immunochemical methods and funtions
DESCRIPTION
immunochemistryTRANSCRIPT
Immunochemical Methods in the Clinical Laboratory
Roger L. Bertholf, Ph.D., DABCCChief of Clinical Chemistry & Toxicology, UFHSC/Jacksonville
Associate Professor of Pathology, University of Florida College of Medicine
ASCP/Bertholf
Name The Antigen
Early theories of antibody formation
• Paul Ehrlich (1854-1915) proposed that antigen combined with pre-existing side-chains on cell surfaces.
• Ehrlich’s theory was the basis for the “genetic theory” of antibody specificity.
• Paul Ehrlich (1854-1915) proposed that antigen combined with pre-existing side-chains on cell surfaces.
• Ehrlich’s theory was the basis for the “genetic theory” of antibody specificity.
The “Template” theory of antibody formation
• Karl Landsteiner (1868-1943) was most famous for his discovery of the A/B/O blood groups and the Rh factor.
• Established that antigenic specificity was based on recognition of specific molecular structures; he called these “haptens”; formed the basis for the “template” theory of antibody formation.
• Karl Landsteiner (1868-1943) was most famous for his discovery of the A/B/O blood groups and the Rh factor.
• Established that antigenic specificity was based on recognition of specific molecular structures; he called these “haptens”; formed the basis for the “template” theory of antibody formation.
Aminobenzene Sulphonate, a Hapten
NH2 NH2 NH2
SO3
SO3
SO3
Ortho Meta Para
Classification of immunochemical methods
• Particle methods
– Precipitation
• Immunodiffusion
• Immunoelectrophoresis
– Light scattering• Nephelometry
• Turbidimetry
• Particle methods
– Precipitation
• Immunodiffusion
• Immunoelectrophoresis
– Light scattering• Nephelometry
• Turbidimetry
• Label methods
– Non-competitive
• One-site
• Two-site
– Competitive
• Heterogeneous
• Homogeneous
• Label methods
– Non-competitive
• One-site
• Two-site
– Competitive
• Heterogeneous
• Homogeneous
Properties of the antibody-antigen bond
• Non-covalent
• Reversible
• Intermolecular forces
– Coulombic interactions (hydrogen bonds)
– Hydrophobic interactions
– van der Waals (London) forces
• Clonal variation
• Non-covalent
• Reversible
• Intermolecular forces
– Coulombic interactions (hydrogen bonds)
– Hydrophobic interactions
– van der Waals (London) forces
• Clonal variation
Antibody affinity
AgAbAgAb
]][[
][
AgAb
AgAbKa
Precipitation of antibody/antigen complexes
• Detection of the antibody/antigen complex depends on precipitation
• No label is involved
• Many precipitation methods are qualitative, but there are quantitative applications, too
• Detection of the antibody/antigen complex depends on precipitation
• No label is involved
• Many precipitation methods are qualitative, but there are quantitative applications, too
Factors affecting solubility
• Size
• Charge
• Temperature
• Solvent ionic strength
• Size
• Charge
• Temperature
• Solvent ionic strength
Zone of equivalence
The precipitin reaction
Pre
cipi
tate
Antibody/Antigen
etc.
Single radial immunodiffusion
Ag
Single radial immunodiffusion
][Agr r
Double immunodiffusion
Örjan Ouchterlony
Developed double immunodiffusion technique in 1948
Double immunodiffusion (Ouchterlony)
Quantitative double immunodiffusion
S1
S2
S3 S4
S5
P
Electroimmunodiffusion
• Why would we want to combine immunodiffusion with electrophoresis?
– SPEED
– Specificity
• Carl-Bertil Laurell (Lund University, Sweden)
– Laurell Technique (coagulation factors)
– “Rocket electrophoresis”
• Why would we want to combine immunodiffusion with electrophoresis?
