HOST DEFENSE SMALL GROUP PROBLEM SOLVING SESSION CLINICAL IMMUNOLOGIC ASSAYS-II

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1 HOST DEFENSE SMALL GROUP PROBLEM SOLVING SESSION CLINICAL IMMUNOLOGIC ASSAYS-II Monday, March 24, :00 PM 4:00 PM Small Group Classrooms LEARNING GOAL Understanding in vitro assessment of immunologic responses that have contemporary clinical relevance BACKGROUND READING Janeway: 7 th edition: P ; An optional article of interest is posted on the Web Forum DEVELOPED BY John A. Robinson, M.D. Page 1

2 IV. Immunologic Assays used for detection and assessment of antibody responses A. Immunofluorescence (fluorescent antibody techniques) - the principle Underlining this method exploits the ability of certain chemical compounds To emit light of restricted wavelength during ultraviolet excitation. Copyright: 2001 From: immunobiology, 5 th edn. Author: Janeway, et al Reproduced by permission of Routledge, Inc., part of h l i G 1. Direct fluorescence. If these light emitting compounds (tags) are linked to antibodies that directly bind to antigens (for example in tissue biopsies) or indirectly bind to a patient s immunoglobulin that has previously bound to antigens in tissue (or test substrates), the specific binding can be documented by direct microscopic visualization during UV excitation. 2. Indirect immunofluorescence. The indirect fluorescent method is useful for detecting and quantitating by titer patient antibodies to various antigens. One widely used assay is for the detection of anti-nuclear antibodies in systemic lupus. Patient serum containing possible antibodies is layered on a substrate. Containing nuclei, incubated, washed and then anti-human Ig-fluorochrome is added. If there were specific anti-nuclear antibody in the patient serum it would bind to the nuclear antigens. This complex, in turn, would then react with antihuman Ig - fluorochrome, and UV light passing through the sample then emits green fluorescence at the site of binding which can be detected by fluorescence microscopy 3. You only need to understand the concept of this method at this time specific ways to use it will become evident in a later Small Group. Page 2

3 V. Techniques that can be used to identify and quantify immune cell populations. A. FLOW CYTOMETRY is a technique that can provide information on the characteristic of single cells in a mixed cell population. This technique also depends on tagging cells with specific light emitting compounds. The cell population to be studied is shepherded into a thin stream of single cells that have been previously incubated with the desired fluorescent- labeled monoclonal (For production, see figure below) or a monospecific antibody probe. Be sure you understand how to use flow cytometry for diagnosis-it is always on the test. 1. Flow cytometry is heavily dependent upon availability of monoclonal antibodies. These reagents have high specificity for individual antigens. Their use is widespread in basic research, clinical testing and now have rapidly growing clinical applications in cancer, inflammatory arthritis and many other diseases. 2. You need to understand how monoclonals are made. Copyright: 2001 From: immunobiology, 5 th edn. Author: Janeway, et al Reproduced : by permission of Routledge, Inc., part a. If you understand how a monoclonal is made, you should immediately be able to see how this technology could be applied to the bird flu case in the prior small group. Page 3

4 3. Back to flow cytometry technique. The single cell queue flows past a laser that emits a light wavelength unique to the characteristics of the fluorochrome. If a cell has bound the antibody-fluorochrome, the emitted wavelength is then photo multiplied and quantitated by computer software. A cartoon of the technique is shown below. A typical plot using two monoclonal antibodies, each with a different light emitting fluorochrome is depicted below on the right. 4. Clinical scenarios where flow analysis can be used a. 84 year male fainted during a church service. A complete blood count in the ER revealed a normal hemoglobin and platelet count but his white blood count was 100,000/mm 3 (normal 4-10,000). b. Set up the flow cytometry experiments to solve the following questions: 1. Lineage of the white blood cells- does he have chronic lymphocytic leukemia? Copyright: 2001 From: immunobiology, 5 th edn. Author: Janeway, et al Reproduced by permission of Routledge, Inc., part of The Taylor Francis Group Page 4

5 2.Phenotype of the white blood cells 3. Clonality of the white blood cells Host Defense 2008 VI. Laboratory Assays Relevant to Transplantation A. Solid Organ Transplantation - with the advent of many new immunosuppressive agents and a better understanding of the genetics of transplantation, solid organ (heart, lung, liver, kidney, pancreas, and now small intestine) transplantation increased almost exponentially until recently. It is now clear that the need for cadaver organs has far outstripped their availability and that significant future increases in transplantation will not occur until either permanent artificial support or xenorgans (different species) are available for replacement. While permanent electrical pump support of left ventricular function in patients with heart failure is now a reality, other artificial organs such as islet cell substitution for whole pancreas or hepatic cell infusion for liver failure are not ready yet. This reality increases the pressure to use available cadaver organs judiciously. In order to do this one must understand the genetics of the major histocompatibility loci - in clinical parlance, the HLA system - and how these genetic loci influence the outcome of an organ transplant. B. Bone Marrow or Stem Cell Transplantation - This form of transplantation is different because, in most circumstances, the bone marrow transplant recipient must be rendered immunologically bankrupt prior to transplantation in order that the donor cells can engraft and provide the recipient with a new immune system. C. Methods available to predict donor organ compatibility 1. Lymphocytotoxic Antibodies - MHC or histocompatibility (HLA) typing techniques are used to detect MHC specificities on mononuclear nucleated cells. The number of matched MHC loci, especially the Class 2 ones (DR), between donor and recipient can be used to predict graft acceptance. The more loci matched the better, but Class 2 (DR) matches are the most strongly predictive. The source of the antibodies used to detect HLA specificities are usually from women with multiple pregnancies who reacted to a paternal HLA antigen or tailored monoclonal antibodies against specific MHC loci. 2. HLA (MHC) cell typing methods. The classic way to determine whether a potential organ recipient has MLC (HLA) antibodies present in their serum is incubate their serum with a panel of lymphocytes of known HLA specificity in individual wells. If there are HLA antibodies in the serum they will bind to their MHC antigen on the lymphocyte cell surface. Page 5

