Preparation method of fluorescence antibody for detecting Newcastle disease virus and solid-phase immunofluorescence detection assay kit

Abstract

The invention relates to a preparation method of fluorescence antibody for detecting a Newcastle disease virus and a solid-phase immunofluorescence detection assay kit. The preparation method comprises the following steps of: respectively deriving R-phycoerythrin (RPE) and an antibody resisting the Newcastle disease virus (NDV) by using a cross-linking agent SPDP (N-succinimidyl-3-(2-pyridyldithiol) propionate), cross-linking the derivatives in a proper molar ratio, and purifying through HPLC (High Performance Liquid Chromatography) to prepare an RPE marked NDV fluorescence antibody. The solid-phase immunofluorescence detection assay kit is formed from the fluorescence antibody, an NDV-resisting antibody, an agarose microsphere, diluted hydrochloric acid and a washing liquid. The assay kit comprises the following detection flows of: coating an activated microsphere with the antibody, washing, combining with a sample to be measured, washing, combining with the fluorescence antibody, fully washing, exciting by blue and green light in a fluorescence microscope, observing and judging the result. The prepared fluorescence antibody has the advantages of high yield, high purity, bright orange fluorescence and good stability; and the assay kit has signal enrichment action by using a spherical carrier and can increase detection sensitivity. The invention is applicable to the rapid detection of the Newcastle disease virus.

Description

technical field [0001] The invention relates to a preparation method of a fluorescent antibody for detecting Newcastle disease virus and a solid-phase immunofluorescence detection kit, belonging to the field of immunofluorescence detection. technical background [0002] Newcastle disease is a severe infectious disease that seriously endangers the breeding industry. It is listed as a category A animal infectious disease by the International Veterinary Bureau. At the same time, it is also a zoonotic disease that threatens human public health. Newcastle disease virus detection is a mandatory inspection item for entry-exit inspection and quarantine of poultry and animal products. The International Veterinary Bureau requires that once Newcastle disease virus is detected in poultry and animal products, it must be destroyed on the spot. Rapid and sensitive detection methods are crucial for the timely diagnosis of epidemics, effective prevention and control, prediction and early war...

AI Technical Summary

Problems solved by technology

[0005] When fluorescent dyes such as RPE are used for fluorescence detection, they need to be cross-linked with biomacromolecules such as antibodies to form fluorescent antibody probes. It is difficult to quantitatively control the shortcomings of protein cross-linking, which often results in low yields of fluorescent markers and mixed markers. There is a large amount of unconjugated fluorescent dyes, which leads to high production costs of labels and reduces the sensitivity of fluorescent detection

Moreover, the high concentration of cross-linking agent will also lead to structural changes of fluorescent dyes, loss of protein activity, and even loss of fluorescence. At the same time, too high concentration of cross-linking agent will also affect the immune activity of antibodies. Concentration, cross-linking method, etc. directly affect the quality of fluorescent probes, thereby affecting the sensitivity of fluorescent detection

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