A kind of human lactoferrin double-antibody sandwich ELISA kit
An enzyme-linked immunoassay and human lactoferrin technology, which is applied in the biological field, can solve the problems of low antibody sensitivity and specificity, and cannot meet the requirements of human lactoferrin, and achieve high sensitivity and specificity.
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Embodiment 1
[0022] This example is used to illustrate the screening of human lactoferrin monoclonal antibody pairs.
[0023] The protein sequence of human lactoferrin (hLF) is shown in NCBI Genbank sequence number: U95626. Human lactoferrin was obtained according to the method in the literature (Penghua Yang, et al., Cattle Mammary Bioreactor Generated by a Novel Procedure of Transgenic Cloning for Large-Scale Production of Functional HumanLactoferrin., PLoS ONE, 2008, 3(10), e3453) (hLF) transgenic dairy cows.
[0024] Intramuscular injection of prolactin needles (purchased from Xi'an Caotan Pharmaceutical Factory, the infertile cow lactation needles, the product is boxed, 10 No. I needles, 3 No. II needles) to transgenic dairy cows for lactation, of which No. I needles were injected intramuscularly continuously 10 days, once a day. One No. II needle was injected on the 13th, 15th, and 17th days respectively. From the 7th day of injection, the udders of the transgenic cows were massag...
Embodiment 2
[0033] Carry out the HRP (horseradish peroxidase) labeling operation of the second monoclonal antibody (human lactoferrin monoclonal antibody 3B4, preservation number is CGMCC NO.10595) according to the product manual of Pierce HRP labeling kit (Cat No31488), obtain Horseradish peroxidase-conjugated secondary monoclonal antibody.
[0034] Enzyme-linked immunosorbent assay (ELISA) detection steps are as follows: (1) coating: with the phosphate buffer that contains 0.08 volume % Tween-20 as coating buffer, the first monoclonal antibody (human lactoferrin monoclonal antibody) The cloned antibody 5C5, whose preservation number is CGMCC NO.10594), was diluted to 5 μg / mL with coating buffer, added to an enzyme-linked plate at 100 μL / well, placed at 37°C for 2 hours, and washed 3 times with coating buffer; (2) blocking: add 150 μL / well of blocking solution (phosphate buffer containing 0.08% by volume Tween-20 and 6% by weight of casein), placed at 37°C for 1 hour, washed with coating...
Embodiment 3
[0039] 140 milks known to be negative for human lactoferrin were collected (including 100 non-transgenic cows collected from a dairy farm in Dachang, Hebei, 20 transgenic cows with human lysozyme in a dairy farm at China Agricultural University and 20 human α-lactalbumin transgenic cows located in the dairy farm of China Agricultural University), and 22 known positive human lactoferrin milk (collected from the dairy farm located in China Agricultural University, transgenic human lactoferrin genetic cows), a total of 162 samples.
[0040] With reference to the same method of Example 2, the sample to be tested is detected, wherein the sample to be tested is listed in Table 2, and the OD value obtained by corresponding detection is also shown in Table 2
[0041] Table 2
[0042]
[0043] According to the data in Table 2, it can be seen that the detection of the milk of transgenic cows with transgenic human lactoferrin in the present invention has a sensitivity of 1000 times o...
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