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Preparation method of R-phycocyanin (RPC)-marked fluorescent anti-antibody

A phycocyanin and anti-antibody technology, applied in the field of immunofluorescence detection, can solve the problem of no RPC

Inactive Publication Date: 2011-02-23
QILU UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no report on the application of RPC to animal disease detection at home and abroad

Method used

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  • Preparation method of R-phycocyanin (RPC)-marked fluorescent anti-antibody
  • Preparation method of R-phycocyanin (RPC)-marked fluorescent anti-antibody
  • Preparation method of R-phycocyanin (RPC)-marked fluorescent anti-antibody

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Embodiment approach 1

[0019] Embodiment 1: Preparation of RPC-labeled anti-chicken IgG fluorescent anti-antibody

[0020] Separation and purification of RPC: Add 6 times the volume of 20mM acetate buffer (pH5.8) to cryopreserved Polytubuleum, swell at 4°C for 24 hours, filter and squeeze through multi-layer gauze to obtain a brown extract, and then add solid ammonium sulfate To a concentration of 60% (w / v), place at 4°C for 24 hours, collect the precipitate by centrifugation, dissolve the precipitate in 20 mM acetate buffer (pH5.8), and then dialyze against 20 mM acetate buffer (pH5.8), Add the dialyzate to the anion exchange column pre-balanced with 20mM acetate buffer (pH5.8) (containing 50mM NaCl), wash off the excess sample and impurities on the anion exchange column with 20mM acetate buffer (pH5.8), and use 20mM acetic acid buffer (pH4.8) (containing 50mM NaCl) can be eluted in one step to obtain a large amount of pure RPC. 620 / A 280 >4.5, the structural formula is (αβ) 3, the molecular we...

Embodiment approach 2

[0027] Embodiment 2: Preparation of RPC-labeled anti-pig IgG fluorescent anti-antibody

[0028] Separation and purification of RPC: The preparation of RPC was separated and purified from Polytubuleum by anion exchange chromatography. Take the cryopreserved Polytubuleum, add 6 times the volume of 20mM acetate buffer (pH5.8), and swell at 4°C for 24h , multi-layer gauze filter, extrude to obtain a brown extract, then add solid ammonium sulfate to a concentration of 60% (w / v), place it at 4°C for 24h, collect the precipitate by centrifugation, and dissolve the precipitate in 20mM acetate buffer (pH5 .8), and then dialyzed against 20mM acetate buffer (pH5.8), the dialysate was added to an anion exchange column pre-equilibrated with 20mM acetate buffer (pH5.8) (containing 50mM NaCl), buffered with 20mM acetate solution (pH5.8) to wash off the excess sample and impurities on the anion exchange column, and a large amount of pure RPC can be obtained by one-step elution with 20mM aceta...

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Abstract

The invention relates to a preparation method of an R-phycocyanin (RPC)-marked fluorescent anti-antibody. A preparation process comprises the following steps of: preparing the RPC by an anion exchange chromatographic method; deriving the RPC and the anti-antibody with difunctional chemical cross-linking agents, namely, N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP) in molar ratios of 20-30:1 and 50-100:1; crosslinking a derivative with a liquid phase in an appropriate molar ratio; and performing high-pressure liquid phase chromatography purification so as to prepare the RPC-marked fluorescent anti-antibody. The RPC-marked fluorescent anti-antibody prepared by the method has high crosslinking efficiency, purity reaching electrophoretic purity, bright red fluorescence and stable fluorescent anti-antibody activity and can be taken as the general fluorescent probe for performing indirect immunofluorescence assay on zoonosis.

Description

Technical field: [0001] The invention relates to a preparation method of an R-phycocyanin (RPC)-labeled fluorescent anti-antibody, belonging to the field of immunofluorescence detection. technical background [0002] Immunofluorescence detection is a marker analysis method with a long history, which has the specificity of antigen and antibody reaction and the sensitivity of fluorescence detection, and is widely used in disease detection and biological research. The specificity and sensitivity of immunofluorescence assays depend on the properties and quality of fluorochromes, antibodies, and fluorescent probes. In practical application, since serum and other biological samples emit background fluorescence with a wavelength of 400-600nm, which overlaps with the fluorescence emission spectrum of traditional fluorescent dyes such as FITC, which seriously reduces the sensitivity of fluorescence detection, resulting in the detection of immunofluorescence within a period of time. ...

Claims

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Application Information

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IPC IPC(8): G01N33/533C07K14/405C07K1/18
Inventor 朱丽萍颜世敢颜士勇
Owner QILU UNIV OF TECH
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