Download biomedica product listLeucine-rich alpha-2-glycoprotein (LRG) ELISA | BI-LRG
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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, heparin plasma (urine and cell culture protocol available)
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Sample volume
100 µl pre-diluted sample / well (5 µl sample)
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Assay time
2 h / 1 h / 30 min
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Sensitivity
LOD: 0.26 ng/ml; LLOQ: 0.5 ng/ml
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Standard range
0 – 64 ng/ml (0 / 2 / 4 / 8 / 16 / 32 / 64)
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Conversion factor
1 ng/ml = 0,0262 nmol/l; MW: 38.178 kDa
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Specificity
Endogenous and recombinant human LRG (leucine-rich alpha-2-glycoprotein)
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Precision
In-between-run (n=9): ≤ 6 % CV
Within-run (n=3): ≤ 3 % CV
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Cross-reactivity
The cross-reactivity of the human LRG ELISA with non-human samples was not tested.
The LRG sequence similarity between human LRG with mouse, rat and pig is 64%, 64% and 71%, respectively.
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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
Product Overview
The leucine-rich alpha-2-glycoprotein (LRG) immunoassay is a 3,5 hour, 96-well sandwich ELISA for the quantitative determination of leucine-rich alpha-2-glycoprotein (LRG) in serum and plasma (EDTA, heparin, citrate). The assay employs human based serum standards and controls to ensure the measurement of biologically reliable data.
The LRG ELISA kit uses highly purified, epitope mapped antibodies.
Principle of the Assay
The leucine-rich alpha-2-glycoprotein (LRG) ELISA kit is a sandwich enzyme immunoassay for the quantitative determination of LRG in serum and plasma (EDTA, heparin, citrate).
A protocol for urine and cell culture supernatants is available.
The figure below explains the principle of the LRG sandwich ELISA:
Capture antibody: polyclonal sheep anti-human LRG antibody
Detection antibody: polyclonal sheep anti-human LRG antibody, HRP-labeled
Target antigen: human LRG (leucine-rich alpha-2-glycoprotein)
In a first step, standards, controls and pre-diluted samples are pipetted into the wells of the microtiter strips, which are pre-coated with polyclonal sheep anti-human LRG antibody. LRG present in the standard/control/sample binds to the pre-coated antibody in the well. All non-specific unbound material is removed in a washing step and the detection antibody (CONJ, polyclonal sheep anti-LRG-HRPO) is pipetted into the wells. After another washing step, the substrate (TMB, tetramethylbenzidine) is added. The enzyme-catalyzed color change of the substrate is directly proportional to the amount of LRG 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 LRG in the sample is determined from the dose response curve. This sample concentration must be multiplied by the dilution factor used for sample preparation to obtain the final sample concentration.
Typical Standard Curve
The figure below shows a typical standard curve for the LRG ELISA assay. The immunoassay is calibrated against recombinant leucine-rich alpha-2-glycoprotein peptide:
Human LRG ELISA Kit Components
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CONTENT |
DESCRIPTION |
QUANTITY |
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PLATE |
Detachable microtiter strips pre-coated with sheep polyclonal anti-LRG antibody |
12 x 8 tests |
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WASHBUF |
Wash buffer concentrate 20x |
1 x 50 ml |
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STD |
Serum-based standards containing varying concentrations of recombinant LRG, lyophilized (0, 2, 4, 8, 16, 32, 64 ng/ml) |
7 vials |
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CTRL |
Serum-based controls with expected concentrations after reconstitution (lyophilized) |
2 vials |
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ASYBUF |
Assay buffer, ready to use |
1 x 115 ml |
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CONJ |
Anti-LRP antibody-HRPO conjugate |
1 x 13 ml |
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SUB |
Substrate (TMB solution) |
1 x 13 ml |
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STOP |
Stop solution |
1 x 7 ml |
Storage instructions: All reagents of the LRG ELISA kit are stable at 4°C until the expiry date stated on the label of each reagent.
Serum, EDTA plasma, and citrate plasma are suitable for use in this assay. Don’t 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 three freeze-thaw cycles. Samples should be mixed well before assaying. We recommend duplicates for all values.
Serum and plasma sample preparation/dilution:
Before assaying: Samples must be diluted 1:4000 with assay buffer (ASYBUF) in 2 steps e.g.:
Transfer 5µl sample to 995µl ASYBUF (yielding a 1:200 dilution) in an Eppendorf tube.
Next, transfer 20µl of the 1:200 diluted sample to 380 µl ASYBUF resulting in a final sample dilution of 1:4000 (in an Eppendorf tube).
Samples with values above STD7 (64 ng/ml) can be diluted with ASYBUF (Assay buffer) using higher dilution factor than 1:4000.
Urine
Note: the experiments performed to measure human LRG (leucine-rich alpha-2-glycoprotein) 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. For more information please refer to the validation data.
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 human LRG (leucine-rich alpha-2-glycoprotein) in 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. For more information please refer to the validation data.
Remove particulates by centrifugation and assay immediately or aliquot and store samples at -25°C or lower. Do not freeze-thaw samples more than five times.
Reagent Preparation
Wash Buffer
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1. |
Bring the WASHBUF concentrate to room temperature. Crystals in the buffer concentrate will dissolve at room temperature. |
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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 and Controls for Serum and Plasma Measurements
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1. |
Pipette 500 µl of distilled or deionized water into each vial. |
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2. |
Leave at room temperature (18-26°C) for 15 min. Vortex gently. |
Reconstituted STDs and CTRLs can be stored at -25°C or lower until expiry date stated on the label. STDs and CTRLs are stable for three freeze-thaw cycles.
Standards for Cell Culture Supernatant Measurements
For the preparation of a cell culture-based standard curve always use the identical cell culture medium (CCM) as used for the experiment.
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1. |
Reconstitute standard 7 (STD7) in 500 µl deionized water. Leave at room temperature (18-26°C) for 15 min and mix well before making dilutions. Use polypropylene tubes. |
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2. |
Mark tubes ccSTD6 to ccSTD1. Dispense 200 µl cell culture medium into each vial. |
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3. |
Pipette 200 µl of STD7 into tube marked as ccSTD6. Mix thoroughly. |
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4. |
Transfer 200 µl of ccSTD6 into the tube marked as ccSTD5. Mix thoroughly. |
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5. |
Continue in the same fashion to obtain ccSTD4 to ccSTD2. CCM serves as the ccSTD1 (0 pg/ml LRG). |
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6. |
Using the prepared standards, follow the protocol as indicated for serum and plasma samples. |
Attention: Supplied STD1-STD7 and controls are only valid for plasma and should not be used for cell culture measurements.
Sample Preparation
Bring samples to room temperature and mix samples gently to ensure the samples are homogenous. We recommend duplicate measurements for all values.
High measuring samples outside of the calibration range of the assay should be diluted in assay buffer.
Assay Protocol
Read the entire protocol before beginning the assay.
