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BASIC PRINCIPLE

The binding of
antibodies (Ab) and
their complementary
Antigens (Ag) forms
the basis of all
immunochemical
techniques.

Kricka LJ, 2001

BASIC PRINCIPLE

The exquisite specificity and the high affinity of


antibodies for specific antigens, coupled with
the unique ability of antibodies to cross link
antigens, allow the identification and
quantitation of specific substances by a variety
of methods.

[pathmicro.med.sc.edu/mayer/ab-ag-rx.htm]

ANTIBODIES AND ANTIGENS

Antibodies (Ab) are immunoglobulins that can


bind specifically to a wide array of natural and
synthetic antigens.
Antigens (Ag) are materials capable of reacting
with an antibody, without necessarily being
capable of inducing antibody formation.

Kricka LJ, 2001

HETEROGENEITY

Immunization with a single antigenic


determinant produces a variety of Ab with
different antibody-combining sites and with a
range of Ab affinity.
This is termed the heterogeneity of the
immune response.

Feldkamp CS. 2003

SPECIFICITY

For every reagent Ab, the specificity of its


immunochemical reactivity is the single
most important factor in the success or
failure of any immunological technique used
in the clinical laboratory.
Specificity refers to the ability of the Ab to
restrict its reaction to a defined group of
molecules.

Feldkamp CS. 2003

CROSS REACTIVITY

Cross reactivity of Ag and Ab is a by product of


the heterogeneity of the immun response.
Thus cross reactivity may result from similar or
identical antigenic determinant in different Ag.
This reaction between Ab and the undesired
Ag is termed Cross reactivity.

Feldkamp CS. 2003

Rheumatic Fever
Complication arising from infection with
Streptococcus pyogenes
antibodies to bacterial proteins (antigen or
streptolysin) cross-react with myocardial
andmuscle proteins.

ANTIBODY AFFINITY

The strength of the binding of a single


antigenic determinant to an antibody is a
function of the closeness of fit and is
called antibody affinity.
Antibody affinity is an expression of the
attraction between molecules of Ab and
Ag.

Feldkamp CS. 2003

ANTIBODY AFFINITY

Reagent Ab generally fall into two categories,


those of high affinity and those of low affinity.
Polyclonal Ab in the reagent maybe a mixture
of both, but reagents in which high affinity are
predominant should be used.
This results in a strong union with the Ag that
is not readily reversible and that will not be
influenced greatly by alteration of the reaction
conditions.

Feldkamp CS. 2003

ANTIBODY AVIDITY

Avidity is a measure of stability of Ab-Ag


complex and is partially dependent on
the affinity of each Ab for its
complementary antigenic determinant.

Feldkamp CS. 2003

ANTIBODY AS REAGENTS

Immunochemical reactions form the basis of a


diverse range of sensitive and specific clinical
assays. In a typical immunochemical analysis,
an antibody is used as a reagent to detect an
antigen of interest.

[http://www.htk-hamburg.com]

ANTIGEN AS ANALYTES

Numerous naturally occurring molecules or Ag


that are proteins, glycoproteins, or lipoproteins
can be detected and measured easily in
biological fluids if specific reagent Ab are
available.
In addition, many small molecules such as
drugs and hormones can be measured.

Feldkamp CS. 2003

Classes of Immunoassay
LABELS

Signal Detection

Precipitation immunoassay

Not required

Naked eye, turbidity,


nephelometry

Particle immunoassay

Blood cells, artificial


particles

Naked eye, pattern analyzer,


spectrophotometry, particle
counting

Radioimmunoassay

Radioisotopes (125I, 3H)

Photon counting

Enzyme immunoassay

Enzymes

Spectrophotometry,
fluorometry, photon
counting

Fluorescent immunoassay

Fluorophores

Photon counting

Chemiluminescent
immunoassay

Chemiluminescent
compounds

Photon counting

(1)

Precipitin and Nephelometric


Immunoassay
Precipitin Reaction
Principles
The precipitate that forms when large complexes of antigen and
antibody combine to form an insoluble lattice.

Limitation

Low limit of sensitivity ( 0.1 0.5 mg/dl)


Condition of temperature, pH and ionic strength of the medium, and
the antibody characteristics of avidity and affinity were affect the
formation of the immune precipitate
(1,2)

Qualitative Precipitant Assay Methods


Single immunodiffusion (Williams, 1970)
Double immunodiffusion (Garvey, 1977)
Double immunodiffusion in two dimension (Williams, 1970)
Electroimmunodiffusion reaction (Ritzmann, 1975)
Immunoelectrophoresis (Rose, 1973)

Semiquantitative Precipitant Assay Methods


Single radial immunodiffusion (Mancini, 1965; Fahey,

1965)

Single dimension electroimmunodiffusion (Axelsen, 1975)


rocket electrophoresis
(1)

Immunonephelometry

Principles

Small aggregates that can scatter light quickly


form, when an antibody and an antigen combine
in solution, and giving a turbid appearance to
the solution

(1,2)

