ASSIGNMENTS CLASS 4. COMPLEMENT. ANTIGEN-ANTIBODY INTERACTIONS - IMMUNOASSAYS

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1 A) Subtopics ASSIGNMENTS CLASS 4. COMPLEMENT. ANTIGEN-ANTIBODY INTERACTIONS - IMMUNOASSAYS 1. Complement nomenclature and functions 2. Complement activation pathways. Membrane attack complex formation. 3. Regulation of Complement 4. Antigen-antibody interactions. B) Objectives 1. To understand the complement role and activation pathways. 2. To learn how the exquisite specificity of antibodies can be used in the clinical laboratory for diagnostic assays that measure either antibodies or antigens and review experimental systems that will be discussed later in the course. C) Keywords Complement, MAC, lectin, C1-inhibitor, affinity, agglutination, prozone, zeta potential, precipitation, immunoelectrophoresis, radial immunodiffusion, nephelometry, radioimmunoassay, ELISA. D) Reading: Khaitov R. M. Immunology (+CD-ROM). GEOTAR-Media Publishing, Moscow, 2008 Abul Abbas, Andrew H. Lichtman, Shiv Pillai. Cellular and Molecular Immunology (7 th ed.) Philadelphia : Elsevier/Saunders, PP 73-74, , Owen J., Punt J, Stranford S. Kuby Immunology. 7th ed. New York: W.H. Freeman and Company, PP , R.S. Geha and Notarangelo, L. Case Studies in Immunology: A Clinical Companion. (6 th ed.) Garland Publishing, New York, Web Resource: Animations: Complement: Monovlonal antibody production: ELISA: Immunoblotting: Complement fixation test: 1

2 Blood grouping: ELISPOT: Flow cytometry (FACS): 2

3 IMMUNOLOGY CLINICAL CORRELATION Complement Assigned for topic: Antigen-antibody interactions immunoassays Required readings (from R.S. Geha and Notarangelo, L. Case Studies in Immunology: A Clinical Companion. (6 th ed.) Garland Publishing, New York, 2012) 32. Factor I Deficiency 33. Deficiency of C8 46. Hemolytic Disease of the Newborn Required readings complement lectures and presented materials. It is highly encouraged to view these clinical cases. Case materials may not be covered in full during lectures, however, all required case study readings contain material that may be tested on exams. WHAT IS COMPLEMENT Complement is a system of more than 30 serum and cell surface proteins that is involved in numerous functions in inflammation and immunity. In conjunction with specific antibodies, it acts as the primary humoral defense system against bacterial and viral infections. Complement activity is heat labile and can be destroyed by heating serum to 56ºC 30 minutes (which inactivates the C3 and C4 proteins as well as other complement components). Most of the complement proteins in the serum are produced by liver hepatocytes. C3 is the most abundant serum complement protein with a normal range of 1.0 to 1.5 mg/ml in healthy individuals. Some of the complement components (e.g. C3, factor B) are acute phase proteins and can increase in concentration two to three fold. Many of the complement proteins have shared sequences, indicating that they evolved by gene duplication and recombination. The complement genes are scattered throughout the human genome; the genes for the proteins C4, C2, and factor B are located within the Major Histocompatibility Complex on chromosome 6. Functions Activation of the complement system results in the production of several different polypeptide cleavage fragments that are involved in five primary biological functions of inflammation and immunity. 1. Cytolysis of foreign organisms (e.g. bacteria) 2. Opsonization and phagocytosis of foreign organisms 3. Activation of inflammation and directed migration of leukocytes 4. Solubilization and clearance of immune complexes 5. Enhancement of humoral immune response THE COMPLEMENT CASCADES Complement activation involves the sequential activation of complement proteins, either by protein-protein interactions or by proteolytic cleavage. At each step, the number of protein molecules activated increases, amplifying the reaction. This sequential reaction is call the 3

