Download biomedica product listHuman IL-6 ELISA Kit | BI-IL6
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Method
Sandwich ELISA, HRP/TMB, 12×8-well detachable strips
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Sample type
Serum, plasma (EDTA, citrate, heparin), cell culture supernatants, urine
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Sample volume
100 µl sample / well
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Assay time
2 h / 1 h / 1h /30 min
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Sensitivity
LOD: 0.28 pg/ml; LLOQ: 0.78 pg/ml (measurable concentrations in serum AND plasma samples!)
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Standard range
0 – 200 pg/ml (0 / 3.12 / 6.25 / 12.5 / 25/ 50 / 100 / 200)
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Precision
In-between-run (n=3): ≤ 6 % CV
Within-run (n=3): ≤ 7 % CV
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Specificity
This assay recognizes recombinant and endogenous (natural) human IL-6
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Validation Data
See validation data tab for: precision, accuracy, dilution linearity, values for healthy donors, etc
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Use
Research use only
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Conversion
1 pg/ml=0.048 pmol/l (MW=20.8 kD)
Human IL-6 ELISA Product Overview
The Biomedica human Interleukin-6 ELISA (IL-6) ELISA kit is a sandwich enzyme immunoassay that has been optimized and fully validated for the quantitative determination of human IL-6 in serum and plasma (EDTA, heparin, citrate). Validation experiments have been performed according to international quality guidelines (ICH/ FDA/ EMEA). Cell culture supernatant and urine samples are compatible with this ELISA. The IL-6 ELISA assay recognizes both natural and recombinant human IL-6. The assay employs highly purified epitope mapped antibodies as well as human serum-based standards and controls.
Human IL-6 ELISA Assay Principle
The figure below explains the principle of the human IL-6 sandwich ELISA:
Capture antibody: recombinant anti-human IL-6 antibody
Detection antibody: polyclonal anti-human IL-6 antibody, Biotin-labeled
Target antigen: human IL-6
This kit is a sandwich enzyme immunoassay for the quantitative determination of human IL-6 (Interleukin-6) in human serum, plasma, cell culture supernatants, and urine samples. In a first step, STD/sample/CTRL are pipetted into the wells, which are pre-coated with the recombinant anti-human IL-6 antibody. Any soluble IL-6 present in the STD/sample/CTRL binds to the pre-coated anti-IL-6 antibody in the well. After incubation, a washing step is applied where all non-specific unbound material is removed. In a next step, the biotinylated anti-IL-6 antibody (AB) is pipetted into the wells and reacts with the IL-6 present in the sample, forming a sandwich. Next, all unbound antibody is removed during another washing step. In the next step, the conjugate (streptavidin-HRPO) is added and reacts with the biotinylated anti-IL-6 antibody. After another washing step, the substrate (Tetramethylbenzidine; TMB) is pipetted into the wells. The enzyme catalysed colour change of the substrate is directly proportional to the amount of IL-6 present in the sample. This colour change is detectable with a standard microtiter plate ELISA reader. A dose response curve of the absorbance (optical density, OD at 450 nm) versus standard concentration is generated, using the values obtained from the standards. The concentration of soluble IL-6 in the sample is determined directly from the dose response curve.
Typical Data
This standard curve and the displayed OD values are for demonstration only. A standard curve should be generated for each assay run.
|
Standard |
IL-6 [pg/ml] |
OD |
CV [%] |
||
|
#1 |
#2 |
Average |
|||
|
STD1 |
0 |
0.056 |
0.059 |
0.058 |
4 |
|
STD2 |
3.125 |
0.140 |
0.129 |
0.135 |
6 |
|
STD3 |
6.25 |
0.216 |
0.207 |
0.212 |
3 |
|
STD4 |
12.5 |
0.354 |
0.366 |
0.360 |
2 |
|
STD5 |
25 |
0.661 |
0.660 |
0.661 |
0 |
|
STD6 |
50 |
1.090 |
1.138 |
1.114 |
3 |
|
STD7 |
100 |
1.970 |
1.835 |
1.903 |
5 |
|
STD8 |
200 |
2.951 |
2.894 |
2.923 |
1 |
The quality control protocol supplied with the kit shows the results of the final release QC for each kit at the production date. ODs obtained by customers may differ due to various influences including a normal decrease of signal intensity throughout shelf life. However, this does not affect the validity of the results, provided an OD of 1.50 or higher is obtained for the standard with the highest concentration, and the measured control values fall into their respective target range (see labels).
IL-6 ELISA Kit Components
All reagents supplied in the human IL-6 ELISA kit are stable at 2-8°C until the expiry date stated on the label of each reagent. Reconstituted reagents must be stored at -25°C until the expiry date.
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CONTENT |
DESCRIPTION |
QUANTITY |
|
PLATE |
Microtiter strips coated with recombinant IL-6 antibody specific for human IL-6 in strip holder packed in an aluminum bag with desiccant |
12 x 8 tests |
|
WASHBUF |
20x wash buffer concentrate, transparent cap |
1 x 50 ml |
|
ASYBUF |
Assay buffer, red cap, ready to use |
1 x 10 ml |
|
STD |
Recombinant IL-6 standards (0 / 3.125 / 6.25/ 12.5 / 25 / 50 / 100 / 200 pg/ml), human serum based, white caps, lyophilized |
8 vials |
|
CTRL |
Control A and B, human serum based, yellow cap, lyophilized, exact concentration is stated on labels |
2 vials |
|
AB |
Polyclonal IL-6 antibody specific for human IL-6, biotin-labeled, green cap, ready to use |
1 x 13 ml |
|
CONJ |
Conjugate (streptavidin-HRPO), brown 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 |
Serum and plasma ( EDTA, citrate, heparin) , cell culture supernatants, and urine samples 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.
Serum & Plasma
Collect venous blood samples by using standardized blood collection tubes. Perform plasma or serum separation by centrifugation according to supplier’s instructions of the blood collection devices. 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 up to five freeze-thaw cycles. Samples should be mixed well before assaying. We recommend duplicates for all values.
Cell Culture Supernatant
Cell culture supernatants should contain at least 1% fetal bovine serum. Remove particles by centrifugation and assay immediately or aliquot and store samples at ≤ -25 °C or lower. Avoid repeated freeze-thaw cycles.
Urine
Aseptically collect the first urine of the day (mid-stream), voided directly into a sterile container. Centrifuge to remove particles matter, assay immediately or aliquot and store at -25°C or lower. Avoid repeated freeze-thaw cycles.
Reagent Preparation
Wash Buffer
|
1. |
Bring the WASHBUF concentrate to room temperature. Crystals in the buffer concentrate will dissolve at room temperature (18-26°C). |
|
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 & controls for serum, plasma, cell culture supernatants and urine measurements
|
1. |
Pipette 500 µl of distilled or deionized water into each standard (STDs) 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. STDs and CTRLs are stable up to five freeze-thaw cycles.
The standards and controls provided in the kit are suitable for all sample types.
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 with analyte concentrations outside of the calibration range of the assay (200 pg/ml) should be diluted with assay buffer (ASYBUF) and remeasured.
