Immunoassays and Antibodies in Analytical Chemistry
IMMUNOASSAYS
Fundamental Questions for an Analytical Chemist
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IMMUNOASSAYS
Introduction
I.
Definition of an immunoassay:
•
An
immunoassay
is an analytical technique which uses naturally occurring
reagents known as antibodies for the selective determination of sample
components
•
Immunoassays are commonly used in a wide variety of areas, especially in
biochemistry and clinical chemistry
II.
Examples of the application of immunoassay include:
•
Drug testing
•
Hormone testing (insulin in diabetic patients)
•
Bacterial or viral testing (AIDS, hepatitis)
•
Environmental testing (herbicides, pesticides)
III.
Advantages of immunoassays are:
•
Inexpensive to perform
•
Highly selective
•
Low limits of detection
•
Can have high-throughput. Often done in batch mode
•
Applicable to the determination of a wide-range of compounds
IMMUNOASSAYS
Antibodies
I.
Definition of an antibody:
•
An
antibody (Ab),
or
immunoglobulin (Ig),
is a member of a family of
glycoproteins that make up part of the body’s immune system.
II.
Basic structure of an antibody:
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IMMUNOASSAYS
Antibodies
II.
Basic structure of an antibody:
•
More realistic graphical representations of an antibody or Ig
IMMUNOASSAYS
Introduction
IV.
Antibody – Antigen Interactions:
•
The body contains between 10
6
and 10
8
types of antibodies
•
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(
A
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)
•
The ability of an antibody to recognize and bind a given antigen depends on
the structure of its binding site
−
Determined by the amino acid sequence of the antibody near the N-terminal ends
of the heavy and light chains
IMMUNOASSAYS
Introduction
IV.
Antibody – Antigen Interactions:
•
The general reaction between a single binding site on the antibody (Ab) and
antigen (Ag) can be written as follows:
−
w
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•
The value of
K
a
is typically in the range of 10
6
to 10
10
M
-1
•
The binding is very selective and only occurs between
Ab
and
Ag
, or
between
Ab
and molecules similar to
Ag
in their three-dimensional structure.
A
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+
A
g
↔
A
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-
A
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K
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IMMUNOASSAYS
Introduction
V.
Antibody Usage:
•
The selectivity of Ab-Ag interaction makes antibodies useful as analytical
reagents for the determination of specific components in mixtures
•
Antibodies are useful as analytical reagents since they can be produced to a
wide variety of substances:
−
For large analytes (> 5,000 MW), antibodies can be produced by directly
injecting the compound into an animal
−
For small analytes (< 5,000 MW), antibodies can also be produced, but require
that the compound first be coupled to a larger molecule, such as a protein, prior
to injections
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IMMUNOASSAYS
Introduction
VI.
Antibody Production -
polyclonal antibodies
:
•
One common method for making antibodies to a substance (antigen) is to
inject the analyte or analyte-protein conjugate into an animal several times
over a period of a few weeks to a few months
IMMUNOASSAYS
Introduction
VI.
Antibody Production –
polyclonal antibodies
:
•
If the agent is a foreign to the animal, the animal will develop antibodies to
the agent and release these antibodies into its blood.
•
After a few months, blood is removed from the animal and the antibodies
produced are collected for use
•
Antibodies produced in this fashion are typically very heterogeneous
−
Recognize a number of different sites on the analyte
−
Binding with a range of affinities (
K
a
)
•
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−
Arise from several different lines of antibody-producing cells within the animal
IMMUNOASSAYS
Introduction
VII.
Antibody Production -
monoclonal
antibodies (mAb)
:
•
Monoclonal antibodies differ from
polyclonal antibodies in that they
are produced by a single cell line
within the body
•
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(
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)
IMMUNOASSAYS
Types of Immunoassays
I.
There are several different ways in which antibodies can be used in the
detection or analysis of an antigen. Some common ways include:
•
Precipitation-based immunoassay
•
Competitive binding immunoassay
•
Sandwich immunoassay
II.
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III.
