Product Specification| Usage | Need to bring your own test equipment
1. Microplate reader (can measure the absorption value of 450nm detection wavelength and 540nm or 570nm correction wavelength)
2. High precision liquid dispenser and disposable suction head
3. Distilled water or deionized water
4. Washing bottle (spray bottle), multi-channel plate washer or automatic plate washer
5. 500mL cylinder
One, preparation before the experiment
1. Sample collection and storage
① Cell culture supernatant: particles should be removed by centrifugation; Test the samples immediately. If the sample is not tested in time after collection, it is recommended that the sample be divided according to the dosage and stored in the refrigerator at -20 ° C to avoid repeated freezing and thawing. Samples may need to be diluted (1×) Dilute.
② Serum: Samples were collected using a serum separation tube (SST) and samples were left at room temperature for 30 minutes. The samples were centrifuged at 1000g for 15 min. Serum was immediately removed and tested immediately. If the sample is not tested in time after collection, it is recommended to repack according to a single dosage and freeze in ≤ -20℃ refrigerator to avoid repeated freezing and thawing. Samples may need to be diluted (1×) Dilute.
③ Plasma: Plasma was collected using EDTA, heparin, or citric acid as an anticoagulant, centrifuged at 1000g for 15 min within 30 min of collection, and tested immediately. If the samples are not detected in time after collection, it is recommended to separate the samples according to the single dosage and freeze them in &le. -20℃ refrigerator to avoid repeated freezing and thawing. Samples may need to be diluted (1×) Dilute.
2 Reagent preparation (Place all reagents and samples at room temperature for 15 minutes before use. It is recommended that all experimental samples and standards do double hole detection )
1× Preparation of washing solution: concentrated washing solution in the kit is 20× Mother liquor, diluted to 1× with distilled water before use; Working liquid. Example: Take 10mL concentrated washing solution +190mL distilled water to 200mL, the actual operation can be calculated first, then make up.
②1× Dilution with buffer preparation: concentrate dilution in the kit with buffer 10× Mother liquor, diluted to 1× with distilled water before use; Working solution. example: Take 3mL of concentrated dilution with buffer +27mL of distilled water to a constant volume of 30mL. In practical operation, the required dilution buffer can be calculated according to the dilution multiple of the sample, and then the preparation can be made.
③ Detection of antibody: the dry powder was centrifuged to the bottom of the tube, and 110uL dilution buffer (1×) was used. Dissolve and let stand at room temperature for 5 minutes to obtain 100× Mother liquor; Dilute to 1× before use; Working solution. Calculate the desired volume by using 100uL per well. example: 10 Wells were used, then take 10uL of 100 times the working concentration of the test antibody, using dilution buffer (1×) Constant volume to 1mL, get 1mL of 1× The working concentration of the detected antibody.
④SA-HRP: SA-HRP is 40× Mother liquor, use dilution buffer before use (1×) Dilute to make 1× Working solution, 100uL required per well. example: used 10 holes, then take 25uL of 40× Mother liquor +975uL dilution buffer (1×) Constant volume to 1mL to obtain 1× of 1mL; The working concentration of the detected antibody.
⑤ Chromogenic agent: according to 100uL per well, calculate the amount needed for this test, take out the corresponding volume of chromogenic agent, avoid light; The removed chromogenic agent is only used on the same day.
⑥ Standards: lyophilized standards with dilution buffer (1×) Redissolve, redissolve volume 1000uL, to obtain a concentration of 1000pg/mL standard mother liquor. Gently shake for at least 5 minutes, it is fully dissolved. Add 300uL dilution buffer (1×) to each dilution tube. . The standard mother liquor is diluted according to the picture below, and each tube must be fully mixed before pipetting to the next tube. The standard mother liquor without dilution can be used as the highest point of the standard curve (1000pg/mL), and the dilution buffer (1×) Can be used as the zero point of the standard curve (0pg/mL).
