Clinical Chemistry Immunoassays

CLINICAL CHEMISTRY
CHAPTER 6
IMMUNOASSAYS
1
• Introduction
– In the last chapter, we discussed a variety of analytical techniques
– In this chapter we’ll add some new techniques … They all involve
the use of antibody – antigen reactions
– Antibody – Antigen reactions have the advantage of being very specific
2
Key Terms
• Antibody • Tracer ( Tag )

• Antigen • Turbidimetry
• Affinity • Haptene
• Avidity • Crossreactivity
• Competitive Immunoassay • Polyclonal
• Heterogeneous Immunoassay • Monoclonal
• Homogeneous Immunoassay • Prozone
• IEP • Postzone
• IFE
• Nephelometry
• Non-competitive Immunoassay
• Postzone
• Prozone
3
• Objectives
– Discuss the basic principles of the following immunoassays
• Immunoelectrophoresis ( IEP )
• Immunofixation Electrophoresis ( IFE )
• Nephelometry and Turbidimetry
• Competitive Immunoassays ( RIA, EIA, FIA )
• Non-competitive Immunoassays
• Fluorescence Polarization
– Discuss different types of tags or labels used in immunoassays

– Classify homogenous, heterogeneous, competitive and
noncompetitive immunoassay techniques
– Discuss methods of separation of free and bound tagged reagents
4
• Immunoelectrophoresis ( IEP )
– Electrophoresis of antigens is followed by the addition of various

antibodies to a parallel trough along the separated proteins
– The antibodies diffuse through the agar and form lines of precipitation
with their respective antigens
– The visible precipitant arcs can be compared to known standards to

identify specific protein bands – Or to detect missing bands
5
Immunoelectrophoresis ( IEP )
Step 1.
Patient plasma is placed in a well and undergoes electrophoresis.
Precipitant lines against 3 proteins
= +
Step 2
Known anti-sera against one or more proteins is
placed in a parallel trough after electrophoresis and
diffuses through the agar.
Visible lines of precipitation form if antibody

antigen reaction occurs.
6
• IMMUNOFIXATION ELECTROPHORESIS ( IFE )
– Antibody is poured over a completed electrophoresis procedure

( performed on an agar surface ) to produce visible precipitation lines
7
• ROCKET ( LAURELL TECHNIQUE )
– Modification of IEP technique
– Antigen ( proteins ) undergo electrophoresis in a supporting agarose gel

with specific antibody previously mixed into the gel
– As antigen moves thru the gel , antigen-antibody complexes form creating

visible precipitation lines in the shape of long arches or “rockets”
– The length of these “rockets” is proportional to the concentration of

antigen
8
• Turbidimetry and Nephelometry
– Light is obstructed by insoluble complexes ( usually antibody – antigen )
– Light is obstructed by these insoluble complexes
– Turbidimetry measures transmitted light

• Photo-detector is placed at 180 degrees from the light source
– Nephelometry measures scattered light

• Photo-detector is placed at 90 degrees from the light source
9
• Labeled Immunoassays
– Antigen or antibody is labeled ( tagged ) with a substance that can be

detected later on and allows for the detection of an antibody – antigen
reaction
– Different binding agents are allowed to attach to substances we want to

measure.
– The type of binding agent defines what type of assay it is
• Antibody Immunoassay
• Transport Protein Competitive Assay
• Hormone receptor Receptor Assay
– Types of tags
• Radioactive isotopes
• Enzymes
• Fluorescent molecules
10
• Competitive Immunoassays
– Competition between tagged and un-tagged antigen for limited antibody
– Tagged antigen Reagent

– Untagged antigen Patient antigen we want to measure
– Specific antibody Reagent
– Let the competition begin !!!
• Mix the three components together

• Allow the antigens to compete for the limited antibody
• Antibody will bind with tagged or un-tagged antigen ( it doesn’t
care )
• Separation Step : Antibody-Antigen complexes are separated from
free antigen
• Tagged antibody-antigen complex is measured
11
– The tagged antigen and antibody from the reagent kit are constant.
– The only variable is the concentration of the patient antigen
( the thing we want to measure )
– A standard curve can be constructed with known antigen

concentrations giving the following general results
• High concentrations of patient antigen means that more of the

antibody-antigen complexes are untagged
• Low concentrations of patient antigen means that more of the

antibody-antigen complexes are tagged
• There is an inverse relationship between patient antigen concentration

and tag activity after the separation process
12
Competitive Labeled Immunoassays ( RIA, FIA, EIA )
A competition between tagged antigens ( reagent ) and

untagged antigens ( patient )for a limited amount of antibody ( reagent )
13
Calculation of RIA / FIA / EIA
The activity of the tag is measured twice :
Before separation step = Total tag activity

