A kind of ELISA method and application of measuring bispecific antibody BSAB in serum

A bispecific antibody and antibody detection technology, which is applied in the chemical field, can solve problems affecting the determination of quantitative lower-line concentration, PK parameter calculation errors, drug development failures, etc., and achieve the effects of shortening the project process, improving sensitivity, and reducing errors

Active Publication Date: 2021-06-08
南京广祺医药科技有限公司
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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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  • A kind of ELISA method and application of measuring bispecific antibody BSAB in serum
  • A kind of ELISA method and application of measuring bispecific antibody BSAB in serum
  • A kind of ELISA method and application of measuring bispecific antibody BSAB in serum

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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 the anti-human hOX40 (Fc Tag) antigen 1mg / mL with 1XPBS solution to 0.5ug / mL. will dilute well

[0075] The anti-human hOX40 (Fc Tag) antigen 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, ...

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Abstract

The invention relates to an ELISA method and application for measuring bispecific antibody BsAb in serum, belonging to the field of chemical technology. The method of the present invention comprises the following steps, 1) coating the micropore plate with anti-human hOX40 (Fc Tag) antigen to obtain the coated micropore plate; 2) adding the BsAb and the sample to be tested into the antibody-coated micropore plate Incubate and bind to form an antigen-antibody complex; 3) Add hPD-L1 (his Tag) to the antibody-coated microwell plate and incubate with the antigen-antibody complex to form an antigen-antibody-detection antibody complex; 4) Add Anti-His HRP-labeled avidin to the antibody-coated microwell plate and incubate with the antigen-antibody-detection antibody complex to form an antigen-antibody-detection antibody-HRP-labeled avidin complex 5) Add a stop solution to stop the reaction and measure the OD value; 6) Calculate the BsAb concentration in the serum of the sample to be tested by using the BsAb standard curve. The invention has the beneficial effects of 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 Patents(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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