Sample Collection, Preparation, and Storage 

1. Serum: After whole blood samples have been stored at room temperature for 2 hours or at 4°C overnight, centrifuge them 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, avoiding repeated freeze-thaw cycles.

2. Plasma: Within 30 minutes of collection, centrifuge the sample at 1000×g for 15 minutes at 2-8°C. Remove the supernatant for testing. EDTA-Na₂ is recommended as an anticoagulant. Avoid using samples with hemolysis or high lipid levels. Store at -20°C or -80°C. 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. Then, add the appropriate volume of PBS (generally a 1:9 mass-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) to the homogenizer and grind thoroughly on ice. To further lyse tissue cells, the homogenate can be ultrasonically disrupted or repeatedly frozen and thawed (keep the homogenate in an ice bath during ultrasonication. Repeat the freeze-thaw cycle twice). Finally, centrifuge the prepared homogenate at 5000×g for 5-10 minutes. Collect the supernatant for testing. (Tissue homogenates should also be tested for protein concentration to obtain a more accurate test substance concentration per milligram of protein.)

4. Cell culture supernatant: Centrifuge the cell supernatant at 1000×g for 20 minutes to remove impurities and cell debris. Collect the supernatant for testing and store at -20°C or -80°C, avoiding repeated freezing and thawing.

5. Urine: Collect the first morning urine (midstream) or a 24-hour urine collection. Centrifuge at 2000×g for 15 minutes, then collect the supernatant. Store the sample at -20°C, avoiding 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 freeze-thaw cycles.

7. Other biological samples: Centrifuge at 1000×g for 20 minutes, then remove the supernatant for testing.

Precautions

1. The sample 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. If samples are to be tested within one week of collection, they can be stored at 4°C. 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 samples to room temperature before experimentation.

Sample Dilution Guidelines

If your test samples require dilution, general dilution guidelines are as follows:

1. 50-fold dilution: One-step dilution. Dispense 5 μL of sample into 245 μL of Standard and Sample Diluent for a 50-fold dilution.

2. 100-fold dilution: One-step dilution. 3. 1000-fold dilution: Two-step dilution. Add 5 μL of sample to 95 μL of standard sample diluent for a 20-fold dilution. Then add 5 μL of the 20-fold diluted sample to 245 μL of standard sample diluent for a 50-fold dilution, for a total of 1000-fold dilution. 4. 100,000-fold dilution: Three-step dilution. Add 5 μL of sample to 195 μL of standard and sample diluent for a 40-fold dilution. Then add 5 μL of the 40-fold diluted sample to 245 μL of standard and sample diluent for a 50-fold dilution. Finally, add 5 μL of the 2000-fold diluted sample to 245 μL of standard and sample diluent for a 50-fold dilution, for a total dilution of 100,000 times. 5. The volume of liquid taken in each dilution step should be no less than 3 μL, and the dilution factor should not exceed 100 times. A sample volume that is too small can easily cause greater errors during mixing. Ensure thorough mixing at each dilution step to avoid foaming.

6. For very high dilution ratios, you can initially dilute with PBS, and then use the standard and sample diluent provided in the kit as the final step.

Sample Dilution Recommendations

1. Normal, fresh serum/plasma samples are recommended (Original solution - 1:2).

2. Due to individual variability, the recommended dilution ratio is for reference only. For actual testing, please estimate the sample concentration range in advance and determine the dilution ratio for the sample to be tested through preliminary experiments.