Human IL-6 ELISA Assay Protocol
|
Read the entire instructions for use before beginning the assay. |
|
All reagents and samples must be at room temperature (18-26°C) before use in the assay. |
| Prepare reagents and samples as instructed. |
|
Mark position for STD/CTRL/SAMPLE (standard/control/sample) on the protocol sheet. |
| Bring unused and prepared components to the storage temperature mentioned in the package insert. |
|
Take microtiter strips out of the aluminium bag. Store unused strips with desiccant at 4°C (2-8°C) in the aluminium bag. Strips are stable until expiry date stated on the label. |
|
1. |
Add 100 ul of STD/SAMPLE/CTRL (Standard/Sample/Control) in duplicate into respective well. Swirl gently. |
|
2. |
Cover tightly and incubate for 2 hours at room temperature (18-26°C). |
|
3. |
Aspirate and wash wells 5x with 300 µl diluted WASHBUF (Wash buffer, natural cap). After final wash, remove remaining WASHBUF by strongly tapping plate against paper towel. |
|
4. |
Add 100 µl AB (biotinylated anti-IL-6 antibody, green cap) into each well, swirl gently. |
|
5. |
Cover tightly and incubate for 1 hour at room temperature (18-26°C). |
|
6. |
Aspirate and wash wells 5x with 300 µl diluted WASHBUF (Wash buffer, natural cap). After final wash, remove remaining WASHBUF by strongly tapping plate against paper towel. |
|
7. |
Add 100 µl CONJ (Conjugate, amber cap) into each well, swirl gently. |
|
8. |
Cover tightly and incubate for 1 hour at room temperature (18-26°C). |
|
9. |
Aspirate and wash wells 5x with 300 µl diluted WASHBUF (Wash buffer, natural cap). After final wash, remove remaining WASHBUF by strongly tapping plate against paper towel. |
|
10. |
Add 100 µl SUB (Substrate, blue cap) into each well, swirl gently. |
|
11. |
Incubate for 30 min at room temperature (18-26°C), in the dark. |
|
12. |
Add 50 µl STOP (Stop solution, white cap) into each well, swirl gently. |
|
13. |
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, pg/ml can be converted into pmol/l by applying a conversion (1 pg/ml = 0.048 pmol/l, MW: 20.8 kDa). Concentrations of high-measuring samples that have been diluted during sample preparation must be multiplied by the dilution factor.
Interleukin-6 (IL-6), also known as B-cell stimulatory factor 2 (BSF-2), CTL differentiation factor (CDF), Hybridoma growth factor or Interferon beta-2 (IFN-beta-2), was successfully cloned by Hirano et al. in 1986 (Hirano T et al.) . The gene is mapped at chromosome 7p21. IL-6 protein is built up by 183 amino acids and has a calculated molecular weight of 23.7 kDa.
It is a pleiotropic, alpha helical protein that is composed of a four-helix bundle (Somers W et al.). It shares 39% sequence identity with mouse and 40% with rat IL-6. IL-6 is phosphorylated at amino acid 81 and it is variably glycosylated by N-linked glycosylation. IL-6 belongs to the IL-6/GCSF/MGF protein family (Rose-John S et al.) whose members share a common use of the gp130 receptor subunit. IL-6 isoforms, with internal deletions, are generated by alternative splicing. The principal cell sources for IL-6 are mononuclear phagocytes, vascular endothelial cells, fibroblasts or other cells.
IL-6 is the ligand for the Interleukin-6 receptor α (IL-6Rα) (Schwantner A et al.) that occurs membrane-bound, but that may also circulate as soluble form generated by alternative splicing or proteolytic cleavage. To induce signaling, IL-6 first forms a complex with the non-signaling IL-6Rα. Subsequent binding to the signal transducing subunit gp130 leads to dimerization of gp130 and finally to the formation of the hexameric signaling complex (Boulanger MJ et al.).
Complexes of IL-6 and soluble IL-6Rα may elicit responses in cells lacking the membrane-bound IL-6Rα but expressing the ubiquitous gp130 coreceptor. This process is known as trans-signaling, it enlarges the spectrum of target cells responding to IL-6 (Mihara M et al.).
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Full Name |
Interleukin-6 (IL-6) |
|
Sequence Length; Molecular Weight |
212 AA; 22-28 kDa (theoretical: 23,7 kDa) |
|
Structure |
alpha-helical (dimer in crystal structure) |
| Cellular localisation |
Secreted |
|
Post-translational modifications |
Phosphorylated, N-glycosylated |
|
Alternative names-Synonymes |
Interleukin-6 (IL6), B-cell stimulatory factor 2 (BSF-2), CTL differentiation factor (CDF), Hybridoma growth factor, Interferon beta-2 (IFN-beta-2) |
|
Entrez/NCBI ID |
3569 |
|
Uniprot ID |
P05231 |
|
Sequence similarities |
Belongs to the IL6/GCSF/MGF family |
|
Expression |
Mononuclear phagocytes, vascular endothelial cells, fibroblasts, etc |
|
Molecular function |
Cytokine activity, growth factor activity, IL-6 receptor binding |
|
Biological function |
Acute phase response, aging, bone remodeling, humoral immune respone, inflammatory response, negative regulation of apoptotic process and many others. |
IL-6 Function
IL-6 is immediately produced in response to infections or tissue injury, and it plays a major role in host defense. After synthesis the principal cellular targets of IL-6 are liver cells where IL-6 leads to the synthesis of acute phase proteins, B cells where proliferation of antibody producing cells is induced, or T cells where differentiation is induced. Signaling is induced by homodimerization of the receptor complex upon IL-6 binding, and subsequent activation of Janus kinases that then phosphorylate tyrosine residues in the cytoplasmic domain of gp130. Two main pathways are activated in the signaling event: the MAPK and the JAK/STAT pathway. IL-6 expression is tightly regulated, and mis-regulation contributes to chronic inflammation and autoimmunity. IL-6 plays an important role in acute phase reaction, inflammation, hematopoiesis, bone metabolism, and cancer progression. (Ramadori G et al., Tanaka T et al., Hou T et al.). Increased IL-6 levels were observed in inflammatory conditions like rheumatoid arthritis, systemic juvenile idiopathic arthritis, castleman’s disease, or sepsis. In this context, pro-inflammatory activities seem to depend mainly on IL-6 trans-signaling via sIL-6Rα. IL-6 also has anti-inflammatory activities that depend on membrane-bound IL-6Rα (Jones SA et al., Calabrese LH et al., Schmidt-Arras D et al.). In healthy individuals, IL-6 levels in the blood are reported in the single-digit pg/ml range. However, during inflammatory states IL-6 levels can increase several thousand-fold.
Targeting of the IL-6 pathway has led to innovative therapeutic approaches for various rheumatic diseases, such as rheumatoid arthritis, juvenile idiopathic arthritis, adult-onset Still's disease, giant cell arteritis and Takayasu arteritis, as well as other conditions such as Castleman disease and cytokine release syndrome. Targeting this pathway has also identified avenues for potential expansion into several other indications, such as uveitis, neuromyelitis optica and, most recently, COVID-19 pneumonia (Choy et al.).
Therapeutic Areas:
- Rheumatoid arthritis
- Systemic juvenile idiopathic arthritis
- Castleman´s disease
- Sepsis
- Cancer
- Metabolic disease
- Heart and cardiovascular disease
- Muscle and bone
- COVID-19 pneumonia
Literature:
Complementary DNA for a novel human interleukin (BSF-2) that induces B lymphocytes to produce immunoglobulin,T. Hirano, K. Yasukawa, H. Harada, Nature. 324 (1986) 73–76
1.9 A crystal structure of interleukin 6: implications for a novel mode of receptor dimerization and signalingW. Somers, EMBO J. 16 (1997) 989–997
Interleukin-6 Family Cytokines,S. Rose-John, Cold Spring Harb. Perspect. Biol. 10 (2018)
Cytokines and the hepatic acute-phase response,G. Ramadori, B. Christ, Semin Liver Dis. 19 (1999) 141–155.
