Escolar Documentos
Profissional Documentos
Cultura Documentos
Laboratory
ASCP/Bertholf
NH2
NH2
NH2
SO3
SO3
SO3
Ortho
Meta
Para
Particle methods
Precipitation
Immunodiffusion
Immunoelectrophoresis
Light scattering
Nephelometry
Turbidimetry
Label methods
Non-competitive
One-site
Two-site
Competitive
Heterogeneous
Homogeneous
Non-covalent
Reversible
Intermolecular forces
Coulombic interactions (hydrogen bonds)
Hydrophobic interactions
van der Waals (London) forces
Clonal variation
Antibody affinity
Ab Ag Ab Ag
[ Ab Ag ]
Ka
[ Ab][ Ag ]
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
Size
Charge
Temperature
Solvent ionic strength
Precipitate
etc.
Zone of equivalence
Antibody/Antigen
Ag
r [ Ag ]
Double immunodiffusion
rjan Ouchterlony
Developed double immunodiffusion technique in 1948
S3
S4
P
S2
S5
S1
Electroimmunodiffusion
Electroimmunodiffusion
+
-
Immunoelectrophoresis
Immunoelectrophoresis
-human serum
Specimen
Immunoelectrophoresis
-
Immunofixation electrophoresis
SPE
IgG
IgA
IgM
Light reflection
0-90
Rate nephelometry
Intensity of scattering
Rate
C1
C2
Time
%Bound label
Antigen concentration
Logistic equation
a
%Bound label
y
c
ad
x
a
c
Slope = b
Logit transformation
a
Y logit y ln
1 y
%Bound label
y d
where y
a d
d
Log antigen concentration
Logit y
Logit plot
%Bound antigen
Antigen concentration
Radioisotope labels
Advantages
Flexibility
Sensitivity
Size
Disadvantages
Toxicity
Shelf life
Disposal costs
Enzyme labels
Advantages
Diversity
Amplification
Versatility
Disadvantages
Lability
Size
Heterogeneity
Fluorescent labels
Advantages
Size
Specificity
Sensitivity
Disadvantages
Hardware
Limited selection
Background
Chemiluminescent labels
Advantages
Size
Sensitivity
S/N
Disadvantages
Hardware
?
Chemiluminescent labels
NH 2
NH 2
N
N
O*
H
+
H
2 H 2 O 2 + OH -
OO-
Pe r ox i dase
+ N2 +
3 H 2O
L um i n o l
NH 2
COO +
COO -
( ma x = 4 3 0 nm )
Chemiluminescent labels
CH 3
Br -
N+
O-
CH 3
N
O
+ H 2 O 2 + OH -
CO 2 H
A c r i d i n i um e s t e r
+ CO 2 + h
CO 2 H
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
Heterogeneous immunoassays
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
Specimen
Substrate
2nd antibody
E
S
E
P
E
Microtiter well
ELISA (variation 1)
Specimen
Labeled antigen
E
S
E
P
E
Microtiter well
ELISA (variation 2)
Labeled antibody
E
Specimen
E
E
E
E
Microtiter well
Coated tube
Coated bead
Solid phase antibody methods
Labeled antigen
Wash
Labeled antibody
E
Fe
Fe
Fe
Fe
Fe
Fe
Fe
Fe
Fe
Aspirate/Wash
Fe
Fe
Fe
Fe
Fe
Electrochemiluminescence immunoassay
(Elecsys system)
Flow cell
Oxidized
Reduced
Fe
ASCEND
Wash
ASCEND
Developer
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
Homogeneous immunoassays
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
EMIT method
S
Enzyme
S
No signal
P
S
Enzyme
Signal
Functional concentration
range
Antigen concentration
Triplet
VR
E1
IC
10-6-10-9 sec
P
E0
10-4-10 sec
Polarized radiation
z
Polarizing
filter
Fluorescence polarization
HO
OH
O
C
in
Fluorescein
Fluorescence polarization
in
HO
OH
But. . .
Slow rotation
HO
OH
O
C
HO
OH
O
C
Polarization maintained
Rapid rotation
Polarization lost
Signal (I /I)
Functional concentration
range
Antigen concentration
Donor
Spontaneous
Acceptor
Monomer
(inactive)
Active tetramer
Donor
Acceptor
No activity
Donor
Acceptor
Active enzyme
Functional concentration
range
Antigen concentration
Fluorescence methods
Electrochemical methods
Enzyme methods
Enzyme channeling immunoassay
Substrate-labeled fluorescence
immunoassay
S
Enzyme
S
No signal
Fluorescence
S
Enzyme
Signal
No signal
Signal
Oxidized
Reduced
Enzyme
No signal
P
P
Enzyme
Signal
Substrate
E1
Product 1
E2
Ag
Product 2
Artificial antibodies
Imparted antigen
specificity on native
globulin by denaturation
and incubation with
antigen.
Fundamentals of antigen/antibody
interaction
Cl
O
NH
3 +
O-
OH
NH2
CH2-CH2-CH2-CH3
Methacrylic acid
+ Porogen
O
H3C
O
NH
N
N
CH3
O
H3C
O
NH
N
N
CH3
O
H3C
O
NH
N
N
CH3
O
H3C
O
NH
N
N
CH3
O
H3C
O
NH
N
N
CH3
O
H3C
O
NH
N
N
CH3
MIPs
In vivo preparation
In vitro preparation
Limited stability
Unlimited stability
Variable specificity
Predictable specificity
General applicability
Limited applicability
Aptamers
Target
Oligonucleotide-Target complex
Unbound oligonucleotides
+ Target
Aptamer candidates
PCR
New oligonucleotide library