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Humanized IgM monoclonal antibody standard substance and preparation method thereof

A monoclonal antibody and standard substance technology, applied in peptide preparation methods, chemical instruments and methods, organic chemistry, etc., can solve problems such as large molecular weight and complex structure

Active Publication Date: 2021-06-15
NAT INST OF METROLOGY CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] However, since the IgM antibody is a multimer, containing multiple subunits with a molecular weight of more than 1000KDa, the heavy chain of each subunit contains multiple glycosylation sites, with complex structure and large molecular weight.
At this stage, there is no standard preparation method for immunoglobulin with such a complex structure and the largest molecular weight

Method used

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  • Humanized IgM monoclonal antibody standard substance and preparation method thereof
  • Humanized IgM monoclonal antibody standard substance and preparation method thereof
  • Humanized IgM monoclonal antibody standard substance and preparation method thereof

Examples

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Embodiment 1

[0066] For acute infectious diseases caused by sudden outbreaks of new viruses, taking the 2019-2020 novel coronavirus pandemic as an example, the development process of the 2019-nCoV new coronavirus N protein human IgM monoclonal antibody solution standard material is demonstrated, and the specific design, The experimental process is as follows:

[0067] 1. Screening and protein expression of human IgM monoclonal antibody against N protein of 2019-nCoV novel coronavirus

[0068] According to the sequence of the novel coronavirus N gene (position 28274-29533) published in NCBI GenBank, the N complete protein is expressed, and the phage display library constructed after immunizing animals with the N protein (the storage capacity is about 10 9 ) to carry out antibody screening on the N antigen to obtain the IgG prototype antibody with a certain affinity (affinity threshold Kd-7 ). The IgG prototype antibody was subjected to affinity maturation, and then the optimized variable r...

Embodiment 2

[0164] For common important infectious diseases, taking AIDS as an example, IgM monoclonal antibody standard substances can also be developed according to the technical scheme of the present invention:

[0165] At present, there are no certified reference materials for AIDS IgM antibodies in China. There are two main reasons:

[0166] 1. It is impossible to obtain and process the serum of AIDS patients as a standard product under daily conditions;

[0167] 2. Even if dozens of AIDS patients’ serum can be obtained, the components in it are very complex, containing only 1% or even less than 0.1% of anti-HIV polyclonal IgM antibodies, which is difficult to accurately characterize and quantify to make it a certified standard substance.

[0168] Therefore, the scheme in Example 1 was used to screen IgG antibodies against AIDS antigens, and humanized transformation and variable region grafting were carried out to obtain humanized IgM antibodies, which were expressed and purified in...

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Abstract

The invention discloses a humanized IgM monoclonal antibody standard substance and a preparation method thereof, and the method comprises the following steps: carrying out phage antibody library screening on a virus antigen to obtain an IgG prototype antibody meeting the affinity threshold requirement; after the affinity of the prototype IgG antibody is mature, grafting an optimized antibody heavy chain variable region to a constant region of a human IgM antibody to form a chimeric heavy chain, and unchanging a Kappa light chain; performing expression and purification to obtain a humanized IgM monoclonal antibody standard substance raw material; carrying out purity characterization, protein activity characterization and structure characterization on the humanized IgM monoclonal antibody standard substance raw material; and on the basis that the purity is fully represented and the structure is confirmed to be consistent with the sequence of a target antibody, determining the value of the humanized IgM monoclonal antibody standard substance by adopting an isotope dilution mass spectrometry method based on amino acid analysis through uniformity and stability inspection.

Description

technical field [0001] The invention relates to the technical field of IgM antibody detection standard substances, in particular to a preparation method of humanized IgM monoclonal antibody standard substances. Background technique [0002] Immunological detection is one of the important methods of clinical detection. It is a specific diagnostic method and is widely used in clinical detection, diagnosis and epidemiological investigation. Known antigens can be used to detect unknown antibodies, and known antibodies can also be used to detect unknown antibodies. Antigens (eg, serological tests). The immunological detection method uses the specific and reversible spatial combination between antigen and antibody to detect the target. According to different detection targets, immunoassay methods can be mainly divided into virus antigen detection and serum IgM / IgG antibody detection; according to different detection principles, they can be divided into colloidal gold method, immu...

Claims

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Application Information

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IPC IPC(8): C07K16/10C07K1/36C07K1/34C07K1/22C07K1/20C07K1/18C07K1/16G01N27/62G01N33/96
CPCC07K16/10C07K16/1045G01N33/6848G01N33/96C07K2317/24C07K2317/92C07K2317/94
Inventor 米薇戴新华胡志上王越珉翟睿方向欧阳艳艳
Owner NAT INST OF METROLOGY CHINA
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