Dracorhodin enzyme linked immunization detection method and application
A technology for hematoxylin and immunization of animals, which is applied in the field of hematoxylin antibody and its preparation, can solve the problems that hematagonin has not been reported, and achieve the effect of low cost and good effect
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Embodiment 1
[0036] Embodiment 1, the synthesis of hematogenin antigen ( figure 1 )
[0037] 1. Synthesis of Compound 1
[0038] Hematine 20 mg, p-aminobenzoic hydrazide 80 mg, dissolved in 95% methanol, heated at 50°C for 24 hours with nitrogen gas, TLC traced the disappearance of raw material reaction (chloroform:methanol:ammonia=95:5:2), PTLC preliminary separation The synthesized hapten was purified, eluted with methanol, and spun to dryness.
[0039] 2. Synthesis of hematoxylin-BSA immunogen and hematoxylin-OVA coating
[0040] 0.02mmol of compound 1 synthesized was dissolved in 5ml of 0.1mol / L hydrochloric acid solution, excessive 1% sodium nitrite solution was added dropwise, and the reaction was continued under stirring at 4°C in the dark, (the addition of sodium nitrite can be carried out with starch potassium iodide test paper monitor, when the test paper turns blue-black, indicating that sodium nitrite is excessive), after the solution turns blue-black, continue to react for...
Embodiment 2
[0041] Example 2, preparation of hematoxylin antibody
[0042] (1) Balb / C mice aged 8-10 weeks were used as experimental animals.
[0043] (2) Experimental immunization dose The basic immunization is 0.1 mg per mouse, the booster immunization dose is 0.15 mg per mouse, dilute the hematoxylin-BSA immune antigen obtained in Example 1 with sterile water, add it after filtering through a sterile filter, etc. The volume of Freund's complete adjuvant is fully emulsified until it is dropped into water without spreading.
[0044] (3) The mice were immunized with the emulsified immune antigen, and the initial immunization was injected intraperitoneally, and the booster immunization was injected subcutaneously at multiple points on the back, and 0.2-0.3 ml was injected.
[0045] (4) After the basic immunization, use incomplete Freund's adjuvant to emulsify the immune antigen, the method is the same as (2), and carry out booster immunization every 2 weeks, starting after the third immun...
Embodiment 3
[0046] Embodiment 3, the detection of mouse serum titer
[0047] Seven days after the fifth immunization, blood was collected from the orbit of the mice, and after standing at room temperature for 1 hour, they were placed in a refrigerator at 4°C overnight. After centrifugation at 5000rpm for 5min, the serum was collected, and the serum titer was detected by indirect non-competitive ELISA (Table 1).
[0048] (1) Coating antigen: Dilute hematogenin-OVA to an appropriate multiple with coating buffer, add 100 μl to each well, put it in a wet box, coat it in a 37°C incubator for 3 hours, and shake dry Well coating solution, washed 5 times with PBST.
[0049] (2) Add the serum to be tested: After diluting the serum by 500, 1000, 2000, 4000, and 8000 times with the sample diluent, add 100ul to each well, put it into a wet box, incubate at 37°C for 30min, and wash 5 times with PBST .
[0050] (3) Add enzyme-labeled secondary antibody: After properly diluting the enzyme-labeled sec...
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