Escherichia coli host residual protein (E.coli P) enzyme-linked immunosorbent assay ( ELISA )
Kit instruction manual
This reagent is for research use only       Purpose: This kit is used to determine the content of E. coli P in E. coli in serum, plasma and related liquid samples.
Experimental principle :
  E. coli host proteins remaining samples (E.coli P) of the present assay kit horizontal double-antibody sandwich method. The residue was purified by Protein E. coli host (E.coli P) antibody-coated microtiter plate wells the immobilized antibody, monoclonal antibody-coated wells were added protein remaining E. coli host (E.coli P), and then The antibody - antigen - enzyme - labeled antibody complex was formed by binding to an HRP- labeled E. coli P antibody, and after thorough washing, the substrate TMB was added for color development. TMB in HRP enzyme blue, yellow and eventually converted to the action of an acid. The color depth is positively correlated with the E. coli P protein in the sample . The absorbance ( OD value) was measured at 450 nm using a microplate reader , and the concentration of E. coli P in the sample was calculated from the standard curve .
Kit composition :
Kit composition | 48 hole configuration | 96- well configuration | save |
Instruction manual | 1 copy | 1 copy | Â |
Sealing film | 2 pieces ( 48 ) | 2 pieces ( 96 ) | Â |
sealed bag | 1 | 1 | Â |
Enzyme label coated plate | 1 × 48 | 1 × 96 | 2 Store at -8 ° C |
Standard: 36ng/L | 0.5ml × 1 bottle | 0.5ml × 1 bottle | Store at 2-8 ° C |
Standard dilution | 1.5ml × 1 bottle | 1.5ml × 1 bottle | Store at 2-8 ° C |
Enzyme standard reagent | 3 ml × 1 bottle | 6 ml × 1 bottle | Store at 2-8 ° C |
Sample diluent | 3 ml × 1 bottle | 6 ml × 1 bottle | Store at 2-8 ° C |
Developer A solution | 3 ml × 1 bottle | 6 ml × 1 bottle | Store at 2-8 ° C |
Developer B solution | 3 ml × 1 bottle | 6 ml × 1 bottle | Store at 2-8 ° C |
Stop solution | 3ml × 1 bottle | 6ml × 1 bottle | Store at 2-8 ° C |
Concentrated washing solution | ( 20ml × 20 times) × 1 bottle | ( 20ml × 30 times) × 1 bottle | Store at 2-8 ° C |
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Sample processing and requirements :
1. Serum: coagulation at room temperature for 10-20 minutes, 20 minutes, centrifugation (2000-3000 rev / min). The supernatant is carefully collected, and if precipitation occurs during storage, it should be centrifuged again.
2. Plasma: should be selected EDTA or citrate as an anticoagulant, mix 10-20 minutes, about 20 minutes centrifugation (2000-3000 rpm / min) according to the requirements of the specimen. The supernatant is carefully collected, and if a precipitate forms during storage, it should be centrifuged again.
3. Urine: a sterile collection tube, centrifuged at about 20 minutes (2000-3000 rev / min). The supernatant is carefully collected, and if a precipitate forms during storage, it should be centrifuged again. The chest and ascites and cerebrospinal fluid are referred to.
4. Cell culture supernatant: When detecting secreted components, collect them with a sterile tube. Centrifugation 20 min (2000-3000 rev / min). Collect the supernatant carefully. When measuring the components in the cells, the cell suspension was diluted with PBS ( pH 7.2-7.4 ), and the cell concentration reached about 1 million /ml . By repeated freezing and thawing, the cells are destroyed and the intracellular components are released. Centrifugation 20 min (2000-3000 rev / min). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again.
5. Tissue specimen: After cutting the specimen, weigh the weight. Add a certain amount of PBS , pH 7.4 . It was quickly frozen and stored in liquid nitrogen for use. The specimen still maintains a temperature of 2-8 ° C after melting . Add a certain amount of PBS ( pH 7.4 ) and homogenize the specimen by hand or homogenizer. Centrifugation 20 min (2000-3000 rev / min). Collect the supernatant carefully. One part of the package is to be tested, and the rest is frozen for use.
6. The specimens should be extracted as soon as possible after collection, and the extraction should be carried out according to the relevant literature. The experiment should be carried out as soon as possible after extraction. If the test cannot be performed immediately, the specimen can be stored at -20 °C , but repeated freezing and thawing should be avoided .
