Mouse C5a ELISA Kit

1. Sample processing and requirements:
1 、 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.
2
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.
3 、 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.
4 、 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.
5 、 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.
6 、 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.
7 、 Cell lysate:
Precooling 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.
8 、 Other biological samples:
1000×g Centrifugation 20 Minutes, take the supernatant to detect.
9 、 Sample Appearance:
The sample should be clear and transparent, and the suspension should be removed by centrifugation.
10 、 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. Sample dilution plan:
Please estimate the concentration range of the sample in advance. If your test sample needs to be diluted, refer to the dilution plan such as Under:
Dilution 100 Times:
One step dilution.
Take 5uL Sample to 495uL Within universal diluent, do 100 Double dilution;
Dilution 1000 Times:
Two-step dilution.
Take 5uL Sample to 95uL Within universal diluent, do 20 Dilute, and then take 5uL 20 Double dilute sample to 245uL Within universal diluent, do 50 Double dilution, total dilution 1000 Times;
Dilution 100000 Times:
Three-step dilution.
Take 5uL Sample to 195uL Within universal diluent, do 40 Dilute, and then take 5uL 40 Double dilute sample to 245uL Within universal diluent, do 50 Time dilution, and finally take 5uL 2000 Double dilute sample to 245uL Within universal diluent, do 50 Double dilution, total dilution 100000 Times;
The amount of liquid taken during each dilution step is not less than 3uL , the dilution factor is not more than 100 Times 。
Each step of dilution should be mixed evenly to avoid foaming.

3. 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,

4. 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 1000pg/mL) And then according to the following concentrations: 1000pg/mL 、 500pg/mL 、 250pg/mL 、 125pg/mL 、 62.5pg/mL 、 31.25pg/mL 、 15.62pg/mL 、 0pg/mL The dilution was performed.
Double dilution method :
Take 7 branch EP Tube , added to each tube 500μL Universal diluent ,1000pg/mL Pipette from the standard working solution 500μL To the first EP Mix evenly in a tube 500pg/mL Standard of Standard working fluid , according to this step, absorb and mix evenly in turn.
The last tube is directly used as a blank hole , no The liquid needs to be drawn from the penultimate tube again 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 ) , now available for use.
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, with a universal diluent 100× concentrate HRP The enzyme conjugate is diluted into 1× Working concentration ( Example: 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).

5. 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
sample addition :
The sample or different concentration standards are respectively according to 100μl Each well is added to the corresponding well , blank hole join 100μL Universal diluent.
After covering the sealing film 37℃ Incubation 60 Minutes.
(Recommendation : Will wait Minimum dilution of test sample with universal diluent 1 Times later, add the enzyme labeled plate for testing.
So that that effect of the matrix is reduce Dealing with the impact of test results Finally, when calculating the sample concentration, it needs to be multiplied by the corresponding dilution factor.
All of the It is recommended to set up double wells for the samples to be tested and standards during the 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.

VI. 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.
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 sample content should be calculated from the standard curve drawn from each experimental data.

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 selected healthy mouse serum, plasma and cell culture supernatant 3 No Mice at the same concentration level C5a , calculate the recovery.

Complement fragment 5a (C5a) is a protein fragment released after the protease C5-convertase cleaves complement component C5 into C5a and C5b fragments. C5a 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. C5a is a chemotactic and antiallergic agent, which is crucial in innate immunity, but is also involved in adaptive immunity. Increased production of C5a has been implicated in some inflammatory diseases.