IMMUNOASSAY

IMMUNOASSAY
A
A
brief
brief
guide
guide
through
through
its
its
history
history
,
,
principles
principles
,
,
practice
practice
and
and
future
future
trends
trends
Helena
Helena
Fingerov
Fingerov
Palack University Palack University Medical Medical School School, Olomouc, , Olomouc, Czech Czech Republic Republic
IMMUNOASSAYS
IMMUNOASSAYS
Highly specific in vitro tests that use antigen-antibody
reaction to detect extremely low concentrations
of a broad range of biologically important substances
in blood and other body fluids
Antigen-antibody reaction - known since the end of the
19
th
ct, precipitation in gel, agglutination or turbidimetry
assays gradually developed until
their potential has fully been appreciated since 1960
when higher sensitivity was achieved by
labeling one of the components
WHY?
WHY?
Because
Because
the
the
principle
principle
made
made
possible
possible
to
to
develop
develop

simple
simple

precise
precise

sensitive
sensitive ( (nano nano- - and and picomolar picomolar range range) )

high
high
throughput
throughput
measurement
measurement
of of more more substances substances than than any any other other analytical analytical technique technique
All
All
immunoassay
immunoassay
require
require
the
the
same
same
key
key
reagents
reagents
One
One
or
or
more
more
antibodies
antibodies
raised
raised
against
against
epitopes
epitopes
believed
believed
to
to
be
be
specific
specific
to
to
the
the
analyte
analyte
in
in
question
question
A
A
label
label
(
(
tracer
tracer
)
)
producing
producing
a
a
measurable
measurable
signal
signal
Calibrators
Calibrators
in a fluid (
in a fluid (
matrix
matrix
)
)
similar
similar
to
to
the
the
patient
patient
s
s
sample
sample
Antibody
Antibody
(
(
antiserum
antiserum
)
)
The The antibody antibody = = immunoglobulin immunoglobulin produced produced by by the the body in body in
response to response to an an invading invading ( (foreign foreign) substance as a part ) substance as a part
of of immune immune response response
Good Good antibodies antibodies possess possess high high specificity specificity and and affinity affinity for for
a a specific specific antigen antigen
The The antibody antibody used used in in immunoassay immunoassay is is usually usually of of the the IgG IgG
class class
Antibody
polyclonal monoclonal engineered
IgG
IgG
structure
structure
Natural
Natural
Antigen
Antigen
Substance Substance that that
naturally
naturally
elicit
elicit immune immune response response
Usually Usually a a larger larger molecule molecule ( (over over 10 10 kD kD) ) with with several several
epitopes
epitopes ( (antigenic antigenic determinants determinants) )
rhFSH
Conjugated
Conjugated
haptens
haptens
Smaller
Smaller
molecules
molecules
(
(
called
called
haptens
haptens
) are
) are
either
either
weakly
weakly
or
or
not not
not not
at
at
all
all
immunogenic
immunogenic
To
To
produce
produce
the
the
antibody
antibody
it
it
is
is
necessary
necessary
to
to
couple
couple
them
them
to
to
an
an
immunogenic
immunogenic
carrier
carrier
(e.g.
(e.g.
BSA,
BSA,
thyreoglobulin
thyreoglobulin
..)
..)
The The optimal optimal molar molar ratio ratio ( (excess excess may may range range from from 10 :1 10 :1
to 80 :1) to 80 :1) is is important important for for production production of of good good antisera antisera
Polyclonal
Polyclonal
antibodies
antibodies
raised raised in in animals animals ( (rabbits rabbits, , sheep sheep, , goat goat) by ) by repeated repeated
immunization immunization
a a mixture mixture of of antibodies antibodies which which may may bind bind to to different different
epitopes epitopes of of the the immunogen immunogen with with different different avidities avidities
Double
Double
antibody
antibody
Raised Raised in in another another species species to to the the primary primary antibody antibody, e.g. , e.g.
sheep sheep ( (goat goat, , donkey donkey) ) anti anti rabbit rabbit ( (mouse mouse, ) , ) IgG IgG
added added in much in much higher higher concentrations concentrations than than primary primary antibody antibody + +
normal normal rabbit rabbit ( (mouse mouse,..) ,..) gamma gamma globulin globulin
Production
Production
of
of
monoclonal
monoclonal
antibodies
antibodies
Injecting Injecting an an antigen antigen into into a host a host
animal animal ( (typically typically a a mouse mouse) )
Isolating Isolating antibody antibody- -producing producing
cells cells (B (B lymphocytes lymphocytes) )
Fusing Fusing immune immune cells cells to to mouse mouse
myeloma myeloma cells cells
Hybridomas Hybridomas are are grown grown in in culture culture
and and produce produce antibodies antibodies
Selecting Selecting hybridomas hybridomas that that
produce produce desired desired antibodies antibodies
Monoclonal
Monoclonal
