14968282
Description
Flashcards by Dennis Jallah, updated more than 1 year ago
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Created by Dennis Jallah
over 5 years ago
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| Question | Answer |
| Chemiluminescence | Emission of light w/o emission of heat as result of a chemical reaction |
| Assay | Procedure where the activity, property, or concentration of an analyte is measured |
| Analyte | Substance determined in analytical procedure |
| Assay Buffer | Solution in which assay is performed in |
| Multiplex | Detection of more than one analyte in a single homogenous sample |
| Pos Control | Highest value or confirmation that assay worked as intended |
| Neg Control | Lowest signal possible in assay |
| Signal to Background Ratio | Ratio of mean signals for positive and negative control |
| Assay Format | Method of performing the assay |
| % Inhibition | Fraction of inhibition of the signal Given by the equation ( 1 - ((exp - neg)/(pos - neg)) |
| Assay Design | Choosing an assay format and approach |
| Assay Development | Optimizing an assay |
| Assay Validation | Characterization of an assay |
| Accuracy | Closeness of value found to accepted reference value |
| Precision | Degree of scatter between series of measurements taken from multiple sampling of the same homogenous sample |
| LOD | lowest concentration of an analyte in a sample that can be detected but not quantified |
| LOQ | Lowest concentration of an analyte in a sample that can be determined with acceptable precision and accuracy |
| Specificity | Measure of degree of interference from other substances |
| Linearity | Ability of method to elicit test results that are directly proportional to analyte concentration within a given range |
| Range | Interval between upper and lower levels of analyte that have been demonstrated to be determined with precision, accuracy, and linearity |
| Ruggedness | Degree of reproducibility of results obtained under varying conditions |
| Robustness | Capacity of method to remain unaffected by small deliberate variations in method parameters |
| Direct Assay | Signal proportional to analyte concentration |
| Competitive Assay | Inverse relationship between signal and concentration of analyte |
| Absorption | Physical process of absorbing light |
| Absorbance | Defined by the mathematical equation: Log10(Io/I) |
| Transmittance | Opposite of absorbance (I/Io) x 100 |
| Fluorescence Quenching | Bimolecular process that reduces fluorescence quantum yield without changing emission spectrum |
| Self quenching | Quenching of one fluorphore molecule by another of the same fluorphore |
| Quencher | Non-fluorescent molecule that accepts energy from fluorphore in close proximity but does not re-emit the energy as light |
| Quantum Yield | Measures fluorphore |
| Choices for detection methods | ELISA RIA Time-Resolved Fluorescence Electro-Chemiluminescent |
| Optimization Parameters for ELISA | Robustness Drift/Edge Effect Number of replicates |
| Assessing an assay | Noise - from instrument Background - from non-specific binding Signal - from the specific and selective binding on the labeled antibody to the analyte |
| Parameters essential for immunoassay validation | accuracy precision lower limit of quantitation upper limit of quantitation linearity reproducibility specificity stability |
| Advantages/Disadvantages of ELISA | Adv: very sensitive, many configurations available, many antibodies and secondary reagents commercially available Disadvantages: time consuming, labor intensive, relatively expensive |
| Epitope | part of antigen that antibody binds |
| Two types of Epitopes | Conformational - non-continuous amino acids that bind to Ab Linear - amino acid sequence that binds to Ab |
| Monoclonal Antibodies | Immunization (inject rodent w/ antigen) Fusion (fuse antibodies w/ immortal B tumor) Cloning (limiting dilution in microwell) Clonal expansion Monoclonals |
| Polyclonal Antibodies | Derived from injecting animals multiple times with antigen and later collecting serum. Run through affinity column with antigen beads to purify Ab. Occurs In vivo |
| Details about Polyclonal | In vivo Recognize multiple epitopes polyclonal antibodies are inexpensive large quantities can be produced Disadvantages: animal dies, supply ends variability in titers and epitope recognition between animals |
| Details about Monoclonal | in vitro immortal in nature recognize single epitopes more specific and selective Disadvantages - takes a long time and requires significant experience Expensive |
| Different ELISA formats | Sandwich - measurements involves the quantification of a substance by its ability to bind to both the capturing and detecting antibodies (linear graph with a slope of 1) Competitive - involves the quantification of a substance by its ability to displace a labeled version of the same analyte (sigmoidal graph) |
| Direct vs Indirect ELISA | Direct - simplest form; doesnt require anti-antibody Indirect - more complex & most common; requires anti-antibody |
| Parameters to optimize a sandwich ELISA | plate type coating method assay buffer choice of Ab pairs Enzyme label Capture Ab conc Detection Ab conc Labled secondary Ab conc # of washes |
| Methods of coating wells with capture antibody or antigen | Passive absorption (most common) Covalent linkage (amino/thiol group) Biological Linkers (biotin:strep; GST/His Tag; protein A coated plate |
| Protein Tags | His - Nickel GST - GSH Protein A - Fc region of antibody Biotin - streptavidin |
| Steps for indirect sandwich ELISA | 1) Coat plate with capture Ab 2) wash plate 3) add blocking solution 4) wash 5) add serum 6) wash 7) add detection antibody 8) wash 9) add enzyme labeled anti-antibody 10) wash 11) add substrate 12) detect |
| Choices of detection method for ELISA | Colorimetric, Fluorescent, and Chemiluminscent substrates |
| 2 Most common types of enzymes used for ELISA | HRP and AP |
| Mechanism of fluorescence | 1) ground state 2) absorbance 3) excitation 4) partial energy loss 5) complete energy loss 6) emission 7) return to ground state |
| Photobleaching | irreversible chemical breakdown of the fluorophore due to exposure to high intensity excitation light, leading to loss of fluorescence |
| Fluorescence detection | 1) excitation source 2) fluorophore 3) wavelength filters to separate Em photons from Ex photons 4) detector of Em photons (translates photons to electrical signal) |
| Monochromator - fluorescence | adv - tunable to any Ex/Em maxima (no need to buy filter for every new fluor); can scan for optimal Ex/Em disadv - lower sensitivity (Ex of fluor is weaker and only measuring one wavelength of light); slower throughput |
| Filter-based - flourscence | adv - high throughput; ultra sensitive (excites and detects greater # fo fluorophore molecules) disadv - have a different set of filters for every fluorophor |
| environmental effects on fluorescence | solvent polarity microenvironment chemical conjugation pH proximity and concentration of quenching species |
| Background signals that limit fluorescence sensitivity | Reagent background (unbound or nonspecifically bound probes) Endogenous sample constituents (autofluorescence) Solutions: Shifting Ex/Em filters or wavelengths to reduce autofluorescence --> reduces signal intensity; increase fluor concentration, thereby increasing signal relative to background; Time-resolved fluorimetry (TRF) |
| Importance of reading Absorbance assay at max wavelength | Signal to noise higher better sensitivity |
| Spectrophotometric assay | course of reaction is plotted as a measure of change in much light the assay solution absorbs. Either increase or decrease as substrate is converted to product |
| Flourescence | cyclic process of fluorescence means high sensitivity |
| Coupled enzyme assays | indirect assay to measure the product of one reaction using another enzyme whose activity is easier to follow -- needs excess secondary enzyme Disadv - more complicated than direct assay; more parameters to optimize; more complicated kinetics; more difficult to troubleshoot |
| Malachite green detection of phosphate | adv - high sensitivity & wide detection range simple homogenous robust and amendable to HTS |
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