Product ID: EIA05314 working principle Content in each kit (96T) Reagents and equipment that are needed but not provided Precautions Washing method: Manually washing the plate: aspirate (not touch the wall) or pry off the liquid in the plate; pour a few layers of absorbent paper on the bench, and take a few times with the plate; use the recommended PBS or TBS wash buffer was injected into the well at least 0.35 ml and soaked for 1-2 minutes. Repeat this process several times as needed. Preparation and storage of samples If not immediately analyzed, they should be stored separately and stored frozen, and avoid repeated freezing and thawing. General Principles of Sample Dilution The user must consult the literature to understand the amount of the test factor in the sample and determine the appropriate dilution factor so that the concentration of the test factor in the diluted sample is within the range of the ELISA kit. The dilution of the sample should be recorded in detail. Reagent preparation and storage B. Biotinylated antibody working solution: Prepared within 2 hours before use. C. Preparation of avidin-peroxidase complex (ABC) working solution: Prepared within 1 hour before use. D. Preparation of TMB working fluid: Prepare within 1 hour before use. Take 1 part of TMB coloring solution A and add 1 part of TMB coloring liquid B. After mixing, it is TMB working solution. Avoid TMB working solution to avoid light preservation. Avoid contact with metal instruments during use. Operating procedure Summary of operating procedures: Add prepared samples and standards and react at 37 ° C for 90 minutes. Wash the plate twice, add biotinylated antibody working solution, and react at 37 ° C for 60 minutes. Wash the plate 3 times, add ABC working solution, and react at 37 ° C for 30 minutes. Wash the plate 5 times, add TMB coloring solution, 37 ° C reaction Add TMB Stop Solution Read OD within 30 minutes Calculate the test range of the test factor content of the specimen: 80 ng / ml → 1.25 ng / ml. REFERENCES 16 Slice CT,Veterinary Radiology,Micro Computed Tomography,Diagnostic Imaging In The Veterinary MinFound Medical Systems Co., Ltd , https://www.minfoundmed.com
Read the instructions thoroughly before use. The enzyme-linked immunosorbent kit is based on the principle of classical enzyme-linked sandwich technology and can measure rat LDL. It can only be used for research purposes and should not be used for medical diagnosis.
LDL is a blood lipoprotein, and low-density lipoprotein is composed of various substances such as cholesterol and phospholipids. LDL is a cholesterol-rich lipoprotein whose cholesterol is mainly derived from cholesterol in high-density lipoprotein transported from CE. At present, there are two ways to find the source of LDL in plasma: 1 The main pathway is transformed from VLDL metabolism; the secondary pathway is directly secreted into the blood after liver synthesis. The degradation of LDL is metabolized by the LDL receptor pathway. The covered lacuna on the surface of the cell membrane is the site of LDL receptor, ie, ApoB100 in LDL is recognized by the receptor, and LDL is bound to the receptor lacuna, and then Membrane separation forms endocytic vesicles, transmembrane H+-ATPase in endocytic vesicles, pH decreases and becomes acid, LDL is separated from receptors and fused with lysosomes, and then enzymatically hydrolyzed to produce cholesterol into transport vesicles, or It is re-esterified by ACAT to accumulate. The ELISA kit provided is a typical sandwich enzyme-linked immunosorbent assay kit (ELISA). The pre-coated antibody is a monoclonal antibody, and the detection phase antibody is a polyclonal antibody, which is labeled with biotin. The sample and the biotin-labeled antibody were sequentially added to the wells of the plate, and washed with PBS or TBS. The peroxidase-labeled avidin reaction was then added; after thorough washing with PBS or TBS, the substrate was developed with TMB. TMB is converted to blue under the catalysis of peroxidase and converted to the final yellow color of zui under the action of an acid. The depth of the color is positively correlated with the factor to be tested in the sample.
Material quantity standard 96T (2vial)
96-well plate 96T (8×12) pre-coated with antibody
Biotinylated antibody 96T (1:100)
ABC (peroxidase-labeled avidin) 96T (1:100)
Sample diluent 96T
Antibody dilution 96T
ABC Diluent 96T
TMB coloring solution A 96T
TMB color developing solution B 96T
TMB Stop Solution 96T
ELISA special washing liquid 96T (1:25)
1. 37 ° C incubator.