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1. |
Bring samples and reagents to room temperature (18-26°C). |
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2. |
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. |
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3. |
Pipette 100 µl STD/CTRL or 100 µl SAMPLE (pre-diluted 1:4000 with ASYBUF) into respective wells. |
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4. |
Cover tightly and incubate for 2 hours at room temperature (18-26°C). |
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5. |
Aspirate and wash wells 5x with 300 µl diluted WASHBUF (Wash buffer). After final wash, remove remaining WASHBUF by strongly tapping plate against paper towel. |
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6. |
Add 100 µl CONJ (Conjugate, amber cap) into each well, swirl gently. |
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7. |
Cover tightly and incubate for 1 hour at room temperature (18-26°C). |
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8. |
Aspirate and wash wells 5x with 300 µl diluted WASHBUF (Wash buffer). After final wash, remove remaining WASHBUF by strongly tapping plate against paper towel. |
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9. |
Add 100 µl SUB (Substrate, blue cap) into each well, swirl gently. |
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10. |
Incubate for 30 min at room temperature (18-26°C) in the dark. |
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11. |
Add 50 µl STOP (Stop solution, white cap) into each well, swirl gently. |
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12. |
Measure absorbance immediately at 450 nm with reference 630 nm, if available. |
Calculation of Results
Construct the standard curve from the OD values of the standards. Use commercially available software or graph paper.
Obtain sample concentrations from the standard curve. The assay was evaluated with logit-log and 4PL curve fitting algorithms.
Different curve fitting methods need to be evaluated by the user.
Urine Protocol
Follow the standard protocol as described for serum and plasma samples. Pipette 100 µl of undiluted urine sample directly into the well of the microtiter plate. If required, dilute samples 1+1 with assay buffer.
Attention: Concentrations defined for controls A and B are only valid for measuring LRG in human serum or plasma. The controls cannot be used for urine sample measurements.
LRG Protein
LRG (leucine-rich alpha-2-glycoprotein) is a glycoprotein with a molecular mass of 38.2 kDa (https://www.uniprot.org/uniprot/P02750). It is encoded by the human gene LRG-1 which is mapped on chromosome 19 at the cytogenetic band 19p13.3. The protein LRG (or also named LRG1) runs at approximately 50 kDa under reducing conditions, as it contains a carbohydrate content of 23% (Haupt et al., 1977). LRG is present in serum, plasma and urine (Kharbanda et al., 2012; Kentsis et al., 2012). LRG is the founding member of the family of leucine-rich repeat proteins (Takahashi et al., 1985). The mature protein consists of 312 amino acids, from Val36 to Gln347, with a leucine content of 66 amino acids. It is folded to eight leucine-rich repeat (LRR) domains of 22 amino acid length, and a C-terminal LRRCT domain with 49 amino acid length (O’Donnell et al., 2002). Human LRG1 shows 62,5% sequence identity with mouse LRG1, and 60,7% with rat LRG1.
LRG binds to the TGFβ accessory receptor endoglin, and in the presence of TGFβ1 this leads to the induction of the TβRII-ALK1-Smad1/5/8 signaling pathway (Wang et al., 2013). TGFβ1 therefore promotes binding of LRG to the proangiogenic ALK1 but inhibits the interaction with angiostatic ALK5. Induced signaling leads to endothelial cell proliferation and blood vessel outgrowth (Wang et al., 2013).
Like many other family members of the LRG family, LRG is described to have multiple binding partners. LRG is described to directly interact with the mitochondrial electron transfer protein cytochrome c (Cummings et al., 2006), whereas the physiological relevance of this interaction in not known yet. LRG further binds to TGFβ1, the most frequently expressed TGFβ isoform.
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Molecular weight |
38.2 kDa |
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Cellular localisation |
Extracellular region or secreted |
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Post-translational modifications |
Not known |
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Alternative names |
LRG1, HMFT1766, LRG, leucine-rich alpha-2-glycoprotein 1, leucine rich alpha-2-glycoprotein 1 |
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Protein Atlas |
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Uniprot ID |
P02750 |
LRG Function
Leucine-rich alpha-2-glycoprotein (LRG) is involved in cell proliferations and immune responses, in cell migration, neovascularization and apoptosis (Zhang et al., 2016; Zhong et al., 2015; Wang et al., 2013). It is a proangiogenic factor which is involved in the regulation of the TGFβ signaling pathway. Upregulation of LRG is described in response to acute phase response in hepatocytes (Shirai et al., 2009). It is further upregulated in patients with inflammatory or neoplastic conditions (Kharbanda et al., 2012).
Tissue distribution of LRG is described to vary, with high-level expression in the liver, lower expression in the heart, and minimal expression in spleen and lung (O’Donnell et al., 2002). LRG is expressed during hematopoiesis. It plays a role in innate immune system as it is upregulated during neutrophil differentiation, packed into peroxidase-negative granules of human neutrophils, and secreted upon activation to modulate the microenvironment (Ai et al., 2008; Druhan et al., 2017). Differential expression of LRG is further associated with certain carcinomas, neurodegenerative disease, aging or autoimmune disease. Also, there is an association of cardiac remodeling (hypertrophy, fibrosis, abnormal vasculature, heart failure) and reduced expression of LRG (Pek et al., 2015; Song et al., 2015).
LRG is potentially a biomarker for a variety of diseases e.g. as an inflammatory biomarker for autoimmune diseases such as rheumatoid arthritis and inflammatory bowel disease (IBD) (Fujimoto M et al.; 2015, Serada et al., 2010). Various groups have shown that LRG is increased in various immune-related diseases such as psoriasis (Nakajima et al., 2017), juvenile idiopathic arthritis (Shimizu M et al., 2017) Kawasaki disease (Kimura, Y. et al. 2017), appendicitis (Kharbanda et al. 2012; Kentsis A et al., 2012; Yap L et al., 2019), and cancers (Yamamoto M et al., 2017; Xie ZB et al., 2019; Otsuru T et al., 2019; Wu JH et al., 2015; Zhang Q et al., 2018; Wang et al. 2015). In addition, LRG may serve as a biomarker for several other disease conditions such as heart failure (Watson CJ et al., 2011), and diabetes-related complications (Liu JJ et al., 2017; Hong Q et al. 2019).
Literature
Isolation and characterization of an unknown, leucine-rich 3.1-S-alpha2-glycoprotein from human serum.
Haupt et al. Hoppe Seylers Z Physiol Chem, 1977; 358 (6):639-46.
Periodicity of leucine and tandem repetition of a 24-amino acid segment in the primary structure of leucine-rich alpha 2-glycoprotein of human serum.
Takahashi N et al. Proc Natl Acad Sci USA, 1985; 82(7): 1906-10.
Molecular characterization and expression analysis of leucine-rich alpha2-glycoprotein, a novel marker of granulocytic differentiation.
O'Donnell LC et al. J Leukoc Biol, 2002; 72(3): 478-85.
LRG1 promotes angiogenesis by modulating endothelial TGF-β signalling.