antibodies
antibodies
derived
derived
from
from
a
a
single cell line,
single cell line,
monoclonals
monoclonals
are
are
specific
specific
for a
for a
single
single
epitope
epitope
on a
on a
multivalent
multivalent
antigen
antigen
hybridoma
hybridoma
cell
cell
lines
lines
can
can
produce
produce
the
the
same
same
antibody
antibody
consistently
consistently
and
and
indefinitely
indefinitely
,
,
monoclonal
monoclonal
antibodies
antibodies
facilitated
facilitated
:
:
manufacturing manufacturing of of immunodiagnostics immunodiagnostics
further further development development and and automation automation of of
immunoassays immunoassays
How
How
does
does
it
it
work
work
?
?
How
How
does
does
it
it
work
work
?
?
Photo: Z. Putz
In 1960 In 1960 the the first first RIA for RIA for insulin insulin ( (Berson Berson & & Yalow Yalow) )
used used
131 131
I I- -insulin as a insulin as a tracer tracer, ,
and and gel gel filtration filtration to to separate separate the the bound bound and and free free fraction fraction
Paper Paper was was originally originally rejected rejected by Science by Science and and J J Clin Clin Invest Invest, , but but later later accepted accepted. .
In In 1977 1977 R. R. Yalow Yalow, R. , R. Guillemin Guillemin and and A.V. A.V. Schally Schally shared shared Nobel Nobel prize prize for for the the
development development of of RIAs RIAs for peptide for peptide hormones hormones
Principle Principle: : competition competition of of unlabeled unlabeled analyte analyte in in sample sample with with
fixed fixed amount amount of of radio radio- -labeled labeled analyte analyte for for limited limited binding binding
sites sites on a on a specific specific Ab Ab
Probably Probably the the most most important important advance advance in in biological biological
measurement measurement in in the the second second half half of of the the 2O 2O
th th
century century
DISCOVERY OF RIA
DISCOVERY OF RIA
Competitive
Competitive
immunoassay
immunoassay
The The less less Ag Ag in in the the sample sample, ,
the the more more labeled labeled Ag Ag can can be be bound bound by by Ab Ab
a) Isotope label (RIA)
b) Enzyme label (EIA)
Calibration curve
Precision profile
Competitive
Competitive
assay
assay
for
for
antibody
antibody
testing
testing
Noncompetitive
Noncompetitive
immunoassay
immunoassay
a a new new assay assay format format in 1968 in 1968
Analyte Analyte in in the the sample sample is is bound bound to to excess excess of of capture capture antibody antibody
immobilized immobilized on solid on solid phase phase ( (testtubes testtubes, , microplates microplates, , etc etc.) .)
So So that that there there always always remain remain unoccupied unoccupied binding binding sites sites
Only Only the the occupied occupied binding binding sites sites can can be be detected detected by by labeled labeled
antibody antibody
The The amount amount of of Ag Ag in in the the sample sample is is directly directly related related to to the the signal signal, ,
e.g. e.g. the the amount amount of of bound bound labeled labeled Ab Ab
Noncompetitive
Noncompetitive
immunoassay
immunoassay
( (also also known known as as sandwich sandwich, , immunometric immunometric, , excess excess reagent reagent assays assays) )
Competitive format
Why
Why
noncompetitive
noncompetitive
immunoassays
immunoassays
are
are
better
better
than
than
competitive
competitive
assays
assays
?
?
higher higher sensitivity sensitivity and and
specificity specificity
universal universal labeling labeling
procedure procedure ( (IgGs IgGs) )
generally generally longer longer shelf shelf- -life life
of of labeled labeled antibodies antibodies
extended extended working working range range
Limitations
Limitations
of
of
noncompetitive
noncompetitive
immunoassays
immunoassays
not not applicable applicable to to small small molecules molecules
more more expensive expensive ( (higher higher consumption consumption of of antibodies antibodies, , isolation isolation
of of pure pure immunoglobulin immunoglobulin necessary necessary) )
hook hook effect effect ( (high high dose dose hook hook effect effect) in ) in some some assays assays
HAMA interference HAMA interference
Categories
Categories
(
(
formats
formats
)
)
of
of
immunoassay
immunoassay
Competitive
Competitive
immunoassays
immunoassays
(limited
(limited
reagent
reagent
assays
assays
)
)
Noncompetitive
Noncompetitive
or
or
immunometric
immunometric
assays
assays
(
(
excess
excess
reagent
reagent
assay
assay
,
,
sandwich
sandwich
assay
assay
)
)
Heterogeneous
Heterogeneous
Assays
Assays
Always
Always
require
require
separation
separation
of
of
the
the
label
label
bound
bound
in
in
the
the
immune
immune
complex
complex
and
and
the
the
free
free
label
label
Double Double antibody antibody + PEG + PEG

