Human Osteopontin (OPN) ELISA Kit

Name: Human Osteopontin (OPN) ELISA Kit
SKU: DEIA2696
Rating: 5 (1 reviews)

Regulatory status: For research use only, not for use in diagnostic procedures.

COA

SDS

Datasheet

Summary

Sample

Cell culture supernates, plasma, urine, and human milk

Species Reactivity

Human

Detection Method

sELISA

Intended Use

For the quantitative determination of human Osteopontin (OPN) concentrations in cell culture supernates, plasma, urine, and human milk.

Contents of Kit

1. Human OPN Microplate, 96 well polystyrene microplate (12 strips of 8 wells) coated with a monoclonal antibody specific for human OPN. Return unused wells to the foil pouch containing the desiccant pack. Reseal along entire edge of the zip-seal. STORAGE OF OPENED/RECONSTITUTED MATERIAL: May be stored for up to 1 month at 2-8 °C.*
2. Human OPN Standard, 1 vial. Recombinant human OPN in a buffered protein base with preservatives. lyophilized. Refer
to the vial label for reconstitution volume. STORAGE OF OPENED/RECONSTITUTED MATERIAL: Aliquot and store at ≤ -20 °C for up to 1 month in a manual defrost freezer.* Avoid repeated freeze-thaw cycles.
3. Human OPN Conjugate, 1 vial. 21 mL/vial of a polyclonal antibody speciffc for human OPN conjugated to horseradish peroxidase with preservatives. STORAGE OF OPENED/RECONSTITUTED MATERIAL: May be stored for up to 1 month at 2-8 °C.*
4. Assay Diluent, 1 via. 11 mL/vial of a buffered protein base with preservatives. May contain a precipitate. Mix well before and during use. STORAGE OF OPENED/RECONSTITUTED MATERIAL: May be stored for up to 1 month at 2-8 °C.*
5. Calibrator Diluent, 1 vial. 21 mL/vial of a buffered protein base with preservatives. STORAGE OF OPENED/RECONSTITUTED MATERIAL: May be stored for up to 1 month at 2-8 °C.*
6. Wash Buffer Concentrate, 1 vial. 21 mL/vial of a 25-fold concentrated solution of buffered surfactant with preservatives. May turn yellow over time. STORAGE OF OPENED/RECONSTITUTED MATERIAL: May be stored for up to 1 month at 2-8 °C.*
7. Color Reagent A, 1 vial. 12 mL/vial of stabilized hydrogen peroxide. STORAGE OF OPENED/RECONSTITUTED MATERIAL: May be stored for up to 1 month at 2-8 °C.*
8. Color Reagent B, 1 vial. 12 mL/vial of stabilized chromogen (tetramethylbenzidine). STORAGE OF OPENED/RECONSTITUTED MATERIAL: May be stored for up to 1 month at 2-8 °C.*
9. Stop Solution, 1 vial, 6 mL/vial of 2 N sulfuric acid. STORAGE OF OPENED/RECONSTITUTED MATERIAL: May be stored for up to 1 month at 2-8 °C.*
* Provided this is within the expiration date of the kit.

Storage

Store the unopened kit at 2-8 °C. Do not use past kit expiration date.

Precision

Intra-Assay Precision (Precision within an assay)
Three samples of known concentration were tested twenty times on one plate to assess intra-assay precision.
Inter-Assay Precision (Precision between assays)
Three samples of known concentration were tested in forty separate assays to assess inter-assay precision. Assays were performed by at least three technicians using two lots of components.

Sensitivity

Fifty-seven assays were evaluated and the minimum detectable dose (MDD) of human OPN ranged from 0.006-0.024 ng/mL. The mean MDD was 0.011 ng/mL.
The MDD was determined by adding two standard deviations to the mean O.D. value of twenty zero standard replicates and calculating the corresponding concentration.