The analyte reacting with the antibody is then detected either directly or through
the use of various chemical labels which produce easy to measure signals
antigen
signal
mAb
IMMUNOASSAYS
Precipitation assays
I.
Use the antibody as a selective precipitation reagent for the determination of
analyte in the sample
•
Involves the use of two or more types of antibodies that bind o different sites
on the same analyte (
i.e.
, polyclonal antibodies)
•
Since each antibody has two binding sites per molecule, this can result in
precipitates being formed between
Ab
and
Ag
•
Maximum precipitation occurs at some optimal
Ab
/
Ag
ratio
Soluble
Complexes
Insoluble
Complexes
Soluble
Complexes
IMMUNOASSAYS
Precipitation assays
I.
To quantitate analyte by this technique, typically take multiple aliquots of sample
and add various amounts of antibody to each sample (
i.e.
, titration)
II.
The amount of precipitate formed for each aliquots is then determined visually,
gravimetry, light scattering measurement,
etc.
IMMUNOASSAYS
Precipitation assays
III.
Technique can be performed in gels by having antibody and analyte diffuse
towards each other from different sections of the gel
•
A concentration gradient of
Ab
and
Ag
is formed in the gel
•
Maximum precipitation will occur at the location where the antibody and
analyte are both present in the correct ratio
IMMUNOASSAYS
Precipitation assays
IV.
Precipitation in gels can be used either quantitatively or quantitatively to analyze
the an analyte in the sample
•
Ouchterlony assay:
−
Qualitative method: formation of precipitate between sample and antibody wells
indicates the sample contains analyte to which antibody binds
Skamel
et al.
(2014):
PLOS ONE
. 10.1371/journal.pone.0113069.g009.
IMMUNOASSAYS
Precipitation assays
IV.
Precipitation in gels can be used either quantitatively or quantitatively to analyze
the an analyte in the sample
•
Radial Immunodiffusion assay:
−
Quantitative method: area of ring within precipitation band is proportional to
concentration of analyte in sample
IMMUNOASSAYS
Precipitation assays
V.
Advantages of precipitation methods
•
Inexpensive-only reagent usually required is antibody
•
Selective-few interferences from other compounds in sample
•
Easy to perform
VI.
Disadvantages of precipitation methods
•
Only useful for fairly high concentration analytes (10-200 mg/L)
•
Long incubation times (hours-days)
•
Can require large amounts of antibody
IMMUNOASSAYS
Competitive binding immunoassays
I.
Q
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d
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g
s
i
t
e
s
(
e
q
u
i
l
i
b
r
i
u
m
m
e
t
h
o
d
)
•
Indirectly measures the amount of analyte in the sample by looking at amount
of labeled analyte it displaces from the antibody
IMMUNOASSAYS
Competitive binding immunoassays
I.
Q
u
a
n
t
i
t
a
t
i
v
e
m
e
t
h
o
d
b
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d
i
n
g
s
i
t
e
s
(
e
q
u
i
l
i
b
r
i
u
m
m
e
t
h
o
d
)
•
A typical calibration curve for the assay
Linear
transform
Ln(antigen concentration)
IMMUNOASSAYS
Competitive binding immunoassays
II.
Advantages of competitive binding immunoassay
•
Can be used with any type of analyte
•
Good limit of detection
−
Theoretical limit: 1/
K
a
or 10
-6
to 10
-10
M
•
Few interference from other compounds in sample
III.
Disadvantages of competitive binding immunoassay
•
Some skill required to obtain optimum conditions for assay
•
Long incubation times (hours-days)
•
Limit of detection ultimately controlled by quality of antibody
−
Antibody binding strength (
K
a
)
−
Detection limit varies between different antibody preparations
•
Usually manual method
IMMUNOASSAYS
Sandwich immunoassays
I.
Quantitative method based on use of two antibodies to detect analyte
•
First antibody extracts analyte from sample
•
Second antibody (containing chemical label) identifies presence of analyte
•
This type of assay measures the amount of analyte in the sample by looking
at the amount of labeled antibody that binds to analyte on the solid support
IMMUNOASSAYS
Sandwich immunoassays
I.
