CD14 Molecule (CD14) ELISA Kit

Antigen: CD14 Molecule (CD14)

Synonyms: CD14

Reactivity: Human

Application: ELISA

Catalog no.: ABIN866766

Additional Information

Detection Method: Colorimetric

Alternative name: CD14

Sample Type: Serum, Plasma, Urine, Tissue Homogenate, Cell Culture Supernatant, Biological Fluids

Plate: Pre-coated Microplate: 96 wells (12 strips x 8 wells) coated with anti-Human CD14.

Components: 1. Pre-coated Microplate: 96 wells (12 strips x 8 wells) coated with anti-Human CD14.
2. Standards: 96 tests (2 vials)
3. Detection Antibody Human CD14: 96 tests (1:100)
4. Avidin-Biotin-Peroxidase Complex (ABC): 96 tests (1:100)
5. Sample Diluent Buffer: 96 tests (2 vials)
6. Antibody Diluent Buffer: 96 tests (1 vial)
7. ABC Diluent Buffer: 96 tests (1 vial)
8. TMB color developing reagent A: 96 tests (1 vial)
9. TMB color developing reagent B: 96 tests (1 vial)
10. TMB stop solution: 96 tests (1 vial)
11.ELISA Special TBS Diluent: 96 tests (1 vial, 1:25)

Characteristics: The ELISA kit is a sandwich enzyme immunoassay for the in vitro quantitative measurement of Human CD14 in serum, plasma, Urine,tissue homogenates, cell culture supernates and other biological fluids.

Specificity: The ELISA Kit shows no cross reactivity with any of the cytokines.

Sensitivity: 156 pg/ml - 10000 pg/ml

Detection Range: 156 pg/ml - 10000 pg/ml

Application Details

Principle: The Human CD14 ELISA Kit is an Vitro enzyme-linked immunosorbent assay for the quantitative measurement of Human CD14 in serum, plasma, tissue, cell culture supernatants and urine. The Human CD14 polyclonal antibodies are precoated onto 96-well plate. Standard and samples are pipetted into the wells and Human CD14 present in a sample is bound to the wells by the immobilized antibody. The biotinylated detection antibodies are added to the wells and then followed by washing with PBS or TBS buffer. After washing away unbound biotinylated antibody, Avidin-Biotin-Peroxidase Complex is pipetted to the wells. The wells are washed again, a TMB substrate solution is added to the wells and the color changes after adding acidic stop solution. The indensity is proportional to the amount of Human CD14 bound and measured at 450nm +/- 10nm.

Reagent Preparation: A) Preparation of the standard: Standard solution should be prepared no more than 2 hours prior to the experiment. Add 1 ml sample diluent buffer into one tube, dissolve the standard thoroughly and make times dilution.
B) Preparation of biotinylated anti-Human 96T.
2. Standards: 96 tests (2 vials)
3. Detection Antibody Human CD14 antibody should be diluted in 1:99 with the antibody diluent buffer and mixed thoroughly.
C) Preparation of Avidin-Biotin-Peroxidase Complex (ABC) working solution: The solution should be prepared no more than 1 hour prior to the experiment.
a. The total volume should be: 0.1ml/well x (the number of wells). (Allowing 0.1-0.2 ml more than total volume)
b. Avidin-Biotin-Peroxidase Complex (ABC) should be diluted in 1:99 with the ABC dilution buffer and mixed thoroughly.
D) Preparation of TMB working solution: transfer 9 volumes of TMB color developing reagent A in one volume of TMB color developing reagent B for 30 minutes in 37° C before using to make TMB substrate, mixing thoroughly.

Sample Preparation: 1. Plate washing. Discard the solution in the plate without touching the side walls. Blot the plate onto paper towels or other absorbent material. Soak each well with at least 0.35 ml PBS or TBS buffer for 1~2 minutes, then discard the rinse solution. Repeat this process t for several times.
2. Sample Preparation and Storage. Store samples to be assayed within 24 hours at 2-8° C. For long-term storage, aliquot and freeze samples at -20° C. Avoid repeated freeze-thaw cycles.
Cell culture supernate, tissue lysate or body fluids: Remove particulates by centrifugation, analyze immediately or aliquot and store at -20° C.
Serum: Allow the serum to clot in a serum separator tube (about 2 hours or 4° C) at room temperature. Centrifuge at approximately 1000 X g for 10 min. Analyze the serum immediately or aliquot and store frozen at -20° C.
Plasma: Collect plasma using heparin, EDTA, citrate as an anticoagulant. Centrifuge for 15 min at 1000 x g within 30 min of collection. Analyze immediately or aliquot and store frozen at -20°C.
Sample Dilution Guideline: User needs to estimate the concentration of the target protein in the sample and select proper dilution factor so that the diluted target protein concentration falls near the middle of the linear regime in the standard curve.

Assay Procedure: The user should decide sample dilution fold by crude estimation of Human CD14 amount in samples.
1. Aliquot 0.1ml per well of the grades Human CD14 standard solutions into the precoated 96-well plate. Add 0.1ml of the sample diluent buffer into the control well. Add 0.1ml of each properly diluted sample of sera, plasma, body fluids, tissue lysates or cell culture supernatants to each empty well.
2. Seal the plate with the cover and incubate at 37° C for 90 min.
3. Remove the cover, discard plate content, and blot the plate onto paper towels or other absorbent material. Do NOT let the wells completely dry at any time, washing twice.
4. Add 0.1ml of biotinylated anti-Human CD14 antibody working solution into each well and incubate the plate at 37° C for 60 min.
5. Wash the plate three times with 0.01M TBS or 0.01M PBS, and each time let washing buffer stay in the wells for 1 min.
6. Add 0.1ml of prepared ABC working solution into each well and incubate the plate at 37° C for 30 min.
7. Wash plate 5 times with 0.01M TBS or 0.01M PBS, and each time let washing buffer stay in the wells for 1-2 min.
8. Add 0.09 ml of prepared TMB color developing agent into each well and incubate plate at 37° C away from light, observe the color at all times, when shades of blue can be seen in the wells with the three-four most concentrated Human CD14 standard solutions. The other wells show no obvious color. Add 0.1ml of prepared TMB stop solution into each well. The color changes into yellow immediately.
9. Read the O.D. absorbance at 450nm in a microplate reader within 30 min after adding the stop solution.
10. The standard curve can be plotted as the relative O.D. 450 of each standard solution (Y) vs. the respective concentration of the standard solution (X). The Human CD14 concentration of the samples can be interpolated from the standard curve.

Application Notes: Detection wavelength: 450 nm

Material not included: 1. Microtiter plate reader in standard size.
2. Polypropylene tubes for diluting and aliquoting standard.
3. Distilled or deionized water.
4. Calibrated, adjustable precision pipettes, preferably with disposable plastic tips

Storage: Store at -20° C for at least 8 months beyond and 4° C for short- term storage. Shipped on cold packs. Avoid multiple freeze-thaw cycles.

Research Area: Stem Cells, Innate Immunity, Hematopoietic Progenitors, Hematopoietic Stem Cells, CD Antigens, Surface Receptors of Immune Cells, Apoptosis/Necrosis, Immunology, Inflammation

Restrictions: For Research Use only

Publications

Product: Smith, Johnson: "Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase." in: Gene, Vol. 67, Issue 1, pp. 31-40, 1988 (PubMed).