Tetracycline ELISA Kit
Tetracycline ELISA Kit
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Product Specification
| Usage |
I. Self-provided test equipment required for the experiment: 1.microplate reader ( 450nm ） 2.High-precision sampler and gun head: 0.5-10μL 、 5-50μL 、 20-200μL 、 200-1000μL 3. 37℃ incubator 4. Distilled or deionized water II. Sample processing and requirements: 1. The detection range of the kit is not equivalent to the concentration range of the substance to be tested in the sample, It is recommended that the concentration of the test substance in the sample be estimated by relevant literature before the experiment and the actual concentration of the sample be determined by pre-experiment. If the concentration of test substance in the sample is too high or too low, dilute or concentrate the sample appropriately. 2. If the tested sample is not among the sample types listed in the instructions, it is recommended to do a pre-experiment to verify its testing effectiveness. 3.Serum: The whole blood sample collected in the serum separation tube was placed at room temperature 2 Hours or 2-8℃ overnight, and then 1000×g centrifugation 20 Minutes, take the supernatant, or place the supernatant in -20℃ Or -80℃ Preserve, but repeated freezing and thawing should be avoided. 4. Plasma: with EDTA Or heparin as an anticoagulant, and the samples are collected after the collection 30 Within minutes at 2-8℃1000×g centrifugation 15 Minutes, take the supernatant to detect, or place the supernatant in -20℃ Or -80℃ Preserve, but repeated freezing and thawing should be avoided. 5. Tissue homogenate: using pre-cooled PBS (0.01M, pH=7.4) Rinse the tissue, remove residual blood (lysed red blood cells in the homogenate will affect the test results), and shear the tissue after weighing. Combine the shredded tissue with the corresponding volume of PBS (generally based on 1:9 The weight-to-volume ratio of, such as 1g Tissue sample corresponding to 9mL of PBS The specific volume can be appropriately adjusted according to the experimental needs and recorded. Recommended in PBS Add protease inhibitor into a glass homogenizer and grind well on ice or homogenizer. To further lyse tissue cells, the homogenate can be ultrasonically disrupted, or repeatedly freezed and thawed. Finally, the homogenate was added to 5000×g centrifugation 5~10 Minutes, take the supernatant for detection. 6. Cell culture supernatant: Please 1000×g centrifugation 20 Minutes, take the supernatant to detect, or place the supernatant in -20℃ Or -80℃ Preserve, but repeated freezing and thawing should be avoided. 7. Cell lysates: pre-cooling for adherent cells PBS Wash gently and then digest with trypsin, 1000×g centrifugation 5 Cells were collected after minutes; Suspended cells can be collected directly by centrifugation. Collected cells were pre-cooled PBS cleaning 3 times, per 1×10^6 Added to cells 150-200μL PBS Resuspension (recommended in PBS Adding a protease inhibitor; If the content is very low, it can be appropriately reduced PBS Volume) and disrupted the cells by repeated freeze-thaw or ultrasound. The extract was placed in 2-8℃ ， 1500×g centrifugation 10 Minutes, take the supernatant for detection. 8. Other biological samples: 1000×g centrifugation 20 Minutes, and the supernatant can be detected. 9. Sample appearance: The sample should be clear and transparent, and the suspended matter should be removed by centrifugation. 10. Sample preservation: if the sample is collected in 1 Those tested within the week can be stored in 4℃ If it cannot be detected in time, please divide it according to the single use amount and freeze it in -20℃ （ 1 tested within months), or -80℃ （ 6 Test within months), avoid repeated freezing and thawing, the hemolysis of the sample will affect the final test result, so the hemolytic sample is not suitable for this test. III. Sample dilution scheme: Please estimate the concentration range of the sample in advance. If your test sample needs to be diluted, refer to the following dilution protocol: Dilution 100 Times: One-step dilution. Take 5μL Sample to 495μL In general diluent, do 100 fold dilution; Dilution 1000 Times: Two-step dilution. Take 5μL Sample to 95μL In general diluent, do 20 Double diluted, and then taken 5μL 20 Double diluted samples to 245μL In general diluent, do 50 Double dilution, total dilution 1000 times; Dilution 100000 Times: Three-step dilution. Take 5μL Sample to 195μL In general diluent, do 40 Double diluted, and then taken 5μL 40 Double diluted samples to 245μL In general diluent, do 50 Double dilution, and finally take 5μL 2000 Double diluted samples to 245μL In general diluent, do 50 Double dilution, total dilution 100000 times; The amount of liquid taken at each step of dilution shall not be less than 3μL The dilution factor does not exceed 100 times. Each step of dilution needs to be mixed evenly to avoid blistering. IV. Preparation before testing: 1. Please advance 10 Minutes Remove the kit from the refrigerator and equilibrate to room temperature. 