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ELISA method for determining bispecific antibody BsAb in serum and application

A bispecific antibody and detection antibody technology, applied in the field of chemistry, can solve the problems of drug development failure, impurity antibody interference, PK parameter calculation error and other problems

Active Publication Date: 2021-01-29
南京广祺医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If the existing method is used, the antibody used is aimed at the Fc segment of the target antibody, and the impurity antibody also contains the same Fc segment. If the cleaning step is not thorough, the method will produce interference from the impurity antibody, which will affect the determination of the lower limit of quantification (LLOQ). That is to say, it will affect the incorrectness of the determination, and the calculation of PK parameters is wrong, especially the half-life calculation (T1 / 2)
Misleading Dosing Frequency and Dosing Produces Toxicity Leads to Drug Development Failure

Method used

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  • ELISA method for determining bispecific antibody BsAb in serum and application
  • ELISA method for determining bispecific antibody BsAb in serum and application
  • ELISA method for determining bispecific antibody BsAb in serum and application

Examples

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Embodiment 1

[0068] Such as figure 1 As shown in Table 1-11, a method for quantitative determination of BsAb in cynomolgus monkey serum comprises the following steps:

[0069] 1 Experiment preparation

[0070] 1.1 Adjust the constant temperature incubator to 37 ℃.

[0071] 1.2 Fill the wash solution bottle with wash buffer, empty the waste solution bottle, and rinse the plate washer.

[0072] 2 Experimental operation

[0073] 2.1 Coating antigen

[0074] Dilute antigen (OX40) 1mg / mL with 1XPBS solution to 0.5ug / mL. will dilute well

[0075] The antigen (hOX40-Fc Tag) was added to the plate, 100ul per well. Paste it with a sealing film, seal it tightly, and place it at 4°C overnight (≥16h).

[0076] 2.2 Closure plate

[0077] Wash overnight coated plates with PBST (0.05%Tween / PBS) 4 times in a plate washer

[0078] (300ul / well). Pat off the residue. Add 300ul of blocking buffer (1%BSA / PBS / 0.05%Tween20) to each well, stick it with a sealing film, seal tightly and place at 37°C for...

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Abstract

The invention relates to an ELISA method for determining a bispecific antibody BsAb in serum and application, and belongs to the technical field of chemistry. The method provided by the invention comprises the following steps: 1) coating a microwell plate with an anti-human hOX40(Fc Tag) antigen to obtain a coated microwell plate; 2) adding BsAb and a to-be-detected sample into the antibody-coatedmicrowell plate, and carrying out incubating and combining to form an antigen-antibody compound; 3) adding hPD-L1 (his Tag) into the antibody-coated microwell plate, and incubating and combining withthe antigen-antibody compound to form an antigen-antibody- detection antibody compound; 4) adding Anti-His HRP labeled avidin into the antibody-coated microwell plate, incubating and combining with the antigen-antibody-detection antibody compound to form an antigen-antibody-detection antibody-HRP labeled avidin compound, and adding a substrate for color development; 5) adding a stop solution to stop the reaction, and determining the OD value; and 6) calculating the BsAb concentration of the to-be-detected sample in the serum through a BsAb standard curve. The method has the beneficial effectsof high detection sensitivity, small error and high specificity.

Description

technical field [0001] The invention relates to an ELISA method and application for measuring bispecific antibody BsAb in serum, belonging to the field of chemical technology. Background technique [0002] Bispecific antibody (BsAb) refers to an artificial antibody that specifically binds two antigens or antigenic epitopes at the same time. Bispecific antibodies do not exist under natural conditions, but are produced through cell fusion or recombinant DNA technology. Because of its specificity and bifunctionality, it has become a research hotspot in the field of antibody engineering, and has broad application prospects in the fields of tumor treatment and autoimmune diseases. In the process of antibody development, it is very important to support the pharmacokinetic (PK) and toxicokinetic (TK) experiments by detecting the concentration of the drug in plasma, and to rapidly advance the progress of the project. Generally, there are four types of ELISA detection methods; 1) d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/58G01N33/543
CPCG01N33/54393G01N33/581G01N33/6854G01N2333/70532G01N2333/70578
Inventor 山莽挺刘莉张小健刘仁杰
Owner 南京广祺医药科技有限公司
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