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Fluorescent immunochromatographic test paper for detecting human hsp90α-2 protein and preparation method thereof

A technique of fluorescent immunochromatography and test paper, which is applied in the fields of peptide chemistry and immunology in Ming Dynasty, and achieves the effects of simple operation, good sensitivity, and improved fluorescence signal-to-background ratio

Active Publication Date: 2017-09-22
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 HSP90α-2 protein

Method used

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  • Fluorescent immunochromatographic test paper for detecting human hsp90α-2 protein and preparation method thereof
  • Fluorescent immunochromatographic test paper for detecting human hsp90α-2 protein and preparation method thereof
  • Fluorescent immunochromatographic test paper for detecting human hsp90α-2 protein and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0106] Example 1: Preparation of HSP90α-2 epitope peptides (1) and (2).

[0107] The preparation method uses chemical synthesis method: the HSP90α-2 epitope peptides (1) and (2) are respectively synthesized 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 2404.35 and 2483.39 respectively, which are determined by mass spectrometry, and the synthesized polypeptide sequences are identified by polypeptide sequence determination.

[0108] 1. Synthesis of HSP90α-2 epitope peptides (1) and (2)

[0109] 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 synthesiz...

Embodiment 2

[0194]Example 2: Link the HSP90α-2 antigen epitope peptides (1) and (2) obtained in Example 1 to carrier proteins to prepare HSP90α-2 antigens (1) and (2), and use the obtained antigens (1) and (2) Animals are immunized separately to prepare specific monoclonal antibodies and polyclonal antibodies using the antigen (1), and specific monoclonal antibodies and polyclonal antibodies are prepared using the antigen (2).

[0195] 1. Preparation of antigen: HSP90α-2 peptides (1) and (2) were respectively linked with carrier protein KLH (keyhole limpet hemocyanin) (obtained from sigma company) by BDB (Bis-diazotizedbenzidine dichloride) method to prepare HSP90α -2 antigens (1) and (2).

[0196] Take HSP90α-2 peptide (1) or (2) 10.0mg, dissolve with 1ml 0.1M PBS buffer (pH7.4); KLH10mg, dissolve with 0.2M borate buffer (pH9.0) 20ml; then Mix the two, 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.

[...

Embodiment 3

[0211] Example 3: Specific identification of human HSP90α-2 monoclonal antibodies (1) and (2)

[0212] Detection was performed by ELISA. Human HSP90α-2 protein, S-100B protein, and neuron-specific enolase NSE (all purchased from Shanghai Lianshuo Co., Ltd.) were used as detection antigens to coat ELISA plates, and the prepared HSP90α-2 monoclonal clones were detected by ELISA respectively. For the specific reaction of antibodies (1) and (2) with the human HSP90α-2 protein, normal BALB / c mouse serum was used as negative control, and PBS solution was used as blank control.

[0213] Results: HSP90α-2 monoclonal antibodies (1) and (2) were positive for HSP90α-2 only (P / N>2.1), but negative for S-100B protein and neuron-specific enolase NSE , indicating that the HSP90α-2 monoclonal antibodies (1) and (2) of the present invention have specificity respectively.

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Abstract

The invention relates to fluorescence immunochromatographic test paper for detection of a human heat shock protein 90 alpha-2 (HSP90 alpha-2) and a preparation method thereof. The test paper detects the human HSP90 alpha-2 by a double-antibody sandwich method, and the double-antibody sandwich method adopts a fluorescent microsphere-labeled first HSP90 alpha-2 monoclonal antibody as a capture antibody, wherein the first HSP90 alpha-2 monoclonal antibody is sourced from one of sequences SEQ ID NO.1 and SEQ ID NO.2 shown in a sequence table; moreover, the double-antibody sandwich method adopts a second HSP90 alpha-2 monoclonal antibody as a detection antibody, wherein the second HSP90 alpha-2 monoclonal antibody is sourced from the other one of the sequences SEQ ID NO.1 and SEQ ID NO.2 shown in the sequence table. The fluorescence immunochromatographic test paper has the advantages of simple and rapid operation, wide detection range, high specificity and good sensitivity.

Description

technical field [0001] The invention belongs to the field of polypeptide chemistry and immunology, and in particular relates to human heat shock protein 90α-2 (HSP90α-2) epitope peptide, HSP90α-2 specific antigen prepared by using the epitope peptide and corresponding monoclonal antibody or Polyclonal antibody, application of said antibody in preparing human HSP90α-2 in vitro diagnostic kit, human HSP90α-2 in vitro diagnostic kit, and a fluorescent immune layer for quantitative detection of human HSP90α-2 protein in analyte Analysis test paper and its preparation method. Background technique [0002] In recent years, the research on tumor markers has attracted the attention of many researchers. It is the common wish of many researchers to find a reliable, non-invasive tumor marker that can reflect the occurrence, proliferation and differentiation of tumors. Through the study of blood and interstitial fluid, some tumor markers have been found to predict the occurrence, proli...

Claims

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Application Information

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