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Human AD7C-NTP antigen determinant polypeptide, antibody and application thereof in diagnosis kit

An antigenic determinant and kit technology, which is applied in the application field of preparing human AD7C-NTP in vitro diagnostic kits, can solve the problems of non-specificity, inconvenient measurement, and no practical value, and achieves strong antigenicity and easy synthesis. Effect

Active Publication Date: 2008-07-09
XUANWU HOSPITAL OF CAPITAL UNIV OF MEDICAL SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, so far, the early diagnosis of AD is very difficult, the reasons are as follows: 1. Cerebrospinal fluid examination is required, and it is inconvenient to obtain materials; 2. Although phosphorylated Tau protein and other skeleton proteins in cerebrospinal fluid increase, they are not specific. There are also changes in systemic diseases; while Aβ42 does not change in the early stage of AD, and decreases with the aggravation of the disease, which is not easy to measure; 3. The determination of other proteins including enzymes has no guiding significance for diagnosis
Therefore, although there are more than 100 reports on the early diagnosis of AD, they are limited to descriptive studies and have no practical value due to the lack of effective biochemical indicators.

Method used

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  • Human AD7C-NTP antigen determinant polypeptide, antibody and application thereof in diagnosis kit
  • Human AD7C-NTP antigen determinant polypeptide, antibody and application thereof in diagnosis kit
  • Human AD7C-NTP antigen determinant polypeptide, antibody and application thereof in diagnosis kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Preparation of two AD7C-NTP epitope polypeptide fragments.

[0042] Two kinds of AD7C-NTP epitope polypeptide fragments that embodiment adopts are polypeptide fragment (3) and polypeptide fragment (5) of the present invention, namely:

[0043] Tyr-Asn-Pro-Gly-Ser-Ser-Asp-Ser-Pro-Ala-Ser-Ala-Ser-Pro,

[0044] Tyr-Arg-Arg-Pro-Pro-Arg-Leu-Ala-Asn.

[0045] The preparation method adopts the chemical synthesis method: the antigenic determinant polypeptide is synthesized by the solid phase method by using the American ABI431A automatic peptide synthesizer. The purity of the peptide was assessed by thin-layer chromatography and high-performance liquid chromatography, and the concentration of the antigenic peptide was determined.

[0046] The two polypeptide fragments are synthesized by a solid-phase method. The main idea of ​​solid-phase peptide synthesis is: first connect the carboxyl group of the carboxyl-terminal amino acid of the peptide chain to be synthesiz...

Embodiment 2

[0104] Example 2: The AD7C-NTP epitope polypeptide fragment obtained in Example 1 was linked to a carrier protein and used as an antigen to immunize animals to prepare specific polyclonal antibodies.

[0105] (1) AD7C-NTP epitope polypeptide fragment (3) Preparation of polyclonal antibody:

[0106] 1. Antigen preparation: the AD7C-NTP polypeptide fragment (3) was linked with the carrier protein hemocyanin (KLH) by the carbodiimide method to prepare the antigen.

[0107] 2. Preparation of antiserum from animals: Take the antigen and 1ml of Freund's complete adjuvant (basic immunization) or Freund's incomplete adjuvant (boost immunization) and fully emulsify it to immunize Balb / c mice, inject intraperitoneally, and each dose is 50- 200μg / ml, intradermally injected at 20 points, and immunized 7-8 times in total. The tenth day after the last immunization, ear blood was taken to measure the antibody titer.

[0108] 3. Determination of antibody titer: The antibody titer was determi...

Embodiment 3

[0120] Example 3: Preparation of AD7C-NTP ELISA kit.

[0121] The AD7C-NTP polyclonal antibody prepared above was used to prepare a diagnostic kit for AD7C-NTP in urine. In this example, the polypeptide fragment (3) polyclonal antibody in the two AD7C-NTP polyclonal antibodies was used as the coating antibody in the kit , the polyclonal antibody of the polypeptide fragment (5) was used as the binding antibody.

[0122] The preparation and operation of the AD7C-NTP ELISA kit are as follows:

[0123] 1. Preparation of various buffers and reagents:

[0124] A. Coating buffer: 0.050M, CB (carbonate buffer) at pH 9.6

[0125] Na 2 CO 3 (MW 105.99) 16.0 grams

[0126] NaHCO 3 (MW 84.01) 29.0 grams

[0127] Dissolve in distilled water to 1000ml.

[0128] B. Sample / wash buffer: PBS-Tween 20 at pH 7.2

[0129] Na 2 HPO 4 .12H2O 58 grams

[0130] K H 2 PO 4 4 grams

[0131] NaCl 100g

[0132] KCl 4 g

[0133] Dissolve in distilled water to 1000ml

[0134] Ad...

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Abstract

The invention relates to a human AD7C-NTP antigen determinant polypeptide antibody and the application on a diagnosis reagent case. The AD7C-NTP antigen determinant polypeptide of the invention is one of polypeptide sections which are stated by the sequence table SEQ ID NO.1 to SEQ ID NO.6. The antibody of the invention is prepared by any AD7C-NTP antigen determinant polypeptide of the invention. The AD7C-NTP antibody of the invention can be applied to prepare an AD7C-NTP external diagnosis reagent case.

Description

technical field [0001] The present invention relates to human AD7C-NTP antigenic determinant polypeptide, corresponding antibody and its application in the preparation of human AD7C-NTP in vitro diagnostic kit. Background technique [0002] In today's increasingly aging population, the incidence of geriatric diseases characterized by neurodegeneration is showing an increasing trend year by year. The problem of aging in my country is particularly severe. On February 23, 2006, the National Office on Aging issued the "Research Report on the Forecast of the Development Trend of China's Population Aging", pointing out that China has entered the aging society in 1999. In many developed countries, it generally takes 50-80 years for the proportion of the population over 65 years old to rise from 5% to 7%, while it took only 18 years for my country to rise from 4.9% in 1982 to 7.2% in 2000, which shows the aging speed of my country’s population. far more than developed countries. Th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06C07K7/08C07K16/18G01N33/577G01N33/53
CPCY02P20/582
Inventor 盛树力王蓉姬志娟朱建安
Owner XUANWU HOSPITAL OF CAPITAL UNIV OF MEDICAL SCI
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