Monkey anti-PEG IgM ELISA Kit
Monkey anti-PEG IgM ELISA Kit
Product Details
Product Details
Product Specification
| Usage | I. Sample Collection and Storage 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 may be tested immediately or aliquoted into single-use amounts and frozen at -20°C or -80°C. 2. Plasma: EDTA-Na₂/K₂ is the recommended anticoagulant. Centrifuge at 1000×g for 15 minutes at 2-8°C within 30 minutes of sample collection and remove the supernatant. The sample may be tested immediately or aliquoted into single-use amounts and frozen at -20°C or -80°C. For information on the use and selection of other anticoagulants, please refer to the Sample Preparation Guide. 3. Tissue Samples: Tissue samples are generally prepared as a homogenate using the following procedures: 1. Place the target tissue on ice and wash with pre-chilled PBS (0.01M, pH 7.4) to remove any residual blood. Weigh the sample and set aside. 2. Grind the tissue homogenate on ice using lysis buffer. The volume of lysis buffer added depends on the weight of the tissue; generally, 9 ml of lysis buffer is used per 1 g of tissue fragment. It is recommended to add a protease inhibitor, such as 1 mM PMSF, to the lysis buffer. 3. The sample can be further processed by ultrasonication or repeated freeze-thaw cycles (cooling should be performed in an ice bath during ultrasonication; repeated freeze-thaw cycles can be repeated twice). 4. Centrifuge the homogenate at 5000 × g for 5 minutes. Retain the supernatant for analysis. Alternatively, aliquot the sample into single-use portions and freeze at -20°C or -80°C. 5. Depending on experimental needs, the total protein concentration of tissue homogenate samples can be measured first to facilitate data analysis. The BCA assay is recommended. Generally, the total protein concentration should be adjusted to 1-3 mg/ml for ELISA testing. Certain tissue samples, such as liver, kidney, and pancreas, contain high concentrations of endogenous peroxidase. This can react with the chromogenic substrate at high sample concentrations, resulting in false positives. Try inactivating the sample with 1% H2O2 for 15 minutes before testing. Note: Lysis buffer is typically PBS, or a medium-strength RIPA lysis buffer should be used. When using RIPA lysis buffer, adjust the pH to 7.3. Avoid using components containing NP-40, Triton X-100, and DTT, as these can severely inhibit the kit's performance. We recommend using 50mM Tris + 0.9% NaCl + 0.1% SDS, pH 7.3. 4. Cell Culture Supernatant: Collect the supernatant and centrifuge at 2500 rpm for 5 minutes at 2-8°C. Collect the clarified cell culture supernatant. Use immediately for analysis or aliquot and freeze at -80°C for later use. 5. Cell Lysis Buffer 1. Harvesting and Lysing Suspended Cells: Centrifuge at 2500 rpm for 5 minutes at 2-8°C to harvest cells. Add pre-chilled PBS, gently mix, and wash. Centrifuge at 2500 rpm for 5 minutes at 2-8°C to harvest cells. Add 0.5-1 ml of cell lysis buffer and an appropriate amount of protease inhibitor (e.g., PMSF, working concentration 1 mmol/L). Place on ice and lyse for 30 minutes to 1 hour, or disrupt with sonication. 2. Harvesting and Lysing Adherent Cells: Aspirate the supernatant and wash three times with pre-chilled PBS. Add 5-1 ml of cell lysis buffer and an appropriate amount of protease inhibitor (e.g., PMSF, working concentration 1 mmol/L). Gently scrape adherent cells with a cell scraper. Transfer the cell suspension to a centrifuge tube, place on ice, and lyse for 30 minutes to 1 hour, or disrupt with sonication. 3. During cell lysis, use a pipette tip to pipette or shake the centrifuge tube intermittently to fully lyse the proteins. If a sticky mass appears, it is DNA, which can be fragmented using ultrasound. (Alternatively, use a 3-5mm ultrasonic probe at 150-300W power. Sonicate the sample on ice, on for 1-2 seconds, off for 30 seconds, for 3-5 cycles.) 4. After lysis or ultrasonication is complete, centrifuge at 10,000 rpm for 10 minutes at 2-8°C. Transfer the supernatant to an EP tube and use immediately for testing, or aliquot the sample into a single-use aliquots and freeze at -80°C until further use. Note: The same precautions apply as for tissue samples. 6. Other Biological Samples: Centrifuge the sample at 1000×g for 20 minutes at 2-8°C. Collect the supernatant for immediate testing, or aliquot into single-use amounts and freeze at -80°C until further use. Other Sample Precautions 1. Blood collection tubes should be disposable, endotoxin-free tubes. Avoid using hemolyzed 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 use for testing. 