Enzyme-linked immunoassay method for quantitatively detecting content of anti-human interleukin 17 monoclonal antibody in serum

Abstract

The invention discloses an enzyme-linked immunoassay method for quantitatively detecting the content of an anti-human interleukin 17 monoclonal antibody in serum. The invention relates to an antibody,a heavy chain and a light chain of the antibody, a corresponding variable domain and a corresponding complementary decision domain, and an enzyme-linked immunoassay method established using the antibody as a secondary antibody in BA-ELISA analysis.

Description

technical field

[0001] The invention belongs to the field of biotechnology, in particular to an enzyme-linked immunoassay method for quantitatively detecting the content of anti-human interleukin-17 monoclonal antibody in serum. Background technique

[0002] Interleukin 17 (IL-17), also known as CTLA-8 or IL-17A, is a pro-inflammatory cytokine that stimulates non-immune cells such as fibroblasts, keratinocytes, epithelial and endothelial cells, synoviocytes, etc. to secrete IL-6 , IL-8, PGE2, MCP-1, CXCL-1 and G-CSF and other cytokines, and also has biological effects such as inducing ICAM-1 surface expression and T cell proliferation. IL-17A is mainly produced by a type of activated CD4+ T cells called Th17 and exerts its effect by binding the complex of IL-17RA and IL-17RC (Toy et al., 2006, J. Immunol.177 (11); 36-39). The anti-human interleukin-17 monoclonal antibody is an IgG1κ-type humanized monoclonal antibody that specifically binds to human interleukin 17A (IL-17A...

Examples

Embodiment 1

[0149] Example 1 Preparation and Purification of Biotin-labeled Secondary Antibody

[0150] Anti-human interleukin-17 monoclonal antibody (F(ab) 2 ) as an antigen for rabbit immunization, and through B cell cloning technology to screen out the monoclonal antibody that can bind to the monoclonal antibody, does not block the binding of the anti-human interleukin-17 monoclonal antibody to its target human interleukin-17, and does not cross-react with other human IgG of the same type non-neutralizing rabbit monoclonal antibody.

[0151] After obtaining the above-mentioned anti-antibody light / heavy chain gene fragment and sequence information, first, design and synthesize the 3' end primer containing the Avi-Tag gene sequence; then use the PCR method to amplify and obtain the 3' end containing Avi-Tag sequence chain gene; by one-step cloning method, the heavy chain gene containing Avi-Tag was inserted into the expression plasmid to construct the heavy chain expression plasmid cont...

Embodiment 2

[0152] Embodiment 2 Carry out performance appraisal to the monoclonal antibody made by embodiment 1

[0153] 1. Identification of binding activity

[0154] Use anti-human interleukin-17 monoclonal antibody and human IgG as antigens to coat the enzyme-linked plate; make the serially diluted monoclonal antibody in Example 1 react with the coated antigen; sequentially add chromogenic antibody; substrate chromogenic solution; terminate solution; for binding activity experiments. The results showed that the monoclonal antibody had good binding activity to the anti-human interleukin-17 monoclonal antibody, and did not cross-react with isotype IgG.

[0155] 2. Identification of neutralizing activity

[0156] Use human interleukin-17 as an antigen to coat the enzyme-linked plate; add the incubation of the quantitative anti-human interleukin-17 monoclonal antibody and the serially diluted monoclonal antibody in Example 1 to react with the coated antigen; add the chromogenic antibody ...

Embodiment 3

[0159] Example 3 Using the "Secondary Antibody" Prepared in Example 1 to Construct an Elisa Kit

[0160] The antigen is interleukin-17 and is immobilized on the enzyme-linked plate; anti-human interleukin-17 monoclonal antibody can bind to the coated antigen; the "secondary antibody" prepared in Example 1 is used as the second antibody; horseradish peroxidase-labeled Streptavidin was used as a chromogenic antibody.