Wang X et al. Nature, 2013; 499(7458): 306-11.
Serum leucine-rich alpha-2-glycoprotein-1 binds cytochrome c and inhibits antibody detection of this apoptotic marker in enzyme-linked immunosorbent assay.
Cummings C et al.Apoptosis, 2006; 11(7): 1121-9.
LRG-accelerated differentiation defines unique G-CSFR signaling pathways downstream of PU.1 and C/EBPepsilon that modulate neutrophil activation.
Ai J et al. J Leukoc Biol, 2008; 83(5): 1277-85.
Leucine Rich α-2 Glycoprotein: A Novel Neutrophil Granule Protein and Modulator of Myelopoiesis.
Druhan LJ et al.PLoS One, 2017; 12; 12(1): e0170261.
Elevation of a novel angiogenic factor, leucine-rich-α2-glycoprotein (LRG1), is associated with arterial stiffness, endothelial dysfunction, and peripheral arterial disease in patients with type 2 diabetes.
Pek SL et al. J Clin Endocrinol Metab, 2015; 100(4): 1586-93.
The role of TGFβ1 and LRG1 in cardiac remodeling and heart failure.
Song W et al. Biophys Rev, 2015; 7(1):91-104.
LRG1 modulates epithelial-mesenchymal transition and angiogenesis in colorectal cancer via HIF-1α activation.
Zhang J et al. J Exp Clin Cancer Res, 2016; 35:29.
Stable knockdown of LRG1 by RNA interference inhibits growth and promotes apoptosis of glioblastoma cells in vitro and in vivo.
Zhong D et al. Tumour Biol, 2015; 36(6):4271-8.
Up-regulation of the expression of leucine-rich alpha(2)-glycoprotein in hepatocytes by the mediators of acute-phase response.
Shirai R et al.Biochem Biophys Res Commun, 2009; 382(4): 776-9.
iTRAQ-based proteomic identification of leucine-rich alpha-2 glycoprotein as a novel inflammatory biomarker in autoimmune diseases.
Serada, S. et al. Ann. Rheum 2010; Dis 69, 770–774.
Leucine-rich alpha-2 glycoprotein is an innovative biomarker for psoriasis.
Nakajima H et al. J Dermatol, 2017; Sci 86, 170–174.
Leucine-rich alpha2-glycoprotein as the acute-phase reactant to detect systemic juvenile idiopathic arthritis disease activity during anti-interleukin-6 blockade therapy: A case series.
Shimizu M et al., Mod Rheumatol, 2017; 27, 833–837.
Identification of candidate diagnostic serum biomarkers for Kawasaki disease using proteomic analysis.
Kimura, Y. et al. Sci Rep, 2017; 7, 43732.
A Novel Noninvasive Appendicitis Score with a Urine Biomarker.
Yap TL. et al. J Pedriatic Surgery, 2019; 54, 1, 91–96.
Overexpression of leucine-rich alpha2-glycoprotein-1 is a prognostic marker and enhances tumor migration in gastric cancer.
Yamamoto M et al. Cancer Sci, 2017; 108, 2052-2060.
LRG-1 Promotes Pancreatic Cancer Growth and Metastasis via Modulation of the EGFR/P38 Signaling.
Xie ZB et al. J Exp & Clin Cancer Res, 2019; 38 (1), 75.
Epithelial‐mesenchymal Transition via transforming growth factor beta in Pancreatic Cancer Is Potentiated by the Inflammatory Glycoprotein Leucine-Rich Alpha-2 Glycoprotein.
Otsuru T et al. Cancer Science, 2019; cas.13918.
Validation of LRG1 as a Potential Biomarker for Detection of Epithelial Ovarian Cancer by a Blinded Study.
Wu JH et al. PloS One, 2015; 10, 3, e0121112..
Leucine-rich alpha-2-glycoprotein-1 is up-regulated in colorectal cancer and is a tumor promoter.
Zhang Q et al. OncoTargets and therapy, 2018; 11, 2745–52. 5.
Proteomic analysis of coronary sinus serum reveals leucine-rich alpha2-glycoprotein as a novel biomarker of ventricular dysfunction and heart failure.
Watson CJ et al. Circ Heart Fail, 2011; 4, 188–197.
Plasma Leucine-Rich alpha-2-Glycoprotein 1 Predicts Rapid eGFR Decline and Albuminuria Progression in Type 2 Diabetes Mellitus.
Liu JJ et al. J Clin Endocrinol Metab, 2017 ; 102, 3683–3691.
LRG1 Promotes Diabetic Kidney Disease Progression by Enhancing TGF-beta-Induced Angiogenesis.
Hong Q et al. J Am Soc Nephrol, 2019; 30, 546–562.
Brain Localization of Leucine-Rich a2-Glycoprotein and Its Role.
Nakajima M et al. Hydrocephalus, Acta Neurochirurgica Supplementum, Vol. 113, DOI: 10.1007/978-3-7091-0923-6_20.
Leucine-rich α-2-glycoprotein is a marker for idiopathic normal pressure hydrocephalus.
Nakajima M et al. Acta Neurochir (Wien). 2011 Jun;153(6):1339-46.
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Cardiovascular Disease
Ventricular dysfunction and heart failure
Proteomic analysis of coronary sinus serum reveals leucine-rich alpha2-glycoprotein as a novel biomarker of ventricular dysfunction and heart failure.
Watson CJ et al. Circ Heart Fail, 2011; 4, 188–197.
Arterial stiffness
Elevation of a novel angiogenic factor, leucine-rich-α2-glycoprotein (LRG1), is associated with arterial stiffness, endothelial dysfunction, and peripheral arterial disease in patients with type 2 diabetes.
Pek SLT et al. J Clin Endocrinol Metab 100, 158, 2015; 6–1593.
Cardiac remodeling after MI
Myeloid cell-derived LRG attenuates adverse cardiac remodeling after myocardial infarction.
Kumagai S et al. Cardiovasc Res, 2016; 109, 272–282. -
Inflammation
Rheumatoid arthritis
Leucine-rich α2 -glycoprotein as a potential biomarker for joint inflammation during anti-interleukin-6 biologic therapy in rheumatoid arthritis.
Fujimoto M. et al. Arthritis Rheumatol, 2015; 67, 2056–2060.Usefulness of serum leucine-rich alpha-2 glycoprotein as a disease activity biomarker in patients with rheumatoid arthritis.
Ha YJ et al. J Korean Med Sci, 2014; 29, 1199–1204.iTRAQ-based proteomic identification of leucine-rich alpha-2 glycoprotein as a novel inflammatory biomarker in autoimmune diseases.
Serada S et al. Ann Rheum Dis, 2010; 69, 770–774.
Psoriasis
Leucine-rich alpha-2 glycoprotein is an innovative biomarker for psoriasis.
Nakajima H et al. J Dermatol, 2017; Sci 86, 170–174.
Juvenile idiopathic arthritis
Leucine-rich alpha2-glycoprotein as the acute-phase reactant to detect systemic juvenile idiopathic arthritis disease activity during anti-interleukin-6 blockade therapy: A case series.
