Establishment And Application Of ELISA Method For Purine Alkaloid Compound Theophylline - 7 - Acetic Acid, Theacrine And Caffeine

Purine alkaloids is a kind of nitrogen-containing compounds with small molecular weight. It is widely distributed in many plants in nature. Pharmacological effects of these compounds are complex. There are a variety of compounds have been developed for clinical drugs, such as caffeine, theophylline and so on. Therefore, to establish the detection method of the content of purine alkaloids in biological samples have important significance.Currently, purine alkaloids commonly used detection method in biological samples, including HPLC, LC-MS, etc. This requires a more cumbersome and complex before sample processing, equipment is more expensive, and for high-throughput sample testing can not be done while testing. Establish enzyme-linked immunosorbent assay (ELISA) by using the purine alkaloids monoclonal antibody. A biological sample testing is simple, you can do a high-throughput, simultaneous detection of trace samples.The topics chosen for purine alkaloids theophylline-7-acetic acid as a hapten, prepared theophylline-7-acetic acid monoclonal antibody, and the establishment of a 7-TAA, theacrine, caffeine ELISA. Were detected by ELISA in sera of mice 7-TAA content, theacrine content, theacrine in mouse blood when drug distribution curves and theacrine organ in the brain of mice Asia, as well as various commercial drugs caffeine.Established the 7-TAA, theacrine, caffeine ELISA method for high-throughput, rapid detection is important.[Objective]1. Preparation of anti-theophylline-7-acetic acid monoclonal antibody.2. Established 7-TAA, theacrine and caffeine ELISA by usuing anti-theophylline-7-acetic acid monoclonal antibody.3. Detected by ELISA in sera of mice 7-TAA content, theacrine content, theacrine in mouse blood when drug distribution curves and theacrine organ in the brain of mice Asia, as well as various commercially available drugs in coffee because content.[Method]1. Synthesis of 7-TAA artificial antigen by the active ester method. Serum antibodies after vaccination. After cell fusion, get the anti-7-TAA monoclonal antibody. Ascites purified using ammonium sulfate bitterness, preparing anti 7-TAA monoclonal antibody2. Establish the ELISA of 7-TAA, theacrine and caffeine.3. Use the ELISA to detecte the 7-TAA contents in blood, the theacrine contents and caffeine contents, and compared with the results of HPLC method.4.10 male mice, according to dose 45 mg · kg-1 to mice orally administered theacrine, respectively, in 10 time points after administration, take blood 10 μL, dissolved in 90 μL phosphate buffer, using ELISA theacrine pharmacokinetics study in mouse blood.5.10 male mice, according to dose 45 mg · kg-1 to mice orally administered theacrine, decapitated 35 min after administration were taken cortex, hippocampus, hypothalamus, brain stem and cerebellum, weighing After adding 1.0 mL PBS buffer, homogenized centrifuged supernatant using ELISA assay theacrine content of mouse brain sub-organs of theacrine initial distribution of organs in the mouse brain Asia.[Results]1. Preparation of 7-TAA successful artificial antigen by MALDI-TOF-MS detection to calculate the coupling ratio of about 25.2. Successfully secreting anti-7-TAA monoclonal antibody cell lines C11B3, secreted antibody specificity and cross-reactivity with theacrine caffeine was 34.0% and 0.3%, and theophylline, theobromine,1,3,9-trimethyl acid no cross-reactivity (cross-reactivity rate <0 .01).3 Establish enzyme-linked immunosorbent assay by 7-TAA. Competitive range of linear is 46.8 ng · mL-1-3000 ng · mL-1, sensitivity 528.43 ng · mL-1, coefficient of variation between the holes difference<7.8%, the difference between the coefficient of variation board <8.5%. The average recovery was 99.59%. Mice blood test results of 7-TAA content is 1188.97 ± 44.9,812.31 ± 43.0,284.70 ± 16.5 ng · mL-1.4 Established enzyme-linked immunosorbent assay by theacrine. Competitive range of linear is 156.25 ng · mL-1-100 μg · mL-1, a sensitivity of 1547.6 ng · mL-1, coefficient of variation between the holes difference< 8.0%, coefficient of variation of the difference between the plates< 7.7%. The average recovery was 99.87%. Mice blood test results of theacrine content is 1254.61 ± 68.8,696.02 ± 33.36,439.57 ± 13.15 ng · mL-1. Detection of mice blood metabolic profile theacrine by ELISA. During 0.5 h, theacrine drug concentration in the blood reaches a peak, the second absorption peak appears at 2.0 h. Detection of theacrine distributed in the brain by ELISA, theacrine highest level among the hypothalamus.5. Established ELISA by caffeine. Competitive range of linear is 7.0 μg · mL-1-500 μg · mL-1, a sensitivity of 165.34 μg · mL-1, coefficient of variation between the holes difference < 8.7%, coefficient of variation of the difference between the plates<6.7%. The average recovery was 100.5%. The detection result of marketed drugs caffeine content is 15.003 ± 0.55,30.294 ± 0.91,32.045 ± 0.78 mg.[Conclusion]Active ester synthesis of 7-TAA artificial antigen to good effect, after cell fusion was successfully prepared anti-7-TAA monoclonal antibody, than established 7-TAA, theacrine and caffeine enzyme-linked immunosorbent assay, mice can be used to detect Serum 7-TAA content, theacrine content, theacrine in mouse blood and theacrine distribution curve when the drug in mouse brain sub-organs, as well as various commercial drugs caffeine content.