Method for detecting human papilloma virus neutralizing antibody

A human papillomavirus and antibody technology, which is applied in the field of human papillomavirus antibody detection, can solve the problems of high cost of the kit, affect the determination of results, and adversely affect the experimental efficiency, and achieves reduction of manual intervention, good sensitivity, and improved detection efficiency. Effect

Inactive Publication Date: 2009-10-07
XIAMEN UNIV +1
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Problems solved by technology

In the application, it is found that the operation of the first method above is more complicated and time-consuming, and requires cell digestion operation, and then detects with FACS one by one. The detection process of a 96-well plate usually takes 2 to 3 hours, which is time-consuming and laborious. The operation of the second method can use an instrument similar to a microplate reader to quickly scan and judge, which is conducive to batch detection, but the cost of the kit is relatively high, and it mainly detects the degree of enzymatic color reaction. When the cells are infected with a small amount of SEAP secretion, the positive / negative value ratio (P / N ratio) of the reading value will be low, which will affect the judgment of the result.
Therefore, in order to improve the P / N ratio, it is necessary to use a larger amount of pseudoviruses, which is not conducive to improving the experimental efficiency.

Method used

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  • Method for detecting human papilloma virus neutralizing antibody
  • Method for detecting human papilloma virus neutralizing antibody
  • Method for detecting human papilloma virus neutralizing antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] The construction of embodiment 1.HPV structural protein expression vector

[0063] HPV virus particles are composed of L1 protein and L2 protein. Studies have shown that co-expressing L1 and L2 proteins in cells can assemble virus-like particles of HPV, and its structural characteristics are similar to those of natural virus particles (Roden RB et al., Journal of Virology, 1996, Volume 70: pp. 5875-5883) . In the present invention, according to the gene data of HPV derived from Genbank, the method of gene synthesis is used to design and synthesize HPV containing different subtypes (here, HPV16, HPV18, HPV6, HPV11, HPV52, HPV58 are included as examples, but other subtypes can also be included. HPV subtype) L1 and L2 protein gene fragments, the 5' end of the fragment is added with a NotI site, and the 3' end is added with an XbaI site; the gene fragment is digested with NotI and XbaI and digested with mammalian cells of the same enzyme The expression vector pCDNA3.1(+) ...

Embodiment 2

[0065] Example 2. Selection of reporter gene expression plasmids

[0066] The present invention adopts spot detection instrument (such as Elispot) to detect the infection effect of HPV pseudovirus or HPV to target cells, so the signal produced by the reporter gene expression product mediated by HPV pseudovirus or HPV after infecting target cells must be able to be detected by spots To be identified and detected by the detection instrument, it is necessary to select a reporter gene whose expression product can lead to a suitable signal. In this example, we take β-galactosidase as an example as a reporter gene, and the expression element of the β-galactosidase reporter gene can be expressed to obtain β-galactosidase after being introduced into the cells. In the presence of X-gal or bluo-gal, etc.), the expressed cells can produce an obvious blue signal, and the generation of this signal does not need the assistance of other light sources, and this signal can be detected by the s...

Embodiment 3

[0069] The preparation of embodiment 3.HPV pseudovirus

[0070] The present invention prepares HPV pseudoviruses in 293FT cells (invitrogen company, catalog no. R700-07) by calcium phosphate multi-plasmid co-transfection method. The expression plasmids capable of expressing HPV L1 and L2 proteins and reporter gene expression plasmids (such as pCMVβ) were co-transfected into 293FT cells, and the cell lysates containing HPV pseudoviruses were collected 48 hours after transfection.

[0071] experimental method:

[0072] 293FT cells were cultured in 10cm cell culture dishes with DMEM medium (supplemented with 10% FBS, 2mM L-glutamine, 0.1mM MEM Non-Essential Amino Acids and 1% penicillin-streptomycin), and the confluence rate was about 80%.

[0073] After 12 hours, each plate of cells was transfected with 40 μg of HPV L1 expression plasmid, 40 μg of reporter gene expression plasmid and 4 μg of HPV L2 expression plasmid. The transfection reagent was calcium phosphate. For the tran...

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Abstract

The invention relates to a high-efficiency and simple method for detecting a human papilloma virus (HPV) neutralizing antibody, which can be applicable to high-throughput detection of the human papilloma virus neutralizing antibody. The method is characterized by using a spot detection method for detecting cells infected by human papilloma virus or pseudotype virus. The invention further discloses applications of the method in screening and identifying the neutralized monoclonal antibody against HPV, evaluating the immunity protectiveness of HPV vaccine, the potency test (ED50 test) of HPV vaccine, etc.

Description

technical field [0001] The invention relates to the field of human papillomavirus antibody detection. More specifically, the present invention relates to a method for detecting human papillomavirus neutralizing antibodies through the combined application of a human papillomavirus pseudovirus with a reporter gene and an automatic scanning instrument. Background technique [0002] Human papillomavirus (HPV) infection has a wide range of epidemics, and the related fatal malignant tumors and various sexually transmitted diseases caused by it are seriously harmful to human health. It is of great significance to develop safe and effective preventive or therapeutic vaccines. The level of neutralizing antibodies against HPV is a key indicator in the research and development of HPV vaccines, so it is very important to establish an efficient, accurate and convenient detection method for neutralizing antibodies. However, due to the strict host specificity of HPV and the close relation...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/68G01N33/533
Inventor 程通郑舟周国栋杜海莲张军夏宁邵
Owner XIAMEN UNIV
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