Shimizu M et al. Mod Rheumatol, 2017; 27, 833–837.
Kawasaki disease
Identification of candidate diagnostic serum biomarkers for Kawasaki disease using proteomic analysis.
Kimura Y et al.Sci Rep, 2017; 7, 43732.
Inflammatory bowel disease
iTRAQ-based proteomic identification of leucine-rich alpha-2 glycoprotein as a novel inflammatory biomarker in autoimmune diseases.
Serada S et al. Ann Rheum Dis, 2010; 69, 770–774.
Ulcerative colitis
Serum leucine-rich alpha-2 glycoprotein is a disease activity biomarker in ulcerative colitis.
Serada S et al. Inflamm Bowel Dis, 2012; 18, 2169–2179.Leucine-rich Alpha-2 Glycoprotein is a Serum Biomarker of Mucosal Healing in Ulcerative Colitis.
Shinzak S et al. J Crohns Colitis, 2017; 11(1): 84–91.
Appendicitis
A Novel Noninvasive Appendicitis Score with a Urine Biomarker.
Yap TL et al. J Pedriatic Surgery, 2019; 54, 1, 91–96.Novel serum and urine markers for pediatric appendicitis.
Kharbanda A Bet al. Acad Emerg Med Off J Soc Acad Emerg Med, 2012; 19, 56–62.Value of Leucine-rich alpha-2-glycoprotein-1 (LRG-1) on diagnosis of acute appendicitis in female patients with right lower-quadrant abdominal pain.
Demirci O L et al. JPMA J Pak Med Assoc, 2017; 67, 1383–1386.Detection and diagnostic value of urine leucine-rich alpha-2-glycoprotein (LRG) in children with suspected acute appendicitis.
Kentsis A et al. Ann Emerg Med, 2012; 60, 78-83. e1. -
Neurological Disorders
Parkinsons
Leucine-rich α2-glycoprotein is a novel biomarker of neurodegenerative disease in human cerebrospinal fluid and causes neurodegeneration in mouse cerebral cortex.
Miyajima M et al. PloS One 8, 2013; e74453.Brain Localization of Leucine-Rich α2-Glycoprotein and Its Role.
Nakajima M et al. Acta neurochirurgica, 2012; Supplement 113: 97–101.
Idiopathic normal pressure hydrocephalus (iNPH)
Leucine-rich α-2-glycoprotein is a marker for idiopathic normal pressure hydrocephalus.
Nakajima M et al. Acta Neurochir, 2011; 153, 1339–1346.
Meningitis (Chong PF et al., 2018)
Leucine-rich alpha-2 glycoprotein in the cerebrospinal fluid is a potential inflammatory biomarker for meningitis.
Chong PF et al. J Neurol Sci, 2018; 15;392:51-55. -
Oncology
Breast cancer
High serum levels of leucine-rich α-2 glycoprotein 1 (LRG-1) are associated with poor survival in patients with early breast cancer.
Göbel A et al. Arch Gynecol Obstet. 2024 Feb 28. Epub ahead of print
Gastric cancer
Overexpression of leucine-rich alpha2-glycoprotein-1 is a prognostic marker and enhances tumor migration in gastric cancer.
Yamamoto M et al. Cancer Sci, 2017; 108, 2052-2060.
Pancreatic cancer
LRG-1 Promotes Pancreatic Cancer Growth and Metastasis via Modulation of the EGFR/P38 Signaling.
Xie ZB et al. J Exp & Clin Cancer Res, 2019; 38 (1), 75.Epithelial‐mesenchymal Transition via transforming growth factor beta in Pancreatic Cancer Is Potentiated by the Inflammatory Glycoprotein Leucine-Rich Alpha-2 Glycoprotein.
Otsuru T et al. Cancer Science, 2019; cas.13918.Clinicopathological Significance of Leucine-Rich α2-Glycoprotein-1 in Sera of Patients With Pancreatic Cancer.
Furukawa K et al. Pancreas, 2015; 44, 93–98.A Plasma-Derived Protein-Metabolite Multiplexed Panel for Early-Stage Pancreatic Cancer.
Fahrmann JF et al. J Natl Cancer Inst, 2019; 1;111(4):372-379.Sequential Validation of Blood-Based Protein Biomarker Candidates for Early-Stage Pancreatic Cancer.
Capello M et al. J Natl Cancer Inst, 2017; 109.Diagnostic performance enhancement of pancreatic cancer using proteomic multimarker panel.
Park J et al. Oncotarget, 2018; 8, 93117–93130.
Ovarian cancer
Validation of LRG1 as a Potential Biomarker for Detection of Epithelial Ovarian Cancer by a Blinded Study.
Wu JH et al. PloS One, 2015; 10, 3, e0121112.Leucine-rich alpha-2-glycoprotein-1 is upregulated in sera and tumors of ovarian cancer patients.
Andersen J D et al. J Ovarian Res, 2010; 3, 21.Identification of O-glycosylated Proteins That Are Aberrantly Excreted in the Urine of Patients with Early Stage Ovarian Cancer.
Mu AKW et al. Int J Mol Sci, 2013; 14, 7923–7931.
Colorectal cancer
Leucine-rich alpha-2-glycoprotein-1 is up-regulated in colorectal cancer and is a tumor promoter.
Zhang Q et al. OncoTargets and therapy, 2018; 11, 2745–52.Leucine-rich alpha-2-glycoprotein-1, relevant with microvessel density, is an independent survival prognostic factor for stage III colorectal cancer patients: a retrospective analysis.
Sun DC et al. Oncotarget, 2017; 8, 66550–66558.LRG1 modulates epithelial-mesenchymal transition and angiogenesis in colorectal cancer via HIF-1α activation.
Zhang J Zhu et al. J Exp Clin Cancer Res, 2016; CR 35, 29.
Hepatocellular carcinoma
LRG1 Expression Indicates Unfavorable Clinical Outcome in Hepatocellular Carcinoma.
Wang CH et al. Oncotarget, 2015; 6, 39: 42118–29.LRG1 suppresses the migration and invasion of hepatocellular carcinoma cells.
Zhang Y et al. Med Oncol Northwood Lond Engl, 2015; 32, 146.
Endometrial carcinoma
LRG1 is an independent prognostic factor for endometrial carcinoma.
Wen SY et al. Tumour Biol. J. Int. Soc. Oncodevelopmental Biol Med, 2014; 35, 7125–7133.
Lung cancer
Identification of urine protein biomarkers with the potential for early detection of lung cancer.
Zhang H et al. Sci Rep, 2015; 5, 11805.Discovery of Lung Cancer Biomarkers by Profiling the Plasma Proteome with Monoclonal Antibody Libraries.
Guergova-Kuras, M et al. Mol Cell Proteomics, 2011; MCP 10.
