Rat TRAP ELISA Kit

style="width: 20.042%; height: 22px;">89-103

Usage	I. Required Equipment: 1. Microplate reader (450nm) 2. High-precision pipettes and pipette tips: 0.5-10uL, 5-50uL, 20-200uL, 200-1000uL 3. 37°C incubator 4. Distilled or deionized water II. Sample Preparation and Requirements: 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 a pilot experiment to determine the actual concentration in the sample. If the analyte concentration in the sample is too high or too low, dilute or concentrate the sample appropriately. If the sample type is not listed in the instructions, it is recommended to conduct a pilot experiment to verify the validity of the test. Serum: Collect whole blood in a serum separator tube and place it at room temperature for 2 hours or at 2-8°C overnight. Then, centrifuge at 1000×g for 20 minutes. Remove the supernatant and store it at -20°C or -80°C. Avoid repeated freezing and thawing. Plasma: Collect the sample using EDTA or heparin as an anticoagulant. Within 30 minutes of collection, centrifuge at 1000×g for 15 minutes at 2-8°C. Remove the supernatant and test it. Alternatively, store it at -20°C or -80°C. Avoid repeated freezing and thawing. Tissue homogenate: Rinse the tissue with pre-chilled PBS (0.01M, pH=7.4) to remove any 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., 1g of tissue sample to 9mL of PBS. The specific volume can be adjusted according to 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 the tissue cells, the homogenate can be sonicated or repeatedly freeze-thawed. Finally, centrifuge the homogenate at 5000×g for 5-10 minutes and remove the supernatant for analysis. Cell Culture Supernatant: Centrifuge at 1000×g for 20 minutes. Remove the supernatant and analyze it immediately, or store it at -20°C or -80°C, but avoid repeated freeze-thaw cycles. Cell Lysis Buffer: Adherent cells are gently washed with pre-chilled PBS, then trypsinized and harvested by centrifugation at 1000×g for 5 minutes. Suspension cells can be directly harvested by centrifugation. Wash the collected cells three times with pre-chilled PBS and resuspend in 150-200 μL of PBS per 1 × 10^6 cells (protease inhibitors are recommended; if the cell count is very low, the PBS volume can be reduced appropriately). Disrupt the cells by repeated freeze-thaw cycles or sonication. Centrifuge the extract at 1500 × g for 10 minutes at 2-8°C, and remove the supernatant for testing. Other biological samples: Centrifuge at 1000 × g for 20 minutes, and remove the supernatant for testing. Sample Appearance: The sample should be clear and transparent, and suspended matter should be removed by centrifugation. Sample Storage: Samples collected for testing within one week can be stored at 4°C. If testing is not possible, aliquot the sample into a single-use aliquot 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. III. Sample Dilution Scheme: Please estimate the sample concentration range in advance. If your test sample requires dilution, the reference dilution scheme is as follows: 100-fold dilution:One-step dilution. Add 5 μL of sample to 495 μL of universal diluent for a 100-fold dilution. 1000-fold dilution:Two-step dilution. Add 5 μL of sample to 95 μL of universal diluent for a 20-fold dilution. Then, add 5 μL of the 20-fold diluted sample to 245 μL of universal diluent for a 50-fold dilution, for a total dilution of 1000-fold. 100,000-fold dilution:Three-step dilution. Add 5 μL of sample to 195 μL of universal diluent and dilute it 40-fold. Then, add 5 μL of the 40-fold diluted sample to 245 μL of universal diluent and dilute it 50-fold. Finally, add 5 μL of the 2,000-fold diluted sample to 245 μL of universal diluent and dilute it 50-fold, for a total dilution of 100,000-fold. For each dilution step, the sample volume should be at least 3 μL, and the dilution factor should not exceed 100-fold. Mix thoroughly during each dilution step to avoid foaming. IV. Pre-Test Preparation: 1. Remove the test kit from the refrigerator 10 minutes in advance and equilibrate to room temperature. 2. Prepare a 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 250 ng/mL). Then dilute to the following concentrations: 250 ng/mL, 125 ng/mL, 62.5 ng/mL, 31.25 ng/mL, 15.62 ng/mL, 7.81 ng/mL, 3.9 ng/mL, and 0 ng/mL. Serial dilution method: Take seven EP tubes and add 500 μL of universal diluent to each tube. Pipette 500 μL of the 250 ng/mL standard working solution into the first EP tube and mix thoroughly to make a 125 ng/mL standard working solution. Repeat this procedure for subsequent tubes. The last tube serves as a blank well; there is no need to pipette liquid from the penultimate tube. See the figure below for details. 3. Preparation of biotinylated detection antibody working solution: 15 minutes before use, centrifuge 100 μL concentrated biotinylated detection antibody at 1000 × g for 1 minute. Dilute the 100 μL concentrated biotinylated detection antibody to a working concentration of 1 μL with universal diluent (e.g., 10 μL concentrated + 990 μL universal diluent). Prepare fresh solution for use. 4. Preparation of Enzyme Conjugate Working Solution: 15 minutes before use, centrifuge 100 μL of concentrated enzyme conjugate at 1000 × g for 1 minute. Dilute the 100 μL concentrated enzyme conjugate to a 1 × working concentration with universal diluent (e.g., 10 μL of concentrate + 990 μL of universal diluent). Prepare immediately. 5. Preparation of 1 × Wash Solution: Dissolve 10 mL of 20 × Wash Solution in 190 mL of distilled water. (Concentrated wash solution removed from the refrigerator may crystallize; this is normal. Allow to stand at room temperature until the crystals have completely dissolved before preparing.) 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 a film sealer 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 plate. This will minimize matrix effects on the test results. The sample concentration should be multiplied by the dilution factor when calculating the final sample concentration. It is recommended to run replicates for all samples and standards.) 3. Add Biotinylated Detection Antibody: Remove the plate and discard the liquid. Do not wash. Add 100 μL of biotinylated detection antibody working solution directly to each well. Cover with a film sealer and incubate at 37°C for 60 minutes. 4. Wash: Discard the liquid and 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. Add Enzyme Conjugate Working Solution: Add 100 μL of enzyme conjugate working solution to each well, cover with a film, and incubate at 37°C for 30 minutes. 6. Wash: Discard the solution and wash the plate five times according to the procedure in step 4. 7. Add Substrate: Add 90 μL of substrate (TMB) to each well, cover with a film, and incubate at 37°C in the dark for 15 minutes. 8. Add Stop Solution: Remove the 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 a correction value. Plot a standard curve for the four-parameter logistic function on double-logarithmic graph paper with concentration as the horizontal axis and OD value as the vertical axis. 2. If the sample OD value is above the upper limit of the standard curve, dilute the sample appropriately and re-measure. Multiply the sample concentration by the corresponding dilution factor when calculating the sample concentration. VII. Kit Performance: 1. Repeatability: Intra-assay coefficient of variation (CV) less than 10%, inter-assay coefficient of variation (CV) less than 10%. 2. Recovery: Rat TRAP was spiked into selected healthy serum, plasma, and cell culture supernatant at three different concentrations, and the recovery was calculated. Sample Type Range (%) Average Recovery (%) Serum (n=8) 84-101 96 Plasma (n=8) 92-105 102 Cell culture supernatant (n=8) 97-108 105
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	87-108	105-115
	Average recovery rate	94	97	108