Pre-Test Preparation

1. Remove the kit from the refrigerator 30 minutes in advance and equilibrate to room temperature. 2. Dilute 25 ml of concentrated washing solution to 1 ml of working solution with double-distilled water. Return the unused portion to 4°C. 3. Standards: Add 1.0 mL of universal diluent for standards and samples to the lyophilized standard. Tighten the cap and let stand for 10 minutes to fully dissolve. Then gently mix (concentration: 100 mIU/mL). Then, serially dilute the standard solution to 100 mIU/mL, 50 mIU/mL, 25 mIU/mL, 12.5 mIU/mL, 6.25 mIU/mL, 3.13 mIU/mL, and 1.57 mIU/mL. Use the standard diluent (0 mIU/mL) as a blank well. Prepare the required amount of standard solution and set aside. It is recommended that the prepared standard solution be added to the sample within 15 minutes; it is not recommended to leave it for an extended period. 4. Biotinylated Antibody Working Solution: Before the experiment, calculate the required amount of biotinylated antibody working solution (calculated as 100 μL/well, and 100-200 μL should be added during the actual preparation). 15 minutes before use, dilute the concentrated biotinylated antibody (1:100) with biotinylated antibody diluent to the working concentration and use it on 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). 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 amount of solution. Do not return any remaining solution to the reagent bottle. Precautions: 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 relatively small and may disperse throughout the tube during transportation. Before use, centrifuge at 1000 × g for 1 minute 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. Crystals may form in the concentrated wash buffer after removal from the refrigerator. This is normal. Dissolve the crystals completely in a water bath or incubator before preparing the wash buffer (do not heat above 40°C). The wash buffer should be at room temperature before use. 4. Samples should be added quickly, preferably within 10 minutes for each addition. To ensure experimental accuracy, replicate wells are recommended. When pipetting reagents, maintain a consistent order of addition from well to well. This will ensure consistent incubation times for all wells. 5. During the wash process, pat dry any remaining wash buffer in the reaction wells on absorbent paper. Do not place filter paper directly into the reaction wells to absorb moisture. Before reading, remove any residual liquid and fingerprints from the bottom of the wells to avoid affecting the microplate reader reading. 6. The chromogen TMB should be protected from direct sunlight during storage and use. After adding the substrate, carefully observe the color change in the reaction wells. If a gradient is already evident, terminate the reaction early to prevent excessive color from affecting the microplate reader reading.

7. All test tubes and reagents used in the experiment are disposable. Reuse is strictly prohibited, as this 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 Regulations.

9. Components from different batches of the kit should not be mixed (except for the wash buffer and the reaction stop solution).

10. The enzyme labeling strips in the kit are removable; please use them in batches according to experimental needs.

Procedure

1. Before beginning the experiment, all reagents should be equilibrated to room temperature and prepared in advance. When diluting reagents or samples, mix thoroughly and avoid foaming. If the sample concentration is too high, dilute it with sample diluent to bring it within the assay range of the kit. 2. Add 100 μL of the standard or sample to be tested (if the sample requires dilution, 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 from 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 from the plate (or wash 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 discard any 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 actual 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 experimental 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: Subtract the OD value of the blank well from the OD value of each standard and sample. If replicate wells are used, the average value should be used for calculation. 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). For intuitive visualization of the results, the graphs present raw data rather than logarithmic values. The OD values of the standard curve may vary due to differences in experimental conditions (such as operator, pipetting technique, plate washing technique, and temperature). The standard curve provided is for reference only. Experimenters need to establish a standard curve based on their own experiments. The OD value of the used sample can be used to calculate the sample concentration on the standard curve, and then multiplied by the dilution factor to obtain the actual concentration of the sample. It is recommended to use professional curve drawing software, such as Curve Expert.

Note:This figure is for reference only

Intraplate precision (precision within the assay): CV%<8%

Three samples of known concentration were tested on 1 plate for 20 Inter-plate precision (CV%): <10% Three samples of known concentration were tested 40 times on three different ELISA plates to assess inter-plate precision.

Recovery rate

Recovery rate experiments were performed by adding rat FSH of known concentration to different samples to obtain the recovery rate range and average recovery rate.

Linearity

The samples spiked with rat FSH were diluted 2-fold, 4-fold, 8-fold, and 16-fold for recovery experiments, and the recovery rate range was obtained.