Mouse IFN-α2(Interferon alpha-2) ELISA Kit
Mouse IFN-α2(Interferon alpha-2) ELISA Kit
Price:
Regular price
$519.05 USD
Regular price
Sale price
$519.05 USD
Unit price
per
For shipping services or bulk orders, you may request a quotation.
Secure checkout with
View full details
Product Details
Product Details
Product Specification
| Usage | I. Required Equipment and Reagents: 1. Microplate reader (450 nm wavelength filter) 2. 37°C incubator (CO2 incubator for cells is not recommended) 3. Automatic plate washer or multichannel pipette/5 mL dropper (for manual plate washing) 4. Precision single-channel (0.5-10 μL, 5-50 μL, 20-200 μL, 200-1000 μL) and multichannel pipettes (pipettes must be calibrated before use). 5. Sterile EP tubes and disposable pipette tips. 6. Blotting paper and sample reservoir. 7. Deionized or distilled water. II. Sample Collection and Storage: The following sample processing steps are simplified. 1. Serum: Incubate whole blood samples at room temperature for 2 hours or at 2-8°C overnight. Centrifuge at 1000×g for 20 minutes and remove the supernatant. The sample can be tested immediately or aliquoted for single use and frozen at -20°C or -80°C. 2. Plasma: EDTA-Na2O2/K2O2 is recommended as the anticoagulant. Centrifuge at 1000×g for 15 minutes at 2-8°C within 30 minutes of sample collection and remove the supernatant. It can be tested immediately or divided into aliquots and frozen at -20°C or -80°C according to the amount used once. 3. Tissue samples Tissue samples are generally made into tissue homogenates. The processing method is as follows: (1) Place the target tissue on ice, wash it with pre-cooled PBS buffer (0.01M, pH=7.4) to remove residual blood, weigh it and set aside. (2) Grind the tissue homogenate with lysis buffer on ice. The volume of lysis buffer added depends on the weight of the tissue. Generally, 9mL of lysis buffer is used for every 1g of tissue fragments. It is also recommended to add protease inhibitors to the lysis buffer, such as 1mM PMSF. (3) It can be further processed by ultrasonic disruption or repeated freeze-thaw (during ultrasonic disruption, an ice bath is required to cool down; repeated freeze-thaw method can be repeated twice). (4) Centrifuge the prepared homogenate at 5000×g for 5 minutes, and keep the supernatant for detection. Or aliquot and freeze at -20°C or -80°C according to the amount used once. (5) According to experimental needs, the total protein concentration of the tissue homogenate sample can be measured first to facilitate data analysis. The BCA method is recommended. Generally, the total protein concentration is adjusted to 1-3 mg/mL for ELISA detection. Some tissue samples, such as liver, kidney, and pancreas, contain high concentrations of endogenous peroxidase. When the sample concentration is high, they will react with the chromogenic substrate and produce false positives. You can try to use 1% H2O2 to inactivate for 15 minutes before testing. Note: PBS buffer is commonly used for lysis buffer, or medium-strength RIPA lysis buffer is used. When using RIPA lysis buffer, the pH value needs to be adjusted to pH 7.3. Avoid using components containing NP-40, Triton X-100, and DTT, as they will seriously inhibit the work of the kit. It is recommended to use 50mM Tris+0.9%NaCI+0.1%SDS, pH 7.3, which can be prepared by yourself. 4. Cell culture supernatant Collect the supernatant, centrifuge at 2500rpm for 5min at 2-8°C, and collect the clarified cell culture supernatant. Use it for detection immediately, or divide it into aliquots and freeze it at -80°C according to the amount used once. 5. Cell lysis buffer (1) Collection and lysis of suspended cells: Centrifuge at 2500rpm for 5min at 2-8°C to collect the cells. Then add pre-cooled PBS and mix gently to wash, centrifuge at 2500rpm for 5min at 2-8°C to collect the cells. Add 0.5-1mL cell lysis buffer and an appropriate amount of protease inhibitor (such as PMSF, working concentration 1mmoL/L), place on ice, and lyse for 30min-1h, or use ultrasonic disruption. (2) Collection and lysis of adherent cells: Aspirate the supernatant and wash three times with pre-cooled PBS. Add 0.5-1mL cell lysis buffer and an appropriate amount of protease inhibitor (such as PMSF, working concentration 1mmoL/L), and gently scrape the adherent cells with a cell scraper. Transfer the cell suspension to a centrifuge tube, place it on ice, and lyse for 30min-1h, or use ultrasonic disruption. (3) During the cell lysis process, you can use the tip of the pipette to blow or shake the centrifuge tube intermittently to fully lyse the protein. If a sticky substance appears, it is DNA, which can be broken by ultrasonication. (Or use an ultrasonic 3-5mm probe, power 150-300W, ultrasonically treat the sample on ice, work for 1-2 seconds, stop for 30 seconds, and repeat 3-5 cycles.) (4) After lysis or ultrasonic disruption is completed, centrifuge at 2-8°C, 10000rpm for 10min, transfer the supernatant to an EP tube, and use it immediately for detection, or divide it into aliquots and freeze it at -80°C for later use. Note: Precautions are the same as for tissue samples. 