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Monoclonal antibody capable of resisting chlamys farrei particle blood corpuscle and preparation method thereof

A monoclonal antibody and granule blood cell technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, anti-animal/human immunoglobulin, etc., can solve the problems of molecular markers tracking immune response and inconclusive conclusions , to achieve the effect of strict, reasonable and feasible preparation technology route, uniform size and good potency

Active Publication Date: 2010-10-20
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the role and status of these two types of blood cells in the function and occurrence have not been definitively determined, mainly because there are no suitable molecular markers to track the process of their occurrence and participation in the immune response

Method used

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  • Monoclonal antibody capable of resisting chlamys farrei particle blood corpuscle and preparation method thereof
  • Monoclonal antibody capable of resisting chlamys farrei particle blood corpuscle and preparation method thereof
  • Monoclonal antibody capable of resisting chlamys farrei particle blood corpuscle and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1: Percoll medium discontinuous density gradient separation of whole blood cells of Chlamys farreri

[0020] 1. Percoll commercial stock solution and 10 times concentration of anticoagulant (0.14M NaCl, 3mM KCl, 8mM NaCl 2 HPO 4 , 1.5mMKH 2 PO 4 , 20mM EDTA, pH 7.4) to prepare Percoll application solution at a ratio of 9:1 by volume, and then dilute Percoll application solution with anticoagulant to different gradients of 50% (v / v), 40%, 30%, 20% and 10%, each gradient 2ml, placed in a Percoll centrifuge tube, 4 ° C overnight;

[0021] 2. Take 8-10 vigorous and healthy Chlamys farreri with normal physique, extract hemolymph from the adductor muscle with a sterilized clean syringe, and mix it with pre-cooled anticoagulant at a volume ratio of 1:1. Centrifuge at 4°C at 760g for 10 minutes, and then resuspend the whole blood cell pellet with anticoagulant to adjust the concentration of the whole blood cell suspension to 10 7 cells / ml;

[0022] 3. Take part of...

Embodiment 2

[0027] Example 2: Preparation of monoclonal antibodies against hemocytes from Chlamys farreri

[0028] 1. Immunization of Mice

[0029] The third layer of cell suspension separated by Percoll was used as the antigen to immunize 4-week-old female BALB / c mice. The dose of each immunization was 0.1ml, and the immunization was divided into 4 times. The interval between the first 2 immunizations was 2 weeks, and the interval between the next 2 immunizations was 1 week. The first 2 times were intraperitoneal injections, and the last 2 times were tail vein injections.

[0030](1) Basic immunization: the third layer of cell suspension is mixed with Freund's complete adjuvant in equal volumes as antigen;

[0031] (2) Booster immunization: mix the suspension of the third layer of cells with Freund's incomplete adjuvant in equal proportions to make the antigen;

[0032] (3) Second booster immunization: the third layer of cell suspension is used as antigen;

[0033] (4) Expansion immun...

Embodiment 3

[0061] Example 3: Verification of the characteristics of the monoclonal antibody 6H7 by flow immunofluorescence

[0062] 1. Take a clean 24-well culture plate, select 2 wells, and add cells at a concentration of 10 7 cells / ml of Chlamys farreri whole blood cell suspension 1ml, then add 1ml of monoclonal antibody 6H7 and myeloma culture supernatant (negative control) respectively, incubate at 37°C for 1 hour, and blow evenly while incubating;

[0063] 2. Transfer the cell suspension in the culture well to a centrifuge tube, centrifuge at 760g for 5min at 4°C, resuspend the cell pellet with anticoagulant, and wash by centrifugation for 3 times;

[0064] 3. Transfer the cell suspension to a 24-well culture plate, add 50 μl of FITC-labeled goat anti-mouse antibody to each well, incubate at 37°C in the dark for 1 hour, and blow evenly while incubating ;

[0065] 4. Transfer the cell suspension in the culture well to a centrifuge tube again, centrifuge at 760g for 5min at 4°C, res...

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Abstract

The invention discloses a monoclonal antibody capable of resisting a chlamys farrei particle blood corpuscle. The monoclonal antibody, which is named as mouse hybridoma cell strain 6H7 and is secreted by hybridoma cells, is collected by China Center for Type Culture Collection with the collection number of CCTCC No. C201042 and the collection date is 24th April, 2010. The hybridoma cells with favor growth vigor have the characteristics of uniform size, full, perfectly round and transparent appearance and exuberant fission; after the hybridoma cells are subject to routine culture in a culture medium, the culture medium comprises a large quantity of the chlamys farrei with high purity, good valence and strong specificity; and the results of detections by an indirect immnnofluotesent method and a streaming immnnofluotesent method show that the chlamys farrei can be combined with the specificity of the chlamys farrei particle blood corpuscle. The invention can be used for studying the generation, differentiation and distribution of the particle blood corpuscle in the process of scallop embryonic development and the conversion and difference between immunologic functions of the particle blood corpuscle and blood corpuscle of other types.

Description

technical field [0001] The invention relates to a monoclonal antibody against Chlamys farreri granule blood cells secreted by hybridoma cells and a preparation method thereof, belonging to the technical field of shellfish cell immunology. Background technique [0002] Shellfish lack immunoglobulin and specific immunity, and their immune defense process is completed by humoral factors in blood cells and hemolymph. Blood cells achieve the purpose of identifying, encapsulating, and removing foreign matter through autolysis, aggregation, phagocytosis, encapsulation, exocytosis, and oxidative killing; blood cells can also regulate auxiliary body fluids by releasing lysins, lectins, and opsonins. Factor immunity, which plays a key role in the process of shellfish resisting external environmental stimuli and foreign pathogenic microorganisms. [0003] At present, the classification of shellfish blood cells is mostly divided into two categories according to their morphology and the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N5/20C12R1/91
CPCC07K16/18
Inventor 邢婧战文斌绳秀珍林听听
Owner OCEAN UNIV OF CHINA
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