Mouse TL ELISA Kit

Usage	I. Required Experimental Equipment 1. Microplate reader (450nm wavelength filter) 2. 37°C incubator (CO2 incubator for cells is not recommended) 3. Automatic plate washer or multichannel pipette/5ml dropper (for manual plate washing) 4. Precision single-channel (0.5-10µL, 5-50µL, 20-200µL, 200-1000µL) and multichannel pipettes (pipettes must be calibrated before use). 5. Sterile EP tubing and disposable pipette tips 6. Blotting paper and sample reservoir 7. Deionized or distilled water II. Sample Preparation and Requirements: Serum: Whole blood samples should be incubated 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 assayed immediately or aliquoted and stored frozen at -20°C or -80°C in a single-use aliquot. Plasma: EDTA-Na2/K2 is recommended as the anticoagulant. Within 30 minutes of sample collection, centrifuge at 1000×g for 15 minutes at 2-8°C and remove the supernatant. The sample can be assayed immediately or aliquoted and stored frozen at -20°C or -80°C in a single-use aliquot. Tissue Samples: Tissue samples are generally homogenized using the following procedures: 1. Place the target tissue on ice and wash with pre-chilled PBS (0.01M, pH 7.4) to remove residual blood. Weigh the sample for later use. 2. Grind the homogenate in lysis buffer on ice. The volume of lysis buffer added depends on the weight of the tissue. Generally, 9 ml of lysis buffer is used for every gram of tissue fragments. It is also recommended to add a protease inhibitor, such as 1 mM PMSF, to the lysis buffer. 3. Further processing can be performed by sonication or freeze-thaw cycles (cooling should be performed in an ice bath during sonication; freeze-thaw cycles can be repeated twice). 4. Centrifuge the prepared homogenate at 5000 × g for 5 minutes and retain the supernatant for testing. Alternatively, aliquot and freeze at -20°C or -80°C according to single-use amounts. 5. Depending on experimental needs, the total protein concentration of the tissue homogenate can be measured to facilitate data analysis. The BCA assay is recommended. The total protein concentration is generally adjusted to 13 mg/ml for ELISA testing. Certain tissue samples, such as liver, kidney, and pancreas, contain high concentrations of endogenous peroxidase. High sample concentrations may cause reactions with the chromogenic substrate, resulting in false positives. Try inactivating the sample with 1% H₂O₂ for 15 minutes before testing. Note: Lysis buffer is typically PBS buffer or a medium-strength RIPA lysis buffer. When using RIPA lysis buffer, adjust the pH to 7.3. Avoid using components containing NP-40, Triton X-100, and DTT, as these will severely inhibit the kit's performance. We recommend 50mM Tris + 0.9% NaCl + 0.1% SDS, pH 7.3. You can prepare your own or contact us to purchase. 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 a single use. Cell Lysis Buffer: 1. Harvesting and Lysing Suspension Cells: Centrifuge at 2500 rpm for 5 minutes at 2-8°C to collect the cells. Then, add pre-chilled PBS and gently mix to wash. Centrifuge at 2500 rpm for 5 minutes at 2-8°C to harvest the cells. Add 0.5-1 ml of cell lysis buffer and an appropriate amount of protease inhibitor (such as 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 0.5-1 ml of cell lysis buffer and an appropriate amount of protease inhibitor (such as PMSF, working concentration 1 mmol/L), and gently scrape the 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, pipette or shake the tube intermittently to fully lyse the proteins. A sticky mass is DNA, which can be disrupted with sonication. (Or use an ultrasonic probe (3-5mm) at 150-300W and sonicate the sample on ice, operating for 1-2 seconds and resting for 30 seconds, for 3-5 cycles.) 4. After lysis or sonication, centrifuge at 10,000 rpm for 10 minutes at 2-8°C. Transfer the supernatant into an EP tube and use immediately for testing, or aliquot and freeze at -80°C for a single use. Note: The same precautions apply as for tissue samples. Other biological samples: Centrifuge the sample at 1,000 × g for 20 minutes at 2-8°C. Collect the supernatant and use immediately for testing, or aliquot and freeze at -80°C for a single use. III. Other Sample Precautions1. Blood collection tubes should be disposable, endotoxin-free tubes. Avoid using hemolyzed or hyperlipidemia samples. 2. Optimal Sample Storage Conditions: Storing at 2-8°C should be less than 5 days, at -20°C should not exceed 6 months, and at -80°C should not exceed 2 years. After these times, samples should be stored 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 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, 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. IV. Sample Dilution Scheme: If your model group samples require a different dilution ratio, please refer to the following general dilution scheme (this scheme is for assays without replicates. If replicates are required, multiply the sample and diluent volumes by the number of replicates): 2-fold dilution (1/2): One-step dilution. Add 60µl of sample to 60µl of sample diluent and mix gently. 5-fold dilution (1/5): One-step dilution. Add 24µl of sample to 96µl of sample diluent and mix gently. 10-fold dilution (1/10): One-step dilution. Add 12µl of sample to 108µl of sample diluent and mix gently. 20-fold dilution (1/20): One-step dilution. Add 6µl of sample to 114µl of sample diluent and mix gently. 50-fold dilution (1/50): One-step dilution. Add 3µl of sample and 47µl of saline (i.e., 0.9% sodium chloride) to 100µl of sample diluent and mix gently. 100-fold dilution (1/100): One-step dilution. Add 3µl of sample and 177µl of saline to 120µl of sample diluent and mix gently. 1000-fold dilution (1/1000): Two-step dilution: First dilute 50-fold (using 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 (using 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 (using saline for all dilutions in the first two steps), and finally dilute 100-fold. Mix gently. Note: For each dilution step, use at least 3 μl of solution, and the dilution factor should not exceed 100x. Mix thoroughly at each dilution step to avoid foaming. 