IL-6 in Inflammation, Immunity, and Disease,T. Tanaka, M. Narazaki, T. Kishimoto, Cold Spring Harb. Perspect. Biol. 6 (2014) a016295–a016295.
Accuracy of serum interleukin (IL)-6 in sepsis diagnosis: a systematic review and meta-analysis,T. Hou, D. Huang, R. Zeng, Z. Ye, Y. Zhang, , Int J Clin Exp Med. 8 (2015) 15238–15245.
Direct Determination of the Interleukin-6 Binding Epitope of the Interleukin-6 Receptor by NMR Spectroscopy,A. Schwantner, A.J. Dingley, S. Özbek, S. Rose-John, J. Grötzinger, , J. Biol. Chem. 279 (2004) 571–576.
Hexameric Structure and Assembly of the Interleukin-6/IL-6 -Receptor/gp130 Complex,M.J. Boulanger, , Science. 300 (2003) 2101–2104.
IL-6/IL-6 receptor system and its role in physiological and pathological conditions.,M. Mihara, M. Hashizume, H. Yoshida, M. Suzuki, M. Shiina, Clin Sci. 122 (2012) 143–159.
Translating IL-6 biology into effective treatments,E. H. Choy, F. De Benedetti, T. Takeuchi, M. Hashizume, M.R. John, T. Kishimoto, Nat Rev Rheumatol Actions. 16 (6) (2020) 335-345.
Therapeutic strategies for the clinical blockade of IL-6/gp130 signaling,S.A. Jones, J. Scheller, S. Rose-John, J. Clin. Invest. 121 (2011) 3375–3383.
IL-6 biology: implications for clinical targeting in rheumatic disease,L.H. Calabrese, S. Rose-John, Nat. Rev. Rheumatol. 10 (2014) 720–727.
IL-6 pathway in the liver: From physiopathology to therapy,D. Schmidt-Arras, S. Rose-John, J. Hepatol. 64 (2016) 1403–1415.
The role of interleukin-6 signalling and its therapeutic blockage in skewing the T cell balance in rheumatoid arthritis: IL-6 signalling in rheumatoid arthritis,K. Schinnerling, J.C. Aguillón, D. Catalán, L. Soto, Clin. Exp. Immunol. 189 (2017) 12–20.
Association of interleukin-6 single nucleotide polymorphisms with juvenile idiopathic arthritis,V. Ziaee, M. Maddah, M.-H. Moradinejad, A. Rezaei, S. Zoghi, M. Sadr, S. Harsini, N. Rezaei, Clin. Rheumatol.
The Role of Interleukin-6 in Castleman Disease,K. Yoshizaki, S. Murayama, H. Ito, T. Koga, Hematol. Oncol. Clin. North Am. 32 (2018) 23–36.
Clinical Significance of Interleukin-6 in the Diagnosis of Sepsis and Discriminating Sepsis Induced by Gram-negative Bacteria:W. Shao, D. Yu, W. Zhang, X. Wang, Pediatr. Infect. Dis. J. 37 (2018) 801–805.
Interleukin-6/STAT3 signaling as a promising target to improve the efficacy of cancer immunotherapyH. Kitamura, Y. Ohno, Y. Toyoshima, J. Ohtake, S. Homma, H. Kawamura, N. Takahashi, A. Taketomi, Cancer Sci. 108 (2017) 1947–1952.
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Cancer
Interleukin-6/STAT3 signaling as a promising target to improve the efficacy of cancer immunotherapyH. Kitamura, Y. Ohno, Y. Toyoshima, J. Ohtake, S. Homma, H. Kawamura, N. Takahashi, A. Taketomi, Cancer Sci. 108 (2017) 1947–1952.<PMID: 28749573
The role of the interleukin (IL)-6/IL-6 receptor axis in cancerM.Y. Taher, D.M. Davies, J. Maher, Biochem. Soc. Trans. (2018) 46(6):1449-1462.PMID: 30467123
The Role of Interleukin-6 in Castleman DiseaseK. Yoshizaki, S. Murayama, H. Ito, T. Koga, Hematol. Oncol. Clin. North Am. 32 (2018) 23–36.PMID: 29157617
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Covid-19 Pneumonia
Translating IL-6 biology into effective treatmentsE. H. Choy F. De Benedetti, T. Takeuchi, M. Hashizume, M.R. John, T. Kishimoto, Nat Rev Rheumatol Actions. 16 (6) (2020) 335-345.
The Role of Cytokines including Interleukin-6 in COVID-19 induced Pneumonia and Macrophage Activation Syndrome-Like DiseaseDennis McGonagle, Kassem Sharif, Anthony O'Regan, Charlie Bridgewood, Review. Autoimmun Rev. (2020) 19(6):102537
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Heart & Cardiovascular disease
The clinical significance of interleukin-6 In heart failure: results from the BIOSTAT-CHF study.G. Markousis-Mavrogenis, J. Tromp, W. Ouwerkerk et al., Eur. J. Heart Fail. (2019) 8: 965-973.
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Rheumatoid Arthritis
The role of interleukin-6 signalling and its therapeutic blockage in skewing the T cell balance in rheumatoid arthritis: IL-6 signalling in rheumatoid arthritisK. Schinnerling, J.C. Aguillón, D. Catalán, L. Soto, Clin. Exp. Immunol. 189 (2017) 12–20.
Association of interleukin-6 single nucleotide polymorphisms with juvenile idiopathic arthritisV. Ziaee, M. Maddah, M.-H. Moradinejad, A. Rezaei, S. Zoghi, M. Sadr, S. Harsini, N. Rezaei, Clin. Rheumatol.
-
Sepsis
Clinical Significance of Interleukin-6 in the Diagnosis of Sepsis and Discriminating Sepsis Induced by Gram-negative BacteriaW. Shao, D. Yu, W. Zhang, X. Wang, Pediatr. Infect. Dis. J. 37 (2018) 801–805.
All 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 Biomedica human Interleukin-6 (IL-6) ELISA kit is calibrated against a highly purified recombinant human IL-6 protein (expressed in human embryonic kidney cell, HEK-293).
The human serum based calibrator is provided in eight lyophilized glass vials in the following concentrations: 0 / 3.125 / 6.25 / 12.5 / 25 / 50 / 100 / 200 pg/ml.
CALIBRATION using WHO standard
The WHO reference reagent IL-6/NIBSC code 89/548 (recombinant DNA, human sequence) was analysed in this human IL-6 ELISA kit.
The equation below can be used to convert the sample values obtained with this kit to approximate WHO/IL-6 /NIBSC 89/548 units:
WHO/NIBSC (89/548) reference (IU/ml) = 0.08 BI-IL-6 value (pg/ml).
Human IL-6 ELISA Detection Limit & Sensitivity
To determine the sensitivity of the IL-6 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 IL-6, with a confidence level of 99%. It is defined as the mean back-calculated concentration of standard 1 (0 pg/ml of IL-6, 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 IL-6, is diluted, measured five times and its concentration back calculated. The lowest dilution, which meets both criteria, is reported as the LLOQ.
