 |
120 |
116 |
||||
Data showing dilution linearity of endogenous endostatin in human EDTA plasma samples:
|
Endostatin [pmol/l] |
% Recovery |
|||||||
|
Sample matrix |
ID |
Reference (1+15) |
1+29 |
1+59 |
1+119 |
1+29 |
1+59 |
1+119 |
|
EDTA plasma |
E1 |
255 |
168 |
85 |
44 |
131 |
134 |
139 |
|
EDTA plasma |
E2 |
185 |
122 |
74 |
38 |
132 |
160 |
163 |
|
EDTA plasma |
E3 |
287 |
167 |
86 |
43 |
117 |
120 |
120 |
|
|
|
|
|
|
Mean |
127 |
138 |
141 |
Data showing recovery of recombinant Endostatin in a human citrate plasma sample:
|
Endostatin [pmol/l] |
% Recovery |
|||||||
|
Sample matrix |
ID |
Reference (1+15) |
1+29 |
1+59 |
1+119 |
1+29 |
1+59 |
1+119 |
|
Citrate plasma |
C1 |
341 |
175 |
77 |
39 |
102 |
91 |
90 |
|
Citrate plasma |
C2 |
238 |
129 |
68 |
31 |
108 |
114 |
104 |
|
Citrate plasma |
C3 |
312 |
177 |
76 |
36 |
114 |
97 |
92 |
|
|
Mean |
108 |
100 |
95 |
||||
Data showing recovery of recombinant Endostatin in human heparın plasma samples:
|
Endostatin [pmol/l] |
% Recovery |
|||||||
|
Sample matrix |
ID |
Reference (1+15) |
1+29 |
1+59 |
1+119 |
1+29 |
1+59 |
1+119 |
|
Heparın plasma |
H1 |
308 |
172 |
85 |
46 |
112 |
111 |
119 |
|
Heparın plasma |
H2 |
308 |
172 |
85 |
46 |
112 |
111 |
119 |
|
Heparın plasma |
H3 |
337 |
207 |
101 |
51 |
123 |
120 |
121 |
|
|
Mean |
116 |
114 |
120 |
||||
Data showing recovery of recombinant Endostatin in human urine samples:
|
Endostatin [pmol/l] |
% Recovery |
|||
|
Sample matrix |
ID |
Reference |
1+2 |
1+29 |
|
Urine |
U1 |
74 |
33 |
132 |
|
Urine |
U2 |
66 |
26 |
115 |
|
Urine |
U3 |
22 |
7 |
97 |
|
Urine |
U4 |
19 |
9 |
138 |
|
Urine |
U5 |
103 |
40 |
116 |
|
Mean |
120 |
|||
Human Endostatin ELISA Specificity
The specificity of an ELISA is defined as its ability to exclusively recognize an analyte meaning that the ELISA antibodies will bind to the target analyte while not binding to other molecules in solution.
The specificity of the human Endostatin ELISA was established through competition experiments, which measures the ability of the antibodies to bind Endostatin exclusively.
Competition of Signal
Competition experiments were carried out by pre-incubating human samples with an excess of capture antibody. 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. If competition is successful, the labelled Endostatin capture antibody is expected to be competed out by the capture antibody added. The mean competition was 98 % for serum and 97 % for EDTA plasma.
|
Endostatin [pmol/l] |
% Competition |
||||
|
Sample matrix |
ID |
Reference |
+ 400 pmol/l |
+ 400 pmol/l |
|
|
Serum |
S1 |
46 |
568 |
9 |
98 |
|
Serum |
S2 |
49 |
635 |
16 |
98 |
|
Serum |
S3 |
54 |
619 |
14 |
98 |
|
Serum |
S4 |
39 |
564 |
14 |
98 |
|
Serum |
S5 |
40 |
489 |
15 |
97 |
|
Serum |
S6 |
54 |
549 |
13 |
98 |
|
Serum |
S7 |
49 |
475 |
11 |
98 |
|
Serum |
S8 |
51 |
746 |
17 |
98 |
|
Mean |
98 |
||||
|
Endostatin [pmol/l] |
% Competition |
||||
|
Sample Matrix |
ID |
Reference |
+ 400 pmol/l |
+ 400 pmol/l |
|
|
EDTA plasma |
E1 |
43 |
487 |
13 |
97 |
|
EDTA plasma |
E2 |
43 |
445 |
13 |
97 |
|
EDTA plasma |
E3 |
44 |
436 |
14 |
97 |
|
EDTA plasma |
E4 |
36 |
388 |
14 |
96 |
|
EDTA plasma |
E5 |
43 |
361 |
15 |
96 |
|
EDTA plasma |
E6 |
48 |
342 |
11 |
97 |
|
EDTA plasma |
E7 |
48 |
378 |
13 |
96 |
|
EDTA plasma |
E8 |
52 |
391 |
12 |
97 |
|
Mean |
97 |
||||
Cross-Reactivity
The assay is specific to human Endostatin (human COL18-derived Endostatin) and shows sequence similarities with COL15A1-derived Endostatin in a BLAST-P query. The assay shows no cross-reactivity with COL15A1.
