Human C5a ELISA Kit
Human C5a ELISA Kit
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Product Specification
| protein | C5a | |||||||||||||||||||||||||||||||||||
| Usage |
Self-prepared test equipment required for the experiment: 1 , plate reader ( 450nm ） 2 , high-precision sampler and gun head: 0.5-10uL 、 5-50uL 、 20-200uL 、 200-1000uL 3 、 37℃℃ Incubator 4 Distilled water or deionized water Sample handling and requirements: The detection range of the kit is not equivalent to the concentration range of the test substance in the sample. It is recommended to estimate the concentration of the test substance in the sample through relevant literature before the experiment and determine the actual concentration of the sample through pre-experiment. If the concentration of the test substance in the sample is too high or too low, please dilute or concentrate the sample appropriately. If the sample tested is not among the sample types listed in the instructions, it is recommended to conduct pre-experiments to verify its detection effectiveness. Serum: Whole blood samples collected in serum separation tubes were placed at room temperature 2 Hour or 2-8℃ Overnight, then 1000×g Centrifugation 20 Minutes, take the supernatant, or place the supernatant in -20℃ Or -80℃ Store, but repeated freezing and thawing should be avoided. Plasma: with EDTA Or heparin as an anticoagulant to collect a sample, and collect the sample after collection 30 Within minutes 2-8℃1000×g Centrifugation 15 Minutes, take the supernatant to detect, or place the supernatant in -20℃ Or -80℃ Store, but repeated freezing and thawing should be avoided. Tissue homogenate: with pre-cooled PBS(0.01M,pH=7.4) The tissue is flushed to remove residual blood (lysed red blood cells in the homogenate can affect the test results), and the tissue is weighed and cut into pieces. Combining the shredded tissue with the corresponding volume PBS (Generally according to 1:9 Weight to volume ratio, such as 1g The tissue samples correspond to 9mL Of PBS The specific volume can be appropriately adjusted according to the experimental needs and recorded. Recommended in PBS Add protease inhibitor) into a glass homogenizer and grind thoroughly on ice or grind in a homogenizer. For further lysis of tissue cells, the homogenate can be sonicated, or freeze-thawed repeatedly. Finally, the homogenate was mixed in 5000×g Centrifugation 5~10 Minutes, take the supernatant for detection. Cell culture supernatant: please 1000×g Centrifugation 20 Minutes, take the supernatant to detect, or place the supernatant in -20℃ Or -80℃ Store, but repeated freezing and thawing should be avoided. Cell lysate: pre-cooling for adherent cells PBS Gently washed, followed by trypsinization, 1000×g Centrifugation 5 Cells were collected after minutes. The suspended cells can be collected directly by centrifugation. The collected cells were pre-cooled with PBS Washing 3 Times, every 1×10^6 Added to cells 150-200μLPBS Resuspension (recommended at PBS Adding a protease inhibitor. If the content is very low, it can be appropriately reduced PBS Volume) and the cells were disrupted by repeated freeze-thaw or sonication. The extracts were mixed in 2-8℃ ， 1500×g Centrifugation 10 Minutes, take the supernatant for detection. Other biological fluids: 1000xg Centrifugation 20 Minutes, take the supernatant to detect. Sample appearance: The sample should be clear and transparent, and the suspended solids should be removed by centrifugation. Sample storage: after sample collection, if 1 Those tested within weeks can be stored in 4℃ , if it cannot be detected in time, please pack it according to the one-time usage amount and freeze it in -20℃ （ 1 Within months), or -80℃ （ 6 Test within months) to avoid repeated freezing and thawing. Hemolysis of the sample will affect the final test result, so hemolyzed samples are not suitable for this test. Sample dilution protocol: Please estimate the concentration range of the sample in advance. If your test sample needs to be diluted, refer to the dilution plan as follows: Dilution 100 Times: One-step dilution. Take 5μL Sample to 495μL Within universal diluent, do 100 Double dilution. Dilution 1000 Times: Two-step dilution. Take 5μL Sample to 95μL Within universal diluent, do 20 Double dilution. Take again 5μL20 Double dilute sample to 245μL Within universal diluent, do 50 Double dilution, total dilution 1000 Times. Dilution 100000 Times: Three-step dilution. Take 5μL Sample to 195μL Within universal diluent, do 40 Dilute, and then take 5μL40 Double dilute sample to 245μL Within universal diluent, do 50 Time dilution, and finally take 5μL 2000 Double dilute sample to 245μL Within universal diluent, do 50 Double dilution, total dilution 100000 Times. The amount of liquid taken during each dilution step is not less than 3μL , the dilution factor is not more than 100 Times. Each step of dilution should be mixed evenly to avoid foaming. Preparations before testing: 1 , please advance 10 Minutes remove the kit from the refrigerator and equilibrate to room temperature. 