Human Fibronectin ELISA Kit

Name: Human Fibronectin ELISA Kit
SKU: DEIA1497
Rating: 5 (1 reviews)

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

COA

SDS

Datasheet

Summary

Sample

serum, plasma and cell culture supernatants

Species Reactivity

Human

Detection Method

sELISA

Intended Use

For the quantitative determination of human Fibronectin concentrations in cell culture supernates, cell lysates, tissue lysates, serum, plasma, saliva, urine, and human milk.

Contents of Kit

1. Human Fibronectin Microplate. 96 well polystyrene microplate (12 strips of 8 wells) coated with a monoclonal antibody speciffc for human Fibronectin. STORAGE OF OPENED/RECONSTITUTED MATERIAL: Return unused wells to the foil pouch containing the desiccant pack. Reseal along entire edge of the zip-seal. May be stored for up to 1 month at 2-8°C.*
2. Human Fibronectin Standard. 2 vials of human Fibronectin in a buffered protein base with preservatives; lyophilized. Refer to the vial label for reconstitution volume. Note: Human sourced material. See Precautions section. STORAGE OF OPENED/RECONSTITUTED MATERIAL: Use a new standard for each assay. Discard after use.
3. Human Fibronectin Conjugate. 21 mL of a polyclonal antibody specific for human Fibronectin 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. 11 mL of a buffered protein base with preservatives. May contain a precipitate. Warm to room temperature, and mix gently to dissolve. If the precipitate does not completely dissolve, mix well during use. STORAGE OF OPENED/RECONSTITUTED MATERIAL: May be stored for up to 1 month at 2-8 °C.*
5. Calibrator Diluent. 21 mL of a buffered protein base with preservatives. Use diluted 1:10 in this assay. STORAGE OF OPENED/RECONSTITUTED MATERIAL: May be stored for up to 1 month at 2-8 °C.*
6. Wash Buffer Concentrate: 21 mL of a 25-fold solution of buffered surfactant with preservative. 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: 12 mL 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: 12 mL of stabilized chromogen (tetramethylbenzidine). STORAGE OF OPENED/RECONSTITUTED MATERIAL: May be stored for up to 1 month at 2-8 °C.*
9. Stop Solution: 6 mL of 2 N sulfuric acid. STORAGE OF OPENED/RECONSTITUTED MATERIAL: May be stored for up to 1 month at 2-8 °C.*
10. Plate Sealers: 4 adhesive strips.
* 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 twenty separate assays to assess inter-assay precision. Assays were performed by at least three technicians using two lots of components

Detection Range

Twenty-eight assays were evaluated and the minimum detectable dose (MDD) of human Fibronectin ranged from 0.062-0.579 ng/mL. The mean MDD was 0.187 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

Fibronectin (FN) is a large modular glycoprotein that is found as a polymeric ffbrillar network in the extracellular matrix (ECM) and as soluble disulffde-linked dimeric protomers in plasma and other body fluids. Fibronectin is a ligand for many molecules including fibrin, heparin, chondroitin sulfate, collagen/gelatin, various integrins and growth factors, myocilin, and Apolipoprotein A. It is involved in multiple cellular processes such as cell adhesion/migration, blood clotting, morphogenesis, tissue repair, and cell signaling. Fibronectin functions are mediated by the insoluble polymeric ffbrils. Conversion of soluble Fibronectin to Fibronectin fibrils in the ECM is initiated by binding to cell surface integrins, resulting in exposure of cryptic epitopes necessary for polymerization. Mature human Fibronectin shares 88% aa sequence identity with mouse and rat Fibronectin.
Fibronectin is made up of three types of homologous structural motifs termed FN type I, type II, and type III repeats. Alternative splicing generates multiple isoforms of Fibronectin which may have insertions of extra type III domains (EDA and EDB) or alteration of the type III connecting segment (IIICS). Differential splicing within the IIICS domain determines the presence of CS1 and CS2 sequences and the sensitivity to proteases. The IIICS domain contains two sites (CS1 and CS2) that interact with Integrin α4β1. The CS1 sequence is not accessible in full length Fibronectin but is exposed by protease digestion, thereby enabling cell adhesion via Integrin α4β1. A fragment from the ffrst type III repeat, known as Anastellin, binds to Fibronectin and induces the formation of high molecular weight disulffde-linked superffbronectin (sFN). sFN exhibits increased protease sensitivity and cellular adhesiveness compared to Fibronectin. Anastellin acts in vivo to inhbit angiogenesis, tumor growth, and metastasis.

Standard Curve

This standard curve is provided for demonstration only. A standard curve should be generated for each set of samples assayed.