 2, operation steps
1. Ready for all the needed reagent and standard; 2 Remove the microplate from the sealed bag that has been balanced to room temperature, and put the unused slat back into the aluminum foil bag and re-seal it; 3. Add 300uL washing solution to the microplate, let it soak for 30 seconds, discard the washing solution and pat the microplate dry on absorbent paper, please use immediately do not let the microplate dry; 4. Add different concentrations of standard, experimental samples or quality control into the corresponding Wells, 100uL for each well. The Wells were sealed with plate adhesive and incubated at room temperature for 2 hours. 5 Suck the liquid out of the plate and wash the plate using a bottle washer, a multi-channel plate washer, or an automatic plate washer. Add 300uL of washing liquid to each well, and then suck the washing liquid out of the plate. Repeat 3 times. Every time you wash the plate, try to absorb the residual liquid to help you get a good test result. At the end of the last plate wash, please blot all the liquid in the plate or invert the plate and pat all the residual liquid in the absorbent paper; 6. Add 100uL detection antibody to each microwell. Seal the reaction Wells with sealer tape and incubate for 2 hours at room temperature; 7. Repeat the plate washing operation of step 5; 8. Add 100 ULSA-HRP to each microwell and incubate for 20 minutes at room temperature. Be careful to avoid light; 9. Repeat step 5 to wash the plate; 10. Add 100uL of color development solution to each microwell, incubate at room temperature for 5-30 minutes, pay attention to avoid light; 11. Add 50uL of termination solution to each microwell, and the color of the solution in the well will change from blue to yellow. If the color of the solution turns green or the color change is inconsistent, tap the microplate to mix the solution evenly; 12. Within 30 minutes after the termination solution is added, the absorbance value at 450nm is measured using a microplate reader and 540nm or 570nm is set as the correction wavelength. If the dual wavelength correction is not used, the accuracy of the results may be affected; 13 Calculation results: The corrected absorbance values (OD450-OD540/OD570), the compound reading were averaged for each standard and sample, and then the average zero standard OD value was subtracted. Standard curves were created by 4-parameter logic (4-PL) curve fitting using computer software. Alternatively, a curve can be generated by plotting the logarithm of the concentration of the standard against the logarithm of the corresponding OD value, and the best fit line can be determined by regression analysis. This process produces an adequate but less accurate fit to the data. If the sample is diluted, the concentration should be multiplied by the dilution.
Note: The standard curve data provided in are for reference only, and the sample content should be calculated according to the standard curve drawn in the same test.
3. Kit parameters
1. Recovery rate: Different levels of Mouse VEGF were incorporated into the cell culture medium samples and the recovery rate was determined. Recovery range in 82-110%, the average recovery rate at 94%. Sensitivity: 2. The minimum measurable Mouse VEGF dose (MDD) is generally less than 1.8 pg/mL. The lowest detectable value was calculated as the corresponding concentration based on the average of the zero absorbance values of 20 standard curves plus two standard deviations. 3. Calibration: The ELISA kit was calibrated with high purity recombinant Mouse VEGF protein expressed by Sf21. 4. Linearity: Four different samples were mixed with high concentrations of Mouse VEGF followed by dilution (1×). Linearity was determined by dilutes the samples to the detection range. 5. Specificity: both native and recombinant Mouse VEGF protein could be detected by this ELISA. The following factors in diluent (1 & times) Configure to a concentration of 50ng/mL to detect cross-reactivity with mouse VEGF. Interference with mouse VEGF was detected by incorporating 50ng/mL of the interfering factor into the intermediate range of recombinant mouse VEGF controls. No significant cross-reaction or interference was observed.
| Recombinant mouse proteins | Recombinant human proteins | | VEGF R2/Fc Chimera | VEGF121 | | VEGF115 | VEGF165 | | VEGF-B167 | VEGF165/pIGF | | VEGF-B186 | | | VEGF-D | |
4, common problem resolution1. The white board (no color), after the completion of color | NO. | Cause | Solution | | 1 | Kit stored improperly; A mixture of different kit reagent | Buy a new kit, pay attention to the storage conditions; Do not mix | | 2 | Endow low temperature, short time | If the temperature is too low, the incubation time is prolonged and the color development time is prolonged | | 3 | Wrong addition or omission of reagents | In strict accordance with the manual steps to add the correct reagents | | 4 | Used to configure the solution container not clean, or there is something wrong with the water | The use of clean containers and qualified distilled water | | 5 | In the process of washing the plate, the soaking time is long, the number of washing the plate is too much, and the impact of washing the plate is large | In strict accordance with the manual operation | | 6 | The temperature of the reagent was not uniform | All reagents were equilibrated at room temperature for 30 minutes | | 7 | Detection of antibody and/or HRP concentrations was too low | Do not dilute at will according to the instructions |
2. Flower plate (blank, negative and positive controls were normal, but the OD value of sample Wells was significantly higher) | NO. | Cause | Solution | | 1 | Fewer washing, inadequate | Wash according to instructions | | 2 | The substrate 3,3',5,5' -tetramethylbenzidine (TMB) is contaminated or exposed to metal ions or oxidants | The use of clean containers and when making up qualified distilled water; Avoid light preservation | | 3 | High incubation temperature and/or excessive incubation time | Control incubation and the enzymatic reaction temperature and time | | 4 | Sample did not change when the spear head, cause cross contamination | Change the tip of each sample | | 5 | Near the hole cross contamination | Vertical clappers, using the appropriate legal pad, avoiding the hole into confetti | | 6 | Samples are endogenous interfering substance | Possible infectious agents were speculated and treated accordingly | | 7 | Sample hemolysis, storage for too long, incomplete agglutination, contaminated by bacteria, blood vessels to add impact | Avoid hemolysis, contamination, too long storage and other phenomena |
