Week 9 Flashcards
What is a heterogeneous immunoassay
-competitive binding assay where you have to seperate the protein bound labelled AG from unbound
-both bound and unbound have the same signal
Criteria for Separation Techniques in a heterogeneous immunoassay
-clean and complete partition of bound and free
-reproducible, efficient and quick
-not influenced by anything else in the assay like plasma or proteins
-doesnt interfere with AG:AB binding
-minimizes non specific binding NSB
-automated
MUST CONSIDER - the environment must be optimized :
-time, temp,
-protein and lipid dependance (matrix)
-pH and ion dependance
-buffer type
-labelled AG trapped in precipitate
What is non specific binding
-when AG binds to substances other than the AB
-can cause loss or retention of unwanted labelled AG
-NSB in high amounts causes errors when measuring final concentration of AG
- can be minimized by blanking
how is NSB different from cross reactivity
how do you know when you have one instead of other
- in NSB - the AG binds so something that isnt the AB like test tub, plate or matrix contaminants. But in cross reactivity the AB binds to AG analogue or look alike IN ADDITION to the AG of interest thereby losing specificity
-results arent compatible with clinical or biochemical findings
-false positive = increased TROP with normal CKMB and no MI
False negative - digoxin with RI but presence of toxicity
what can cause NSB
-analyte variants with different epitopes
-small analytes with similar chemical structures
-Heterophilic (multi-specific, cross-linking) Abs, anti-animal Ab (HAMA = Human Anti-Mouse Antibody)
Heterophilic is an Ab response to an Ag other than
the specific one you want to measure for example
AB against GAS can react with heart tissue
-Labeled contaminants
-Adsorption of label to assay tube walls
What separation methods used in heterogenous immunoassays
-Precipitation (chemical/immunological) of the bound fraction
-Liquid-phase / Solid-phase adsorption
-Differential migration of bound and free
-Column separation: ion exchange, gel filtration
Miscellaneous: electrophoresis
“adsorption” = sticking on; attachment of a substance to the surface of another material
“absorption” = taking up
What is Liquid-phase Adsorption
-Adsorption occurs from surface atoms having free functional groups that attract molecules
Non specific
-when free unreacted AG is adsorbed on particles of activated charcoal or dextran coated charcol which can be removed by settling or centrifugation
Molecular charge - ion exchange resins helping with purification and decontamination
Advantages: cheap, fast, simple
Disadvantages: natural adsorbent binds everything, so time of contact is critical
What is solid phase adsorption -Immobilized detection Ab Dis/advantages
-Second Ab directed against the primary Ab is attached to a solid support:
wall of reaction tube
outer surface of insoluble materials
cellulose
magnetic latex beads
Centrifugation or magnetized separation
Advantages
speeds separation
highly specific
Disadvantages
expensive
What is chemical precipitation
Precipitating the AB:AG* from reaction by adding denaturing agent
-protein-precipitating agent can be organic solvent like polyethylene glycol, alcohols (methanol, ethanol), acetone, Salts , or organic acids - Picric, tannis or trichoroacetic
-denaturing agents can precipitate the bound complex while the free remains in the supernatant
What are the advantages and dis for chemical precipitation
- AD
Cheap, fast, simple, reproducible, flexible
DIS
-Can disrupt Ab:Ag binding: Ag may “fall off”
-Time and temperature dependent
-High NSB: due to a change in conformation that might alter the binding characteristics of the Ab
-Cannot be used for protein Ag (hormones): may precipitate the Ag (but can be used for drugs)
What is solid phase adsorption -Immobilized capture Ab
Dis/advantages
most popular
Immobilized capture Ab
-Ag* and Ag compete for binding sites on a capture Ab that has been attached to a solid support inner surface of plastic tubes
-wells of microtitre plates
-Washed and detected “in place”
Advantages
fast, no centrifugation
automated
high separating efficiency
Disadvantages
expensive: increased cost of reagent (magnetic particles, beads, fiber mesh).
