Phycoerythrin-labeled anti-STI (soybean trypsin inhibitor) antibody, preparation method and use thereof
A phycoerythrin and labeling technology, applied in chemical instruments and methods, instruments, peptides, etc.
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Embodiment 1
[0025] Embodiment 1: the preparation of soybean protein STI standard substance
[0026] Grind soybeans into powder with a high-speed drug grinder, pass through a 60-mesh sieve, weigh 100 g of powder, add 300 mL of acetone and stir for 2 h, filter with silk cloth, and discard the acetone layer. The residue was washed with 80% ethanol, centrifuged at 8000 g for 20 min, and the supernatant was discarded, and this was repeated 4 times. The resulting precipitate was soaked in 0.08 mol / L H 2 SO 4 Centrifuge at 6000 g for 15 min, discard the supernatant, and resuspend the pellet in 0.125 mol / L H 2 SO 4 , stirred for 4 h, centrifuged at 8000 g for 20 min, filtered through silk cloth, and discarded the precipitate. The supernatant was adjusted to pH 4.7 with 1 mol / L NaOH, allowed to stand for 0.5 h, centrifuged at 8000 g for 20 min, and the precipitate was discarded. The supernatant was added with 70% saturation (NH 4 ) 2 SO 4 , placed at 4 °C for 2 h, centrifuged at 8000 g fo...
Embodiment 2
[0028] Example 2 Preparation of anti-soybean protein STI monoclonal antibody
[0029] Select 5 female BALB / c mice aged 6-8 weeks (Shanghai Slack Experimental Animal Co., Ltd.), and treat the purified soybean protein STI obtained in Example 1 at 110 °C for 30 min (100 μL) with The volume of complete Freund's adjuvant (Sigma) was mixed to obtain the antigen, and the dose of 100 μg antigen was injected intraperitoneally into each mouse; after that, the same dose of antigen was boosted every 2 weeks; after three times of immunization, the mice were Blood was collected from the tail of the mouse, and the titer of mouse serum was detected by indirect ELISA, that is, the antigen was coated on the microtiter plate at a concentration of 2 μg / mL, coated overnight at 4 °C, washed 5 times with 200 μL / well TBST, and passed through 5 times. % skimmed milk was incubated at 37 °C for 1.5 h, washed twice with 200 μL / well TBST, and the mouse antiserum was mixed with 1:200, 1:1000, 1:5000, 1:10...
Embodiment 3
[0033] Example 3 Preparation of phycoerythrin-labeled anti-STI antibody
[0034] Phycoerythrin treatment: The ratio of R-phycoerythrin to SPDP is 1:80, react on a constant temperature shaker, 100 rpm / min, and react at 25°C for 1 hour. After the reaction is completed, the excess SPDP is removed through a desalting column and concentrated with a 10kDa membrane ; The above-mentioned SPDP-labeled R-phycoerythrin was treated with DTT, and the reaction concentration of DTT and the SPDP-labeled R-phycoerythrin was 1:100 in molar ratio, and reacted on a shaking table at 100 rpm / min at 25°C for 30 min, After the reaction, excess DTT was removed through a desalting column, and concentrated with a 10kDa membrane to obtain treated phycoerythrin.
[0035] Treatment of anti-STI monoclonal antibody: the molar ratio of anti-STI monoclonal antibody to SPDP was 1:80, and reacted on a shaker at 100 rpm / min at 25 °C for 1 h. The membrane was concentrated to obtain the treated anti-STI monoclona...
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