2 , Standard gradient working solution preparation: add 1mL Universal diluent into lyophilized standard and let stand 15 Minutes until it is completely dissolved and then gently mix (the concentration is 500ng/mL ） And then according to the following concentrations: 500ng/mL 、 250ng/mL 、 125ng/mL 、 62.5ng/mL 、 31.25ng/mL 、 15.62ng/mL 、 7.81ng/mL 、 0ng/mL The dilution was performed. Double dilution method : Take 7 branch EP Tube , added to each tube 500uL Universal diluent ,500ng/mL Pipette from the standard working solution 500uL To the first EP Mix evenly in a tube 250ng/mL Standard Working Solution , according to this step, absorb and mix evenly in turn. The last tube is directly used as a blank hole , there is no need to suck liquid from the penultimate tube, as shown in the figure below.  3 、 HRP Preparation of anti-resistance detection working solution: before use 15 Minutes will 100× concentrate HRP Detect resistance to 1000×g Centrifugation 1 Minutes, with a universal diluent 100× concentrate HRP Detection resistance dilution to 1× Working concentration ( Example :10μL Concentrate +990μL Universal diluent ) , now available for use. 4 、 1× Wash liquid preparation: Take 10mL 20× Wash liquid to 190mL In distilled water (the concentrated washing liquid taken out of the refrigerator may have crystals, which is a normal phenomenon. It can be left at room temperature and prepared after the crystals are completely dissolved). Operation steps: 1 Equilibration from room temperature 10 After minutes, remove the required slats from the aluminum foil bag, and seal the remaining slats with ziplock bag and put them back 4℃ 。 2 , Adding samples: respectively add samples or different concentration standards according to 100μL Each well is added to the corresponding well, and the blank well is added 100μL Universal diluent. After covering the sealing film 37℃ Incubation 60 Minutes. (Recommendation: minimum dilution of sample to be tested with universal diluent 1 Times later, add the enzyme labeled plate for testing. So as to reduce the influence of matrix effect on the test results, and finally, the sample concentration needs to be multiplied by the corresponding dilution factor when calculating. It is recommended to set up double wells for all samples and standards to be tested during testing). 3 Plate washing: discard the liquid and add to each well 300μL1x Wash liquid, stand 1 Minutes, throw off the washing liquid, pat dry on absorbent paper, and repeat washing the plate 3 Times (the plate can also be washed with a plate washing machine). 4 Add HRP Anti-resistance test: After washing the plate, add directly to each well 100μLHRP Check the resistance to working fluid and cover the sealing film 37° Incubation 60 Minutes. 5 Plate washing: discard the liquid according to the steps 3 Washing method, wash plate 5 Times. 6 Add substrate: add per well 90μL Substrate (TMB) Covered with a sealing film, 37℃ Incubation protected from light 15 Minutes. 7 Add stop solution: take out the enzyme label plate and add it directly to each well 50μL Stop solution, immediately in 450nm Wavelength measurement of each well OD Value. Calculation of experimental results: Result judgment: 1 , calculate the average of the standard and sample replica well OD Value and subtract the blank hole's OD Values as correction values. Taking concentration as the abscissa, OD Value is ordinate , draw the standard curve of the four-parameter logic function on the double logarithmic coordinate paper. 2 If the sample OD If the value is higher than the upper limit of the standard curve, the test should be retested after appropriate dilution and multiplied by the corresponding dilution factor when calculating the sample concentration.  Note: This figure is for reference only, and the sample content should be calculated from the standard curve drawn from each experimental data.Kit Performance: 1 Repeatability: the coefficient of variation in the plate is less than 10% , the interplate coefficient of variation is less than 10% 。 2 Recovery rate: adding to selected healthy human serum, plasma and cell culture supernatant 3 People with different concentration levels C5a , calculate the recovery.
