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How do enzymes function?
Lower the activation energy of a reaction
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Phases of Enzyme reaction with brief explanation. Which is clinically relevant?
- Lag Phase: secondary reactions may occur
- Linear Phase: [S] >> [E] (enzymes saturated)
- zero order (rate independent of [S])
- Measurements made in this area
- Depletion/Inhibition: [S] ~[E]
- first order (rate depends on [S])
- Product accumulates or substrate is no longer readily available
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What is Km?
- Km = [S] at 1/2Vmax
- Low Km = high affinity = less concentration will produce results
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Velocity of enzyme rxn equation
V = Vmax[S]/Km[S]
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What happens in competitive enzyme inhibition?
- Inhibitor binds at active site (competes)
- Can be overcome by increased [S]
- Vmax does not change, but Km is increased
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Isoenzymes vs Isoforms
- Isoenzymes: multiple forms of the same enzyme (same catalytic f(x)) derived from distinct genes
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What units are used for enzyme measurements?
- International Units
- 1 IU = 1umol/minute substrate consumed or product formed
- **Determining enzyme activity, but actually measuring product formation/substrate removal
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NAD(P)+ vs NAD(P)H absorbance
- 340nm
- NAD+ gives no abs
- NADH has peak abs
- Used frequently due to this distinct differentiation
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Enzyme measurement types - equilibrium vs kinetic (compare/contrast)
- Equlibrium (end-point): substrate is completely consumed
- May need to trap product to approach completion
- Less sensitive to variables (rate is irrelevant as long as we complete the rxn)
- Kinetic: measures at specific times, the more points the better
- Uncommon, but used for decreasing absorbance rxn (starting w/ NADH -> NAD+)
- Requires highly-controlled conditions (temp will alter the rate of rxn)
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Spectrophotometer instrument overview w/ brief explanation
- Source: Polychromatic light
- tungsten lamp (320nm+), deuterium (UV-Vis)
- Monochromator: Prism (old), Diffraction grating (common), filters
- Cuvets: plastic (not UV), glass (both), quartz (UV, rare)
- Photodetector: Photomultiplier tube, Photodiode, Diode Array (must common, best)
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Formula for % transmittance
- %T = Is/Io x 100
- Is = intensity of light transmitted through sample
- Io = intesntiy of incident light
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Beer's Law (equations)
- A = 2 - log(%T) = abc
- A = absorbance
- a = absorptivity (constant)
- b = path length (constant)
- c = concentration
- *NOTE - If A = 0, %T = 100; if A = 2 %T = 1 (logarithmic)
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List what must occur for Beer's law to be followed
- Incident radiation is monochromatic
- Solvent absorption insignificant
- Solute concentration is within certain limits
- No optical interferent is present
- No chemical reaction occurs within solute
- No stray light exists
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What is the optimal range for measurement's that use Beer's Law?
0.1-0.7 A (20-80 %T) has least amount of error
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Describe the reflectance method
- Dry reagant slide is hydrated by sample to initiate reaction
- Reaction occurs on a thin film matrix w/ highly relfective surface
- Monochromatic light is reflected to detector
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Atomic absorption instrument overview w/ brief explanation
- Hollow cathode: element-specific lamp
- Flame/graphite tube: heat generates ground-state atoms from rest of sample
- Monochromator: isolates flame light (stray) from HC (desired)
- Detector
- **Typically used for metals, one element at a time
- **NOTE - this works by detecting a decrease in light from the source, because it is absorbed by the atom in question
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Fluorescence theory, basics of instrumentation
- Absorbance of monochromatic light is followed by emission of a longer wavelength (lower energy)
- Two monochromators required (excitation, emission) which increases sens and spec
- Measured at a 90 degree angle to reduce incident light
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Turbidimetry/Nephelometry compare/contrast
- Both measure scattered light by large particles (eg immune complexes)
- Scattered light is the same wavelength as incident light
- Turbidimetry: measures decrease in transmitted light
- Same geometry as spectophotometer (can use typical analyzer)
- S:N ratio may be an issue (head-on geometry)
- Nephelometry: measure increase in off-axis light
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Potentiometry - theory, common tests, water analogy
- Measures electrical potential difference between two electrodes (half cells)
- unknown cell compared to reference cell (AgCl)
- Used for pH, pCO2, Ion-selective electrodes (Na+, K+, Cl-, Li+, Ca2+)
- Analogous to water pressure (PSI)
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How is pCO2 measured?