General Description

Osteopontin (OPN), also known as early T lymphocyte activation 1 (Eta-1), is a secreted multifunctional glyco- phosphoprotein with roles in bone metabolism, immune regulation, tissue remodeling, cell survival, and tumor progression. Gene structure and chromosomal location identify OPN as a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family that also includes bone sialoprotein (BSP), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP), enamelin (ENAM), and matrix extracellular phosphoglycoprotein (MEPE). Human OPN is synthesized as a 314 amino acid (aa) precursor protein with a predicted 16 aa signal peptide and a highly unusual mature protein sequence, containing 75 acidic aa and 33 potential Ser/Thr phosphorylation sites. Although the predicted molecular weight of OPN is 33 kDa, phosphorylation and N- and O-glycosylation may allow it to appear as large as 75 kDa. Variability in post-translational modiffcations can inffuence the activity of OPN. OPN contains a classic integrin binding site (RGD) that binds integrins αvβ1, αvβ3, αvβ5, α5β1, and α8β1. MMP-3, MMP-7, or thrombin cleavage separates the integrin-binding domain in the N-terminal fragment and the C-terminal CD44 binding domain. Proteolytic cleavage enhances OPN adhesion by revealing an additional adhesion site (SVVYGLR in the human protein) recognized by integrins α4β1, α4β1, and α9β1.
Osteopontin, meaning "bone bridging", is highly expressed in mineralized tissues. It is also expressed in other tissues including cartilage, kidney, vascular tissues, activated macrophages, lymphocytes, and epithelia. In addition to being incorporated in the matrix of mineralized connective tissues, secreted OPN is found in various biological fluids including blood, milk, urine, and seminal ffuid. A portion of cell expressed OPN is also retained intracellularly. In vitro, OPN stimulates the adhesion of osteoclasts to bone, and bone resorption is blocked by inhibition of this interaction. Knockout mice have outwardly normal bone development but exhibit deffcient postnatal bone resorption in several contexts, supporting a role for OPN in osteoclast function. In kidney epithelia, OPN is upregulated by high concentrations of oxalate and inhibits calcium oxalate crystal nucleation and growth. In endothelial and smooth muscle cells, OPN is upregulated by high phosphate concentration and during atherosclerosis; binding of OPN to hydroxyapatite inhibits calciffcation of blood vessels and heart valves.
OPN expression by macrophages and T cells is upregulated by inffammatory mediators including LPS, NO, IL-1β, and TNF-α. OPN regulates macrophage differentiation and recruitment. It also functions as a chemotactic factor and co-stimulator of T cells and may act as a Th1 cytokine, stimulating IL-12 production. OPN knockout mice exhibit deffcient Th1 responses and are susceptible to bacterial and viral infection. OPN production by macrophages is upregulated at sites of tissue remodeling including the placenta, endometrium, and myocardium post-infarction. OPN is expressed by many tumor types and plays a role in tumor progression and metastasis. In cancer patients, increased serum OPN concentration has been associated with increased tumor burden. Binding of OPN to speciffc variants of the hyaluronan receptor CD44, possibly mediated by integrins, can stimulate cell migration and metastatic potential of tumors. OPN may also be upregulated during autoimmune processes such as rheumatoid arthritis or in granulomatous lesions in response to infections such as tuberculosis.

Standard Curve

Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density (O.D.).
Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the human OPN concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data.
If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.

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Documents

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References

Comprehensive analysis of gene expression profiles associated with proliferative diabetic retinopathy

EXPERIMENTAL AND THERAPEUTIC MEDICINE

Authors: Gong, Meng-Ting; Li, Wen-Xing; Zhang, Qing; Lv, Wen-Wen; He, Zheng-Hong; Zhou, Shu-Li; Zhang, Hui; Wang, Jing; He, Kan

Abstract

Proliferative diabetic retinopathy (PDR) is characterized by neovascularization on the surface of the retina or the optic disc, which is associated with environmental and genetic factors. However, its regulatory mechanism remains to be fully elucidated, particularly at a multiomics level. In the present study, a comprehensive analysis was performed of the gene expression profile of fibrovascular membranes (FVMs) associated with PDR, including an analysis of differentially expressed genes, functional enrichment, and regulation of transcription factors (TFs). As a result, novel marker genes of PDR were identified, including flavin containing monooxygenase 2. Furthermore, several common or specific genes, pathways and TFs have been recovered for active and inactive FVMs. In the present study, lymphoid enhancer binding factor 1 (LEF1) was identified as an upregulator in active and inactive FVMs, which is capable of activating or repressing target genes, including claudin 2, secreted phosphoprotein 1 (SPP1), and aristaless-like homeobox 4. It was demonstrated that the Wnt/-catenin effector LEF1 regulating SPP1 is potentially important in PDR. The results of the present study may provide novel insights into the molecular mechanisms underlying the pathophysiology of PDR.

Seven immune-related genes prognostic power and correlation with tumor-infiltrating immune cells in hepatocellular carcinoma

CANCER MEDICINE

Authors: Liu, Tiantian; Wu, Hao; Qi, Jianni; Qin, Chengyong; Zhu, Qiang

Abstract

Background Given poor prognosis and the lack of efficient therapy for advanced hepatocellular carcinoma, immunotherapy has emerged as an increasingly important role. However, there were few reports on the correlation between immune-related genes and HCC. The purpose of this study is to construct a novel immune-related gene-based prognostic signature for HCC and to explore the potential mechanisms. Methods We organized expression data of 374 HCC samples and 50 nontumor samples from TCGA database. A robust signature was constructed by Cox regression analysis based on the immune-related genes, which were filtered by differential genes analysis and Cox regression analysis. Then, the correlation analysis between the signature and clinical characteristics was conducted. And the signature was validated in ICGC database. Furthermore, the relationships between immune cell infiltration and the signature were explored by bioinformatics analysis. Results Seven genes-based model (Risk score = BIRC5 * 0.0238 + FOS * 0.0055 + DKK1 * 0.0085 + FGF13 * 0.3432 + IL11 * 0.0135 + IL17D * 0.0878 + SPP1 * 0.0003) was constructed eventually and it was proved to be an independent prognostic factor for HCC patients. The signature-calculated risk scores were shown to be positively correlated with the infiltration of these five immune cells, including macrophages, neutrophils, CD8+T, dendritic, and B cells. And the results suggested that high amplication of BIRC5, FGF13, IL11, IL17D, and SPP1 were more likely correlated with immune cell infiltration. Finally, PPI network, TFs-based regulatory network and gene enrichment plots were performed to show potential molecular mechanisms. Conclusion We construct a robust immune-related gene-based prognostic signature with seven genes and explore potential mechanisms about it, which may contribute to the immunotherapy research for HCC.