6. Other Biological Samples Centrifuge the sample at 1000×g for 20 minutes at 2-8°C. Collect the supernatant and use it immediately for testing, or aliquot the amount for single use and freeze at -80°C for later use. III. Other Sample Precautions: 1. Blood collection tubes should be disposable, endotoxin-free tubes. Avoid using hemolytic or hyperlipidemic samples. 2. Optimal Sample Storage Conditions: Storing at 2-8°C for less than 5 days, at -20°C for no more than 6 months, and at -80°C for no more than 2 years. After these times, store in liquid nitrogen. When thawing frozen specimens, to minimize damage to the sample by ice crystals (0°C), rapidly thaw in a 15-25°C water bath. After thawing, centrifuge to remove the precipitate and mix thoroughly before testing. 3. The detection range of the kit is not equivalent to the concentration range of the analyte in the sample. If the analyte concentration in the sample is too high or too low, please dilute or concentrate the sample appropriately. 4. If the sample being tested is unique and reference data is unavailable, preliminary experiments are recommended to verify its validity. 5. The recombinant protein may not be compatible with the capture or detection antibodies in the kit, resulting in non-detection. IV. Recommended Sample Dilution Scheme: Please refer to the included package insert or contact us for information on samples, dilution ratios, and background information. Matrix components in serum/plasma can affect test results; dilution with Sample Diluent should be at least 2-fold (1/2). If your model group samples require a different dilution ratio, please refer to the following general dilution scheme (this scheme is for non-replicate wells. If replicate wells are required, multiply the sample and diluent volumes by the number of replicate wells): 2-fold (1/2) dilution: One-step dilution. Add 60uL of sample to 60uL of Sample Diluent and mix gently. 5-fold dilution (1/5): One-step dilution. Add 24uL of sample to 96uL of sample diluent and mix gently. 10-fold dilution (1/10): One-step dilution. Add 12uL of sample to 108uL of sample diluent and mix gently. 20-fold dilution (1/20): One-step dilution. Add 6uL of sample to 114uL of sample diluent and mix gently. 50-fold dilution (1/50): One-step dilution. Add 3uL of sample and 47uL of saline (i.e., 0.9% sodium chloride) to 100uL of sample diluent and mix gently. 100-fold dilution (1/100): One-step dilution. Add 3uL of sample and 177uL of saline to 120uL of sample diluent and mix gently. 1000-fold dilution (1/1000): Two-step dilution: First dilute 50-fold (use normal saline for all dilutions in this step), then dilute 20-fold. Mix gently. 10,000-fold dilution (1/10,000): Two-step dilution: First dilute 100-fold (use normal saline for all dilutions in this step), then dilute 100-fold. Mix gently. 100,000-fold dilution (1/100,000): Three-step dilution: First dilute 50-fold, then dilute 20-fold (use normal saline for the first two steps), then dilute 100-fold. Mix gently. Note: For each dilution step, dispense at least 3 μL of solution, and the dilution factor should not exceed 100-fold. Mix thoroughly at each dilution step to avoid foaming. V. Pre-Assay Reagent Preparation: Remove the test kit from the refrigerator 20 minutes in advance and equilibrate to room temperature (18-25°C). If the kit is to be used multiple times, only remove the ELISA strips and standards needed for the current experiment. The remaining ELISA strips and standards should be stored according to the specified conditions. 1. Wash Solution: Dilute 30 ml of concentrated wash solution (15 ml for 48T) to 750 ml (375 ml for 48T) with deionized or distilled water (recommended ultrapure water with a resistivity of 18 MΩ) and mix thoroughly. Alternatively, dilute an appropriate amount of concentrated wash solution to 25 times the volume as needed for the experiment and mix thoroughly. Return the unused solution to 2-8°C. If crystals form in the concentrated wash solution, heat it in a 40°C water bath (the heating temperature should not exceed 50°C) until the crystals completely dissolve. Mix thoroughly before use. It is best to use the prepared wash solution within the same day. Any remaining solution can be stored at 2-8°C for up to 48 hours. 