Citations

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Documents

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QA & Reviews

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References

Expression kinetics of human periodontal ligament fibroblasts in the early phases of orthodontic tooth movement

JOURNAL OF OROFACIAL ORTHOPEDICS-FORTSCHRITTE DER KIEFERORTHOPADIE

Authors: Schroeder, Agnes; Bauer, Kathrin; Spanier, Gerrit; Proff, Peter; Wolf, Michael; Kirschneck, Christian

Abstract

Purpose Human periodontal ligament (hPDL) fibroblasts play a crucial mediating role in orthodontic tooth movement (OTM). In this study, we investigated the expression kinetics of genes associated with OTM in its early phase to obtain better insight into the timing and regulation of molecular and cellular signalling and transformation processes occurring in compressive areas of the periodontal ligament during OTM. Methods Adherent hPDL fibroblasts were stimulated with physiological orthodontic compressive forces of 2 g/cm(2) for 24, 48, 72, and 96 h under cell culture conditions. At each time point, we quantified relative gene expression of genes involved in bone remodelling (ALPL), inflammation (COX2, IL-6), extracellular matrix reorganization (COL1A2, P4HA1, FN1, MMP8) and angiogenesis (VEGF-A) by means of RT-qPCR as well as protein expression of osteoclastogenesis-regulating RANK-L and OPG relative to pressure-untreated controls incubated for corresponding time periods. In addition, coculture experiments with osteoclast precursor cells were performed to determine the extent of hPDL-fibroblast-mediated osteoclastogenesis (TRAP staining). Results As primary response to compressive forces within 24 h, we observed an induction of genes associated with angiogenesis, inflammation, osteoblastogenesis, and the remodelling of the extracellular matrix, with RANK-L expression at first slightly inhibited and only increased after 48 h. Major hPDL-mediated osteoclastogenesis was observed after 72 h with minor, non-RANK-L-dependent osteoclastogenesis occurring as early as 24 h after compressive force application. Conclusions hPDL fibroblasts seem to play a major mediating role in the early phase of OTM with a differentiated, time-dependent regulation and expression pattern of cytokines and other mediators.

The contribution of chronic intermittent hypoxia to OSAHS: From the perspective of serum extracellular microvesicle proteins

METABOLISM-CLINICAL AND EXPERIMENTAL

Authors: Zhang, Huina; Yang, Fan; Guo, Yichen; Wang, Li; Fang, Fang; Wu, Hao; Nie, Shaoping; Wang, Yifan; Fung, Man-Lung; Huang, Yu; Deng, Haiteng; Qin, Yanwen; Ma, Xinliang; Wei, Yongxiang

Abstract

Objective: Obstructive sleep apnea hypopnea syndrome (OSAHS) is an independent risk factor for many clinical complications. However, how OSAHS cause multiple organ injury and initiate inter-organ communication remains unclear. Moreover, despite it is well-recognized that chronic intermittent hypoxia (CIH) is a main feature of OSAHS, specific contribution of CIH to overall OSAHS-initiated pathological complications remains unclear. This study aimed to use an unbiased proteomic approach to determine whether OSAHS alters protein profiles of serum extracellular microvesicles (SEMVs) and how CIH contributes to such alterations. Methods: Tandem mass tag (TMT)-labeled quantitative proteomics assay was used to compare the differentially expressed proteins (DEPs) in SEMVs of OSAHS patients and non-OSAHS subjects. To evaluate the contribution of CIH to OSAHS, CIH rodent model was constructed and the same comparative proteomics study was performed in SEMVs from CIH and normoxia rats. The similarity and disparity of DEPs and DEPs-related functions predicted by bioinformatics tools were compared in above-mentioned two models, and several DEPs were selected and further verified by ELISA or Western blotting. Results: TMT-labeled quantitative proteomics assay unravels 32 DEPs in OSAHS patient SEMVs from a total of 560 human SEMV proteins identified. Four DEPs, namely C-reactive protein (CRP), Haptoglobin (HP),Fibronectin (FN1) and Platelet factor 4 (PF4), were further verified by ELISA and three of them (CRP, FN1 and Hp) showed significant difference in expression level between OSAHS and non-OSAHS groups. In SEMVs of rat CIH model, 121 DEPs out of 723 proteins were identified. By comparing the DEPs identified from the two models. 3 proteins (CRP and FN1 and F13a1) were found identical with the same alteration pattern (CRP was upregulated, FN1 and Fl3a1 were downregulated) in SEMVs from OSAHS patients and CIH rats, which were further verified by Western blotting. Computational functional analysis further revealed the common and distinct DEP-involved pathways under OSAHS or CIH status. Conclusions: This study provides the first evidence that OSAHS causes significant alteration in SEMV protein composition, which may contribute to OSAHS-triggered multiple organ injury and organ-to-organ communication. Moreover, we have demonstrated that CIH is the primary contributor for increased inflammatory protein expression in SEMV. As CRP is being increasingly recognized not only as a marker but also a mediator of inflammatory response to tissue injury, increased SEMV CRP in CIH/OSAHS may play an important role in OSAHSinduced tissue injury, suggesting SEMV CRP might be a therapeutic target against OSAHS-related complications. (C) 2018 Elsevier Inc. All rights reserved.