– SPEED
– Specificity
• Carl-Bertil Laurell (Lund University, Sweden)
– Laurell Technique (coagulation factors)
– “Rocket electrophoresis”
Electroimmunodiffusion
+
-
Immunoelectrophoresis
• Combines serum protein electrophoresis with immunometric detection
– Electrophoresis provides separation
– Immunoprecipitation provides detection
• Two related applications:
– Immunoelectrophoresis
– Immunofixation electrophoresis
• Combines serum protein electrophoresis with immunometric detection
– Electrophoresis provides separation
– Immunoprecipitation provides detection
• Two related applications:
– Immunoelectrophoresis
– Immunofixation electrophoresis
Immunoelectrophoresis
Specimen
-human serum
+
-
Immunoelectrophoresis
P C P C P C
+
-
Immunofixation electrophoresis
SPE IgG IgA IgM
Particle methods involving soluble complexes
• The key physical property is still size
• Measurement is based on how the large antibody/antigen complexes interact with light
• The fundamental principle upon which the measurement is made is light scattering
• Two analytical methods are based on light scattering: Nephelometry and Turbidimetry
• The key physical property is still size
• Measurement is based on how the large antibody/antigen complexes interact with light
• The fundamental principle upon which the measurement is made is light scattering
• Two analytical methods are based on light scattering: Nephelometry and Turbidimetry
Light reflection
- -+
Molecular size and scattering
Distribution of scattered radiation
Nephelometry vs. Turbidimetry
0°-90°
Inte
nsit
y of
sca
tter
ing
Time
Rate nephelometry
Rate
C2
C1
Additional considerations for quantitative competitive binding immunoassays
• Response curve
• Hook effect
• Response curve
• Hook effect
Competitive immunoassay response curve%
Bou
nd la
bel
Antigen concentration
%Bound vs. log concentration
Logistic equation
%B
ound
labe
l
Log antigen concentration
a
d
c
Slope = b
d
cx
a
day b
Logit transformation
%B
ound
labe
l
Log antigen concentration
a
d
y
yyY
1lnlogit
da
dyy
where
Logit plotL
ogit
y
Log antigen concentration
High dose “hook” effect%
Bou
nd a
ntig
en
Antigen concentration
Analytical methods using labeled antigens/antibodies
• What is the function of the label?
– To provide a means by which the free antigens, or antigen/antibody complexes can be detected
– The label does not necessarily distinguish between free and bound antigens
• What is the function of the label?
– To provide a means by which the free antigens, or antigen/antibody complexes can be detected
– The label does not necessarily distinguish between free and bound antigens
Analytical methods using labeled antigens/antibodies
• What are desirable properties of labels?
– Easily attached to antigen/antibody
– Easily measured, with high S/N
– Does not interfere with antibody/antigen reaction
– Inexpensive/economical/non-toxic
• What are desirable properties of labels?
– Easily attached to antigen/antibody
– Easily measured, with high S/N
– Does not interfere with antibody/antigen reaction
– Inexpensive/economical/non-toxic
The birth of immunoassay
• Rosalyn Yalow (1921-) and Solomon Berson described the first radioimmunoassay in 1957.
• Rosalyn Yalow (1921-) and Solomon Berson described the first radioimmunoassay in 1957.
Radioisotope labels
• Advantages
– Flexibility
– Sensitivity
– Size
• Advantages
– Flexibility
– Sensitivity
– Size
• Disadvantages
– Toxicity
– Shelf life
– Disposal costs
• Disadvantages
– Toxicity
– Shelf life
– Disposal costs
Enzyme labels
• Advantages
– Diversity
– Amplification
– Versatility
• Advantages
– Diversity
– Amplification
– Versatility
• Disadvantages
– Lability
– Size
– Heterogeneity
• Disadvantages
– Lability
– Size
– Heterogeneity
Fluorescent labels
• Advantages
– Size
– Specificity
– Sensitivity
• Advantages
– Size
– Specificity
– Sensitivity
• Disadvantages
– Hardware
– Limited selection
– Background
• Disadvantages
– Hardware
– Limited selection
– Background
Chemiluminescent labels
• Advantages
– Size
– Sensitivity
– S/N
• Advantages
– Size
– Sensitivity
– S/N
• Disadvantages
– Hardware
– ?
• Disadvantages
– Hardware
– ?