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.Comparison with other human LRG ELISA assays
1. ICH Q2(R1) Validation of Analytical Procedures: Text and Methodology
2. EMEA/CHMP/EWP/192217/2009 Guideline on bioanalytical method validation
3. Bioanalytical Method Validation, Guidance for Industry, FDA, May 2018
Calibration
The leucine-rich alpha-2-glycoprotein (LRG) ELISA kit is calibrated against recombinant human LRG protein (P02750 - Uniprot ID).
Human LRG ELISA Detection Limit & Sensitivity
To determine the sensitivity of the LRG 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 LRG, with a confidence level of 99%. It is defined as the mean back-calculated concentration of standard 1 (0 pmol/l of LRG, 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 standards containing LRG, 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 LRG ELISA:
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LOD |
0.26 ng/ml |
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LLOQ |
0.5 ng/ml |
Human LRG 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 (intra-assay precision) was assessed by measuring two samples of known concentrations three times within one LRG ELISA lot by one operator.
|
ID |
n |
Mean LRG [ng/ml] |
SD [ng/ml] |
CV [%] |
|
Sample 1 |
3 |
3.9 |
0.1 |
2 |
|
Sample 2 |
3 |
31.7 |
0.8 |
3 |
In-Between-Run Precision
In-between-run presicion (intra-assay precision) was assessed by measuring two samples of known concentrations nine
times with three kits from two different LRG ELISA lots on different days by two different operators.
|
ID |
n |
Mean LRG [ng/ml] |
SD [ng/ml] |
CV [%] |
|
Sample 1 |
9 |
4.0 |
0.2 |
5 |
|
Sample 2 |
9 |
32.0 |
1.9 |
6 |
Human LRG 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 LRG ELISA was measured by adding recombinant LRG to clinical samples containing a known concentration of endogenous LRG. 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 LRG ELISA in different sample matrices.
|
% Recovery |
|||||
|
Sample matrix |
n |
+ 32 ng/ml |
+ 6.4 ng/ml |
||
|
Mean |
Range |
Mean |
Range |
||
|
Serum |
5 |
96 |
91 - 99 |
86 |
77 - 92 |
|
EDTA plasma |
5 |
89 |
82 - 95 |
80 |
65 - 89 |
|
Citrate plasma |
2 |
100 |
99 - 100 |
96 |
87 - 106 |
|
Heparın plasma |
2 |
91 |
88 - 94 |
90 |
83 - 97 |
Data showing recovery % recovery of recombinant LRG in human serum samples:
|
LRG [ng/ml] |
% Recovery |
|||||
|
Sample matrix |
ID |
Reference |
+ 32 ng/ml |
+ 6.4 ng/ml |
+ 32 ng/ml |
+ 6.4 ng/ml |
|
Serum |
s1 |
6.3 |
38.1 |
11.3 |
99 |
77 |
|
Serum |
s2 |
6.3 |
36.7 |
11.6 |
95 |
83 |
|
Serum |
s3 |
6.4 |
37.3 |
12.0 |
97 |
88 |
|
Serum |
s4 |
5.2 |
36.0 |
10.8 |
96 |
87 |
|
Serum |
s5 |
4.5 |
33.7 |
10.4 |
91 |
92 |
|
Mean |
96 |
86 |
||||
|
Min |
91 |
77 |
||||
|
Max |
99 |
92 |
||||
Data showing % recovery of recombinant LRG in human EDTA plasma samples:
|
LRG [ng/ml] |
% Recovery |
|||||
|
Sample matrix |
ID |
Reference |
+ 32 ng/ml |
+ 6.4 ng/ml |
+ 32 ng/ml |
+ 6.4 ng/ml |
|
EDTA plasma |
e1 |
5.0 |
35.5 |
10.7 |
95 |
88 |
|
EDTA plasma |
e2 |
7.1 |
33.4 |
12.1 |
82 |
79 |
|
EDTA plasma |
e3 |
5.9 |
35.2 |
11.1 |
91 |
81 |
|
EDTA plasma |
e4 |
7.1 |
33.8 |
11.3 |
83 |
65 |
|
EDTA plasma |
e5 |
3.3 |
33.0 |
9.0 |
93 |
89 |
|
Mean |
89 |
80 |
||||
|
Min |
82 |
65 |
||||
|
Max |
95 |
89 |
||||
Data showing % recovery of recombinant LRG in human heparin plasma samples:
|
LRG [ng/ml] |
% Recovery |
|||||
|
Sample matrix |
ID |
Reference |
+ 32 ng/ml |
+ 6.4 ng/ml |
+ 32 ng/ml |
+ 6.4 ng/ml |
|
Heparın plasma |
h1 |
3.5 |
33.7 |
9.7 |
94 |
97 |
|
Heparın plasma |
h2 |
6.0 |
34.0 |
11.3 |
88 |
83 |
|
|
|
|
|
Mean |
91 |
90 |
|
|
|
|
|
Min |
88 |
83 |
|
|
|
|
|
Max |
94 |
97 |
Data showing % recovery of recombinant LRG in human citrate plasma samples:
|
LRG [ng/ml] |
% Recovery |
|||||
|
Sample matrix |
ID |
Reference |
+ 32 ng/ml |
+ 6.4 ng/ml |
+ 32 ng/ml |
+ 6.4 ng/ml |
|
Citrate plasma |
c1 |
2.5 |
34.3 |
9.3 |
99 |
106 |
|
Citrate plasma |
c2 |
3.7 |
35.7 |
9.3 |
100 |
87 |
|
|
|
|
Mean |
100 |
96 |
|
|
|
|
|
Min |
99 |
87 |
|
|
|
|
|
Max |
100 |
106 |
|
Human LRG ELISA Dilution Linearity & Parallelism
Tests of dilution linearity and parallelism ensure that both, endogenous and recombinant samples containing LRG, behave in a dose-dependent manner and are not affected by matrix effects. Dilution linearity assesses the accuracy of measurements in diluted clinical 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
Dilution linearity was assessed by serially diluting human serum and plasma samples spiked with recombinant LRG in assay buffer.
