Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

HPV pseudovirus, kit thereof and method for detecting HPV neutralizing antibodies

A pseudovirus and kit technology, applied in the field of immunology, can solve the problems of long operation cycle, high background value, limited HPV genotype, etc., and achieve the effects of less sample demand, objective result interpretation and standardized methods

Active Publication Date: 2013-10-02
NAT INST FOR FOOD & DRUG CONTROL +4
View PDF2 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantages of this method are: the test operation is complicated, the operation cycle is long (about 3 months), and the experimenter's proficiency is high, so it is not suitable for large-scale sample testing.
The disadvantages of this method are: the applicable HPV genotypes are very limited, and human keratinocytes need to be cultivated, which is difficult and complicated to operate
The disadvantages of this method are: Direct ELISA can only initially measure the total antibody titer, but not the neutralizing antibody titer
The disadvantage of this method is: when using this method to detect neutralizing antibodies, the neutralizing antibodies in the serum to be tested that do not compete with the HRP-labeled neutralizing monoclonal antibody for a specific epitope in the VLP cannot be detected, so in the competitive inhibition ELISA method The HRP-labeled neutralizing monoclonal antibody should be the monoclonal antibody against the main neutralizing epitope of VLP
This method is finally quantified by measuring the percentage of GFP-expressing cells in all cells by flow cytometry. Its disadvantages are: the operation is relatively complicated, and it is difficult to achieve high-throughput detection. Epidemiological Investigation
Its disadvantages are: the background value of target cell SEAP is high, it is difficult to prepare high-titer pseudoviruses, although high-throughput can be achieved, the operation is relatively complicated, and the test cost is high, so it is not suitable for large-scale clinical trials or epidemiological investigation of clinical samples
[0015] In short, the current test method is difficult to detect the level of neutralizing antibodies in large-scale clinical samples, so it is urgent to develop a method with high sensitivity, good specificity, short cycle time and high-throughput detection

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • HPV pseudovirus, kit thereof and method for detecting HPV neutralizing antibodies
  • HPV pseudovirus, kit thereof and method for detecting HPV neutralizing antibodies
  • HPV pseudovirus, kit thereof and method for detecting HPV neutralizing antibodies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] The preparation of embodiment 1 pseudovirus

[0072] 1 Structural gene expression plasmid and reporter plasmid co-transfected 293FT cells

[0073] at 75cm 2 293FT cells were inoculated into 15ml DMEM complete medium (containing 1% double antibody penicillin streptomycin solution (purchased from Hyclone Company), 1% L-glutamine (purchased from Hyclone Company), 1% non- Essential amino acids (purchased from Hyclone, 10% FBS (purchased from Invitrogen)) at 37°C, 5% CO 2 cultured in an incubator. When the cells grow to a confluence rate of more than 85%, the cell culture medium is aspirated, and 6ml of PBS is added to wash the cells, then the PBS is aspirated, and 3ml of 0.05% trypsin is added (purchased from Hyclone Company, which is pressed by PBS solution) Dilute the 293FT cells at a volume ratio of 1:5) to digest the 293FT cells, resuspend the cells with DMEM complete medium for 5 minutes and count them.

[0074] Press 4×10 5 cells / ml inoculated the cells in 75cm ...

Embodiment 2

[0111] HPV virus neutralizing antibody detection in embodiment 2 serum

[0112] The World Health Organization (WHO) pointed out in its "Guidelines for the Evaluation of Quality, Safety and Efficacy of HPV VLP Vaccines" that neutralization experiments are the "gold standard" for evaluating whether antibodies induced by HPV vaccines have protective effects.

[0113] In this experiment, after immunizing rabbits with HPV L1 antigens of the above-mentioned 6 genotypes (New Zealand big-eared white, provided by the Laboratory Animal Production and Supply Office of China Food and Drug Control Institute), the same type of pseudovirus and different types of pseudovirus were used to immunize rabbits. The sera after immunization were detected separately, and the steps were as follows:

[0114] (1) Spread 293TT cells on a 96-well culture plate, add 100 μl of 3.0×10 2 293TT cells / μl in complete DMEM medium at 37°C, 5% CO 2 Incubate for 6 hours in the incubator.

[0115] (2) According to ...

Embodiment 3

[0133] Embodiment 3 methodological verification and parameter optimization

[0134] 1 Selection of cell volume:

[0135] Add 100 μl of cultures of 293TT cells in DMEM complete medium at concentrations of 75 cells / μl, 150 cells / μl, 300 cells / μl and 600 cells / μl to the culture wells of a 96-well cell culture plate . At 37°C, 5% CO 2 Incubate in the incubator for 6 hours, then add an equal volume (100 μl) of pseudovirus dilution, mix well and place at 37°C, 5% CO 2 incubator for a total of 72 hours.

[0136] After co-incubation, transfer 15 μl of the culture supernatant in the cell culture plate to the corresponding wells of the chemiluminescence detection plate, and then add 15 μl of the chemiluminescence detection reagent (same as above) to each well of the chemiluminescence detection plate, and Immediately read in a chemiluminescent detector (same as above).

[0137] (1) Effect of cell volume on virus titer detection:

[0138] The pseudovirus extract obtained in Example ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to the immunology field, and concretely relates to an HPV pseudovirus, an HPV pseudovirus-containing kit and a method for detecting HPV neutralizing antibodies thereby.

Description

technical field [0001] The invention relates to the field of immunology, and more specifically relates to a HPV pseudovirus, a kit containing the HPV pseudovirus and a method for detecting HPV neutralizing antibodies by using the HPV pseudovirus. Background technique [0002] Human papillomavirus (HPV) belongs to the papillomaviridae family (papillomaviridae), which is a non-enveloped double-stranded circular DNA virus. After complete sequencing, the classified HPV can be divided into more than 100 genotypes [1 ,2]. According to its carcinogenicity, it can be divided into "low risk" type and "high risk" type. The former includes HPV6, HPV11, etc., which mainly cause benign genital warts and low-grade cervical epithelial necrosis (CIN) [3]; the latter includes HPV16, HPV18, etc., and their infection is the main cause of cervical cancer [4-6] . In most cases, the immune system clears HPV before it can cause harm, and more than 95 percent of genital HPV infections resolve wi...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01G01N33/53
Inventor 王佑春聂建辉黄维金吴雪伶
Owner NAT INST FOR FOOD & DRUG CONTROL
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com