Sample Stability
The stability of endogenous Endostatin was tested by comparing Endostatin measurements in samples that had undergone three freeze-thaw cycles.
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 measured directly after sample collection. The mean recovery of sample concentration after three freeze-thaw cycles is 99 %.
|
Endostatin [pmol/l] |
% Recovery after 3 freeze/thaw cycles |
||||
|
Sample matrix |
ID |
Reference |
2x |
3x |
|
|
Serum |
S1 |
52 |
53 |
50 |
104 |
|
Serum |
S2 |
94 |
93 |
93 |
99 |
|
Citrate plasma |
C1 |
82 |
84 |
76 |
93 |
|
Citrate plasma |
C2 |
75 |
74 |
76 |
101 |
|
Mean |
99 |
||||
Sample Values
Serum, Plasma and Urine Endostatin Values in Apparently Healthy Individuals
To provide expected values for circulating Endostatin, a panel of samples from apparently healthy donors was tested.
A summary of the results is shown below:
| Sample matrix |
n |
Median Endostatin [pmol/l] |
|
Serum |
59 |
51 |
|
Citrate plasma |
30 |
47 |
|
Urine |
798 |
20 |
It is recommended to establish the normal range for each laboratory.
Endostatin Values in a Renal Disease Panel
In addition to samples from apparently healthy donors, a panel of urine samples from renal disease patients was tested.
A summary of the results is shown below:
|
Endostatin [pmol/l] |
||||
|
Sample type |
n |
Median |
Minimum |
Maximum |
|
Apparently healthy |
41 |
45 |
11 |
179 |
|
Renal disease patients |
22 |
182 |
13 |
806 |
Endostatin Values in a Cardiovascular Disease Panel
A comparison of apparently healthy individuals and patients with cardiovascular disease shows that endostatin is significantly increased in CVD patients:
A summary of the results is shown below:
|
Endostatin [pmol/l] |
||||
|
Sample type |
n |
Median |
Minimum |
Maximum |
|
Apparently healthy |
30 |
47 |
33 |
79 |
|
CVD patients |
20 |
387 |
286 |
568 |
Matrix Comparison
To assess whether all tested matrices behave the same way in the Endostatin ELISA, concentrations of Endostatin were measured in EDTA, heparın and citrate plasma samples prepared from a panel of 20 donors with CVD indications. Each individual donated blood in all tested sample matrices.
Endostatin was measured in EDTA, heparın and citrate plasma matrices from 20 different individual donors. A summary table of Endostatin levels is shown below:
|
Endostatin [pmol/l] |
|
|||
|
Sample ID |
EDTA plasma |
Citrate plasma |
Heparın plasma |
CV (%) |
|
#1 |
317 |
461 |
375 |
19 |
|
#2 |
385 |
539 |
459 |
17 |
|
#3 |
462 |
624 |
532 |
15 |
|
#4 |
304 |
337 |
394 |
13 |
|
#5 |
318 |
351 |
381 |
9 |
|
#6 |
375 |
- |
453 |
13 |
|
#7 |
250 |
603 |
286 |
51 |
|
#8 |
260 |
320 |
337 |
13 |
|
#9 |
309 |
352 |
365 |
9 |
|
#10 |
479 |
539 |
519 |
6 |
|
#11 |
276 |
317 |
377 |
16 |
|
#12 |
307 |
341 |
305 |
6 |
|
#13 |
235 |
316 |
326 |
17 |
|
#14 |
372 |
417 |
434 |
8 |
|
#15 |
640 |
551 |
568 |
8 |
|
#16 |
386 |
369 |
406 |
5 |
|
#17 |
270 |
300 |
319 |
8 |
|
#18 |
230 |
270 |
438 |
35 |
|
#19 |
384 |
371 |
438 |
9 |
|
#20 |
236 |
324 |
301 |
16 |
|
Mean |
15 |
|||
A figure of Endostatin levels in various sample matrices is shown below:
-
Endostatin Is an Independent Risk Factor of Graft Loss after Kidney Transplant.
Chu, C., Hasan, A.A., Gaballa, M.M.S., Zeng, S., Xiong, Y., Elitok, S., Krämer, B.K., Hocher, B., 2020. AJN 51, 373–380.
- Validation of an enzyme-linked immunosorbent assay (ELISA) for quantification of endostatin levels in mice as a biomarker of developing glomerulonephritis.
Wallwitz, J., Aigner, P., Gadermaier, E., Bauer, E., Casanova, E., Bauer, A., Stoiber, D., 2019. PLoS One 14.
PMID: 31404120; PMCID: PMC6690585
- Higher Parathyroid Hormone Level Is Associated With Increased Arterial Stiffness in Type 1 Diabetes.
Zobel, E.H., Theilade, S., Scholten, B.J. von, Persson, F., Tarnow, L., Lajer, M., Hansen, T.W., Rossing, P., 2017. Diabetes Care 40, e32–e33.
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Kanbay, Mehmet, Baris Afsar, Dimitrie Siriopol, Hilmi Umut Unal, Murat Karaman, Mutlu Saglam, Mustafa Gezer, 2016 European Journal of Internal Medicine : 81–87.
