Analysis method for extracting immune polypeptide in biological sample based on immunoprecipitation method

A technology of immunoprecipitation and immune peptides, which is applied in the field of analysis of immune peptides in biological samples based on immunoprecipitation, can solve problems such as polymer contamination, enrichment and peptide loss, and achieve high specificity and effectiveness. The effect of high application prospects

Pending Publication Date: 2022-01-18
上海中科新生命生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are two common methods of purification and impurity removal. 1. Use a molecular weight filter to separate peptides from HLA protein molecules, but this operation will lead to the loss of enriched peptides and may also introduce polymer contamination.
2. Set up a liquid phase gradient by reversed-phase HPLC to separate peptides from larger complex components, but this operation requires large-scale instruments such as liquid chromatography and fraction collectors

Method used

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  • Analysis method for extracting immune polypeptide in biological sample based on immunoprecipitation method
  • Analysis method for extracting immune polypeptide in biological sample based on immunoprecipitation method
  • Analysis method for extracting immune polypeptide in biological sample based on immunoprecipitation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Extraction and mass spectrometric identification of immune polypeptides from HCC1937 cells.

[0030] The reagents used in this example were purchased from Sigma unless otherwise noted, including triethanolamine, DMP cross-linking agent, Tris, acetonitrile, trifluoroacetic acid, and acetic acid. Protein A sepharose 4B, protease inhibitors, pepstatinprotease inhibitors and PBS were purchased from Thermo Fisher Scientific. The desalting column SepPak C18 Cartridges was purchased from Waters. Econo-column empty columns were purchased from Bio-Rad.

[0031] An analysis method for extracting immune polypeptides in biological samples based on immunoprecipitation, which first coats the specific antibody (W6 / 32 antibody) corresponding to the target immune polypeptide to magnetic beads through cross-linking reagents, and then enriches through magnetic bead precipitation The target immune polypeptide (HLA-peptide in HCC1937 cell lysate), the enriched target immune pol...

Embodiment 2

[0045] Example 2: Identification results of immunological peptides in tissues of 5 patients and distribution diagrams of peptide lengths.

[0046] The method used in the second embodiment is the same as that in the first embodiment, only the cell line samples are replaced with patient tissue samples.

[0047] 5 patient tissue samples (liver cancer tissue) were taken for extraction of immune polypeptides. In addition to the difference between the tissue lysis method and the cell line, the preparation of the immunoaffinity column, the immunoaffinity binding, elution, purification and desalting of the lysate, the detection and data analysis of mass spectrometry are all consistent with Example 1, and the tissue lysis method details as follows:

[0048]Each tissue needs more than 200mg, add about 20mL of cell lysate, then homogenize with tissue magnetic beads, and incubate on ice for 1 hour, and the solution can be pipet or inverted every 10 minutes. Then, put the lysed tissue sa...

Embodiment 3

[0050] Example 3: Verification of the enriched HLA in Example 1 and Example 2.

[0051] The acrylamide electrophoresis gel 10% NuPAGE precast gel, 10× reducing agent, 4× loading solution, 20× electrophoresis buffer, western blot imprinting membrane and filter paper used in this example were all purchased from Thermo Company; the molecular weight of prestained protein The marker was purchased from Bio-rad Company; the primary anti-beta 2-microglobulin rabbit recombinant antibody (anti-beta 2microglobulin) was purchased from Abcam Company; the secondary antibody horseradish peroxidase-labeled goat anti-rabbit IgG was purchased from Beyontian Company; electrophoresis The instrument MiniGel Tank and iBlot2 dry transfer system were purchased from Thermo Company.

[0052] Take one part of each of the effluents 2 in Examples 1 and 2, dissolve them in PBS, measure the protein concentration with OD280, and take 200ng for SDS-PAGE running gel and WB analysis to verify the success of the...

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Abstract

The invention provides an analysis method for extracting immune polypeptide in a biological sample based on an immunoprecipitation method, and belongs to the technical field of immune polypeptide analys. The method comprises the steps that an immunoaffinity column is prepared through a cross-linking reagent, then lysate of the biological sample is subjected to immunoprecipitation, so that an HLA-peptide fragment compound is obtained through enrichment, and then simple two-step elution is conducted through a C18 small column. Impurity removal and desalination of the immune polypeptide can be achieved at the same time through the steps, recovered components can be subjected to mass spectrometric detection after being concentrated, efficient extraction of the immune polypeptide in biological samples such as cells and tissues can be achieved, the sample pretreatment process does not depend on complex instruments, purification and desalination of the immune polypeptide can be completed, and the time required for operation is greatly shortened.

Description

technical field [0001] The invention belongs to the technical field of immune polypeptide analysis, and in particular relates to an analysis method for extracting immune polypeptides in biological samples based on an immunoprecipitation method. Background technique [0002] Cancer, as a malignant tumor, is one of the diseases with a very high mortality rate in both developed and developing countries, and the number of new patients is increasing every year. Therefore, researchers all over the world are looking for ways to cure cancer. Due to the side effects of traditional chemotherapy and radiotherapy, along with the research on cancer-related pathways, more molecular targeted therapy has been applied to cancer treatment. With the continuous development and improvement of immunotherapy technology, it is the most popular and promising method for treating cancer in recent years. Tumor vaccine technology was rated as one of the top ten technological breakthroughs in 2019 by M...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/6848G01N2560/00
Inventor 阮宏强李紫艺张惠萍
Owner 上海中科新生命生物科技有限公司
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