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.

Mac rophage

Virus particles

Macrophage

T-cell

Antigen

Antibody

antigen-presenting molecule

B-cell

There are five classes of antibodies, we will focus on the IgG class.

IgG IgD

IgM

IgAIgE

Antigen binding site

Fab region

Fc region

Fab region

Antigen binding site Disulfide bonds

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 A Antigen B

Antigen fits correctlyAntigen does not fit

Production of antibodies1. 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 a long 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 3. Anticoagulant is added to the blood to prevent clot formation.

Step 4. Blood sample is centrifuged to sediment the blood cells

Plasma containingantibodies andother proteins

Pelleted blood cells

Step 5. P lasma is heated to 65 C to destroy contam inating proteins leaving the antibodies intact

o

Polyclonal antibodies

Production of antibodies

2. Monoclonal antibodies – a myeloma cell is fused with an antibody-producing 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 2. Myeloma cells are grown in cell culture

HybridomaMyeloma

Antibodyproducing

cell

+

Step 3. Antibody producing cells are isolated from the animal's spleen.Step 1. Inoculate animal with specific

antigen to stimulate antibody production

Step 5. The hybridoma cell that produces the correct antibody is selected

Y

YY

Y

YYY

YY

Y Y

Step 6. The hybridomas are grown in cell culture and the antibodies are harvested from the supernatant

Step 1. F irst animal (mouse) is immunized with target antigen

Target antigen(protozoan)

Step 2. Mouse monoclonal antibodies are produced specific for the target antigen

Step 3. Mouse monoclonal antibody is in jected into a second animal (goat).

Step 4. Goat-antimouse monoclonal antibodies (antiglobulins) are produced.

Production of antibodies3. 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).

Signal molecule

Antig lobulin

Target antigen(protozoan)

Primary antibody

Microscope slide

Step 5. Antiglobulin is labeled with a signal molecule and indirect immunoassay is used to detect the target antigen

+

self

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:cheapsensitiverapid

Giardia (left) and Cryptosporidium (right)– Fluorescent Antibody StainingH.D.A. Lindquist, USEPA

microtiter plate

Indirect ELISA

4) add enzyme-linked Ab

5) incubate and wash

6) perform enzyme assay and measure color

1) coat wells with Ag

2) add sample (Ab) to each well

3) incubate and wash

Steps of the ELISA assay

4) add enzyme-linked Ab

5) incubate and wash

6) perform enzyme assay and measure color

1) coat wells with Ab

2) add sample (Ag) to each well

3) incubate and wash

Direct ELISA steps

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:

Inorganicsnitrate

cadmium lead mercury calcium cobalt nickel zinc

Other organic contaminants

PCP (pentachlorophenol)PCB (polychlorinated biphenyls)BTEX (benzene, toluene, ethylbenzene)PAH (polyaromatic hydrocarbons)TNT

Detection limits

Water – low ug/L (ppb)

Soil – high ug/L to low mg/L (ppb – ppm)

Pesticides

Atrazine2,4-DMetolachlorParaquatAldicarbCarbarylCarbofuanProcymidone

Alachlor

1. sample containing 2,4-D is extracted

2. enzyme-linked 2,4-D is added

3. antibody-linked magnetic beads are added

2,4-D

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

Immunoassay for chemical contaminants

These kits are based on the competitive ELISA reaction.

-Ab2,4-D

2,4-D

-Ab

• Assay sensitivity is in the low ug/L (ppb)

• Assay is rapid

• Assay is easy to perform

• Accepted by EPA

Advantages of Immunoassays:

• Assay is cost-effect ($20/sample)

• 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