Detection method for anti-CD20 monoclonal antibody binding activities

A monoclonal antibody and detection method technology, applied in the field of biomedicine, can solve the problems of lack of methods, different methods, unstandardized methods, etc., and achieve the effects of high precision, good specificity and strong specificity

Active Publication Date: 2013-04-10
GENOR BIOPHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantages of this method are: ①. The fluorescent labeling efficiency is difficult to standardize. If the labeling is incomplete, it is easy to cause the unlabeled anti-CD20 monoclonal antibody to compete with the fluorescently labeled anti-CD20 monoclonal antibody to bind to the cell surface antigen together with the sample to be tested. True reaction antigen-antibody binding efficiency; ②, this method is a competitive binding experiment, which can only reflect the strength of the ability of the sample to be tested and the reference product to compete with CD20 on the cell surface, and the method is stable, repeatable, and reliable There is also a lack of systematic research, and it is impossible to truly evaluate the binding activity of the sample to be tested.
The other is the indirect detection method. Although commercial fluorescently labeled FITC anti-Human IgG is also used for detection, the method has not been standardized, and it is mostly used for monoclonal antibody screening in the research and development stage, that is, to identify negative and positive results. There is a lack of exclusive methods for the method. There are also different evaluations on the experimental results, and it is difficult to guarantee the accuracy and reliability of the test results.

Method used

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  • Detection method for anti-CD20 monoclonal antibody binding activities
  • Detection method for anti-CD20 monoclonal antibody binding activities
  • Detection method for anti-CD20 monoclonal antibody binding activities

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Anti-CD20 Monoclonal Antibody Binding Activity Detection

[0045] Using the anti-CD20 monoclonal antibody test sample (prepared by Jiahe Bio-Pharmaceutical Co., Ltd.) as the material, the above-mentioned method was used to detect its binding activity.

[0046] The result is as figure 1 , the anti-CD20 monoclonal antibody that specifically binds to CD20 on the surface of Raji cells can combine with FITC Mouse anti-Human IgG to generate fluorescence, and the fluorescence intensity increases with the increase of the concentration of the bound anti-CD20 monoclonal antibody, FITC The higher the fluorescence intensity, the peak shape gradually shifted to the right; while the negative control cells did not bind to the anti-CD20 monoclonal antibody, so the position of the peak shape remained unchanged, no right shift occurred, and the fluorescence intensity was extremely low.

[0047] Such as image 3 , the curve fitting of the reference product and the sample to be...

Embodiment 2

[0048] Example 2 Detection of Anti-CD20 Monoclonal Antibody Binding Activity (Specificity Evaluation)

[0049] Human IgG1: Remicade (product of Johnson&Johnson Co.), which does not target the CD20 target, was used as the sample to be tested, and its binding activity was detected by the above-mentioned method.

[0050] The result is as figure 2 , the monoclonal antibody that does not target CD20 does not bind to cells with high expression of CD20 even at the same dilution concentration as the reference product, and the average fluorescence intensity is almost equal to that of the negative control. It can be seen that the detection method of the present invention can specifically detect the binding activity of the monoclonal antibody against the CD20 target.

Embodiment 3

[0051] Example 3 Detection of Anti-CD20 Monoclonal Antibody Binding Activity (Precision Evaluation)

[0052] The precision of the method was evaluated with anti-CD20 monoclonal antibody drug sample (prepared by Jiahe Biopharmaceutical Co., Ltd.) as the material. First, 1 experimenter uses the following method to conduct parallel experiments on the same sample to be tested 5 times to investigate the repeatability of the method; then 2 experimenters use the following method to detect the binding activity of the same sample to be tested, and the personnel who investigate the method Error; Finally, 1 experimenter used the following method to detect the binding activity of the same sample to be tested within 3 different working days, and investigated the inter-day difference of the method.

[0053] The results are shown in Table 1. One experimenter repeated the experiment 5 times on the same sample to be tested, and the RSD of the test result was 2 >0.95 (the curve fitting diagram ...

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Abstract

The invention belongs to the technical field of biological medicines and particularly relates to a detection method for anti-CD20 monoclonal antibody binding activities. The method comprises the following steps: a. taking a logarithmic growth phase high-expression CD20 cell, carrying out cell counting, and adjusting the cells to proper cell density for planking; b. carrying out series gradient dilution on a reference and a sample to be detected, and adding culture plates planked with the cells in sequence for a period of time; c. collecting the cells, and adding secondary antibody incubation marked with fluorescein isothiocyanate (FITC) for a period of time; and d. detecting by a flow cytometry to obtain the bonding activities of the sample to be detected. With the adoption of the detection method, the requirements in checking aspects of specificity, precision, linearity, range, accuracy and durability and the like can be satisfied; and the method can be effectively used for detecting the anti-CD20 monoclonal antibody binding activities.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for detecting the binding activity of an anti-CD20 monoclonal antibody. Background technique [0002] In recent years, significant progress has been made in clinical trials of monoclonal antibodies and their oriented treatment of non-Hodgkin's lymphoma, among which anti-CD20 monoclonal antibody preparations are widely used and effective. The anti-CD20 monoclonal antibody Rituximab (Rituximab, trade name Rituxan) developed by Genentech and IDEC in the United States was approved by the FDA in 1997 and was the first monoclonal antibody for the treatment of B-cell non-Hodgkin's lymphoma. Antibody drugs. Rituxan is an IgG1 (kappa) chimeric monoclonal antibody, which contains the variable region of the mouse anti-human CD20 monoclonal antibody heavy chain and light chain, the constant region of the human IgG1 (Kappa) heavy chain and light chain, and the Fc par...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N15/14
Inventor 刘培培王少雄吕品陈香
Owner GENOR BIOPHARMA
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