Solid
Solid
phase
phase
systems
systems
Coated Coated tubes tubes, , microplates microplates, , beads beads, , etc etc
Magnetic Magnetic particles particles
Solid
Solid
phase
phase
RIA
RIA
kit
kit
(DHEA
(DHEA
-
-
S)
S)
procedure procedure
standard standard curve curve
expected expected values values
in in age age and and sex sex groups groups
Labels
Labels
,
,
tracers
tracers
Radioactive
Radioactive
isotopes
isotopes

3 3
H
H
used
used
in
in
competitive
competitive
binding
binding
assays
assays
before
before
the
the
era
era
of
of
immunoassays
immunoassays

131 131
I
I
used
used
in
in
the
the
first
first
RIAs
RIAs
(t
(t
1/2 1/2
8
8
days
days
)
)

125 125
I
I
with
with
a
a
longer
longer
half
half
-
-
life
life
(60
(60
days
days
)
)
soon
soon
replaced
replaced
131 131
I isotope for use in
I isotope for use in
RIAs
RIAs
RADIOACTIVE LABELS (
RADIOACTIVE LABELS (
125 125
I,
I,
3 3
H)
H)
High
High
sensitivity
sensitivity
of
of
detection
detection
No
No
interferences
interferences
Small
Small
molecular
molecular
size
size
Simple
Simple
labeling
labeling
Low
Low
cost
cost
Enviromental
Enviromental
risk (
risk (
waste
waste
disposal
disposal
)
)
Dedicated
Dedicated
instrumentation
instrumentation
Separation
Separation
step
step
necessary
necessary
Short
Short
shelf
shelf
-
-
life
life
Difficult
Difficult
to automate
to automate

ALTERNATIVE LABELS
ALTERNATIVE LABELS
Enzymes
Enzymes ( (alkaline alkaline phosphatase phosphatase, , horse horse raddish raddish
peroxidase peroxidase and and others others) )
Fluorescent
Fluorescent
substances
substances (fluorescein, (fluorescein,
lanthanide lanthanide chelates chelates) )
Luminiscent
Luminiscent
substances
substances ( (substituted substituted
isoluminol isoluminol, , acridinium acridinium esters esters) )
Particles
Particles (latex (latex particles particles, , colloidal colloidal gold gold, , Eu Eu chelate chelate
nanoparticles nanoparticles) )
Detection
Detection
of
of
enzyme
enzyme
labels
labels
Low Low sensitivity: sensitivity: Absorbance Absorbance measurement measurement
chromogenic chromogenic substrates substrates
High High sensitivity sensitivity: : Light Light emission emission measurement measurement
chemiluminiscent chemiluminiscent substrates substrates ( (peroxidase peroxidase + + luminol luminol + + enhancer enhancer) )
or or alkaline alkaline phosphatase phosphatase + + adamantyl adamantyl- -1,2 1,2- -dioxetane dioxetane phenyl phenyl
phosphate phosphate) )
nonfluorescent nonfluorescent substrates substrates that that are are converted converted to to fluorescent fluorescent
products products (4 (4- -methylumbelliferyl methylumbelliferyl phosphate phosphate) )
Sensitivity gain
ELISA
ELISA
E
E
nzyme
nzyme
L
L
abeled
abeled
I
I
mmuno
mmuno
S
S
orbent
orbent
A
A
ssay
ssay
probably probably the the most most popular popular format format
microplate
individual strips or wells
in a frame
ELISA
ELISA
readers
readers
Fluorogenic
Fluorogenic
ELISA
ELISA
Test specific module
FLUORESCENT LABELS
FLUORESCENT LABELS
Fluorescein
Fluorescein and and
fluorescein
fluorescein
isothiocyanate
isothiocyanate
(FITC)
(FITC) used used for for labeling labeling antibodies antibodies in in
histochemistry
histochemistry
Background
Background
fluorescence
fluorescence is is a a problem problem in in biological biological
specimens specimens
Solution
Solution
:
:
time
time
-
-
resolved
resolved
fluoroimmunoassays
fluoroimmunoassays
using using long long- -lived lived fluorescence fluorescence of of lanthanide lanthanide chelates chelates ( (lifetime lifetime
in in micro microseconds seconds) ) label label signal signal is is measured measured after after background background
fluorescence has fluorescence has decayed decayed ( (lifetime lifetime in in nano nanoseconds seconds) )
DELFIA
DELFIA