The following values were determined for the human IL-6 ELISA:
|
LOD |
0.28 pg/ml |
|
LLOQ |
0.78 pg/ml |
Human IL-6 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 two samples of known concentrations three times within one IL-6 ELISA lot by one operator.
|
ID |
n |
Mean IL-6 [pg/ml] |
SD IL-6 [pg/ml] |
CV (%) |
|
Sample 1 |
3 |
6.5 |
0.5 |
7 |
|
Sample 2 |
3 |
50.5 |
0.7 |
1 |
In-Between-Run Precision
In-between-run precision was tested by measuring two samples of known concentrations three times within different IL-6 ELISA kit lots by different operators.
|
ID |
n |
Mean IL-6 [pg/ml] |
SD IL-6 [pg/ml] |
CV (%) |
|
Sample 1 |
3 | 6.3 | 0.4 | 6 |
|
Sample 2 |
3 | 50.5 | 2.4 | 5 |
Human IL-6 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 human Interleukin-6 (IL-6) ELISA was measured by adding recombinant human IL-6 to samples containing a known concentration of endogenous IL-6. 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 IL-6 ELISA in different sample matrices:
|
|
|
% Recovery |
|||||
|
Sample Matrix |
n |
+100 pg/ml rec. IL-6 |
+50 pg/ml rec. IL-6 |
+25 pg/ml rec.IL-6
|
|||
|
Mean |
Range |
Mean |
Range |
Mean |
Range |
||
|
Serum |
6 |
113 |
103-122 |
112 |
103-120 |
96 |
88-103 |
|
EDTA plasma |
6 |
111 |
107-116 |
109 |
103-113 |
99 |
93-104 |
|
Citrate plasma |
2 |
111 |
106-115 |
99 |
97-102 |
102 |
99-105 |
|
Heparin plasma |
2 |
107 |
105-109 |
103 |
103-104 |
95 |
89-100 |
|
|
|
|
|
+50 pg/ml |
|
||
|
Cell culture supernatant |
2 |
|
|
97 |
90-103 |
|
|
|
|
|
|
|
+50 pg/ml |
|
||
|
Urine |
5 |
|
|
102 |
95-110 |
|
|
Data showing % recovery of recombinant IL-6 in human serum samples:
|
ID |
IL-6 [pg/ml] |
% Recovery |
|||||
|
Reference |
+100 pg/ml |
+50 pg/ml |
+25 pg/ml |
+100 pg/ml |
+50 pg/ml |
+25 pg/ml |
|
|
S1 |
2.7 |
104.4 |
52.8 |
26.6 |
103 |
103 |
97 |
|
S2 |
2.5 |
114.1 |
53.7 |
24.8 |
113 |
105 |
90 |
|
S3 |
1.4 |
119.7 |
57.6 |
26.1 |
119 |
114 |
99 |
|
S4 |
2.5 |
113.7 |
61.1 |
26.4 |
112 |
120 |
97 |
|
S5 |
0.5 |
122.3 |
56.9 |
26.2 |
122 |
113 |
103 |
|
S6 |
17.7 |
116.2 |
66.8 |
37.6 |
107 |
116 |
88 |
|
|
Mean R[%] |
113 |
112 |
96 |
|||
Data showing % recovery of recombinant IL-6 in human EDTA plasma samples:
|
ID |
IL-6 [pg/ml] |
% Recovery |
|||||
|
Reference |
+100 pg/ml |
+50 pg/ml |
+25 pg/ml |
+100 pg/ml |
+50 pg/ml |
+25 pg/ml |
|
|
E1 |
2.1 |
110.7 |
57.4 |
26.1 |
110 |
113 |
97 |
|
E2 |
2.7 |
116.9 |
52.8 |
25.5 |
116 |
103 |
93 |
|
E3 |
0.7 |
107.6 |
55.5 |
25.3 |
107 |
110 |
99 |
|
E4 |
2.9 |
113.4 |
56.0 |
27.2 |
112 |
109 |
99 |
|
E5 |
0.6 |
111.1 |
55.2 |
26.4 |
111 |
110 |
104 |
|
E6 |
0.3 |
110.1 |
55.4 |
25.5 |
110 |
111 |
101 |
|
|
Mean R[%] |
111 |
109 |
99 |
|||
Data showing % recovery of recombinant IL-6 in human citrate plasma samples:
|
ID |
IL-6 [pg/ml] |
% Recovery |
|||||
|
Reference |
+100 pg/ml |
+50 pg/ml |
+25 pg/ml |
+100 pg/ml |
+50 pg/ml |
+25 pg/ml |
|
|
C1 |
2.1 |
115.6 |
52.2 |
28.1 |
115 |
102 |
105 |
|
C2 |
1.7 |
107.3 |
49.1 |
26.3 |
106 |
97 |
99 |
|
|
Mean R[%] |
111 |
99 |
102 |
|||
Data showing % recovery of recombinant IL-6 in human heparin plasma samples:
|
ID |
IL-6 [pg/ml] |
% Recovery |
|||||
|
Reference |
+100 pg/ml |
+50 pg/ml |
+25 pg/ml |
+100 pg/ml |
+50 pg/ml |
+25 pg/ml |
|
|
H1 |
0.9 |
105.8 |
52.2 |
25.8 |
105 |
104 |
100 |
|
H2 |
0.9 |
109.4 |
51.7 |
23.1 |
109 |
103 |
89 |
|
|
Mean R[%] |
107 |
103 |
95 |
|||
Human IL-6 ELISA Dilution Linearity & Parallelism
Tests of dilution linearity and parallelism ensure that both, endogenous and recombinant samples containing IL-6 behave in a dose-dependent manner and are not affected by matrix effects. Dilution linearity assesses the accuracy of measurements in diluted humanl samples spiked with known concentrations of recombinant analyte. By contrast, parallelism refers to dilution linearity in clinical samples and provides evidence that endogenous analyte behaves in the same way as the recombinant one /likewise to the recombinant analyte. Dilution linearity and parallelism are assessed for each sample type and should be within 20% of the expected concentration.
Dilution linearity was assessed by serially diluting human serum and plasma samples spiked with recombinant IL-6 in assay buffer.
The figure and table below show the mean recovery and range of serially diluted recombinant IL-6 in several sample matrices:
|
|
|
% Recovery of recombinant IL-6 in diluted samples |
|||||
|
Sample Matrix |
n |
1+1 |
1+3 |
1+7 |
|||
|
Mean |
Range |
Mean |
Range |
Mean |
Range |
||
|
Serum |
5 |
93 |
83-100 |
95 |
85-104 |
96 |
91-104 |
|
EDTA plasma |
5 |
95 |
85-104 |
98 |
93-104 |
99 |
94-106 |
|
Citrate plasma |
2 |
93 |
93-94 |
98 |
94-102 |
88 |
86-89 |
|
Heparin plasma |
2 |
99 |
94-104 |
107 |
103-111 |
94 |
94-94 |
|
Cell culture supernatant |
2 |
93 |
90-97 |
95 |
92-97 |
96 |
94-98 |
|
Urine |
1 |
89 |
|
99 |
|
89 |
|
Data showing dilution linearity of recombinant IL-6 spiked into human serum and plasma samples (ref) containing endogenous IL-6.