The figure and table below show the mean recovery and range of serially diluted recombinant LRG in several sample matrices:
|
|
|
|
% Recovery of recombinant LRG in diluted samples |
|||||
|
Sample matrix |
n |
1+1 |
1+3 |
1+7 |
||||
|
Mean |
Range |
Mean |
Range |
Mean |
Range |
|||
|
Serum |
6 |
98 |
94 - 103 |
89 |
83 - 95 |
87 |
75 - 93 |
|
|
EDTA plasma |
6 |
102 |
96 - 110 |
101 |
91 - 106 |
95 |
91 - 102 |
|
|
Citrate plasma |
2 |
102 |
99 - 104 |
85 |
84 - 87 |
77 |
76 - 77 | |
|
Heparın plasma |
2 |
98 |
97 - 99 |
100 |
98 - 101 |
91 |
89 - 92 | |
Calculation of dilution linearity of spiked serum samples:
|
LRG [ng/ml] |
% Recovery |
||||||||
|
Sample matrix |
ID |
Reference +32 ng/ml |
ref |
1+1 |
1+3 |
1+7 |
1+1 |
1+3 |
1+7 |
|
Serum |
s1 |
38.1 |
6.3 |
18.3 |
7.9 |
3.6 |
96 |
83 |
75 |
|
Serum |
s2 |
36.7 |
6.3 |
18.0 |
7.9 |
3.9 |
98 |
86 |
85 |
|
Serum |
s3 |
37.3 |
6.4 |
17.5 |
8.4 |
4.1 |
94 |
90 |
87 |
|
Serum |
s4 |
36.0 |
5.2 |
16.9 |
7.8 |
4.0 |
94 |
87 |
89 |
|
Serum |
s5 |
33.9 |
5.8 |
17.4 |
8.0 |
3.9 |
103 |
94 |
93 |
|
Serum |
s6 |
33.7 |
4.5 |
17.2 |
8.0 |
3.8 |
102 |
95 |
91 |
|
|
Mean |
98 |
89 |
87 |
|||||
|
|
Min |
||||||||
|
|
Max |
||||||||
Calculation of dilution linearity of spiked EDTA plasma samples:
|
LRG [ng/ml] |
% Recovery |
||||||||
|
Sample matrix |
ID |
Reference +32 ng/ml |
ref |
1+1 |
1+3 |
1+7 |
1+1 |
1+3 |
1+7 |
|
EDTA plasma |
e1 |
35,5 | 5.0 | 17.1 | 8.1 | 4.1 | 96 | 91 | 92 |
|
EDTA plasma |
e2 |
33.4 | 7.1 | 18.1 | 8.9 | 3.9 | 109 | 106 | 94 |
|
EDTA plasma |
e3 |
35.2 | 5.9 | 17.5 | 8.5 | 4.2 | 99 | 97 | 95 |
|
EDTA plasma |
e4 |
33.8 |
7.1 |
16.8 |
8.9 |
4.3 |
100 |
106 |
102 |
|
EDTA plasma |
e5 |
33.0 |
3.3 |
16.0 |
8.2 |
3.8 |
97 |
99 |
91 |
|
EDTA plasma |
e6 |
31.6 |
4.9 |
17.3 |
8.4 |
3.7 |
110 |
106 |
95 |
|
|
Mean |
102 | 101 | 95 | |||||
|
|
Min |
96 | 91 | 91 | |||||
|
|
Max |
110 | 106 | 102 | |||||
Calculation of dilution linearity of spiked Heparin plasma samples:
|
LRG [ng/ml] |
% Recovery |
|||||||||
|
Sample matrix |
ID |
Reference +32 ng/ml |
ref |
1+1 |
1+3 |
1+7 |
1+1 |
1+3 |
1+7 |
|
|
Heparın plasma |
h1 |
33.7 |
3.5 |
16.6 |
8.5 |
3.9 |
99 |
101 |
92 |
|
|
Heparın plasma |
h2 |
34.0 |
6.0 |
16.5 |
8.4 |
3.8 |
97 |
98 |
89 |
|
|
|
|
|
|
|
Mean |
98 |
100 |
91 |
||
|
|
|
|
|
|
Min |
97 |
98 |
89 |
||
|
|
|
|
|
|
Max |
99 |
101 |
92 |
||
Calculation of dilution linearity of spiked Citrate plasma samples:
|
LRG [ng/ml] |
% Recovery |
|||||||||
|
Sample matrix |
ID |
Reference +32 ng/ml |
ref |
1+1 |
1+3 |
1+7 |
1+1 |
1+3 |
1+7 |
|
|
Citrate plasma |
c1 |
34.3 | 2.5 | 17.9 | 7.4 | 3.3 | 104 | 87 | 77 | |
|
Citrate plasma |
c2 |
35.7 |
3.7 |
17.7 |
7.5 |
3.4 |
99 |
84 |
76 |
|
|
|
|
|
|
|
|
Mean |
102 |
85 |
77 |
|
|
|
|
|
|
|
|
Min |
99 |
84 |
76 |
|
|
|
|
|
|
|
|
Max |
104 |
87 |
77 |
|
Parallelism
Experiment:
Parallelism was assessed by serially diluting human samples containing endogenous LRG with assay buffer.
The table below shows the mean recovery and range of serially diluted endogenous LRG in several sample matrices:
|
|
|
|
% Recovery of recombinant LRG in diluted samples |
|||||
|
Sample matrix |
n |
1+1 |
1+3 |
1+7 |
||||
|
Mean |
Range |
Mean |
Range |
Mean |
Range |
|||
|
Serum |
6 |
116 |
108 - 121 |
117 |
100 - 132 |
116 |
110 - 128 |
|
|
EDTA plasma |
6 |
107 |
101 - 114 |
101 |
93 - 111 |
96 |
87 - 104 |
|
|
Citrate plasma |
1 |
118 |
|
111 |
110 |
|||
|
Heparın plasma |
1 |
115 |
|
113 |
109 |
|||
Data showing dilution linearity of endogenous LRG in human serum samples:
|
LRG [ng/ml] |
% Recovery |
||||||||
|
Sample matrix |
ID |
Reference |
1+1 |
1+3 |
1+7 |
1+1 |
1+3 |
1+7 |
|
|
Serum |
s1 |
23.4 |
13.7 |
7.0 |
3.4 |
117 |
121 |
117 |
|
|
Serum |
s2 |
25.3 |
15.5 |
8.4 |
4.1 |
123 |
132 |
128 |
|
|
Serum |
s3 |
16.0 |
8.6 |
4.0 |
2.2 |
108 |
100 |
111 |
|
|
Serum |
s4 |
19.0 |
10.5 |
5.6 |
2.6 |
110 |
117 |
110 |
|
|
Serum |
s5 |
20.0 |
12.1 |
5.7 |
2.8 |
121 |
114 |
114 |
|
| Serum |
s6 |
23.4 |
13.7 |
7.0 |
3.4 |
117 |
121 |
117 |
|
|
|
|
|
|
Mean |
116 |
117 |
116 |
||
|
|
|
|
|
Min |
108 |
100 |
110 |
||
|
|
|
|
|
Max |
123 |
132 |
128 |
||
Data showing dilution linearity of endogenous LRG in human EDTA plasma samples:
|
LRG [ng/ml] |
% Recovery |
||||||||
|
Sample matrix |
ID |
Reference |
1+1 |
1+3 |
1+7 |
1+1 |
1+3 |
1+7 |
|
|
EDTA plasma |
e1 |
19.6 |
10.1 |
4.7 |
2.2 |
103 |
97 |
90 |
|
|
EDTA plasma |
e2 |
20.2 |
10.9 |
5.2 |
2.5 |
108 |
103 |
99 |
|
|
EDTA plasma |
e3 |
17.7 |
9.8 |
4.4 |
2.2 |
110 |
100 |
98 |
|
|
EDTA plasma |
e4 |
18.4 |
9.8 |
4.6 |
2.2 |
107 |
100 |
94 |
|
|
EDTA plasma |
e5 |
24.0 |
13.7 |
6.7 |
3.1 |
114 |
111 |
104 |
|
| EDTA plasma |
e6 |
6.9 |
3.5 |
1.7 |
0.8 |
101 |
99 |
96 |
|
|
|
|
|
|
Mean |
107 |
101 |
96 |
||
|
|
|
|
|
Min |
101 |
93 |
87 |
||
|
|
|
|
|
Max |
114 |
111 |
104 |
||
Data showing recovery of recombinant LRG in a human citrate plasma sample:
|
LRG [ng/ml] |
% Recovery |
||||||||
|
Sample matrix |
ID |
Reference |
1+1 |
1+3 |
1+7 |
1+1 |
1+3 |
1+7 |
|
|
Citrate plasma |
c1 |
19.0 |
11.2 |
5.3 |
2.6 |
118 |
111 |
110 |
|
Data showing recovery of recombinant LRG in a human heparin plasma sample:
|
LRG [ng/ml] |
% Recovery |
||||||||
|
Sample matrix |
ID |
Reference |
1+1 |
1+3 |
1+7 |
1+1 |
1+3 |
1+7 |
|
|
Heparin plasma |
H1 |
20.5 |
11.7 |
5.8 |
2.8 |
115 |
113 |
109 |
|
Characterization of the Antibodies
Epitope Mapping
Antibodies were characterized by epitope mapping of linear epitopes with microarray technology and by the determination of binding kinetics with biolayer interferometry.
