Hybridoma cell strain C11-6F7 as well as produced human cytomegalovirus (HCMV) monoclonal antibody and application thereof

A hybridoma cell line and monoclonal antibody technology, applied in biochemical equipment and methods, microbes, biological material analysis, etc., can solve the problems of low specificity, low sensitivity, and inability to solve active infection, etc., and achieve high purity , the effect of excellent performance

Active Publication Date: 2018-12-07
GUANGDONG HECIN SCI INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, domestic methods for detecting HCMV mainly include fluorescent quantitative PCR or ELISA. Although the nucleic acid detection kit has the characteristics of high sensitivity and strong specificity, the detection signal is nucleic acid (including latent infection), and it is impossible to distinguish between active infection and latent infection. Infection, which does not resolve the issue of an active infection
Antibodies used in immunoassays such as ELISA are polyclonal antibodies, which have problems such as purity and stability cannot be guaranteed, so there are disadvantages such as low sensitivity and low specificity for its application in the detection of active infection

Method used

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  • Hybridoma cell strain C11-6F7 as well as produced human cytomegalovirus (HCMV) monoclonal antibody and application thereof
  • Hybridoma cell strain C11-6F7 as well as produced human cytomegalovirus (HCMV) monoclonal antibody and application thereof
  • Hybridoma cell strain C11-6F7 as well as produced human cytomegalovirus (HCMV) monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Obtaining of HCMV-pp65-C214 monoclonal antibody

[0026] Prepare anti-HCMV-pp65-C214 monoclonal antibody according to the following steps:

[0027] 1. Preparation of immunogen: The protein sequence encoded by UL83 was found from NCBI, and the 214 amino acids at the C-terminal were optimized according to the prokaryotic expression codons to synthesize the gene sequence, connected to the prokaryotic expression vector pet30a, and induced by IPTG. After the inclusion body was crushed by ultrasonic waves, the precipitate was dissolved in 8M urea and then purified by a nickel column. The miscellaneous proteins were washed with 20mM imidazole, and the proteins eluted with 50mM, 100mM, and 200mM imidazole were collected respectively. The eluted products were identified by SDS-PAGE. The electrophoresis results are shown in the figure As shown in 1, the one marked PP65-C214 in the figure is the target protein eluted from 200mM imidazole, and the electrophoresis picture...

Embodiment 2

[0030] Embodiment 2: Ascites titer and sensitivity measured by indirect ELISA

[0031] 2.1 Ascites titer: The antigen PP65-C214 obtained in Example 1 was diluted with carbonate buffer solution and coated on a 96-well plate, 100ng / well, incubated at 37°C for 2 hours, and then PBS -T Wash the plate once. Add PP65-C214 ascites with different dilutions obtained in Example 1 and incubate at 37°C for 30 minutes, set up negative control wells, positive control wells (3E4) and blank control wells (antibody dilution), and wash the plate with PBS-T after incubation for 5 minutes Second-rate. Dilute the HRP-labeled antibody (goat anti-mouse IgG-HRP) by 1000 times with enzyme label diluent, 100 μl per well. Incubate at 37°C for 30min. After incubation, the plate was washed 5 times with PBS-T. Color development, liquid A: liquid B = 1:1, ready to use. 100 μl per well, incubate at 37°C for 15 minutes; add stop solution, 50 μl / well, to terminate the color reaction, and detect the absorb...

Embodiment 3

[0037] Example 3 Specific detection of ascites and further verification by Western Blot

[0038] 3.1 Specificity detection: PP65-N347 antigen and PP65-C214 antigen encoded by the N-terminal 1041bp of pp65 gene were respectively coated, and the cross-reaction of 6F7 antibody to PP65-N347 was detected by competitive ELISA method to detect antibody specificity. Coat the 96-well plate after diluting with salt coating solution, 100 / 50ng per well, incubate at 37°C for 2 hours, and wash the plate once with PBS-T after the incubation. Add monoclonal antibody, incubate at 37°C for 30min, blank control (antibody diluent). After incubation, the plate was washed 5 times with PBS-T. Dilute the HRP-labeled antibody (goat anti-mouse IgG-HRP) 1000 times with 100 μl per well. Incubate at 37°C for 30min. After incubation, the plate was washed 5 times with PBS-T. Chromogenic solution A: Chromogenic solution B = 1:1, ready to use. 100 μl per well, incubated at 37°C for 15 minutes; adding sto...

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Abstract

The invention provides a hybridoma cell strain C11-6F7 as well as a produced human cytomegalovirus (HCMV) monoclonal antibody and an application thereof. The hybridoma cell strain C11-6F7 is preservedin the China center for type culture collection (CCTCC) with the preservation number CCTCC NO: C2017196. The HCMV monoclonal antibody can be applied to an immunoassay method. The invention also provides a kit comprising the HCMV monoclonal antibody. PP65-C214 protein is expressed by utilizing truncated 642 genes at C terminal of a PP65 gene, so that the expression difficulty of recombinant antigen is reduced while the cost is reduced and the obtained recombinant antigen has high purity; and the monoclonal antibody which has high sensitivity and specificity is applied to the immunofluorescenceand flow cytometry techniques and can be used for detecting infected cells and reflecting the active state of HCMV infection.

Description

technical field [0001] The invention relates to the field of a monoclonal antibody, in particular to a hybridoma cell line C11-6F7 and the HCMV monoclonal antibody produced therefrom and application thereof. Background technique [0002] Human cytomegalovirus (human cytomegalovirus, HCMV) is a double-stranded linear DNA virus of the genus Herpesvirinae. It can be transmitted through the placenta or reproductive tract, and can also be transmitted through breast milk, blood, saliva, etc. After infection, it is carried for life. The virus has a wide range of target cells and can invade various organs of the body, so the clinical symptoms are complex and changeable. The infection rate of cytomegalovirus in developing countries is more than 90%, which is quite common. Although most adult infected persons do not have obvious clinical symptoms (latent infection), it will bring serious symptoms to fetuses, newborns or individuals with immunodeficiency. serious illness. HCMV is one...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/08G01N33/577G01N33/569
CPCC07K16/088G01N33/56994G01N33/577G01N2333/045
Inventor 李小锋李晨阳刘铭龙邓国亮李园枚庞妙桦
Owner GUANGDONG HECIN SCI INC
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