Chemiluminescent labels
+ 2H2O2 + OH -
COO -
COO -
O -
O -
+ h ( max = 4 3 0 nm )
+ N2 + 3H2O
NH2
L um i n o l
P e r o x i d a s e
O
O
N
NH
NH2
H
O
O*NH2
Chemiluminescent labels
CH3
N+
CO2H
O O
B r -
Ac r i d i n i um e s t e r
O -
CO2H
+ H2O2 + OH -+ + CO2 + h
O
CH3
N
Introduction to Heterogeneous Immunoassay
• What is the distinguishing feature of heterogeneous immunoassays?– They require separation of bound and free ligands
• Do heterogeneous methods have any advantage(s) over homogeneous methods?– Yes
• What are they?– Sensitivity– Specificity
• What is the distinguishing feature of heterogeneous immunoassays?– They require separation of bound and free ligands
• Do heterogeneous methods have any advantage(s) over homogeneous methods?– Yes
• What are they?– Sensitivity– Specificity
Heterogeneous immunoassays
• Competitive
– Antigen excess
– Usually involves labeled competing antigen
– RIA is the prototype
• Competitive
– Antigen excess
– Usually involves labeled competing antigen
– RIA is the prototype
• Non-competitive
– Antibody excess
– Usually involves secondary labeled antibody
– ELISA is the prototype
• Non-competitive
– Antibody excess
– Usually involves secondary labeled antibody
– ELISA is the prototype
Enzyme-linked immunosorbent assay
Microtiter well
E E E E E
Specimen 2nd antibodyE
Substrate
S P
ELISA (variation 1)
Microtiter well
Specimen Labeled antigenE
EEE
S P
ELISA (variation 2)
Microtiter well
Specimen Labeled antibodyE
E E E E
EEE
Automated heterogeneous immunoassays
• The ELISA can be automated
• The separation step is key in the design of automated heterogeneous immunoassays
• Approaches to automated separation
– immobilized antibodies
– capture/filtration
– magnetic separation
• The ELISA can be automated
• The separation step is key in the design of automated heterogeneous immunoassays
• Approaches to automated separation
– immobilized antibodies
– capture/filtration
– magnetic separation
Immobilized antibody methods
• Coated tube
• Coated bead
• Solid phase antibody methods
• Coated tube
• Coated bead
• Solid phase antibody methods
Coated tube methods
Specimen Labeled antigen
Wash
Coated bead methods
Microparticle enzyme immunoassay (MEIA)
Labeled antibodyE
E E
S P
Glass fiber matrix
Magnetic separation methods
Fe
Fe
FeFe
Fe
Fe
FeFe
Fe
Magnetic separation methods
Fe Fe FeFe Fe
Aspirate/Wash
Electrochemiluminescence immunoassay (Elecsys™ system)
Flow cell
Fe
Oxidized
Reduced
ASCEND (Biosite Triage™)
ASCEND
Wash
ASCEND
Developer
Solid phase light scattering immunoassay
Introduction to Homogeneous Immunoassay
• What is the distinguishing feature of homogeneous immunoassays?
– They do not require separation of bound and free ligands
• Do homogeneous methods have any advantage(s) over heterogeneous methods?
– Yes
• What are they?
– Speed
– Adaptability
• What is the distinguishing feature of homogeneous immunoassays?
– They do not require separation of bound and free ligands
• Do homogeneous methods have any advantage(s) over heterogeneous methods?
– Yes
• What are they?
– Speed
– Adaptability
Homogeneous immunoassays
• Virtually all homogeneous immunoassays are one-site
• Virtually all homogeneous immunoassays are competitive
• Virtually all homogeneous immunoassays are designed for small antigens
– Therapeutic/abused drugs
– Steroid/peptide hormones
• Virtually all homogeneous immunoassays are one-site
• Virtually all homogeneous immunoassays are competitive
• Virtually all homogeneous immunoassays are designed for small antigens
– Therapeutic/abused drugs
– Steroid/peptide hormones
Typical design of a homogeneous immunoassay
No signal
Signal
Enzyme-multiplied immunoassay technique (EMIT™)
• Developed by Syva Corporation (Palo Alto, CA) in 1970s--now owned by Behring Diagnostics
• Offered an alternative to RIA or HPLC for measuring therapeutic drugs
• Sparked the widespread use of TDM
• Adaptable to virtually any chemistry analyzer
• Has both quantitative (TDM) and qualitative (DAU) applications; forensic drug testing is the most common use of the EMIT methods
• Developed by Syva Corporation (Palo Alto, CA) in 1970s--now owned by Behring Diagnostics
• Offered an alternative to RIA or HPLC for measuring therapeutic drugs
• Sparked the widespread use of TDM
• Adaptable to virtually any chemistry analyzer
• Has both quantitative (TDM) and qualitative (DAU) applications; forensic drug testing is the most common use of the EMIT methods
EMIT™ method
Enzyme
S
S P
No signal
SignalEnzyme
S
EMIT™ signal/concentration curveS
igna
l (en
zym
e ac
tivi
ty)
Antigen concentration
Functional concentration range
Fluorescence polarization immunoassay (FPIA)
• Developed by Abbott Diagnostics, about the same time as the EMIT was developed by Syva
– Roche marketed FPIA methods for the Cobas FARA analyzer, but not have a significant impact on the market
• Like the EMIT, the first applications were for therapeutic drugs
• Currently the most widely used method for TDM
• Requires an Abbott instrument
• Developed by Abbott Diagnostics, about the same time as the EMIT was developed by Syva
– Roche marketed FPIA methods for the Cobas FARA analyzer, but not have a significant impact on the market
• Like the EMIT, the first applications were for therapeutic drugs
• Currently the most widely used method for TDM
• Requires an Abbott instrument
Molecular electronic energy transitions
E0
E4E3
E2
E1
Singlet
Triplet
A
VR
F
IC
P
10-6-10-9 sec
10-4-10 sec
Polarized radiation
z
y
x
Polarizingfilter
Fluorescence polarization
OHO OH
C
O
O
Fluoresceinin
Orientation of polarized radiation is maintained!
out (10-6-10-9 sec)
Fluorescence polarization
OHO
OH
C
O
O
Rotational frequency 1010 sec-1
in
Orientation of polarized radiation is NOT maintained!
out (10-6-10-9 sec)
But. . .
