immunoassays Flashcards
what are immunochemical assays and what are they used to measurement
involve the binding of an antibody (Ab) with an antigen (Ag) to detect an analyte used to measure analytes in low concentrations (< 1 µmol/L) that are difficult to measure by traditional methods examples: proteins hormones metabolites therapeutic drugs drugs of abuse nucleic acids
what is the molecule of interest in immunoassays
either antigens or antibodies
what is the functional domain
On antibodies the functional domain us the F( ab) portion
- this is the part that can bind to a site on the antigen
what is the most common immunochemical reagent
IgG is the most common immunochemical reagent
what is the region that binds a complementary Ab
an antigenic determinant or epitope
immunogen
a substance capable of inducing an immune response & the formation of an Ab
Affinity
the strength of a single antibody - antigen interaction (IgG)
affinity is measured using a Hapten ( Hp)
( low MW Ag with only one epitope )
Avidity
strength of all interactions combined (IgM)
cross reactivity
When an Ab reacts with an Ag that is structurally similar to the Ag that induced Ab production
- the greater the similarity the greater the Rxn
Labelled immunoassays
labeled product is detected
a label is attached to the Ag or Ab
label can be radioactive element, fluorescent dye, or enzyme
more sensitive technique
Unlabelled immunoassays
nothing is attached to the Ag or Ab ( no label used )
Turbidimetry or Nephelometry is used for direct measurement ( in solution)
A precipitation pattern can be looked at in agar gel
less anlytically sensitive technique
types of binding reagents in labelled immunoassays
if binding reagent is an antibody = immunoassay ( majority)***
if binding reagent is a receptor ( ex. estrogen receptor) = receptor assay
if binding reagent is a transport protein ( ex. TGB) = Competitive protein-binding assay
Enzyme labels
most common :
horseradish peroxidase ( HRP)
alkaline phosphatase (ALP)
glucose- 6- phosphate dehydrogenase
enzymes: biological catalysts that increase rate of conversion & arent consumed in reaction
enzymes can catalyze many substrates molecules
enzyme activity can be monitored by the amount of product formed or the effect of the product on a coupled reaction
fluorescent labels
compounds that absorb radiant energy of one wavelength & emit radiant energy of a longer wavelength
emitted light is detected at an angle of 90 degrees from the path of excitation light by a fluorometer or a modified spectrophotometer ( 2 monochromators )
florescein ( a fluorescent label) can be substituted for an enzyme label
measure fluorescence
Luminescent labels
Emit a photon of light as a result of electrical, biochemical, or chemical reactions
Luminol
- 1st chemiluminescent label used in immunoassays
- emits light under alkaline conditions
Acridinium Esters
- triple-ringed organic molecule
- linked by an ester bond to an organic chain
Radioactive labels
radioactive elements as the label
measures radioactivity
largely replaced by non-isotopic labels
competitive immunoassays
labelled Ag* competes with an unlabelled patient Ag for a limited number of binding sites ( equal competition )
labelled Ag concentration id constant & limited
as concentration of patient Ag increases, more binds to Ab resulting in less bind of labelled Ag*
Ag* is inversely proportional to patient analyte
2 types:
heterogenous - requires separation
homogenous - no separation
competitive labelled immunoassay steps
- test sample ( quality control, calibrator or patient ) is added to test tube
- labelled Ag & Ab reagents are added
- incubation
- separation of free labelled ( unbound Ag)
- bound labeled Ag is measured
non-competitive immunoassays
also known as immunometric assays
labelled reagent Ab is used to detect Ag
excess labelled Ab is required to ensure that the labeled Ab reagent does not limit the reaction
concentration of Ag is directly proportional to the bound labeled Ab
examples:
IRMA - immunoradiometric assay
IEMA- immunoezymatic assay
non-competitive one site assay
only one antigenic site involved ( epitope)
labelled Ab ( in excess) is added to the patient sample
resulting Ab - Ag complex is precipitated
the amount of label in the precipitate is measured
direct relationship ***
as patient Ag increases radioactivity in ppt increases or enzyme activity in ppt increases
