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Poly-clonal antibody for specifically identifying protein A, as well as preparation method and application thereof

A polyclonal antibody and protein recognition technology, which is applied in the field of polyclonal antibody and its preparation, can solve problems affecting immune detection, etc., and achieve the effects of easy breeding, high expression yield, and guaranteed expression

Inactive Publication Date: 2019-01-11
武汉百意欣生物技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, if the antibody product is contaminated with Protein A, it may also affect the results of the immunoassay

Method used

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  • Poly-clonal antibody for specifically identifying protein A, as well as preparation method and application thereof
  • Poly-clonal antibody for specifically identifying protein A, as well as preparation method and application thereof
  • Poly-clonal antibody for specifically identifying protein A, as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Preparation of polyclonal antibody specifically recognizing protein A

[0027] 1. Epitope Analysis

[0028] DNAStar software was used for epitope analysis, and the analysis conclusion showed that the antigenicity of the sequence shown in SEQ ID No.1 was good, and the antigenic determinants were mainly concentrated in the N-terminal 19 amino acids and the C-terminal 45 amino acids. According to the results of the antigenic determinant analysis, Consider prokaryotic expression of SEQID No.1, and design specific primers based on the sequence.

[0029] 2. Construction of expression vector

[0030] 1. Acquisition of protein A target fragment

[0031] In Beijing Tianyi Huiyuan Biotechnology Co., Ltd., the DNA sequence used for the construction of the prokaryotic expression vector was synthesized by the method of whole gene synthesis, that is, the nucleotide sequence shown in SEQ ID No.1 was synthesized.

[0032] 2. PCR amplification

[0033] With the nucleotide...

Embodiment 2

[0051] Embodiment 2: ELISA method is used to quantitatively detect the content of protein A in the test sample

[0052] Protein A affinity chromatography is widely used in the purification of antibodies. In this method, Protein A is bound to the purification medium by covalent coupling. The shedding of Protein A during purification is a major problem in drug research. The polyclonal antibody of the present invention that specifically recognizes protein A is applied to the residual level of protein A in the quantitative detection test sample, and it is also convenient for the protein A purification medium manufacturer to monitor the shedding characteristics of protein A under specific conditions.

[0053] In this example, the principle of double-antibody sandwich enzyme-linked immunoassay (sandwich ELISA) was used to detect the residue of Protein A, and the microwell plate was coated with a monoclonal antibody that specifically recognized protein A to form a solid-phase antibody...

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Abstract

The invention relates to the technical field of biological science and detection reagents, and particularly relates to a polyclonal antibody for specifically identifying protein A, as well as a preparation method and application thereof. The method comprises the following steps: performing epitope analysis on antigen of protein A, amplifying a gene segment capable of generating stronger immunogenic site with a proper primer, preparing a recombinant protein segment by adopting the amino acid sequence of the site, and immunizing an animal with the segment to obtain an antibody for specifically targeting the site. A colibacillus expression system is adopted in recombinant protein preparation, expression of recombinant protein can be well ensured, the expression yield is high, and the polyclonal antibody for specifically identifying the protein A can be prepared in large scale. The polyclonal antibody for specifically identifying protein A can be applied to quantitative detection of residual level of protein A in a tested sample, and facilitates protein A purifying medium manufacturers to detect the shedding characteristic of protein A under a specific condition.

Description

technical field [0001] The present invention relates to the technical field of biological science and detection reagents, more specifically, relates to a polyclonal antibody specifically recognizing protein A and its preparation method and application. Background technique [0002] Protein A is the abbreviation of staphylococcus protein A (SPA), which is a protein isolated from the cell wall of Staphylococcus aureus. Protein A mainly has five antibody-binding functional domains, and each domain consists of approximately 58 amino acid residues (1, Karen L.A, Julia D.B, Emily S.S. aureus IgG-binding proteins SpA and Sbi: Host specificity and mechanisms of immune complex formation, Molecular Immunology, 2008, 45: 1600-1611; 2. Feng Shaojiao, Liang Hao, Song Shuliang, Staphylococcus protein A activity mechanism and modern application, Chemistry of Life, 2008, Vol. 28 No. 6, 748-751). [0003] In the purification process of monoclonal antibody drugs, protein A-based affinity chr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C07K16/12G01N33/68
CPCC07K16/1271C07K2317/11C12N15/70G01N33/68G01N2333/31
Inventor 吴刚谢胜平易红波
Owner 武汉百意欣生物技术有限公司
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