The peptide-specific coating antibody binds to a linear epitope in the N-terminal region of LRG. Multiple linear epitopes recognized by the polyclonal detection antibody are distributed over the whole LRG sequence and are located in the N- and C-terminus, as well as within the leucine-rich repeats. Both antibodies bind to LRG with low dissociation rate constants.
Human LRG ELISA Specificity
The specificity of an ELISA is defined as its ability to exclusively recognize the analyte of interest.
The specificity of the LRG 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 LRG.
This assay recognizes recombinant and endogenous human LRG.
Competition of Signal
Competition experiments were carried out by pre-incubating human samples containing endogenous LRG 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 was 99%.
| LRG [ng/ml] | % Competition | |||
| Sample matrix | ID | Reference | Reference+ capture AB | |
| Serum | s1 | 5 | 0.1 | 99 |
| Serum | s2 | 7 | 0.0 | 100 |
| Serum | s3 | 20 | 0.1 | 99 |
| EDTA plasma | e1 | 16 | 0.1 | 99 |
| Citrate plasma | c1 | 15 | 0.1 | 100 |
| Heparın plasma | h1 | 13 | 0.0 | 100 |
| Mean | 99 | |||
Isoforms
There are no known isoforms of leucine-rich alpha-2-glycoprotein (LRG).
Cross-Reactivity
The LRG sequence similarity between human LRG with mouse, rat and pig is 64%, 64% and 71%, respectively.
The cross-reactivity of the human LRG ELISA with non-human samples was not tested.
Sample Stability
Serum, EDTA plasma, heparin 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
Sample Preparation
We recommend separating plasma or serum by centrifugation as soon as possible, e.g. 20 min at 2,000 x g, preferably at 4°C (2-8°C). Samples can be stored at 4°C (2-8°C) overnight. For long term storage, aliquot the acquired plasma or serum samples and store at -25°C or lower.
Freeze-Thaw Stability
The stability of endogenous leucine-rich alpha-2-glycoprotein (LRG) 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 four freeze-thaw cycles is 95%.
| Sample ID | LRG [ng/ml] | % Recovery referred to reference | |||
| Reference | 1x F/T | 5x F/T | 1x F/T | 5x F/T | |
| #s1 | 15.4 | 14.6 | 15.1 | 95 | 98 |
| #s2 | 8.1 | 7.2 | 8.7 | 89 | 107 |
| #s3 | 20.8 | 16.8 | 16.3 | 81 | 78 |
All samples should undergo a maximum of five freeze-thaw cycles.
Samples can be subjected to five freeze-thaw cycles.
Benchtop Stability
The benchtop stability of endogenous leucine-rich alpha-2-glycoprotein (LRG) was tested by comparing LRG 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 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 98%.
LRG concentrations of samples stored at -25°C (reference), at room temperature (RT) or overnight (o.n.) at 4°C:
| Sample ID | LRG [ng/ml] | % Recovery referred to reference | |||
| Reference | 3h @RT | o.n.@4°C | 3h @RT | o.n.@4°C | |
| #s1 | 15.4 | 15.2 | 16.0 | 99 | 104 |
| #s2 | 8.1 | 7.9 | 8.5 | 98 | 105 |
| #s3 | 20.8 | 19.2 | 17.7 | 92 | 85 |
| Mean | 96 | 98 | |||
Sample Values
Leucine-rich alpha-2-glycoprotein (LRG) values in apparently healthy individuals
To provide values for circulating leucine-rich alpha-2-glycoprotein (LRG), a panel of samples from apparently healthy donors was tested.
Each individual donated blood for all tested sample matrices.
|
|
|
LRG [ng/ml] |
||
|
Sample matrix |
n |
Mean |
Range |
Median |
|
Serum |
18 |
27.7 |
19.2 - 40.2 |
27.5 |
|
EDTA plasma |
22 |
28.6 |
17.3 - 42.5 |
27.9 |
|
Heparın plasma |
22 |
24.7 |
15.1 - 34.4 |
23.9 |
|
Citrate plasma |
22 |
31.8 |
18.8 - 47.3 |
31.1 |
We recommended establishing the normal range for each laboratory.
Leucine-rich alpha-2-glycoprotein (LRG) values in disease panels
In addition to samples from apparently healthy donors, panels of samples from patients with heart disease (HD), rheuma and arthrose, as well as patients with kidney diseases were tested.
Summary of the results:
|
|
|
LRG [ng/ml] |
||
|
Samples |
n |
Mean |
Range |
Median |
|
Controls |
14 |
25.0 |
16.3 - 36.7 |
26.1 |
|
HD NHYA 2 |
12 |
30.4 |
19.2 - 47.9 |
32.3 |
|
HD NHYA 2 |
14 |
32.0 |
22.6 - 48.1 |
27.8 |
|
HD NHYA 2 |
8 |
38.3 |
27.6 - 59.3 |
37.3 |
|
Apparently healthy |
18 |
27.7 |
19.2 - 40.2 |
27.5 |
|
Rheuma cohort |
18 |
53.5 |
33.0 - 79.0 |
51.0 |
|
Arthrose cohort |
16 |
39 |
12.0 - 89.0 |
33.0 |
|
Kidney disease |
16 |
54.7 |
36.0 - 90.0 |
47.0 |
Matrix Comparison
To assess whether all tested matrices behave the same way in the concentrations of leucine-rich alpha-2-glycoprotein (LRG) were measured in serum, EDTA, heparın, and citrate plasma samples prepared from 10 apparently healthy donors. Each individual donated blood in all tested sample matrices.
