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Monoclonal antibody and preparation method thereof

A monoclonal antibody and antibody purification technology, which is applied in botany equipment and methods, biochemical equipment and methods, chemical instruments and methods, etc., can solve the problems of inability to produce influenza virus diagnostic reagents, etc., and achieve excellent reactivity and stable performance , the effect of high antigen titer

Inactive Publication Date: 2018-08-17
INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Colloidal gold test strips for accurate diagnosis of influenza virus infection and antigen ELISA kits for detecting influenza A virus are inseparable from influenza monoclonal antibodies with stable performance and high antigen titer. Domestic biopharmaceutical companies do not have Good monoclonal antibody, so it cannot produce useful influenza virus diagnostic reagents

Method used

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  • Monoclonal antibody and preparation method thereof
  • Monoclonal antibody and preparation method thereof
  • Monoclonal antibody and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1 Antigen Preparation

[0016] 1. Prepare the protein sequence matrix protein 1 [Influenza Avirus (A / PuertoRico / 8 / 1934 (H1N1))] of M1 gene, such as SEQ NO.1, numbered as C0131-Trx:

[0017]

[0018]

[0019] The preparation method is to insert the expression vector into the M1 gene, and insert the specified vector: pet32a

[0020] The M1 gene sequence is shown as SEQ NO.2:

[0021]

[0022]

[0023]

[0024]

[0025] Positive clone identification: After sequencing verification, the synthetic sequence is correct.

[0026] 2. Sample expression. Select 6 single clones from the transformation plate and inoculate 3ml of resistant medium respectively; culture to OD600nm0.5-0.6, add 0.5mM IPTG at 28°C to induce expression for 3.5 hours; collect the bacteria by centrifugation, sonicate, and detect the expression by SDS-PAGE. Analysis of small sample expression results: the protein is expressed in both the supernatant and the inclusion body, and solu...

Embodiment 2

[0029] Embodiment 2 animal immunization

[0030] 1 animal

[0031] Five Balb / C mice aged 5-8 weeks were immunized with the recombinant protein C0131-Trx and the viral protein mixture C0131-H9 provided by the customer, respectively.

[0032] 2 adjuvants

[0033] Freund's complete adjuvant was used for the first main injection, and Freund's incomplete adjuvant was used for subsequent booster injections. Both were fully mixed with an equal volume of antigen before injection.

[0034] 3 immunization methods. Multiple injections on the back. Main injection of 100ug antigen / rat, booster injection of 50ug antigen / rat.

[0035] immune cycle

[0036]

[0037]

[0038] 4. Antiserum detection. A small amount of blood was collected from the mouse tail vein to prepare antiserum. The titer of antiserum was detected by indirect ELISA method.

[0039]

[0040]

[0041] Reaction conditions: antigen coating: 37 degrees 2h

[0042] Closed: 37 degrees 1.5h

[0043] Serum ant...

Embodiment 3

[0047] Example 3 Cell Fusion and Subcloning

[0048] 1. Myeloma Cell Preparation

[0049] One week before fusion, revive SP2 / 0 cells and culture them to logarithmic phase normally.

[0050] 2. Splenocyte Preparation

[0051] The mice to be fused were selected and sacrificed by cervical dislocation on the day of fusion, the spleen was taken, and splenocytes were collected and counted according to the standard procedure.

[0052] 3. Cell Fusion

[0053] Mix myeloma cells and spleen cells at a ratio of 1:3-1:10, perform cell fusion operation according to the standard procedure, and then culture with HAT DMEM complete medium. Hybridoma cells can be seen 3 days after fusion, and replace them on the 7th day. 1 / 2HAT complete medium, change 1 / 2HT medium on the 8th day. Screening assays begin approximately 10 days after fusion.

[0054] Results of cell fusion: after fusion, cultured with HAT selective medium, observed under a microscope, many growing hybridoma cells were seen, pro...

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Abstract

The invention provides an influenza virus monoclonal antibody and a preparation method thereof. The influenza virus monoclonal antibody having stable performance and high antigen titer is prepared by:gene synthesis, carrier construction, expression of purified M1 recombinant protein, preparation of a monoclonal antibody strain by using the recombinant protein as an antigen, preparation of asciticfluid with the monoclonal antibody, and purification of the monoclonal antibody.

Description

technical field [0001] The invention relates to a monoclonal antibody, in particular to a preparation method of the influenza virus monoclonal antibody. Background technique [0002] Influenza virus is an important seasonal infectious disease. Several major outbreaks in history have caused huge damage and loss to human health and economy. Since the discovery of influenza virus, people have devoted themselves to the diagnosis, prevention and control of influenza virus. Influenza A virus has the characteristics of high variability and numerous subtypes, which brings certain difficulties to its prevention and control. Rapid and accurate diagnosis of influenza can provide time for proper treatment and prevention and control of influenza. Colloidal gold test strips for accurate diagnosis of influenza virus infection and antigen ELISA kits for detecting influenza A virus are inseparable from influenza monoclonal antibodies with stable performance and high antigen titer. Domestic...

Claims

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Application Information

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IPC IPC(8): C07K14/11C07K16/10C12N15/13
CPCC07K14/005C07K16/10C12N2760/16022
Inventor 许传田鲁梅崔宁黄庆华杨少华刘畅
Owner INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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