6 Complement is then added to all the wells. Lymphocytes that have bound antibody on their MHC will be lysed. Since the MHC is known for each lymphocyte well, the exact anti-mhc specificity can be determined. 3. Tissue Typing Laboratories are now using flow cytometry, ELISA and molecular methods to decrease time of analysis and increase sensitivity and decrease costs but you need to understand the fundamentals of detecting HLA antibodies.. 4. The Mixed lymphocyte reactions ("In vitro") transplants: a. Description of the Mixed Lymphocyte Culture (MLC) The primary concept in this assay is that differences in the HLA class II (particularly HLA-DR) antigens between the donors of the cells used in the assay will stimulate T lymphocytes to synthesize DNA and divide. The MLC is designed to quantitate the amount of cell division as measured by newly synthesized DNA in responder lymphocytes when exposed to irradiated stimulator lymphocytes. The MLC requires peripheral blood lymphocytes from two individuals. Responder and irradiated stimulator lymphocytes are mixed together in the MLC. The stimulator lymphocytes are irradiated to prevent them from acting simultaneously as stimulator and responder cells. A MLC consisting of a mixture of responder lymphocytes and irradiated responder lymphocytes is conducted simultaneously. The cultures are incubated for a period of 5 days at 37 C, at which time a radioactive precursor of DNA, tritiated thymidine ( 3 H-TdR) is added to each of the cultures. After 18 hours of additional incubation, the cellular DNA containing the incorporated 3 H-TdR is assayed for radioactivity. As shown in the example in this diagram, the responder lymphocytes recognize the irradiated stimulator lymphocytes and incorporate a significant amount of 3 H-TdR. In contrast, the responder lymphocytes do not respond to irradiated responder lymphocytes and do not incorporate radioactive thymidine. This typing assay thus measures the ability of responder lymphocytes to recognize non-self-hla-d antigens on stimulator cells and is a measure of the HLA-DR antigen difference between the donors of the responder and the stimulator lymphocytes. Page 6

7 Copyright: 2001 From: immunobiology, 5 th edn. Author: Janeway, et al Reproduced by permission of Routledge, Inc., part of The Taylor Francis Group b. Design an MLC that will be helpful for selecting the best family donor for a bone marrow transplant in a sibling c. Design an MLC that will be helpful for selecting the best family donor for a kidney transplant in a sibling VII. ASSAYS OF THE VERY NEAR FUTURE BASED ON MOLECULAR IMMUNOLOGIC METHODS. This section is for the sake of knowledge only and will give you the basis for exciting dinner conversations with family, loved ones and non-medical people. YOU WILL NOT BE TESTED ON THIS SECTION. Terrorism has sparked a flurry of extremely creative applications of nanotechnology/molecular immunology for the rapid detection of pathogens. The two following methods are only the tip of the iceberg. Additional information on them is posted on the Forum. A 1. B lymphocytes have been engineered as pathogen sensors by inserting genes for: a. bioluminescent jellyfish proteins that emit light when their surface antibody is cross-linked. b. and using monoclonal antibody technology to produce antibody to the specific pathogen of interest. Page 7

8 2. The sensitivity and speed of the assay are spectacular. Anthrax specific B cells, when exposed to as few as 50 units of the organism from a nasal swab, emit light that can be easily detected with available sensors within 5"! PATHOGEN SENSING B CELLS B B-light B-light & Ab LIGHT DETECTOR Jellyfish light Protein gene Use monoclonal antibody Technique for specific ab B-light & Ab B-light & Ab PATHOGEN B. Nanotechnology uses minute iron and gold particles that increase the sensitivity a million fold over current assays. 1. The technique exploits magnetic properties of iron particles that are encased in plastic coat that has been coated with a monoclonal antibody against a protein of interest. 2. The coated iron particles are reacted with the protein and also with gold nanoparticles coated with a polyclonal antibody to protein (the sandwich technique similar to ELISA assays) and short strands of DNA that act like a "bar code". The iron particles that have migrated to the magnetic field are isolated, the DNA snipped off and the "bar code" read by ultrasensitive DNA techniques. Page 8

9 NANOIMMUNOLOGY MONOCLONAL ANTIBODIES IRON PARTICLE IRON PARTICLE Concentrate with magnet Target protein Gold particle, antibodies Snip DNA and read the code DNA bar code More Nanotechnology that will be used in your time These nanobeads can be infused into patient and target a tumor with tumor specific antibody localization, then release chemotherapeutic drugs into the tumor.they can also carry an MRI imaging agent so tumor can be found. Page 9

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