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Ultra-Sensitive Immunoassay

#4004


Ultra-Sensitive Immunoassay for Early Diagnosis of Cancer

Tech Area / Field

  • BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
  • MED-DID/Diagnostics & Devices/Medicine

Status
3 Approved without Funding

Registration date
01.10.2009

Leading Institute
Engelhardt Institute of Molecular Biology, Russia, Moscow

Supporting institutes

  • Research Center of Molecular Diagnostics and Theraphy, Russia, Moscow

Collaborators

  • Centre for Addition and Mental Helth / The Krembil Family Epigenetics Laboratory, Canada, ON, Toronto\nForschungszentrum fur Medizintechnik und Biotechnologie e.V., Germany, Bad Langensalza\nKarolinska Institute / Department of Microbiology, Tumor and Cell Biology, Sweden, Stockholm

Project summary

Numerous studies demonstrated the existence of multiple genes, which are overexpressed in particular tumor type, and thus can be used as diagnostic and/or prognostic markers of cancer. Identification of tumor markers and quantitative analysis of their expression profiles at different stages of the disease open a possibility of:
  • early serological diagnosis of cancer based on detection of tumor specific proteins secreted in blood circulation, using ultra-sensitive immunoassays
  • accurate prediction of tumor development and disease outcome based on detection of mRNA or protein expression in biopsies or surgical material (van't Veer L.J. et al. Gene expression profiling predicts clinical outcome of breast cancer. Nature, 2002, 415 (6871), 530-6).

Early diagnosis and prognosis of tumors are crucial for successful treatment of cancer patients and can lead to substantial increase in patient’s survival rate (van’t Veer L.J. and Bernards R. Enabling personalized cancer medicine through analysis of gene-expression patterns. Nature, 452 (7187), 564-70, 2008). However, early diagnosis of cancer is an extremely complicated task. Though it can be achieved by positron-emission tomography (PET), such an approach is extremely expensive and can not be widely used in clinical practice.

Project Goal

The main goal of this project is to develop an ultra-sensitive serological immunoassay, which allows detection of several hundred antigen molecules in a biological sample. This will open a possibility of an early, serological diagnosis of cancer, as well as infectious, autoimmune, and other diseases in patients at risk. The efficiency of the developed immunoassay will be tested by its application to serum diagnosis of colon cancer.

Significant increase in immunoassay sensitivity will be achieved by simultaneous application of three technical innovations:

  • significant increase in antibody affinity
  • complete removal of all components of the analyzed sample (except for the antigen) by “stringent wash” of antigen-antibody complexes by chaotropic buffer
  • development of a new modification of immuno-PCR protocol, based on synthesis of DNA reporter of new generation and a novel method of conjugation of antibodies to DNA reporter.

Staff participants of the project from the Engelhardt Institute of molecular biology and Russian research center of molecular diagnosis and treatment have tens of publications in international journals in the field of immunology, development of PCR technologies, identification of tumor suppressor genes and markers of different types of tumors, and early diagnosis of cancer (see the list of publications). Several project participants worked abroad for substantial periods of time in university medical schools, hospitals, and cancer centers (Memorial Sloan-Kettering Cancer Center, New York, USA; Karolinsky Institute, Stockholm, Sweden; Cold Spring Harbor Laboratory, Cold Spring Harbor, USA; University of Pennsylvania, Philadelphia, USA).

Experimental objectives:

The experimental objectives of the project are:

  • production of high affinity sequence specific antibodies to linear epitopes of a given antigen from polyclonal antiserum with dissociation constant 10-9 – 10-11 M
  • radical increase of ELISA-test sensitivity
  • synthesis of DNA reporter of new generation and development of a new method of its conjugation to antibodies
  • development of an ultra-sensitive immuno-PCR methodology, allowing detection of several antigen molecules in a biological sample, using DNA reporter of new generation
  • development of an immunoassay for early serum diagnosis of colon cancer in patients at risk, using previously identified serological markers of colon cancer.

The following methodologies will be used in this project:
  • immunochemistry – immunization, selection of high affinity antibodies from antiserum by affinity chromatography, production of hybridomas, immunoprecipitation, Western blotting, ELISA, immuno-PCR
  • protein chemistry – protein purification by FPLC chromatography, production of DNA-antibody conjugates (according to a new technology developed by project participants), synthesis of peptides, peptide purification by HPLC, conjugation of peptides to carrier protein
  • recombinant DNA technologies – cloning, amplification, and DNA sequencing, protein expression with His6-tag, synthesis of DNA reporter of new generation
  • PCR technologies – immuno-PCR, RT-PCR, single molecule PCR
  • collection of samples – collection and storage of blood samples from healthy donors and patients with the diagnosis: colon cancer, histological analysis of surgical materials (normal and tumor, using a protocol approved by a medical council), compiling of a computerized tumor registry.

In case of successful completion of the project, we will apply for patents in order to protect our intellectual property rights on newly developed ultra-sensitive immunoassays of new generation for early serological diagnosis of colon cancer. We intend to license the patents to interested companies in order to optimize automate and distribute the tests on commercial basis. The realization of the current project will provide a research opportunity to the scientists of the Russian scientific center of molecular diagnosis and treatment, prior involved in development of means of biological defense, to reorient their efforts and to apply their knowledge and experience to cancer research in public interest.


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