Quantitative method based on use of two antibodies to detect analyte
•
A typical calibration curve for the assay
Concentration of Analyte
Response
IMMUNOASSAYS
Sandwich immunoassays
II.
Advantages of sandwich immunoassay
•
Linear calibration curve
•
Lower limits of detection possible than with competitive binding immunoassay
−
< 10
-12
M
•
Greater selectivity than competitive binding assay
−
Two antibodies instead of one are used to recognize analyte
•
Shorter incubation times than competitive binding assay (hours vs. days)
•
Less susceptible to variations in quality of antibody preparation then
competitive binding assay
III.
Disadvantages of competitive binding immunoassay
•
Only useful for large analytes
−
1000 to 2000 MW
−
Requires enough room on molecule to bind two antibodies simultaneously
•
Requires multiple antibodies per analyte
•
Usually manual method
IMMUNOASSAYS
Labels for Immunoassays
I.
The selectivity of a competitive binding assay depends on the
specificity of the antibody
•
The use of a chemical label is also required
•
Several types of chemical labels have been used in immunoassays
IMMUNOASSAYS
Learning Objectives:
1.
The student should be familiar with the general definitions and advantages of
“immunoassays” and some examples of the application of this field.
2.
The student should be familiar with important features, structure and the production of
antibodies and the intrinsic value to immunoassays.
3.
The student should be familiar with the differences between monoclonal and polyclonal
antibodies
4.
The student should be familiar with the details of the antibody-antigen binding interaction
5.
The student should be familiar with the different types of immunoassays, be able to describe
how the assays function, and understand their advantages and disadvantages:
Precipitation-based immunoassay Competitive binding immunoassay Sandwich immunoassay
Ouchterlony assay Radial Immunodiffusion assay
4.
The student should be familiar with the different labels for immunoassays, including how
the label is measured and the limit of detection:
Radiolabels
Fluorescent
Enzymatic Chemiluminescent
Immunoassays are analytical techniques utilizing antibodies to selectively determine components in samples. Antibodies, crucial in the immune system, bind antigens through specific interactions. The binding affinity is high, making immunoassays valuable for diverse applications like drug testing and environmental analysis.
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IMMUNOASSAYS Fundamental Questions for an Analytical Chemist How do you rapidly, cheaply and easily detect a single analyte present in a complex heterogeneous mixture (e.g., blood, soil, etc.) ? Use a naturally occurring or synthetic analog of a molecule (antibody, aptamer, etc.) that has a high affinity to a specific ligand (or analyte)
IMMUNOASSAYS Introduction I. Definition of an immunoassay: An immunoassay is an analytical technique which uses naturally occurring reagents known as antibodies for the selective determination of sample components Immunoassays are commonly used in a wide variety of areas, especially in biochemistry and clinical chemistry Examples of the application of immunoassay include: Drug testing Hormone testing (insulin in diabetic patients) Bacterial or viral testing (AIDS, hepatitis) Environmental testing (herbicides, pesticides) Advantages of immunoassays are: Inexpensive to perform Highly selective Low limits of detection Can have high-throughput. Often done in batch mode Applicable to the determination of a wide-range of compounds II. III.
IMMUNOASSAYS Antibodies I. Definition of an antibody: An antibody (Ab), or immunoglobulin (Ig), is a member of a family of glycoproteins that make up part of the body s immune system. Basic structure of an antibody: II. The above antibody consists of four polypeptides-two identical heavy chains (H) and two identical light chains (L) connected by disulfide bonds. These are arranged in a Y -shaped structure ending with two identical sites that recognize and bind a given foreign agent or antigen
IMMUNOASSAYS Antibodies II. Basic structure of an antibody: More realistic graphical representations of an antibody or Ig
IMMUNOASSAYS Introduction IV. Antibody Antigen Interactions: The body contains between 106 and 108 types of antibodies Each antibody has the ability to bind to a different foreign agent, or antigen (Ag) The ability of an antibody to recognize and bind a given antigen depends on the structure of its binding site Determined by the amino acid sequence of the antibody near the N-terminal ends of the heavy and light chains
IMMUNOASSAYS Introduction IV. Antibody Antigen Interactions: The general reaction between a single binding site on the antibody (Ab) and antigen (Ag) can be written as follows: Ka Ab + Ag Ab-Ag where Ka is the binding or association equilibrium constant The value of Ka is typically in the range of 106 to 1010 M-1 The binding is very selective and only occurs between Ab and Ag, or between Ab and molecules similar to Ag in their three-dimensional structure.