2. Standard specification microplate reader.
3. Automatic washing machine.
4. Single or multi-channel adjustable pipettes and their tips.
5. Distilled water, volumetric flask, etc.
6. Clean test tubes and Eppendof tubes.
1. Allow room temperature to equilibrate for at least 30 minutes before opening the kit.
2. When preparing various reagents, remember to mix!
3. When the user first uses the kit, the various reagent tubes should be centrifuged for a few minutes to allow the reagents to collect at the bottom of the tube.
4. It is recommended that all standards and samples be tested in duplicate.
5. It is necessary to strictly avoid drying of the microplate during operation. Drying will quickly deactivate the biological components on the microplate!
6. To avoid cross-contamination, avoid reusing the tips and tubes in your hand.
Automatic washing: If there is an automatic washing machine, it should be used in the formal experiment after skilled use.
Serum - Blood was collected in a clean tube, coagulated at room temperature for 2 hours or overnight at 4 ° C, centrifuged at 1000 x g for 10 minutes, and serum was collected. Immediately analyze or dispense and store at -20 °C.
Cell culture, tissue homogenate, cerebrospinal fluid, body fluid - centrifuge to remove the precipitate, immediately analyze or dispense and store at -20 °C.
A. Dilution and use of standards: Prepare within 2 hours before use. Add 500 ul of sample dilution to the lyophilized tube, dissolve thoroughly, remove 250 ul and add to 750 ul of sample dilution, mix and dilute.
The diluted standard was used within 2 hours.
1. According to the need of 0.1ml per hole to calculate the total amount (in the actual preparation should be prepared 0.1-0.2ml).
2. The working solution was prepared in a ratio of 10 ul of biotin-labeled antibody plus antibody dilution 990 ul. Mix gently.
1. According to the need of 0.1ml per hole to calculate the total amount (0.1-0.2ml should be prepared when the actual preparation).
2. The working solution was prepared in a ratio of 10 ul of avidin-peroxidase complex (ABC) plus ABC dilution 990 ul. Mix gently.
1. Determine the number of well-coated antibody plate wells required for this assay.
2. 0.1 ml of each of the diluted standards was sequentially added to a row of 7 wells, and only 1 sample of the dilution was added as a control. The treated specimens were added in 100 ul per well.
3. The plate was capped and reacted at 37 ° C for 90 minutes.
4. After the reaction, the liquid in the microplate is sucked by an automatic washing machine; or the liquid in the microplate is removed, and then the absorbent paper is photographed a few times. Wash twice.
5. The prepared biotin antibody working solution was added in order of 0.1 ml per well. The reaction was carried out at 37 ° C for 60 minutes.
6. Wash 3 times with 0.01 M TBS or 0.01 M PBS, soak for about 1 minute each time.
7. The prepared ABC working solution was added in order of 0.1 ml per well. The reaction was carried out at 37 ° C for 30 minutes.
8. Wash with 0.01 M TBS or 0.01 M PBS 5 times, soak for 1-2 minutes each time.
9. 0.1ml per well was added to the TMB working solution in turn, and the reaction was avoided at 37 °C in the dark. During the reaction, it was necessary to observe frequently. When the naked eye showed the first 3-4 holes of the standard, there was a clear gradient blue, and the difference between the 3-4 holes was not When it is obvious, TMB stop solution can be added at 0.1 ml/well. (The color reaction is not longer than 30 minutes).
10. The OD value was measured at 450 nm using a microplate reader.
11. Find the corresponding concentration on the coordinates based on the absorbance of the sample. Users can also use a variety of application software to calculate. It should be remembered that since the sample is diluted N times, its actual concentration should be ×N.
Prepare reagents, samples and standards
Sensitivity: <0.3ng/ml
Specificity: There is no cross-reactivity between the system and other cytokines.
Storage: -20°C [4°C in short term (eg two weeks)]
Validity: 12 months (-20 ° C).
Uses: For the in vitro quantitative analysis of liquid specimens (universal type).
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