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A pair of monoclonal antibodies capable of specifically recognizing hcv NS3 protein and its application

A monoclonal antibody, specific technology, applied in the field of molecular biology and immunology, to achieve great application prospects and promotion value, high sensitivity and specificity, and reduce the effect of missed detection

Active Publication Date: 2022-01-07
山东莱博生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After searching, there are few reports about monoclonal antibodies with high affinity and specific binding to NS3 antigens or the application of using them to develop HCV antigen detection reagents or kits

Method used

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  • A pair of monoclonal antibodies capable of specifically recognizing hcv NS3 protein and its application
  • A pair of monoclonal antibodies capable of specifically recognizing hcv NS3 protein and its application
  • A pair of monoclonal antibodies capable of specifically recognizing hcv NS3 protein and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1: Preparation of HCV NS3 monoclonal antibody

[0028] 1.1 Antigen preparation

[0029] According to the HCV NS3 protein sequence (ID: O92530.3) on NCBI GenBank, TMHMM software was used to analyze the transmembrane domain of NS3 protein, and IEDB software was used to analyze the immunogenicity, hydrophobicity and surface accessibility of NS3 protein, and found that NS3 protein 226- The 480aa amino acid has strong antigenicity, hydrophilicity and surface accessibility, and can change the polarity of the corresponding sequence through amino acid mutation. After the above analysis, the final screening determined that the recombinant HCV NS3 antigen polypeptide sequence is: 226-480aa (the amino acid sequence is shown in SEQID NO.9). The short peptide and the N-terminal coupled human IL-1β protein short peptide (IL-1β-NS3) were synthesized by Jinan Boshang Biotechnology Co., Ltd.

[0030] 1.2 Animal immunity

[0031] Take the above-mentioned synthetic antigen, ...

Embodiment 2

[0048] Example 2: Purification of monoclonal antibodies

[0049] Wash the column with ultrapure water for about 5 column volumes; wash the column with equilibrium solution for about 5 column volumes (flow rate is 1.5ml / min). The crude antibody extract was diluted 2-3 times with buffer, filtered through a 0.45 μm microporous membrane, and then combined with protein A in the filler (1.5ml / min). Rinse the column with equilibration solution until the baseline is equilibrated, at least 10 column volumes. Use the eluent for antibody elution (flow rate 1.5ml / min). When the UV value is higher than 0.1, start to collect the flow-through liquid. When the UV value rises above 2.0, it starts to fall. When the UV value is lower than 0.1, stop receiving flow. The collected flow-through solution is the truncated HCV NS3 monoclonal antibody solution. Concentrate the collected solution to 15 ml using an ultrafiltration concentrator tube. The eluent continues to wash the affinity column unti...

Embodiment 3

[0052] Example 3: Antibody identification

[0053] 3.1 Potency identification of monoclonal antibodies

[0054] The synthetic HCV NS3 antigen short peptide was taken to coat the microtiter plate, and the titer of the purified monoclonal antibody was detected by indirect ELASA after blocking with 5% BSA solution. The ascitic fluid samples containing the above monoclonal antibodies were serially diluted, and PBS was used as a negative control, 100 μl per well, added to the microtiter plate, and incubated at 37°C for 1 hour. After washing with TBST, add 1:4000 diluted HRP-labeled secondary antibody, 100 μl per well, and incubate at 37°C for 1 hour. After washing with TBST, TMB substrate was added to develop color for 20 min, and then the reaction was terminated. Microplate reader reads OD 450 values, and the test results are shown in Table 1.

[0055] 3.2 Subtype identification

[0056] Use the antibody typing kit to identify the subtype of the monoclonal antibody, in which ...

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Abstract

The invention discloses two monoclonal antibodies capable of specifically recognizing HCV NS3 protein used in pairs, wherein the antibody is named 3A10B5, the amino acid sequence of its light chain is shown in SEQ ID NO.1, and the amino acid sequence of its heavy chain is As shown in SEQ ID NO.2; another antibody named 7E3B6, the amino acid sequence of its light chain is shown in SEQ ID NO.3, and the amino acid sequence of its heavy chain is shown in SEQ ID NO.4. The invention also discloses the application of the monoclonal antibody as a coating antibody and a detection antibody paired in the preparation of a hepatitis C virus antigen ELISA detection reagent or kit. Experiments have proved that the binding of the monoclonal antibody of the present invention to the NS3 antigen in the serum of HCV infected patients has high specificity and affinity, and has important application value for the screening of hepatitis C virus.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and immunology, and relates to two paired monoclonal antibodies capable of specifically recognizing HCV NS3 protein and applications thereof. Background technique [0002] Hepatitis C virus (HCV) infection is the main cause of hepatitis C in humans. According to statistics, there are currently about 170 million people infected with hepatitis C virus in the world, and about 70% of the infected people will develop chronic infection, and 5% of them will aggravate into liver cirrhosis or hepatocellular carcinoma. Currently, there is no vaccine available to prevent HCV infection. Therefore, the use of highly sensitive and specific detection methods to screen relevant populations is an effective measure to control HCV transmission and detect virus carriers as early as possible. People's health and maintaining public safety are of great significance. [0003] HCV is a single-stranded RNA viru...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/10C12N15/13C12N5/20C07K14/18G01N33/577G01N33/576G01N33/569
CPCG01N33/56983G01N33/5767G01N33/577C07K14/005C07K16/109C07K2317/33G01N2333/186G01N2469/10C12N2770/24222
Inventor 欧兰香徐明杰解光宁陈振任素平朱之炜王佳颖王岩李红霞李敏原月
Owner 山东莱博生物科技有限公司
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