Polyclonal antibody of phosphorylating and corresponding non-phosphorylating protein in application for preparing reagent for disease diagnosis, and preparation method

A phosphorylated protein and polyclonal antibody technology, applied in biological testing, material inspection products, measuring devices, etc., can solve problems such as few, harmful to the health of operators and the environment, and unsuitable for clinical diagnosis in hospitals

Active Publication Date: 2006-02-01
南京川博生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] Identification of phosphorylation sites by radioisotope labeling is also a traditional method for studying phosphorylated proteins, but it is difficult to apply in tissue samples, and radioisotopes are harmful to the health of operators and the environment
Although non-isotopes have been used instead of radioactive isotopes for labeling, this method can only analyze one target protein at a time, and it is not suitable for clinical diagnosis in hospitals.
[0016] In addition, anti-specific amino acid phosphorylated protein antibodies have only been used in the research of phosphorylated proteins for several years, and have not been used in clinical diagnosis, mainly because there are not many types of anti-specific amino acid phosphorylated protein antibodies that can be produced on a large scale. , the production of many antibodies that can theoretically be used for disease diagnosis is very low, and can only be used for research

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Polyclonal antibody preparation method: 1. Synthetic polypeptide: According to the amino acid sequence of c-Jun found in the Swiss-Prot site, select the sequence near serine at position 73, design and artificially synthesize two 14 peptides, the two polypeptide sequences Similarly, a Cys is added to the N-terminal or C-terminal of the polypeptide chain, and the serine at the 7th position (equivalent to the 73rd position of c-Jun) is calculated as the phosphorylated amino acid at the N-terminal of one polypeptide chain.

[0048]2. Use bifunctional coupling agent sulfosuccinimide 4-[N methylmaleic acid]-1-carboxycyclohexane (Sulfo-SMCC) as coupling agent, and keyhole limpet hemocyanin (KLH ) on the free amino group to activate it. The specific method is to redissolve KLH in double distilled water, add Sulfo-SMCC (mass ratio: KLH / SMCC is 4 / 1), stir slowly at room temperature for 60 minutes, and then use a G25 desalting column to remove uncoupled SMCC and collect the protei...

Embodiment 2

[0071] Example 2 is basically the same as Example 1, but with the following changes: the two artificially synthesized polypeptides are changed to: design and synthesize according to the amino acid sequences on both sides of the 63 serine site in the amino acid sequence of the c-Jun protein, and the length is changed to 13 amino acid.

Embodiment 3

[0072] Example 3 is basically the same as Example 1, but with the following changes: the two artificially synthesized polypeptides are changed to: design and synthesize according to the amino acid sequence on both sides of the 243 serine site of c-Jun protein, and the length is changed to 15 amino acids.

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PUM

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Abstract

A method for preparing multicloning antibody includes synthesizing polypeptide, connecting the coupling agent with keyhole limpet hemocyanin for forming hapten ¿C carrier protein system, immunizing rabbit then picking up its blood for separating out serum, purifying out multicloning antibody of anti ¿C specific site amino acid phosphorylation protein and anti ¿C corresponding non phosphorylation protein after two steps of antigen affinity chromatography .

Description

technical field [0001] The present invention relates to a new application of a protein polyclonal antibody and a preparation method thereof, in particular to the application of an anti-specific site amino acid phosphorylated polyclonal antibody and the corresponding non-phosphorylated protein polyclonal antibody in the preparation of disease diagnostic reagents and its preparation method. The antibody preparation method of the present invention is actually a method for preparing reagents that can be applied to the diagnosis of diseases. These diseases include diseases associated with the state and degree of protein phosphorylation, such as tumors, nervous system diseases, geriatric diseases, etc. The diagnostic methods include Enzyme-linked immunoassay (ELISA), Western Blot and immunohistochemistry (IHC). Background technique [0002] The current methods of using immune methods to diagnose diseases generally distinguish between normal cells and diseased cells by detecting s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/531G01N33/53
Inventor 彭早元张薇林世康胡云龙
Owner 南京川博生物技术有限公司
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