7. Samples containing NaN3 could not be detected because NaN3 inhibited horseradish peroxidase ( HRP ) activity.
Steps
1. Standard dilution loaded product: 10 are plates disposed bore hole the standard Coated ELISA, the first and second holes each with standard 100μ l, then add in the first standard, a second hole 50μ l dilution, mix; from the first hole, second hole depicting 100μ l are applied to the third and fourth apertures, then at the third, fourth holes each standard dilution 50μ l mix; then in the third hole and the fourth hole from each standard dilution 50μ l to discard, from each of 50μ l are applied to the fifth and sixth holes, and then were added to the fifth, sixth hole 5OuI solution, mix; are applied 50μ l depicting the seventh, the eighth well, and then were added 50μ l standard dilution in the seventh and eighth holes from the fifth, sixth holes, after mixing taken from the seventh, eighth holes each 50μ l was added to the ninth, tenth hole, and then were added 50μ l standard dilution holes in the ninth or tenth, seventh and the eighth hole after mixing in depicting 50μ l discarded. (Diluted sample volume of each hole are 50μ l, concentrations were 24n g / L, 16n g / L, 8n g / L, 4n g / L, 2n g / L).
2. Loading: Set blank holes separately (the blank control wells are not added with the sample and the enzyme standard reagent, the other steps are the same), and the sample holes to be tested . Coated ELISA plates before adding the sample holes sample diluent 40μ l, and then combined with the test sample 10μ l (final dilution is 5-fold). Add sample to the bottom of the wells, and try not to touch the wall of the hole and mix gently.
3. Incubation: After sealing with a sealing film , incubate at 37 °C for 30 minutes .
4. dosing: 30 (20 times the 48T) fold concentrated fold diluted solution was washed with distilled water and 30 alternate (48T of 20 times).
5. Washing: Carefully remove the sealing film, discard the liquid, dry it , fill each well with the washing liquid, let stand for 30 seconds, then discard it, repeat 5 times, and pat dry.
6. Enzyme: enzyme reagent added to each well except 50μ l, blank wells.
7. Incubation: The operation is the same as 3 .
8. Washing: The operation is the same as 5 .
9. Color development: first add color developer A50μ l , then add color developer B50μ l , gently shake and mix, and color at 37 °C for 15 minutes.
10. Termination: 50 μl of stop solution was added to each well to terminate the reaction (when the blue color turned yellow) .
Determination: The blank as zero, 4 50 nm wavelength sequentially measuring absorbance (OD) of each well. The measurement should be carried out within 15 minutes after the addition of the stop solution .
Precautions:
1 . The kit should be taken out from the refrigerated environment and should be used at room temperature for 15-30 minutes before use. If the enzyme label is unsealed after opening, the slats should be stored in a sealed bag.
2 . Washing buffer may be crystallized, can be heated in a water bath solubilization dilution, washing does not affect the results.
3 . The sampler should be used for each step, and the accuracy should be corrected frequently to avoid test errors. It is best to control the loading time within 5 minutes. If the number of specimens is large, it is recommended to use a gun.
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 substance to be tested in the specimen is too high (the OD value of the sample is larger than the OD value of the first hole of the standard well ), please first dilute the sample dilution with a certain multiple ( n times) and then measure it. When calculating, multiply the total dilution by the total dilution. Multiple (×n×5).
5 . The sealing film is intended for single use only to avoid cross-contamination.
6 . Please keep the substrate away from light.
7 . Strictly follow the instructions of the manual, the test results must be based on the microplate reader reading .
8 . All samples, washings and various wastes should be treated as infectious materials.
9 . The different batch components of this reagent must not be mixed.
10. If it is different from the English manual, the English manual shall prevail.
Calculation :
Take the standard density as abscissa, OD value of the ordinate, a standard curve is plotted on graph paper, according to the sample OD Isolated from the standard curve values corresponding concentrations; multiplied by the dilution factor; or calculated linear regression equation of the standard curve with the concentration and the OD value of the sample OD value in the equation to calculate the concentration of the sample, and then by The dilution factor is the actual concentration of the sample.
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Kit performance:
1. The linear regression coefficient of the sample and the expected concentration correlation coefficient R value is 0.95 or more.
2. Within and within the batch should be less than 9% and 11% respectively
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Storage conditions and expiration date:
1. The kit of preservation:; 2-8 ℃.
2 . Validity: 6 months
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