3. The detection range of the test kit does not equate to the concentration range of the analyte in the sample. If the concentration of the analyte 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 no reference data is available, it is recommended to conduct a preliminary experiment to verify its validity. II. Recommended Sample Dilution Scheme Matrix components in serum/plasma can affect test results. Sample dilution should be at least 2-fold (1/2) with Sample Diluent! If your model group samples require a different dilution ratio, please refer to the following general dilution scheme (this scheme is for testing without replicates. If replicates are required, multiply the sample and diluent volumes by the number of replicates): 2-fold (1/2): One-step dilution. Add 60µl of sample to 60µl of Sample Diluent and mix gently. 5-fold (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 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 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 saline for the first two steps), then dilute 100-fold. Mix gently. Note: Use at least 3 μL of solution for each dilution step, and the dilution factor should not exceed 100-fold. Mix thoroughly at each dilution step to avoid foaming. 3. 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 will be used multiple times, remove only the ELISA strips and standards needed for this experiment. The remaining ELISA strips and standards should be stored according to the specified conditions. 1. Wash Solution: Dilute 30ml of concentrated wash solution (15ml for 48T) to 750ml (375ml for 48T) with deionized or distilled water (recommended ultrapure water with a resistivity of 18MΩ) and mix thoroughly. Alternatively, dilute the concentrated wash solution to 25 times its volume as needed for the experiment and mix thoroughly. Return any unused solution to a 2-8°C container. 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 are completely dissolved. Mix thoroughly before use. It is best to use the prepared wash solution the same day. Any remaining volume can be stored at 2-8°C for no more than 48 hours. 2. Standards: 1. Centrifuge the standard tube at 1000×g for 1 minute, allowing the liquid to flow to the bottom of the tube. Label this tube as "Zero tube." 2. Serial dilution: Prepare seven separate EP tubes and label them 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. Add 0.3ml of the standard solution from the Zero tube to the 1/2 tube and mix thoroughly. Add another 0.3ml of the standard solution from the 1/2 tube to the 1/4 tube and mix thoroughly. Add 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 sample diluent. At this point, the concentrations of the standards in the eight EP tubes, from the Zero tube to the Blank tube, are 20ng/ml, 10ng/ml, 5ng/ml, 2.5ng/ml, 1.25ng/ml, 0.625ng/ml, 0.312ng/ml, and 0ng/ml, respectively. Note: The dissolved standard in tube 0 should be stored at 2-8°C and used within 12 hours.The diluted working solutions of other gradient standards should be used within 2 hours. 3. HRP-Antigen Working Solution: Prepare within 30 minutes before the experiment. Prepare immediately before use. Not suitable for long-term storage. 1. Calculate the total volume of working solution required: 100 μl/well × number of wells. (It is best to prepare 100-200 μl more than the total volume.) 2. Centrifuge at 1000 × g for 1 minute to collect the concentrated HRP-antigen at the bottom of the tube. 3. Dilute the concentrated HRP-antigen 1/100 with Antigen Diluent and mix thoroughly. (For example, add 10 μl of concentrated HRP-antigen to 990 μl of Antigen Diluent). 4. Procedure: Thoroughly mix samples and reagents when diluting. It is recommended to create a standard curve for each test. 1. Set up the standard, sample, and blank wells and record their positions. To reduce experimental error, it is recommended to set up duplicate wells for standards and samples. Wash the plate twice before adding 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 film and incubate at 37°C for 90 minutes. (Add the solution to the bottom of the microplate and gently shake to mix. Avoid contact with the tube walls and foaming.) 3. Wash the plate 3 times: Remove the cover film, aspirate or shake off the liquid in the microplate, and tap the plate 2-3 times on clean absorbent paper. Add 350µl 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 this wash step 3 times. 