Mouse RETN ELISA Kit

Usage	I. Sample Handling and Requirements: 1. The detection range of the kit is not equivalent to the concentration range of the analyte in the sample. Before the experiment, it is recommended to estimate the analyte concentration in the sample based on relevant literature and conduct preliminary experiments to determine the actual concentration. If the analyte concentration in the sample is too high or too low, please dilute or concentrate the sample appropriately. 2. If the sample type to be tested is not listed in the instructions, it is recommended to conduct preliminary experiments to verify the validity of the assay. 3. Serum: Whole blood collected in a serum separator tube should be allowed to stand at room temperature for 2 hours or at 2-8°C overnight. Then, centrifuge at 1000 × g for 20 minutes and remove the supernatant. Alternatively, the supernatant can be stored at -20°C or -80°C, but avoid repeated freezing and thawing. 4. Plasma: Collect the sample using EDTA or heparin as an anticoagulant. Centrifuge the sample at 1000 × g for 15 minutes at 2-8°C within 30 minutes of collection. The supernatant can be assayed or stored at -20°C or -80°C. Avoid repeated freezing and thawing. 5. Tissue Homogenization: Rinse the tissue with pre-chilled PBS (0.01M, pH 7.4) to remove residual blood (lysed red blood cells in the homogenate may affect the test results). Weigh the tissue and mince it. Add the minced tissue to the appropriate volume of PBS (generally a 1:9 weight-to-volume ratio, e.g., 1 g of tissue sample to 9 mL of PBS. The specific volume can be adjusted based on experimental needs and recorded. It is recommended to add protease inhibitors to the PBS) in a glass homogenizer and grind thoroughly on ice or in a homogenizer. To further lyse tissue cells, the homogenate can be sonicated or repeatedly frozen and thawed. Finally, centrifuge the homogenate at 5000 × g for 5-10 minutes and remove the supernatant for analysis. 6. Cell Culture Supernatant: Centrifuge at 1000 × g for 20 minutes. Remove the supernatant for analysis or store at -20°C or -80°C, but avoid repeated freezing and thawing. 7. Cell Lysis Buffer: Gently wash adherent cells with ice-cold PBS, then trypsinize and harvest by centrifugation at 1000 × g for 5 minutes. Suspension cells can be harvested directly by centrifugation. Wash harvested cells three times with ice-cold PBS and resuspend in 150-200 μL of PBS per 1 × 106 cells (it is recommended to add protease inhibitors to the PBS; if the cell count is very low, reduce the PBS volume appropriately). Disrupt the cells by repeated freezing and thawing or sonication. Centrifuge the extract at 1500 × g for 10 minutes at 2-8°C, and remove the supernatant for testing. 8. Other biological samples: Centrifuge at 1000 × g for 20 minutes, and remove the supernatant for testing. 9. Sample Appearance: Samples should be clear and transparent, and suspended matter should be removed by centrifugation. 10. Sample Storage: Samples collected for testing within one week can be stored at 4°C. If testing cannot be performed promptly, aliquot the sample into a single-use amount and freeze at -20°C (for testing within one month) or -80°C (for testing within six months). Avoid repeated freeze-thaw cycles. Hemolysis of the sample can affect the final test results, so hemolyzed samples are not suitable for this test. II. Sample Dilution Protocol: Please estimate the sample concentration range in advance. If your test sample requires dilution, the reference dilution protocol is as follows: 100-fold dilution: One-step dilution. Add 5µL of sample to 495µL of universal diluent for a 100-fold dilution. 1000-fold dilution: Two-step dilution. Add 5uL of sample to 95uL of universal diluent and dilute 20-fold. Then add 5uL of the 20-fold diluted sample to 245uL of universal diluent and dilute 50-fold, for a total dilution of 1000-fold. 100,000-fold dilution: Three-step dilution. Add 5uL of sample to 195uL of universal diluent and dilute 40-fold. Then add 5uL of the 40-fold diluted sample to 245uL of universal diluent and dilute 50-fold. Finally, add 5uL of the 2000-fold diluted sample to 245uL of universal diluent and dilute 50-fold, for a total dilution of 100,000-fold. The volume of each dilution step should be no less than 3uL, and the dilution factor should not exceed 100-fold. Mix thoroughly at each dilution step to avoid foaming. III. Required experimental equipment: 1. Microplate reader (450nm) 2. High-precision pipettes and tips: 0.5-10uL, 5-50uL, 20-200uL, 200-1000uL 3. 37°C incubator 4. Distilled or deionized water IV. Preparation before testing: 1. Please remove the test kit from the refrigerator 10 minutes in advance and equilibrate to room temperature. 