PCT (Procalcitonin) and CRP (C-Reactive Protein) double-label time resolution fluorescence immunoassay method for simultaneously detecting bacterial meningitis and viral meningitis

A viral meningitis and bacterial technology, applied in the biological field, can solve the problems of difficulty in meeting the needs of early diagnosis of meningitis, poor sensitivity, etc., and achieve the effect of shortening the time of disease diagnosis, reducing anxiety and panic, and reducing the molecular weight of antigens

Active Publication Date: 2017-02-15
王燕 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are many detection methods for PCT and CRP, their sensitivity is poor and it is difficult to meet the needs of clinical early diagnosis of meningitis

Method used

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  • PCT (Procalcitonin) and CRP (C-Reactive Protein) double-label time resolution fluorescence immunoassay method for simultaneously detecting bacterial meningitis and viral meningitis
  • PCT (Procalcitonin) and CRP (C-Reactive Protein) double-label time resolution fluorescence immunoassay method for simultaneously detecting bacterial meningitis and viral meningitis
  • PCT (Procalcitonin) and CRP (C-Reactive Protein) double-label time resolution fluorescence immunoassay method for simultaneously detecting bacterial meningitis and viral meningitis

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0028] 1. Preparation of PCT-CRP recombinant antigen

[0029] PCT and CRP specific sequences are selected and combined to obtain PCT-CRP recombinant antigen, the nucleotide sequence of which is as follows:

[0030] GCCCTGGAAAGCAGCCCGGCCGATCCGGCCACCCTGAGCGAAGATGAAGGCGGCGGCGGCGCCATTGGCGTGGGCGCCCCGGGCAAAAAACGCGATATGAGCAGCGATGGCGGCGGCGGCAAAGCCCCGCTGACCAAAACCGCTGAAAAGCCTTTACCSEGTGTGCCTGCATGGCGGCGGCGCAACTTTGAAGGCAGCCANOGCCTGGTGGGC as nucleotides in GATATTG. The present invention obtains the above-mentioned PCT-CRP recombinant antigen through a large number of preliminary studies and preliminary experiments, through screening and optimizing combination of specific fragments related to PCT and CRP antigens.

[0031]The above-mentioned PCT-CRP recombinant polypeptide was prepared and expressed by genetic engineering technology, its amino acid sequence is: ALESSPADPATLSEDEGGGGAIGVGAPGKKRDMSSDGGGGKAPLTKPLKAFTVCLHGGGGNFEGSQSLVGDIGNVN, and its amino acid sequence is shown in SEQ ID NO:1. ...

Embodiment 1

[0043] Example 1 A PCT and CRP double-labeled time-resolved fluorescent immunoassay kit for simultaneous detection of bacterial and viral meningitis

[0044] 1. Preparation of solid-phase coated antibodies: Dilute PCT and CRP antibodies to 3 μg / ml and 4 μg / ml with 50 mmol / L, pH 9.6 carbonate buffer, respectively, and add 100 μl / well to a 96-well plate , overnight at 4°C, pour off the coating solution, add blocking solution at 200 μl / well, block overnight, pour off the blocking solution, wash and pat dry, and store in a vacuum at -20°C.

[0045] 2. Eu 3+ and Sm 3+ Preparation of labeled antibody: Add 0.5 mg of labeled antibody into a centrifuge tube with filter membrane from Millipore Company, and centrifuge at 8000 r / min for 6 min. Then labeling buffer (50mmol / L, NaCO 3 , pH 9.0) were washed 6 times. 200 μl of labeled antibody and 50 μg of europium (or samarium) labeling reagent were thoroughly mixed and shaken at room temperature overnight.

[0046] 3. Eu 3+ and Sm 3+ ...

Embodiment 2

[0049] Example 2 A PCT and CRP double-labeled time-resolved fluorescent immunoassay method for simultaneous detection of bacterial and viral meningitis

[0050] The present invention uses the kit described in Example 1 to quantitatively detect PCT and CRP levels. The detection operation steps are: add 25 μl of PCT / CRP reference standard or serum to be tested on the coated enzyme-linked plate, and then add 200 μl of analysis buffer solution, incubated with shaking for 1h, washed with washing solution for 4 times, and then added Eu 3+ -PCT monoclonal antibody with Sm 3+ - CRP monoclonal antibody mixture 200 μl / well, incubate with shaking for 1 hour, wash with washing solution 6 times, finally add 200 μl / well of enhancement solution and shake for 5 minutes, then detect on a time-resolved fluorescence detector.

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Abstract

The invention discloses a PCT (Procalcitonin) and CRP (C-Reactive Protein) double-label time resolution fluorescence immunoassay method for simultaneously detecting bacterial meningitis and viral meningitis. A recombinant polypeptide antigen is included and the amino acid sequence is shown as SEQ ID NO: 1. The recombinant polypeptide antigen is used as an immunogen, and a PCT antibody and a CRP antibody are obtained by screening respectively through immune treatment; double-label time resolution fluorescence immunoassay is carried out. The method disclosed by the invention is high in sensitivity, is rapid, is simple and convenient and is easy to popularize clinically. The whole detection time of a kit utilizing the method is less than 2 hours, and suspected patients with the meningitis can be rapidly screened at an early stage. Reliable evidences are provided for specific treatment of children with the meningitis; pains of the children with the meningitis are alleviated and the diagnosis time of diseases is shortened; valuable time for the specific treatment is won, so that medical cost and a lot of manpower and material resource expenditures are saved, and worries and panics of parents are also alleviated.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a PCT and CRP double-labeled time-resolved fluorescent immunoassay method for simultaneously detecting bacterial and viral meningitis. Background technique [0002] Central nervous system infection is a common and frequently-occurring disease in pediatrics. Bacterial meningitis and viral meningitis and encephalitis are the most common diseases, among which bacterial meningitis is the most common, accounting for about 75% of central nervous system infections. Bacterial meningitis is a serious infectious disease with high mortality and sequelae. Among them, meningococcus, influenza bacillus, pneumococcus, Escherichia coli and other Gram-positive bacilli are the pathogenic bacteria of bacterial meningitis , which requires acute care, and any delay in diagnosis and treatment will result in permanent disability and death. Viral meningitis is a group of diffuse inflammatory sy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/6408
Inventor 王燕张立成
Owner 王燕
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