4. Add HRP-Antigen Working Solution: Add 100 μL of HRP-Antigen Working Solution to each well. Apply the overlay and incubate at 37°C for 30 minutes. 5. Wash the plate 5 times: Remove the overlay and wash the plate 5 times with wash buffer, following the procedure in step 3. 6. Add TMB chromogenic substrate: Add 90 μL of TMB chromogenic substrate to each well, apply the film, and incubate at 37°C in the dark for 10-20 minutes. Preheat the microplate reader 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 it must not exceed 30 minutes. When a good blue gradient appears in the standard wells, the reaction can be terminated. 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.) 7. Add reaction stop solution: After color development, the liquid in the wells should not be discarded. Add 50ul of reaction stop solution to each well. The color will immediately change from blue to yellow. The order of adding the stop solution is the same as the order of adding TMB substrate. 8. Measurement of OD value: 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 value is the OD450 value minus the OD570 or OD630 value. This method can correct and remove the OD value of non-colorogenic substances, thereby obtaining more accurate test results. If the microplate reader does not have a wavelength of 570nm or 630nm, the original OD450 value can be used.)
1. Take the average OD450 value of the standard and sample replicate wells (use the original OD450 value or the calibration reading value), and then subtract the OD450 value of the blank well as the calculated value. 2. Using concentration as the horizontal axis and OD450 value as the vertical axis, use the four-parameter equation 4PL to plot a standard curve (excluding the blank well values when plotting). Alternatively, use the microplate reader's built-in plotting software (such as SkanIt RE software for Thermo FC microplate readers) or Curve Expert 1.3 or 1.4 professional software (available for free download from our website) to plot the standard curve. 3. Substitute the sample's OD450 value into the standard curve to calculate the sample concentration. If the sample has been diluted, multiply by the appropriate dilution factor. Experimental Data and Standard Curve This product has been tested by the Quality Control Department and meets the performance requirements in the user manual. (Laboratory humidity: 20%-60%, temperature: 18°C-25°C). Before color development, equilibrate TMB to 37°C. After adding 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.
 Precision 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.
Linearity The samples spiked with appropriate concentrations of Anti-PEG IgM were diluted 2-fold, 4-fold, and 8-fold to obtain the recovery range.
Stability The unopened kit was subjected to stability experiments at 37°C and 2-8°C to obtain stability data.
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| Sensitivity | 0.188ng/ml | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Species Reactivity | Monkey | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Theory | This kit uses a capture ELISA assay, with a 3-hour test duration. The ELISA plate included in the kit is pre-coated with Anti-Monkey IgM (u chain). HRP-antigen is used as the detection source. The standard and appropriately diluted test sample are added to the corresponding wells, incubated, and unbound components are washed away. HRP-antigen is then added, which binds to the Anti-PEG IgM bound to the Anti-Monkey IgM (u chain). After washing away unbound components, TMB chromogenic substrate is added. TMB develops a blue color under the catalysis of horseradish peroxidase (HRP), which turns yellow upon addition of the reaction stop solution. The OD value is measured at 450 nm using a microplate reader, and the concentration of Anti-PEG IgM in the sample is calculated by plotting a standard curve. The concentration of the target substance is directly proportional to the OD450 value. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Synonym | Monkey anti-PEG(Polyethylene glycol) IgM ELISA Kit | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Detection Type | Used for in vitro quantitative detection of Anti-PEG IgM in serum, plasma, cell culture supernatant or other biological samples. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Composition |
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