CD14 Kit ELISA

N° du produit ABIN5664985

Détail du produit CD14 Kit ELISA

Antigène: CD14

Reactivité: Souris

Méthode de détection: Colorimetric

Type de méthode: Sandwich ELISA

Gamme de detection: 3 ng/mL - 50 ng/mL

Seuil minimal de détection: 3 ng/mL

Application: ELISA

Fonction: The mouse CD14 kit has been developed for the quantitative measurement of natural and recombinant mouse CD14 in serum, plasma and culture medium.

Type d'échantillon: Plasma, Serum, Urine

Analytical Method: Quantitative

Specificité: No reaction with human, rabbit, horse, pork, bovine or rat CD14 antibodies

Sensibilité: Normal CD14 range in healthy mice: (0.3 - 6ug/ml) n= 10, Interassay variation coefficient: 9.8% till 17.8 depending of concentration, Intraassay variation coefficient: 6.9%, n=10 serum samples, Effective range: 5 -50 ng/ml

Attributs du produit: A mixture of monoclonal antibodies specific for mouse sCD14 is coated at modules. In the first step the precoated modules will be incubated with the antigen (standard or sample) together with a POD-labelled antibody specific for mouse sCD14. During this incubation, mouse CD14 is captured by solid bound antibody. Unbound material present in the sample is removed by washing. Revelation step includes TMB as chromogen. The enzyme reaction is stopped by the addition of sulphuric acid (O.25M) and the absorption at 450 nm is measured with a spectrophotometer. A standard curve is obtained by plotting the absorptions versus the corresponding concentrations of the known standards. The mouseCD14 concentration of samples with unknown concentrations, which are run concurrently with the standards, can be determined from the standard curve. The dilution step of sample with second antibody is incorporated in standard curve.

Ingrédients: 1x Precoated ELISA modules, detecting antibody (POD-labelled monoclonal antibody), Mouse CD14-standard, Reference serum, PBS, Dilution Buffer ,Tween 20, Stopping solution, Substrate solution

Matériel non inclus: Orbital shaker, Micro plate reader for measurement absorbance at 450 /620 nm, Precision pipettes with disposable tips, 10-1000 ul adjustable multiwell pipettes

Information d'application

Indications d'application: Preparation of reagents: Use all reagents for assay at room temperature. (A) Wash Buffer: Dissolve 1 tablet PBS (vial 5) in 200 ml distilled water-add 100 ul Tween 20 (vial 7) , store at room temperature. Prepared wash buffer is stable for 4 weeks at refrigerator. (B) PBS: (Phosphate balanced salt solution) Dilute 1 tablet of vial 5 in 200 ml distilled water. Store and use at room temperature. (C) Sample dilution buffer: Dissolve content of vial 6 with 50 ml PBS (Buffer C) and add 50ul Tween 20 from vial 7. Use buffer at room temperature. This buffer is 1-2 weeks stable at 4oC. (D) Detecting ab dilution buffer: Add whole content of the vial10 to 10ml PBS (Buffer B). Prepare just before use. Store remaining buffer after reconstitution at -20oC. (E) Detection antibody: Add 500 ul detecting ab dilution buffer (D) to vial 2, mix carefully and than dissolve 250ul of this vial 2 in 8 ml dilution buffer for detecting ab (D). Prepare just before use. (F) Reference mouse serum lyophilized: Add 10ul distilled water to vial 4 for solubility and secondly dilute the whole content of vial 4 with 1490 ul dilution buffer for samples in a new vial (C). Pipette 50ul/well. This represents a dilution of 1:150. The mCD14 content of this reference serum is 2.7

Volume d'échantillon: 100 μL

Durée du test: 2 h

Plaque: Pre-coated

Protocole: The sCD14 Kit is a solid phase sandwich Enzyme-Linked-Immunosorbent-Assay (ELISA). A mixture of monoclonal antibodies specific for mouse sCD14 is coated at modules. In the first step the precoated modules will be incubated with the antigen (standard or sample) together with a POD-labelled antibody specific for mouse sCD14. During this incubation, mouse CD14 is captured by solid bound antibody. Unbound material present in the sample is removed by washing. Revelation step includes TMB as chromogen. The enzyme reaction is stopped by the addition of sulphuric acid (O.25M) and the absorption at 450 nm is measured with a spectrophotometer. A standard curve is obtained by plotting the absorptions versus the corresponding concentrations of the known standards. The mouseCD14 concentration of samples with unknown concentrations, which are run concurrently with the standards, can be determined from the standard curve. The dilution step of sample with second antibody is incorporated in standard curve.

