| I. Sample Collection, Preparation, and Storage 1. Serum: After placing whole blood samples at room temperature for 2 hours or at 4°C overnight, centrifuge at 1000×g for 20 minutes. Remove the supernatant for testing. Blood collection tubes should be disposable, pyrogen-free, and endotoxin-free. Store at -20°C or -80°C and avoid repeated freezing and thawing. 2. Plasma: Within 30 minutes of collection, centrifuge at 1000×g for 15 minutes at 2-8°C. Remove the supernatant for testing. EDTA-Na2 is recommended as the anticoagulant to avoid hemolysis or hyperlipidemia. Store at -20°C or -80°C and avoid repeated freezing and thawing. 3. Tissue Homogenization: Take an appropriate amount of tissue and wash it in pre-chilled PBS (0.01M, pH 7.0-7.2) to remove blood (lysed red blood cells in the homogenate will affect the measurement results). After weighing, mince the tissue and mix it with the appropriate volume of PBS (generally a 1:9 weight-to-volume ratio; the specific volume can be adjusted according to experimental needs and recorded. It is recommended to add protease inhibitors to the PBS). Pour the mixture into a glass homogenizer and grind thoroughly on ice. To further lyse tissue cells, the homogenate can be sonicated or freeze-thawed repeatedly (keep the sonication in an ice bath and repeat the freeze-thaw cycle twice). Finally, centrifuge the homogenate at 5000×g for 5-10 minutes. Remove the supernatant for analysis. 4. Cell Culture Supernatant: Centrifuge the cell supernatant at 1000×g for 20 minutes to remove impurities and cell debris. Remove the supernatant for testing and store at -20°C or -80°C, but avoid repeated freezing and thawing. 5. Urine: Collect the first morning urine (midstream) or 24-hour urine collection. Centrifuge at 2000×g for 15 minutes, collect the supernatant, and store the sample at -20°C. Avoid repeated freezing and thawing. 6. Saliva: Collect the sample using a saliva sample collection tube, then centrifuge at 1000×g for 15 minutes at 2-8°C. Remove the supernatant for testing, or aliquot and store at -20°C. Avoid repeated freezing and thawing. 7. Other biological samples: Centrifuge at 1000×g for 20 minutes, collect the supernatant, and store at -20°C. Avoid repeated freezing and thawing. Notes:1. Samples should be clear and transparent, and suspended matter should be removed by centrifugation. Hemolysis of the sample will affect the results, so hemolyzed samples should not be used. 2. Samples can be stored at 4°C if tested within one week of collection. If testing cannot be performed promptly, aliquot the sample into single-use portions and freeze at -20°C (for testing within one month) or -80°C (for testing within three to six months). Avoid repeated freeze-thaw cycles. Bring the sample to room temperature before experimenting. 3. If the concentration of the test substance in your sample is higher than the highest value of the standard, please dilute it appropriately based on the actual situation (it is recommended to conduct a pilot experiment to determine the dilution factor). 2. Pre-Assay Preparation 1. Remove the test kit from the refrigerator 30 minutes in advance and equilibrate to room temperature. 2. Dilute 25 μg of concentrated wash buffer to 1 μg of working solution with double-distilled water. Return any unused portion to 4°C. 3. Standards: Add 1.0 mL of Universal Diluent for Standards & Samples to the lyophilized standard. Tighten the cap and let stand for 10 minutes to fully dissolve. Gently mix to achieve a concentration of 5000 pg/mL. Perform serial dilutions to 5000 pg/mL, 2500 pg/mL, 1250 pg/mL, 625 pg/mL, 312.5 pg/mL, 156.25 pg/mL, and 78.13 pg/mL. Use the standard diluent (0 pg/mL) as a blank well. Prepare the required amount of standard solution and set aside. It is recommended to add the prepared standard solution to the sample within 15 minutes; do not allow it to sit for extended periods. 