Rat Insulin ELISA Kit

5. Urine: Collect your 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. 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 the sample is to be tested within one week of collection, it 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 the sample to room temperature before the experiment.

3. If the concentration of the test substance in your sample is higher than the highest value of the standard, perform an appropriate dilution based on the actual situation (it is recommended to conduct a pilot experiment to determine the dilution factor).

II.Preparation

1. Remove the test kit from the refrigerator 30 minutes in advance and equilibrate to room temperature. 2. Dilute 25 μg/mL of concentrated wash buffer to 1 μg/mL of working solution with double-distilled water. Return any unused solution to 4°C. 3. Standards: Add 1.0 mL of Universal Standard & Sample Diluent to the lyophilized standard. Tighten the cap and let stand for 10 minutes until fully dissolved. Then gently mix (concentration: 1000 pg/mL). Then, serially dilute the standard to 1000 pg/mL, 500 pg/mL, 250 pg/mL, 125 pg/mL, 62.5 pg/mL, 31.25 pg/mL, and 15.63 pg/mL. Use the standard diluent (0 pg/mL) as a blank well. Prepare the standard sample according to your desired volume and set aside. It is recommended to add the prepared standard sample within 15 minutes and not to leave it for too long. 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 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. 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 when used.

4. Samples should be added quickly. Each addition should preferably be controlled within 10 minutes. To ensure the accuracy of the experiment, it is recommended to use duplicate wells. When pipetting reagents, keep the same addition order from one well to another. This will ensure that the incubation time of all wells is the same.

5. During the washing process, the washing solution remaining in the reaction well should be patted dry on absorbent paper. Do not place the filter paper directly into the reaction well to absorb water. Before reading, be sure to remove the residual liquid and fingerprints at the bottom to avoid affecting the reading of the microplate reader.

6. The color developer TMB should be avoided 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 the gradient is already obvious, please stop the reaction in advance to avoid the color being too dark and affecting the reading of the microplate reader.

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 washing 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). Be careful not to create bubbles. Add the sample to the bottom of the ELISA plate well, avoiding contact with the sides. Gently shake to mix. Cover the plate or seal with 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, soaking for 1-2 minutes. Spin off the liquid in the plate (or wash with a microplate washer). After the final wash, pat the plate dry on absorbent paper. 4. Add 100 μL of biotin 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, soak for 1-2 minutes, and discard the liquid in the plate (or use a plate washer to wash the plate). 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 the liquid in the plate (or use a plate washer to wash the plate). 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 as appropriate 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 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. Preheat the instrument and set the assay program before use. Calculation of Results 1. 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.

2. For ease of calculation, although concentration is the independent variable and OD value is the dependent variable, we still 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) when drawing the graph. At the same time, for the intuitiveness of the experimental results, the figure provides the original data rather than the logarithmic value. Due to different experimental operating conditions (such as operators, pipetting techniques, plate washing techniques and temperature conditions, etc.), the OD value of the standard curve will vary. The standard curve provided is for reference only, and the experimenter needs to establish a standard curve based on his own experiment. 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.

Intra-assay precision (within-assay precision): CV% <8%

Three samples of known concentration were tested 20 times on each ELISA plate to assess intra-assay precision.

Inter-assay precision (between-assay precision): CV% <10%

Three samples of known concentration were tested 40 times on each of three different ELISA plates to assess inter-assay precision.

• Recovery

Recovery experiments were performed by adding rat INS at known concentrations to different samples to obtain the recovery range and average recovery.

• 1. If the entire kit is stored at -20°C, please place it at 4°C the night before the experiment.
• 2. Concentrated wash buffer may precipitate salts if stored at low temperatures. Warm it in a water bath to aid dissolution during dilution.
• 3. A small amount of water-like substance may be present in the wells of a newly opened ELISA plate. This is normal and will not affect the experimental results.
• 4. This kit is for laboratory research and development use only and is not intended for use on humans or animals.
• 5. Reagents should be treated as hazardous substances and should be handled with care and disposed of properly.
• 6. Always wear gloves, a lab coat, and protective glasses to avoid contact between skin and eyes with the stop solution and TMB. In case of accidental contact, rinse thoroughly with water.