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Method for preparing allophycocyanin-marked fluorescent antinuclear antibody

An allophycocyanin and anti-antibody technology, applied in the field of immunofluorescence detection, can solve the problems of difficult separation and purification of APC, difficult quantitative control of protein, low yield of APC labeling, etc. The effect of high coupling efficiency

Inactive Publication Date: 2011-02-23
QILU UNIV OF TECH
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Problems solved by technology

[0005] APC is an excellent dye that emits red fluorescence, but due to the difficulty in separation and purification of APC, poor stability, and quantitative control during protein cross-linking, resulting in low yield of APC labeling, high production costs, and even inactivation of APC and loss of fluorescence, limiting Application of this excellent dye in clinical detection
There is no report on the application of APC-labeled fluorescent antibody in the detection of animal diseases at home and abroad

Method used

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  • Method for preparing allophycocyanin-marked fluorescent antinuclear antibody
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  • Method for preparing allophycocyanin-marked fluorescent antinuclear antibody

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Embodiment approach 1

[0023] Embodiment 1: Preparation method of APC-labeled anti-chicken IgG fluorescent anti-antibody

[0024] Preparation of allophycocyanin (APC): APC was isolated and purified from Spirulina platensis. Add 5 times the volume (v / w) of 20mM phosphate buffer (pH6.8-7.0) to the algae cells, freeze and thaw three times, centrifuge at 10000rpm at 4°C, add ammonium sulfate to the supernatant to a final concentration of 60% (w / v), placed in a refrigerator at 4°C for 24 hours, centrifuged, the precipitate was dissolved in 20mM phosphate buffer (pH7), dialyzed in 20mM phosphate buffer (pH7), and the dialysate was chromatographed on a DEAE Sepharose Fast Flow anion exchange column. The exchange column was pre-equilibrated with 20mM acetate buffer (pH5.0, containing 50mM NaCl), the eluent was 20mM acetate buffer (pH3.6, containing 50mM NaGl), and the elution rate was 60mL / h, and the sky blue liquid was collected as Purified allophycocyanin from Spirulina. The purity of the purified Spir...

Embodiment approach 2

[0031] Embodiment 2: Preparation method of APC-labeled anti-pig IgG fluorescent anti-antibody

[0032] Preparation of allophycocyanin (APC): APC was isolated and purified from Spirulina platensis. Add 5 times the volume (v / w) of 20mM phosphate buffer (pH6.8-7.0) to the algae cells, freeze and thaw three times, centrifuge at 10000rpm at 4°C, add ammonium sulfate to the supernatant to a final concentration of 60% (w / v), placed in a refrigerator at 4°C for 24 hours, centrifuged, the precipitate was dissolved in 20mM phosphate buffer (pH7), dialyzed in 20mM phosphate buffer (pH7), and the dialysate was chromatographed on a DEAE Sepharose Fast Flow anion exchange column. The exchange column was pre-equilibrated with 20mM acetate buffer (pH5.0, containing 50mM NaCl), the eluent was 20mM acetate buffer (pH3.6, containing 50mM NaCl), the elution rate was 60mL / h, and the sky blue liquid was collected as Purified allophycocyanin from Spirulina. The purity of the purified Spirulina AP...

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Abstract

The invention relates to a method for preparing an allophycocyanin(APC)-marked fluorescent antinuclear antibody, which comprises: separating and purifying spirulina APC by anion exchange chromatography; crosslinking APC and antinuclear antibody derivatives in a liquid phase in a proper molar ratio; and preparing the APC-marked fluorescent antinuclear antibody by purification by high-pressure liquid chromatogram. The fluorescent antinuclear antibody prepared by the method has high crosslinking efficiency, high purity, bright red fluorescence, high stability and high sensibility, and can be used as a common fluorescent probe for indirect immunofluorescent detection of infectious diseases in animals such as chickens and pigs.

Description

technical field [0001] The invention relates to a preparation method of a fluorescent anti-antibody labeled with Allophycocyanin (APC), and belongs to the field of immunofluorescence detection. technical background [0002] Immunofluorescence detection is a marker analysis method with a long history, which combines the specificity of antigen and antibody reactions 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 development of fluorescence detection methods...

Claims

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Application Information

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IPC IPC(8): G01N33/533C07K14/405C07K1/18
Inventor 朱丽萍颜世敢姚强吕爱杰赵守山
Owner QILU UNIV OF TECH
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