4. Biotinylated Antibody Working Solution: Calculate the required volume of biotinylated antibody working solution before use (based on 100 μL/well; add 100-200 μL more). 15 minutes before use, dilute the concentrated biotinylated antibody (1:100) with Biotinylated Antibody Diluent to the working concentration. Use the same day. The dilution principle is to add 1 μL of concentrated biotinylated antibody to 99 μL of biotinylated antibody diluent and mix thoroughly with a pipette. 5. Enzyme Conjugate Working Solution: Before the experiment, calculate the required volume for the experiment (based on 100 μL/well; add 100-200 μL more when preparing). 15 minutes before use, dilute the concentrated HRP enzyme conjugate (1:100) with enzyme conjugate diluent to the working concentration for use that day. The dilution principle is to add 1 μL of concentrated enzyme conjugate to 99 μL of enzyme conjugate diluent and mix thoroughly with a pipette. 6. TMB Substrate - Use a pipette to aspirate the required volume of solution. Do not pour any remaining solution back into the reagent bottle. Notes: 1. Before using the kit, ensure that all components are dissolved and mixed thoroughly. Discard any unused standard after reconstitution. 2. Concentrated biotinylated antibody and enzyme conjugate solutions are small in size and may disperse throughout the tube during transportation. Centrifuge at 1000 × g for 1 minute before use to allow any liquid on the tube walls or cap to settle to the bottom. Mix the solution by carefully pipetting 4-5 times before use. Prepare the standard, biotinylated antibody working solution, and enzyme conjugate working solution according to the required volume and use the corresponding diluents. Do not mix them. 3. Concentrated wash solution may crystallize after removal from the refrigerator. This is normal. Dissolve the crystals completely in a water bath or incubator before preparing the wash solution (do not heat above 40°C). The wash solution should be at room temperature when used. 4. Samples should be added quickly, preferably within 10 minutes each time. To ensure experimental accuracy, it is recommended to use replicates. When pipetting reagents, maintain a consistent order of addition from one well to another. This will ensure the same incubation time for all wells. 5. During the wash process, any wash solution remaining in the reaction wells should be patted dry on absorbent paper. Do not place filter paper directly into the reaction wells to absorb water. Before reading, be sure to remove any residual liquid and fingerprints at the bottom to avoid affecting the microplate reader reading. 6. The color developer TMB should be protected from direct exposure to strong light during storage and use. After adding the substrate, pay attention to the color change in the reaction well. If a gradient is already obvious, stop the reaction early to avoid excessive color that affects the microplate reader reading. 7. The test tubes and reagents used in the experiment are disposable. Reuse is strictly prohibited, otherwise it will affect the experimental results. 8. Please wear a lab coat and latex gloves for proper protection during the experiment, especially when testing blood or other body fluid samples. Please follow the National Biological Laboratory Safety Protection Regulations. 9. Components of the kit from different batches cannot be mixed (except for wash buffer and reaction stop solution). 10. The enzyme label strips in the kit are detachable. Please use them in batches according to experimental needs.