D Dissociation issociation E Enhanced nhanced L Lanthanide anthanide F Fluoro luoroI Immuno mmunoA Assay ssay
LUMINISCENCE HISTORY
LUMINISCENCE HISTORY
1667 1667 bioluminiscence bioluminiscence was was recognized recognized
1887 1887 first first luminiscent luminiscent substances substances known known
1947 1947 first first application application of of firefly firefly luciferase luciferase
1967 1967 first first immunoassay immunoassay utilizing utilizing luminol luminol
1982 1982 acridinium acridinium ester ester used used for for the the first first time time as a as a
label label in a in a manual manual assay assay
MAGICLITE by Ciba Corning
In
In
1990,
1990,
Ciba
Ciba
Corning
Corning
launched
launched
the
the
world
world
s
s
first
first
fully
fully
automated
automated
immunoassay
immunoassay
system
system
with
with
Random
Random
Access,
Access,
ACS:180
ACS:180
ADVIA CENTAUR
ADVIA CENTAUR
output
output
240
240
tests
tests
/
/
hour
hour
MODERN PARTICLE LABELS
MODERN PARTICLE LABELS
Immunochromatographic
Immunochromatographic
tests
tests
,
,
membrane
membrane
tests
tests
,
,
lateral
lateral
flow
flow
or
or
one
one
step
step
tests
tests
(in dipstick or device format)
How does it work?
Ab coated particle
(coloured latex, colloidal
gold) binds Ag and is
captured by immobilised
Ab
Excess of labeled Ab
binds to the second Ab
(anti- mouse Ab)
Some
Some
alternative
alternative
labels
labels
made
made
it
it
possible
possible
to
to
develop
develop
Homogeneous
Homogeneous
immunoassays
immunoassays
Heterogeneous
Heterogeneous
assays
assays
(
(
require
require
separation
separation
of
of
bound
bound
Ab
Ab
-
-
Ag
Ag
complex
complex
)
)
Homogeneous
Homogeneous
assays
assays
(do not
(do not
require
require
this
this
separation
separation
, as
, as
signal
signal
is
is
changed
changed
when
when
the
the
label
label
is
is
bound
bound
in
in
the
the
Ab
Ab
-
-
Ag
Ag
complex
complex
Moreless
Moreless
outdated
outdated
HOMOGENEOUS ENZYME IMMUNOASSAYS
HOMOGENEOUS ENZYME IMMUNOASSAYS
EMIT
EMIT
(
(Enzyme Enzyme Modified Modified Immunoassay Immunoassay Technology), Technology), the the
active active site site of of the the enzyme enzyme label label is is blocked blocked when when bound bound
FPIA
FPIA (Fluorescence (Fluorescence Polarization Polarization ImmunoAssay ImmunoAssay) ) rotation rotation
of of fluorescent fluorescent label label is is slower slower when when bound bound
Competitive Competitive assays assays for for small small molecules molecules in in relatively relatively high high
concentration concentration TDM TDM
The The only only isotopic isotopic homogenous homogenous immunoassay immunoassay: :
SPA SPA ( (Scintillation Scintillation Proximity Proximity Assay Assay), ),
3 3
H H labelled labelled small small
hapten hapten gets gets into into the the proximity proximity of of a a scintilator scintilator encapsuled encapsuled
in in the the solid solid phase phase with with immobilized immobilized antibody antibody
Modern
Modern
HOMOGENEOUS ENZYME IMMUNOASSAY
HOMOGENEOUS ENZYME IMMUNOASSAY
automate
Cryptor
MESSAGE: IMMUNOASSAYS
MESSAGE: IMMUNOASSAYS
are are unique unique in in using using antibodies antibodies as as analytical analytical reagents reagents
are are indirect indirect analytical analytical tests tests
the the intensity intensity of of a a signal signal in a in a sample sample is is compared compared with with the the signal signal
generated generated by a by a simultaneously simultaneously measured measured calibrator calibrator
calibrators calibrators should should be be in a proper in a proper matrix matrix to to mimic mimic the the
sample sample
traceability traceability of of calibrator calibrator to reference to reference preparation preparation should should be be
documented documented
International International Reference Reference Preparations Preparations
reference reference methods methods do not do not exist exist in many in many cases cases
1
1
st st
generation
generation
immunoassays
immunoassays

