Our brief introduction into the topic of Immune Testing is intended to allow you to become familiar with a selection of technologies that are in use in diagnostic laboratories, and play a critical role in the diagnosis of many infectious diseases. Immune testing may be used in places where culturing of the infectious agent in the microbiology laboratory is not practical, due to time constraints or due to difficulty in culturing the organism. I do not expect the class to be able to carry out these assays or interpret these assays, but you should understand the basis of the techniques described in class. An understanding of these techniques, at least at the basic level, will be a necessary and essential part of your jobs during your daily interactions with patients and their families. Here is a summary of the techniques described in class, but I do expect this summary to not be used in lieu of a reading of the appropriate section in Bauman.
All of these assays either measure the presence of an antigen or the presence of antibody that has been made in response to an antigen. In the case of measuring an antigen, a patient sample is collected (potentially infected with a specific agent) and the antigen is detected using a commercially available monoclonal or polyclonal antibody. The presence of the antibody is then measured, and the amount of bound antibody is correlated with the amount of antigen originally present. Please recall the difference between monoclonal and polyclonal antibodies.
In the agglutination assay, a sample containing antigen is mixed with an antibody. As discussed in class, immunoglobulins have the ability to bind two copies of the same antigen per antibody molecule, at least for IgG, serum IgA, and IgE isotypes. In the image to the right (appropriated from the Wikipedia article on agglutination) a sample of a patient’s blood has been mixed in each of the circles with an antibody sample. In the circle to the left, the antibody recognizes the “A” antigen, and in the circle to the right, the antibody recognizes the “B” antigen. Since the positive agglutination reaction is only seen with the “A” antibody, the patient would have blood type A. We will use the agglutination assay in Lab 9 in order to determine whether or not an unknown bacterium is Staphylococcus aureus or the related bacterium S. epidermidis.
In the Immunofluorescence assay, a sample containing antigen is mixed with an antibody. In this assay, frequently the sample will be a patient biopsy of tissue, which will be examined using the microscope. The antigen containing sample is mixed with an antibody which will recognize the antigen, but this time the antibody has been tagged with a chemical which allows it to glow under ultraviolet light. The presence of the glowing material is assessed as a positive immunofluorescence result. The immunofluorescence assay has the advantage of being able to detect small amounts of an antigen that might not be detectable using other methods. In the image to the left, cells infected with measles virus have been directly detected in a patient sample. The glowing green cells are infected with the virus, and the orange cells throughout the field are uninfected cells.
In the Western Blot, the procedure is much the same as immunofluorescence. A patient sample containing an antigen is collected, but this time the sample is separated by polyacrylamide gel electrophoresis, or PAGE. Following separation of proteins, an antibody sample is used to detect the presence of a specific protein antigen. Alternatively, a known sample of antigen can be subjected to electrophoresis, and a patient sample potentially containing an antibody can be used to measure antibody/antigen interactions. In both cases, the procedure will show up as a series of bands, which represent specific proteins reacting with the antibody solution. Control samples are included, so that the technician can be sure that the presence of a specific band is due to the presence of a specific antigen in the patient sample. The Western Blot is used as a definitive test for several infectious diseases, including Lyme Disease.
Finally, in the Enzyme Linked Immunoadsorbant Assay, or ELISA, a sample of known antigen is deposited uniformly onto the surface of a 96 well plastic tray. Patient samples containing antibody are mixed into each well in different dilutions, and positive and negative antibody controls are also added to wells. An antibody solution capable of detecting antibody bound to antigen is added to each well, and the intensity of the color in the respective wells is taken as a measure of the amount of reactive antibody in each well. In the example to the left, each well of an ELISA plate (there are 96 wells in total) has been filled with a combination of antigen and antibody. The intensity of the green color is an indication of how much antibody is present, and therefore how much antigen is present. The darker wells have more antigen. This technique can be used to measure the amount of an antigen that a patient might have in his or her serum, or to measure how much antibody they have made against a specific antigen target at a given time. The amount of antibody that has been made is referred to as the titer of antibody (you may have heard of ‘virus titer‘ used as a measure of how much virus a patient has in their system.)
One more thing before the weekend; we will again have a review session. If you think that a face to face review would be beneficial, vote in the poll.