III.Operation Procedure 1. Before beginning the experiment, all reagents should be equilibrated to room temperature and all reagents should be prepared in advance. When diluting reagents or samples, mix thoroughly and try to avoid foaming during mixing. If the sample concentration is too high, dilute it with sample diluent to bring the sample within the detection range of the kit. 2. Add 100 μL of the standard or sample to be tested (if the sample needs to be diluted, refer to the sample dilution guidelines for dilution methods). Be careful not to create bubbles. Add the sample to the bottom of the plate well, avoiding contact with the well walls. Gently shake to mix. Cover the plate with a film or film and incubate at 37°C for 80 minutes. To ensure the validity of the experimental results, use a fresh standard solution for each experiment. 3. Discard the liquid in the wells, spin dry, and wash the plate three times. Wash each well with 200 μL of wash buffer, soak for 1-2 minutes, and spin off the liquid in the plate (or wash the plate using a plate washer). After the final wash, pat the plate dry on absorbent paper. 4. Add 100 μL of biotinylated antibody working solution to each well (can be prepared 15 minutes in advance), cover the plate with film, and incubate at 37°C for 50 minutes. 5. Discard the liquid in the wells and wash the plate three times. Wash each well with 200 μL of wash buffer, soaking for 1-2 minutes. Discard the liquid in the plate (or wash the plate using a plate washer). After the final wash, pat the plate dry on absorbent paper. 6. Add 100 μL of enzyme conjugate working solution to each well (can be prepared 15 minutes in advance) and incubate at 37°C for 50 minutes. 7. Discard the liquid in the wells and wash the plate five times. Wash each well with 200 μL of wash buffer, soak for 1-2 minutes, and shake off any remaining liquid from the plate (or wash using a plate washer). After the final wash, pat the plate dry on absorbent paper. 8. Add 90 μL of TMB chromogenic substrate solution to each well and incubate at 37°C in the dark for 20 minutes (shorten or extend the time depending on the color development, but do not exceed 30 minutes). 9. Add 50 μL of stop solution to each well to terminate the reaction (the blue color will immediately turn yellow). The stop solution should be added in the same order as the color developer. To ensure accurate results, add the stop solution as soon as possible after the substrate reaction time expires. 10. Immediately measure the optical density (OD) of each well using a microplate reader at a wavelength of 450 nm. The instrument should be preheated and the assay program set before use. Calculation of Results 1. The OD value of the blank well should be subtracted from the OD value of each standard and sample. If replicate wells are used, the average value should be used for calculation. 2. For ease of calculation, although concentration is the independent variable and OD value is the dependent variable, the graphs use the OD value of the standard as the horizontal axis (X-axis) and the concentration of the standard as the vertical axis (Y-axis). To ensure intuitiveness of the experimental results, the graphs present raw data rather than logarithmic values. Due to differences in experimental operating conditions (such as operator, pipetting technique, plate washing technique, and temperature), the OD values of the standard curve will vary. The provided standard curve is for reference only; experimenters should establish their own standard curve. The sample concentration can be calculated from the OD value of the used sample on the standard curve. This value is then multiplied by the dilution factor to obtain the actual sample concentration. It is recommended to use professional curve drawing software, such as curve expert.
Concentration (ng/mL) | OD | Corrected OD | 5000 | 2.268 | 2.171 | 2500 | 1.72 | 1.623 | 1250 | 1.174 | 1.077 | 625 | 0.892 | 0.795 | 312.5 | 0.507 | 0.41 | 156.25 | 0.305 | 0.208 | 78.13 | 0.261 | 0.164 | 0 | 0.097 | 0.000 |
Note: This figure is for reference only.
Precision
Intra-plate precision (within-assay precision): CV% <8% Three samples of known concentrations were tested 20 times on a single ELISA plate to assess intra-plate precision. Inter-plate precision (between-assay precision): CV% <10% Three samples of known concentrations were tested 40 times on three different ELISA plates to assess inter-plate precision.
Recovery Recovery experiments were performed by spiking different samples with known concentrations of rabbit MYO to determine the recovery range and average recovery.
Sample Type | Recovery Range | Average Recovery | Serum(n=5) | 85-99% | 92% | EDTA plasma (n=5) | 83-97% | 90% | heparin plasma(n=5) | 80-92% | 86% |
Linearity The samples spiked with rabbit MYO were diluted 2-fold, 4-fold, 8-fold, and 16-fold to perform recovery experiments and obtain the recovery rate range.
Sample type | 1:2 | 1:4 | 1:8 | 1:16 | Serum (n=5) | 88-104% | 86-102% | 79-93% | 89-97% | EDTA plasma (n=5) | 85-94% | 87-101% | 82-95% | 82-93% | heparin plasma (n=5) | 95-103% | 79-96% | 93-106% | 87-98% |
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