The usage of the ApoC3 assay kit may vary depending on the type of kit (such as ELISA, immunoturbidimetry, etc.) and specific brand. The following is a general usage method for an ELISA based apolipoprotein C3 assay kit, but please note that the specific steps should refer to the instructions of the kit used:
1、 Collection and preservation of specimens
Serum: Whole blood samples should be left at room temperature for 2 hours or overnight at 4 ℃, centrifuged at 1000 × g for 20 minutes, and the supernatant can be collected for detection. Alternatively, the samples can be stored at -20 ℃ or -80 ℃, but repeated freezing and thawing should be avoided.
Plasma: EDTA or heparin can be used as anticoagulants. Within 30 minutes after sample collection, centrifuge at 1000 × g at 2-8 ℃ for 15 minutes to obtain the supernatant for detection. Alternatively, store the sample at -20 ℃ or -80 ℃ and avoid repeated freezing and thawing.
Other biological specimens, such as cell culture supernatants, should also be centrifuged appropriately to obtain the supernatant and stored as needed.
2、 Preparation and storage of reagents
Kit composition: usually includes enzyme-linked immunosorbent assay (ELISA) plate, standard, sample diluent, biotinylated antibody working solution, enzyme conjugate working solution, washing solution, substrate solution, termination solution, etc.
Storage of reagents: All reagents should be stored according to the label instructions and used within their expiration date. Before use, it is necessary to return to room temperature (if applicable).
The preparation of reagents, such as washing solution, biotinylated antibody working solution, enzyme conjugate working solution, etc., should be diluted and prepared according to the instructions.
3、 Operation steps
Sample addition: Set blank holes, standard holes, and test sample holes separately. Add standard and sample diluent to blank wells, add different concentrations of standard to standard wells, and add test samples to test wells. Be careful not to generate bubbles when adding samples, and try not to touch the hole wall as much as possible.
Incubation: After adding the sample, cover the enzyme-linked immunosorbent assay (ELISA) plate with a film and incubate it at 37 ℃ for a certain period of time (such as 90 minutes).
Washing: Discard the liquid in the well, thoroughly wash the enzyme-linked immunosorbent assay plate with washing solution, and pat dry. The number of washes and the duration of each wash should be carried out according to the instructions.
Add biotinylated antibody working solution: Add a certain amount of biotinylated antibody working solution to each well and incubate again.
Washing: Same as step 3.
Enzyme conjugate working solution: Add a certain amount of enzyme conjugate working solution to each well and continue incubation.
Washing: Same as step 3.
Add substrate solution: Add substrate solution to each well and incubate in the dark for a certain period of time (such as 15 minutes). Pay attention to the color change, and stop when there is a significant gradient in the standard hole.
Add termination solution: Add termination solution to each well to terminate the reaction, and the color will change (such as blue to yellow).
Read OD value: Immediately measure the optical density (OD value) of each well using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450nm.
4、 Result Calculation and Analysis
Draw a standard curve: Draw a standard curve based on the concentration of the standard substance and the corresponding OD value.
Calculate sample concentration: Find the corresponding concentration on the standard curve based on the OD value of the sample to be tested.
5、 Precautions
Operating standards: During the experimental process, strict adherence to sterile operating standards should be followed to avoid cross contamination.
Reagent usage: Do not mix reagents of different batches and use them within their expiration date.
Sample processing: The sample should be clear and transparent, and any suspended solids should be removed by centrifugation. Avoid using substandard samples such as hemolysis and hyperlipidemia.
Thoroughly wash: When washing the enzyme-linked immunosorbent assay (ELISA) plate, it should be thoroughly dried to avoid the formation of bubbles and residual washing solution.
Result interpretation: The experimental results should be comprehensively judged based on clinical information and other examination results.
Please note that the above steps are for reference only. Please be sure to follow the instructions of the purchased reagent kit when using it. If you have any questions or concerns, please contact the supplier or professional of the reagent kit for consultation in a timely manner.
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