After separation step = Bound tag activity ( antibody – antigen complex )
Note that the separation process removes all unbound ( free ) tag from the testing
% B = B / T x 100
The ratio of the Bound activity to the Total activity ( B / T ) decreases as the
concentration of the patient’s ( untagged ) antigen increases.
Using Standard solutions of known antigen concentrations, the % B is plotted against

the concentrations of the Standards
14
Example of an Competitive Assay Standard Curve
B/T
0
Concentration
15
ELISA ( Enzyme Linked Immunosorbent Assay )
- Antibody is adsorbed onto a solid surface

- Tagged and untagged antigens compete for limited antibody
- Separation is achieved by pouring off excess unbound ( free ) antigen
- Enzymatic activity is inversely proportional to patient antigen concentration
16
EMIT ( Enzyme Multiplied Immunoassay Technique )
Enzyme tag
Homogenous technique - no separation step of antibody - bound

and free antigen
Steric hindrance : Antibody binding to the enzyme–tagged–antigen
inhibits enzymatic activity
Patient antigen concentration is inversely proportional to enzyme

activity
17
Immunometric Technique
- Immunometric techniques utilize a tagged antibody
- Patient antigen concentration is proportional to measured tag activity
18
Fluorescence Polarization Immunoassay
• Competitive Immunoassay
• Homogenous assay – No separation step required
• Fluorescent – tagged antigen ( reagent ) and untagged antigen ( patient )

compete for specific antibody in a curvet
• The curvet is exposed to polarized fluorescent light
• Large molecules ( tagged - antigen – antibody complexes ) emit polarized

light, where as smaller molecules ( free tagged antigens ) do not
• The amount of polarized light emitted is inversely proportional to the

concentration of patient ( untagged ) antigen
• Fluorescence Polarization is used by the ABBOTT TDX analyzer, commonly

used for Therapeutic Drug Monitoring ( TDM )
19
Example of Polarized and “Normal” Light
- Normal light has wavelengths that occur in all planes
- A polarizing filter blocks all planes except one, but the wavelength is unchanged
20
Tagged antigen
Fluorescence Polarization Untagged antigen
Antibod
y
( Low concentration of patient antigen )
Most of the limited antibody will bind with fluorescent

tagged antigen. Large bound molecules will increase
emission of polarized light. 21
Tagged antigen
Untagged antigen
Fluorescence Polarization Antibody
( High concentration of patient antigen )
Most of the limited antibody will bind with untagged patient antigen.
Free unbound fluorescent antigen will decrease emission of polarized
light. 22
TOP 10
23

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CLINICAL CHEMISTRY

– In the last chapter, we discussed a variety of analytical techniques

– Antibody – Antigen reactions have the advantage of being very specific

• Antibody • Tracer ( Tag )

– Discuss the basic principles of the following immunoassays

– Discuss different types of tags or labels used in immunoassays

– Electrophoresis of antigens is followed by the addition of various

– The visible precipitant arcs can be compared to known standards to

Precipitant lines against 3 proteins

Visible lines of precipitation form if antibody

– Antibody is poured over a completed electrophoresis procedure

– Modification of IEP technique

– Antigen ( proteins ) undergo electrophoresis in a supporting agarose gel

– As antigen moves thru the gel , antigen-antibody complexes form creating

– The length of these “rockets” is proportional to the concentration of

– Light is obstructed by insoluble complexes ( usually antibody – antigen )

– Light is obstructed by these insoluble complexes

– Turbidimetry measures transmitted light

– Nephelometry measures scattered light

– Antigen or antibody is labeled ( tagged ) with a substance that can be

– Different binding agents are allowed to attach to substances we want to

– Competition between tagged and un-tagged antigen for limited antibody

– Tagged antigen Reagent

– Let the competition begin !!!

• Mix the three components together

– A standard curve can be constructed with known antigen

• High concentrations of patient antigen means that more of the

• Low concentrations of patient antigen means that more of the

• There is an inverse relationship between patient antigen concentration

A competition between tagged antigens ( reagent ) and

The activity of the tag is measured twice :

Before separation step = Total tag activity

Using Standard solutions of known antigen concentrations, the % B is plotted against

- Antibody is adsorbed onto a solid surface

Homogenous technique - no separation step of antibody - bound

Patient antigen concentration is inversely proportional to enzyme

- Immunometric techniques utilize a tagged antibody

- Patient antigen concentration is proportional to measured tag activity

• Homogenous assay – No separation step required

• Fluorescent – tagged antigen ( reagent ) and untagged antigen ( patient )

• The curvet is exposed to polarized fluorescent light

• Large molecules ( tagged - antigen – antibody complexes ) emit polarized

• The amount of polarized light emitted is inversely proportional to the

• Fluorescence Polarization is used by the ABBOTT TDX analyzer, commonly

- Normal light has wavelengths that occur in all planes

Most of the limited antibody will bind with fluorescent

( High concentration of patient antigen )

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