Anti-porcine hemoglobin hybridoma cell strain, and monoclonal antibody and application of anti-porcine hemoglobin hybridoma cell strain

A hybridoma cell line, porcine hemoglobin technology, applied in the field of immunology, can solve the problems of complex, uncomfortable and rapid detection, and achieve the effects of specificity, high titer, and good sensitivity

Active Publication Date: 2019-03-01
BIOLOGY INST OF HEBEI ACAD OF SCI +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, laboratory methods are used for hemoglobin in pig blood, but they are relatively complicated and not suitable for rapid detection in large quantities on site. Therefore, it is necessary to develop a simple, fast, sensitive and efficient detection method

Method used

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  • Anti-porcine hemoglobin hybridoma cell strain, and monoclonal antibody and application of anti-porcine hemoglobin hybridoma cell strain
  • Anti-porcine hemoglobin hybridoma cell strain, and monoclonal antibody and application of anti-porcine hemoglobin hybridoma cell strain
  • Anti-porcine hemoglobin hybridoma cell strain, and monoclonal antibody and application of anti-porcine hemoglobin hybridoma cell strain

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Example 1 Preparation of anti-porcine hemoglobin hybridoma cell line and monoclonal antibody.

[0025] (1) Preparation of hybridoma cell lines

[0026] Select 4 fast-moving female BLAB / c mice aged 6-8 weeks, dilute 40 μg of porcine hemoglobin with normal saline to 0.5 mL for the first time, mix the diluted porcine hemoglobin as antigen with an equal volume of Freund’s complete adjuvant Together, mix well, and directly perform subcutaneous multi-point injection, and then replace Freund's complete adjuvant with Freund's incomplete adjuvant during the immunization process, the immunization dose is 40 μg / monkey, and the immunization is boosted every 2 weeks. The immunization process is shown in Table 1 As shown, blood was collected 7 days after 3 immunizations, and the serum antibody titer of immunized mice was detected by indirect ELISA method, and antiserum inhibition was measured by blocking ELISA. Fusion with mouse myeloma SP2 / 0 cells, and then subcloning positive well...

Embodiment 2

[0031] Example 2 Characterization of the anti-porcine hemoglobin monoclonal antibody of the present invention

[0032] (1) Antibody titer determination

[0033] Use 5ug / mL porcine hemoglobin as the coating source, coat the detection plate, and dilute the purified anti-porcine hemoglobin monoclonal antibody at 1:200, 1:400, 1:800, ... 1:409600 as the primary antibody Add it into the well of the microtiter plate, incubate, then add HRP-labeled goat anti-mouse secondary antibody, then use TMB to develop color, and use sulfuric acid as the stop solution to stop the reaction. The result shows that the purified anti-porcine hemoglobin monoclonal antibody When the concentration is 1mg / ml, the titer reaches 1:819200. Ascites titer also reached 1:819200, see figure 1 .

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Abstract

The invention relates to an anti-porcine hemoglobin hybridoma cell strain, and a monoclonal antibody and an application of the anti-porcine hemoglobin hybridoma cell strain, and belongs to the technical field of immunology. The anti-porcine hemoglobin hybridoma cell strain PHb-3B4 has a collection number of CCTCC NO (China Center for Type Culture Collection number): C201784. The anti-porcine hemoglobin monoclonal antibody is generated by the anti-porcine hemoglobin hybridoma cell strain; a subtype is IgG1 (immune globulin G1); the valence is 1:819200; an affinity constant is 2.8*10<8>L/mol; and cross reaction rates of the monoclonal antibody with bovine hemoglobin and human hemoglobin are less than 0.1%. The anti-porcine hemoglobin monoclonal antibody is used for detecting porcine hemoglobin, and has the advantages of high valence, good sensitivity and high specificity. The porcine hemoglobin monoclonal antibody can be used for developing an immunological diagnostic reagent related tothe porcine hemoglobin, such as a colloidal gold test strip, a fluorescence detection test strip and an ELISA (enzyme-linked immuno sorbent assay) reagent kit.

Description

technical field [0001] The invention relates to a hybridoma cell line and its monoclonal antibody and application, in particular to an anti-porcine hemoglobin hybridoma cell line and its monoclonal antibody and application, belonging to the technical field of immunology. Background technique [0002] With the rapid development of the pig industry, the types of pig diseases are also increasing, which makes it not uncommon for the society to buy and slaughter sick and dead pigs privately, and the situation of sick and dead pork evading quarantine and entering the market is not uncommon. The requirements for product hygiene and quality are also getting higher and higher, so the suggestion and identification of sick and dead pork on the market are increasingly receiving widespread attention. Up to now, the detection methods of dead pork on the market are mainly laboratory inspection and sensory inspection, but the quickness, sensitivity and accuracy of these methods cannot well ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/18G01N33/72G01N33/577
CPCC07K16/18C07K2317/33C07K2317/92G01N33/721
Inventor 吴萌李翠娜李春生王增利韩庆安董维亚程华陈英珠李亚璞张小兵董超
Owner BIOLOGY INST OF HEBEI ACAD OF SCI
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