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Mesothelin immunohistochemical detection kit

An immunohistochemistry and kit technology, which is applied in the field of immunohistochemistry kits for detecting mesothelin

Active Publication Date: 2019-12-03
REMEGEN CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is still a lack of immunohistochemical antibody reagents and related detection methods that can quickly and effectively detect the expression of mesothelin protein that can meet the needs of the market.

Method used

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  • Mesothelin immunohistochemical detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1: Preparation of anti-MSLN monoclonal antibody

[0052] (1) Establishment of hybridoma cell lines

[0053] 1. Preparation of experimental materials:

[0054] The MSLN-ECD (Cat. No. FCL1589) protein fragment of G&P Biosciences was used as the immunogen. This fragment contains 317 amino acids and is located in the Glu296-Ser592 region of the MSLN (UniProt accession#Q13421, isoform 2) sequence; the medium used includes but is not limited to HAT, HT selection medium, DEME medium; experimental animals include Balb / c mice; adjuvants used are emulsified Freund's complete adjuvant and emulsified Freund's incomplete adjuvant.

[0055] 2. Animal immunization

[0056] Basic immunization: Equal volume of antigen and complete Freund's adjuvant are fully emulsified, subcutaneously injected at 50 μg / mouse, and 2 Balb / c mice are immunized.

[0057] Booster immunization: antigen emulsified with Freund's incomplete adjuvant; 3 days before cell fusion, direct intraperitonea...

Embodiment 2

[0068] Example 2: Optimization of Experimental Conditions for Anti-MSLN Monoclonal Antibody 3-2G6

[0069] (1) Antigen retrieval conditions were optimized by evaluating different antigen retrieval buffers. Restoration condition 1 (Cit6.0) refers to using acidic pH6.0 citrate buffer for high-pressure restoration for 2.5 minutes. Restoration condition 2 (Tris 9.0) refers to high-pressure restoration with alkaline pH9.0 Tris-EDTA buffer for 2.5 minutes. Repair condition 3 (Tryp) refers to repairing with 0.25% trypsin repair solution at 37° C. for 10 min. like figure 2 Shown in A: In these tested conditions, the staining effect of Cit 6.0 after high-pressure repair is better than that of other repair methods, specifically showing that the target cells (tumor cells) are clearly stained, and there is no non-specific staining of interstitial cells. Therefore, this method was finally selected as the antigen retrieval method suitable for this antibody.

[0070] (2) The staining in...

Embodiment 3

[0072] Embodiment 3: the selection of antibody diluent

[0073] We conducted a series of evaluations on the various components of the 3-2G6 antibody dilution, including matrix, salt ion concentration, pH value, protective protein concentration, surfactants, preservatives, etc.

[0074] In terms of matrix, use pH 7.4, 0.05M TRIS buffer and PB (Phosphate Buffer) as buffers to dilute 3-2G6 antibody for immunohistochemical experiments, according to image 3 The result of A shows: the use of Tris buffer makes non-specific background staining very obvious;

[0075] In terms of salt ion concentration, after adding 0.015-1.2M NaCl to 0.05M TRIS and PB buffer respectively, it was found that as the ion concentration increased, the non-specific staining disappeared, and when the ion concentration increased to above 0.15M, the specific staining intensity also increased. Then lower. Under the premise of other conditions being the same, too high salt ion concentration will lead to a decre...

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Abstract

The application relates to a mesothelin (MSLN) immunohistochemical detection kit, which contains a MSLN monoclonal antibody 3-2G6.

Description

technical field [0001] The invention relates to an immunohistochemical kit for detecting mesothelin (MSLN). Background technique [0002] Mesothelin (MSLN) is a cell surface glycoprotein. The mesothelin gene encodes a 71KD proprotein, which is modified and cleaved into two segments, namely the 41KD glycosylated phosphatidylinositol cell membrane-anchored protein and the 30KD free fragment called megakaryocyte-promoting factor. Studies have confirmed that the 41KD membrane anchor protein contains a binding region with another tumor marker CA125 protein, which may be related to cell adhesion, so the anchor protein may contribute to tumor metastasis and lead to unsatisfactory prognosis . The latest research shows that: MSLN is highly expressed in almost all mesotheliomas and pancreatic cancers, most ovarian cancers and lung cancers, and rarely expressed in normal tissues. Although the function of Mesothelin is not yet clear, both the cell membrane binding region and the free...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/68G01N33/531
CPCG01N33/577G01N33/6863G01N33/531G01N2333/47
Inventor 毛海燕于占娇肖童雨李壮林房健民
Owner REMEGEN CO LTD
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