Fluorescence polarization immunoassay
OHO OH
C
O
O
Polarization maintainedSlow rotation
OHO OH
C
O
O
Rapid rotation
Polarization lost
FPIA signal/concentration curveS
igna
l (I
/I)
Antigen concentration
Functional concentration range
Cloned enzyme donor immunoassay (CEDIA™)
• Developed by Microgenics in 1980s (purchased by BMC, then divested by Roche)
• Both TDM and DAU applications are available
• Adaptable to any chemistry analyzer
• Currently trails EMIT and FPIA applications in market penetration
• Developed by Microgenics in 1980s (purchased by BMC, then divested by Roche)
• Both TDM and DAU applications are available
• Adaptable to any chemistry analyzer
• Currently trails EMIT and FPIA applications in market penetration
Cloned enzyme donor
Donor
Acceptor
Monomer(inactive)
Active tetramer
Spontaneous
Cloned enzyme donor immunoassay
Donor
Acceptor
Donor
Acceptor
No activity
Active enzyme
CEDIA™ signal/concentration curveS
igna
l (en
zym
e ac
tivi
ty)
Antigen concentration
Functional concentration range
Other approaches to homogeneous immunoassay
• Fluorescence methods
• Electrochemical methods
• Enzyme methods
• Enzyme channeling immunoassay
• Fluorescence methods
• Electrochemical methods
• Enzyme methods
• Enzyme channeling immunoassay
Substrate-labeled fluorescence immunoassay
Enzyme
S
S Fluorescence
No signal
SignalEnzyme
S
Fluorescence excitation transfer immunoassay
Signal
No signal
Electrochemical differential polarographic immunoassay
Oxidized
Reduced
Prosthetic group immunoassay
Enzyme
Enzyme
P
P
S P
Signal
No signal
Enzyme channeling immunoassay
Ag
E1
E2
Substrate
Product 1
Product 2
Artificial antibodies
• Immunoglobulins have a limited shelf life
– Always require refrigeration
– Denaturation affects affinity, avidity
• Can we create more stable “artificial” antibodies?
– Molecular recognition molecules
– Molecular imprinting
• Immunoglobulins have a limited shelf life
– Always require refrigeration
– Denaturation affects affinity, avidity
• Can we create more stable “artificial” antibodies?
– Molecular recognition molecules
– Molecular imprinting
History of molecular imprinting
• Linus Pauling (1901-1994) first suggested the possibility of artificial antibodies in 1940
• Imparted antigen specificity on native globulin by denaturation and incubation with antigen.
• Linus Pauling (1901-1994) first suggested the possibility of artificial antibodies in 1940
• Imparted antigen specificity on native globulin by denaturation and incubation with antigen.
Fundamentals of antigen/antibody interaction
O
O-
O
O-
NH 3
+CH2-CH2-CH2-CH3
OH
N
NH2
Cl
Molecular imprinting (Step 1)
N
NO N
NH
O
H3C
CH3
N
NO N
NH
O
H3C
CH3
Methacrylic acid+ Porogen
Molecular imprinting (Step 2)
N
NO N
NH
O
H3C
CH3
N
NO N
NH
O
H3C
CH3
Molecular imprinting (Step 3)
N
NO N
NH
O
H3C
CH3
N
NO N
NH
O
H3C
CH3
Cross-linking monomerInitiating reagent
Molecular imprinting (Step 4)
Comparison of MIPs and antibodies
• In vivo preparation
• Limited stability
• Variable specificity
• General applicability
• In vivo preparation
• Limited stability
• Variable specificity
• General applicability
• In vitro preparation
• Unlimited stability
• Predictable specificity
• Limited applicability
• In vitro preparation
• Unlimited stability
• Predictable specificity
• Limited applicability
Antibodies MIPs
Immunoassays using MIPs
• Therapeutic Drugs: Theophylline, Diazepam, Morphine, Propranolol, Yohimbine (2-adrenoceptor antagonist)
• Hormones: Cortisol, Corticosterone
• Neuropeptides: Leu5-enkephalin
• Other: Atrazine, Methyl--glucoside
• Therapeutic Drugs: Theophylline, Diazepam, Morphine, Propranolol, Yohimbine (2-adrenoceptor antagonist)
• Hormones: Cortisol, Corticosterone
• Neuropeptides: Leu5-enkephalin
• Other: Atrazine, Methyl--glucoside
Aptamers
1014-1015 random sequences Target
Oligonucleotide-Target complex
Unbound oligonucleotides
Aptamer candidates
PCR
New oligonucleotide library
+ Target