Data and graph for apparently healthy donors are shown below:
|
|
LRG [ng/ml] | % CV | |||
| Donor ID | Serum | EDTA plasma | Citrate plasma | Heparın plasma | All matrices |
| #1 | 40.2 | 35.6 | 32.1 | 33.9 | 8 |
| #2 | 29.5 | 29.9 | 26.0 | 38.8 | 15 |
| #3 | 31.6 | 30.5 | 25.1 | 35.0 | 12 |
| #4 | 19.7 | 17.3 | 16.7 | 18.8 | 7 |
| #5 | 37.2 | 42.5 | 33.5 | 44.5 | 11 |
| #6 | 32.9 | 23.9 | 22.2 | 28.6 | 10 |
| #7 | 37.4 | 36.8 | 30.4 | 47.3 | 16 |
| #8 | 31.2 | 32.0 | 27.3 | 35.1 | 9 |
| #9 | 27.7 | 20.6 | 19.5 | 22.8 | 14 |
| #10 | 27.3 | 31.0 | 21.6 | 29.2 | 13 |
| Mean | 11 | ||||
Figure showing matrix comparison of LRG sample concentrations between serum, EDTA plasma, heparın plasma, and citrate plasma in an apparently healthy cohort (n=10).
Comparison with other human LRG ELISA assays
Assay characteristics of different human LRG ELISA assays
| BIOMEDICA |
Another MANUFACTURER |
|
| Method |
Sandwich ELISA |
Sandwich ELISA |
|
Sample type |
Serum, EDTA plasma, citrate plasma, heparin plasma (urine and cell culture protocol available) |
Plasma, serum, CSF, urine |
|
Sample volume |
100 µl pre-diluted sample / well |
100 µl pre-diluted sample / well |
|
Assay time |
2 h / 1 h / 30 min |
Overnight / 30 min / 30 min |
|
Assay range |
0 – 64 ng/ml (0 / 2 / 4 / 8 / 16 / 32 / 64) Assay range optimized for clinical samples, no additional testing required. Pre-dilution of samples 1:4000. |
1.56 – 100 ng/ml (pre-dilution of samples 1:2000 -1:10000) |
|
Sensitivity |
LOD: 0.26 ng/ml; LLOQ: 0.5 ng/ml |
0.17 ng/ml |
|
Specificity |
Endogenous and recombinant human LRG 100%. |
Human LRG 100% |
|
Antibodies |
Epitope-mapped antibodies Capture antibody: polyclonal sheep anti-human LRG antibody Detection antibody: polyclonal sheep anti-human LRG antibody, HRP-labeled Target antigen: human LRG (leucine-rich alpha-2-glycoprotein) |
Capture antibody: polyclonal rabbit anti-human LRG antibody Detection antibody: polyclonal rabbit anti-human LRG antibody, HRP-labeled Target antigen: human LRG (leucine-rich alpha-2-glycoprotein) |
|
Standard matrix |
Human serum matrix containing recombinant human LRG 7 ready to use standards |
Matrix not indicated 1 stock standard vial containing recombinant human LRG |
|
Values of apparently healthy samples |
Serum mean (n=18): 27.5 µg/ml Plasma mean (n=22): 27.9 µg/ml |
Not indicated |
|
Controls |
2 controls (high and low) included | Not indicated |
|
Validation |
According to FDA/ICH/EMEA guidelines |
Not indicated |
|
Use |
RUO | RUO |
Comparison of human sample concentrations measured with different LRG ELISA Assays
The Biomedica LRG ELISA (Cat. No. BI-LRG) was compared with the an ELISA kit from another manufacturer. The same panel of samples, consisting of 52 samples (healthy and diseased), was tested.
Correlation of LRG values: measured with two LRG ELISA assays - Biomedica versus another manufacturer in 52 samples (healthy + diseased)
|
Pearson coefficient |
0.85 |
|
P value |
0.00001 |
|
P value summary |
**** |
Conclusion:
- Serum and plasma LRG concentrations are approximately 3-5 fold lower in the Biomedica assay than when measured with the assay from another manufacturer.
- Good correlation between both assays – Pearson correlation coefficient R = 0.85, p < 0.00001.
Table showing human LRG concentrations measured with the Biomedica LRG ELISA and an LRG ELISA assay from another manufacturer:
|
ALL SAMPLES |
|||
|
n = 52 |
|||
|
Sample ID |
Biomedica (BI-LRG) |
Other (JP27769) |
|
| Cohorts |
LRG, µg/ml |
LRG, µg/ml |
|
| Apparently healthy (AH) samples | AH1 | 33 | 147 |
| AH2 | 29 | 104 | |
| AH3 | 25 | 138 | |
| AH4 | 33 | 130 | |
| AH5 | 33 | 213 | |
| AH6 | 23 | 112 | |
| AH7 | 27 | 158 | |
| AH8 | 25 | 83 | |
| AH9 | 33 | 112 | |
| AH10 | 28 | 65 | |
| AH11 | 29 | 90 | |
|
Nephrology (N) samples |
N12 | 43 | 157 |
| N13 | 64 | 183 | |
| N14 | 79 | 436 | |
| N15 | 40 | 177 | |
| N16 | 46 | 159 | |
| N17 | 42 | 191 | |
| N18 | 55 | 275 | |
| N19 | 44 | 150 | |
|
Unspecific hospital panel (UHP) samples |
UHP20 | 46 | 63 |
| UHP21 | 38 | 139 | |
| UHP22 | 42 | 131 | |
| UHP23 | 69 | 526 | |
| UHP24 | 28 | 56 | |
| UHP25 | 38 | 63 | |
| UHP26 | 23 | 31 | |
| UHP27 | 28 | 79 | |
| UHP28 | 64 | 195 | |
| UHP29 | 32 | 53 | |
| UHP30 | 80 | 416 | |
|
Cardiology (C) samples |
C31 | 33 | 117 |
| C32 | 30 | 124 | |
| C33 | 44 | 138 | |
| C34 | 29 | 59 | |
| C35 | 74 | 266 | |
| C36 | 27 | 69 | |
| C37 | 48 | 133 | |
| C38 | 55 | 309 | |
| C39 | 47 | 284 | |
| C40 | 69 | 384 | |
| C41 | 38 | 164 | |
| C42 | 35 | 88 | |
| C43 | 42 | 164 | |
| C44 | 30 | 86 | |
| C45 | 26 | 91 | |
|
Rheuma (R) samples |
R46 | 59 | 333 |
| R47 | 53 | 284 | |
| R48 | 70 | 373 | |
| R49 | 39 | 161 | |
| R50 | 38 | 192 | |
| R51 | 53 | 283 | |
| R52 | 45 | 192 | |
| Pearson | 0.85 | ||
|
p value |
< 0.00001 |
||
Novel ELISA for the quantification of human LRG..
NOVEL ELISA for the quantification of human LRG.