2. Preparation of standard gradient working solution: Add 1mL Universal diluent into lyophilized standard and allow to stand 15 Minutes until it is completely dissolved and then gently mix (concentration of 2000pg/mL ), followed by the following concentrations: 2000pg/mL 、 1000pg/mL 、 500pg/mL 、 250pg/mL 、 125pg/mL 、 62.5pg/mL 、 31.25pg/mL 、 0pg/mL Dilution was performed. Double ratio dilution method: take 7 branch EP Tubes, each tube added 500μL Universal diluent, 2000pg/mL Pipette from the standard working fluid 500μL To the first branch EP Mix well in tube to prepare 1000pg/mL Follow this procedure to suck and mix the standard working solution. The last tube is directly used as a blank hole, and there is no need to draw liquid from the penultimate tube, as shown in the following figure.  4. Preparation of enzyme conjugate working solution: before use 15 Minutes will 100× The enzyme conjugate was concentrated in 1000×g centrifugation 1 Minutes, in a general dilution 100× The concentrated enzyme conjugate is diluted to 1× Working concentration ( Example: 10μL Concentrate +990μL Universal diluent ) , ready for use. 5.1× Washing liquid preparation: Take 10mL 20× Washing liquid to 190mL In distilled water (the concentrated washing liquid taken out of the refrigerator may have crystallization, which is a normal phenomenon. It can be placed at room temperature and prepared after the crystallization is completely dissolved ) 。 V. Operation steps: 1. Equilibration from room temperature 10 After a minute, take out the required slats from the aluminum foil bag, and seal the remaining slats with a ziplock bag and put them back 4℃ 。 2. Sample addition: the samples or standards with different concentrations are separately determined according to 50μL Each well was added to the corresponding well, blank wells were added 50μL Universal dilution, followed by each well 50μL Biotin- Antibody working fluid. After covering the sealing plate film 37℃ Incubation 60 Minutes. （ Recommendation: Minimum dilution of sample to be tested with universal diluent 1 Then adding into the enzyme label plate for testing, Thereby reducing the influence of matrix effect on the test results, and finally multiplying the corresponding dilution factor when calculating the sample concentration. All samples to be tested and standards are recommended to set up double wells in the test). 3. Wash the plate: discard the liquid and add each well 300μL 1x Washing liquid, stand 1 Minutes, throw off the washing liquid, pat dry on absorbent paper, and repeat washing the plate 3 Time (plate washer can also be used to wash the plate). 4. Enzyme adding conjugate working solution: each well is added 100μL Enzyme conjugate working solution, covered with sealing membrane 37℃ Incubation 30 Minutes. 5. Wash the plate: discard the liquid according to the steps 3 Washing method, washing plate 5 Twice. 6. Adding substrate: Adding per well 90μL Substrate (TMB) Covered with a sealing film, 37℃ Incubation in the dark 15 Minutes. 7. Adding stop solution: Take out the enzyme label plate and directly add to each well 50μL Stop solution, immediately in 450nm The wavelength of each well was measured OD Value. VI. Calculation of experimental results: Result Judgment: 1. Calculate the average of standard and sample double wells OD Value and subtract the blank well's OD Value as a correction value. Taking the concentration as the abscissa, OD The value is the ordinate , draw a standard curve of a four-parameter logistic function on double logarithmic paper. 2. If the sample OD If the value is above the upper limit of the standard curve, the test should be retested after appropriate dilution and multiplied by the corresponding dilution factor when calculating the sample concentration.  VII. Kit performance: 1. Repeatability: Intraplate coefficient of variation is less than 10% The inter-plate coefficient of variation is less than 10% 。 2. Recovery rate: Add to selected healthy serum, plasma and cell culture supernatant 3 Different concentration levels Tetracycline , recovery was calculated.