[0161] experiment procedure:

[0162] Coating: human interleukin-17 was diluted to 1 μg / ml with 1×PBS, 100 μl / well, and coated overnight at 2-8°C;

[0163] Blocking: 200 μl / well blocking solution, blocking at room temperature for 2 hours;

[0164] Adding samples: Dilute the MRD 20 times with the diluent before adding the standard and quality control samples; 100 μl / well, incubate at room temperature for 2 hours;

[0165] Add secondary antibody: add the "secondary antibody" prepared in Example 1 to diluent to 10ng / ml, 100μl / well, incubate at room temperature for...

technical field

[0001] The invention belongs to the field of biotechnology, in particular to an enzyme-linked immunoassay method for quantitatively detecting the content of anti-human interleukin-17 monoclonal antibody in serum. Background technique

[0002] Interleukin 17 (IL-17), also known as CTLA-8 or IL-17A, is a pro-inflammatory cytokine that stimulates non-immune cells such as fibroblasts, keratinocytes, epithelial and endothelial cells, synoviocytes, etc. to secrete IL-6 , IL-8, PGE2, MCP-1, CXCL-1 and G-CSF and other cytokines, and also has biological effects such as inducing ICAM-1 surface expression and T cell proliferation. IL-17A is mainly produced by a type of activated CD4+ T cells called Th17 and exerts its effect by binding the complex of IL-17RA and IL-17RC (Toy et al., 2006, J. Immunol.177 (11); 36-39). The anti-human interleukin-17 monoclonal antibody is an IgG1κ-type humanized monoclonal antibody that specifically binds to human interleukin 17A (IL-17A...

Examples

Embodiment 1

[0149] Example 1 Preparation and Purification of Biotin-labeled Secondary Antibody

[0150] Anti-human interleukin-17 monoclonal antibody (F(ab) 2 ) as an antigen for rabbit immunization, and through B cell cloning technology to screen out the monoclonal antibody that can bind to the monoclonal antibody, does not block the binding of the anti-human interleukin-17 monoclonal antibody to its target human interleukin-17, and does not cross-react with other human IgG of the same type non-neutralizing rabbit monoclonal antibody.

[0151] After obtaining the above-mentioned anti-antibody light / heavy chain gene fragment and sequence information, first, design and synthesize the 3' end primer containing the Avi-Tag gene sequence; then use the PCR method to amplify and obtain the 3' end containing Avi-Tag sequence chain gene; by one-step cloning method, the heavy chain gene containing Avi-Tag was inserted into the expression plasmid to construct the heavy chain expression plasmid cont...

Embodiment 2

[0152] Embodiment 2 Carry out performance appraisal to the monoclonal antibody made by embodiment 1

[0153] 1. Identification of binding activity

[0154] Use anti-human interleukin-17 monoclonal antibody and human IgG as antigens to coat the enzyme-linked plate; make the serially diluted monoclonal antibody in Example 1 react with the coated antigen; sequentially add chromogenic antibody; substrate chromogenic solution; terminate solution; for binding activity experiments. The results showed that the monoclonal antibody had good binding activity to the anti-human interleukin-17 monoclonal antibody, and did not cross-react with isotype IgG.

[0155] 2. Identification of neutralizing activity

[0156] Use human interleukin-17 as an antigen to coat the enzyme-linked plate; add the incubation of the quantitative anti-human interleukin-17 monoclonal antibody and the serially diluted monoclonal antibody in Example 1 to react with the coated antigen; add the chromogenic antibody ...

Embodiment 3

[0159] Example 3 Using the "Secondary Antibody" Prepared in Example 1 to Construct an Elisa Kit

[0160] The antigen is interleukin-17 and is immobilized on the enzyme-linked plate; anti-human interleukin-17 monoclonal antibody can bind to the coated antigen; the "secondary antibody" prepared in Example 1 is used as the second antibody; horseradish peroxidase-labeled Streptavidin was used as a chromogenic antibody.

[0161] experiment procedure:

[0162] Coating: human interleukin-17 was diluted to 1 μg / ml with 1×PBS, 100 μl / well, and coated overnight at 2-8°C;

[0163] Blocking: 200 μl / well blocking solution, blocking at room temperature for 2 hours;

[0164] Adding samples: Dilute the MRD 20 times with the diluent before adding the standard and quality control samples; 100 μl / well, incubate at room temperature for 2 hours;

[0165] Add secondary antibody: add the "secondary antibody" prepared in Example 1 to diluent to 10ng / ml, 100μl / well, incubate at room temperature for...

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