ABSTRACT Tumor markers are molecules that can be detected and dosed as indicators of a normal biological process, of a pathological process and of a pharmacological response to a therapeutic intervention. Once the therapeutic area and the pathology to work on are identified, the industrial development for the application of the tumor marker in the diagnostic field, involves the development of the production process of this molecule to be used in the analysis and validation of the analytical assay. The work developed during the PhD was dedicated to the development of a potential enzyme immunoassay for the diagnosis of colorectal cancer (CRC). It is possible to divide this work into several stages, ranging from the development of the production process of the recombinant CoA-1, to the production and selection of monoclonal antibodies against this protein and to the development of an ELISA test, can be applied to the diagnosis of CRC. The first part of the process has been dedicated to the optimization production of the recombinant CoA-1, fused with glutathione S transferase in E. coli. The kinetics of the production was analyzed and the possible transfer to a bioreactor for the medium or large-scale production, such as the Wave system. A protocol was evaluted for the extraction of the fusion protein CoA-1, contained in the cytoplasmic space. In order to produce monoclonal antibodies against the CoA-1, devoid of GST, a protocol for removal of GST by proteolysis was established by evaluating the kinetics of reaction with the protease Factor Xa. The product CoA-1 was used in immunization protocols and in the development and validation of the diagnostic kit. The selection of hybridoma clones, obtained after the immunization and somatic fusion, was performed by ELISA test and Western blot. The purpose of this study was to create a portfolio of specific antibodies against CoA-1, to be used in the development of diagnostic kit. All the antibodies have showed different affinities towards CoA-1 in the ELISA sandwich, in which they were used alternatively as a capture or detection antibody. Several experiments were conducted using decreasing concentrations of antigen recombinant CoA-1, in order to identify the pair of antibodies with higher sensitivity. This allowed the identification of the pair of mAbs 23C4/E5- 3A8/C2, with a cut-off detection of antigen CoA-1 of 1 pg/l. This pair of mAbs has been selected for the validation of the analytical method in which different parameters were considered, such as linearity, accuracy, precision, sensitivity, limit of quantitation (LOQ), specificity, the range of the curve and the recovery percentage. Therefore ELISA sandwich tests were performed repeatedly, with the same concentration of the antigen CoA-1, in the range from 1 to 15 pg/l. This way the analytical test to use as a potential diagnostic test has been validated. Moreover, the degree of interference by the biological matrices has been estimated on the developed ELISA sandwich test. At last, it has been described the development of the large scale production process of selected antibodies to by employed in the analytical test, by transferring the culture of ibridoma in the Wave bioreactor. In synthesis, this thesis presents the results of the industrial approach for the generation and validation of an assay for early diagnosis of CRC, by employing the tumor marker CoA-1.

STUDIO DI FATTIBILITA¿ E SVILUPPO INDUSTRIALE PER L¿APPLICAZIONE DI UN NUOVO MARCATORE TUMORALE NELLA DIAGNOSTICA PRECOCE DEL CARCINOMA DEL COLON-RETTO / M. Chimienti ; tutore: S. Della Bella ; correlatore: M. L. Nolli ; coordinatore: M. Clerici. Universita' degli Studi di Milano, 2012 Feb 07. 24. ciclo, Anno Accademico 2011.

STUDIO DI FATTIBILITA¿ E SVILUPPO INDUSTRIALE PER L¿APPLICAZIONE DI UN NUOVO MARCATORE TUMORALE NELLA DIAGNOSTICA PRECOCE DEL CARCINOMA DEL COLON-RETTO

M. Chimienti
2012

Abstract

ABSTRACT Tumor markers are molecules that can be detected and dosed as indicators of a normal biological process, of a pathological process and of a pharmacological response to a therapeutic intervention. Once the therapeutic area and the pathology to work on are identified, the industrial development for the application of the tumor marker in the diagnostic field, involves the development of the production process of this molecule to be used in the analysis and validation of the analytical assay. The work developed during the PhD was dedicated to the development of a potential enzyme immunoassay for the diagnosis of colorectal cancer (CRC). It is possible to divide this work into several stages, ranging from the development of the production process of the recombinant CoA-1, to the production and selection of monoclonal antibodies against this protein and to the development of an ELISA test, can be applied to the diagnosis of CRC. The first part of the process has been dedicated to the optimization production of the recombinant CoA-1, fused with glutathione S transferase in E. coli. The kinetics of the production was analyzed and the possible transfer to a bioreactor for the medium or large-scale production, such as the Wave system. A protocol was evaluted for the extraction of the fusion protein CoA-1, contained in the cytoplasmic space. In order to produce monoclonal antibodies against the CoA-1, devoid of GST, a protocol for removal of GST by proteolysis was established by evaluating the kinetics of reaction with the protease Factor Xa. The product CoA-1 was used in immunization protocols and in the development and validation of the diagnostic kit. The selection of hybridoma clones, obtained after the immunization and somatic fusion, was performed by ELISA test and Western blot. The purpose of this study was to create a portfolio of specific antibodies against CoA-1, to be used in the development of diagnostic kit. All the antibodies have showed different affinities towards CoA-1 in the ELISA sandwich, in which they were used alternatively as a capture or detection antibody. Several experiments were conducted using decreasing concentrations of antigen recombinant CoA-1, in order to identify the pair of antibodies with higher sensitivity. This allowed the identification of the pair of mAbs 23C4/E5- 3A8/C2, with a cut-off detection of antigen CoA-1 of 1 pg/l. This pair of mAbs has been selected for the validation of the analytical method in which different parameters were considered, such as linearity, accuracy, precision, sensitivity, limit of quantitation (LOQ), specificity, the range of the curve and the recovery percentage. Therefore ELISA sandwich tests were performed repeatedly, with the same concentration of the antigen CoA-1, in the range from 1 to 15 pg/l. This way the analytical test to use as a potential diagnostic test has been validated. Moreover, the degree of interference by the biological matrices has been estimated on the developed ELISA sandwich test. At last, it has been described the development of the large scale production process of selected antibodies to by employed in the analytical test, by transferring the culture of ibridoma in the Wave bioreactor. In synthesis, this thesis presents the results of the industrial approach for the generation and validation of an assay for early diagnosis of CRC, by employing the tumor marker CoA-1.
7-feb-2012
Settore MED/04 - Patologia Generale
marcatore tumorale ; carcinoma del colonretto ; anticorpi monoclonali
DELLA BELLA, SILVIA ANGELA MARIA
CLERICI, MARIO SALVATORE
Doctoral Thesis
STUDIO DI FATTIBILITA¿ E SVILUPPO INDUSTRIALE PER L¿APPLICAZIONE DI UN NUOVO MARCATORE TUMORALE NELLA DIAGNOSTICA PRECOCE DEL CARCINOMA DEL COLON-RETTO / M. Chimienti ; tutore: S. Della Bella ; correlatore: M. L. Nolli ; coordinatore: M. Clerici. Universita' degli Studi di Milano, 2012 Feb 07. 24. ciclo, Anno Accademico 2011.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/169914
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