Human body beta-amyloid protein detection kit and application thereof

An amyloid and amyloid technology, applied in the field of molecular biology, can solve the problem that the exact mechanism of AD pathogenesis is not completely clear, and achieve the effects of being easy to master and popularize, not requiring high laboratory conditions and equipment, and high sensitivity

Active Publication Date: 2014-10-29
苏州依科赛生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, the exact mechanism of AD pathogenesis is not fully understood

Method used

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  • Human body beta-amyloid protein detection kit and application thereof
  • Human body beta-amyloid protein detection kit and application thereof
  • Human body beta-amyloid protein detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Construction and screening of single-chain antibodies by phage display technology

[0046] 1 Experimental materials

[0047] 1.1 Cells and cell lines

[0048] E. coli TG1, used for the transformation of pCANTAB-5E; E. coli HB2151, for scFv expression.

[0049] 1.2 Plasmid vector

[0050] pCANTAB-5E: purchased from Pharmacia (acquired by GE), vector map see figure 1 .

[0051] 1.3 Reagents

[0052] Trizol total RNA extraction kit, M-MLV reverse transcriptase, and RNase inhibitors were purchased from Invitrogen. Tag DNA polymerase, T4 DNA ligase and endonuclease were purchased from NEB Company. DNA gel recovery kit was purchased from Tiangen Company. M13KO7 helper phage was purchased from Pharmacia (acquired by GE).

[0053] 1.4 Primer sequences for gene amplification (see Table 1)

[0054] Table 1 List of primer sequences for gene amplification

[0055]

[0056]

[0057] 2 Experimental methods

[0058] 2.1 Animal immunity

[0059]After...

Embodiment 2

[0095] Example 2 Assembly of Human Beta Amyloid Precursor Protein Kit

[0096] After ELISA screens out a suitable single-chain antibody, it is assembled into a human β-amyloid precursor protein kit. The kit includes antibody-coated strips, standards, concentrated biotinylated antibodies (single-chain antibody-labeled biotin), concentrated enzyme conjugates, reagent diluents, concentrated washing solutions, chromogenic reagents, and stop solutions. The specific composition is shown in Table 1.

[0097] Table 1 The composition of the human β-amyloid precursor protein kit of the present invention

[0098]

[0099] The preparation of the antibody-coated strips comprises the following steps: Dilute the mouse anti-human β-amyloid precursor protein single-chain antibody prepared by the present invention to 1 μg / ml with a phosphate buffer solution with a pH value of 7.2, and add it on the coated plate , seal the plate with sealing paper, place the coated plate on the plate wash...

Embodiment 3

[0148] Embodiment 3 Stability and sensitivity test of kit of the present invention

[0149] The stability and sensitivity of the human β-amyloid precursor protein kit of the present invention and the human β-amyloid precursor protein kit (invitrogen, product number KHB3442) currently on the market were compared.

[0150] 1 Experimental method

[0151] (1) Dilute the mouse anti-human β-amyloid precursor protein antibody to 1 μg / ml with phosphate buffer, add 50 μl per well on the coated plate, and place it at 4°C overnight;

[0152] (2) Block the coated plate with 200 μl of phosphate buffer blocking solution containing bovine serum albumin 2% (W / V) and sucrose 3% (W / V);

[0153] (3) Add the standard and biotinylated anti-human AβTotal antibody at the same time, 50 μl per well, and repeat each concentration gradient three times; the AβTotal in the standard will combine with the biotinylated anti-human AβTotal antibody and The monoclonal antibody coated on the microtiter plate...

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Abstract

The invention relates to a human body beta-amyloid protein detection kit and application thereof. The human body beta-amyloid protein detection kit is prepared by the following steps: firstly, immunizing a mouse by use of antigen beta-amyloid precursor protein, separating out obvious lymph nodes in the spleen and the body of the mouse after confirming successful immunization, extracting total RNAs (Ribose Nucleic Acids), performing reverse transcription on the total RNAs to obtain cDNAs (complementary Desoxvribose Nucleic Acids), and amplifying the light and heavy chain genes of an antibody by virtue of PCR (Polymerase Chain Reaction); secondly, assembling and amplifying scFvDNA and constructing pCANTAB5E-scFv recombinant plasmid; thirdly, converting the plasmid to obtain a single-chain antibody phage library, and screening out to obtain target antibodies; and finally, assembling the target antibodies to form the human body beta-amyloid protein detection kit. The detection range of the screened antibodies is relatively wide from 0 to 1,000pg / ml, the linear correlation coefficient of the kit is high, the detection sensitivity of the kit can be 3.5pg / ml, and the kit achieves high stability; during detection, the reaction patterns of a two-step method and a room temperature oscillating method can be adopted, with low requirements on the laboratory conditions and the equipment.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a detection kit for human beta amyloid protein and its application. Background technique [0002] Population aging is a major social problem facing all countries in the world today, and has attracted widespread attention from the international community. According to relevant statistics, my country's big cities such as Beijing and Shanghai have entered the aging city, and my country will enter the aging society in the first half of the next century. The mental and physical health problems of the elderly have increasingly become the focus of attention of the whole society. Alzheimer's disease is a manifestation of brain dysfunction in the elderly, characterized by mental decline, behavior and personality changes. Typical clinical symptoms include memory, abstract thinking, and disorientation, accompanied by decreased social mobility. The main manifestations are forgetf...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCA61K2039/505C07K16/18G01N33/6896G01N2333/4709
Inventor 郭亚楠秦俭陈刚
Owner 苏州依科赛生物科技股份有限公司
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