Calculation of dilution linearity of spiked serum samples:
|
ID |
IL-6 [pg/ml] |
% Recovery |
|||||
|
Reference |
1+1 |
1+3 |
1+7 |
1+1 |
1+3 |
1+7 |
|
|
S1 |
126.8 |
63.1 |
30.9 |
14.7 |
100 |
97 |
93 |
|
S2 |
114.1 |
50.6 |
26.3 |
14.8 |
89 |
92 |
104 |
|
S3 |
119.7 |
57.7 |
29.0 |
14.3 |
96 |
97 |
96 |
|
S4 |
113.7 |
54.7 |
29.7 |
14.3 |
96 |
104 |
96 |
|
S5 |
124.7 |
51.6 |
26.4 |
14.2 |
83 |
85 |
91 |
|
|
Mean R[%] |
93 |
95 |
96 |
|||
Calculation of dilution linearity of spiked EDTA plasma samples:
|
ID |
IL-6 [pg/ml] |
% Recovery |
|||||
|
Reference |
1+1 |
1+3 |
1+7 |
1+1 |
1+3 |
1+7 |
|
|
E1 |
110.7 |
57.8 |
28.9 |
14.7 |
104 |
104 |
106 |
|
E2 |
116.9 |
49.5 |
28.3 |
14.2 |
85 |
97 |
97 |
|
E3 |
111.1 |
50.4 |
25.8 |
13.4 |
91 |
93 |
96 |
|
E4 |
111.4 |
52.7 |
26.0 |
14.1 |
95 |
93 |
101 |
|
E5 |
119.3 |
60.7 |
30.7 |
14.0 |
102 |
103 |
94 |
|
|
Mean R[%] |
95 |
98 |
99 |
|||
Calculation of dilution linearity of spiked citrate plasma samples:
|
ID |
IL-6 [pg/ml] |
% Recovery |
|||||
|
Reference |
1+1 |
1+3 |
1+7 |
1+1 |
1+3 |
1+7 |
|
|
C1 |
115.6 |
54.2 |
29.4 |
12.9 |
94 |
102 |
89 |
|
C2 |
107.3 |
49.9 |
25.2 |
11.5 |
93 |
94 |
86 |
|
|
Mean R[%] |
93 |
98 |
88 |
|||
Calculation of dilution linearity of spiked heparin plasma samples:
|
ID |
IL-6 [pg/ml] |
% Recovery |
|||||
|
Reference |
1+1 |
1+3 |
1+7 |
1+1 |
1+3 |
1+7 |
|
|
H1 |
105.8 |
49.6 |
27.2 |
12.4 |
94 |
103 |
94 |
|
H2 |
109.4 |
56.8 |
30.3 |
12.9 |
104 |
111 |
94 |
|
|
Mean R[%] |
99 |
107 |
94 |
|||
[/collapsible-block title="Show Individual Measurements"]
Parallelism
Parallelism was assessed by serially diluting samples containing endogenous IL-6 with assay buffer.
|
|
|
% Recovery of endogenous IL-6 in diluted samples |
|||||
|
Sample Matrix |
n |
1+1 |
1+3 |
1+7 |
|||
|
Mean |
Range |
Mean |
Range |
Mean |
Range |
||
|
Serum |
5 |
93 |
82-100 |
90 |
86-94 |
89 |
84-93 |
|
EDTA plasma |
5 |
99 |
92-104 |
96 |
76-114 |
92 |
86-98 |
|
Citrate plasma |
2 |
103 |
101-106 |
100 |
92-109 |
99 |
92-106 |
|
Heparin plasma |
2 |
104 |
104-104 |
90 |
84-95 |
89 |
88-90 |
|
Cell culture supernatant |
2 |
95 |
89-101 |
102 |
88-117 |
98 |
88-108 |
|
Urine |
2 |
96 |
95-98 |
108 |
97,120 |
108 |
93-124 |
Data showing dilution linearity of endogenous IL-6 in human serum samples:
|
ID |
IL-6 [pg/ml] |
% Recovery |
|||||
|
Reference |
1+1 |
1+3 |
1+7 |
1+1 |
1+3 |
1+7 |
|
|
S1 |
40.3 |
19.4 |
9.1 |
4.7 |
96 |
90 |
93 |
|
S2 |
44.4 |
18.1 |
9.9 |
4.9 |
82 |
89 |
88 |
|
S3 |
91.9 |
41.6 |
21.5 |
10.0 |
91 |
94 |
87 |
|
S4 |
39.9 |
20.0 |
9.1 |
4.2 |
100 |
91 |
84 |
|
S5 |
45.8 |
22.3 |
9.6 |
5.2 |
97 |
86 |
91 |
|
|
Mean R[%] |
93 |
90 |
89 |
|||
Data showing dilution linearity of endogenous IL-6 in human EDTA plasma samples:
|
ID |
IL-6 [pg/ml] |
% Recovery |
|||||
|
Reference |
1+1 |
1+3 |
1+7 |
1+1 |
1+3 |
1+7 |
|
|
E1 |
74.2 |
34.1 |
14.1 |
8.2 |
92 |
76 |
88 |
|
E2 |
22.0 |
11.0 |
5.2 |
2.7 |
100 |
95 |
98 |
|
E3 |
38.1 |
18.1 |
8.8 |
4.1 |
95 |
92 |
86 |
|
E4 |
9.8 |
5.0 |
2.8 |
1.1 |
102 |
114 |
90 |
|
E5 |
31.4 |
16.3 |
8.0 |
3.8 |
104 |
102 |
97 |
|
|
Mean R[%] |
99 |
96 |
92 |
|||
Data showing recovery of endogenous IL-6 in human citrate plasma samples:
|
ID |
IL-6 [pg/ml] |
% Recovery |
|||||
|
Reference |
1+1 |
1+3 |
1+7 |
1+1 |
1+3 |
1+7 |
|
|
C1 |
59.3 |
29.9 |
13.7 |
6.8 |
101 |
92 |
92 |
|
C2 |
147.8 |
78.2 |
40.1 |
19.5 |
106 |
109 |
106 |
|
|
Mean R[%] |
103 |
100 |
99 |
|||
Data showing recovery of endogenous IL-6 in human heparin plasma samples:
|
ID |
IL-6 [pg/ml] |
% Recovery |
|||||
|
Reference |
1+1 |
1+3 |
1+7 |
1+1 |
1+3 |
1+7 |
|
|
H1 |
144.7 |
75.1 |
34.5 |
16.0 |
104 |
95 |
88 |
|
H2 |
92.1 |
48.0 |
19.4 |
10.4 |
104 |
84 |
90 |
|
|
Mean R[%] |
104 |
90 |
89 |
|||
[/collapsible-block title="Show Individual Measurements"]
IL-6 ELISA Specificity
The specificity of an ELISA is defined as its ability to exclusively recognize the analyte of interest.
The specificity of the IL-6 ELISA was shown by characterizing both the capture and the detection antibodies through epitope mapping. In addition, the specificity of the ELISA was established through competition experiments, which measure the ability of the antibodies to exclusively bind IL-6.
This assay recognizes recombinant and endogenous (natural) human IL-6 and detects free circulating IL-6 as well as receptor-bound IL-6.
The recombinant anti-human IL-6 capture antibody detects a structural epitope near the receptor binding site of the IL-6 molecule. Four linear epitopes of the polyclonal anti-human IL-6 detection antibody are spread throughout the IL-6 protein.
Competition of Signal
Competition experiments were carried out by pre-incubating human samples containing endogenous IL-6 with an excess of capture antibody (AB). The concentration measured in this mixture was then compared to a reference value, which was obtained from the same sample without the pre-incubation step. Mean competition in serum and plasma samples is 99%.
|
|
|
IL-6 [pg/ml] |
% Competition |
|
|
Sample Matrix |
ID |
Reference |
+ capture AB |
|
|
Serum |
S1 |
1.4 |
0.0 |
95 |
|
Serum |
S2 |
61.4 |
0.3 |
99 |
|
Serum |
S3 |
41.5 |
0.3 |
99 |
|
Serum |
S4 |
33.2 |
0.6 |
98 |
|
EDTA plasma |
E1 |
18.9 |
0.2 |
99 |
|
EDTA plasma |
E2 |
27.6 |
0.1 |
99 |
|
Citrate plasma |
C1 |
45.6 |
0.0 |
100 |
|
Heparin plasma |
H1 |
81.0 |
0.0 |
100 |
|
|
|
|
Mean R[%] |
99 |
Cross Reactivity
CROSS REACTIVITY with non-human samples
This human IL-6 ELISA kit cannot be used for the detection of IL-6 in rat, mouse, and porcine samples.