2. Preparation of Gradient Standard Working Solution: Add 1 mL of Universal Diluent to the lyophilized standard. Let stand for 15 minutes to completely dissolve, then gently mix (concentration 1000 pg/mL). Then dilute to the following concentrations: 1000 pg/mL, 500 pg/mL, 250 pg/mL, 125 pg/mL, 62.5 pg/mL, 31.25 pg/mL, 15.62 pg/mL, and 0 pg/mL. Serial Dilution Method: Add 500 μL of Universal Diluent to each of seven EP tubes. Pipette 500 μL of the 1000 pg/mL standard working solution into the first EP tube and mix thoroughly to make a 500 pg/mL standard working solution. Repeat this procedure for subsequent tubes. The last tube is directly used as a blank well, and there is no need to draw liquid from the penultimate tube, as shown in the figure below. 3. Preparation of biotinylated antibody working solution: Centrifuge the concentrated biotinylated antibody at 1000×g for 1 minute 15 minutes before use. Dilute the 100× concentrated biotinylated antibody to a 1× working concentration with universal diluent (e.g., 10 μL concentrate + 990 μL universal diluent). Prepare the solution for immediate use. 4. Preparation of enzyme conjugate working solution: 15 minutes before use, centrifuge 100 μL concentrated enzyme conjugate at 1000 μg for 1 minute. Dilute 100 μL concentrated HRP enzyme conjugate to 1 μL working concentration with universal diluent (e.g., 10 μL concentrate + 990 μL universal diluent). Prepare and use immediately. 5. Preparation of 1× Wash Buffer: Dissolve 10 mL of 20× Wash Buffer in 190 mL of distilled water. (Concentrated Wash Buffer removed from the refrigerator may crystallize; this is normal. Allow to stand at room temperature until the crystals have completely dissolved before preparing the solution.) V. Procedure: 1. Remove the desired strips from the aluminum foil bag after equilibration at room temperature for 10 minutes. Seal the remaining strips in a ziplock bag and return them to 4°C. 2. Sample Addition: Add 100 μL of sample or standard of varying concentrations to the corresponding wells. Add 100 μL of Universal Diluent to the blank wells. Cover with film and incubate at 37°C for 60 minutes. (Recommendation: Dilute the sample to be tested at least 1-fold with universal diluent before adding it to the ELISA plate for testing. This will minimize the impact of matrix effects on the test results. The sample concentration should be multiplied by the corresponding dilution factor when calculating the final sample concentration. It is recommended to run replicates for all samples and standards.) 3. Adding Biotinylated Antibody: Remove the ELISA plate and discard the liquid. Do not wash. Add 100 μL of Biotinylated Antibody Working Solution directly to each well. Cover with a film sealer and incubate at 37°C for 60 minutes. 4. Washing: Discard the liquid. Add 300 μL of 1x Wash Solution to each well. Let stand for 1 minute. Shake off the wash solution and pat dry on absorbent paper. Repeat this process three times (a plate washer can also be used). 5. Adding Enzyme Conjugate Working Solution: Add 100 μL of Enzyme Conjugate Working Solution to each well. Cover with a film sealer and incubate at 37°C for 30 minutes. 6. Wash: Discard the liquid and wash the plate five times as in step 4. 7. Add substrate: Add 90 μL of substrate (TMB) to each well, cover with a film sealer, and incubate at 37°C in the dark for 15 minutes. 8. Add stop solution: Remove the ELISA plate and add 50 μL of stop solution directly to each well. Immediately measure the OD value of each well at 450 nm. VI. Calculation of experimental results: 1. Calculate the average OD value of the standard and sample replicates and subtract the OD value of the blank well as the correction value. Plot the standard curve of the four-parameter logistic function on double-logarithmic graph paper, using concentration as the horizontal axis and OD value as the vertical axis. 2. If the sample OD value is higher than the upper limit of the standard curve, dilute the sample appropriately and retest. Multiply the sample concentration by the corresponding dilution factor when calculating the sample concentration. Typical Data and Reference Curves: The following data and curves are for reference only. Experimenters should establish a standard curve based on their own experiments. Concentration (pg/mL) 1000 500 250 125 62.5 31.25 15.62 0 OD  value 2.26 1.49 1.03 0.7 0.46 0.32 0.23 0.08 Corrected OD  Value 2.18 1.41 0.95 0.62 0.38 0.24 0.15 -
Sample type	Range (%)	Average recovery rate (%)
Serum(n=8)	84-101	96
Plasma (n=8)	92-105	102
Cell culture supernatant (n=8)		105

3. Linear Dilution: Add high concentration of mouse Resistin to 4 selected healthy mouse serum, plasma and cell culture supernatant, and dilute within the dynamic range of the standard curve to evaluate linearity.

