A Japanese blood fluke ovum antibody magnetic particle EILSA detecting method
A technology of magnetic particles and schistosomiasis, which is applied in the direction of measuring devices, analytical materials, color/spectral characteristics measurement, etc., can solve the problems of increasing operational complexity, inability to detect antibodies, and inability to use universally, so as to save detection time, good repeatability, The effect of easy operation
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Embodiment 1
[0030] Example 1: Sj-SEA is coupled with surface amino (COOH-) magnetic particles to prepare a magnetic separation reagent
[0031] Take 100 mg of magnetic particles containing carboxyl (COOH-) active groups on the surface and wash them three times with 0.1 M MES (2-[N-morpholino] ethanesulfonic acid), 10 ml of pH 4.5-5 solution. The magnetic particles were resuspended in 1ml of this solution, and 2mg of Sj-SEA was added and mixed evenly. Add 100μl 10mg / ml EDC solution, mix well and react at room temperature for 2h. After washing the magnetic beads three times with 10 ml of 0.01 M PBS pH7.4 solution containing 1% bovine serum albumin (BSA), the solution was used to prepare a 2.5 mg / ml magnetic separation reagent working solution.
Embodiment 2
[0032] Example 2: Sj-SEA coupled alkaline phosphatase (ALP), preparation of enzyme-labeled reagents
[0033] Take 5 mg of SJ-SEA antigen, concentrate it to 5 mg / ml, add 10 μl of 13.76 mg / mL activator 2-Iminothiolane HCl (2IT) solution, place at room temperature for 20 minutes, add glycine to terminate the activation reaction, and place at room temperature for 5 minutes. Use Sephadex G25 column to desalt and collect protein elution peaks.
[0034] Take 5 mg of ALP solution, add 50 μl of 6.69 mg / mL Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) solution, leave it at room temperature for 30 minutes, add glycine to terminate the activation reaction, and leave it at room temperature for 5 minutes. Use Sephadex G25 column to desalt and collect protein elution peaks.
[0035] The activated Sj-SEA was mixed with the activated ALP, left at room temperature for 10 hours, then separated and purified using a Supperdex200 gel chromatography column, and the second and thi...
Embodiment 3
[0038] Example 3: Schistosoma japonicum egg antibody magnetic particle separation enzyme immunoassay
[0039] Materials and Instruments
[0040] (1) Magnetic separation reagent: see the preparation of the above-mentioned magnetic separation reagent;
[0041] (2) Enzyme-labeled reagent: see the preparation of the above-mentioned enzyme-labeled reagent;
[0042] (3) Washing solution: 500mL (produced by Beijing Beiaikang Biotechnology Co., Ltd.);
[0043] (4) Substrate solution (PMP solution): 300mL (produced by Beijing Bei Aikang Biotechnology Co., Ltd.);
[0044] (5) Stop solution: 600mL (produced by Beijing Bei Aikang Biotechnology Co., Ltd.);
[0045] (6) National reference product of Schistosoma japonicum: 15 positive reference products (P1-P15), 15 negative reference products (N1-N15), 1 sensitivity reference product (P16), 1 precision reference product (P17), Purchased from China Institute for the Control of Pharmaceutical and Biological Products.
[0046] (7) Serum s...
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