Sample Stability
Serum and plasma (EDTA , citrate , heparin) cell culture supernatants, and urine samples 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 Interleukin-6 (IL-6) was tested by comparing samples that had undergone five 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 five freeze-thaw cycles is 95%.
Sample OD values after freeze-thaw (F/T) cycles:
|
IL-6 [OD] |
|
% Recovery |
||||||
|
Sample Matrix |
ID |
Reference |
1x F/T |
3x F/T |
5x F/T |
1x F/T |
3x F/T |
5x F/T |
|
Serum |
S1 |
1.09 |
1.10 |
1.09 |
1.09 |
101 |
100 |
100 |
|
Serum |
S2 |
0.41 |
0.45 |
0.43 |
0.33 |
110 |
98 |
82 |
|
EDTA plasma |
E1 |
0.45 |
0.43 |
0.43 |
0.44 |
95 |
95 |
98 |
|
Citrate plasma |
C1 |
0.18 |
0.18 |
0.17 |
0.18 |
98 |
95 |
99 |
|
Heparin plasma |
H1 |
0.53 |
0.52 |
0.50 |
0.51 |
98 |
94 |
97 |
|
|
Mean R[%] |
101 |
97 |
95 |
||||
All samples should undergo a maximum of five freeze-thaw cycles.
Benchtop Stability
The benchtop stability of endogenous Interleukin-6 (IL-6) was tested by comparing IL-6 measurements in human samples that had been stored at different temperatures.
For the assessment of the benchtop stability, a set of human samples was aliquoted and stored 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.
Sample OD values of samples stored at -25°C (reference), at room temperature (RT) or overnight (o.n.) at 4°C:
|
|
|
IL-6 [pg/ml] |
% Recovery |
|||
|
Sample Matrix |
ID |
Reference |
o.n. @4°C |
o.n. @4°C |
||
|
Serum |
S1 |
1.09 |
1.07 |
0.97 |
98 |
89 |
|
Serum |
S2 |
0.41 |
0.46 |
0.39 |
114 |
97 |
|
EDTA plasma |
E1 |
0.45 |
0.45 |
0.44 |
99 |
98 |
|
Citrate plasma |
C1 |
0.18 |
0.17 |
0.17 |
96 |
97 |
|
Heparin plasma |
H1 |
0.53 |
0.51 |
0.49 |
96 |
92 |
|
|
Mean R [%] |
101 |
95 |
|||
Sample Values
IL-6 Values in Apparently Healthy Individuals
Serum / Plasma: IL-6 was measured in samples from apparently healthy donors (no medical histories were available).
|
|
|
IL-6 [pg/ml] |
% Detectable |
||
|
Sample Matrix |
n |
Mean |
Range |
Median |
|
|
Serum |
48 |
1.73 |
0.30 – 4.36 |
1.50 |
100 |
|
EDTA plasma |
26 |
1.01 |
0.01 – 2.69 |
0.98 |
100 |
|
Citrate plasma |
14 |
1.86 |
0.01 – 2.10 |
0.71 |
100 |
|
Heparin plasma |
11 |
1.52 |
0.00 – 2.41 |
0.60 |
91 |
It is recommended to establish the normal range for each laboratory.
IL-6 values measured in serum and plasma (EDTA, citrate, heparin) from samples of ten apparently healthy donors (matched-paired samples).
|
|
|
IL-6 [pg/ml] |
% Detectable |
||
|
Sample Matrix |
n |
Mean |
Range |
Median |
|
|
Serum |
10 |
1.12 |
0.58-1.56 |
1.19 |
100 |
|
EDTA plasma |
10 |
0.64 |
0.01-1.45 |
0.53 |
100 |
|
Citrate plasma |
10 |
0.71 |
0.01-1.32 |
0.68 |
100 |
|
Heparin plasma |
9 |
0.62 |
0.00-2.41 |
0.48 |
90 |
Interleukin-6 (IL-6) Values in Disease Panels
In addition to samples of apparently healthy donors, panels of samples from patients with heart disease, kidney diseases, as well as a panel of unselected hospital patients were tested.
IL-6 values measured in apparently healthy individuals and in subjects with chronic kidney disease (CKD).
IL-6 values measured in apparently healthy individuals and in subjects with aortic stenosis, arthritis and melanoma:
Summary of the results:
|
|
|
IL-6 [pg/ml] |
||
|
Samples / Matrix |
n |
Mean |
Range |
Median |
|
Apparently Healthy Panel / Serum |
48 |
1.7 |
0.3-4.3 |
1.5 |
|
Aortic Stenosis Panel / Serum |
18 |
6.5 |
0.8-42.6 |
2.0 |
|
Arthritis Panel I / Serum |
13 |
40.5 |
6.5-79.4 |
41.9 |
|
Arthritis Panel II / Serum |
32 |
18.5 |
0.4-270 |
3.4 |
|
Melanoma Panel / Serum |
32 |
6.0 |
0-40.9 |
2.5 |
|
Nephro-CKD Panel/ EDTA plasma |
28 |
4.5 |
1.0-15.2 |
4.1 |
Matrix Comparison
To assess whether all tested matrices behave the same way Interleukin-6 (IL-6) was measured in serum and plasma (EDTA, citrate, heparin) from samples of ten apparently healthy donors (matched-paired samples).
|
|
|
IL-6 [pg/ml] |
% Detectable |
||
|
Sample Matrix |
n |
Mean |
Range |
Median |
|
|
Serum |
10 |
1.12 |
0.58-1.56 |
1.19 |
100 |
|
EDTA plasma |
10 |
0.64 |
0.01-1.45 |
0.53 |
100 |
|
Citrate plasma |
10 |
0.71 |
0.01-1.32 |
0.68 |
100 |
|
Heparin plasma |
10 |
0.62 |
0.00-2.41 |
0.48 |
90 |
Measurment of human IL-6 in Urine Samples - Performance check
Urine:
Aseptically collect the first urine of the day (mid-stream), voided directly into a sterile container. Centrifuge to remove particles, assay immediately or aliquot and store at -25°C or lower. Urine samples were not normalized to creatinine values.
|
Sample Matrix Urine |
IL-6 [pg/ml] |
|
Donor 1. Apparently healthy |
0.9 |
|
Donor 2. Apparently healthy |
0.7 |
|
Donor 3. Apparently healthy |
1.5 |
|
Donor 4. Apparently healthy |
0.0 |
|
Donor 1. Kidney disease |
122.6 |
|
Donor 2. Kidney disease |
24.6 |
|
Donor 3. Kidney disease |
36.6 |
|
Donor 4. Kidney disease |
2.1 |
|
Donor 5. Kidney disease |
13.9 |
Urine samples were not normalized to creatinine values.