5. Pre-Assay Preparation: Remove the 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. Store the remaining ELISA strips and standards under 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, mix thoroughly, and return any unused solution to 2-8°C. If crystals form in the concentrated wash solution, heat it in a 40°C water bath (do 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 wash solution can be stored at 2-8°C for no more than 48 hours. 2. Standards: 2.1. Centrifuge the lyophilized standard tube at 10,000×g for 1 minute. Label it "Zerotube." 2.2. Add 1 mL of sample diluent to the lyophilized standard tube, tighten the cap, and let it stand at room temperature for 2 minutes. Gently mix by inverting several times (or add 1 mL of sample diluent, let it stand for 1-2 minutes, then mix by low-speed vortexing for 3-5 seconds). Centrifuge at 1,000×g for 1 minute and collect the liquid at the bottom of the tube. 2.3. Serial Dilution: Take another seven 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. Then, add 0.3 ml of the standard solution from the Zero tube to the 1/2 tube and mix thoroughly. Then, add 0.3 ml of the standard solution from the 1/2 tube to the 1/4 tube and mix thoroughly. Then, add 0.3 ml 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 Zerotube to Blank are 1000ng/ml, 500ng/ml, 250ng/ml, 125ng/ml, 62.5ng/ml, 31.25ng/ml, 15.625ng/ml, and 0ng/ml, respectively. Note: Please store the dissolved Zerotube standard at 2-8°C and use within 12 hours. Use diluted working solutions of other gradient standards within 2 hours. 3. Biotin-Antibody Working Solution: Prepare the working solution within 30 minutes before the experiment and prepare it immediately before use. It is not suitable for long-term storage. 3.1. Reconstitution: Centrifuge at 2000×g for 1 minute to collect the concentrated biotin-antibody at the bottom of the tube. Add 70 μl of purified water to the biotin-labeled antibody tube. Dissolve completely and mix thoroughly. Centrifuge at 1000×g for 1 minute and store at 2-8°C. 3.2. Calculate the total volume of working solution required: 50 μl/well × number of wells. (It is best to prepare 100-200 μl more than the total volume.) 3.3. Dilute the concentrated biotin-antibody 1/100 with Antibody Diluent and mix thoroughly. (For example, add 10 μl of concentrated biotin-antibody to 990 μl of antibody diluent.) 4. HRP-Streptavidin (SABC) Working Solution: Prepare within 30 minutes before the experiment. Prepare immediately before use. Do not store for long periods. 4.1. Calculate the total volume of working solution required: 100 μl/well × the number of wells. (It is best to prepare 100-200 μl more than the total volume.) 4.2. Centrifuge at 1000 × g for 1 minute to collect the concentrated SABC at the bottom of the tube. 4.3. Dilute the concentrated SABC 1/100 with SABC diluent and mix thoroughly. (For example, add 10 μl of concentrated SABC to 990 μl of SABC diluent.) VI. Procedure: Completely mix the sample and reagents when diluting. It is recommended to create a standard curve for each test. 1. Assign wells for standards and samples to be tested and record their positions. To minimize experimental error, it is recommended to assign duplicate wells for standards and samples. Before adding samples, wash the plate twice with wash buffer. 2. Sample Addition: Add 50 μl of standard or sample to the appropriate wells. Immediately add 50 μl of the prepared biotinylated detection antibody working solution to each well. Gently shake the plate for 1 minute to mix. Apply the cover film and incubate at 37°C for 45 minutes. (Add the solution to the bottom of the microplate, avoiding contact with the sides and bubbles.) 3. Wash the plate three times: Remove the cover film, aspirate or shake off the liquid in the plate, and tap it 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 it 2-3 times on absorbent paper. Repeat this wash step three times. 4. 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.) 5. Wash the plate five times: Remove the cover film and wash the plate five 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 cover film and incubate at 37°C in the dark for 10-20 minutes. Preheat the plate 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 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. 7. 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. The order of adding Stop Solution is the same as that of adding TMB substrate. 8. OD Measurement: Immediately read the OD450 value at 450 nm using a microplate reader. (If your microplate reader has a selectable calibration wavelength, set it to 570 nm or 630 nm. 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 570 nm or 630 nm wavelength, use the raw OD450 value.) VII. Calculation of Experimental Results: Calculation of Results: 1. Take the average OD450 value of the replicate wells for the standard and sample (use either the original OD450 value or the corrected reading). 2. Use the four-parameter equation (4PL) to plot a standard curve, with concentration as the horizontal axis and the average OD450 value as the vertical axis. 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's concentration. If the sample has been diluted, multiply by the dilution factor. Instructions for different methods of plotting the standard curve: 1. Linear Plot: One axis represents antigen concentration, and the other represents the OD450 reading. The R² value is often used to determine the good fit, with values greater than 0.99 indicating 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 concentration versus reading. This approach typically produces an S-shaped curve with a more even distribution of data points. 3. Logarithmic/Logarithmic Plot: This provides good linearity in the low to mid-range concentration range, but can be prone to loss of linearity at the upper end of the range. 4. Four- or Five-Parameter Equation (4PL or 5PL) Curve: This approach is more complex, taking into account additional parameters, such as maxima and minima, and therefore requires 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 will be suitable for most ELISA calibration standard curves. 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 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. 8. Kit performance:
Category	Intra-plate coefficient of variation			Inter-plate coefficient of variation
Sample	1	2	3	1	2	3
Quantity	20	20	20	20	20	20
Average (ng/ml)	30.53	122.5	508	124.3	124.3	502
Standard deviation	1.35	5.93	24.23	1.39	5.82	21.79
Coefficient of variation (%)	4.41	4.84	4.77	4.53	4.68	4.34

