Immunoassay method based on fluorescein and luciferase bioluminescent reaction and application thereof
A bioluminescence, immunoassay technology, applied in analytical materials, measuring devices, instruments, etc.
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[0080] Preparation of biotinylated antibodies
[0081] Mix 4.52 mg / mL monoclonal antibody recognizing PCT with 5 mg / mL biotin at a molar ratio of 1:20, dissolve in 300 μL PBS buffer solution, shake at room temperature for 1 hour, and use a 10kD ultrafiltration centrifuge tube under the condition of 1000 r / g after the reaction Centrifuge for 10 min, then wash once with PBS, and finally dissolve the reagent in the ultrafiltration tube in 300 μL PBS, and finally store it at 4°C for future use.
[0082]In the following examples, based on the ultra-sensitive response of the luciferin-luciferase-ATP" bioluminescence system to changes in ATP concentration and the ultra-fast enzymatic catalysis speed, the bioluminescence system was used to detect alkaline phosphate in the reaction system Enzyme (AlkalinePhosphatase, ALP). This method uses ATP as the substrate of ALP, and under alkaline conditions, ALP can efficiently degrade ATP into adenosine monophosphate, and adenosine monophosphat...
Embodiment 1
[0083] Example 1 Detection of model protein based on "luciferin-luciferase-ATP" bioluminescence system (goat anti-human IgG)
[0084] 1. Draw a standard curve
[0085] (1) Human IgG was diluted 2000 times with carbonate buffer solution (pH=9.6, 0.01M) to a concentration of 10 μg / mL, coated on an ELISA plate, overnight at 4°C, and washed with PBST (containing 1‰ Tween-20 in PBS buffer) and washed 3 times; 150 μL of 3% BSA blocking solution was used to block the reaction plate, reacted at 37°C for 2 hours, and washed 3 times with PBST washing solution; the goat anti-human IgG standard solution was diluted to different concentrations, each 150 μL, react at 37°C for 1 hour; wash with PBST washing solution for 3 times, react at 37°C for 1 hour; wash with PBST washing solution for 3 times; dilute ALP-labeled rabbit anti-goat secondary antibody (1mg / mL) 1000 times, and place on the reaction plate Add 150 μL, react at 37°C for 1 hour; wash once with PBST washing solution and twice wi...
Embodiment 2
[0089] Example 2 Detection of methylamphetamine in urine based on "luciferin-luciferase-ATP" bioluminescence system
[0090] 1. Draw a standard curve
[0091] (1) Dilute the complete antigen (concentration: 2mg / mL) formed by coupling methylamphetamine to BSA 4000 times with carbonate buffer solution (pH=9.6, 0.01M), and pour it into each well of a 96-well ELISA plate Add 100 μL of the above-mentioned diluted solution, coat overnight at 4°C, wash with PBST three times, and block with 100 μL of 3% BSA at 37°C for 2h.
[0092] Dilute the methylamphetamine standard solution to a concentration of 500, 200, 100, 50, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0 ng / mL, and add it to the above ELISA plate respectively, 50 μL per well, and 50 μL of antibody (1 mg / mL diluted 1000 times) solution recognizing methamphetamine drug was added to each well, and reacted at 37° C. for 1.5 h.
[0093] Wash 4 times with PBST, then dilute ALP-labeled horse anti-mouse secondary antibody (Ab2-ALP) 2000 times, a...
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