Chapter 12 - Immunological methods Objectives Be able to define the terms antibody and antigen. Understand the structure of an IgG antibody. Be able to give a brief description of the production of polyclonal and monoclonal antibodies, and antiglobulins. Be able to describe measurement of antibody-antigen complexes (immunofluorescence, direct and indirect ELISA. Be able to describe competitive ELISA and its application to measuring chemical contaminats. Be able to give a brief description of affinity chromotography, western blotting and immunoprecipitation.

Antigen Antibody Virus particles Macrophage B-cell T-cell antigen-presenting molecule T-cell Antibody

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

The B cells can make a unique antibody for each antigen presented 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.

Production of antibodies Polyclonal antibodies – a mix of many different antibodies that recognize different determinants on an antigen. This mix makes standardization of assays difficult.

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.

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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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Detection of the antibody-antigen complex Direct or indirect immunofluorescence 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

Direct ELISA steps Indirect ELISA Steps of the ELISA assay 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

In the 1970’s, the first antibodies against pesticides were developed 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 Atrazine 2,4-D Metolachlor Paraquat Aldicarb Carbaryl Carbofuan Procymidone Alachlor Inorganics Other organic contaminants PCP (pentachlorophenol) PCB (polychlorinated biphenyls) BTEX (benzene, toluene, ethylbenzene) PAH (polyaromatic hydrocarbons) TNT nitrate cadmium lead mercury calcium 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 2,4-D -Ab 2. enzyme-linked 2,4-D is added 2,4-D 3. antibody-linked magnetic beads are added -Ab 4. a magnetic field is applied beads are collected the enzyme substrate is added and color is produced depending on the amount of enzyme linked to the beads

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