4 complement cascade. Many complement proteins are present as zymogens which are activated either by conformational changes or by proteolytic cleavage by other complement proteins. Activation of these zymogens results in specific serine protease activities that are capable of cleaving other complement proteins, producing the complement cascade. ACTIVATION Complement activation is initiated by the presence of antigen-antibody complexes (Classical Pathway), foreign cell surfaces (Alternative Pathway), or by mannose on pathogenic organisms (Lectin Pathway). 4

5 CLASSICAL PATHWAY ACTIVATION -Primarily by IgG and IgM immune complexes -IgM > IgG3 > IgG1> IgG2 -IgG4, IgA, IgD, and IgE do not activate Activation of the classical pathway requires the local reaction of antibodies with two or more antigenic sites. These Ag-Ab complexes may consist of a single IgM molecule bound to two or more antigenic sites, or two or more human IgG molecules (IgG1, IgG2, or IgG3) bound to epitopes. Such a complex could (for example) occur on a bacterial cell surface or in an aggregate of antibodies with soluble antigens. Ag-Ab reaction causes conformational changes in CH2 of IgG and CH3 of IgM, permitting the binding of C1q. Binding of two or more arms of C1q causes conformational changes that lead to cleavage and activation of the bound zymogens C1r and C1s. 5

6 Activated C1s can cleave C4 and C2 into large (C4b and C2b) and small (C4a and C2a) fragments. C4 is cleaved first, and approximately 1% binds to a nearby surface via a covalent linkage. C2 can complex with surface bound C4b and can be cleaved by C1s. The resulting C4bC2b complex is the classical pathway C3 convertase, and has the ability to specifically cleave C3 into large (C3b) and small (C3a anapylatoxin) fragments. C3 is the most abundant complement protein and plays a pivotal role in complement activation. Many molecules can be cleaved into C3b and C3a. Cleavage results in exposure of the labile thiolester bond in C3b, permitting some to bind covalently to proteins and carbohydrates on cell surfaces. The C3a anaphylatoxin is released into the blood and mediates many important inflammatory activities that will be discussed later. Some of the C3b binds to C4bC2b to form C4b2b3b, which is the C5 convertase. This complex by the C2b protease subunit will cleave C5 into big (C5b) and small (C5a anaphylatoxin) subunits. C5a is released into the blood and as C3a mediates many important inflammatory activities. C5a on a molar basis is 100 times more potent than C3a. The classical pathway of complement can also be activated by the serum mannose binding lectin complex (MBL-MASP). This complex is structurally similar to the C1 complex. However, instead of binding to immune complexes it binds to directly to polysaccharides on gram-negative bacteria. The mannose binding lectin is C1q-like in structure and the MBL associated proteases (MASP) are similar to C1r and C1s. MBL-MASP on binding bacterial surfaces can cleave C4 and C2 thereby activating the remainder of the classical pathway. 6

7 ALTERNATIVE PATHWAY ACTIVATION The main difference between the classical and alternative pathways is that the initiation of the classical requires an activating substance. The alternative pathway, by contrast, runs continuously and spontaneously at low levels in the blood plasma. The alternative pathway activation occurs when C3b binds to a surface that lacks inhibitors that block complement activity, such as most bacterial cell surfaces. Certain plastic surfaces, like those initially used in heart-lung machines and dialysis machines, also activate the alternative pathway with obvious deleterious effects. Because antibody is not necessary, the alternative pathway represents an innate immune response and can react as soon as bacteria enter the body. The alternative pathway is also important in amplifying reactions initiated by the classical pathway. The low level activation of C3 that allows the alternative pathway to be activated is called the tick-over model. The thiolester bond in C3 is spontaneously hydrolyze at low rates yielding a C3b-like [C3(H2O)] molecule that now has a binding site for factor B exposed. The bound factor B is attacked by factor D, which cleaves it into Ba and Bb fragments. The Ba fragment is released, while the Bb fragment remains noncovalently associated with C3(H2O), forming an initial C3 convertase. The Bb subunit of this convertase has serine protease activity specific that can now specifically cleave additional C3 molecules into C3a and C3b fragments. If a activator surface is nearby, such as a bacterial surface, then the newly formed C3b molecule can covalently attach and bind factor B. The bound factor B is cleaved by factor D and the surface-bound C3 convertase (C3bBb) then attacks another native C3 molecule and so on. The activating surface (bacteria) thus accelerates a reaction that in its absence occurs at a slow rate. 7

