Chloramphenicol chemiluminescence enzyme-linked immunodetection kit
A chemiluminescence enzyme and immunodetection technology, applied in the field of immunological detection, can solve the problems of the use of imported reagents, the high price of chemiluminescence instruments, and the inability to popularize the chemiluminescence immunoassay method, and achieve the effect of high sensitivity
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Embodiment 1
[0042] Example 1: Preparation of immunogen, coating agent and enzyme-labeled monoclonal antibody
[0043] (1) Chloramphenicol hapten preparation
[0044] A. Dissolve chloramphenicol in methanol, add 5% Pd / C, pass in hydrogen, keep a certain pressure, react at room temperature for 2 hours, filter to remove Pd / C, evaporate the solvent to obtain a light yellow viscous liquid, obtained chloramphenicol hapten. The molar ratio of Pd / C to chloramphenicol was 1:10. Adjust the pH value of the target substance obtained above to 1-2 with hydrochloric acid at 0-5°C, and add 0.1-1mol / L NaNO dropwise under stirring. 2 solution to make the starch potassium iodide test paper turn blue, then add 0.1-1mol / L urea solution dropwise to make the starch potassium iodide test paper light blue, then add 0.1-1mol / L NaOH solution to adjust the pH value to 7-9, and obtain the clear liquid as Solution A is ready for use.
[0045] B. Measure 21.8 mL of solution A and add it to 3 mL of DMF, pre-cool at ...
Embodiment 2
[0057] Embodiment 2: the establishment of ELISA detection method
[0058] (1) Optimization of antibody and coated antigen concentration (square matrix)
[0059] Serially dilute each coated antigen longitudinally at 160.0 μg / mL, 80.0 μg / mL, 40.0 μg / mL, 20.0 μg / mL, 10.0 μg / mL, 5.0 μg / mL, 2.5 μg / mL, 1.25 μg / mL Coat the ELISA plate with 100 μL / well, place it in a 37°C incubator for 2 hours, and then pat it dry; seal it with 150 μL / well blocking solution, place it in a 37°C incubator for 2 hours, wash the plate once, and pat it dry; add 50 μL / well A series of diluted enzyme-labeled chloramphenicol monoclonal antibodies (1:1000 to 1:512000), incubate at room temperature (20-25°C) for 15 minutes, wash the plate five times, and pat dry for the last time; add 100 μL / well of chemiluminescence solution , to measure the luminescence value. Specificity determination was carried out with the coating antigen concentration and antibody dilution having obvious gradient changes in luminescenc...
Embodiment 3
[0069] Embodiment 3: Chemiluminescent ELISA kit for detecting chloramphenicol
[0070] (1) The composition of the chemiluminescent ELISA kit for detecting chloramphenicol
[0071] A, the solid phase carrier (elisa plate) that is coated with coating former (CAP-OVA);
[0072] B, Chloramphenicol standard solution: 0pg / mL, 20pg / mL, 60pg / mL, 180pg / mL, 540pg / mL and 1620pg / mL.
[0073] C, enzyme-labeled chloramphenicol antibody solution: the monoclonal antibody prepared by immunizing animals with artificial immune antigen (CAP-BSA), and the obtained chloramphenicol antibody is labeled with horseradish peroxidase and diluted to 1: 16000 working concentration.
[0074] D, luminescent solution: A solution is tris(hydroxymethyl)aminomethane solution with luminol content of 0.01M and p-cresol content of 0.001M pH=8.8, B solution is 100mL solution containing 2.1g of citric acid, anhydrous Na 2 HPO 4 2.82g, a solution of 0.75% urea hydrogen peroxide in 0.64mL. Mix liquid A and liquid...
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