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Fluorescence immune chromatography test paper for detecting human ApoE-[epsilon]4 protein and preparation method thereof

A fluorescence immunochromatography and test paper technology, which is applied in the field of fluorescent immunochromatography test paper for detecting human ApoE-ε4 protein and its preparation, can solve the problem of lack of human ApoE protein, etc., so as to improve detection sensitivity and result reliability, operation The effect of simple, rapid and timely diagnosis of the disease

Active Publication Date: 2016-05-25
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 ApoE protein

Method used

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  • Fluorescence immune chromatography test paper for detecting human ApoE-[epsilon]4 protein and preparation method thereof
  • Fluorescence immune chromatography test paper for detecting human ApoE-[epsilon]4 protein and preparation method thereof
  • Fluorescence immune chromatography test paper for detecting human ApoE-[epsilon]4 protein and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0106] Example 1: Preparation of ApoE-ε4 epitope peptides (1) and (2).

[0107] The preparation method uses chemical synthesis method: ApoE-ε4 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 2187.32 and 2909.12 respectively, which are determined by mass spectrometry, and the synthesized polypeptide sequences are identified by polypeptide sequence determination.

[0108] 1. Synthesis of ApoE-ε4 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 synthesized with...

Embodiment 2

[0197] Embodiment 2: the ApoE-ε4 antigen epitope peptide (1) and (2) obtained in Example 1 are connected with carrier protein to prepare ApoE-ε4 antigen (1) and (2), utilize gained antigen (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).

[0198] 1. Preparation of antigen: ApoE-ε4 peptides (1) and (2) were respectively linked with carrier protein KLH (keyhole limpet hemocyanin) (obtained from sigma company) by BDB (Bis-diazotizedbenzidinedichloride) method to prepare ApoE- ε4 antigen (1) and (2).

[0199] Take 10.0mg of ApoE-ε4 peptide (1) or (2), dissolve it with 1ml 0.1MPBS buffer (pH7.4); dissolve 10mg of KLH with 20ml of 0.2M borate buffer (pH9.0); Mix them, cool to 0°C, take BDBCl 2110 μL, reacted at room temperature for 1.5 h, dialyzed overnight, then aliquoted, and stored at -20 °C.

[...

Embodiment 3

[0214] Example 3: Specific Identification of Human ApoE-ε4 Monoclonal Antibody (1) and (2)

[0215] Detection was performed by ELISA. Human ApoE-ε4 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 ApoE-ε4 monoclonal clones were detected by ELISA respectively. For the specific reaction of antibodies (1) and (2) with the human ApoE-ε4 protein, normal BALB / c mouse serum was used as negative control, and PBS solution was used as blank control.

[0216] Results: ApoE-ε4 monoclonal antibodies (1) and (2) only react positively with ApoE-ε4 (P / N>2.1), but negatively react with S-100B protein and neuron-specific enolase NSE , indicating that the ApoE-ε4 monoclonal antibodies (1) and (2) of the present invention have specificity respectively.

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Abstract

The invention relates to a fluorescence immune chromatography test paper for detecting human ApoE-[epsilon]4 protein and a preparation method thereof. The test paper is used for detecting the human ApoE-[epsilon]4 protein by virtue of a double-antibody sandwich method, and in the double-antibody sandwich method, a first ApoE-[epsilon]4 monoclonal antibody is labeled with fluorescence microspheres serves as a trapping antibody and is sourced from one of sequences indicated in sequence tables of SEQ ID NO.1 and SEQ ID NO.2; a second ApoE-[epsilon]4 monoclonal antibody serves as a detection antibody, and is sourced from the other one of the sequences indicated in the sequence tables of SEQ ID NO.1 and SEQ ID NO.2. The fluorescence immune chromatography test paper is simple and rapid to operate, wide in detection range, high in specificity and good in sensitivity.

Description

technical field [0001] The invention belongs to the field of polypeptide chemistry and immunology, and in particular relates to human apolipoprotein E-ε4 (ApoE-ε4) epitope peptide, ApoE-ε4 specific antigen prepared by using the epitope peptide and corresponding monoclonal antibody or Polyclonal antibody, application of said antibody in preparation of human ApoE-ε4 in vitro diagnostic kit, human ApoE-ε4 in vitro diagnostic kit, and a fluorescent immune layer for quantitative detection of human ApoE-ε4 protein in analyte Analysis test paper and its preparation method. Background technique [0002] Alzheimer's disease (AD) is a primary brain degenerative disease, which has become the fourth leading killer of human beings after cardiovascular disease, cancer and stroke. According to the current medical level, the treatment of AD can only delay the progression of the disease in early patients, and there is no effective treatment method for patients in the middle and late stages....

Claims

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Application Information

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Patent Type & Authority Applications(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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