Bispecific antibody biological activity and titer detection method and application thereof

A bispecific antibody and biological activity technology, which is applied in the field of bispecific antibody biological activity and titer detection, can solve the problems of bispecific antibody inapplicability and inability to screen out bispecific antibody, so as to save experiments Effects of reagent cost, manpower reduction, and low reaction volume

Active Publication Date: 2017-08-18
SHANGHAI WUXI BIOLOGIC TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method mainly uses the Fc end of monoclonal antibody or fusion protein for detection and screening, but this method cannot screen out complete bispecific antibodies, and is not applicable to bispecific antibodies

Method used

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  • Bispecific antibody biological activity and titer detection method and application thereof
  • Bispecific antibody biological activity and titer detection method and application thereof
  • Bispecific antibody biological activity and titer detection method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Example 1, for detecting the integrity and biological activity of the light chain:

[0089] 1) Add cell supernatant, Anti-Lambda-Eu in 384 microwell plate 3+ cryptate (anti-Lambda light chain antibody coupled to europium crypt), Anti-his-XL665 (anti-His antibody linked to XL665), recombinant human CD4 protein with his tag (ie, antigen A), and mix well. Incubate at room temperature for 2 hours or more or overnight with anti-CD4 and anti-ENV bispecific antibodies as standard (final concentration: 0-250 ng / mL).

[0090] 2) Set the fluorescence wavelength: Excitation 313nm, Emission 665nm / 620nm, cutoff 630nm / 590nm, and read the fluorescence value.

[0091] 3) Perform four-parameter fitting according to the ratio of Em650 / Em620 (Em665 / Em620*104) fluorescence and the concentration of the standard to obtain a standard curve, and calculate the concentration of the sample to be tested according to the standard curve. standard curve as figure 1 shown. figure 1 The middle abs...

Embodiment 2

[0093] Example 2, for detecting the integrity and biological activity of light chain and heavy chain:

[0094] 1) Add cell supernatant, Anti-Lambda-Eu to 384 microwell plate #1 3+ cryptate (cryptate-coupled anti-Lambda light chain antibody), Anti-(h)Fc-d2 (d2-linked anti-human IgG Fc antibody), mix well. Incubate at room temperature for 2 hours or more or overnight with the target bispecific antibody as a standard.

[0095] 2) Add cell supernatant, Anti-(h)Fc-d2 (anti-human IgG Fc antibody linked to d2), Anti-his-Eu in 384 microwell plate 3+ cryptate (anti-His antibody conjugated to europium crypt), CD4 protein (his tag), mix well. Incubate at room temperature for 2 hours or more or overnight with the target bispecific antibody as a standard.

[0096] 3) Set the fluorescence wavelength: Excitation 313nm, Emission 665nm / 620nm, cutoff 630nm / 590nm, and read the fluorescence value.

[0097] 4) Perform four-parameter fitting according to the ratio of Em650 / Em620 (Em665 / Em620*...

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Abstract

The invention discloses a bispecific antibody biological activity and titer detection method. The method includes: detecting two antigen binding sites of a bispecific antibody at the same time; performing four-parameter fitting according to a fluorescence ratio and the concentration of a standard sample to obtain a standard curve; calculating the concentration of a to-be-tested sample according to the standard curve. By adoption of the method, intact antibody modules and cell strains high in bispecific binding activity and expression can be screened out more effectively; the method has the advantage of low reaction volume, and experiment reagent cost can be saved maximally; the method can be widely popularized and applied to CLD clone high-throughput screening.

Description

technical field [0001] The present invention relates to a method for detecting the biological activity and titer of a bispecific antibody, in particular to a method for evaluating and screening the biological activity and titer of a bispecific antibody using an improved homogeneous time-resolved fluorescence technique (HTRF) method of detection. Background technique [0002] Bispecific antibodies are a class of artificial antibodies that contain two specific antigen-binding sites. Except for the Fc end, the Fab segments in bivalent antibodies have different specificities. In the prior art, the bispecific antibody biological activity (binding activity) is mainly through the ELISA method, but the ELISA method requires repeated cleaning and multiple steps during the experiment, which is cumbersome and time-consuming, and it is impossible to use one test to explain the two specificities. Sexual binding activity. [0003] Homogeneous Time Resolved Fluorescence (HTRF) can measur...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64G01N33/53
CPCG01N21/6486G01N33/53
Inventor 陈敏贾晓青周伟昌陈智胜
Owner SHANGHAI WUXI BIOLOGIC TECH CO LTD
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