Acccuracy
Data showing % recovery of recombinant IL-6 in human urine samples:
|
ID |
IL-6 [pg/ml] |
% Recovery |
|
|
Reference |
+50 pg/ml |
||
|
U1 |
372 |
60.5 |
107 |
|
U2 |
265 |
80.7 |
120 |
|
U3 |
312 |
60.7 |
121 |
|
U4 |
343 |
58.3 |
115 |
|
U5 |
171 |
59.4 |
119 |
|
|
|
Mean R [%] |
116 |
Dilution linearity, parallelism
Data showing dilution linearity of endogenous IL-6 in human urine samples:
|
Sample ID |
IL-6 [pg/ml] |
R [%] |
|||
|
Ref |
1+1 |
1+3 |
1+1 |
1+3 |
|
|
U1 |
124.8 |
54.8 |
27.0 |
88 |
86 |
|
U2 |
13.9 |
7.2 |
2.6 |
103 |
n.d. |
|
U3 |
41.7 |
24.9 |
12.0 |
120 |
115 |
|
|
|
|
Mean R [%] |
103 |
101 |
Competition experiments were carried out by pre-incubating human urine samples containing endogenous IL-6 with an excess of capture antibody (AB). The concentration measured in this mixture was then compared to a reference value, which was obtained from the same sample without the pre-incubation step. Mean competition in urine samples was 100%.
Competition of endogenous signal:
|
Sample ID |
IL-6 [pg/ml] |
R [%] |
|
|
Reference |
+AB |
||
|
U1 |
147 |
0.0 |
100 |
|
U2 |
16.6 |
0.0 |
100 |
|
U3 |
28.6 |
0.0 |
100 |
|
U4 |
36.1 |
0.0 |
100 |
|
U5 |
9.5 |
0.0 |
100 |
|
|
|
Mean R [%] |
100 |
Measurment of human IL-6 in Cell Culture Supernatants - Performance check:
Cell Culture Supernatants (CCS):
Two human breast cancer cell lines MDA-MB-231, MCF-7 and a human macrophage cell line 4TL9.R were cultured in DMEM/Ham’s F12 and RPMI, respectively, and supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were grown in a humidified atmosphere of 95% air and 5% CO2 for 48 hours. Aliquots of the cell culture supernatants were removed, centrifuged to remove particles, and assayed for levels of human IL-6.
|
Sample Matrix CCS |
IL-6 [pg/ml] |
|
CCS - MDA-MB-231 |
146.6 |
|
CCS - MCF-7 |
18.5 |
|
CCS - 4TL9.R |
0.44 |
|
Medium- DM-F-12 (with supplements) |
0.0 |
|
Medium-RPMI (with supplements) |
0.0 |
Accuracy
Recombinant IL -6 was spiked into samples by using STD7 (100pg/ml) or STD8 (200pg/ml).
Two concentration levels were generated using the following protocol:
-50pg/ml spike: STD7 volume ratio of 1+1.
-25pg/ml spike: STD8 volume ratio of 7+1.
|
CCS |
ID |
IL-6 [pg/ml] |
R [%] |
|||
|
Reference |
50 |
25 |
50 |
25 |
||
|
MDA-MB-231 |
CCS#1 |
146.6 |
118.2 |
n.d. |
90 |
n.d. |
|
MCF-7 |
CCS#2 |
18.5 |
57.9 |
40.7 |
97 |
98 |
n.d.: not determined
Dilution linearity
Dilution linearity of recombinant analyte in cell culture supernatants.
Samples were spiked with STD 7 (100pg/ml, +50pg/ml) and diluted with dilution medium (ASYBUF).
|
CCS |
ID |
IL-6 [pg/ml] |
% Recovery |
|||||
|
Ref |
1+1 |
1+3 |
1+7 |
1+1 |
1+3 |
1+7 |
||
|
MDA-MB-231 |
CCS#1 |
118.2 |
53.1 |
27.1 |
13.9 |
90 |
92 |
94 |
|
MCF-7 |
CCS#2 |
57.9 |
28.0 |
14.1 |
7.1 |
97 |
97 |
98 |
|
|
|
|
|
|
Mean R [%] |
93 |
95 |
96 |
Dilution linearity of endogenous analyte in cell culture supernatants.
Performance was tested in conditioned media (48h) and diluted with dilution medium (ASYBUF).
|
CCS |
ID |
IL-6 [pg/ml] |
% Recovery |
|||||
|
Ref |
1+1 |
1+3 |
1+7 |
1+1 |
1+3 |
1+7 |
||
|
MDA-MB-231 |
CCS#1 |
146.6 |
65.0 |
32.2 |
16.1 |
89 |
88 |
88 |
|
MCF-7 |
CCS#2 |
18.5 |
9.3 |
5.4 |
2.5 |
101 |
117 |
108 |
|
|
|
|
|
|
Mean R [%] |
95 |
102 |
98 |
Competition of IL-6 in cell culture supernatants
Competition experiments were carried out by pre-incubating human cell culture supernatnant samples containing endogenous IL-6 with an excess of capture antibody (AB). The concentration measured in this mixture was then compared to a reference value, which was obtained from the same sample without the pre-incubation step. Mean competition in cell culture supernatants was 98%.
Calculated according to calibration curve prepared from supplied ELISA standards (resuspended in DMEM Medium + supplements).
|
CCS |
ID |
IL-6 |
R [%] |
||
|
Ref |
+ CAB |
|
|||
|
MDA-MB-231 |
CCS#1 |
146.6 |
0.4 |
100 |
|
|
MCF-7 |
CCS#2 |
18.5 |
0.8 |
96 |
|
|
|
|
|
Mean R [%] |
98 |
|
Comparison with another human IL-6 ELISA assay
Assay Characteristics of two different human IL-6 ELISA assays
|
|
BIOMEDICA |
Another MANUFACTURER |
|
Method |
Sandwich ELISA Streptavidin-HRPO/TMB, 12x8-well detachable strips |
Sandwich ELISA HRPO/TMB, 12x8-well detachable strips |
|
Sample type |
Serum, EDTA plasma, heparin plasma, citrate plasma, cell culture supernatants, urine |
Serum, plasma, cell culture supernates |
|
Sample volume |
100 µl sample / well |
100 µl / well 200 µl (cell culture) / well |
|
Assay time |
2 h / 1 h / 1 h / 30 min |
2 h / 2 h / 30 min |
|
Assay range |
0 –200 pg/ml (0 / 3.125 / 6.25 / 12.5 / 25 / 50 / 100 / 200) |
0 – 300 pg/ml |
|
Sensitivity |
LOD: 0.28 pg/ml; LLOQ: 0.78 pg/ml (measurable concentrations in serum AND plasma samples) |
MDD: 0.7 pg/ml |
|
Specificity |
Assay recognizes recombinant and endogenous (natural) human IL-6. |
Assay recognizes natural and recombinant human IL-6.
|
|
Antibodies |
Epitope-mapped antibodies Capture antibody: recombinant IL-6 antibody specific for human IL-6 Detection antibody: polyclonal IL-6 antibody specific for human IL-6, streptavidin-HRPO-labeled |
Capture antibody: monoclonal antibody specific for human IL-6 Detection antibody: polyclonal IL-6 antibody specific for human IL-6, HRPO-labeled |
|
Standard matrix |
Serum based matrix containing recombinant IL-6 8 ready to use standards, lyophilized |
Protein based matrix containing recombinant IL-6 1 stock standard, lyophilized |
|
Values of apparently healthy samples |
Serum median (n=48): 1.5 pg/ml 100 % detectable
EDTA-plasma mean (n=26): 0.9 pg/ml 100 % detectable |
Serum/plasma median (n=40): 33 samples < 3.13 pg/ml
3.13 pg/ml < 7 samples < 12.5 pg/ml
|
|
Controls |
2 controls (high and low) included |
Not included |
|
Validation |
According to FDA/ICH/EMEA guidelines |
Not indicated |
|
Use |
RUO |
RUO |
Comparison of human sample concentrations measured with different human IL-6 ELISA Assays
The Biomedica human IL-6 ELISA kit (Cat. No. BI-IL6) was compared with an ELISA kit from another manufacturer. The same panel of samples, consisting of 36 samples (healthy and diseased), were tested.
