Detection of collagen triple helix repeat containing-1 and nuclear factor (erythroid-derived 2)-like 3 in colorectal cancer

BMC Clinical Pathology

María-Jose Rodriguez²  Paloma Rueda²  Carmen Vela²  Elisabet Rosell¹  Julita García²  Tamara Ruiz²  Nuria de Roja²  Margarita García²  Lissett Lopez²  Marco Palma³ 

[1]Oryzon Genomics, S.A., Barcelona, Spain

[2]Inmunología y Genética aplicada, S.A., Madrid, Spain

[3]Inmunología y Genética Aplicada, SA, Calle Hermanos García Noblejas, 39 - 28037 Madrid, Spain

关键词: DELFIA assay;    DAS-ELISA;    Double antibody sandwich enzyme-linked immunosorbent assay;    NFE2L3;    CTHRC1;    Nuclear factor (erythroid-derived 2)-like 3;    Triple Helix Repeat Containing-1;    Biomarker;    Cancer biomarker;    Cancer;    CRC;    Colorectal cancer;   

Others  :  1085479
DOI  :  10.1186/1472-6890-12-2

 received in 2011-07-13, accepted in 2012-02-09,  发布年份 2012

【 摘 要 】

Background

Collagen Triple Helix Repeat Containing-1 (CTHRC1) and Nuclear factor (erythroid-derived 2)-like 3 (NFE2L3) may be useful biomarker candidates for the diagnosis of colorectal cancer (CRC) since they have shown an increase messenger RNA transcripts (mRNA) expression level in adenomas and colorectal tumours when compared to normal tissues.

Methods

To evaluate CTHRC1 and NFE2L3 as cancer biomarkers, it was generated and characterised several novel specific polyclonal antibodies (PAb), monoclonal antibodies (MAbs) and soluble Fab fragments (sFabs) against recombinant CTHRC1 and NFE2L3 proteins, which were obtained from different sources, including a human antibody library and immunised animals. The antibodies and Fab fragments were tested for recognition of native CTHRC1 and NFE2L3 proteins by immunoblotting analysis and enzyme-linked immunosorbent assay (ELISA) in colorectal cell lines derived from tumour and cancer tissues.

Results

Both, antibodies and a Fab fragment showed high specificity since they recognised only their corresponding recombinant antigens, but not a panel of different unrelated- and related proteins.

In Western blot analysis of CTHRC1, a monoclonal antibody designated CH21D7 was able to detect a band of the apparent molecular weight of a full-length CTHRC1 in the human colon adenocarcinoma cell line HT29. This result was confirmed by a double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) with the monoclonal antibodies CH21D7 and CH24G2, detecting CTHRC1 in HT29 and in the colon adenocarcinoma cell line SW620.

Similar experiments were performed with PAb, MAbs, and sFab against NFE2L3. The immunoblot analysis showed that the monoclonal antibody 41HF8 recognised NFE2L3 in HT29, and leukocytes. These results were verified by DAS-ELISA assay using the pairs PAb/sFab E5 and MAb 41HF8/sFab E5.

Furthermore, an immunoassay for simultaneous detection of the two cancer biomarkers was developed using a Dissociation-Enhanced Lanthanide Fluorescent Immunoassay technology (DELFIA).

Conclusions

In conclusion, the antibodies obtained in this study are specific for CTHRC1 and NFE2L3 since they do not cross-react with unrelated- and related proteins and are useful for specific measurement of native CTHRC1 and NFE2L3 proteins. The antibodies and immunoassays may be useful for the analysis of CTHRC1 and NFE2L3 in clinical samples and for screening of therapeutic compounds in CRC.

【 授权许可】

   
2012 Palma et al; licensee BioMed Central Ltd.

【 图 表 】

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