Human NOD-like receptor-1 (NOD-1) enzyme-linked immunoassay (ELISA)
Kit instruction manual
This reagent is for research purposes only: This kit is used to determine the content of NOD-like receptor-1 (NOD-1) in human serum, plasma and related liquid samples.
Experimental principle:
This kit uses the double antibody sandwich method to determine the level of human NOD-like receptor-1 (NOD-1) in the specimen. Microporous plates were coated with purified human NOD-like receptor-1 (NOD-1) antibody to make solid-phase antibodies, and NOD-like receptor-1 (NOD-1) was added to the microwells coated with mAb in turn. It is then combined with HRP-labeled NOD-like receptor-1 (NOD-1) antibody to form an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, the substrate TMB is added for color development. TMB is converted into blue under the catalysis of HRP enzyme, and into the final yellow under the action of acid. The color depth is positively correlated with NOD-like receptor-1 (NOD-1) in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of human NOD-like receptor-1 (NOD-1) in the sample was calculated by a standard curve.
Kit composition:
Kit composition
48 hole configuration
96-well configuration
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Instructions
1 serving
1 serving
Sealing film
2 pieces (48)
2 pieces (96)
sealed bag
1
1
Enzyme coated plate
1 × 48
1 × 96
Store at 2-8 ℃
Standard product: 135ng / L
0.5ml × 1 bottle
0.5ml × 1 bottle
Store at 2-8 ℃
Standard dilution
1.5ml × 1 bottle
1.5ml × 1 bottle
Store at 2-8 ℃
Enzyme reagent
3 ml × 1 bottle
6 ml × 1 bottle
Store at 2-8 ℃
Sample diluent
3 ml × 1 bottle
6 ml × 1 bottle
Store at 2-8 ℃
Developer A liquid
3 ml × 1 bottle
6 ml × 1 bottle
Store at 2-8 ℃
Developer B liquid
3 ml × 1 bottle
6 ml × 1 bottle
Store at 2-8 ℃
Stop solution
3ml × 1 bottle
6ml × 1 bottle
Store at 2-8 ℃
Concentrated washing liquid
(20ml × 20 times) × 1 bottle
(20ml × 30 times) × 1 bottle
Store at 2-8 ℃
Sample processing and requirements:
1. Serum: The blood will naturally coagulate at room temperature for 10-20 minutes and centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully and centrifuge again if a precipitate appears during storage.
2. Plasma: EDTA, sodium citrate or heparin should be selected as anticoagulant according to the requirements of the specimen, mixed for 10-20 minutes, and centrifuged for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again.
3. Urine: collected in a sterile tube and centrifuged for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, centrifuge again. Pleural and ascites, cerebrospinal fluid reference implementation.
4. Cell culture supernatant: When detecting secreted components, collect with a sterile tube. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. When detecting the components inside the cells, dilute the cell suspension with PBS (PH7.2-7.4), and the cell concentration will reach about 1 million / ml. Through repeated freezing and thawing, the cells are destroyed and the intracellular components are released. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again.
5. Organize the specimen: after cutting the specimen, weigh it. Add a certain amount of PBS, PH7.4. Quickly freeze and save with liquid nitrogen for later use. After the specimen melts, it still maintains a temperature of 2-8 ° C. Add a certain amount of PBS (PH7.4) and homogenize the specimen with a manual or homogenizer. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. After aliquoting, a portion is to be tested, and the rest is frozen for future use.
Steps
1. Dilution and loading of standard products: set 10 standard wells on the enzyme-coated plate, add 100 μl of standard products in the first and second wells, and then add standard products in the first and second wells 50μl of diluent, mix well; then take 100μl from the first well and the second well and add them to the third and fourth wells respectively, and then add 50μl of standard diluent to the third and fourth wells respectively, mix well; Then take 50μl each in the third and fourth wells and discard it, then add 50μl each to the fifth and sixth wells, and then add 50ul of the standard dilution solution to the fifth and sixth wells respectively, and mix well; After mixing, take 50μl from the fifth and sixth wells and add them to the seventh and eighth wells respectively. Then add 50μl of the standard dilution solution to the seventh and eighth wells respectively. Take 50μl from the eight wells and add them to the ninth and tenth wells. Then add 50μl of the standard dilution solution to the ninth and tenth wells. After mixing, take 50μl from the ninth and tenth wells and discard. (After dilution, the volume of each well is 50 μl, and the concentrations are 90 ng / L, 60 ng / L, 30 ng / L, 15 ng / L, 7.5 ng / L).
