Rat Ab1-40 ELISA Kit

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).

II. 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. Standard: 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 (concentration: 1000 pg/mL). Then, perform serial dilutions 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 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 for each experiment (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; 100-200 μL should be added 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 - Pipette the required amount of solution. Do not pour any remaining solution back into the reagent bottle.

Note:

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 concentrated enzyme conjugate are relatively small 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 buffer may crystallize 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. Sample addition should be rapid, ideally within 10 minutes for each addition. To ensure experimental accuracy, replicate wells are recommended. When pipetting reagents, maintain a consistent order of addition from one well to the next. This will ensure consistent incubation times for all wells.

5. During the wash process, any remaining wash solution 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 remaining 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 changes in the reaction wells. If a gradient is already evident, terminate the reaction early to avoid excessive color changes that may affect 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. 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. Kit components from different batches must not be mixed (except for the wash solution and the reaction stop solution).

10. The ELISA strips in the kit are removable. Please use them in batches according to experimental needs.

III.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 as much as possible. If the sample concentration is too high, dilute it with sample diluent to bring it within the detection 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 ELISA plate well, avoiding contact with the well walls. Gently shake to mix. Cover the plate or cover 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, soak for 1-2 minutes, and discard the liquid from 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 from 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 from 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 any liquid from the wells and wash the plate five times. Wash each well with 200 μL of wash buffer, soaking for 1-2 minutes. 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 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 test program set up before use.

Result Calculation

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, we 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 plotting. Also, 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 a standard curve based on their own experiments. 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.

Note: This figure is for reference only

Precision

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

Three samples of known concentrations were tested 20 times on one ELISA plate to assess intra-plate precision.

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

Three samples of known concentrations were tested 40 times on three different ELISA plates to assess inter-plate precision.

Recovery rate

Recovery rate experiments were performed by adding rat Ab1-40 of known concentration to different samples to obtain the recovery rate range and average recovery rate.

Linearity

The samples spiked with rat Ab1-40 were diluted 2-fold, 4-fold, 8-fold, and 16-fold for recovery experiments, and the recovery rate range was obtained

• 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.