2. Standards: (1) Centrifuge the freeze-dried standard tube at 10,000×g for 1 minute. Mark it as Zero tube. (2) Add 1 mL of sample diluent to the freeze-dried standard tube, tighten the tube cap, let it stand at room temperature for 2 minutes, and gently mix it by inverting it several times (or add 1 mL of sample diluent, let it stand for 1-2 minutes, and then mix it with a low-speed vortex for 3-5 seconds). Centrifuge at 1,000×g for 1 minute and collect the liquid at the bottom of the tube. (3) Gradient dilution: Take another 7 EP tubes and mark them as 1/2, 1/4, 1/8, 1/16, 1/32, 1/64 and blank. First, add 0.3 mL of sample diluent to each EP tube. Then take 0.3 mL of Zero tube standard solution and add it to the 1/2 tube and mix thoroughly. Transfer 0.3mL of the standard solution from the 1/2 tube to the 1/4 tube and mix thoroughly. Transfer another 0.3mL of the standard solution from the 1/4 tube to the 1/8 tube and mix thoroughly, and so on. Note that the blank EP tube contains only the sample diluent. At this point, the concentrations of the standards in the eight EP tubes, from the zero tube to the blank tube, are 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, 31.25pg/mL, 15.625pg/mL, 7.813pg/mL, and 0pg/mL, respectively.  3. Biotin-antibody working solution: Prepare within 30 minutes before the experiment and prepare it immediately before use. It is not suitable for long-term storage. (1) Calculate the total volume of working solution required: 100ul/well×number of wells. (It is best to prepare 100uL-200uL more than the total volume) (2) Centrifuge at 1000×g for 1 minute to collect the concentrated biotin-antibody at the bottom of the tube. (3) Dilute the concentrated biotin-antibody with antibody diluent at a ratio of 1/100 and mix thoroughly. (For example, add 10uL of concentrated biotin-antibody to 990uL of antibody diluent) 4. HRP-streptavidin (SABC) working solution: Prepare within 30 minutes before the experiment, prepare immediately before use, and not suitable for long-term storage. (1) Calculate the total volume of working solution required: 100uL/well×number of wells. (It is best to prepare 100uL-200uL more than the total volume.) (2) Centrifuge at 1000×g for 1 minute to collect the concentrated SABC at the bottom of the tube. (3) Dilute the concentrated SABC with SABC diluent at a ratio of 1/100 and mix thoroughly. (For example, add 10uL of concentrated SABC to 990uL of SABC diluent) VI. Summary of operation steps:  Wash the plate: Wash the plate twice. Do not soak. Step 2: Add 100uL of biotin-antibody working solution, apply the cover film, and incubate at 37°C for 60 minutes. Wash: Wash the plate three times, soaking for 1 minute each time. Step 3: Add 100uL of HRP-streptavidin (SABC) working solution, apply the cover film, and incubate at 37°C for 30 minutes. Wash: Wash the plate five times, soaking for 1 minute each time. Step 4: Add 90uL of TMB chromogenic substrate. Apply the cover film, and incubate at 37°C for 10-20 minutes (please use the TMB chromogenic control method). Step 5: Add 50uL of reaction stop solution. Immediately read the OD450 value at 450nm and calculate. VII. Detailed Procedure: When diluting samples and reagents, mix them thoroughly. It is recommended to create a standard curve for each test. 1. Assign standard, sample, and blank wells and record their positions. To minimize experimental error, it is recommended to set up duplicate wells for standards and samples. 2. Sample Addition: Add 100 μL of each gradient standard to the standard wells, 100 μL of appropriately diluted test sample to the sample wells, and 100 μL of sample diluent to the blank wells. Apply the overlay and incubate at 37°C for 90 minutes. (Add the solution to the bottom of the microplate and gently shake to mix, avoiding contact with the tube walls and causing bubbles.) 3. Wash the plate twice: Remove the overlay, aspirate or shake off the liquid in the ELISA plate, and tap the plate 2-3 times on clean absorbent paper. Add 350 μL of wash buffer to each well without soaking, discard the liquid in the wells, and tap the plate 2-3 times on absorbent paper. Repeat this wash step twice. 4. Add Biotin-Antibody Working Solution: Add 100 μL of Biotin-Antibody Working Solution to each well. Apply the cover film and incubate at 37°C for 60 minutes. 5. Wash the plate 3 times: Remove the cover film, aspirate or shake off the liquid in the plate, and tap the plate 2-3 times on clean absorbent paper. Add 350 μL of Wash Buffer to each well, soak for 1 minute, discard the liquid in the well, and tap the plate 2-3 times on absorbent paper. Repeat the wash step 3 times. 6. Add HRP-Streptavidin (SABC): Add 100 μL of SABC working solution to each well. Apply the cover film and incubate at 37°C for 30 minutes. (At the same time, place the entire bottle of TMB in a 37°C incubator to equilibrate.) 