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Heat-resistant protective agent for universal monoclonal antibody

A technology of monoclonal antibody and heat-resistant protective agent, which is applied in the direction of antibodies, medical preparations of non-active ingredients, metabolic diseases, etc., and can solve problems such as unsatisfactory IgM and IgA monoclonal antibodies

Active Publication Date: 2021-08-24
CHINA INST OF VETERINARY DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In addition, due to certain differences in the molecular structure of different antibody subtypes, since the IgM molecule is a pentamer polymerized by five monomer molecules, and the IgA molecule is a dimer, antibodies with good protective effects against IgG monoclonal antibodies Thermoprotectants may not be ideal for IgM and IgA mAbs

Method used

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  • Heat-resistant protective agent for universal monoclonal antibody
  • Heat-resistant protective agent for universal monoclonal antibody
  • Heat-resistant protective agent for universal monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Screening and preparation of monoclonal antibodies in Brucella

[0028] (1) Take the extracted purified brucellol polysaccharide 10 μg in 0.1 ml of physiological saline, and the like of the equivalend-based complete adjuvant is subcutaneously injected with 8 weeks old BALB / C mice. Immunization was immunized by the same dose after 14 days of immunization, and immunized three times in the same dose. Enhance immunity without adding adjuvants with the same dose antigen without adding adjuvants in 2 to 3 days before fusion.

[0029] (2) The spleen cells of mice were fused in accordance with the standard procedure according to the standard procedure.

[0030] (3) Collection of hybridoma cells were collected from 7 to 10 days after fusion, and the 96-liplavogenic plates used by LPS package were used as indirect enzyme-linked immunosorbent assays and LPS had a strong reaction-positive hybridoma cell line.

[0031] (4) Three subclones were subcloned by a limited dilution...

Embodiment 2

[0035] Example 2 - Established a method for determination of Brochillium single-resistance

[0036] (1) Lipopolysaccharide package is quantified by antigen. The basic steps are as follows:

[0037] 1) Dilution of the construction of standard curve samples. The commercial LPS standard was diluted into different dilutions such as 1 mg / ml, 100 μg / ml, 10 μg / ml, 1 μg / ml, 100 ng / ml, 10 ng / ml.

[0038] 2) Test LPS purity determination. The LPS was prepared into an initial concentration of 1 mg / ml according to the weighing result, and diluted to 100 μg / ml concentration. In step 1), 100 μl of each dilution LPS solution was added to each of the detection holes of the 96-porous enzyme panel, and the absorbance was read at a wavelength of 529 nm. 3) Establish OD of the dilution of LPS standards 529 Value, and establish a standard curve between concentrations and absorbance. 4) Calculate the concentration of LPS according to the standard curve and the LPS absorbance of Brurate....

Embodiment 3

[0050] Example 3 - Singleclonon antibody titer assay

[0051] The basic steps are as follows:

[0052] (1) ELISA plate was added to 100 μl of (1 μg / ml) Brochobacterial lipid polysaccharide (LPS) package overnight.

[0053] (2) After washing, 100 μl of PBS containing 5% degreasing was added to 37 ° C for 2 hours, and the washed washing was used as an ELISA plate.

[0054] (3) After the prepared ascites were diluted with a PBS solution 1: 1000, it was diluted to 1: 64000 times.

[0055] (4) Add a diluted monoclonal antibody, 100 μl per well, oscillated with mixing for 5 min.

[0056] (5) After incubation of 30 min at 37 ° C, the reaction plate was taken out, and the reaction liquid was discarded, and 300 μl of 1 × wash solution was added, and after washing 3 times, dry.

[0057] (6) After the HRP labeled sheep, IgG was diluted with PBS 1: 10000, 100 μl of 10 min, 37 ° C for 30 min, washed 3 times, dried.

[0058] (7) Immediately after the substrate coloration was added to the ELIS...

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Abstract

The invention relates to a heat-resistant protective agent for a universal monoclonal antibody. Compared with monoclonal antibody heat-resistant protective agents containing different components, the invention finds that trehalose has more excellent protection on monoclonal antibodies at normal temperature and low temperature; 0.1% of tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl) is added in the formula of the protective agent, so that the stability of the monoclonal antibody in a high-temperature environment is enhanced, 10% of glycerol and 0.25% of mannose are added, so that the protective effect on IgM and IgA subtype monoclonal antibodies is enhanced, and the effective preservation time of the monoclonal antibody is greatly prolonged by the universal heat-resistant protective agent disclosed by the invention; cold storage and normal-temperature preservation of different antibody subtype monoclonal antibodies and related products in the storage and transportation process are achieved, and the storage life of the monoclonal antibodies can be prolonged to 18 months or above at the temperature of 2-8 DEG C, 15 months at the temperature of 25 DEG C and 16 days at the temperature of 37 DEG C.

Description

Technical field [0001] The present invention relates to a monoclonal antibody heat resistant protective agent and a preparation method thereof, and is a biological product field. Background [0002] The monoclonal antibody (simply antibody) is a height produced by a single B cell clone, only for antibodies of a particular antigenic epitope. It is usually prepared by hybridoma techniques such that its cell line can be proliferated long-term unlimited, and can stabilize and secrete specific antibodies. In the 1970s, the first use of hybridous tumors was used to screen for a single anti-cell. The monoclonal antibody was widely used in the field of disease diagnosis and treatment, and cell biology research. There are already many monoclonal antibodies to chronic Prevention and treatment of metabolic diseases and infectious diseases. [0003] Compared to traditional multi-resistance, the monoclon has a single, high specificity, convenient preparation, and high preparation of antibody ...

Claims

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Application Information

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IPC IPC(8): A61K47/42A61K47/26A61K47/18A61K47/10A61K39/395A61P3/00
CPCA61K47/42A61K47/26A61K47/18A61K47/10C07K16/1221A61P3/00A61K2039/505C07K2317/35
Inventor 冯宇秦玉明彭小薇丁家波范学政朱良全沈青春靳继惠
Owner CHINA INST OF VETERINARY DRUG CONTROL
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