- Severinghaus electrode (potentiometry)
- pH electrode in HCO3- buffered solution separated from sample by CO2 permeable membrane
- CO2 crosses membrane, causes H+ release, lowers pH
- *NOTE - this test always contains an internal reference electrode
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Amperometry - theory, common tests, water analogy
- Measures current at a fixed applied potential
- Ox/Red potential applied to solid electrode produces current
- pO2 (Clark electrode)
- Analagous to instantaneous water flow (L/min)
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Coulometry - theory, common tests, water analogy
- Measures total charge between electrodes over time
- "titration where the titrant is electically generated" eg. Ag+ produced until all Cl- is bound in AgCl. Stops when free Ag+ is detected
- Chloride meter
- Analagous to cumulative flow (L)
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What are colligative properties? How can they me measured?
- Properties dependent upon number, not nature
- Freezing Point depression
- Vapor pressure depression
- Osmotic pressure (not used)
- Boiling point (not practical for clinical)
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Molarity vs Molality
- Molarity = mol/L
- Molality = mol/kg solvent (measured in lab)
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Freezing point depression theory
- One mol /kg lowers fp 1.86C
- Lower temp below 0 (supercool)
- Induce crystallization (vibration)
- Monitor temp as it slowly rises
- Temp plateus when sample begins to melt until completely thawed
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Vapor point depression theory/Osmalal gap
- Paper disc soaked w/ sample placed in closed chamber
- Temp measured when dew point is reached
- PROBLEM - assumes all vapor is water
- Osmolal gap: calculated osmolality as a surrogate test for ethanol, isoprop, etc
- gap = measured (freezing point) - calculated
- gap > 15 is considered significant
- VAPOR PRESSURE UNDERESTIMATES osmolality in the presence of alcohols and is not recommended
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Why is equivalence so important in an AgAb rxn?
- Ab excess: all antigenic sites are covered, lattice formation inhibited
- Equivalence: 2-3 Ab per antigen, producing a lattice (ppt)
- Ag excess: all antibody sites are saturated (2 ag per Ab), lattice formation inhibited
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What analytes are typically measured using immunochemistry?
Proteins, hormones, drugs
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Competitive immunoassay theory, adv/dis
- Patient sample (Ag) competes with labeled compound (Ag*)
- Decreasing signal corresponds with increasing analyte
- Adv: can measure large or small molecules
- Dis: low [Ab] required, less specific than sandwich, limited dynamic range
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Heterogenious vs Homogenous Immunoassays
- Hetero: free-labeled Ag must be removed
- RIA, ELISA, coated beads, etc
- Homo: Separation not required, binding of AgAb alters the label in some way
- EMIT, CEDIA, etc
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Homogeneous Immunoassays (EMIT and CEDIA) basic theory
- EMIT: hapten is bound to enzyme, Ab is added
- drug present = Ab binds to free drug and enzyme activity occurs
- drug absent = Ab binds to enzyme and sterically prevents activity
- CEDIA: enzyme in 2 recombinable fragments, one fragment has bound hapten, Ab added
- drug present = Ab binds to free drug, enzyme combines, activity occurs
- drug absent = Ab binds to one fragment, enzyme cannot recombine
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Fluorescence polarization theory and basic instrumentation
- Excitation light is polarized
- Free fluorescent ligands rotate quickly and depolarize light
- Bound fluorescent ligands rotate slowly and continue the polarized emission
- Emission polarizer only allows detection of polarized light
- 90 degree angle reading
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Sandwich immunometric assay theory, adv/dis
- Capture Ab bound to solid phase, labeled (free) tracer Ab recognized by system
- Must wash away unbound tracer Ab
- Adv: increased specificity, no limit to [Ab] (can drive rxn by increasing), greater dynamic range
- Dis: can only measure proteins (bivalent Ag), hook effect, interfering Ab, Monovalent epitope
- hook-effect = antigen excess causes underestimation of [ ]
- Interfering Abs (mouse or other animal) in sample may give false pos or false neg
