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Mass spectrum-based epitope positioning method of in vitro diagnostic reagent antibody

A technology of in vitro diagnosis and positioning method, which is applied in measurement devices, material analysis by electromagnetic means, instruments, etc., can solve the problems of differences in binding sites, mutual recognition of quantitative results, and insufficient traceability process, so as to ensure accuracy. , the effect of easy operation

Pending Publication Date: 2022-03-01
NAT INST OF METROLOGY CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0004] IVD reagents are often tested in the form of kits. Many domestic and foreign manufacturers of IVD reagents can produce a variety of detection kits for clinical testing items, which greatly enriches the selectivity of IVD reagents, but At the same time, the quantitative results of the same type of kits from different manufacturers cannot be mutually recognized, which brings great challenges
The reason for this phenomenon is that on the one hand, the manufacturer’s traceability process is not standardized enough; on the other hand, because IVD kits are usually designed based on the principle of immunity, the antibodies used in different brands of kits are often different. , which leads to differences in their antigen-binding sites to be tested, and this difference leads to inconsistencies in the quantitative results of these kits

Method used

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  • Mass spectrum-based epitope positioning method of in vitro diagnostic reagent antibody
  • Mass spectrum-based epitope positioning method of in vitro diagnostic reagent antibody
  • Mass spectrum-based epitope positioning method of in vitro diagnostic reagent antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Such as figure 1 As shown, a mass spectrometry-based epitope mapping method for in vitro diagnostic reagent antibodies, the specific steps are:

[0054] 1. Antigen and kit antibody preparation

[0055] Select a kit for epitope analysis, prepare an antigen solution with a purity of more than 90% for the detection items of the kit, and determine its concentration. Antigens were stored in a -80°C freezer until relevant experiments.

[0056] Determine the composition type of the kit antibody (multi-antibody pairing or single antibody), collect all antibodies in the selected kit according to the type, and determine the type (multi-antibody / monoclonal antibody) and concentration of each antibody. Each antibody was stored in a -80°C freezer until the relevant experiment.

[0057] 2. Antigen purity and aggregation state analysis

[0058] 2.1 Antigen purity analysis

[0059] The epitope mapping method based on hydrogen-deuterium exchange mass spectrometry usually requires t...

Embodiment 2

[0119] The method of Example 1 was used to locate the epitope of the antibody of the whole process C-reactive protein assay kit of a domestic company, and the specific experiment was as follows:

[0120] 1. Preparation of C-reactive protein (CRP) and a company's whole-process C-reactive protein assay kit antibody

[0121] A natural CRP solution (Beijing Deoping Biotechnology Co., Ltd.) with a purity of 95% was prepared at a concentration of 2.05 mg / mL. Jiuqiang kit contains a polyclonal antibody with a concentration of 13.66mg / mL. The above samples were stored in a -80 °C freezer before the experiment.

[0122] 2. Antigen purity and aggregation state analysis

[0123] 2.1 Antigen purity analysis (such as figure 2 )

[0124] SDS-page gel electrophoresis was used to verify the purity of CRP.

[0125] The specific operation method of SDS-page is as follows:

[0126] 1) Fix the glass tank, check for leaks with deionized water, prepare a separation gel with a suitable concen...

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Abstract

The invention discloses an in vitro diagnostic reagent antibody epitope localization method based on mass spectrum, which comprises the following steps: analyzing the interaction of various antibodies in a kit and a detected antigen through surface plasma resonance to obtain the antibody capable of directly interacting with the detected antigen; an immune complex of an antigen to be detected and an antibody is prepared through on-ice incubation, and then the incubation time, the incubation concentration and the binding proportion of the immune complex are determined through size exclusion chromatography. And finally, obtaining a peptide map of an antigen to be detected through an on-line enzyme digestion technology, analyzing the immune complex by using a hydrogen-deuterium exchange technology based on mass spectrum, and obtaining an epitope map of the antibody in the kit through data processing. According to the present invention, the epitope positioning of the antibody in the in vitro diagnostic kit is achieved by using the brand new method; the method has the characteristics of rapidness, sensitivity, high throughput, less sample consumption, no analysis quality upper limit and the like, and can be applied to epitope positioning analysis of most kits on the market.

Description

technical field [0001] The invention relates to the technical field of in vitro diagnosis, in particular to an epitope localization method for an antibody of an in vitro diagnostic reagent. Background technique [0002] In vitro diagnostic (IVD) reagents refer to products used for in vitro detection of human samples in the process of disease prediction, prevention, diagnosis, treatment monitoring, prognosis observation and health status evaluation. [0003] At present, in vitro diagnostic reagents are basically required for disease prevention, clinical diagnosis and treatment, such as the three routine tests of blood, urine and stool, the identification of virus or bacterial infection, heart, liver, kidney, blood vessels, and immune function tests. Therefore, improving the performance of IVD reagents is an important basis for ensuring the correctness of relevant laboratory results and diagnosis. [0004] IVD reagents are often tested in the form of kits. Many domestic and f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/447G01N27/62G01N30/02
CPCG01N27/447G01N30/02G01N27/62
Inventor 宋德伟孙浩峰刘健仪
Owner NAT INST OF METROLOGY CHINA
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