non competitive two site assay to detect Ag. (Sandwich)
also called Ag capture assay
- immobilized Ab captures patient Ag
- washing removes unreacted molecules
- labeled Ab is added
- labeled Ab binds to captured Ag
- washing removes free labeled Ab
- signal from bound labeled AB is proportional to Ag captured
non competitive two site sandwich assay to detect Ab
- immobilized Ag captures patient Ab
- washing removes unreacted molecules
- labeled Ab is added
- labeled Ab binds to captured Ab
- washing removes free labeled Ab
- signal from bound labeled Ab is proportional to the Ab captured
Separation techniques
free labeled reactants must be distinguished from bound labeled reactant
Heterogeneous immunoassays:
- physical separation needed ( bound labeled Ag from free labeled Ag)
- methods; adsorption, precipitation, interaction with solid phase
- ex: RIA, EIA, FIA, ELISA
Homogenous immunoassays:
- no physical separation step needed
separation techniques - Adsorption
uses particles to trap small antigens ( labeled or not)
a mixture of charcoal & cross-linked dextran is mis commonly used
charcoal: porous , combines with small molecules to remove them fom solution
dextran: prevents nonspecific protein binding to charcoal
other adsorbents: silica, ion exchange resin, Sephadex
After adsorption, sample is centrifuged. Free labeled Ag is found in the precipitate
Adsorption
most common method in immunoassay analyzers:
- binding of captured Ab to paramagnetic particles ( PMP)
Addition of sample containing the analyte & other constituents to capture Ab containing paramagnetic particles
Application of a magnetic to adhere capture antibodies & analyte to the side of reaction chamber. unbound constituent is removed by aspiration. removal of magnet & addtion of signal antibody dacilitate measurement
Separation Techniques - Precipitation
non-immune precipitation - occurs when the environent is altered, effecting the solubility of protein - compounds used to precipitate protein: ammonium sulfate sodium sulfate Polyethylene glycol ( PEG) ethanol - after centrifugation , bound labeled Ag will be in precipitate, free labeled Ag will be in supernatant
immune precipitation
- Add a 2nd Ab that will bind with the original one ( primary Ab) -becomes insoluble
- also called double -Ab or second-Ab method
Separation techniques- solid phase
solid phase is an inert surface to which reagent Ag or Ab is attached
free labeled reagent is separated from bound labeled through washing
advantages: easier to perform & to automate,
require less manipulation & time
disadvantages: require a large amount of reagent Ag or Ab to coat the solid
phase surface
interferences with sandwich immunoassays
subject to false pos & false neg interferences
other Abs can bind to both capture & labeled Abs producing an analytical signal when the analyte isn’t even present
ex. human anti-mouse antibodies (HAMA), heterophile Abs
HAMA can form in individuals exposed to mouse Ags
heterophile Abs are formed from patients who have autoimmune disease or other disorders
Herterophile or HAMA interference in sandwich immunoassays
analytical signal in the presence of the analyte
the capture Ab attcehed tp a solid support , binds to the analyte at one epitope of the analyte
the labled Ab is added & binds to the analyte at a 2nd analyte epitope
false positive signal in the presence of an interfering antibody
the interfering antibody binds to both the capture & labl antibodies, forming a “sandwich “ in the absence of the analyte
the hook effect interference with sandwich immunoassay
increased concentrations of Ag in a sample reduce the analytical signal ( similar to postzone effect)
when the concentration of an analyte is within the dynamic range:
- there is excess capture Ab
- and excess labelled Ab
all analyte is bound & none is free
excess free unbound Ab is washed away
resulting signal is proportional to amount of captured labeled Ab
if analyte concentration is above the dynamic range pf the assay:
- all capture Ab is bound with the analyte
- excess antigen us free in solution & binds to excess lablled Ab
- thee bound labeled Abs are now occupied & cannot bind to the analyte -bound capture Ab
- free analyte -bound labeled Ab is washed away
- 4 units remain ( same as last ex,) despite having a much higher analyte concentration
how to reduce the hook effect