What are the advantages and dis for immunological precipitation
-usually the method of choice
-Double ab - a second precipitating AB from DIFFERENT species (anti rabbit or goat)
AD
-Very specific (excellent partitioning of bound and free label)
-Involves an immune reaction so no precipitation of other assay components
-Low NSB
-Versatile
Disadvantages
Additional processing steps; longer assay time because of incubation/washes
What is differential migration
-using physical forces to separate
-not practical for routine and cant be easily automated
-free and bound complexes move at different rates and can be separated by different forces like:
Centrifugation: gravitational force
Electrophoresis: electrical force & net molecular charge
Ion exchange chromatography: molecular charge
Gel filtration: size exclusion chromatography; molecular size (bound flows through, free is trapped in pores of the gel)
What environmental factors must be in place for successful heterogenous immunoassay separation
pH
Ionic strength
Ionic species
Contaminants
pH
-must be optimized for both Ab and Ag
Ionic strength/molarity
-as low as possible to avoid “salting out” of protein
Ionic species
-salts inhibit binding of similarly-charged ligands (cationic salts and cations, anionic salts and anions)
Contaminants
-cross-reactants (drug metabolites, compounds with similar structures, e.g. “digoxin-like” factors)
matrix (positive or negative effect on results)
What environmental factors must be in place for successful heterogenous immunoassay separation
-Sensitivity, specificity of immunoassays
-Ab:Ag binding (may affect k1 or k-1 and alter the propensity to form a complex or to dissociate)
k = strength of binding
-Signal efficiency of label (especially if label is an enzyme)
-Test conditions must be optimized and carefully controlled
-Temperature affects efficiency of Ab:Ag binding
What is a RADIOIMMUNOASSAY (ria) and how does it work
-first immunoassay to be developed
-Ag or Ab labeled with radioactivity and used in competitive and non-competitive format
-Unlabeled Ag from patient sample competes with Ag* for Ab binding sites
-Separate bound from free Ag* and measure bound Ag* (counts per minute/cpm)
-Amount of radioactivity measured is indicative of the amount of analyte present
Radiolabelling of Ag may change reactivity; try labelling the antibody
Antibodies are more stable proteins and easier to label without damaging function
What is data reduction
-when numerical data is made simpler
-reduces the number of data records and used to produce data and statistics
- Once the bound fraction is separated from the free fraction, a dose-response curve is prepared. Y-axis is the dependent variable and the x-axis is the independent variable
RIA Measurement
What does the graph look like
on linear it is a curve opp to log curve
on semi log paper it would be linear
What is the signal? Counts per minute (cpm)
Concentration of what? Analyte, ligand, antigen
measure bound labelled Ag*, so radioactive signal is inversely proportional to [Ag]
What are ImmunoRadiometric assays (Irma)
Radioisotopically labeled Ab assays
-capture Ab is immobilized to a solid phase
-Ag from patient’s sample is bound by this Ab
-add a second radiolabeled detection Ab (Ab) that binds to a different epitope
-the Ag is sandwiched between the two Abs
-separate by washing away unreacted Ab
-signal directly related to [Ag]
-The detection Ab is labeled (Ab*)
-Non-competitive (reagent is in excess)
-Heterogeneous (separation required)
-Measure bound Ab*, so radioactive signal is directly proportional to [Ag]
Format? Non-competitive heterogeneous.
Why is signal directly related to patient [Ag]? The Ab captures everything: no competition. In order to do that, reagents must be in excess.
LINEAR
What’s the difference between RIA and IRMA? What is labeled?
RIA is competitive and the Ag is labeled. IRMA is noncompetitive and the Ab is labeled.
What are enzyme immunoassays
-enzyme labels used instead of radioactive labels
-easy, non hazardous like alkaline phosphatase or horseradish peroxidase
-enzyme catalyzes production of colored end product so it can be visualized and quantitated
-use ELISA to separate specific AG from non specific complexes
-Separation done through binding the Ag or capture Ab on a solid support (microtiter plate, latex bead, magnetic bead)
-Solid matrix allows for separation through repeated washings to minimize NSB
-quick detection of disease specific Ag or AB in pt serum
Competitive using labeled Ag
Competitive using labeled Ab
Non-competitive to detect Ag
Non-competitive to detect Ab
What is the ELISA competitive design and how it is similar to RIA
Ab is immobilized. The Ag is still labeled.
What is ELISA - DIRECT
-direct and most common ; heterogeneous
-used to detect AB in virues and parasites
-can quantitate Abs
-Capture Ab is immobilized to a solid phase
-Ag from the patient’s sample is bound by this Ab
-Add a second Enzyme-labeled detection Ab (Ab-E) that binds to a different epitope; the Ag is sandwiched between the two Abs
-Separate by washing away unreacted Ab-E
-Add enzyme substrate, which is catalytically converted to product
-Signal (absorbance) directly related to [Ag] results in chromophoric product
difference between IRMA is that Detection Ab is now labeled with an enzyme that needs substrate.
What is immunochromatography
-plastic cassette with membrane attached to a chamber where the sample is added. There is an absorbent material under the membrane that pulls the liquid reagents through along the membrane separating non reacted components from membrane AB-AG complex
-Capture Ab is immobilized onto a surface of a porous membrane (nitrocellulose, nylon, Teflon)
-Sample passes along the membrane
What is Enzyme multiplied immunoassay techniques (EMIT)
-pt AG competes with Ag-E for AB binding sites
-when AB binds Ag-E there is enzyme activity so bound and free AG-E produce different signals
-homogenous so no separation needed
-when enzyme substrate is added it is catalytically converted to product
-used to measure small molecular weight drugs, hormones or metabolites
-Absorbance signal is directly related to AG concentration
-the amount of drug present is proportional to the inhibition of substrate reaction
What is Microparticle Enzyme ImmunoAssay (meia)
-isolates AB/AG complexes on a solid phase surface of microparticles
-Automate measurement of large molecules such as markers associated with cardiac, fertility, cancer, metabolic, hepatitis and thyroid testing
-Non-competitive, heterogeneous
