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Kit for detecting phenotypes of circulating tumor cells

A tumor cell and kit technology, applied in the field of immunoassay detection, can solve the problems of missed CTC screening, inaccurate results, false positives, etc., and achieve the effects of improved sensitivity, high reaction efficiency, and sensitive detection.

Active Publication Date: 2016-08-03
SHANGHAI MAJORBIO BIO PHARM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Density and molecular size can easily lead to missed screening of CTCs, but based on EpCAM, only epithelial phenotype CTCs can be detected. At the same time, normal cells expressing the same marker EpCAM will lead to false positives, and phenotypic heterogeneity will lead to false negatives, and the results are not accurate. precise

Method used

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  • Kit for detecting phenotypes of circulating tumor cells
  • Kit for detecting phenotypes of circulating tumor cells
  • Kit for detecting phenotypes of circulating tumor cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1E-cadherin-oligonucleotide probe

[0041] (1) Aldehyde modification of oligonucleotide probes

[0042] 50 nmol of Oligo1 (oligonucleotide) was prepared into a solution with 0.1 M pH 7.4 phosphate buffer. Weigh 500nmol of SFB (N-succinimidyl 4-formylbenzoate), dissolve it with anhydrous DMF (N,N-dimethylformamide), react at room temperature for 2.5h, and purify through a column to obtain Oligo -FB (aldehyde modified oligonucleotide).

[0043] Detection of the concentration of Oligo-FB: detection of A by Nanodrop spectrophotometer 260 value, the calculated concentration of Oligo-FB was 0.65nmol / μL.

[0044] Detection of aldehyde group modification rate: Use quantitative 2-hydrazinopyridine-2-hydrochloride solution to detect the aldehyde group modification rate. Take the above-mentioned Oligo-FB and add it to the 2-hydrazinopyridine-2-hydrochloride solution, shake and mix well, and react at 37°C for 1 hour. The absorbance value at 360nm detected by Nanodrop...

Embodiment 2

[0057] The QPCR amplification specific detection of embodiment 2Oligo molecule

[0058] Oligo1-13 molecules were diluted to three concentrations of 25000 molecules / μl, 83333 molecules / μl and 250000 molecules / μl. Then, taking Oligo1-3 as an example, mix Oligo1-3 at the same concentration to obtain mixed samples A, B, and C, as shown in Table 2.

[0059] Table 2 Example 2 sample formula

[0060]

[0061] The above samples were configured with QPCR amplification solution according to Table 3 to obtain a QPCR amplification kit, and then Oligo1-3 was subjected to QPCR amplification according to the procedures shown in Table 4, and Oligo1-3 in mixed samples A, B, and C Each was subjected to QPCR amplification, using the extended Oligo1-3 as a template, and the Ct values ​​of the amplification results are shown in Table 5. Among them, the extension primer is RT-P, its 3' end is complementary to the 3' end of the oligonucleotide, the 5' end can form a hairpin structure, and the m...

Embodiment 3

[0076] Embodiment 3 makes standard curve

[0077] The same result can be obtained with Oligo1-13. In order to simplify the process, the following examples use Oligo1-3 as an example.

[0078] According to the steps of Example 1, Oligo2 was modified with 5 times the molar equivalent of SFB to obtain Oligo-FB, and EpCAM was hydrazine-modified with 10 times the molar equivalent of SANH to obtain EpCAM-SANH, and the molar ratio was 10:1 EpCAM-Oligo2 was obtained after reacting Oligo-FB with EpCAM-SANH at room temperature for 16 hours.

[0079] According to the steps of Example 1, Oligo3 was modified with 20 times the molar equivalent of SFB to obtain Oligo-FB, and Cadherin-11 was hydrazino-modified with 50 times the molar equivalent of SANH to obtain Cadherin-11-SANH. The molar ratio Cadherin-11-Oligo3 was obtained after 8:1 Oligo-FB reacted with Cadherin-11-SANH at room temperature for 24 hours.

[0080] E-cadherin-Oligo1, EpCAM-Oligo2 and Cadherin-11-Oligo3 were subjected to Q...

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Abstract

The invention provides a kit for detecting phenotypes of circulating tumor cells. The kit comprises an antibody-oligonucleotide probe obtained by respectively coupling E-cadherin, Cadherin-11 and EpCAM antibodies with different oligonucleotides, an amplification primer and buffer solution used for performing PCR amplification on the antibody-oligonucleotide probe. The kit also comprises a fluorescence probe. According to the kit disclosed by the invention, the E-cadherin, Cadherin-11 and EpCAM are taken as markers to perform parallel detection on content of each antigen in CTC, so that the content of each phenotype in CTC is analyzed, and each phenotype of CTC is distinguished.

Description

technical field [0001] The invention relates to the field of immunoassay detection, in particular to a kit for detecting the phenotype of circulating tumor cells. Background technique [0002] Circulating tumor cells (CTCs) refer to tumor cells released into the peripheral blood circulation from the primary tumor or metastases of solid tumors spontaneously or due to diagnostic and therapeutic procedures, usually in the form of single cells or cell clusters (also known as circulating tumor microemboli, CTM). present in the circulatory system. Metastasis is the main cause of cancer-related death, and CTCs are regarded as the seeds of metastasis. In order to acquire motility and invasiveness, CTC will lose certain epithelial cell phenotypes (including morphology, surface antigens, gene expression, etc.) , Epithelial-Mesenchymal Transition). Most malignant tumor cells undergo EMT during the process of breaking away from the primary tumor. Therefore, CTCs have different pheno...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/574
CPCG01N33/57488
Inventor 蔡红东李静陈昌岳邓文斌甘广利张祥林
Owner SHANGHAI MAJORBIO BIO PHARM TECH
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