Mouse C3 ELISA Kit

1. Sample processing and requirements:
1 、 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.
2 、 Plasma: use 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.
3 、 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.
4 、 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.
5 、 Other biological samples: 1000×g Centrifugation 20 Minutes, take the supernatant to detect.
6 、 Sample Appearance: The sample should be clear and transparent, and the suspension should be removed by centrifugation.
7 、 Sample Preservation: 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.
2. Self-prepared test equipment required for the experiment:
1 , plate reader ( 450nm ）
2 , high-precision pipettes and tips: 0.5-10uL 、 5-50uL 、 20-200uL 、 200-1000uL
3 、 37℃ Incubator
4 Distilled water or deionized water,
3. Preparation work before testing:
1 , please advance 10 Minutes remove the kit from the refrigerator and equilibrate to room temperature.
2 、 Standard gradient working solution preparation : join 1mL Universal Diluent into Lyophilized Standard , let stand 15 Minutes until it is completely dissolved and then gently mix ( The concentration is 1000ng/mL) And then according to the following concentrations: 1000ng/mL 、 500ng/mL 、 250ng/mL 、 125ng/mL 、 62.5ng/mL 、 31.25ng/mL 、 15.62ng/mL 、 0ng/mL The dilution was performed.
Double dilution method : Take 7 branch EP Tube , added to each tube 500μL Universal diluent ,1000ng/mL Pipette from the standard working solution 500μL To the first EP Mix evenly in tube Cheng 500ng/mL Standard Working Solution , according to this step, absorb and mix evenly in turn. The last one   Tube directly as a blank hole No longer need to draw 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, with a universal diluent  100× The concentrated biotinylated antibody was diluted into 1× Working concentration ( Example: 10uL   Concentrate +990uL   Universal diluent )   , used on the same day.
4 、 Enzyme Knot Combine thing Work Work Liquid match Manufacture : Before use 15 Minutes will 100× Concentrated enzyme conjugate in 1000×g   Centrifugation 1 Minutes, in the universal diluent 100× concentrate HRP   The enzyme conjugate is diluted into 1× Working concentration ( Example: 10uL   Concentrate +990uL   Universal diluent )   , used on the same day.
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).
4. 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 Min. ( suggest   : Will wait Minimum dilution of test sample 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 Add substrate: add substrate 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.
5. Calculation of experimental results:
Result judgment ：
1 , calculate the average of the standard and sample replica well OD Value and subtract the blank hole 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.
Typical data and reference curves:
The following data and curves are for reference only , the experimenter needs to establish a standard curve according to his own experiment.

attention   : This picture is for reference only   , the specimen content should be calculated from the standard curve drawn by the same test standard.

7. 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 : added to the serum and plasma of selected healthy mice 3 Mice at different concentration levels C3 , calculated recovery

Complement protein 3, often simply referred to as C3, is a protein of the immune system that is encoded by a gene called C3. It plays a central role in the activation of the complement system. One form of C3 convertase, also known as C4b2a, is formed from isomers of the activated forms of C4 and C2. C4b2a catalyzes the cleavage of the C3 protein into C3a and C3b, produced upon activation by the classical pathway as well as the lectin pathway. C3a is an acytoxin and a precursor to some cytokines such as ASP, while C3b is a progrowth agent. Factor I can cleave C3b into C3c and C3d, the latter of which plays a role in enhancing B cell responses. In the alternative complement pathway, C3 is cleaved by C3bBb, another form of C3 convertase consisting of activated C3 (C3b) and factor B (Bb). Once C3 is activated to C3b, it exposes a reactive thioester, enabling the peptide to be covalently attached to any surface capable of providing a nucleophile, such as a primary amine or hydroxyl group. The activated C3 can then interact with factor B. Factor B is then activated by factor D to form Bb. The resulting complex, C3bBb, is known as an alternative pathway (AP) C3 convertase.