Antigen protein specifically bound with tyrosine phosphatase antibody

A tyrosine phosphatase, specific binding technology, applied in the field of antigen-antibody protein, truncated tyrosine phosphatase, can solve low efficiency, insufficient specificity, long, at least 6 hours, or even overnight reactions, etc. question

Active Publication Date: 2015-04-29
SHENZHEN NEW INDS BIOMEDICAL ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional radioimmunoassay or ELISA method takes a long time to detect, at least 6 hours, or even overnight reaction to complete the test. It mainly relies on a series of operations such as pure manual sample addition, which has low efficiency and insufficient specificity.

Method used

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  • Antigen protein specifically bound with tyrosine phosphatase antibody
  • Antigen protein specifically bound with tyrosine phosphatase antibody
  • Antigen protein specifically bound with tyrosine phosphatase antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Expression of hIA-2 and truncated hIA-2 genes

[0031] (1) Main reagents and equipment

[0032] PCR kits (including Taq enzyme, buffer, dNTP, T4 ligase) and DNA markers were purchased from full gold; protein markers were purchased from Beyond Biotech; BamH I, Xho I, EcoR I, Nco I, HindⅢ and other restriction internal Dicer was purchased from Takara (Dalian Bao Biology); calf intestinal alkaline phosphatase (CIAP) kit (including components: calf intestinal alkaline phosphatase, alkaline phosphatase buffer) was purchased from Takara; gel recovery reagent Kits and plasmid extraction kits were purchased from OMEGA; kanamycin and ampicillin were purchased from BBI; IPTG was purchased from BBI; PCR primers and recombinant plasmid sequencing were completed by BGI or Invitrogen; other reagents were provided by Shenzhen New Industry Biomedical Engineering Co., Ltd. Inc. provided.

[0033] The ABI Veriti PCR instrument was purchased from ABI, the horizontal electrophoresis i...

Embodiment 2

[0046] Determination of the sensitivity and specificity of the 14 antigens expressed by the above insect cells to the hIA-2 antibody

[0047] The principle of the chemiluminescent immune sandwich method: the serum sample to be tested, the buffer solution and the antigen-coated magnetic microspheres are added to the reaction cup for reaction, and at 37°C, the antibody in the serum sample to be tested and the antigen coated on the magnetic microspheres are immune In the case of a magnetic field, the nano-magnetic beads will be magnetized rapidly, and the complex will be washed to obtain the complex of the antigen and the antibody to be tested. Other components will be washed away, and then the labeled protein A-NHS-ABEI will be added. Protein A can Quickly and specifically bind to the C-terminus of the antibody to be tested to form a "sandwich" immune complex. When the detected antibody content in the serum sample is more, the relative light intensity RLU detected by the instrum...

Embodiment 3

[0068] Comparison of prokaryotic and eukaryotic expressed proteins for detection of type 1 diabetes

[0069] The truncated protein N2 of hIA-2 was expressed in Escherichia coli, and the truncated DNA segment of hIA-2 was connected to the pGEX4T-1 expression vector, and the expression strain of Escherichia coli was BL21(DE3). After the expression product is purified by GST tag, the GST tag is cut off with Thrombin, and the untagged target protein is obtained by affinity chromatography.

[0070] The sensitivity and specificity of prokaryotic expressed N2 to hIA-2 antibody were determined.

[0071] The symbol for the prokaryotic expression segment of hIA-2 truncated form is PN2, which corresponds to N2 for eukaryotic expression.

[0072] The detection platform and scheme details are the same as in Example 1, and the results are shown in Table 4.

[0073] Table 4

[0074]

[0075] The results of PN2 in Table 4 are compared with the results of N2. The non-specific binding of ...

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Abstract

The invention relates to the technical field of antigen-antibody protein, and particularly relates to the technical field of truncated tyrosine phosphatase (IA-2) for detecting diabetes mellitus. One aim of the invention is to screen a truncated antigen protein specifically bound with IA-2 antibody, and the other aim of the invention is to develop antigen protein which is capable of fulfilling a chemiluminiscence closed system and applicable to the chemiluminiscence closed system. According to the antigen protein specifically bound with the IA-2 antibody, the antigen protein is one of 600th-979th, 604th-979th, 604th-969th, 600th-969th and 709th-969th truncated proteins. The invention further provides the truncated protein or IA-2 full-length protein which is coated with nano-magnetic beads, and a kit comprising the protein.

Description

technical field [0001] The present invention relates to the technical field of antigen-antibody protein, in particular to the technical field of truncated tyrosine phosphatase (IA-2) for detecting diabetes. Background technique [0002] Diabetes mellitus is a metabolic disease characterized by hyperglycemia caused by absolute or relative insufficiency of insulin secretion, which is affected by various genetic and environmental factors. According to the 1997 American Diabetes Association and the 1999 World Health Organization (WHO) classification and diagnostic criteria for diabetes, diabetes can be divided into type I, type II, special type diabetes and gestational diabetes. [0003] During the immune destruction of type 1 diabetes, various autoantibodies against islet cell self-antigens are produced in patients. Since the 1970s, a variety of insulin autoantibodies have been discovered, such as: islet cell antibody (ICA), islet cell surface antibody (ICSA), insulin antibody...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N21/76G01N33/6893G01N2800/042
Inventor 何海华戚永跃任印玲吴森仁袁锦云李武
Owner SHENZHEN NEW INDS BIOMEDICAL ENG
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