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A high-throughput detection method for human papillomavirus neutralizing antibodies

A technology of human papillomavirus and detection method, which is applied in the field of high-throughput detection of human papillomavirus neutralizing antibodies, can solve the problem of high background value, difficulty in detecting the level of neutralizing antibodies in large-scale clinical samples, and inapplicable epidemics research questions

Active Publication Date: 2017-11-14
NAT INST FOR FOOD & DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of this method is that 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
Disadvantages: 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 clinical trials Epidemiological investigation of samples
Disadvantages: This method can only detect neutralizing antibodies against one type of pseudovirus at a time, and the current HPV vaccines are all multivalent vaccines composed of multiple types of antigens (GSK's 2-valent vaccine, Merck's 4-valent and 9 There are 2-valent, 3-valent, 4-valent, 6-valent, 9-valent and 13-valent vaccines in China). For the detection of different types of antibodies, only modified pseudoviruses can be prepared separately. detection
The current test methods are difficult to detect the level of neutralizing antibodies in large-scale clinical samples, especially for the detection of neutralizing antibodies in the clinical evaluation of multivalent HPV vaccines, it is urgent to develop a method with high sensitivity, good specificity, short cycle time and high throughput detection method

Method used

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  • A high-throughput detection method for human papillomavirus neutralizing antibodies
  • A high-throughput detection method for human papillomavirus neutralizing antibodies
  • A high-throughput detection method for human papillomavirus neutralizing antibodies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0130] Preparation of pseudovirus

[0131] Co-transfection of structural gene expression plasmid and reporter plasmid into 293FT cells

[0132] (1) Trypsinization of 293FT cells, counting, 6 × 10 6 Cells / 15ml inoculated on 75cm 2 Cell culture flasks, 37 °C, 5% CO 2 cultured in an incubator.

[0133] (2) When the cell confluency reached about 50%, the structural gene expression plasmids (p16LLw and p18LLw) and fluorescent protein reporter plasmids (PCDNA3-GFP and pRwB) were co-transfected into 293FT cells using Lipofectamine2000.

[0134] (3) Dissolve 15 μg each of the structural gene expression plasmid and reporter plasmid in 1.875 ml medium without resistance and bovine serum, and mix gently.

[0135] (4) Take 75 μl of Lipofectamine2000, dissolve it in 1.875ml of non-resistance and bovine serum-free medium, mix gently, and let stand at room temperature for 5 minutes. This process should not exceed 25 minutes.

[0136] (5) Add the Lipofectamine 2000 mixture into the plasm...

Embodiment 2

[0183] Example 2 Correlation between Fluorospot spot count and flow cytometry positive cell ratio

[0184] The classic method for determining the detection results of HPV pseudovirus neutralizing antibodies based on green fluorescent protein is calculated according to the proportion of positive cells by flow cytometry. The HPV16 and HPV18 pseudoviruses that respectively comprise green fluorescent protein (EGFP) and red fluorescent protein (RFP) prepared in Example 1 carry out virus titration, then count respectively with Fluorospot and flow cytometer, then carry out linear regression analysis, 4 kinds of pseudoviruses The linear correlation coefficient R of the detection results of the virus 2 The values ​​are above 0.99 ( figure 2 ), indicating that for the counting of two fluorescent protein pseudoviruses, fluorescent spot counting and flow cytometry have good consistency.

[0185] Selection of virus amount: For two different samples, in the case of different virus amount...

Embodiment 3

[0193] Detection of clinical samples after vaccine immunization: using the mixed detection of two pseudoviruses obtained in Example 1 and a separate detection method of pseudoviruses, 10 clinical vaccine immunization samples were detected respectively, and the correlation of the detection results of the two methods was compared , for the detection of HPV16 and HPV18 neutralizing antibodies, both methods showed good correlation, R 2 All greater than 0.95.

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Abstract

The invention relates to the field of biotechnology, in particular to a high-throughput detection method for human papilloma virus (HPV) neutralizing antibodies. According to the detection method, an HPV pseudovirus containing different EGFP and RFP reporter genes is constructed, and pseudoviruses containing different reporter genes are constructed and mixed to detect different neutralizing antibodies. According to the method, operation is easy and convenient, the detection period is short and only contains 72 hours, sensitivity is high, repeatability is good, result interpretation is objective, high throughput is achieved, the demand quantity of samples is small, detection cost is low, the method can be standardized, interlaboratory popularization is facilitated, neutralizing antibodies used for various types of viruses can be detected simultaneously, and the real situation formed after vaccine immunity can be more approached.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a high-throughput detection method for human papillomavirus neutralizing antibodies. Background technique [0002] Human papillomavirus (HPV) belongs to the papillomaviridae family (papillomaviridae), which is a non-enveloped double-stranded circular DNA virus. The HPV classified after complete sequencing can be divided into more than 100 genotypes. 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); the latter includes HPV16, HPV18, etc., and their infection is the main cause of cervical cancer. In most cases, the immune system clears HPV before it can cause harm, and more than 95 percent of genital tract infections heal within 3 to 5 years. But there are also a small number of HPV infections that cannot be clea...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569
CPCG01N33/56983G01N2333/025
Inventor 王佑春聂建辉黄维金吴雪伶
Owner NAT INST FOR FOOD & DRUG CONTROL