Molecular Diagnostics (Subject) / PP-3&4 (Lesson)

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• Types of Antibody labeling -enzymes -fluorophore -luminescent -radioisotope
• Color development in ELISA depends on which factors of the Enzyme? -pH dependent -temperature -substrate -cofactors -stability of reaction product -stability of enzyme
• ELISA: Homologous & heterologous Homo = all reagents come from same species hetero = reagents come from different species?
• ELISA: competitive assay unlabeled analyte (sample) competes with labeled analyte signal decreases the higher the analyte conc. in the sample
• ELISA: Competitive / one-step vs two-step all in one tube (limited amount of Ab) vs Analyte of Interest with excess non-labeled antibody/antigen and then add into the second tube with labeled antibody/antigen (more sensitive)
• ELISA: Homogenous & Heterogenous Hetero: require removal of unbound ab-ag* complex homo: don't need that step: used for small analytes (drugs)
• EMIT Enzyme multiplied immunoassay technique free drug competes with enzyme labeled derivate with antibody case1: enzyme labeled derivate is bound to Ab = enzyme blocked case2: drug is bound to Ab = enzymes free to digest substrate
• Disadvantages of Immunoassays Time consuming Labor intensive Hard to automate
• Alpha LISA one step ELISA no washing very sensitive Excitation bead - Ab - Ag(big boy) - Ab - Acceptor bead
• ELISA Enzyme linked immunosorbent assay
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• LUMINEX 10x10 beads each with distinct color & antigen one laser detects which kind of bead another laser detects how much of analyte is bound the only sandwich is measured proteins, nucleic acids, enzymes & receptors
• Advantages Flow Cytometry Fast quantitative multiple colors positive/negative discrimination differentiation cell sorting (Fluorescence activated cell sorter [FACS]
• FCM Applications Immunophenotypic nucleic acid analysis cell sorting platelet function analysis cell function analysis cytokines
• SNP single nucleotide polymorphism affect recognition site of restriction enzymes
• TRP tandem repeat polymorphism microsatellites: 1-4bp repeats over 100bp minisatellites: 10-50bp copy number variation: 1kb-1mb
• DNA/RNA hybridization attachment of target or probe to solid matrix add the labeled and denatured target/probe if it fits I sits (Sequence homology) detection of hybridized target
• FISH Fluorescence in situ hybridization label probe with fluorescent dye and hybridize with target on chromosome localization of DNA on chromosome Detects: added/removed genes - translocation - telomere/centromere abnormalities
• RFLP Restriction Fragment Length Polymorphism polymorphisms change restriction sites legal proceedings/paternity testing/hereditary disease cheap&fast needs lots of DNA
• AFLP Amplification fragment length polymorphism restriction of gDNA ligation of digested gDNA with adaptor sequences
• AFLP advantages & disadvantages pro: don't need to know the sequence  highly reproducible many loci analyzed simultaneously little DNA needed con: complex
• OLA oligonucleotide ligation assays hybridize two oligos if perfect match oligos ligate different stuffer determines product length 
• MLPA Multiplex ligation-dependent probe amplification improves on OLA detect up to 45 sequences at once detects mutlifactorial diseases
• capillary electrophoresis one tube= one lane less diffusion /differences in temp

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