2. Add samples: set up blank wells (the blank control wells do not add samples and enzyme reagents, the rest of the steps are the same) and the sample wells to be tested. Add 40μl of sample diluent to the test sample well of the enzyme-coated plate, and then add 10μl of the sample to be tested (the final dilution of the sample is 5 times). Add the sample and add the sample to the bottom of the well of the microplate, try not to touch the wall of the well, shake gently to mix.
3. Incubation: Seal the plate with a sealing plate and incubate at 37 ° C for 30 minutes.
4. Mixing solution: Dilute 30 times (20 times of 48T) concentrated washing liquid with distilled water 30 times (20 times of 48T) and then use.
5. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with the washing liquid, let it stand for 30 seconds and then discard, repeat 5 times and pat dry.
6. Add enzyme: add 50μl of enzyme label reagent to each well, except blank well.
7. Incubation: The operation is the same as 3.
8. Washing: The operation is the same as 5.
9. Color development: add 50μl of developer A to each well, then add 50μl of developer B, mix gently, and develop at 37 ° C in the dark for 15 minutes.
10. Termination: Add 50μl of stop solution to each well to stop the reaction (at this time the blue will turn to yellow).
11. Determination: Measure the absorbance (OD value) of each well in sequence with the blank air conditioner at zero and 450 nm wavelength. The measurement should be carried out within 15 minutes after adding the stop solution.
Precautions:
1. The kit should be equilibrated at room temperature for 15-30 minutes before being taken out of the refrigerated environment. If the enzyme label coated plate is unopened, the strip should be stored in a sealed bag.
2. Crystals may be precipitated in the concentrated washing liquid, which can be heated and dissolved in a water bath during dilution, and the results will not be affected during washing.
3. The sampler should be used at each step of sample addition, and the accuracy should be regularly checked to avoid test errors. It is best to control the sampling time within 5 minutes. If there are many specimens, it is recommended to use a volley gun to add samples.
4. Please make a standard curve at the same time of each measurement, it is best to make a double hole. If the content of the test substance in the specimen is too high (the OD value of the sample is greater than the OD value of the first well of the standard well), please first dilute it with a certain multiple (n times) of the sample diluent and then determine it. When calculating, please multiply the total dilution Multiple (× n × 5).
5. The sealing film is limited to one-time use to avoid cross-contamination.
6. Please keep the substrate away from light.
7. Strictly follow the instructions, and the test results must be determined by the microplate reader.
8. All samples, washing liquids and various wastes should be treated as infectious agents.
9. The components of different batches of this reagent shall not be mixed.
10. If there is any difference with the English manual, the English manual shall prevail.
Calculation:
Taking the concentration of the standard as the abscissa and the OD value as the ordinate,
Draw a standard curve on coordinate paper, according to the OD of the sample
The value is determined by the standard curve; then multiplied by the dilution
Multiple; or calculate the standard using the concentration and OD value of the standard
The linear regression equation of the quasi-curve, the OD value of the sample
Substitute into the equation, calculate the sample concentration, and multiply by the dilution
The multiple is the actual concentration of the sample.
Kit performance:
1. The correlation coefficient R between the linear regression of the sample and the expected concentration is above 0.95.
