Universal method for detecting total concentration of monoclonal antibody drug of targeted soluble protein in monkey serum

A monoclonal antibody, general-purpose technology, applied in the direction of color/spectral characteristic measurement, analysis by causing chemical reaction of materials, material analysis by observing the influence on chemical indicators, etc., can solve time-consuming cost and reagent cost , method development failure, method performance not up to standard and other problems, to achieve the effect of reducing time cost and reagent cost, ensuring accuracy and reliability, and significant economic benefits

Active Publication Date: 2021-07-09
苏州西山中科药物研究开发有限公司
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Both the specific indirect method and the specific double-antibody sandwich method can only detect completely free and partially bound monoclonal antibody drugs but not fully bound monoclonal antibody drugs, so the obtained detection data cannot reflect the actual situation. Blood drug concentration, especially when there are high levels of targeted soluble proteins in the body, which makes it impossible to fully observe and evaluate the pharmacokinetic and toxicokinetic characteristics of this type of monoclonal antibody drug; in addition When the specific indirect method or specific double antibody sandwich method is used to detect the blood concentration of monoclonal antibody drugs targeting different soluble proteins, methodological development work needs to be carried out separately, which will also consume a lot of time and cost. Reagent costs and the risk of method development failure or developed method performance not up to specification

Method used

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  • Universal method for detecting total concentration of monoclonal antibody drug of targeted soluble protein in monkey serum
  • Universal method for detecting total concentration of monoclonal antibody drug of targeted soluble protein in monkey serum
  • Universal method for detecting total concentration of monoclonal antibody drug of targeted soluble protein in monkey serum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] A general-purpose enzyme-linked immunoassay method for detecting the total concentration of IgG4 monoclonal antibody drugs targeting complement C5 protein in cynomolgus monkey serum:

[0035] 1. Main test steps

[0036] Coating: the mouse anti-human IgG monoclonal antibody that can specifically bind to human IgG1, IgG2, IgG3 and IgG4 subtypes was prepared with pH9.4-pH9.8 carbonate buffer to prepare 2μg / mL coating antibody Add 100 μL of working solution to each well, seal the plate with a sealing film, and incubate at 2-8°C for 14-20 hours;

[0037] Plate washing: wash the microplate plate 3 times with 0.05% PBST (1×PBS buffer containing 0.05% Tween 20), 300 μL / well, without soaking, after the last wash, pat the microplate plate on absorbent paper Dry;

[0038] Sealing: Add 250 μL of 0.05% PBST containing 0.2% I-Block to each well, seal the plate with a sealing film, place at 37±1°C, and incubate at 400 rpm for 1h±5min;

[0039] Plate washing: wash the plate with 0.0...

Embodiment 2

[0064] A general-purpose enzyme-linked immunoassay method for detecting the total concentration of IgG2 monoclonal antibody drugs targeting complement C5 protein in cynomolgus monkey serum:

[0065] 1. Main test steps

[0066] The main test steps are the same as in Example 1 except that the prepared concentrations of the standard curve calibration standard sample and the quality control substance are different.

[0067] 2. Experimental results

[0068] 1) Standard curve result

[0069] In this example, the standard curve contains calibration standards at 9 concentration levels: 200000.000ng / mL, 160000.000ng / mL, 80000.000ng / mL, 40000.000ng / mL, 20000.000ng / mL, 10000.000ng / mL, 5000.000ng / mL mL, 2500.000ng / mL, 1250.000ng / mL; 200000.000ng / mL is the upper limit of quantification (ULOQ), 2500.000ng / mL is the lower limit of quantitation (LLOQ), and 1250.000ng / mL is the anchor point. The calibration standard sample was prepared from the mixed serum of cynomolgus monkeys used for med...

Embodiment 3

[0083] A general-purpose enzyme-linked immunoassay method for detecting the concentration of completely free monoclonal antibody drugs targeting an IgG1 type monoclonal antibody drug targeting an unknown membrane protein in cynomolgus monkey serum:

[0084] 1. Main test steps

[0085] The main test steps are the same as those in Example 1 except that the number of standard samples calibrated by the standard curve, the prepared concentration and the prepared concentration of the quality control substance are different.

[0086] 2. Experimental results

[0087] 1) Standard curve result

[0088] In this example, the standard curve contains calibration standards at 8 concentration levels: 50000.000ng / mL, 40000.000ng / mL, 20000.000ng / mL, 10000.000ng / mL, 6000.000ng / mL, 3000.000ng / mL, 1500.000ng / mL mL, 750.000ng / mL; 50000.000ng / mL is the upper limit of quantification (ULOQ), 1500.000ng / mL is the lower limit of quantitation (LLOQ), and 750.000ng / mL is the anchor point. The calibrati...

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Abstract

The invention provides a universal method for detecting the total concentration of a monoclonal antibody drug targeting soluble protein in monkey serum. The method comprises the following steps: adding a universal coated antibody working solution into an elisa plate, sealing the plate, and incubating overnight; adding the confining liquid into an elisa plate, sealing the plate, incubating, washing the plate with washing liquid, adding the diluted correction standard sample, blank monkey mixed serum, a quality control product and a monkey serum sample to be detected into the elisa plate, sealing the plate, and incubating; washing the plate with washing liquor, adding the universal enzyme-labeled detection antibody working solution into the enzyme-labeled plate, sealing the plate, and incubating; washing the plate with washing liquor, adding a substrate of enzyme into the elisa plate, and incubating the elisa plate at room temperature for color development; and adding sulfuric acid into the elisa plate to stop color development, reading the absorbance value of each hole of the elisa plate by using an elisa reader, and calculating the concentration value. The detection method provided by the invention can accurately and reliably determine the total drug concentration of the monoclonal antibody of the targeted soluble protein in monkey serum and reflect the actual blood drug concentration.

Description

technical field [0001] The present invention relates to the field of preclinical bioanalysis of drugs, in particular to the field of preclinical pharmacokinetics and toxicokinetics sample analysis of monoclonal antibody drugs, and in particular to a monoclonal antibody drug targeting soluble proteins in monkey serum. A general method of concentration. Background technique [0002] Because of its high selectivity, strong specificity, and low toxic and side effects, monoclonal antibody drugs have become the focus of research and development in the field of biopharmaceuticals at home and abroad, and can be used to treat tumors, autoimmune diseases, nervous system diseases, ophthalmic diseases, etc. Diseases that seriously threaten human life and health have very broad commercial value and application prospects. At present, monoclonal antibody drugs are developed based on the IgG framework. According to the subtype of the antibody, monoclonal antibody drugs can be divided into ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/31G01N21/78
CPCG01N21/31G01N21/78
Inventor 王晓秋金超莫胤康
Owner 苏州西山中科药物研究开发有限公司
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