Karteikarten
Assay Tech - Exam 1
Karten in diesem Stapel (62)
- 1ChemiluminescenceEmission of light w/o emission of heat as result of a chemical reaction
- 2AssayProcedure where the activity, property, or concentration of an analyte is measured
- 3AnalyteSubstance determined in analytical procedure
- 4Assay BufferSolution in which assay is performed in
- 5MultiplexDetection of more than one analyte in a single homogenous sample
- 6Pos ControlHighest value or confirmation that assay worked as intended
- 7Neg ControlLowest signal possible in assay
- 8Signal to Background RatioRatio of mean signals for positive and negative control
- 9Assay FormatMethod of performing the assay
- 10% InhibitionFraction of inhibition of the signal
Given by the equation ( 1 - ((exp - neg)/(pos - neg)) - 11Assay DesignChoosing an assay format and approach
- 12Assay DevelopmentOptimizing an assay
- 13Assay ValidationCharacterization of an assay
- 14AccuracyCloseness of value found to accepted reference value
- 15PrecisionDegree of scatter between series of measurements taken from multiple sampling of the same homogenous sample
- 16LODlowest concentration of an analyte in a sample that can be detected but not quantified
- 17LOQLowest concentration of an analyte in a sample that can be determined with acceptable precision and accuracy
- 18SpecificityMeasure of degree of interference from other substances
- 19LinearityAbility of method to elicit test results that are directly proportional to analyte concentration within a given range
- 20RangeInterval between upper and lower levels of analyte that have been demonstrated to be determined with precision, accuracy, and linearity
- 21RuggednessDegree of reproducibility of results obtained under varying conditions
- 22RobustnessCapacity of method to remain unaffected by small deliberate variations in method parameters
- 23Direct AssaySignal proportional to analyte concentration
- 24Competitive AssayInverse relationship between signal and concentration of analyte
- 25AbsorptionPhysical process of absorbing light
- 26AbsorbanceDefined by the mathematical equation: Log10(Io/I)
- 27TransmittanceOpposite of absorbance (I/Io) x 100
- 28Fluorescence QuenchingBimolecular process that reduces fluorescence quantum yield without changing emission spectrum
- 29Self quenchingQuenching of one fluorphore molecule by another of the same fluorphore
- 30QuencherNon-fluorescent molecule that accepts energy from fluorphore in close proximity but does not re-emit the energy as light
- 31Quantum YieldMeasures fluorphore
- 32Choices for detection methodsELISA
RIA
Time-Resolved Fluorescence
Electro-Chemiluminescent - 33Optimization Parameters for ELISARobustness
Drift/Edge Effect
Number of replicates - 34Assessing an assayNoise - from instrument
Background - from non-specific binding
Signal - from the specific and selective binding on the labeled antibody to the analyte - 35Parameters essential for immunoassay validationaccuracy
precision
lower limit of quantitation
upper limit of quantitation
linearity
reproducibility
specificity
stability - 36Advantages/Disadvantages of ELISAAdv: very sensitive, many configurations available, many antibodies and secondary reagents commercially available
Disadvantages: time consuming, labor intensive, relatively expensive - 37Epitopepart of antigen that antibody binds
- 38Two types of EpitopesConformational - non-continuous amino acids that bind to Ab
Linear - amino acid sequence that binds to Ab - 39Monoclonal AntibodiesImmunization (inject rodent w/ antigen)
Fusion (fuse antibodies w/ immortal B tumor)
Cloning (limiting dilution in microwell)
Clonal expansion
Monoclonals - 40Polyclonal AntibodiesDerived from injecting animals multiple times with antigen and later collecting serum. Run through affinity column with antigen beads to purify Ab. Occurs In vivo
- 41Details about PolyclonalIn 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 - 42Details about Monoclonalin vitro
immortal in nature
recognize single epitopes
more specific and selective
Disadvantages - takes a long time and requires significant experience
Expensive - 43Different ELISA formatsSandwich - 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) - 44Direct vs Indirect ELISADirect - simplest form; doesnt require anti-antibody
Indirect - more complex & most common; requires anti-antibody - 45Parameters to optimize a sandwich ELISAplate type
coating method
assay buffer
choice of Ab pairs
Enzyme label
Capture Ab conc
Detection Ab conc
Labled secondary Ab conc
# of washes - 46Methods of coating wells with capture antibody or antigenPassive absorption (most common)
Covalent linkage (amino/thiol group)
Biological Linkers (biotin:strep; GST/His Tag; protein A coated plate - 47Protein TagsHis - Nickel
GST - GSH
Protein A - Fc region of antibody
Biotin - streptavidin - 48Steps for indirect sandwich ELISA1) 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 - 49Choices of detection method for ELISAColorimetric, Fluorescent, and Chemiluminscent substrates
- 502 Most common types of enzymes used for ELISAHRP and AP
- 51Mechanism of fluorescence1) ground state
2) absorbance
3) excitation
4) partial energy loss
5) complete energy loss
6) emission
7) return to ground state - 52Photobleachingirreversible chemical breakdown of the fluorophore due to exposure to high intensity excitation light, leading to loss of fluorescence
- 53Fluorescence detection1) excitation source
2) fluorophore
3) wavelength filters to separate Em photons from Ex photons
4) detector of Em photons (translates photons to electrical signal) - 54Monochromator - fluorescenceadv - 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 - 55Filter-based - flourscenceadv - high throughput; ultra sensitive (excites and detects greater # fo fluorophore molecules)
disadv - have a different set of filters for every fluorophor - 56environmental effects on fluorescencesolvent polarity
microenvironment
chemical conjugation
pH
proximity and concentration of quenching species - 57Background signals that limit fluorescence sensitivityReagent 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) - 58Importance of reading Absorbance assay at max wavelengthSignal to noise higher
better sensitivity - 59Spectrophotometric assaycourse 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
- 60Flourescencecyclic process of fluorescence means high sensitivity
- 61Coupled enzyme assaysindirect 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 - 62Malachite green detection of phosphateadv - high sensitivity & wide detection range
simple
homogenous
robust and amendable to HTS