Fluorescent immunochromatographic test paper for detecting human gfap protein and preparation method thereof

A technology of fluorescent immunochromatography and test paper, which is applied in the direction of anti-animal/human immunoglobulin, biological testing, chemical instruments and methods, etc., to achieve the effects of optimizing preparation conditions, wide detection range, and improving fluorescence signal-to-background ratio

Active Publication Date: 2016-10-05
深圳市安群生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently no fluorescent immunochromatographic test strip for human GFAP protein

Method used

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  • Fluorescent immunochromatographic test paper for detecting human gfap protein and preparation method thereof
  • Fluorescent immunochromatographic test paper for detecting human gfap protein and preparation method thereof
  • Fluorescent immunochromatographic test paper for detecting human gfap protein and preparation method thereof

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preparation example Construction

[0055] The preparation method of the GFAP epitope peptide of the present invention can be a chemical synthesis method: the antigen epitope peptide is synthesized by a solid-phase method using an American ABI431A type polypeptide automatic synthesizer. The molecular weights of the antigenic epitope peptides (1) and (2) of the present invention are 2690.27 and 1979.35 respectively, which can be determined by mass spectrometry, and the synthesized antigenic epitope peptide sequences are identified by polypeptide sequence determination. The purity of the peptides was evaluated by thin-layer chromatography and high-performance liquid chromatography, and the concentration of the epitope peptide was determined.

[0056] 2. GFAP antigen

[0057] The present invention also provides a GFAP antigen prepared by coupling one of the human GFAP antigen epitope peptides (1) and (2) of the present invention with a carrier protein. Specifically, the present invention provides GFAP antigens (1)...

Embodiment 1

[0107] Example 1: Preparation of GFAP epitope peptides (1) and (2).

[0108] The preparation method uses chemical synthesis method: GFAP antigen epitope peptides (1) and (2) are synthesized respectively by solid-phase method using the American ABI431A automatic peptide synthesizer. The purity of the epitope peptide was assessed by high performance liquid chromatography, and the concentration of the peptide was determined. The molecular weights of the antigenic epitope peptides (1) and (2) of the present invention are 2690.27 and 1979.35 respectively, which are determined by mass spectrometry, and the synthesized polypeptide sequences are identified by polypeptide sequence determination.

[0109] 1. Synthesis of GFAP epitope peptides (1) and (2)

[0110] The above peptides were synthesized by 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 synthesized with ...

Embodiment 2

[0198] Embodiment 2: the GFAP antigen epitope peptide (1) and (2) obtained in Example 1 are connected with carrier protein respectively to prepare GFAP antigen (1) and (2), utilize gained antigen (1) and (2) respectively Animals are immunized to prepare specific monoclonal and polyclonal antibodies using the antigen (1), and specific monoclonal and polyclonal antibodies are prepared using the antigen (2).

[0199] 1. Antigen preparation: GFAP peptides (1) and (2) were respectively linked with carrier protein KLH (keyhole limpet hemocyanin) by BDB (Bis-diazotizedbenzidine dichloride) method to prepare GFAP antigens (1) and (2) .

[0200] Take 10.0mg of GFAP peptide (1) or (2), dissolve it with 1ml 0.1M PBS buffer (pH7.4); dissolve KLH10mg with 20ml of 0.2M borate buffer (pH9.0); then mix the two Mix, cool to 0°C, take BDBCl 2 110 μL, reacted at room temperature for 1.5 h, dialyzed overnight, then aliquoted, and stored at -20 °C.

[0201] In the present embodiment, the formul...

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Abstract

The invention relates to a fluorescent immunochromatographic test paper for detecting a human GFAP protein, and a preparation method thereof. The test paper detects the human GFAP protein through a double antibody sandwich technology, the double antibody sandwich technology adopts a fluorescent microsphere-labeled first GFAP monoclonal antibody as a capture antibody, and the first GFAP monoclonal antibody is from one of a sequence represented by SEQ ID NO.1 in a sequence table and a sequence represented by SEQ ID NO.2 in the sequence table; and the double antibody sandwich technology adopts a second GFAP monoclonal antibody as a detection antibody, and the second GFAP monoclonal antibody is from the other one of the sequence represented by SEQ ID NO.1 in the sequence table and the sequence represented by SEQ ID NO.2 in the sequence table. The fluorescent immunochromatographic test paper has the advantages of simple operation, rapidness, wide detection range, high specificity and good sensitivity.

Description

technical field [0001] The invention belongs to the field of polypeptide chemistry and immunology, and specifically relates to a human glial fibrillary acidic protein (GFAP) epitope peptide, a GFAP specific antigen prepared by using the epitope peptide, and a corresponding monoclonal antibody or polyclonal antibody. The use of the antibody in the preparation of a human GFAP in vitro diagnostic kit, the human GFAP in vitro diagnostic kit, and a fluorescent immunochromatographic test paper for quantitatively detecting human GFAP protein in a test object and a preparation method thereof. Background technique [0002] In recent years, the research on neurobiochemical markers has attracted the attention of many researchers. It is the common wish of many researchers to find a reliable, non-invasive biochemical marker that can reflect the extent and prognosis of central nervous system damage. Through the study of blood and cerebrospinal fluid (CSF), some biochemical markers have be...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68C07K14/47C07K16/18
CPCC07K14/47C07K16/18G01N33/533G01N33/6803
Inventor 朱建安
Owner 深圳市安群生物工程有限公司
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