Particle Immunoassay
(Agglutination assay)
Clumping and sedimentation of an antigen after reaction
with antibody

Principles
Passive particles aglutination (indirect aglutination)
Active particles aglutination (direct aglutination)
Aglutination inhibition

Limitation
Only semiquantitative
(1,2)

Particle Immunoassay
(Agglutination assay)

Hemagglutination
Gelatin Particle Agglutination
Latex Agglutination
Latex Turbidimetric Assay
Particle-Counting Immunoassay

(1)

Aglutination Assay

(3)

Passive particles aglutination


(indirect aglutination)

(1)

hCG agglutination inhibition

(3)

hCG agglutination inhibition

Radioimmunoassay
Principles
Competitive binding reaction to antibodies
between labeled antigens and non labeled
antigens

(1)

Radioimmunoassay
Advantages
1.
2.
3.

Precision and high sensitivity


Ease of isotope conjugation
Stability against interference from the assay among
other

Disadvantages
1.
2.

Short shelf life of the reagents


the need against hazardous radioactivity
(1,4)

Radioimmunoassay

(3)

Sandwich RIA

(1)
cpm=counts per minute

Enzyme Immunoassay
Sensitive technique that uses an enzymeantibody-antigen combination absorbed onto
the sides of a test wall

(3)

Enzyme Immunoassay
Advantages
1.
2.
3.
4.

Sensitive assay can be developed by the amplification effect of


enzymes
Reagents are relatively cheap and can have a long shelf life
A wide variety of assay configurations can be developed
Equipment can be inexpensive and is widely available

Disadvantages
1.
2.
3.

Measurement of enzyme activity can be more complex than


measurement of the activity of some types of radioisotopes
Enzyme activity can be affected by plasma constituens
Not as sensitive as radioimmunoassay

(2)

Competitive assaay

Non Competitive assaay

Heterogeneous Enzyme
Immunoassay (need
separation of B/F fraction)

(1)

Heterogeneous Enzyme
Immunoassay
Colorimetric Enzyme Immunoassay

Enzyme reaction is performed by using chromogenic


substrates to develop a color by prime catalitic reaction
Fluorescent Enzyme Immunoassay
Identical to other EIAs except that they use fluorescent
substrate. This assay generate a signal intensity that is
at least one order of magnitude greater than colometric
EIAs
Chemiluminescent Enzyme Immunoassay
Use chemiluminescent substrate (HRP, AP, etc) that
react with various enzymes employed as labels.

(1)

Enzyme-Linked Immunosorbent Assay (ELISA) - Multi-Lingual Captions.mp4

Homogeneous Enzyme
Immunoassay

Do not require washing steps


Less sensitive than heterogeneous EIAs
Classified as competitive and non
competitive binding assay

(1,5)

Homogeneous Enzyme
Immunoassay
Enzyme-Multiplied Immunoassay Technique

(1)

Substrate-Labeled Fluorescent Immunoassay

(1)

Enzyme Inhibitory Homogeneous Immunoassay

(1)

Cloned Enzyme Donor Immunoassay

(1)

Fluorescent Immunoassay
Fluorescent compounds as immunochemical labels to
detect antigens in tissue section

Clasification

Heterogeneous Fluorescent Immunoassay


- Fluoroimmunometric
- Radial Partition Immunofluorometric Assay
- Time-Resolved Fluoroimmunoassay
Homogeneous Fluorescent Immunoassay
- Fluorescence Polarization Assay
- Fluorescence Excitation Transfer Immunoassay
- Fluorescent Protection Immunoassay

(1,5)

Fluorescent Immunoassay

(3)

Fluorescent Polarization Immunoassay

(1)

Chemiluminescent
Immunoassay

Use chemiluminescence molecules as


labels
Labels of chemiluminescent compounds
produce light electrochemically on the
surface of electrodes and can be applied
to homogeneous assay formats

(1)

Chemiluminescent Immunoassay

(3)

Rapid and Simple Test Devices for


Point of Care Testing

Immunofiltration Assay Devices


Immunochromatographic Devices

(1)

Rapid and Simple Test Devices for


Point of Care Testing

(1)

Rapid and Simple Test Devices for


Point of Care Testing

(1)

Rapid and Simple Test Devices for


Point of Care Testing

(1)

Reference
1.

2.
3.
4.
5.

Ashihara, Y.; Kasahara, Y.; Nakamura, RM; Immunoassay and


Immunochemistry; Clinical Diagnostic and Management by
Laboratory Methods; 20th ed; 821-849; WB. Saunders Company;
2001.
Carpenter, AB; Antibody-Based Methods; Clinical Laboratory
Immunology; 6-48; ASM Press; 2002.
Stepp, AC; Laboratory Procedures; 95-97; 238-293; The CU
Mosby Company; 1989.
Bassion, S; Immunological Reactions; Clinical Chemistry; 153164; WB Saunders Company; 1998.
Hurtubise, PE et.al; Immunochemical Techniques; Clinical
Chemistry; 165-206; WB Saunders Company; 1998.