2. Recovery rate:
Sample Type Range (%) Average Recovery (%) Serum (n=8) 85-99 93 Plasma (n=8) 90-102 98 Cell culture supernatant 87-102 95
3. Linear dilution:
Sample type 1:2 1:4 1:8 Serum((n=10) 86-99% 86-98% 87-98% EDTA plasma (n=10) 92-103% 90-100% Heparin plasma (n=10) 86-103% 88-100% 85-98% 4. Stability:
Stability tests were performed on the unopened kit at 37°C and 2-8°C to obtain stability data.

Test kit (n=5)	37°COne month	2-8°CSix months
Average value (%)	80	95-100

Sensitivity 9.375ng/ml Species Reactivity Mouse Theory This kit uses a competitive ELISA assay, with a 2-hour test duration. The microplate provided in the kit is pre-coated with TC. The TC to be tested in the sample or standard competes with the TC pre-coated on the microplate for binding to a fixed amount of biotin-labeled anti-TC antibody. Free components are washed away. HRP-streptavidin (SABC) is added, and biotin specifically binds to streptavidin to form an immune complex. Unbound conjugates are washed away, and TMB chromogenic substrate is added. TMB exhibits a blue color when catalyzed by horseradish peroxidase (HRP) and turns yellow after addition of the reaction stop solution. The OD value is measured at a wavelength of 450 nm using a microplate reader. Using curve equation software, the OD450 value of the sample is compared with the standard curve to determine the concentration of TC in the sample. The concentration of the target substance is inversely proportional to the OD450 value. Synonym Mouse TC (Total cholesterol) ELISA Kit Detection Type Competition Law Composition

Name	9 6 T Configuration  style="height: 10px; width: 29.98%; text-align: center;" ELISA Plate	8 wells x 12 strips	Place unused wells in a zippered aluminum foil bag, add desiccant, and seal for storage. Store at 2-8°C for 1 month and at -20°C for 6 months. Lyophilized Standards 2 vials Place unused standards in the desiccant bag. Store at 2-8°C for 1 month and at -20°C for 6 months. Concentrated Biotin-Antibody (Lyophilized) 1 tube 100X Concentrated HRP-Streptavidin width="19.0000%"> 120uL	2-8°C (light protection)
TMB chromogenic substrate	1 bottle 10ml
Purified water	200ul	2-8°C
Sample diluent	1 bottle of 20ml
Antibody diluent	1 bottle of 10ml
22px;">	Reaction stop solution		1 bottle of 10ml
Concentrated detergent 25X	1 bottle of 30ml
Laminate	5 pictures