Préparation des réactifs:

A Wash Buffer: Dissolve 1 tablet PBS (vial 5) in 200 mL distilled water-add 100 μL Tween 20 (vial 7) , store at room temperature. Prepared wash buffer is stable for 4 weeks at refrigerator. B PBS: (Phosphate balanced salt solution) Dilute 1 tablet of vial 5 in 200 mL distilled water. Store and use at room temperature. C Sample dilution buffer: Dissolve content of vial 6 with 50 mL PBS (Buffer C) and add 50 μL Tween 20 from vial 7. Use buffer at room temperature. This buffer is 1-2 weeks stable at 4°C. D Detecting ab dilution buffer: Add whole content of the vial10 to 13 mL PBS (Buffer B). Prepare just before use. Store remaining buffer after reconstitution at -20°C E Detecting antibody: Firstly add 500 μL dilution buffer for detecting antibody (D) to vial 2 for solubility (=0.23 μg/mL IgG) , mix carefully and secondly add the 250 μL of this vial 2 in a new vial containing 12 mL of D. Prepare just before use. I F Reference mouse serum lyophilized: Add 10 μL distilled water to vial 4 for solubility and secondly dilute the whole content of vial 4 with 2990 μL dilution buffer for samples (C) in a new vial. Pipette 50 μL/well. This represents a dilution of 1:300. The mCD14 content of this reference serum is 4.3 ± 2.2 μg/mL. G Mouse CD14-standard lyophilized: Firstly pipette 30 μL distilled water to the vial 3 for reconstitution and secondly pipette the whole reconstituted content of vial 3 in a new vial with 770 μL sample dilution buffer (C) and mix carefully. This is vial a. For standard curve prepare vial b-f. Prepare just before use.

Prélèvement de l'échantillon: Serum, plasma and other human LBP containing solutions are suitable for use in the test.Samples containing a visible precipitate must be clarified prior to use in the assay. Lipemic and haemolysed probes are not possible. Samples should be frozen at -20°C for a long-term storage.

Préparation de l'échantillon:

The mouseCD14 concentration of samples with unknown concentrations, which are run concurrently with the standards, can be determined from the standard curve. The dilution step of sample with second antibody is incorporated in standard curve.

Procédure de l'essai:

ASSAY PROCEDURE FOR ""ONE STEP"" ASSAY Let all reagents reach room temperature and mix thoroughly 1. Samples and detecting antibody Add 50 μL of standards (G) vial b-f (50, 25, 12.5, 6.25, 3.12 ng/mL), reference (F) or diluted samples in duplicate into the corresponding wells as well as 50 μL detecting antibody (E). Incubate for 1.5 hours at room temperature with shaking. 2. 3 x washing with 250 μL Wash Buffer/well (A). Remove the Wash Buffer carefully after each wash. 3. Substrate Add 100 μL Substrate (vial 9) to each well. Incubate 14 ± 1 min at room temperature without shaking in the dark up to strong colour change to blue is visible. 4. Stopping Add 100 μL stopping solution (vial 8) to each well. Tape plate gently to mix, now colour is yellow 5. Read absorbance of wells at 450 nm (reference wave length 620).

Calcul des résultats:

Remediate the optical density (OD) with blank, calculate the mean of corrected OD of standard duplicates, reference serum and the samples. Design a standard curve by plotting the OD means of standards (a-f) (y-axis) and the LBP concentration (x-axis). Calculate the LBP concentration from the mean OD of samples from the standard curve and multiply with dilution factor.

Précision du teste: interassay vc 10%, intra assay vc 6%

Restrictions: For Research Use only

Stockage

Agent conservateur: Without preservative

Précaution d'utilisation: protect your eyes

Stock: 4 °C

Stockage commentaire: Short time store at 2-8°C, Long time storage of lyophilized reference serum and standard at -20°C or -80°C, detecting monoclonal can be stored at 2-8°C

Références pour CD14 Kit ELISA (ABIN5664985)

Alaish, Smith, Timmons, Greenspon, Eyvazzadeh, Murphy, Shea-Donahue, Cirimotich, Mongodin, Zhao, Fasano, Nataro, Cross: "Gut microbiota, tight junction protein expression, intestinal resistance, bacterial translocation and mortality following cholestasis depend on the genetic background of the host." dans: Gut microbes, Vol. 4, Issue 4, (2013) (PubMed).

Détail du antigène

Antigène: CD14

Autre désignation: CD14 (CD14 Produits)

Synonymes: CD14 molecule Kit ELISA, CD14 antigen Kit ELISA, CD14 Kit ELISA, Cd14 Kit ELISA

Sujet: Background: The CD14 glycoprotein, gp 55, is present on most monocytic and macrophages like cell types: monocytes, macrophages, weekly at surface of neutrophiles like Kupffer cells, pleural phagocytic cells and dendritic reticular cells. CD14 is also observed on granulocytes and activated or transformed B-cells. Furthermore CD14 is present in a soluble form in human serum, urine and other body fluids. The CD14 Molecule has been reported to be a receptor for endotoxin. CD14 is anchored to cells by linkage to glycosylphosphatidylinositol (GPI) and functions as a high affinity receptor for LPS-LBP (lipopolysaccharide binding protein)-complexes.

Poids moléculaire: ~50kDa

ID gène: 12475

NCBI Accession: NP_033971

UniProt: P10810

Pathways: Signalisation TLR, Activation of Innate immune Response, Cellular Response to Molecule of Bacterial Origin, Toll-Like Receptors Cascades