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Fluorescence immunochromatography test paper for detecting human Lp-PLA2 proteins and preparation method of fluorescence immunochromatography test paper

A fluorescence immunochromatography, lp-pla2 technology, applied in biological testing, measuring devices, analytical materials, etc., to achieve the effects of optimized preparation conditions, wide detection range, and improved fluorescence signal-to-background ratio

Active Publication Date: 2014-12-24
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 Lp-PLA2 protein

Method used

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  • Fluorescence immunochromatography test paper for detecting human Lp-PLA2 proteins and preparation method of fluorescence immunochromatography test paper
  • Fluorescence immunochromatography test paper for detecting human Lp-PLA2 proteins and preparation method of fluorescence immunochromatography test paper
  • Fluorescence immunochromatography test paper for detecting human Lp-PLA2 proteins and preparation method of fluorescence immunochromatography test paper

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0111] Example 1: Preparation of Lp-PLA2 epitope peptides (1) and (2).

[0112]The preparation method uses chemical synthesis method: Lp-PLA2 antigenic 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 1690.09 and 1623.92 respectively, which are determined by mass spectrometry, and the synthesized polypeptide sequences are identified by polypeptide sequence determination.

[0113] 1. Synthesis of Lp-PLA2 epitope peptides (1) and (2)

[0114] 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 synthes...

Embodiment 2

[0202] Embodiment 2: the Lp-PLA2 antigen epitope peptide (1) and (2) obtained in Example 1 are linked with carrier protein to prepare Lp-PLA2 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).

[0203] 1. Antigen preparation: Lp-PLA2 peptides (1) and (2) were respectively linked with carrier protein KLH (keyhole limpet hemocyanin) by BDB (Bis-diazotizedbenzidine dichloride) method to prepare Lp-PLA2 antigen (1) and (2).

[0204] Take 10.0mg of Lp-PLA2 peptide (1) or (2), dissolve it with 1ml 0.1M PBS buffer (pH7.4); dissolve 10mg of KLH with 20ml of 0.2M borate buffer (pH9.0); 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.

[0205] In the present embod...

Embodiment 3

[0219] Example 3: Specific identification of human Lp-PLA2 monoclonal antibodies (1) and (2)

[0220] Detection was performed by ELISA. Human Lp-PLA2 protein, fibrinogen, and C-reactive protein (all purchased from Shanghai Lianshuo Company) were used as detection antigens to coat ELISA plates, and the prepared Lp-PLA2 monoclonal antibodies (1) and (2) For the specific reaction with the human Lp-PLA2 protein, normal BALB / c mouse serum was used as negative control, and PBS solution was used as blank control.

[0221] Result: Lp-PLA2 monoclonal antibody (1) and (2) only react positively (P / N>2.1) with Lp-PLA2 respectively, and are negative with fibrinogen, C-reactive protein reaction, illustrate that the present invention Lp-PLA2 monoclonal antibodies (1) and (2) have specificity respectively.

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Abstract

The invention relates to fluorescence immunochromatography test paper for detecting human Lp-PLA2 proteins and a preparation method of the fluorescence immunochromatography test paper. The test paper is used for detecting the human Lp-PLA2 proteins by virtue of a two-antibody sandwich method, and in the two-antibody sandwich method, a first Lp-PLA2 monoclonal antibody which 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 Lp-PLA2 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 immunochromatography 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 a human lipoprotein-associated phospholipase A2 (Lp-PLA2) epitope peptide, an Lp-PLA2 specific antigen prepared by using the epitope peptide, and a corresponding monoclonal antibody or Polyclonal antibody, use of said antibody in preparing human Lp-PLA2 in vitro diagnostic kit, human Lp-PLA2 in vitro diagnostic kit, and a fluorescent immune layer for quantitative detection of human Lp-PLA2 protein in analyte Analysis test paper and its preparation method. Background technique [0002] Cardiovascular and cerebrovascular diseases have always been recognized as one of the biggest killers of human health and the leading cause of death worldwide. The number of people who die from cardiovascular and cerebrovascular diseases in my country is as high as 3 million or more every year, and there are more than 60 million patients. In recent years, with the co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577
CPCG01N33/566G01N33/92
Inventor 朱建安
Owner 深圳市安群生物工程有限公司
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