Preparation method of human parvovirus antigen and rapid detection kit prepared for detecting parvovirus B19 type antibody through antigen

A parvovirus and human technology, applied in the field of clinical medical detection, can solve the problems of difficult in vitro culture of B19 virus, long detection time, and troublesome operation, and achieve the effect of obvious treatment prompting effect, high immunogenicity and strong specificity.

Inactive Publication Date: 2017-01-04
LANZHOU YAHUA BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because the B19 virus is not easy to culture in vitro, and the antigen preparation is also difficult, it is difficult for the serological detection of B19 to be widely used.
However, electron microscope observation of virus particles requires electron microscope equipment, which has a small application range and troublesome operation. In addition, the selection of observation points may be disturbed by human factors.
In addition, radioimmunoassay (RIA), enzyme immunoassay (EIA), immunofluorescence (IF), western blotting (WB) and enzyme-linked immunosorbent assay (ELISA) in molecular biology detection require professionals to operate , requires equipment for interpretation, the detection time is long, and it is difficult to promote in clinical application, popularization, on-site detection, etc.
[0006] At present, there is no convenient method for rapid detection of human parvovirus

Method used

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  • Preparation method of human parvovirus antigen and rapid detection kit prepared for detecting parvovirus B19 type antibody through antigen
  • Preparation method of human parvovirus antigen and rapid detection kit prepared for detecting parvovirus B19 type antibody through antigen
  • Preparation method of human parvovirus antigen and rapid detection kit prepared for detecting parvovirus B19 type antibody through antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Recombinant expression, structure renaturation and purification of recombinant human parvovirus VP2 protein antigen

[0049] Pediatric human parvovirus VP2 gene refers to the GenBank sequence NC_000883.2, selects part of the gene for synthesis, and removes the signal peptide sequence during synthesis. The expression vector is pET30a, and the enzyme cleavage site is EcoR I / XhoI when connecting. ), transformed into BL21 (DE3), the total expressed recombinant protein was 314aa, the molecular weight was 35.2 kDa, and the isoelectric point was 9.43. The recombinant protein is expressed in the form of inclusion body and can be purified by Ni column.

[0050] Construction and identification of the recombinant expression vector: EcoRI and XhoI were used to double-enzyme digest the human parvovirus VP2 target gene fragment and pET30a plasmid respectively. E. coli For DH5α competent cells, the transformed colonies were picked to extract plasmids for identification by...

Embodiment 2

[0053] Example 2 Preparation of a gold-labeled rapid detection kit for human parvovirus antibody IgG / IgM

[0054] The recombinant human parvovirus VP2 protein obtained above is used as a detection antigen, and the detection line is coated on a nitrocellulose membrane; refer to figure 2 , prepare human parvovirus antibody gold label rapid detection reagent, its composition comprises: be provided with on the backing board 10 and be provided with the water-absorbing layer 4 of sample loading end, detection layer 8 and water-absorbing layer 9, between detection layer and sample-loading end water-absorbing layer 4 There is a gold-labeled anti-human parvovirus antibody layer 5 between them, and a detection line 7 and a quality control line 6 are coated on the detection layer 8 . Wherein, the water-absorbing layer 4 at the sample loading end and the water-absorbing layer 9 at the water-absorbing end are made of multi-layer filter paper: the detection layer 8 is a nitrocellulose memb...

Embodiment 3

[0058] Example 3 Determination of Human Parvovirus Antibody

[0059] Take 10 μL of serum or plasma sample and drop it into the sample well 2 of the test plate 1, then add 100 μL of the sample diluent into the sample well 2, and observe the test result in the observation window 3, and the observation result is valid within 20 minutes. If the sample contains anti-human parvovirus antibodies, two red lines appear in the detection line and quality control line in the observation window, and the test result is judged as positive; If a red line is seen at the position of the quality control line, the test result is judged as negative; if no red line is visible in the observation window, the test result is invalid.

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Abstract

The invention relates to a human parvovirus gene engineering artificial expression antigen. A method for preparing the antigen comprises the steps that part of gene sequences of a human parvovirus VP2 protein antigen are artificially synthesized, a prokaryotic expression vector is constructed, the human parvovirus VP2 protein antigen is expressed by escherichia coli, an inclusion body is renatured by adopting a dialysis method, a gradient dilution method and a gel chromatography method, and the recombinant human parvovirus VP2 protein antigen with a three-dimensional structure and immunocompetence is obtained. A rapid detection method for a human parvovirus antibody comprises the step of applying the human parvovirus VP2 protein antigen. A rapid detection kit for detecting the human parvovirus antibody comprises the human parvovirus VP2 protein antigen and can be directly applied to whole blood detection. The kit comprises a rheumatoid factor disposal pad and can remove rheumatoid factors in a sample and directly detect IgM in the sample. The human parvovirus antigen has the high specificity. The method for preparing the antigen, the method for rapidly determining the human parvovirus antibody and the kit for rapidly determining the human parvovirus antibody are provided.

Description

technical field [0001] The invention belongs to the field of clinical medical detection, relates to immunochromatographic detection technology, in particular to a human parvovirus antigen for diagnosing human parvovirus infection, a method for preparing the antigen, and a rapid detection kit for detecting human parvovirus with the antigen . Background technique [0002] Typical diseases caused by Human Parvovirus B19 (HPV) are erythema infectiosum and acute arthropathy. Generally, infected persons are self-limited and can recover by themselves. However, the virus can cause aplastic crisis in some blood diseases and immunocompromised patients, and can cause fetal hydrops or even stillbirth in pregnant women. In addition to these common clinical symptoms, it often causes respiratory tract infection symptoms such as acute respiratory inflammation. In most countries, B19 virus infection usually occurs in children, and about 50% of children do not have anti-B19 antibodies by th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/015G01N33/68G01N33/569
CPCC07K14/005C12N2750/14222G01N33/56983G01N33/6854G01N2469/20
Inventor 李克生杜惠芬
Owner LANZHOU YAHUA BIOTECH
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