IL-6 reorganization monoclonal antibody and preparation method and application thereof

A monoclonal antibody, IL-6 technology, applied in the field of bioengineering, can solve the problems of high cost of experimental animal culture, unfavorable industrial production, low fusion efficiency, etc., achieve high operability and promotion value, solve the loss of antibody genes, and satisfy Effects of Antibody Stability and Reproducibility Requirements

Inactive Publication Date: 2020-01-07
CUSABIO TECH LLC
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method has always had the disadvantages of low fusion efficiency, large randomness, low positive rate during the experiment, and the need for multiple cell fusions, thereby increasing the workload; in addition, the in vivo cultivation of hybridoma cells needs to be completed with the help of experimental animals. The cost is high, and the large individual differences in animals may easily lead to batch-to-batch differences, which is not conducive to industrial production

Method used

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  • IL-6 reorganization monoclonal antibody and preparation method and application thereof
  • IL-6 reorganization monoclonal antibody and preparation method and application thereof
  • IL-6 reorganization monoclonal antibody and preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0034] Main experimental steps:

[0035] 1. Expression and purification of IL-6 protein: Escherichia coli system was used to express and purify IL-6 protein, and at the same time characterize the purity, concentration and activity of the antigen, and select high purity and high activity as the immunogen.

[0036] 2. Prepare splenocytes: immunize Balb / c mice with the above antigens, and take the spleens of the mice after the immunization to make a single cell suspension.

[0037] 3. Obtaining single cells: Use antibodies against mouse CD45R / B220, CD19, CD27, and CD38 to label cell surface antibodies, and screen out memory B cells and plasma cells by flow cytometry.

[0038] 4. Detection of antibody titer: Spread the above-screened cells into 384 microwell plates for culture, then add the culture supernatant to the microwell plate coated with antigen in advance, detect the cell lines that secrete specific antibodies, and screen out the cells that can Antibody clones that specif...

Embodiment 2

[0050] Take the purified antibody and add it to a 96-well plate pre-coated with protein G, then add biotin-labeled IL-6 protein and streptavidin-labeled HRP to measure antibody affinity (Table 2). Select the pairing, then detect the IL-6 calibration curve, and screen out the appropriate pairing antibodies, that is, cell numbers 2, 3, and 5. The corresponding plasmid was stably transfected into CHO cells, and then repeated production of 3 batches of antibodies to determine the production process.

Embodiment 3

[0052] Use No. 2 and No. 3 as capture antibodies to coat 96 microwell plates, and No. 5 antibody-labeled HRP as the labeled antibody, detect 30 human serum samples on the chemiluminescent platform, and compare the detection results with the hospital values. The results are as follows: figure 1 and figure 2 shown.

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Abstract

The invention provides a preparation method of an IL-6 reorganization monoclonal antibody. The preparation method comprises the following steps of performing immunization on a mouse with IL-6, after immunization, taking the spleen of the mouse, and preparing an unicellular suspension; screening memory B cells and pulp cells with a flow cytometry instrument; respectively culturing the obtained cells, adding the obtained culture supernatant in a culture device coated with the IL-6, continuing performing culturing, detecting cell strains secreting a specific antibody, and screening the antibody which can specially recognize antigen protein, and performing cloning; extracting total RNA on the obtained cells strains, performing reverse transcription to obtain cDNA, amplifying the variable regions of a heavy chain and a light chain of the antibody, and performing separate connection to a carrier; and then performing cell transfection, and producing the IL-6 reorganization monoclonal antibody. Through screening once, a large quantity of antibody sequences can be obtained, the gene information of the antibodies can be obtained, and the problems that cells in a hybridoma technique is low infusion rate and the antibody gene is lost, are solved.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to IL-6 recombinant monoclonal antibody and its preparation method and application. Background technique [0002] Interleukin-6 (Interleukin-6), referred to as Interleukin 6 (IL-6), is a pleiotropic cytokine with a wide range of functions. The determination of serum IL-6 concentration is widely used in the diagnosis of diseases such as surgery, stress response, and infection. At present, the determination of serum IL-6 concentration mainly uses immunological methods such as chemiluminescence and immunofluorescence chromatography. The content of IL-6 in the normal human body is extremely low (<7pg / ml), so higher requirements are put forward for the core raw material in the detection kit --- antibodies, and the development of high-affinity antibodies is what the market needs. [0003] At present, most of the monoclonal antibodies used in diagnostic kits are secre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/24C12N15/70G01N33/68G01N33/577
CPCC07K16/248C12N15/70G01N33/577G01N33/6869G01N2333/5412
Inventor 郑雪松华权高沈鹤霄罗绍祥陈莹谭华菊舒芹
Owner CUSABIO TECH LLC
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