Chapter 12 - Immunological methods
Objectives
1) Be able to define the terms antibody and antigen.
2) Understand the structure of an IgG antibody.
3) Be able to give a brief description of the production of polyclonal
and monoclonal antibodies, and antiglobulins.
4) Be able to describe measurement of antibody-antigen complexes
(immunofluorescence, direct and indirect ELISA.
5) Be able to describe competitive ELISA and its application to
measuring chemical contaminats.
6) Be able to give a brief description of affinity chromotography,
western blotting and immunoprecipitation.
Virus particles
Antigen
Macrophage
antigen-presenting molecule
Macrophage
T-cell
Antibody
B-cell
There are five classes of antibodies, we will focus on the IgG class.
IgG
IgD
IgM
IgE
IgA
The B cells can make a unique antibody for each antigen presented.
It is estimated that there is the potential to produce up to 1 x 1010
structurally different IgG antibodies.
Antigen binding site
Fab region
Disulfide bonds
Antigen binding site
Fab region
Fc region
Antigen A
Antigen does not fit
Antigen B
Antigen fits correctly
Production of antibodies
1.
Polyclonal antibodies – a mix of many different antibodies that recognize
different determinants on an antigen. This mix makes standardization of
assays difficult.
Step 2. Blood containing the antibody
along with blood cells and other
proteins is collected from the
animal.
Step 1. Animal is inoculated with
several doses of antigen
over several weeks to
induce production of antibodies.
Step 4. Blood sample is centrifuged
to sediment the blood
cells
Plasmacontaining
antibodies and
other proteins
Pelleted blood
cells
Step 3. Anticoagulant is added
to the blood to prevent clot
formation.
Step 5. Plasma is heated to 65oC to
destroy contaminating
proteins leaving the antibodies intact
Polyclonal antibodies
Production of antibodies
2. Monoclonal antibodies – a myeloma cell is fused with an antibodyproducing cell to create a hybridoma cell capable of producing a single
antibody. This is a more expensive process than producing polyclonals
but is the cornerstone for a variety of drug/hormone/chemical assays
that are routinely available.
Step 1. Inoculate animal with specific
antigen to stimulate antibody
production
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Production of antibodies
3. Antiglobulins – these are antibodies to an antibody. The use of fluorescently
or chemically-labeled antiglobulins makes it easy to detect antibodies in
assays like ELISA (see later).
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Signal molecule
self
Antiglobulin
Primary antibody
Target antigen
(protozoan)
Microscope slide
Detection of the antibody-antigen complex
1. Direct or indirect immunofluorescence
2. Direct ELISA (detects antigen)
useful for - environmental samples
- medicine
drug testing
hormone testing
3. Indirect ELISA (detects antibody)
useful for - Treponema palladium (syphilis)
- Feline leukemia virus
- HIV
Advantages of ELISA:
cheap
sensitive
rapid
Giardia (left) and Cryptosporidium (right)– Fluorescent Antibody Staining
H.D.A. Lindquist, USEPA
Steps of the ELISA assay
Direct ELISA steps
Indirect ELISA
1) coat wells with Ab
1) coat wells with Ag
2) add sample (Ag) to each well
2) add sample (Ab) to each well
3) incubate and wash
3) incubate and wash
4) add enzyme-linked Ab
4) add enzyme-linked Ab
5) incubate and wash
5) incubate and wash
6) perform enzyme assay and measure
color
6) perform enzyme assay and measure
color
microtiter plate
In the 1970’s, the first antibodies against pesticides were developed. Using
these antibodies, the ELISA assay was modified and developed for use in
monitoring chemical contaminants in the environment. The technology has
been further refined to the point that commercial kits are now available for
detection of many different contaminants.
Immunoassay kits available for:
Pesticides
Alachlor
Atrazine
2,4-D
Metolachlor
Paraquat
Aldicarb
Carbaryl
Carbofuan
Procymidone
Inorganics
Other organic contaminants
nitrate
PCP (pentachlorophenol)
cadmium
PCB (polychlorinated biphenyls)
lead
BTEX (benzene, toluene, ethylbenzene)
mercury
PAH (polyaromatic hydrocarbons)
calcium
TNT
cobalt
nickel
zinc
Detection limits
Water – low ug/L (ppb)
Soil – high ug/L to low mg/L (ppb – ppm)
These kits are based on the competitive ELISA reaction.
Immunoassay for chemical contaminants
1. sample containing 2,4-D is extracted
2. enzyme-linked 2,4-D is added
2,4-D
3. antibody-linked magnetic beads are added
-Ab
-Ab
4. a magnetic field is applied beads are collected
5. the enzyme substrate is added and color is produced
depending on the amount of enzyme linked to the beads
2,4-D
2,4-D
Advantages of Immunoassays:
• Assay sensitivity is in the low ug/L (ppb)
• Assay is rapid
• Assay is easy to perform
• Assay is cost-effect ($20/sample)
• Accepted by EPA
• Assay is portable
Disadvantages of Immunoassays:
• cross reaction – an antibody may cross react with similar
structures. This is a problem with PAHs and with the BTEX
compounds. So usually, BTEX are measured as a combination.
• can be difficult to analyze multiple solutes