8 LYTIC PATHWAY-FORMATION OF THE MEMBRANE ATTACK COMPLEX (MAC) After the C3 convertase cleaves C3 to generate C3b, the next step in either the classical, lectin, or alternative pathways is the binding of C3b to the C3 convertase complex, changing it to a C5 convertase, which catalyzes the proteolytic cleavage of the C5 protein. Cleavage of C5 to C5a and C5b represents the first step of the lytic pathway. The small C5a fragment is released into the blood and is the most potent complement anaphylatoxin. The large C5b molecule binds proteins C6 and C7. The complex C5b67 has hydrophobic regions that permit it to insert into the lipid bilayer nearby cell membranes. Subsequent binding of C8 permits some leakage of cell contents, causing slow lysis. This process is accelerated by binding of multiple C9 molecules, which assemble to form a protein channel through the membrane. C9 is analogous to perforins produced by cytolytic T cell and NK cells. C5b6789 is called the Membrane Attack Complex (MAC). MAC formation is an important mechanism for eliminating bacteria resistant to intracellular killing by phagocytes, such as Neisseria species. 8

9 REGULATION OF COMPLEMENT ACTIVATION Complement activation is a tightly regulated series of reactions, that without control would result in the inappropriate activation on normal host cells. This would result in excess inflammatory mediators and by direct lysis of host cellular membranes by MAC. This does not normally happen because there exist several complement inhibitors in serum as well as on the surface of host cells. COMPLEMENT RECEPTORS There are several characterized complement receptors that are involved in binding complement activation and degradation products. They are expressed on various cell types and are involved in mediating many of the biological functions attributed to complement. 9

10 C5a Receptor (C5aR;CD88) is a seven transmembrane G-protein coupled receptor expressed primarily on neutrophils and macrophages. Also found on hepatocytes and various tissue epithelial cells. Causes smooth muscle contraction, histamine release from mast cells and vasodilation. Will modulate the hepatic acute phase response. It also is a potent chemoattractant for neutrophils, monocytes, macrophages, and eosinophils. C3a Receptor (C3aR) also seven transmembrane receptor. Tissue distribution currently being worked out. Causes smooth muscle contraction, histamine release from mast cells, and vasodilation. Chemoattractant for eosinophils but not neutrophils. BIOLOGICAL FUNCTIONS OF COMPLEMENT 1. CYTOLYSIS OF FOREIGN ORGANISM BY C5B-9 MAC COMPLEX 2. OPSONIZATION AND PHAGOCYTOSIS C3b, C3bi is coated on microorganisms (opsonization) Receptors for C3b (CR1) and C3bi (CR3) on macrophages and neutrophils can then bind the complement coated bacteria facilitating the phagocytosis reaction 10

