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Molecular Diagnostics (Fach) / course 10 (Lektion)
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- Definition of MDx The use of DNA, RNA and/or protein to test specific status of healthy or sick/affected individuals (disease satus) Enabled by genomic, proteomic and metabolomic technology
- predictive application treatment by chance of a disease
- Diagnostic application detection of a disease
- prognostic application predict the outcome of a disease
- therapeutic application response to a treatment of a disease
- Beacon assay ultrasensitive PCR
- Line probe assay PCR and reverse hybridisation
- characteristics of a good test sensitive, specific, simple, accurate
- sensitivity percentage of false negative samples
- specificity percentage of false positive samples
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- RNA/DNA based techniques Sequencing, PCR, restriction digest, FISH, RAPD, DNA fingerprinting, PCR oligonucleotide ligation assay, Microarray
- Protein-based techniques ELISA, FCS, mass spectrometry
- What can be measured with proteins? altered expression post-translational modifications antibodies metaboloites
- mass-pec principle separation based on mass to charge ratio sample is ionised, ions are separated and detected data fingerprint is compared to database
- advantages of mass-spec more quantitative no cross-reactivity very sensitive, can detect small differences
- affinity constant KA = [Ab-Ag] / [Ab] * [Ag]
- Polyclonal AB serum of an immunised animal, many different antibodies => heterogenous
- monoclonal AB B-cell fused with myeloma cells =Y hybridomas one clone is selected for amplification
- Mono vs Polyclonal AB (Advantages and Disadvantages) Monoclonal: + one paratope, highly specific, reproducible - labour intensive and expensive, less sensitive to denatured proteins Polyclonal: + several paratopes, high affinity and signal amplification (disadvantage for quantification) - batch to batch variation, no uniform response, cross-reactivity
- pregnancy test previously: agglutination assay now: ELISA presence of HCG
- Direct vs. Indirect ELISA direct: + cheap and fast, less cross-reactivity - primary AB has to be labelled =Y purification of AB and conjugate Indirect: + big variety of AB available, high sensitivity, - cross-reactivity, extra incubation and washing
- development of an ELISA (components needed) plates, coating AB blocking solution Optimal dilutions (checkerboard) Detection
- purpose of blocking solution prevents unspecific binding
- CBT for optimisation of reagents horizontal: Antigen dilution Vertical: Antibody dilution ideal: linear decrease
- ELISA applications determination of serum AB concentration pregnancy test toxicology: detect presence of drugs, food alergens