• Human Pro-C3 ELISA Kit
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Human Pro-C3 ELISA Kit

Human Pro-C3 ELISA Kit

Catalog Number: abs551775 Application: ELISA Reactivity: Human Conjugation:
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$368 USD
$368 USD
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Product Details

Product Specification

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 Minutes. 37℃
4 Distilled water or deionized water

Sample handling and requirements:
2 Hour or 4℃ Overnight, then 1000×g 20 Minutes, take the supernatant, or place the supernatant in -20℃ Or -80℃
Plasma: with EDTA Or heparin as an anticoagulant to collect specimens, and collect the specimens after collection 30 2-8℃1000×g Centrifugation 15 -20℃ Or -80℃℃
Tissue homogenate: with pre-cooled PBS 0.01M,pH=7.4 ) Rinse the tissue to remove residual blood (lysed red blood cells in the homogenate will affect the measurement result), and break the tissue after weighing. Combine the fragmented 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 ground thoroughly on ice. 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 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 150-200uL PBS 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.
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.
Other biological fluids: 1000xg Centrifugation 20 Minutes, take the supernatant to detect.

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 60ng/mL ） And then at the following concentrations: 60ng/mL 、 30ng/mL 、 15ng/mL 、 7.5ng/mL 、 3.75ng/mL 、 1.875ng/mL 0.9375ng/mL 、 0ng/mL The dilution was performed.
Double dilution method : Take 7 branch EP Tube , added to each tube 500uL Universal diluent ,60ng/mL Pipette from the standard working solution 500uL To the first EP Mix evenly in a tube 30ng/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 Preparation of biotinylated antibody detection working solution: before use 15 Min. The concentrated biotinylated antibody was concentrated in 1000×g   Centrifugation 1 Minutes, in the universal diluent  100× The concentrated biotinylated antibody was diluted into 1× Working concentration (ex: 10uL   Concentrate +990uL   Universal Diluent)    , now available for use.
4 Preparation of enzyme conjugate working solution: before use 15 Minutes will 100× Concentrated enzyme conjugate in 1000×g   Centrifugation 1 Minutes, with a universal diluent 100× concentrate HRP   The enzyme conjugate is diluted into 1× Working concentration (ex: 10uL   Concentrate +990uL   Universal Diluent)    , now available for use.
5 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 100ul Each well is added to the corresponding well, and the blank well is added 100uL 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 Add biotinylated antibody: take out the enzyme plate, discard the liquid without washing. Directly add biotinylated antibody working solution to each well 100uL , after covering the sealing film 37℃ Incubation 60 Minutes.
4 Plate washing: discard the liquid and add to each well 300uL 1x 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).
5 Adding enzyme conjugate working solution: adding enzyme conjugate working solution to each well 100uL , after covering the sealing film 37℃ Incubation 30 Minutes.
6 Plate washing: discard the liquid according to the steps 4 Washing method, wash plate 5 Times.
7 Substrate addition: substrate is added per well ( TMB ） 90uL Covered with a sealing film, 37℃ Incubation protected from light 15 Minutes.
8 Add stop solution: take out the enzyme label plate and directly add stop solution to each well 50uL , 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.
Theory This kit adopts double antibody sandwich enzyme-linked immunosorbent assay (ELISA)。 To pre-coated Pro-C3 A sample, a standard product and a biotin-labeled detection antibody are sequentially added into a microhole for capturing the antibody, HRP TMB Color development, TMB HRP ) and the final yellow color under the action of acid. Shades of color and in the sample Pro-C3 There is a positive correlation. Use a microplate reader in 450nm 0D Value), calculate sample concentration.
Source Human
Synonym Human Pro-C3 ELISA Kit
Detection Type Double antibody sandwich method
Composition a
Name 9 6 T  match   set remark
Pre-coating 96 Well plate 8 ×12 Strip without
Standard 2 branch
Dilute as per instructions
Universal diluent
2×20mL
without
Concentrated biotinylated antibody ( 100× ）  
120uL
Dilute as per instructions
Concentrated enzyme conjugate ( 100×
120uL
Dilute as per instructions
20× Washing liquid
2×10mL
Dilute as per instructions
Bottom （ TMB ）
10mL
without
Stop liquid
6mL
without
Sealing film
4 Zhang
without
Instructions
1 Share
without
Background Fibrosis causes the release of certain extracellular matrix protein fragments into the blood circulation. I.e. precisely cleaved Ⅲ Collagen propeptide (Pro-C3) Mild and moderate disease can be distinguished. Pro-C3 Could become a promising blood parameter to be included in future studies for monitoring disease progression and ultimately for evaluating potential anti-fibrotic therapies.
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 prior to use 20-25℃ 。 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 on the bottom of the plate, otherwise it will affect 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 bright 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 it is possible to spread the disease, all samples should be managed well, and the samples and testing devices should be handled according to the prescribed procedures.
Storage Temp. Unopened kit, 4°C Storage, Expiration Date 6 Months.
Test Range 0.93-60ng/mL
Applications Serum, plasma, tissue homogenates, cell lysates, cell culture supernatants and other biological fluids