11 3. ACTIVATION OF INFLAMMATION AND CHEMOTAXIS OF LEUKOCYTES BY COMPLEMENT ANAPHYLATOXINS (C3A, C4A, C5A) All three peptides mediate: 1. smooth muscle contraction 2. histamine release from mast cells and 3. increase vascular permeability. C5a on binding C5aR mediates chemoattraction of neutrophils, monocytes, macrophages, and eosinophils. C3a is a chemoattractant for eosinophils but not neutrophils. 4. SOLUBILIZATION AND CLEARANCE OF IMMUNE COMPLEXES One of the major roles complement plays is the solubilization and clearance of immune complexes from the circulation. First, C3b and C4b can covalently bind to the Fc region of insoluble immune complexes, disrupting the lattice, and making them soluble. C3b and C4b bound to the immune complex is recognized by the CR1 receptor on erythrocytes facilitating their transport to the liver and spleen. In the liver and spleen the immune complexes are removed and phagocytized by macrophage-like cells. The RBCs are returned to the circulation. Individuals deficient in the early complement components cannot make C3b and C4b. They are therefore predisposed to immune complex diseases such as systemic lupus erythematosus. 11

12 5. ENHANCEMENT OF THE IMMUNE RESPONSE CR2 (CD21) is expressed on B-cells and follicular dendritic cells. This receptor binds the C3d fragment of C3. C3b coated on antigens will be broken down eventually to C3d and C3c fragments by factor I. The C3c fragment is released into the blood with no know function. The C3d fragment remains covalently bound to the antigen. Coating of antigens with C3d facilitates their delivery to germinal centers rich in B and follicular dendritic cells. CR2 also is part of the B-cell co-receptor complex. Binding of C3d coated antigens to CR2 leads to signaling through CD19. Animals deficient in C3 have an impaired immune response to T dependent antigens. 12

13 COMPLEMENT DEFICIENCIES AND ASSOCIATED ABNORMALITIES Human deficiencies in many of the complement proteins have been described. These deficiencies are usually attributable to inherited mutated genes. Genetic deficiencies in classical and alternative pathway components, including C1q, C1r, C4, C2, properdin, and factor D have all been described. C2 deficiency is the most common of the complement deficiencies. Deficiencies in the early classical pathway proteins predispose individuals to the development of systemic lupus erythematosus (SLE). The reason for this is not completely clear, but it is at least partly do to the inability of these individuals to clear immune complexes readily. Because of its central importance in killing bacteria, homozygous C3 deficiency can be lethal, especially in young children if it is not diagnosed. Deficiencies in the terminal component predispose these individuals to recurrent bacterial infections with Neisserial species. Dear Immunology students, There is confusion between designation of the correct term used for the complement C3 convertase, with discrepancies between some sources. We will use the nomenclature provided by the lecturer and use what is listed in the syllabus. Here is our understanding. The nomenclature for complement has undergone revision so that the large, target-bound fragment is consistently given the 'b' designation, while the small, soluble fragment is called 'a'. Thus over the last 10 years texts have begun to reverse 2a and 2b, which were initially named incorrectly by this convention. According to the current (but not universally accepted) nomenclature, the C3 convertase is C4b2b and C4a and C2a are the released fragments; Coico, et al. apparently have not yet adapted this change. 13

14 You will see other opinions in nomenclature, primarily from older texts that have not adopted the new naming structure. Therefore, due to a change in nomenclature in order to maintain the a=smaller and b=larger scheme, the correct C3 Convertase is C4b2b. ANTIGEN-ANTIBODY INTERACTIONS, IMMUNE ASSAYS, EXPERIMENTAL SYSTEMS PRIMARY INTERACTIONS BETWEEN ANTIBODY AND ANTIGEN Antigens and antibodies interact as the result of multiple weak, non-covalent reactions. You should now review these interactions from the Immunogens and Antigens lecture. Due to the relative weakness of these forces, Ab-Ag reactions can be readily dissociated by: a) low or high ph b) by high salt concentrations c) by chaotropic ions. SECONDARY INTERACTIONS BETWEEN ANTIBODY AND ANTIGEN. AGGLUTINATION REACTIONS Definition: The term agglutination infers aggregation of insoluble particles. Aggregation of red blood cells or bacterial cells is routinely used for estimation of the concentration of antibodies in a serum taken from a patient or experimental animal. Definition: The term titer is used to describe the highest dilution of that serum that will agglutinate a standard amount of the cells (i.e. 50 ul of a 1% suspension). PROZONE-Agglutination reactions can sometimes exhibit the phenomenon of prozone. This occurs because very high concentrations of antibodies can totally saturate all epitopes on each cell added so that no cross linking occurs. As the concentration of antibodies is lowered by dilution in succeeding tubes, the numbers of cellular epitopes and antibodies then reach a ratio where effective agglutination occurs. ZETA POTENTIAL-An electrical potential between two like charged particles prevents them from physically associating. The short distance between Fab arms of IgG molecules may not overcome this repulsion, but the larger IgM molecule might be sufficiently large to overcome zeta potential. The high sialic acid density on the surface of red cells is difficult to overcome and the size, coupled with the multivalency, of IgM makes it more efficient as an agglutinator of red cells. COOMBS TEST-The Coombs test can overcome zeta potential by using a second layer of antibodies to bridge cells. If the red cell is coated with IgG antibodies, an antiglobulin 14