- some commercial assays are designed to be immune to the hook effect
- some will produce a warning flag to say the sample must be diluted to obtain accurate results
- some labs perform solutions automatically on certain assays when values are expected to be high ( ex. hCG in pregnant women )
- use of chimeric & humanized Abs minimizes or eliminates HAMA & heterophile Ab interferences (p.170)
- chimeric Abs
- engineered so antigen recognition site (Fab) originates from a mouse
- constant portion contains sequence from human species
examples of competitive heterogenous labeled immunoassays
RIA; radioimmunassay
- radioisotope label
EIA; enzyme immunoassay
- enzyme label
FIA; fluoresence immunoassay
- fluorochrome label
ELISA; enzyme-linked immunosorbent assay
- enzyme labeled
Radioimmunoassay (RIA)
uses radiolabeled Ag* ( tracer)
steps:
- a limited amount of Ab is added to labeled Ag* & patient’s serum (Ag)
-equal competition of Ab occurs
-increase patient Ag = decreased amount of labeled Ag* to attach to Ab
- separate bound label from free label Ag
- measure radioactivity of bound label (in precipitate)
increase patient Ag = lower reactivity in precipitate
Radioimmunoassay (RIA) disadvantages ( not typically used)
high cost of radioactive waste disposal
short reagent life ( radioactive decay)
instrument costs
labor intensive
licensing - radioactivity
health risk
enzyme immunoassay (EIA)
Ag is labeled with a stable enzyme
- horseradish peroxidase, ALP, G6PD
steps:
- enzyme labeled Ag & patient Ag are mixed with known Ab
- incubated
- both compete for Ab
- separate bound from free enzyme-labeled Ag
- measure
- photometer, fluorometer, luminometer
higher the patient Ag = lower the bound enzyme-labeled Ag
=lower the color ( absorbance)
Florescence immunoassay ( FIA)
label is a fluorescent compound
-fluorescein
disadvantage:
-background fluorescence can interfere ( generated by components in the assay system)
UV light absorbed - visible light emitted
measure fluorescence
higher the patient Ag = lower the fluorescence
Enzyme -linked immunosorbent Assay ( ELISA)
use an enzyme as a label
use a solid phase as the separation technique
four formats available:
- competitive assay using labeled Ag
- competitive assay using labeled Ab
- non-competitive assay to detect Ag
- non-competitive assay to detect Ab
we use this method
ELISA steps
steps:
- Ab is adsorbed onto solid support
- patient Ag & enzyme - labeled Ag added to Ab
- incubate
- wash; decant to remove free enzyme -labeled Ag
- Add substrate for enzyme
- measure product
higher patient Ag = lower bound enzyme-labeled Ag & lower the color ( absorbance )
Note** common source of error is insufficient washing. This leads to a falsely increased color ( more labelled Ag present) & falsely decreased patient results
ELISA disadvantages
usually use enzyme or fluorescence detection which is less sensitive than chemiluminescence
more labor intensive than modern clinical assays
( some labs do have automated plate washing & reading stations)
Examples of competitive homogenous labeled immunoassays
do not require separation of free & bound labeled reagent
used labeled Ag *
examples:
EMIT - enzyme multiplied immunoassay technique
FPIA- fluorescence polarization immunoassay
Enzyme- multiplied immunoassay technique ( EMIT)
-often used to measure drugs
-enzyme- labeled drug & patient serum ( free drug) are added to a limited amount of Ab
- bound enzyme - labeled drug is inactivated by the antibody ( no enzymatic activity ), so no separation step is required
- measure free enzyme-labeled drug **
- direct relationship between concentration of drug inpatient & activity of
free labeled drug
-as the concentration of unlabeled Ag increases ( from patient), less enzyme-labeled Ag can bind to the Ab
- more labeled Ag is free, & enzymatic activity is greater
EMIT steps
when enzyme- labeled Ag is bound t the Ab, the enzyme activity is inhibited
free patient Ag binds to the Ab & prevents Ab binding to the labeled antigen
the substrate indicates the amount of free labeled antigen
Fluorescence polarization immunoassay ( FPIA)
uses a fluorescent label
baed on fluorescent polarization - uses polarized light to excite the label
polarized light:
- created when light passes through special filters
- consists of parallel light waves oriented in one plane
when polarized light is used to excite the label, emitted light can be polarized or depolarized