| | Theory | Double antibody sandwich enzyme-linked immunosorbent assay was used in this kit. Specificity of anti VEGF antibody in mice pre package is on the high affinity enzyme label plate. The standard substance, test sample and biotinylated detection antibody were added into the microplate Wells. After incubation, the VEGF present in the sample combined with the solid-phase antibody and detection antibody to form immune complexes. After washing to remove not combined with material, by adding horseradish peroxidase labeled chain mildew avidin (Streptavidin - HRP). After washing, adding chromogenic substrate, dark color. The reaction was terminated by adding the termination solution, and the absorbance value was measured at 450nm wavelength (reference correction wavelength 540nm or 570nm). | | Composition | | Component | Size | Once opened, diluted or heavy soluble reagent expiration date | | Mouse VEGF Microplate | 1 | Unused slats can be stored at 2-8 ° C for up to 30 days after being sealed back in aluminum foil bags with desiccants | | Mouse VEGF standard substance | 2 | Dissolves, calculate the dosage repackaging, can be in - 20 ° C storage for 14 days | | Mouse VEGF detect antibody | 1 | Enrichment dissolved after the volume, can be in 2-8 ° C storage for 14 days | | 40×SA-HRP | 1 | 40× concentration can be stored at 2-8°C; 1 x working concentration is not recommended to save | | 10 x condense dilute with buffer | 1 | After opening, can be in 2-8 ° C stored for 30 days | | Color-substrate solution | 1 | | Stop buffer | 1 | | 20 x inspissation washing buffer | 1 | | Sealing film | 3 | Room temperature storage, to avoid contamination, do not repeat use |
| | Background | Vascular endothelial growth factor (VEGF or VEGF-A), also known as vascular permeability factor (VPF), is A potent regulator of neovascularization and angiogenesis in fetuses and adults. Vascular endothelial growth factor is PDGF family, the family proteins there are eight characteristics of conservative and cystine nodes through a reverse parallel disulfide bond dimers (4). After selective splicing, VEGF spliceosome has different amino acid lengths. VEGF120, VEGF164, VEGF188 in mice; Human VEGF VEGF121, VEGF145, VEGF164, VEGF183, VEGF189 and VEGF206. VEGF165 was the most expressed and active isoform in human, followed by VEGF121 and VEGF89. The same may be true of mice. With the exception of VEGF120 and VEGF121, the other isoforms of VEGF contain the basic heparin-binding domain and do not diffuse freely. Mice VEGF164 and corresponding protein amino acid 97% homology of rats; The homology with human or porcine VEGF was 89%, with bovine VEGF 88%, and with cat, horse, and dog VEGF 90%. VEGF is expressed in a variety of cells and tissues, including skeletal muscle and cardiomyocytes, hepatocytes, osteoblasts, neutrophils, macrophages, keratinocytes, brown adipocytes, CD34+ stem cells, endothelial cells, cellulose cells, vascular smooth muscle cells, and so on. VEGF expression is induced by hypoxia and cytokines, including IL-1, IL-6, IL-8, oncostatinM and tumor necrosis factor-α. Expression of VEGF isoforms also varies during development and in adults. "The dimer of
VEGF binds to two related tyrosine kinase receptors, VEGFR1 (also called FLt-1) and VEGFR2 (Flk-1/KDR)." Binding of VEGF to its receptor induces homodimerization and autophosphorylation of the latter. These receptors possess seven extracellular immunoglobulin-like domains. Vascular endothelial cells and some non-endothelial cells have VEGF receptor expression. "Although VEGF has the highest affinity for VEGFR1, VEGFR2 is the major factor regulating the angiogenic activity of VEGF." VEGF165 also binds to the semaphoring receptor and neuropilin-1, thereby promoting the complex formation with VEGFR2.
VEGF is famous for its participation in angiogenesis. During embryonic development, VEGF regulates the proliferation, migration and survival of endothelial cells, thereby regulating the density and volume of blood vessels, but it does not play a role in the pattern of blood vessel formation. VEGF promotes bone formation through the recruitment of osteoblasts and chondrocytes, and it is also a monocyte chemotactic factor. In the postpartum period, VEGF maintains the integrity of vascular endothelial cells and is a potent mitogen for large/small vascular endothelial cells. In adults, VEGF plays a role mainly in wound repair and in the female reproductive cycle. In disease tissues, VEGF promote the permeability of blood vessels. Thus, VEGF is involved in the metastatic process of tumors by promoting extravasation and tumor angiogenesis. Various therapeutic strategies aimed at blocking VEGF activity are being used to control VEGF-induced tumor angiogenesis. The level of circulating VEGF correlates with the severity of autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, and systemic lupus erythematosus. | | General Notes | 1. Please use the kit within the validity period.
2. Components of different kits and different batch kits should not be mixed.
3. If the sample value is greater than the highest value of the standard curve, the sample should be diluted with (1×) The samples were diluted and retested. If the cell culture supernatant sample needs to be diluted, except for the last step, diluent (1×) is used. "In addition to dilution, cell culture medium may be used for other intermediate dilutions."
4. The difference of the test results can be caused by a variety of factors, including the operation of the experimenter, the use of the pipettor, the washing technique, the reaction time or temperature, the storage of the kit, etc.
5. The termination solution in the kit is acidic. Please protect your glasses, hands, face and clothes when using it.
6. For scientific research only, not for in vitro diagnosis. | | Storage Temp. | Unopened kit, 2-8° C Storage | | Test Range | 15.6pg/mL-1000pg/mL |
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