ELISA Kit for the Quantitative Analysis of Human high sensitive CRP

The human CRP ELISA (enzyme-linked immunosorbent assay) kit is used for detection of human CRP in cell culture supernatants,human serum and plasma.THE ELISA KIT IS FOR RESEARCH USE ONLY. Please read this instruction manual carefully and check out the material provided before use, and you can contact with our company if any questions. You can enter our website or call us for other aim.

Introduction

C-Reactive Protein (CRP) is the prototypical acute phase protein in humans and is an important mediator of immune host defense (1, 2) of the pentraxin family (4-6).. Human CRP precursor is a  224 amino acids protein.The mature human CRP protein has 206 amino acids that are noncovalently linked to form the homo pentameric ring structure .

Serum concentration of CRP increases significantly in cases of both infectious and noninfectious inflammation, of tissue damage and necrosis and in the presence of malignant tumors. In response to infection, inflammation or tissue damage, the level of CRP in human serum can increase 1,000 fold within 24~48 hours.

CRP exhibits Ca2+ dependent binding to ligands. Phosphocholine (PCh), a constituent of many bacterial and fungal walls, is a principal ligand of CRP. CRP also binds to the membrane of injured cells, membrane and nuclear components of necrotic and apoptotic cells. Upon binding with the ligands, CRP is recognized by C1q and initiates the activation of complement cascade. Ligand bound CRP also binds to Fcγ RI and Fcγ RIIa on phagocytes and activates phogocytotic responses.CRP produced exclusively in the liver. Interleukin-6 is the mediator for the synthesis by the hepatocytes . IL-6,IL-1 and glucocorticoids are the major inducer of the CRP gene.

Principles of the Test

The kit is a solid sandwich enzyme-linked immunosorbent assay for detection of human CRP. An anti-human CRP monoclonal antibody has been absorbed onto the wells of the microtiter strips provided. Samples including specimens or standards were pipetted into wells. The human CRP in specimens or standards would be captured by the coated antibody and the free others were removed by washing. The human CRP biotin-conjugated antibody were added and binds to human CRP captured by the first antibody, which formed a sandwich. Streptavidin-HRP would be added and binds to the biotin conjugated antibody, then free Streptavidin-HRP would be removed during a wash step. After this, substrate solution would be added and catalyzed by the HRP, and a coloured product is formed. The intensity of the colored product is used to calculate in proportion to the amount of human CRP in the original.

Materials provided with the kits:

Specimen Collection

1.Collecting specimen as following:

A.The particulate of the cell culture supernatants should be removed before use.

B.Serum was obtained from clot at room temperature.

C.Please collect plasma with EDTA.

D.Assay immediately or store samples at －20℃. Avoid free-thaw cycles.

2.Antiseptic and anticoagulant should not appear in Serum samples.

3.Any particulate should be removed from samples before use.

4. Do not use grossly hemolyzed or lipemic samples.

Note: Strongly recommend that the serum and plasma samples should be diluent as doubling dilution before use.

Precautions for use:

1.Please storage the Kit at 2～8℃。

2. Washing buffer concentrate may have crystal in low temperature, and you can melt its in water-bath before use.

3. Please discard the dissolved standard after 3 days for use.

4. Avoid contact of substrate solution with oxidizing agents and metal.

5. Usage of  disposable pipette tips avoid microbial contamination or cross-contamination of reagents or specimens.

6. Do not mix or substitute reagents with those from other lots or other sources.

7. To ensure the adequate mixture of added reagents, please tap gently the plate after the wells were filled with liquid.

8. Incubation temperature should be 25～28℃.

9. Wash step was crucial for whole assay process.

10. Duplicate wells of the same sample were recommended in assay process.

11. Avoid the foam while pour the liquid into wells.

12. For serum or plasma samples ,the biotin-conjugated antibody should be incubate for at least 90 minutes.

Reagent Preparation

1.The reagents should be warmed up to room temperature before use. The remanent reagents must reseal and put into refrigeratory again as soon as possible.

2. Dilute 1ml of wash buffer Concentrate into 19ml deionized or distilled water to work.

3. If you have a 5x standard diluent, please dilute it with double steaming water or deionized water.

4. Add standard dilution solution to the bottle according to the volume of the label and wait15 minutes for complete dissolution. And in turn add the half concentration diluent by standard diluent .

Wash step:

Automated microplate washer or operating by pipette: Each well should be pour into300ul wash buffer and soak 15 or 30 seconds,then be aspirated, five times process were repeated. After the last wash, remove remaining wash buffer by aspirating.Invert the plate and blot it against clean paper towels.

Materials Required But Not Provided

1. pipettes and pipette tips

2. Microwell strip reader capable of reading at 450 nm (540 nm as optional reference wave length)

3. automated microplate washer

4.Glass-distilled or deionized water

Assay procedure

1.The needed strips were putted into the frame, the remains were returned into foil pouch and resealed.

2.Blank well were recommended, which only color reagent and stop solution be added. It is suggested that each testing with gradient density of standard for standard curve.

3.Add 100ul of standard or sample. then cover with the Plate Covers provided.Incubate for 120 minutes at room temperature. If is plasma serum samples, different sample dilution ratio is different, generally range in 5000 ~ 100000 times, if there is no definite scope, advice from 5000 times dilution, if the sample concentration is too high, more than test scope, please increase after diluted times dilution test again。4.Five times wash process were repeated.

5.Add 100ul of detetion antibody. Cover with the Plate Covers provided.Incubate for 1 hour at room temperature.

6.Five times wash process were repeated.

7.Add 100ul of Streptavidin-HRP. Cover with the Plate Covers provided. Lucifugal incubation for 20 minutes at room temperature.

8.Five times wash process were repeated.

9.Add 100ul of TMB，Lucifugal incubation for 20 minutes at room temperature.

10.Add 50ul of stop solution to each well, determine the optica density of each well within 10 minutes.  

Calculation of Results

1.Duplicates should be within 20 per cent of the mean. Average absorbance values for each set of duplicate samples were used as detection results.

2.The blank absorbance values of subtract should be deducted.

3.Drawing a best fit curve through the points of graph. Draw the standard curve by plotting assayed OD value (on the Y axis) vs. concentration (on the X axis). The sample concentration was obtained based on its OD value founding in the standard concentration curve.

4.If the values obtained are not within the expected range of the standard, Samples should be dilute and assay again.

Typical Data and Standard Curve

Human high sensitive CRP standard curve

 

Sensitivity, Specificity, Repeatability

Sensitivity: repeated assays were evaluated and the minimum detectable dose was 18. 4pg/ml.

Repeatability: The coefficient of variation between wells or plates is less than 10 per cent.