in FPIA what does emission depend on
emission depends on rotation & size of the label
small molecules ( free florescent labeled Ag)
- rotate rapidly & randomly
- interrupt polarized light
large molecules ( bound fluorescent labeled Ag)
- rotate slowly
- emit polarized light parallel to excitation polarized light
FPIA steps
Steps:
labeled Ag & patient Ag compete for limited Ab sites
polarized light is used to excite labeled Ag
light emission from sample depends on the size of the labeled molecules
- when no unlabeled patient Ag is present, labeled Ag will occupy all Ab sites, creating large complexes that rotate slowly & emit a high level of polarized light
- as the unlabeled patient Ag increases, less labeled Ag can bind to the Ab sites. this leases more labeled Ag free to rotate rapidly & emit less polarized light
- inverse relationship between patient Ag & polarization emission **
Unlabelled immunoassays
in an immunoassay, an antibody molecule recognizes & binds to an antigen
Ag + Ab = Ag-Ab complex
when the Ag-Ab complex reaches a sufficient size, it becomes insoluble & precipitates
Standard precipitin Curve
the precipitation curve is a model that describes what occurs when antigens & antibodies are mixed in different ratios
there are 3 stages in the development of Ag-Ab complexes :
Antibody excess
- low Ag concentrations
- complexes are soluble
Zone of equivalence
- optimal proportions
- maximum amount of immunocomplexes precipitate out
Antigen excess
- high Ag concentration
- complexes are soluble
Ag-Ab Reactions
Ab excess
- all antigenic sites are covered with Ab
- lattice formation is inhibited
equivalence
- 2-3 Ab molecules for each Ag present
- maximum lattice formation
- maximum precipitation
Ag excess
- All Ab sites saturated with Ag
- No precipitation is formed
- postzone
Standard precipitin curve
for accurate results, dilutions of the sample should be made to move the result out of the prozone area & into the area of assay linearity
Measurement of immune complexes
In the zone of Ab excess:
- Patient (Ag) is directly proportional to the # of complexes formed
- soluble Ag-Ab complexes can be measured in solution **
- measured by nephelometry ( light scatter) or turbidimetry ( light transmitted)
In zone of equivalence ( optimal proportions )
- maximum lattice formation & precipitation occurs
- insoluble complexes can be measured by the formation of visible precipitin bands in agar gel ***
Immune precipitation methods
Methods in Gel:
Passive methods
- double diffusion ( ouchterlony)
- radial immunodiffusion ( RID)
Electrophoresis
- cross immunoelectrophoresis( CRIE)
- Counterimmunoelectrophoresis (CIE)
- immunoelectrophoresis ( IEP)
- immunofixation electrophoresis ( IFE)
- rocket technique
Methods in Solution:
- turbidimetry
- nephelometry
Immunoprecipitation in gel
- the movement of Ab & soluble Ag in agar to get a visible precipitate
- not commonly performed in the clinical lab today
- gel is made up of dilute agarose ( <1%) in an aqueous buffer
- gel provides a semi-solid medium through which the soluble Ag & Ab can pass
- precipitated complexes are easier to see in gel than in liquid suspension ( advantage )
Double Diffusion ( Ouchterlony Technique )
agarose is solidified on a solid surface
wells are cut into the agarose to which soluble Ag & Ab are applied
diffusion occurs & the pattern of the precipitation band can be interpreted to determine if the test specimens are:
- identical
- cross-reactive
- non-identical
this method can be used to detect Abs associated with autoimmune diseases
Double Diffusion ( Ouchterlony Technique ) Pattern interpretation
Pattern identity
- antibodies in the antiserum react with both the antigens
- a single continuous line of precipitation occurs
- the antibodies cannot distinguish between the 2 antigens i.e the two antigens
are immunological identical
Pattern non- identity
- if the antigens have no similarity, the precipitation lines will cross
Pattern of partial identity
- 2 samples have some epitopes that overlap but complete identity doesnt occur
- 2 lines partially join & form a “Spur”
- the spur is thought to result form the determinants present in one antigen but lacking in the other antigen ( antigens are partially similar)
Radial Immunodiffusion (RID)
single diffusion technique used to quantitate protein ( the Ag )