15 antiserum can be added (Definition: a serum containing antibodies specific for the Fc region of IgG) and it can then cross-link the IgG antibodies previously bound to the cell thereby agglutinating the red cells. Direct Coombs Test In this assay, patient blood that is suspected of having antibodies already bound to the red cell (i.e. blood from a baby at risk for Erythroblastosis fetalis) is mixed with the antiglobulin serum and positive agglutination is diagnostic for the presence of anti-rh antibodies bound to the red cells. Indirect Coombs Test This is to detect the presence in serum of a non-agglutinating antibody. For example, serum from a pregnant patient suspected of having circulating IgG anti-rh antibodies is mixed with Rh+ red cells, then the antiglobulin is added. Positive agglutination is then diagnostic for the presence of anti-rh in patient serum, indicating that the fetus is at risk for erythroblastosis fetalis. 15

16 PASSIVE AGGLUTINATION Passive agglutination is a way to use the extraordinary sensitivity of agglutination assays to detect antibodies specific for soluble antigens such as thyroglobulin to help diagnose Hashimoto s disease, for example. In this assay, purified soluble thyroglobulin is attached to something particulate such as micro-latex beads or red cells. Then sera containing suspected antibodies specific for thyroglobulin can be titered in a standard agglutination format. If red blood cells are used as the particle, the assay is usually called passive hemagglutination to acknowledge the red cell as the carrier of the antigen. PRECIPITATION REACTIONS Precipitation Reaction in Solution (Fluid Phase Reactions) Antigen-Antibody reactions that result in the formation of visible precipitation of the reactants are classed as secondary manifestations of Ag-Ab reactions. This reaction provided the first quantitative assay for antibody, but is rarely used today. However, understanding the Ab-Ag interactions that lead to this reaction is important, as the immune complexes formed are also found in vivo. In this reaction, various amounts of soluble antigen are added to a fixed amount of serum containing antibody. As illustrated in the figure below, when small amounts of Ag are added, Ab-Ag complexes are formed with excess Ab, and each molecule of Ag is bound by Ab and cross-linked to other Ab molecules. When enough Ag is added, ALL of the antigen and antibody complex and fall out as precipitate (the zone of equivalence). When an excess of Ag is added only small Ag-Ab complexes form (no crosslinking) and the precipitate is reduced. This reaction is affected by the number of binding sites that each Ab has for antigen, and the maximum number of Abs that can be bound by an antigen or particle at one time. This is defined as the valence of the antigen or antibody (see figure below) and valence of Ab and Ag has to be > 2 or precipitation will not occur. It is important to note that the valence of an Ag is almost always less than the number of epitopes on an Ag, since steric considerations limit the number of distinct antibody molecules that can bind to a single antigen at any one time. PRECIPITATION REACTIONS IN GELS Often, precipitation reactions are used for analysis in situations where quantitation is important but in other situations only a qualitative answer is necessary. These latter reactions can be done in a gel matrix that slows down the rate of diffusion of reactants and holds the precipitate in the gel web so that it is effectively immobilezed for visualization either directly or 16

