Purpose | This immunoassay kit allows for the in vitro quantitative determination of porcine C-Reactive Protein, CRP concentrations in cell culture supernates, serum, plasma and other biological fluids. |
Sample Type | Cell Culture Supernatant, Serum, Plasma, Biological Fluids |
Analytical Method | Quantitative |
Detection Method | Colorimetric |
Specificity | This assay recognizes recombinant and natural porcine CRP. |
Cross-Reactivity (Details) | No significant cross-reactivity or interference was observed. |
Sensitivity | The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest detectable concentration that could be differentiated from zero. |
Characteristics | Sus scrofa,Pig,C-reactive protein,CRP,PTX1 |
Components | Reagent (Quantity ): Assay plate (1×20ml), Standard (2), Sample Diluent (1 × 20ml), Assay Diluent A (1x10ml), Assay Diluent B (1x10ml), Detection Reagent A (1 × 120μl), Detection Reagent B (1 × 120μl), Wash Buffer (25 x concentrate) (1 × 30ml), Substrate (1x10ml), Stop Solution (1x10ml), Plate sealer for 96 wells (5), Instruction (1) |
Material not included | Luminometer. Pipettes and pipette tips. EP tube Deionized or distilled water. |
Background | C-reactive protein (CRP) has been regarded as an acute phase reactant in serum. It consists of five single subunits, which noncovalently linked and assembled, as a cyclic pentamer with a mol. Wt. Range of 110-140 kDa. CRP has been found to be increased in serum of patients with a wide variety of diseases including infections by gram-positive and gram-negative bacteria, acute phase of rheumatoid arthritis, abdominal abscesses, inflammation of bile ducts, myocardial infarction, and malignant tumors. CRP may be found in patients with Guillain-Barre syndrome and multiple sclerosis, certain viral infections, tuberculosis, acute infectious hepatitis, many other necrotic and inflammatory diseases, burned patients, and after surgical trauma. Although the detection of elevated levels of CRP in the serum is not specific for any particular disease, it is useful indicator of inflammatory processes. CRP levels rise in serum within hours of the onset of inflammation, reach a peak during the acute stage and decrease with resolution of inflammation trauma. The detection of CRP is a more reliable and sensitive indicator of the inflammatory process than the erythrocyte sedimentation rate, which may also be influenced by physiological changes not associated with an inflammation process. |
Gene ID | 3078 |
Sample Volume | 100 μL |
Plate | Pre-coated |
Protocol | The microtiter plate provided in this kit has been pre-coated with an antibody specific to CRP. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for CRP and Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB substrate solution is added to each well. Only those wells that contain CRP, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of CRP in the samples is then determined by comparing the O.D. of the samples to the standard curve. |
Reagent Preparation |
Bring all reagents to room temperature before use. Wash Buffer - If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. Dilute 20 mL of Wash Buffer Concentrate into deionized or distilled water to prepare 500 mL of Wash Buffer. Standard - Reconstitute the Standard with 1.0 mL of Sample Diluent. This reconstitution produces a stock solution of 100 ng/mL. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions. The undiluted standard serves as the high standard (100 ng/mL). The Sample Diluent serves as the zero standard (0 ng/mL). Detection Reagent A and B - Dilute to the working concentration specified on the vial label using Assay Diluent A and B (1:100), respectively. |
Sample Collection | Serum - Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at approximately 1000 x g. Remove serum and assay immediately or aliquot and store samples at -20 °C. Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 x g at 2 - 8 °C within 30 minutes of collection. Store samples at ≤ -20 °C. Avoid repeated freeze-thaw cycles. Note: Citrate plasma has not been validated for use in this assay. |
Assay Procedure |
Allow all reagents to reach room temperature. Arrange and label required number of strips. |
Calculation of Results |
Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the HS-CRP IGM concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor. 4. |
Restrictions | For Research Use only |
Handling Advice |
1. The kit should not be used beyond the expiration date on the kit label. 2. Do not mix or substitute reagents with those from other lots or sources. 3. If samples generate values higher than the highest standard, further dilute the samples with the Assay Diluent and repeat the assay. Any variation in standard diluent, operator, pipetting technique, washing technique,incubation time or temperature, and kit age can cause variation in binding. 4. This assay is designed to eliminate interference by soluble receptors, ligands, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded. |
Storage | 4 °C/-20 °C |
Storage Comment | The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20 °C upon being received. The other reagents can be stored at 4 °C. |