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| Sensitivity | 3.8ng/mL | |||||||||||||||||||||||||||||||||||
| Theory | This kit uses double antibody sandwich enzyme-linked immunosorbent assay (ELISA). To the microwells pre-coated with human complement component 5a (C5a) capture antibody, sample, standard, and HRP-labeled detection antibody were sequentially added, incubated and washed in the middle, and colored by substrate TMB, which was converted to blue under the catalysis of peroxidase (HRP), and to final yellow under the action of acid. There was a positive correlation between the depth of color and the human complement component 5a (C5a) in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450nm, and the sample concentration was calculated. | |||||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||||
| Synonym | Human Complement Component 5a ELISA Kit | |||||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||||
| Description | Specificity: Human C5a can be detected in the sample without significant cross-reaction with its analogs. | |||||||||||||||||||||||||||||||||||
| Composition |
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| Background | Complement component 5a (C5a) is a protein fragment released after the protease C5-convertase cleaves complement component C5 into C5a and C5b fragments. C5a, another cleavage product of C5, acts as a highly inflammatory peptide that encourages complement activation, forms MAC, attracts innate immune cells, and releases histamine in allergic reactions. The origin of C5 is in hepatocytes, but its synthesis can also be found in macrophages, where it may cause a local increase in C5a. It is a chemotactic and anti-allergic agent; It is crucial in innate immunity, but it is also related to adaptive immunity. Increased production of C5a has been implicated in some inflammatory diseases. | |||||||||||||||||||||||||||||||||||
| General Notes | 1. Carry out incubation in strict accordance with the specified time and temperature to ensure accurate results. All reagents must reach room temperature 20-25 °C prior to use. Store reagents in refrigeration immediately after use. 2. Incorrect plate washing may lead to inaccurate results. Make sure to drain the liquid from the wells as much as possible before adding the substrate. Do not allow the wells to dry out during incubation. 3. Eliminate the residual liquid and fingerprints at the bottom of the plate, otherwise it will affect the OD value. 4. The substrate color development solution should be colorless or very light in color, and the substrate solution that has turned blue cannot be used. 5. Avoid cross-contamination of reagents and specimens to avoid wrong results. 6. Avoid direct exposure to strong light during storage and incubation. 7. Any reaction reagent cannot come into contact with the bleaching solvent or the strong gas emitted by the bleaching solvent. Any bleaching component will destroy the biological activity of the reaction reagents in the kit. 8. Expired products cannot be used, and components with different item numbers and batch numbers cannot be mixed. 9. Recombinant proteins from sources other than the kit may not match the antibodies in this kit and are not recognized. 10. If the disease may be spread, all samples should be managed well, and the samples and testing devices should be handled according to the prescribed procedures. |
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| Storage Temp. | Unopened kit, stored at 4 °C, shelf life 6 months. | |||||||||||||||||||||||||||||||||||
| Test Range | 7.81-500ng/mL | |||||||||||||||||||||||||||||||||||
| Applications | Serum, plasma, tissue homogenates, cell lysates, cell culture supernatants and other biological fluids |