17 with the aid of various staining methods. Several qualitative and quantitative methods are in wide use in medicine today for analysis of numerous hormones, enzymes, toxins, and for analysis of the products of the immune system itself. All methods described below will be designated as qualitative or quantitative methods. OUCHTERLONY DOUBLE DIFFUSION ASSAY The Ouchterlony Assay was developed by Orjan Ouchterlony in the 1950 s and is still in widespread use. It has two important features: a) it is inexpensive to use b) it can be used to compare the relatedness of two antigens (Antigenically, are they totally different, are they the same, or only similar?). The assay is called a Double Diffusion assay because both the antigen and antibodies are diffusing. It is a qualitative assay. Format: Reagents are put in wells made in a thin layer of agar or agarose made up in physiological buffer. The molecules in each well then diffuse slowly into the agar in a radial fashion (diffusion in a circular fashion with an ever-increasing radius). Thus, antigen and antibody slowly diffuse toward one another. A positive result will be that a thin opaque precipitate line or band will form in the agar at right angles to a line connecting the centers of the two wells and it will usually be symmetrical, extending the same distance either side of the line connecting the well centers. The presence of a line is a qualitative assay for the presence of either antibody in the antiserum (using a standard antigen solution) or for the presence of antigen (using a standard antiserum). The most widespread use of the Ouchterlony technique is for comparison of antigens. It has also been used in forensic medicine and in a variety of diagnostic assays. RADIAL IMMUNODIFFUSION Radial immunodiffusion is a type of agar gel precipitation technique that is quantitative. Once again, it relies on having a standardized antiserum or standard antigen on hand in order to analyze the unknown sample. IMMUNOELECTROPHORESIS Immunoelectrophoresis is a variation of the Ouchterlony double diffusion in gel technique. It is designed to analyze complex protein mixtures containing many different antigens. Immunoelectrophoresis is a qualitative assay. It is also used in medical research for following the different steps of a purification protocol to show the disappearance of unwanted proteins when purification of one component from a mixture is desired. NEPHELOMETRY Nephelometry is a widely used methodology for accurately measuring quantities of the Ig classes in serum. Obviously, dramatic increases or decreases in quantities of these could contribute to diagnosis of numerous diseases. In this assay, proteins in the sample react with specific antibody (ie an anti-ige antibody). The mixture is placed in a tube and inserted into the Nephelometer. When light passes through the suspension that contains aggregated particles, a portion of the light is scattered. The scattered light is measured and compared with stored 17

18 standards. Thus, this is a quantitative method using liquid-phase precipitation principles. It can be applied to measuring any soluble substance provided specific antisera are available. WESTERN BLOTS Also called immunoblotting-a mixture of antigens is usually separated by electrophoresis on a gel, transferred onto a medium such as nitrocellulose that binds proteins tightly and then antibodies that have an enzyme covalently attached are poured on the nitrocellulose. Substrate for the enzyme is added, turns colors when enzyme is present, and the colored line shows that the antigen was present. IMMUNOASSAYS DIRECT BINDING IMMUNOASSAYS In first step radioactive antigen is reacted with a limited amount of antibody. In the second step, unlabelled test antigen is mixed with the labeled antigen prior to addition of the antibody. The amount of labeled antigen bound to antibody is then reduced by a factor related to the ratio of labeled to unlabeled antigen in the mixture. The unlabeled antigen effectively competes for available antibody since it is identical immunologically, but unlabeled. A standard curve of inhibition can be generated using precisely known amounts of unlabeled antigen and then test samples containing unknown concentrations of antigen can be analyzed and simply read off the standard curve to find the concentration of antigen in the unknown. SOLID-PHASE IMMUNOASSAYS There are a group of assays in which the antigen or the antibody is coated on the surface of a plastic microplate and sensitive indicators such as radioactivity or enzymatic action are used to detect the presence of Ag or of Ab. There are 5 of these assays classified in two groups according to the types of antigens being analyzed: soluble or cellular. A. SOLUBLE ANTIGENS RADIOIMMUNOASSAY--There are many different formats for doing radioimmunoassay (RIA). Only one will be described here, that is to detect antigen. In this format the following steps are done: a) free antigen is first coated onto the surface of plastic plates and the excess is removed by rinsing out the wells with buffer solutions. b) the remaining plastic surface is then blocked by adding an irrelevant protein solution and washing c) antiserum is added to the plate, incubated and then washed out. This leaves the plate with Ab bound to the Ag that is, in turn, bound (noncovalently) to the plastic. 18