- monovalent epitope - multiple subunits may have varying epitopes and this may alter results depending on which epitope is being monitored (eg. hCG from tumors vs preg)
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Chemiluminescence theory, adv
- Emission of light by chemical rxn (wavelength and # photons per label very specific)
- Adv: no light source OR monochromator required
- Dominant automated IA label
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Immunometric vs Competitive Later Flow Immunoassays
- Immunometric: color at test line = positive
- Test line = anti-analyte Ab
- Control line - anti-IgG Ab
- Au conjugate = anti-analyte
- Competitive: Color at test line = negative
- Positive sample binds probe Ab preventing color at test line
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General chromatography theory
- Separation using mobile and stationary phases
- Mobile phase can be altered during test
- More interaction with solid phase = slower elution
- More interaction with solid phase = faster elution
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Basic explanation of the following types of chromatography - partition, adsorption, ion-exchange, size exclusion, affinity
- Partition: stationary phase = nonpolar bonded "liquid", mobile phase = solvent or heated gas
- eg. gas chromatography or HPLC
- Adsorption: stationary phase = solid w/ non-specific adsorption (eg H-bonding), mobile phase = liquid or gas
- eg. TLC
- Ion-Exchange: stationary phase = cationic/anionic, mobile phase = altering pH or ionic strength
- eg. HPLC (abn hgb, HgbA1c)
- Size exclusion: stationary phase = polymer "beads" w/ controlled pore size, mobile = ? smaller particles enter and are significantly slowed
- not used in clinical lab (protein purification)
- Affinity: stationary phase = f(x) group that specifically attracks coumpound of interest, mobile = take everything except compound of interest (binary)
- eg. HbA1c, LSD purification
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Thin Layer Chromatography mobility calculation
- mobility defined by Rf
- Rf = spot migration / solvent migration
- Rf is characteristic, but not necessarily unique
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Gas chromatography instrumentation layout, mobility calculation
- Injection port (volitilizes sample), column oven (controls temp/affinity for mobile), column, detector (FID = organics, NPD = N & P, MS
- Separates samples based on volatility as temp increases
- mobility defined by retention time relative to internal standard (RRT)
- RRT = RT / RTstd
- RRT is characteristic, but not necessarily unique
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HPLC basics
- Smaller particles, higher pressure, higher performance
- Reversed phases: nonpolar stationary, polar mobile
- Gradient elution by increasing solvent strength (MeOH, MeCN)
- Detection by UV-Vis, Electrochem, MS
- *NOTE - besides MS most detectors are highly specific
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Automated Analytical Steps
- Sample ID
- Sample and reagent pipetting
- Reaction mixture mixing
- Incubation/Timing
- Signal Detection
- Reaction vessel washing
- Result computation
- Result entry
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Reagant-Blanked Endpoint vs Sample-Blanked Endpoint vs Blanked Rate rxn
- RB: Single blank for all samples
- does not correct for any sample abs (lipemia, ichterus, etc)
- Only a reading at the endpoint
- SB: Requires 2 reagents, rgt1 is identical to rgt2 except missing a crucial component
- rgt1 is added to allow "side rxns" to complete
- blank is taken at this time (with all components present)
- rgt2 is added and rxn occurs
- second abs is taken at the completion of this rxn and compared to the blank
- Rate: blank is same as SB, except several abs are taken as rxn progresses (linear phase)
- should be a line, and is ΔA/ΔT (slope is related to concentration)
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Linear assay calibration vs non-linear assay calibration w/ examples of each type of assay
- Linear: typically 2-point (zero and midpoint)
- eg. photometric, electrochemical
- Non-linear: multi-point calibration w/ curve-fitting algorithsms
- master curve shape determined by mfgr
- 2 calibrator points assign position of [ ] / response plot
- eg. immunoassays, turbidimetric
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Determine unknown concentration using linear calibration curve
- slope = (y2 - y1)/(x2 - x1)
- [ ] = (Abs - Abs0)/slope
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