competitive
competitive
immunoassay
immunoassay
RIA, EIA, FIA, LIA, RIA, EIA, FIA, LIA, often often in in- -house, house, manual manual double double antibody antibody and and
solid solid phase phase assays assays, , commercial commercial kits kits .) .)
2
2
nd nd
generation
generation
immunoassays
immunoassays

noncompetitive
noncompetitive
immunometric
immunometric
assays
assays
(IRMA, ELISA, (IRMA, ELISA, lateral lateral flow flow assays assays, , automated automated assays assays, ,
random random access access automates automates, , consolidation consolidation with with clinical clinical
chemistry chemistry .) .)
3
3
rd rd
generation
generation
immunoassays
immunoassays

microspot
microspot
analysis
analysis
,
,
biochip
biochip
arrays
arrays
,
,
multiple
multiple
parameter
parameter
testing
testing
3rd
3rd
generation
generation
immunoassays
immunoassays
multiple
multiple
parameter
parameter
testing
testing
nanotechnologies
nanotechnologies
Factors
Factors
impacting
impacting
analytical
analytical
performance
performance
Antibodies
Antibodies
specificity specificity, , avidity avidity, type , type
Labels
Labels
sensitivity sensitivity of of detection detection, , size size, stability, , stability, interferences interferences, ,
background background noise noise
Format
Format ( (competitive competitive or or noncompetitive noncompetitive) )
Separation
Separation
system
system(in (in heterogeneous heterogeneous formats formats) )
Automation
Automation
eliminating eliminating human human error error
MAIN INTERFERENCES IN IMMUNOASSAYS
MAIN INTERFERENCES IN IMMUNOASSAYS
Competitive
Competitive
assays
assays
false false positive positive results results due due to to cross cross- -reacting reacting molecules molecules
( (typical typical example example: E : E
2 2
or or testosterone testosterone direct direct assays assays) )
remedy remedy: : improving improving sensitivity sensitivity of of primary primary antibody antibody
Noncompetitive
Noncompetitive
assays
assays
HAMA, auto HAMA, auto- - and and heterophilic heterophilic antibodies antibodies, , rheumatoid rheumatoid
factors factors - - both both false false positive positive and and false false negative negative results results
( (typical typical examples examples: : hCG hCG, , myoglobin myoglobin) )
remedy remedy: : heterophil heterophil blocking blocking reagent reagent
Complexed Complexed (PSA), (PSA), dimers dimers ( (prolactin prolactin), ), fragments fragments ( (hCG hCG), ),
different different degree degree of of glycosylation glycosylation, , etc etc
Many
Many
analytes
analytes
are
are
heterogenic
heterogenic
It may result in discrepant findings
between assays from different
manufacturers
Sometimes ability to measure
different molecular forms can
increase diagnostic value of the test
(PSA/freePSA, hCG subunits or
hyperglycosylated hCG)
Parameters
Parameters
of
of
Assay
Assay
Quality
Quality
Analytical Analytical: :
Accuracy Accuracy: : ability ability to to measure measure the the correct correct concentration concentration
( (comparison comparison with with reference reference method method?) ?)
Precision Precision ( (reproducibility reproducibility): ): acceptable acceptable coeficient coeficient of of variance variance
Detection Detection limit limit (sensitivity): (sensitivity): lowest lowest concentration concentration different different from from
zero zero
Diagnostic Diagnostic: :
Sensitivity: Sensitivity: % % of of true true positive positive results results
If If high high, , then then a negative a negative result result practically practically exclude exclude the the diagnosis diagnosis
( (SnN SnNout out) )
specificity specificity: : % % of of true true negative negative results results
If If high high, , then then a positive a positive result result practically practically include include the the diagnosis diagnosis
( (SpP SpPin in) )
Immunoassays
Immunoassays
in
in
the
the
past
past
And
And
today
today
Communication Communication between between clinicians clinicians and and
laboratorians laboratorians is is crucial crucial for for the the benefit benefit of of patients patients! !