Unlock instant, AI-driven research and patent intelligence for your innovation.

Polyclonal antibody composition and preparation method and application thereof in pathological detection

A composition and multi-antibody technology, which is applied in the field of multi-antibody composition and its preparation, can solve the problems of no obvious reduction in polymer steric hindrance, poor sensitivity, and high cost

Pending Publication Date: 2021-04-16
HANGZHOU BIOLYNX TECH CO LTD
View PDF4 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, most of the pathologically enhanced secondary antibodies currently on the market have not had major improvements in polymer structure design and preparation process, specifically reflected in: the number of enzymes and antibodies per unit volume has not increased significantly; the steric hindrance of the polymer has not significantly decreased, resulting in Less sensitive and more expensive

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Polyclonal antibody composition and preparation method and application thereof in pathological detection
  • Polyclonal antibody composition and preparation method and application thereof in pathological detection
  • Polyclonal antibody composition and preparation method and application thereof in pathological detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Example 1 Preparation of Reagents After Primary Antibody

[0086] 1. Preparation of rabbit polyclonal antibodies

[0087] (1) 30 mL of blood was collected from multiple unimmunized 3-month-old New Zealand white rabbits, and centrifuged to obtain supernatant to obtain rabbit serum.

[0088] (2) Pass the rabbit serum through a 0.45 μm filter membrane, add an equal volume of PBS, and mix well.

[0089] (3) The rabbit serum mixture was passed through a protein A-coupled purification packing column, and then equilibrated with an equal volume of pH 8.0 equilibration buffer.

[0090] (4) Add an elution buffer of pH 2.5 to the purification column for elution, and obtain the eluted product after elution of 3-5 column volumes.

[0091] (5) Add 10% volume of Tris-Hcl neutralization buffer at pH 12 to the eluted product to obtain a rabbit polyclonal antibody solution, and measure the antibody concentration.

[0092] 2. Preparation of mouse polyclonal antibodies

[0093] (1) 5 m...

Embodiment 2 1

[0108] Example 2 Reagent affinity purification after primary antibody

[0109] In order to further improve the performance of the reagents after the primary antibody, a new process was used in this example to perform affinity purification of the rabbit polyclonal antibody and the mouse polyclonal antibody. The steps are as follows:

[0110] (1) Adjust the concentration of rabbit polyclonal antibody and mouse polyclonal antibody to 4 mg / mL, mix 1 mL with Freund's immune adjuvant 1:1, and inject 2 mL of the mixture into the back muscles of goats for immunization.

[0111] (2) Repeat the immunization once every 4 weeks. After the eighth immunization, take 200 mL of goat blood, and get the supernatant after centrifugation to obtain about 90-110 mL of goat anti-rabbit and goat anti-mouse serum.

[0112] (3) Pass the serum through a 0.45 μm filter membrane, add an equal volume of PBS, and mix well.

[0113] (4) Pass the goat anti-rabbit serum mixture through the affinity purificatio...

Embodiment 3 1

[0127] Example 3 Reagent exclusion human cross-optimization after primary antibody

[0128] Pass the goat anti-rabbit and goat anti-mouse polyclonal antibodies through the affinity purification packing column coupled with human polyclonal antibodies (Shanghai Jingdu Biology, 10mg / tube), collect and flow through, and the flow-through is the high-efficiency polyclonal antibodies after adsorption Goat anti-rabbit polyclonal antibody and goat anti-mouse polyclonal antibody were used to determine the antibody concentration.

[0129] The goat anti-rabbit polyclonal antibody adsorbed by the human polyclonal antibody was cross-detected, and the results were as follows: image 3 As shown, there is still some crossover between polyclonal antibody and human IgG before adsorption (EC50 value 409), and there is basically no crossover between polyclonal antibody and human polyclonal antibody after adsorption (EC50 value 0.000158). It shows that the adsorbed goat anti-rabbit polyclonal anti...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a polyclonal antibody composition, and belongs to the field of antibody immunodetection. The polyclonal antibody composition comprises a first polyclonal antibody and a second polyclonal antibody, the first polyclonal antibody is a goat-anti-rabbit polyclonal antibody, and the second polyclonal antibody is a goat-anti-mouse polyclonal antibody. The invention further discloses a preparation method of the polyclonal antibody composition and a Fab' fragment of a third polyclonal antibody capable of being combined with the polyclonal antibody composition . The Fab' fragment is coupled with a polymer PAMAM, and the polymer PAMAM is combined with an enzyme-labeled protein. The invention also discloses application of the polyclonal antibody composition and the Fab' fragment in preparation of a two-step pathological detection kit and detection. According to the invention, immunohistochemical detection can be directly carried out on a tissue slice, and the detection is simple and convenient; and meanwhile, due to the multiple amplification advantage of two-step pathological detection, the detection sensitivity is higher, and the specificity is higher.

Description

technical field [0001] The invention belongs to the field of antibody immunodetection, and in particular relates to a multi-antibody composition, a preparation method thereof and an application in pathological detection. Background technique [0002] Immunohistochemistry is the application of the basic principle of immunology - antigen-antibody reaction, that is, the principle of specific binding of antigen and antibody, through chemical reaction to make the chromogenic agent (fluorescein, enzyme, metal ion, isotope) of the labeled antibody develop color. The determination of antigens (polypeptides and proteins) in tissue cells, their localization, qualitative and relative quantitative research, is called immunohistochemistry or immunocytochemistry. [0003] In immunohistochemical detection technology, the complex of antibody and enzyme (secondary antibody) plays an extremely important role in the specificity and sensitivity of immunoassay. At present, the polymer enzyme-la...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/42C07K16/06C07K1/36C07K1/34C07K1/22C07K1/16G01N33/577G01N33/574G01N33/535
Inventor 刘浩李明振蔡宁
Owner HANGZHOU BIOLYNX TECH CO LTD