A kind of human lactoferrin sandwich ELISA detection kit and its application
A technology of human lactoferrin and detection kits, which is applied in biological testing, measuring devices, material inspection products, etc., can solve the problems of low detection sensitivity and high detection sensitivity, and achieve the effects of high sensitivity, high purity and rapid results
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Embodiment 1
[0070] The preparation of embodiment 1hLF monoclonal antibody
[0071] 1. Preparation of mouse antibody
[0072] Select 10 healthy female Balb / c mice from the same batch of 6 weeks old as experimental animals, and randomly divide them into 2 groups, with 5 mice in the experimental group (1#, 2#, 3#, 4#, 5# mice), Control group 2. The experimental group was immunized with hLF, and PBS buffer was used instead of hLF as a negative control. Each mouse was subcutaneously injected with a total of 100 μL of antigen emulsified with adjuvant (250 μL of PBS dissolved 400 μg of antigen and mixed with 250 μL of Freund’s incomplete adjuvant, a total of 500 μL, each mouse was injected with 100 μL). The first basic immunization dose was 80 μg per mouse, and the booster immunization dose was 40 μg per mouse. Freund’s complete adjuvant was used for emulsification during basic immunization, and Freund’s incomplete adjuvant was used for emulsification during booster immunization. Booster immu...
Embodiment 2
[0179] Application of embodiment 2hLF monoclonal antibody
[0180] Western Blot detection of genetically modified milk and milk powder samples
[0181] Take genetically modified milk, milk powder and non-genetically modified milk and milk powder for testing. Concentration of milk powder: 5% (5g of milk powder per 100ml solution), and the loading volume is 20μl. The steps are the same as hLFWesternBlot detection.
[0182] The test results of the samples showed that the antibody 3D12 only reacted with genetically modified milk and milk powder, but not with non-genetically modified milk and milk powder. See Figure 4 .
Embodiment 3
[0183] The preparation of embodiment 3 polyclonal antibody
[0184] 1. Preparation of rabbit immune serum
[0185] Select 4 healthy female Japanese white rabbits from the same batch, and randomly divide them into two groups, the experimental group and the control group. Three rabbits in the experimental group are immunized with hLF, and one rabbit in the control group is immunized with PBS buffer instead of hLF. The total amount of injection is 2mL of antigen emulsified with adjuvant, 5mg per rabbit, and the third booster immunization, 5mg per rabbit, after 7 days, blood is collected from the ear vein to detect the antibody titer in the serum (indirect ELISA method), If the titer does not reach 1:10 4 , OD1.0 or more, then boost the immunization. When the antibody titer has reached 1:10 4 On the 10th day afterward, rabbit blood was collected from the heart, serum was separated, and stored at -20°C for future use.
[0186] 2. Extraction of polyclonal antibodies
[0187] Us...
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