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An immunofiltration method and immunofiltration device for detecting serum fucoglycoprotein
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A technology of immune diafiltration and serum fucus, which is applied in the field of protein detection, can solve the problems of expensive reagents, increased cumbersome operation, cumbersome operation, etc., and achieve the effect of short detection time, not easy to break, and good stability
Inactive Publication Date: 2019-01-25
BEIJING YOUAN HOSPITAL CAPITAL MEDICAL UNIV
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Among them, plant lectin affinity immunoelectrophoresis technology and The i30 detection system has high technical requirements, cumbersome operation, and expensive reagents, which limit its popularization and application
However, the glycosyl capture spin column increases the complexity of the operation because the sample processing and detection are carried out separately.
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Embodiment 1
[0055] Embodiment 1 Preparation and use process of immunofiltration device
[0056] Reagents and instruments used in the experiment: prokaryotic expression of AFP (US abcam company); lentilin (Sigma company); nitrocellulose membrane (GE Healthcare), horseradish peroxidase-labeled rabbit-derived antibody (US abcam company); HRP Chromogenic substrate solution (Millipore, USA).
[0057] PBS formula: sodium chloride (NaCl) 8g, potassium chloride (KCl) 0.2g, disodium hydrogen phosphate (NaCl) 2 HPO 4 ) 1.44g, potassium dihydrogen phosphate (KH 2 PO 4 ) 0.24g, adjust the pH value to 7.4, and set the volume to 1L.
[0058] PBST formulation: PBS, 1L+Tween-20, 1mL.
[0059] Nitrocellulose membrane (GE Healthcare), each immunofiltration device contains 1 detection area, each detection area can detect one serum and one AFPL3 marker.
[0060] In each detection area, 0.5 μL each of mouse-derived prokaryotic expression human AFP (abcam company in the United States) and lentilin Sigma ...
Embodiment 2
[0067] The prepared immunofiltration was used to detect the AFP L3 tumor marker in the dynamic serum samples of the healthy control group and the liver cancer experimental group.
[0068] At room temperature, after diluting 4 times with 2.5 μL of serum sample, add it dropwise on the membrane of the immunofiltration device, and use the characteristics of lentilin and fucose to bind lentilin to fucose in serum A lentilin-antigen complex is formed; after 5 seconds, the serum fluid permeable membrane is absorbed by the absorbent material beneath the membrane.
[0069] Add 10 μL of PBST dropwise to wash, remove non-specific binding, repeat 5 times. Then add horseradish peroxidase-labeled rabbit primary antibody diluted in PBS, the rabbit-derived antibody binds to the AFP antigen in the serum, lentilin-(AFP) fucose-rabbit-derived horseradish peroxidase-labeled antibody complex The rabbit antibody is combined with the positive control AFP on the immobilized membrane at the same time...
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Abstract
The invention relates to an immune diafiltration method and an immune diafiltration device for detecting serum fucoglycoprotein. The method comprises the following steps: (1) compounding alpha-fetoprotein antigen and lentilin on a microporous membrane; ( 2) drop serum samples onto the microporous membrane to combine fucosylated alpha-fetoprotein in the sample with lentilin on the microporous membrane; (3) drop enzyme-labeled The alpha-fetoprotein polyclonal antibody is added to the chemiluminescence substrate to obtain the detection result; the invention also provides an immunofiltration device for detecting serum fucoglycoprotein. The present invention utilizes the microporous filter membrane to realize one-time simultaneous detection of multiple antigens or antibodies in serum samples in a relatively short period of time, and the detection process can be completed in only 3-5 minutes, and has the advantages of short detection time, low cost, Good stability and other advantages.
Description
technical field [0001] The invention relates to the technical field of protein detection, in particular to an immune diafiltration method and an immune diafiltration device for detecting serum fucoglycoprotein. Background technique [0002] The sugar chain structure of alpha fetoprotein (AFP) produced by primary liver cancer and benign liver diseases such as hepatitis and cirrhosis is very different. The sugar index is much higher. Fucose has the property of binding to lentilin. AFP can be divided into AFP-L1, AFP-L2 and AFP-L3 according to its (fucosyl) affinity for lentil lectin. Among them, AFP-L1 mainly comes from benign liver diseases, AFP-L2 mainly comes from pregnant women, and AFP-L3 is the fucose glycosylated form of alpha-fetoprotein, which mainly comes from Hepatocellular Carcinoma (HCC). In 2005, FDA officially approved AFP-L3 as one of the markers of primary liver cancer. AFP-L3 has high specificity and sensitivity in early diagnosis, differential diagnosis,...
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