Presentation on theme: "Chapter 12 - Immunological methods"— Presentation transcript:
1Chapter 12 - Immunological methods ObjectivesBe 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.
3There are five classes of antibodies, we will focus on the IgG class.
4The 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.
5Production of antibodies Polyclonal antibodies – a mix of many different antibodies that recognize different determinants on an antigen. This mix makes standardization of assays difficult.
6Production 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.
8Production 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).
10Detection of the antibody-antigen complex Direct or indirect immunofluorescenceDirect ELISA (detects antigen)useful for - environmental samples- medicinedrug testinghormone testing3. Indirect ELISA (detects antibody)useful for - Treponema palladium (syphilis)- Feline leukemia virus- HIVAdvantages of ELISA:cheapsensitiverapid
12Direct ELISA steps Indirect ELISA Steps of the ELISA assay 1) coat wells withAb1) coat wells withAg2) addsample (Ag)to each well2) addsample (Ab)to each well3) incubate and wash3) incubate and wash4) addenzyme-linked Ab4) addenzyme-linked Ab5) incubate and wash5) incubate and wash6) perform enzyme assay and measurecolor6) perform enzyme assay and measurecolor
13In 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:PesticidesAtrazine2,4-DMetolachlorParaquatAldicarbCarbarylCarbofuanProcymidoneAlachlorInorganicsOther organic contaminantsPCP (pentachlorophenol)PCB (polychlorinated biphenyls)BTEX (benzene, toluene, ethylbenzene)PAH (polyaromatic hydrocarbons)TNTnitratecadmiumleadmercurycalciumcobaltnickelzincDetection limitsWater – low ug/L (ppb)Soil – high ug/L to low mg/L (ppb – ppm)
14These kits are based on the competitive ELISA reaction. Immunoassay for chemical contaminants1. sample containing 2,4-D is extracted2,4-D-Ab2. enzyme-linked 2,4-D is added2,4-D3. antibody-linked magnetic beads are added-Ab4. a magnetic field is applied beads are collectedthe enzyme substrate is added and color is produced depending on the amount of enzyme linked to the beads
15Advantages of Immunoassays: Assay sensitivity is in the low ug/L (ppb)Assay is rapidAssay is easy to performAssay is cost-effect ($20/sample)Accepted by EPAAssay is portableDisadvantages of Immunoassays:cross reaction – an antibody may cross react with similarstructures. This is a problem with PAHs and with the BTEXcompounds. So usually, BTEX are measured as a combination.can be difficult to analyze multiple solutes