Sensitivity 1.7 ng/mL Species Reactivity Rat 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 a capture antibody against rat anti-tartrate acid phosphatase (TRAP). 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 rat anti-tartrate acid phosphatase (TRAP) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. Synonym Rat Acid Phosphatase 5, Tartrate Resistant (TRAP) ELISA Kit Detection Type Double antibody sandwich method Composition

Name	9 6 T Configuration  style="height: 10px; width: 29.98%; text-align: center;" width="29.9800%">Remarks
Pre-coated 96-well enzyme plate	8 holes×12 strips	None
Standard	2 bottles	Dilution according to the instructions
Universal diluent	2×20mL	None
Concentrated biotinylated detection antibody 100×	120μL	Concentrated enzyme conjugate 100×	120μL	Dilution according to the instructions
20×Washing solution	2×10mL	Dilute according to instructions
Substrate (TMB)	10mL	None
Stop solution	6mL	none
Sealing film	4 sheets	None
Instructions	1 serving	None

Background Tartrate-resistant acid phosphatase (TRAP), also known as ACP5, is a glycosylated monomeric metalloproteinase expressed in mammals. It has a molecular weight of approximately 35 kDa, an alkaline isoelectric point (7.6-9.5), and optimal activity under acidic conditions. TRAP is synthesized as a latent proenzyme and is activated by proteolytic cleavage and reduction. It differs from other mammalian acid phosphatases in its resistance to tartrate inhibition and molecular weight. Storage Temp. Unopened test kit, stored at 4°C, has a shelf life of 6 months. Test Range 3.90-250ng/mL Applications Serum, plasma, tissue homogenates and other biological fluids