7. Wash the plate 5 times: Remove the cover film and wash the plate 5 times with Wash Buffer, following the procedure in step 5. 8. Add TMB Chromogenic Substrate: Add 90 μL of TMB Chromogenic Substrate to each well, apply the cover film, and incubate at 37°C in the dark for 10-20 minutes. Turn on the microplate reader and preheat for 15 minutes. (Note: Do not use the sample reservoir used to prepare HRP conjugates. The reaction time can be shortened or extended depending on the actual color development, but should not exceed 30 minutes. Terminate the reaction when a clear blue gradient appears in the standard wells. The color intensity should not be too weak or too strong. For precise control of the color development method, please refer to the relevant documents and QR code on page 2 of the instruction manual.) 9. Add Stop Solution: After color development, do not discard the liquid in the wells. Add 50 μL of Stop Solution to each well. The color will immediately change from blue to yellow. Add the Stop Solution in the same order as for adding the TMB substrate. 10. Measure OD: Immediately read the OD450 value at 450 nm using a microplate reader. (If your microplate reader has a selectable calibration wavelength, set it to 570nm or 630nm. The calibration reading is the OD450 value minus the OD570 or OD630 value. This method corrects and removes the OD value of non-chromogenic substances, resulting in more accurate test results. If your microplate reader does not have a 570nm or 630nm wavelength, use the raw OD450 value.) VIII. Calculation of Results: 1. Take the average OD450 value of the replicate wells for the standard and sample (using the raw OD450 value or the calibration reading) and subtract the OD450 value of the blank well for the calculated value. 2. Use the four-parameter equation 4PL to plot the standard curve, with concentration as the horizontal axis and OD450 value as the vertical axis (excluding the blank well values when plotting). You can also use the plotting software provided with your microplate reader (such as SkanIt RE software for Thermo FC microplate readers) or Curve Expert 1.3 or 1.4 professional software to plot the standard curve. 3. Substitute the sample OD450 value into the standard curve to calculate the sample concentration. If the sample has been diluted, multiply by the dilution factor. IX. Explanation of Different Methods for Drawing a Standard Curve: 1. Linear Plot: One axis represents the antigen concentration, and the other represents the OD450 reading. The R² value is typically used to determine the good fit; values greater than 0.99 indicate a very good fit. However, linear plots often compress data points at the lower end of the curve, leading to inaccurate calculations. 2. Semi-logarithmic Plot: This helps offset the compression caused by linear plots. A semi-logarithmic plot uses the logarithm of the concentration versus the reading. This method typically produces an S-shaped curve with a more even distribution of data points. 3. Logarithmic/Double-log Plot: This provides good linearity in the low to mid-range concentration range. However, linearity can be lost at the high end of the range. 4. Four- or Five-Parameter Equation (4PL or 5PL) Curves: This method is more complex and takes into account additional parameters, such as maxima and minima, requiring more complex calculations. 4PL assumes symmetry around the inflection point, while 5PL accounts for asymmetry and is generally more suitable for immunoassays. If your software allows, 4-PL and 5-PL curves are suitable for most ELISA calibration standard curves. X. Experimental Data and Standard Curve: This product has been tested and meets the performance specifications in the user manual. (Laboratory humidity 20%-60%, temperature 18°C-25°C). Before color development, equilibrate TMB to 37°C. After adding it to the ELISA plate wells, incubate at 37°C in the dark for 15 minutes. ) Due to differences in specific experimental environments and operations, the following experimental data and standard curve are for reference only. Experimenters should establish a standard curve based on their own experiments.
 Intra-plate precision: Low, medium, and high concentration samples were each tested 20 times on the same ELISA plate. Inter-plate precision: Low, medium, and high concentration samples were each tested 20 times on three ELISA plates.
A certain amount of IFN-α2 was spiked into the sample, and the recovery was calculated by comparing the measured value with the expected amount of IFN-α2 in the sample.
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
