Lipopolysaccharide Binding Protein (LBP) ELISA Kit

Antigen: Lipopolysaccharide Binding Protein (LBP)

Synonyme: MGC22233, Ly88, MGC124626, LBP, LPSBP, MGC134541, BmCTL21

Reaktivität: Human

Applikation: ELISA

Produktnummer: ABIN370809

Produktbeschreibung

Weitere Bezeichnung: Lipopolysaccharide-binding Protein (LBP)

Spezifität: Cross reactivities: porcine-, rabbit-, cattle-, dog-, horse LBP. Specificity: specific for free LBP

Sensitivität: Normal LBP range: in healthy blood donors: (5-15 ug/ml). Interassay variation coefficient: 9.8 till 17.8 depending of concentration. Intraassay variation coefficient: 6.1%. Effective range: 5 -50ng/ml, linear till 25ng/ml.

Anwendungen

Prinzip: The human LBP kit has been developed for the quantitative measurement of natural and recombinant human LBP in serum, plasma and culture medium. The human LBP Kit is a solid phase sandwich Enzyme Linked-Immunosorbent Assay (ELISA). Monoclonal antibody specific for human LBP is used for coating (precoated and blocked modules). In the first step the plate will be incubated with the antigen (standard or sample). During this incubation, human LBP is captured by solid bound antibody. Unbound material present in the sample is removed by washing. Now the plate will be incubated with a POD-labelled antibody specific for human LBP (second incubation). Revelation step includes TMB as chromogen. The enzyme reaction is stopped by the addition of stopping solution 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 human LBP concentration of samples with unknown concentrations, which are run concurrently with the standards, can be determined from the standard curve.

Protokoll: Let all reagents reach room temperature and mix thoroughly. (1) Samples: Pipette 100 ul of standards (50, 25, 12.5, 6.25, 3.12, 1.5 ng/ml= vial a-f) or diluted samples in duplicate into the corresponding wells of precoated modules (1) and incubate for one hour at room temperature and shaking. (2) 3 x washing with Wash Buffer (A). (3) Detection antibody: Pipette 100 ul detecting antibody (E, vial 2) to each well and incubate at room temperature for 1 hour at shaker.4. 3 x washing with Wash Buffer (A). (5) Substrate: Pipette 100 ul substrate solution (D, vial 9) to each well. Incubate 10 to 12 min in the dark at room temperature without shaking (depending from temperature in the lab). (6) Blocking: Pipette 100ul blocking solution (vial 8) to each well. Tap plate gently to mix. Read absorbance of wells at 450 nm (reference wave length 620). (8) Calculate the LBP concentration: Calculate the mean of optical density (OD) of standard duplicates, reference serum and the samples. Design a standard curve by plotting tthe OD means of standards (b-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.

Applikationshinweise: Preparation of reagents (Recommendations for 1 plate): (A) Wash Buffer: PBS/ Tween: Dissolve 1 Tablet Phosphate buffered saline (PBS, vial 5) in 200 ml distilled water - add 100 ul Tween 20 (vial 7). (Prepared wash buffer is stable for 4 weeks in refrigerator). (B) PBS: Dilute 1 Tablet of vial 5 in 200 ml distilled water. (C) Dilution buffer: Dissolve content of vial 6 with 50 ml PBS (Buffer B) and add 50ul Tween 20 from vial 7. This buffer is 1-2 weeks stable at -20oC. Attention! Use buffer for assay at room temperature. Alternatively: 250mg BSA +25ml PBS+25ul Tween 20. (D) Substrate: Vial 9 Ready for use. Mix carefully (E) Detection antibody: Vial 2 ready for use. Mix carefully. (F) Reference serum: Pipette 30ul distilled water to the vial 4 for reconstitution. For assay pipette the whole content of reconstituted vial 4 to 7970 ul dilution buffer (C) and pipette of this 100ul/well. This represents final dilution of 1:800. The reference serum contains 12.3

Bestandteile: Precoated ELISA modules (1), detecting antibody (POD-labelled monoclonal antibody to human LBP) "Ready for use

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

Lagerung: Short time store at 2-8°C, Long time storage of vial 3 and 4 at -20°C or -80°C, Detecting monoclonal can be stored at 2-8°C

Forschungsgebiet: Immunologie

Beschränkungen: Nur für Forschungszwecke einsetzbar

Bilder

Standard curve