3. Linear dilution: respectively in the selected 4 High concentration of healthy serum, plasma and cell culture supernatant was added Tetracycline Linearity was assessed by dilution within standard curve kinetics.
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| Sensitivity | 14.23pg/mL | |||||||||||||||||||||||||||||||||||
| Theory | This kit uses competitive enzyme-linked immunosorbent assay (ELISA). A sample, a standard, a Biotin-labeled antibody, and a HRP enzyme conjugate were sequentially added to microwells previously coated with Tetracycline antigen, incubated and washed in between, and developed color with a substrate TMB, which was converted to blue under the catalysis of peroxidase (HRP) and to final yellow under the action of acid. There was an inverse correlation between shade of color and Tetracycline in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the sample concentration was calculated. | |||||||||||||||||||||||||||||||||||
| Source | Universal | |||||||||||||||||||||||||||||||||||
| Synonym | General TetracyclineELISA Kit | |||||||||||||||||||||||||||||||||||
| Detection Type | Competition law | |||||||||||||||||||||||||||||||||||
| Composition |
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| Background | Tetracycline is an oral antibiotic in the Tetracycline class that is used to treat a variety of infections, including acne, cholera, brucellosis, plague, malaria, and syphilis. Common side effects include vomiting, diarrhea, rash, and loss of appetite. Use during pregnancy may cause harm to the fetus. It works by inhibiting protein synthesis by bacteria. Tetracycline inhibits protein synthesis by blocking the attachment of charged tRNA to the P-site peptide chain. Tetracycline blocks the A site, so no hydrogen bonds are formed between amino acids. Tetracycline binds to the 30S and 50S subunits of microbial ribosomes. Therefore, it prevents the formation of peptide chains. Even when the drug is withdrawn, the effect is generally not inhibitory and irreversible. | |||||||||||||||||||||||||||||||||||
| General Notes | 1. Incubate strictly according to the specified time and temperature to ensure accurate results. All reagents must reach room temperature 20-25°C prior to use. Preserve reagents in refrigeration immediately after use. 2. Incorrect washing may lead to inaccurate results. Be sure to absorb as much liquid as possible from the wells before adding the substrate. Do not allow microwells to dry out during incubation. 3. Eliminate the residual liquid and finger prints at the bottom of the board, otherwise it will affect the OD value. 4. The substrate chromogenic solution should be colorless or very light in color, and the substrate solution that has turned blue cannot be used. 5. Avoid cross-contamination of reagents and specimens to avoid wrong results. 6. Avoid direct exposure to strong light during storage and incubation. 7. Any reaction reagent cannot contact the bleaching solvent or the strong gas emitted by the bleaching solvent. Any bleaching ingredient will destroy the biological activity of the reaction reagents in the kit. 8. Expired products cannot be used, and components with different item numbers and batch numbers should not be mixed. 9. Recombinant proteins from sources other than the kit may not match the antibodies of this kit and not be recognized. 10. If it is possible to spread diseases, all samples should be managed well, and the samples and detection devices should be processed according to the prescribed procedures. |
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| Storage Temp. | Unopened kit, stored at 2-8°C, shelf life 6 months. | |||||||||||||||||||||||||||||||||||
| Test Range | 31.2-2000pg/mL | |||||||||||||||||||||||||||||||||||
| Applications | Serum, plasma, tissue homogenates, cell culture supernatants, cell lysates and other biological fluids |