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Method for screening hybridoma cells secreting specific monoclonal antibodies, and application thereof

A hybridoma cell and monoclonal antibody technology, applied in the field of immunological detection, can solve the problems of time-consuming and labor-intensive, inability to evaluate, and missed selection of strong positive cells, so as to reduce the number of cloning, shorten the screening time, and shorten the The effect of filter time

Active Publication Date: 2015-10-14
广东忠信生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is not only time-consuming and labor-intensive, but also has the following disadvantages: (1) When screening the wells of multi-cell clones (fusion wells), the ELISA method can only evaluate whether the wells contain positive clones, and cannot evaluate which clone is positive Cloning; (2) During the cloning process, ELISA can only evaluate the overall situation of all cells in the cell well after antibody secretion, and cannot directly evaluate the cell variation in the plate well. (3) In the re-cloning process, when ELISA is combined with limiting dilution method to clone cells, only a very small number of cells (50-100 cells) are left for plate screening, while most During this process, due to cell variation and competition, strong positive cells are likely to be missed; (4) After a single hybridoma cell well is cloned several times, a large number of cells need to be processed and tested hole, heavy workload, trivial
These factors are not conducive to the screening of positive cell lines

Method used

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  • Method for screening hybridoma cells secreting specific monoclonal antibodies, and application thereof
  • Method for screening hybridoma cells secreting specific monoclonal antibodies, and application thereof
  • Method for screening hybridoma cells secreting specific monoclonal antibodies, and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0033] (1) Coupling and identification of Oleyl-PEG4000-NHS with antigen

[0034] 1) Coupling of Oleyl-PEG4000-NHS and BSA

[0035] Weigh 33mg of BSA, dissolve in 3.3mL of 15mM, pH 7.4 PBS (the PBS used below is the same as here), and stir evenly with a stirrer to obtain a BSA solution. Next, 10 mg of Oleyl-PEG4000-NHS (purchased from Japan NF Company) was weighed and dissolved in 1 mL of PBS to obtain an Oleyl-PEG4000-NHS solution. The Oleyl-PEG4000-NHS solution was slowly added to the stirred BSA solution, and stirred at room temperature for 3 hours to obtain the Oleyl-PEG4000-NHS-BSA solution.

[0036] 2) Coupling of Oleyl-PEG4000-NHS-BSA and CPAOZ

[0037] ① Take 500μl of Oleyl-PEG4000-NHS-BSA solution, add it to 500μl of PBS, and put it in an ice box to stir.

[0038]②The derived CPAOZ (according to the literature "Zhou Kenan et al. Preparation of monoclonal antibody to furazolidone metabolite and its new immunochromatographic detection test strip [J]. Chinese Journal ...

Embodiment 2

[0053] (1) Coupling and identification of Oleyl-PEG4000-NHS with antigen

[0054] 1) Coupling of Oleyl-PEG4000-NHS and CKMB

[0055] Weigh 10mg Oleyl-PEG4000-NHS and dissolve it in 1ml PBS, dilute to 1mg / ml with PBS to get 1mg / ml Oleyl-PEG4000-NHS solution; then take 500μl 1mg / ml Oleyl-PEG4000-NHS into the bottle and put it in the ice box After stirring, take 500 μl of 1 mg / ml CKMB antigen and slowly add it to the above solution and stir overnight. After 12 hours, ultrafilter with a 30KD ultrafiltration tube to obtain Oleyl-PEG4000-NHS-CKMB solution. Then perform filter sterilization and store at 4°C.

[0056] 2) Identification of Oleyl-PEG4000-NHS-CKMB

[0057] With embodiment 1. Result: SDS-PAGE electrophoresis (such as image 3 shown) and UV-visible spectroscopic scanning identification (such as Figure 4 shown) showed that Oleyl-PEG4000-NHS-CKMB was obtained.

[0058] (2) Screening of Creatine Kinase Isozyme MB (CKMB) Hybridoma Cell Clusters

[0059] Before the cell...

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Abstract

The invention discloses a method for screening hybridoma cells secreting specific monoclonal antibodies, and an application thereof. The method comprises the following steps: coupling Oleyl-PEG4000-NHS with Ag to obtain Oleyl-PEG4000-NHS-Ag; adding Oleyl-PEG4000-NHS-Ag in cloning of hybridoma cells to be screened, culturing, removing the above obtained supernatant, and cleaning; adding a fluorescent substance-labeled anti-mouse second antibody, incubating, removing the obtained supernatant, and cleaning; and adding an incomplete medium, observing cell masses under visible light and fluorescence by using an inverted fluorescence microscope, carrying out negative and positive labeling, adding a methyl cellulose semisolid medium to plate holes containing positive cell masses, transferring positively labeled cells to a new cell culture plate, and culturing to obtain the hybridoma cells secreting the specific monoclonal antibodies. The method has the advantages of rapidness, simplicity and high accuracy, and can be used for rapid screening to obtain the hybridoma cells secreting the specific monoclonal antibodies.

Description

technical field [0001] The invention belongs to the field of immunological detection, in particular to a method and application for screening hybridoma cells secreting specific monoclonal antibodies. Background technique [0002] In 1975, German scholars Kohler and Milstein invented hybridoma technology. They successfully fused myeloma cells and antibody-producing B lymphocytes into hybridoma cells. This synthetic hybridoma cell can produce monoclonal antibodies directed against a specific antigenic determinant. The establishment of hybridoma technology has created a new era of antibody preparation, provided new tools for the diagnosis, prevention and treatment of clinical diseases, and promoted the development of many disciplines such as immunology, basic and clinical medicine. [0003] Monoclonal antibodies have the characteristics of high purity, strong specificity, high titer, little or no cross-reactivity, etc., and have been widely used in the diagnosis of diseases, d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/577G01N33/58
CPCG01N33/56966G01N33/577G01N33/582
Inventor 唐勇蓝彩凤李秀清
Owner 广东忠信生物科技有限公司
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