IMMUNOASSAYS Introduction V. Antibody Usage: The selectivity of Ab-Ag interaction makes antibodies useful as analytical reagents for the determination of specific components in mixtures Antibodies are useful as analytical reagents since they can be produced to a wide variety of substances: For large analytes (> 5,000 MW), antibodies can be produced by directly injecting the compound into an animal For small analytes (< 5,000 MW), antibodies can also be produced, but require that the compound first be coupled to a larger molecule, such as a protein, prior to injections Five classes of antibodies
IMMUNOASSAYS Introduction VI. Antibody Production - polyclonal antibodies : One common method for making antibodies to a substance (antigen) is to inject the analyte or analyte-protein conjugate into an animal several times over a period of a few weeks to a few months
IMMUNOASSAYS Introduction VI. Antibody Production polyclonal antibodies: If the agent is a foreign to the animal, the animal will develop antibodies to the agent and release these antibodies into its blood. After a few months, blood is removed from the animal and the antibodies produced are collected for use Antibodies produced in this fashion are typically very heterogeneous Recognize a number of different sites on the analyte Binding with a range of affinities (Ka) Heterogeneous antibodies are known as polyclonal antibodies Arise from several different lines of antibody-producing cells within the animal
IMMUNOASSAYS Introduction VII. Antibody Production - monoclonal antibodies (mAb): Monoclonal antibodies differ from polyclonal antibodies in that they are produced by a single cell line within the body All monoclonal antibodies from the same cell line recognize the same site on an analyte and bind with an identical binding affinity (Ka)
IMMUNOASSAYS Types of Immunoassays I. There are several different ways in which antibodies can be used in the detection or analysis of an antigen. Some common ways include: Precipitation-based immunoassay Competitive binding immunoassay Sandwich immunoassay All of these techniques use the specificity of antibodies as a means of selectively recognizing an analyte in the sample The analyte reacting with the antibody is then detected either directly or through the use of various chemical labels which produce easy to measure signals II. III. signal mAb antigen
IMMUNOASSAYS Precipitation assays I. Use the antibody as a selective precipitation reagent for the determination of analyte in the sample Involves the use of two or more types of antibodies that bind o different sites on the same analyte (i.e., polyclonal antibodies) Since each antibody has two binding sites per molecule, this can result in precipitates being formed between Ab and Ag Maximum precipitation occurs at some optimal Ab/Ag ratio Soluble Complexes Insoluble Complexes Soluble Complexes
IMMUNOASSAYS Precipitation assays I. To quantitate analyte by this technique, typically take multiple aliquots of sample and add various amounts of antibody to each sample (i.e., titration) The amount of precipitate formed for each aliquots is then determined visually, gravimetry, light scattering measurement, etc. II.
IMMUNOASSAYS Precipitation assays III. Technique can be performed in gels by having antibody and analyte diffuse towards each other from different sections of the gel A concentration gradient of Ab and Ag is formed in the gel Maximum precipitation will occur at the location where the antibody and analyte are both present in the correct ratio
IMMUNOASSAYS Precipitation assays IV. Precipitation in gels can be used either quantitatively or quantitatively to analyze the an analyte in the sample Ouchterlony assay: Qualitative method: formation of precipitate between sample and antibody wells indicates the sample contains analyte to which antibody binds Skamel et al. (2014): PLOS ONE. 10.1371/journal.pone.0113069.g009.