2. The batch and approval shall be less than 9% and 11% respectively
examination range:
5ng / L –120 ng / L
Storage conditions and validity period:
1. Store the kit: 2-8 ℃.
2. Validity: 6 months
Common problems and solutions in ELISA The ELISA test has been widely used in clinic with the characteristics of high sensitivity and good specificity, but each link in the operation has a great influence on the detection effect of the test. If you do not pay attention, it may be Causes incomplete color development, flower plates and other results. I summarize the causes and solutions of the problems that often occur in various links of the operation in order to bring some inspiration to the peers and improve the quality of the experiment.
The following is an analysis of the reasons that may affect the results in the Elisa test operation, and the corresponding solutions are given. 1 Select the reagents. Select high-quality detection reagents, strictly follow the reagent instructions, and equilibrate the reagents at room temperature for 30-60 minutes before operation.
2Possible reasons for sample addition: 1) Serum or plasma specimens are separated if the sample is not well separated; 2) In manual operation, too many sample plates cause too long waiting time after the sample is placed in the incubator (especially the indoor temperature is high) 3) When the sample is added and the enzyme reagent is added, the enzyme splashes out of the hole. Solution: 1) The sample is serum: it is best to store the blood naturally for 1-2 hours, and then centrifuge it at 3000 rpm for 15 minutes; the sample is plasma: you must use a blood sample collection tube containing anticoagulant, and you must reverse it immediately after blood collection The blood collection tube is mixed 5-10 times, and after a period of time, centrifuged at 3000 rpm for 15 minutes; if it is detected within a few days, it can be placed in a 2-8 ° C refrigerator, and if it is stored, it is placed in a low temperature refrigerator at -20 ° C. 2) Put the sample into the incubator in time. 3) After adding the enzyme reagent, gently blot the surface of the enzyme plate with absorbent paper to dry. 4) If you use AT or other fully automatic sample addition, it is best to choose FAME or other post-processing equipment plus enzyme reagents. 5) When there are many specimens, please operate in batches.
3 Possible reasons for incubation: 1) No seal or cover is added during incubation, which evaporates the specimen or diluent and is adsorbed on the wall of the well, which is difficult to clean thoroughly; 2) The incubation time is artificially extended, resulting in non-specific binding around the reaction well , Difficult to clean thoroughly. Solution: 1) Stick the cover or cover; 2) strictly control the operation time according to the instructions.
4 Possible reasons for washing the plate: 1) Manually wash the plate, and the liquid between the holes crosses. 2) When using a semi-automatic plate washing machine to wash the plates, the amount of washing liquid is insufficient, resulting in incomplete plate washing; the plate washing needle is blocked and the suction is incomplete; the plate washing is not smooth, resulting in poor plate washing effect. 3) Too many reaction plates cause long waiting time for plate washing. Solution: 1) Ensure that the washing liquid is filled into the holes and the plate washing needle is unobstructed. After washing the plate, it is best to pat dry on absorbent paper (choose clean, no or less dust absorbent material); 2) Reasonably arrange, Or use more washing machines.
5 Possible reasons for color development: 1) The developer has been left for too long after the preparation or the expired developer has been used; 2) When the developer is added, it splashes out of the hole and causes the liquid to return. Solutions: 1) The developer is prepared as soon as possible before use, insisting on not using the expired developer, and the light blue TMB developer is not used by the naked eye; 2) keep the developer from flowing out when adding the sample; 3) A. Liquid B should avoid contact with metal instruments.
6 Possible reasons for termination, such as more bubbles generated when the termination solution is added, leading to an increase in false positives. Therefore, avoid adding bubbles when adding the stop solution.
7 Reading the plate, such as the bottom of the plate is not clean when reading the plate, etc. The microplate should be clean.
Therefore, ensure that the enzyme-labeled plate is not exposed to hypochlorous acid during the entire operation process; as far as possible, the ELISA detection standard should be automated to effectively improve the detection quality.
In actual operation, in addition to selecting good reagents, you must strictly follow the operation steps, at the same time make indoor quality control, inter-room quality assessment, and rigorous work style to test each specimen to ensure the quality of the test. At present, a considerable number of domestic units have automatic microplate readers, which plays an important role in realizing standardized ELISA testing and improving the quality of testing.
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