| Usage |
Specimen Requirements
1. Specimens should be extracted as soon as possible after collection, following the relevant literature. Experiments should be performed as soon as possible after extraction. If experiments cannot be performed immediately, specimens can be stored at -20°C, but repeated freeze-thaw cycles should be avoided.
2. Samples containing sodium nitrate (NaN3) cannot be assayed, as NaN3 inhibits horseradish peroxidase (HRP) activity.
Procedure
1. Dilution of the Standard: This kit provides one standard sample at full strength. Users can dilute the sample in a small test tube according to the following chart.
80ng/L |
No. 5 standard |
Add 150μl to the original standard of 150μl 40ng/L Standard Dilution |
40ng/L Standard No. 4 |
Add 150µl of Standard No. 5 to 150µl of Standard Dilution |
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20ng/L
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Standard No. 3 |
150μl of Standard No. 4 was added to 150μl of Standard Dilution Buffer |
10ng/L |
Standard No. 2 |
150µl of Standard No. 3 was added to 150µl of Standard Dilution |
5ng/L |
Standard No. 1 |
150µl of Standard No. 2 was added to 150µl of Standard Dilution 2. Sample Addition: Set up blank wells (blank control wells without sample or enzyme-linked reagent; all other steps remain the same), standard wells, and test sample wells. Accurately add 50 μl of standard to the enzyme-linked coated plate. First, add 40 μl of sample diluent to the test sample wells, followed by 10 μl of the test sample (final sample dilution is 5-fold). Add the sample to the bottom of the plate well, avoiding contact with the well walls. Gently shake to mix. 3. Incubation: Seal the plate with sealing film and incubate at 37°C for 30 minutes. 4. Preparation: Dilute the 30x concentrated wash buffer 30x with distilled water and set aside. 5. Wash: Carefully remove the sealing film, discard the liquid, spin dry, fill each well with wash buffer, let it sit for 30 seconds, then discard. Repeat this process 5 times and pat dry. 6. Add enzyme: Add 50µl of enzyme-labeled reagent to each well, excluding the blank well. 7. Incubation: Same as in 3. 8. Wash: Same as in 5. 9. Color development: First add 50µl of Color Developer A to each well, then add 50µl of Color Developer B. Gently shake to mix. Develop at 37°C in the dark for 10 minutes. 10. Stop: Add 50µl of Stop Buffer to each well to terminate the reaction (the blue color will immediately turn yellow). 11. Measurement: Use the blank well as the zero setting and measure the absorbance (OD) of each well sequentially at 450 nm. Measurements should be performed within 15 minutes after adding the stop solution. Calculation: Draw a standard curve on graph paper with the standard concentration as the horizontal axis and the OD value as the vertical axis. Based on the sample OD value, determine the corresponding concentration from the standard curve; then multiply by the dilution factor. Alternatively, use the standard concentration and OD value to calculate the linear regression equation for the standard curve. Substitute the sample OD value into the equation to calculate the sample concentration. Multiply this by the dilution factor to obtain the actual sample concentration.
 Standard Curve |
| Species Reactivity |
Mouse |
| Theory |
This kit uses a double-antigen sandwich assay to measure the level of mouse anti-cyclic citrullinated peptide (CCPAb) antibodies in samples. Purified antigen is coated on a microplate to form a solid-phase antigen. Anti-cyclic citrullinated peptide (CCPAb) antibodies are then added sequentially to the coated wells. Anti-cyclic citrullinated peptide (CCPAb) antibodies then bind to HRP-labeled antigens, forming an antigen-antibody-enzyme-labeled antigen complex. After thorough washing, the substrate TMB is added for color development. TMB converts to blue under the catalysis of HRP and to yellow under the action of acid. The intensity of the color is positively correlated with the level of anti-cyclic citrullinated peptide (CCPAb) antibodies in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader, and the concentration of mouse anti-cyclic citrullinated peptide (CCPAb) antibodies in the sample is calculated using a standard curve. |
| Detection Type |
Used to determine the content of anti-cyclic citrullinated peptide antibodies (anti-CCPAb) in mouse serum, plasma and related liquid samples. |
| Composition |
Serial number |
Component name |
|
1 |
30 Concentrated washing solution |
20ml×1 bottle |
6 |
Chromogen B solution |
6ml×1/bottle |
2 |
Enzyme labeled reagent |
6ml×1 bottle |
7 |
Stop solution |
6ml×1 bottle |
3 |
Enzyme-coated plate |
12 wells×8 strips |
8 |
Standard (160ng/L) |
0.5ml×1 bottle |
4 |
Sample diluent |
6ml×1 bottle |
9 |
Standard diluent |
1.5ml×1 bottle |
5 |
Developer A Liquid |
6ml×1 bottle |
11 |
Sealing film |
2 photos |
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| General Notes |
1. After removing the kit from the refrigerator, allow it to equilibrate at room temperature for 1 hour before use. If the enzyme-coated plate is opened but not completely used, store it in a sealed bag.
2. Crystals may form in the concentrated wash solution. Warming in a water bath during dilution can aid dissolution. This will not affect the results during washing.
3. Use a pipette for each sample addition step and frequently calibrate its accuracy to avoid experimental error. The time for each addition should ideally be within 5 minutes. For large numbers of samples, using a dispenser is recommended.
4. Develop a standard curve with each measurement, preferably in duplicate. If the analyte concentration in the sample is too high (the OD value of the sample is greater than the OD value of the first standard well), dilute the sample a certain number of times (n) with sample diluent before measurement. When calculating the total dilution factor (×n×5), multiply the final dilution factor.
5. Plate sealing film is for single use only to avoid cross-contamination.
6. Protect the substrate from light. 7. Strictly follow the instructions for use. Test results must be based on the readings of the microplate reader. 8. All samples, wash solutions, and waste should be treated as infectious agents. 9. Components from different batches of this reagent must not be mixed. |
| Storage Temp. |
Unopened test kit, stored at 2-8°C, has a shelf life of 6 months. |
| Test Range |
3ng/L - 90ng/L |
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