Table showing human IL-6 concentrations measured with the Biomedica human IL-6 ELISA and a human IL-6 ELISA assay from another manufacturer:
|
ALL SAMPLES |
|||
|
n = 36 |
|||
|
Sample ID |
Biomedica (BI-IL6) |
Other |
|
|
Cohorts: |
|
IL-6 [pg/ml] |
IL-6 [pg/ml] |
|
Apparently healthy cohort serum samples
|
AH s1 |
0.1 |
0.6 |
|
AH s2 |
1.1 |
1.3 |
|
|
AH s3 |
3.5 |
3.3 |
|
|
AH s4 |
0.2 |
1.3 |
|
|
AH s5 |
1.0 |
1.3 |
|
|
AH s6 |
2.4 |
2.6 |
|
|
AH s7 |
0.5 |
0.9 |
|
|
AH s8 |
1.2 |
1.8 |
|
|
AH s9 |
1.9 |
2.5 |
|
|
|
Mean |
1.31 |
1.75 |
|
Rheuma cohort
serum samples
|
Rs1 |
46.3 |
37.3 |
|
Rs2 |
41.9 |
32.6 |
|
|
Rs3 |
55.1 |
45.0 |
|
|
Rs4 |
22.9 |
20.7 |
|
|
Rs5 |
20.8 |
12.7 |
|
|
Rs6 |
79.4 |
58.0 |
|
|
|
Mean |
44.4 |
34.4 |
|
Cardiology panel (Aortic stensosis) serum samples
|
Cs1 |
3.4 |
4.1 |
|
Cs2 |
6.4 |
6.7 |
|
|
Cs3 |
7.9 |
8.2 |
|
|
Cs4 |
42.6 |
37.0 |
|
|
Cs5 |
28.6 |
25.5 |
|
|
|
Mean |
17.8 |
16.3 |
|
Unselected hospital panel Serum samples
|
Us1 |
24.4 |
18.5 |
|
Us2 |
29.0 |
18.7 |
|
|
Us3 |
24.9 |
18.5 |
|
|
Us4 |
6.1 |
3.9 |
|
|
Us5 |
34.8 |
23.3 |
|
|
Us6 |
21.9 |
16.1 |
|
|
|
Mean |
23.5 |
16.5 |
|
Unselected hospital panel EDTA plasma
|
Uep1 |
19.5 |
18.2 |
|
Uep2 |
10.0 |
12.6 |
|
|
Uep3 |
7.5 |
6.1 |
|
|
Uep4 |
4.9 |
3.6 |
|
|
Uep5 |
34.2 |
24.4 |
|
|
Uep6 |
34.7 |
25.1 |
|
|
|
Mean |
18.5 |
15.0 |
|
Unselected hospital panel Citrate plasma |
Uc1 |
91.3 |
64.6 |
|
Uc2 |
20.3 |
15.4 |
|
|
Uc3 |
145.7 |
96.8 |
|
|
Uc4 |
10.7 |
8.3 |
|
|
|
Mean |
67.0 |
46.3 |
IL-6 ELISAs Comparison - Conversion to NIBSC/WHO units
Concentrations of samples obtained in the Biomedica IL-6 ELISA and the competitor IL-6 ELISA were converted to approximate NIBSC/WHO 89/548 units according to the equation:
- NIBSC/WHO 89/548 approximate value (IU/ml) = 0.131 x obtained value (pg/ml) in competitor IL-6 ELISA
- NIBSC/WHO 89/548 approximate value (IU/ml) = 0.08 x obtained value (pg/ml) in Biomedica IL-6 ELISA
|
Biomedica IL-6 ELISA (#BI-IL6) |
Competitor IL-6 ELISA |
ratio |
|
|
Sample ID |
IL-6 [pg/ml] |
IL-6 [pg/ml] |
|
|
#S1 |
1.95 |
2.42 |
0.81 |
|
#S2 |
2.32 |
2.45 |
0.95 |
|
#S3 |
1.99 |
2.42 |
0.82 |
|
#S4 |
0.49 |
0.51 |
0.95 |
|
#S5 |
2.79 |
3.05 |
0.91 |
|
#S6 |
1.75 |
2.11 |
0.83 |
|
#S7 |
3.70 |
4.89 |
0.76 |
|
#S8 |
3.35 |
4.27 |
0.79 |
|
#S9 |
4.41 |
5.89 |
0.75 |
|
#S10 |
1.83 |
2.71 |
0.68 |
|
#S11 |
1.66 |
1.67 |
1.00 |
|
#S12 |
6.35 |
7.59 |
0.74 |
|
#S13 |
0.27 |
0.53 |
0.51 |
|
#S14 |
0.51 |
0.88 |
0.58 |
|
#S15 |
0.63 |
1.07 |
0.59 |
|
#S16 |
3.41 |
4.85 |
0.70 |
|
#S17 |
2.29 |
3.34 |
0.69 |
|
#H1 |
2.83 |
3.57 |
0.79 |
|
#H2 |
7.45 |
14.08 |
0.53 |
|
#H3 |
0.47 |
0.8 |
0.59 |
|
#E1 |
1.56 |
2.39 |
0.65 |
|
#E2 |
0.80 |
1.65 |
0.48 |
|
#E3 |
0.60 |
0.80 |
0.75 |
|
#E4 |
0.39 |
0.48 |
0.82 |
|
#E5 |
2.74 |
3.19 |
0.86 |
|
#E6 |
2.78 |
3.29 |
0.84 |
|
#E7 |
11.03 |
13.45 |
0.82 |
|
#C1 |
7.30 |
8.46 |
0.86 |
|
#C2 |
1.62 |
2.02 |
0.80 |
|
#C3 |
11.65 |
12.69 |
0.92 |
|
#C4 |
0.86 |
1.08 |
0.79 |
|
#U1 |
9.81 |
12.2 |
0.80 |
|
#U2 |
1.97 |
2.99 |
0.66 |
|
#U3 |
0.32 |
0.52 |
0.61 |
|
#CCM1 |
13.12 |
13.95 |
0.94 |
|
#CCM2 |
1.63 |
2.26 |
0.72 |
Abbreviations: S (serum), H (heparin plasma), E (EDTA plasma),
C (citrate plasma), U (urine), CCM (cell culture supernatants)
Conclusion:
Concentration values obtained in the Biomedica IL-6 ELISA converted to IU/ml using NIBSC/WHO 89/548 measure very similar to the competitor IL-6 ELISA in all matrices.
Correlation of both assays is excellent - Pearson correlation coefficient R = 0.978, p < 0.00001.
Comparison of cell culture supernatant sample concentrations in human breast cancer cell lines measured with different human IL-6 ELISA Assays:
The Biomedica IL-6 ELISA (#BI-IL6) was compared with an ELISA kit from another manufacturer.
|
Cell Culture Supernatatants (CCS) |
||
|
n = 2 |
||
|
Sample ID |
Biomedica (# BI-IL6) |
Other ELISA |
|
|
IL-6 [pg/ml] |
IL-6 [pg/ml] |
|
CCS - MDA-231 |
164.0 |
106.5 |
|
CCS - MCF-7 |
20.3 |
17.3 |