IMMUNOASSAYS Precipitation assays IV. Precipitation in gels can be used either quantitatively or quantitatively to analyze the an analyte in the sample Radial Immunodiffusion assay: Quantitative method: area of ring within precipitation band is proportional to concentration of analyte in sample
IMMUNOASSAYS Precipitation assays V. Advantages of precipitation methods Inexpensive-only reagent usually required is antibody Selective-few interferences from other compounds in sample Easy to perform VI. Disadvantages of precipitation methods Only useful for fairly high concentration analytes (10-200 mg/L) Long incubation times (hours-days) Can require large amounts of antibody
IMMUNOASSAYS Competitive binding immunoassays I. Quantitative method based on competition between analyte in sample and a fixed amount of labeled analyte for a limited number of antibody binding sites (equilibrium method) Indirectly measures the amount of analyte in the sample by looking at amount of labeled analyte it displaces from the antibody Unlabeled antigen Unlabeled antigen displaces labeled antigen
IMMUNOASSAYS Competitive binding immunoassays I. Quantitative method based on competition between analyte in sample and a fixed amount of labeled analyte for a limited number of antibody binding sites (equilibrium method) A typical calibration curve for the assay Linear transform Ln(antigen concentration)
IMMUNOASSAYS Competitive binding immunoassays II. Advantages of competitive binding immunoassay Can be used with any type of analyte Good limit of detection Theoretical limit: 1/Ka or 10-6 to 10-10 M Few interference from other compounds in sample III. Disadvantages of competitive binding immunoassay Some skill required to obtain optimum conditions for assay Long incubation times (hours-days) Limit of detection ultimately controlled by quality of antibody Antibody binding strength (Ka) Detection limit varies between different antibody preparations Usually manual method
IMMUNOASSAYS Sandwich immunoassays I. Quantitative method based on use of two antibodies to detect analyte First antibody extracts analyte from sample Second antibody (containing chemical label) identifies presence of analyte Unlabeled antigen Solid support antigen sandwiched between two antibodies This type of assay measures the amount of analyte in the sample by looking at the amount of labeled antibody that binds to analyte on the solid support
IMMUNOASSAYS Sandwich immunoassays I. Quantitative method based on use of two antibodies to detect analyte A typical calibration curve for the assay Response Concentration of Analyte
IMMUNOASSAYS Sandwich immunoassays II. Advantages of sandwich immunoassay Linear calibration curve Lower limits of detection possible than with competitive binding immunoassay < 10-12 M Greater selectivity than competitive binding assay Two antibodies instead of one are used to recognize analyte Shorter incubation times than competitive binding assay (hours vs. days) Less susceptible to variations in quality of antibody preparation then competitive binding assay III. Disadvantages of competitive binding immunoassay Only useful for large analytes 1000 to 2000 MW Requires enough room on molecule to bind two antibodies simultaneously Requires multiple antibodies per analyte Usually manual method
IMMUNOASSAYS Labels for Immunoassays I. The selectivity of a competitive binding assay depends on the specificity of the antibody The use of a chemical label is also required Several types of chemical labels have been used in immunoassays Limit of Detection Type of Label Example Measurement Principal I125 10-13 M Radiolabels Radioactive delay 10-10 M Fluorescein, Rhodamine Fluorescence Fluorescent 10-13 M Time-resolved fluorescence Rare earth chelates 10-11 M Horse radish peroxidase Formation of colored product by enzyme Enzymatic 10-13 M Acridinium esters, luminol Light production by chemical reaction Chemiluminescent
IMMUNOASSAYS Learning Objectives: 1. The student should be familiar with the general definitions and advantages of immunoassays and some examples of the application of this field. 2. The student should be familiar with important features, structure and the production of antibodies and the intrinsic value to immunoassays. 3. The student should be familiar with the differences between monoclonal and polyclonal antibodies 4. The student should be familiar with the details of the antibody-antigen binding interaction 5. The student should be familiar with the different types of immunoassays, be able to describe how the assays function, and understand their advantages and disadvantages: Precipitation-based immunoassay Competitive binding immunoassay Sandwich immunoassay Ouchterlony assay Radial Immunodiffusion assay 4. The student should be familiar with the different labels for immunoassays, including how the label is measured and the limit of detection: Radiolabels Fluorescent Enzymatic Chemiluminescent
