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Preparation method of latex microsphere immunochromatography test paper based on haemophilus influenza (Hi) surface protein

A technology of Haemophilus influenzae and surface protein, applied in the direction of anti-bacterial immunoglobulin, immunoglobulin, chemical instruments and methods, etc., can solve problems such as difficult to meet rapid identification, affect the effect of amplification, and not suitable, etc. Achieve the effects of low preparation cost, extended shelf life, and strong antigenicity

Active Publication Date: 2019-12-06
HUBEI UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003]At present, the method for detecting the pathogen in the respiratory tract is mainly based on the traditional method, that is, the separation and identification method. Difficult to meet the needs of rapid identification; the PCR technology developed in recent years is a fast, sensitive, and specific technology, but at present this technology still relies on the pre-enrichment step of the traditional method, and the enrichment solution often There are also PCR inhibitors, which affect the effect of amplification
At the same time, this technology also requires professional testing equipment, which is not suitable for bedside testing

Method used

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  • Preparation method of latex microsphere immunochromatography test paper based on haemophilus influenza (Hi) surface protein
  • Preparation method of latex microsphere immunochromatography test paper based on haemophilus influenza (Hi) surface protein
  • Preparation method of latex microsphere immunochromatography test paper based on haemophilus influenza (Hi) surface protein

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Experimental program
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Embodiment 1

[0059] Preparation of Haemophilus influenzae surface protein (D15+PCP) antibody:

[0060] 1.1) Cloning of Haemophilus influenzae d15pcp fusion gene

[0061] Obtain the peptides with the most abundant epitopes in the extracellular domain of Haemophilus influenzae surface protein D15 and PCP (the accession numbers in the NCBI protein database are AAX87955 and AAX88288, respectively) and find the gene coding sequence to optimize its gene coding sequence. And these two sequences are connected with the coding sequence of flexible connecting peptide (ggsggsggsggs) to form a fusion gene. At the same time, the 5'restriction site NdeI was introduced into the fusion gene, and the termination signal TAA and restriction site BamHI were introduced into the 3'end of the fusion gene to chemically synthesize the entire gene sequence, which was recorded as d15pcp. The full sequence of the gene and the encoded amino acid sequence are shown in the sequence table. Specifically, the protein sequences...

Embodiment 2

[0090] Preparation of Haemophilus influenzae surface protein (Pe+pilA) antibody:

[0091] 2.1) Cloning of Pepil fusion gene of Haemophilus influenzae

[0092] Obtain the peptides with the most abundant epitopes in the extracellular domain of the surface proteins Pe and pilA of Haemophilus influenzae (the accession numbers in the NCBI protein database are AGT37361 and AAX12377, respectively) and find the gene coding sequence to optimize the gene coding sequence. And the two sequences are connected with the coding sequence of rigid linking peptide (eaaakeaaak) to form a fusion gene. At the same time, at the 5'end of the fusion gene, the restriction site NdeI was introduced, and the termination signal TAA and restriction site BamHI were introduced into the 3'end of the fusion gene, and the complete gene sequence was chemically synthesized, which was recorded as Pepil. The full sequence of the gene and the encoded amino acid sequence are shown in the sequence table. Specifically, the...

Embodiment 3

[0122] Preparation of latex microsphere marker for Haemophilus influenzae surface protein (D15+PCP) antibody:

[0123] 3.1) Activation of latex microspheres

[0124] Take 1mL of 10% red carboxylated polystyrene latex microsphere (100nm) solution, add 9mL 2-(N-morpholino)ethanesulfonic acid (MES) buffer (0.1mol / L MES, pH8.5 ) And mix well; prepare 10mg / mL N-hydroxysuccinimide (NHS) and 10mg / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide salt with MES buffer Salt (EDC) solution;

[0125] Add 1mL NHS solution and 1mL EDC solution to polystyrene latex microspheres (100nm) solution in turn, mix slowly at room temperature for 30 minutes, centrifuge at 19000g for 20 minutes after incubation, remove the supernatant, and use 10mL borax buffer for precipitation ( 0.1mol / L Na 2 B 4 O 7 , PH8.5) resuspension, shaking, ultrasonic treatment (ultrasonic breaker product model: YJ92-IIDN, power 50W, working time 2s, interval time 3s, alarm temperature 60℃, total time 30min), it becomes the activa...

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Abstract

The invention relates to a preparation method of latex microsphere immunochromatography test paper based on haemophilus influenza (Hi) surface protein. The test paper consists of a sample pad, a binding pad, a nitrocellulose membrane, a water absorbing pad and a PVC plate, wherein an anti-Hi surface protein polyclonal antibody coupled by colored latex microspheres is sprayed on the binding pad, and the nitrocellulose membrane is coated with a detection line of the anti-Hi surface protein polyclonal antibody and a quality control line of a goat anti-rabbit IgG antibody. When an added sample contains Hi, the Hi firstly reacts with latex-rabbit anti-hi surface protein polyclonal antibody to form a complex, the complex is captured when migrating to the detection line coated with the Hi surfaceprotein polyclonal antibody, the detection line has a corresponding color, and whether the sample contains Hi or not can be detected. The test paper has the advantages of rapidness, convenience, highsensitivity and good specificity.

Description

Technical field [0001] The invention belongs to the field of biological detection, and specifically relates to a preparation method of a latex microsphere immunochromatographic test paper based on the surface protein of Haemophilus influenzae. Background technique [0002] Haemophilus influenzae (Hi) is a Gram-negative bacillus with no motility. It was discovered in 1892 by Dr. Favor during the epidemic of influenza. It is generally an aerobic organism, but it can grow into a facultative anaerobic organism. Haemophilus influenzae was initially mistaken for the cause of influenza, but it was not until 1933 when the viral pathogen of influenza was discovered that this misunderstanding was eliminated. Haemophilus influenzae generally has six strains, called type a, type b (also known as type B), type c, type d, type e and type f. Diseases naturally produced by Haemophilus influenzae only occur in humans. In infants and children, Haemophilus influenzae type b can cause bacteremia...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70C07K16/12G01N33/68G01N33/569G01N33/543
CPCC07K14/285C07K16/1242C07K2319/00C12N15/70G01N33/54313G01N33/56911G01N33/68
Inventor 杨波陶冶胡征王毅
Owner HUBEI UNIV OF TECH