procedure:
- monospecific antiserum is added to liquefied agarose & cooled in a plate
- wells are cut into the gel
- QC, standards & patient samples ( containing Ag) are added to the wells
- Ag diffuses out of the wells & binds to the Ab forming a concentric precipitin ring
- diameter of the rings are measured & related to the concentration of the Ag in the well
- a standard curve is constructed to determine concentrations of QC & patients
Radial Diffusion Agar Plate
if ring is larger than the highest standard, the sample should be diluted & retested
Immunoelectrophoresis ( IEP)
Used to ID monoclonal proteins via electrophoresis & immunodiffusion
procedure:
- electrophoresis is used to separate proteins in patient sample
- Abs is placed in troughs running parallel to the separate protein
- Abs & proteins both diffuse - precipitate where they meet in optimal proportions ( precipitin arcs)
- the plate is stained ( amido black ), destained & dried
- interpretation is made by comparing the patient sample arcs with the arcs of the normal QC sample
Immunoelectrophoresis ( IEP) Antiseras
heavy & light chain type must be determined to evaluate monoclonal gammopathies
antisera used :
- antihuman whole serum ( mixture of Abs against major serum proteins )
- Antihuman IgG (y- chain specific)
- Antihuman IgM ( µ- chain specific )
- Antihuman IgA ( alpha- chain specific )
- antihuman lambda & kappa
Immunofixation electrophoresis ( IFE)
used to classify monoclonal proteins in serum, urine, or CSF
ex. identify the type of Ig being produced in Multiple Myeloma
procedure:
- patient sample is applied to all 6 lanes of an agarose gel ( 1 gel/patient)
- electrophoresis separates the proteins
- all lanes except the reference lane are flooded with specific Abs
- if an Ab recognizes a protein, precipitation will occur
- the agarose gel is stained & dried
- interpretation is based on the migration & appearance of the bands
monoclonal proteins appears as a discrete band ( both heavy & light chain occur at same position )
polyclonal proteins will appear as a diffuse band
rocket technique(Laurell Technique)
another immune precipitation method
also known as electroimmunoassay
•Procedure:
•Ab is mixed with agarose
•Ag (calibrators, patient serum) are placed in wells and undergo
electrophoresis.
•As Ag moves toward the anode, it reacts with the reagent Ab, forming a
rocket-shaped precipitin line.
• The height of the precipitin line is proportional to the Ag concentration.
•A calibration curve is obtained from the heights of the calibration rockets
•Heights of the patient samples are compared to the calibration curve.
**Only good for Ags migrating
to the anode
Crossed Immunoelectrophoresis (CRIE) (Two-Dimensional Electrophoresis)
•Variation of Immunoelectrophoresis (IEP)
•Ag mixture (patient) is separated by electrophoresis in 1 direction
•Then individual ags are pulled into the agar containing Abs by
electrophoresis in a 2nd direction.
Counterimmunoelectrophoresis (CIE)
- Qualitative test
- Detects bacterial antigens in:
- Blood
- Urine
- CSF
- Two parallel lines are cut into agarose gel.
- Ab is placed in one line; Ag placed in the other
- Voltage is applied that causes the Ag and Ab to move towards each other.
- A precipitin line forms where they meet.
Detection of Fluid Phase Ag-Ab Complexes
•Use an instrument to detect soluble Ag-Ab complexes in solution as they
interact with light.
•Complexes act as particles in suspension that can scatter light.
•The size of the particles determines the type of scatter.
Turbidimetry
- Measures light transmitted.
- Designed to measure the light passing through a solution.
- The photodetector is placed at an angle of 180o from the incident light
Nephelometry
•Measures light scattered.
•Measures light at an angle other than 180o from the incident light.
•Most measure forward light scattered at less than 90o because the
sensitivity is increased.
Notes Regarding Turbidimetry and Nephelometry
•All reagents/sera must be clear of particles that could scatter light.
•Can use endpoint or kinetic measurements.
•Endpoint:
•Measurement is taken at the start (background signal) and then at a set
time later (endpoint signal).
•Kinetic:
•Rate of complex formation is continuously monitored
•Peak rate is determined and related to concentration of Ag
•Concentrations are determined using a calibration curve.