Dilution ratio	Recovery rate (%)	Serum	Plasma	Cell culture supernatant
1:2	range	84-95	88-96	90-110
	Average recovery rate	91	93	96
1:4	Range	89-103	87-108	105-115
	Average recycling rate	94	98	108

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8. Problem Analysis:

Problem Description	Possible Causes	Corresponding Countermeasures
Incorrect dilution of the standard	Ensure that the standard is dissolved and diluted according to the recommended method
Inaccurate pipetting	Calibrate the pipette regularly and check the seal of the pipette tip
	Weak or no color	Incubation time is insufficient	Ensure incubation time
Incorrect incubation temperature		Incubate at the recommended temperature
Check the pipette and strictly follow the operating procedures
Incorrect dilution		Check the reagent dilution procedure
Mix enzyme conjugate and substrate and check by colorimetric reaction
OD value is low	Microplate reader settings are incorrect	Check instrument wavelength
	No stop solution was added.	Add appropriate amount of stop solution.
		Waiting time for plate reading was too long.	Read the plate in time.
		The sample content is too high. 32.0773%; height: 22px; text-align: center;" height="32">Determine the appropriate dilution factor through preliminary experiments
		The sample content is too low	Determine the appropriate dilution factor through preliminary experiments
The background is high		The color developing solution is contaminated	Replace the color developing solution
	The color developing time is too long	Control the color developing time
	The antibody or enzyme conjugate is diluted incorrectly	Use the recommended dilution method
	Enough washing times and adding sufficient detergent

Theory This kit uses a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Sample, standard, biotin-labeled detection antibody, and HRP conjugate are sequentially added to microwells pre-coated with mouse resistin capture antibody. After incubation and washing, the sample is developed using the substrate TMB. TMB converts to blue under the catalysis of HRP and to yellow under the action of acid. The intensity of the color is positively correlated with the amount of mouse resistin in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. Source Mouse Synonym Mouse Resistin (RETN) ELISA Kit Description Resistin is a 108-amino acid propeptide that is cleaved before secretion. Resistin present in peripheral blood is a dimer composed of two 92-amino acid polypeptides linked by a disulfide bond at Cys26. Resistin may be a key link between obesity and insulin resistance. It acts on skeletal muscle cells, stem cells, and adipocytes, reducing their sensitivity to insulin. On the other hand, obesity significantly suppresses resistin expression, but resistin also appears to inhibit adipogenesis. Composition

Name	9 6 T Configuration  style="height: 10px; width: 29.98%; text-align: center;" width="29.9800%">Storage Conditions after Opening
Pre-coated 96-well ELISA plate	8 wells ×12 strips	-20℃
Pre-coated 96-well ELISA plate	8 wells ×12 strips	-20℃
	2 vials	-20℃, use on the same day after reconstitution
Universal Diluent	2×20mL	2-8℃
Concentrated biotinylated detection antibody (100×)	120uL	-20℃
Concentrated enzyme conjugate (100×)	120uL	-20℃ (avoid light)
2×10mL	2-8℃
Substrate (TMB)	10mL	2-8℃ (protect from light)
Stop solution	6mL	2-8℃
Sealing film	4 pictures	None
Instructions	1 copy	None

General Notes 1. Strictly adhere to the specified incubation time and temperature to ensure accurate results. All reagents must be at room temperature (20-25°C) before use. Refrigerate reagents immediately after use.
2. Improper plate washing may result in inaccurate results. Ensure that all liquid in the wells is aspirated thoroughly before adding substrate. Do not allow the wells to dry out excessively during the entire process.
3. Clean the bottom of the plate of any residual liquid and fingerprints, as this may affect the OD value.
4. The substrate developer solution should be colorless; substrate solution that has turned blue should not be used.
5. Avoid cross-contamination of reagents and samples to prevent erroneous results.
6. Avoid direct exposure to strong light during storage and incubation.
7. Do not expose any reagents to bleaching agents or the strong fumes emitted by bleaching agents. Any bleaching agent will destroy the biological activity of the reagents in the kit.
8. Do not use expired products, and do not mix components from different product numbers and batches.
9. Recombinant proteins from sources other than the kit may not be compatible with the antibodies in this kit and will not be recognized.
10. If there is a possibility of disease transmission, all samples should be managed properly and the samples and testing devices should be handled according to the prescribed procedures. Storage Temp. Store at 2-8°C. Test Range 15.62-1000 pg/mL