RESUMO
A high throughput protein biomarker discovery tool has been developed based on multiplexed proximity ligation assays in a homogeneous format in the sense of no washing steps. The platform consists of four 24-plex panels profiling 74 putative biomarkers with sub-pm sensitivity each consuming only 1 µl of human plasma sample. The system uses either matched monoclonal antibody pairs or the more readily available single batches of affinity purified polyclonal antibodies to generate the target specific reagents by covalently linking with unique nucleic acid sequences. These paired sequences are united by DNA ligation upon simultaneous target binding forming a PCR amplicon. Multiplex proximity ligation assays thereby converts multiple target analytes into real-time PCR amplicons that are individually quantified using microfluidic high capacity qPCR in nano liter volumes. The assay shows excellent specificity, even in multiplex, by its dual recognition feature, its proximity requirement, and most importantly by using unique sequence specific reporter fragments on both antibody-based probes. To illustrate the potential of this protein detection technology, a pilot biomarker research project was performed using biobanked plasma samples for the detection of colorectal cancer using a multivariate signature.
Assuntos
Biomarcadores Tumorais/sangue , Neoplasias Colorretais/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos Glicosídicos Associados a Tumores/sangue , Antígeno Carcinoembrionário/sangue , Neoplasias Colorretais/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Interleucina-8/sangue , Masculino , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular , Análise Multivariada , Inibidor Secretado de Peptidases Leucocitárias/sangue , Estatísticas não Paramétricas , Inibidor Tecidual de Metaloproteinase-1/sangue , Fator A de Crescimento do Endotélio Vascular/sangueRESUMO
The REvolutionary Approaches and Devices for Nucleic Acid analysis (READNA) project received funding from the European Commission for 41/2 years. The objectives of the project revolved around technological developments in nucleic acid analysis. The project partners have discovered, created and developed a huge body of insights into nucleic acid analysis, ranging from improvements and implementation of current technologies to the most promising sequencing technologies that constitute a 3(rd) and 4(th) generation of sequencing methods with nanopores and in situ sequencing, respectively.
Assuntos
Biotecnologia/métodos , DNA/análise , DNA/genética , Animais , Química Click , Exoma/genética , Humanos , Espectrometria de Massas , Análise de Sequência de DNARESUMO
Recent outbreaks of avian influenza in different parts of the world have caused major economic losses for the poultry industry, affected wildlife seriously and present a significant threat even to human public health, due to the risk for zoonotic transmission. The ability to recognize avian influenza viruses (AIVs) early is of paramount importance to ensure that appropriate measures can be taken quickly to contain the outbreak. In this study, the performance of a proximity ligation assay (PLA) for the detection of AIV antigens in biological specimens was evaluated. It is shown that PLA: (i) as a novel principle of highly sensitive antigen detection is extending the arsenal of tools for the diagnosis of AIV; (ii) is very specific, nearly as sensitive as a commonly used reference real-time PCR assay, and four orders of magnitude more sensitive than a sandwich ELISA, utilizing the same antibody; (iii) avoids the necessity of nucleic acids extraction, which greatly facilitates high-throughput implementations; (iv) allows the use of inactivated samples, which safely can be transported from the field to diagnostic laboratories for further analysis. In summary, the results demonstrate that PLA is suited for rapid, accurate and early detection of AIV.