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Principle
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The mouse CD14 kit has been developed for the quantitative measurement of natural and recombinant mouse CD14 in serum, plasma and culture medium. 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.
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Protocol
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Let all reagents reach room temperature and mix thoroughly. (1) Samples and detecting antibody: Add 50 ul 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 50ul detecting antibody (E). Incubate for 1.5 hours at room temperature with shaking. 6. 3 x washing with 250ul Wash Buffer/well (A). Remove the Wash Buffer carefully after each wash. 7. Substrate: Add 100 ul Substrate (vial 9) to each well. Incubate 14
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Application Notes
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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
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Components
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Precoated ELISA module (1 plate), detecting antibody (POD-labelled polyclonal antibodies to mouse CD14) (1 vial), CD14-standard (recombinant mouse CD14, lyophilized) (1 vial), Reference serum (2.7
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Material not included
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Orbital shaker, Micro plate reader for measurement absorbance at 450 /620 nm, Precision pipettes with disposable tips, 10-1000 ul adjustable multiwell pipettes
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Storage
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Short time store at 2-8°C, Long time storage of vials 3-4 at -20°C or -80°C. The test kit is stable for some days at room temperature as well as 3 days at 37°C. Test kit is stable 3 days at 37°C, 1 week at room temperature, 1 year at refrigerator if standard and reference are stored at minus 200C.
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Research Area
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Stem Cells, Innate Immunity, Hematopoietic Progenitors, Hematopoietic Stem Cells, CD Antigens, Surface Receptors of Immune Cells, Apoptosis/Necrosis, Immunology, Inflammation
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Restrictions
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For Research Use only
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Alternatives 