19 d) a radioactive indicator is added which recognizes the Ab but not the Ag. (There is a protein called Protein A derived from Staphylococcus aureus that reacts specifically with the Fc region of most vertebrate IgG molecules. The Protein A can be radiolabeled. ENZYME LINKED IMMUNOADSORBENT ASSAY (ELISA) The ELISA assay is quite similar to the RIA except that the indicator reagent used in ELISA is not radioactive. Instead, the indicator (Protein A) is coupled to an enzyme molecule that converts added substrates to a colored product that can be detected spectrophotometrically due to the color change. The assay is done as diagrammed below: ELISPOT assays Variation of the ELISA method. Incubate with cells instead of soluble antibody. The # of spots after addition of detection antibody and precipitable substrate = the number of cells secreting a specific antibody, thus can be used to determine the frequency of antigen specific B cells. Also used for T cell assays (e.g. the number of T cells producing a cytokine, as illustrated below). Used in biomedical research. 19

20 B. CELLULAR ANTIGENS IMMUNOFLUORESCENCE It is sometimes of diagnostic value to determine if a particular antigen is found on or in the cells of a particular tissue. In this case, assays are needed that can be performed directly on biopsies of tissue and seen using a microscope. The method originally developed by Albert Coons and his colleagues at Harvard involves covalent attachment of fluorescent organic compounds to specific antibodies that then can be used to detect the antigen in question. The fluorescent compounds excite at different wavelengths. This is a highly sensitive and specific assay, and cells individual cells can be stained with up to 12 different compounds. 1. Direct Immunofluorescence-The antibody specific for the antigen in question is directly labeled with the fluorophor and used to identify the antigen. 2. Indirect Immunofluorescence-This is similar to the Coombs reaction discussed earlier (review that if necessary). It is a two step method in which the unlabeled antibody specific for the antigen in question is reacted first with the tissue and the excess antibody is washed away. Then the slide is flooded with a fluorescent anti-ig (preferably Fc specific). This method has the advantage that it is significantly more sensitive than the Direct method. 20

21 IMMUNOHISTOCHEMISTRY is a similar technique. Instead of fluorescent labels, the detection antibodies are labeled with enzymes such as horseradish peroxidase or alkaline phosphatase (these are also used in ELISA). Addition of substrate then colors the membranes of the cells expressing the antigen of interest. FLUORESCENCE ACTIVATED CELL SORTING (FACS) ANALYSIS FACS analysis is used to identify, and sometimes purify, one cell subset from a mixture of cells. This is an extremely effective tool to identify and/or isolate specific cell subsets. The organic fluorescent compounds attached to the detection antibodies are excited by different fluorescent wavelengths, and all emit at different wavelengths as well, allowing for specific detection of the markers. Current instrumentation can detect up to 15 different antigens on one cell (though most investigators use 4 colors at most). Sorting of cells can also be accomplished using antibodies coupled to magnetic beads (magnetic activated cell sorting, or MACS). The cells are then placed over a magnetized column, and any cells with labeled antibody bound to them can be isolated from the unbound population. LYMPHOCYTE FUNCTION ASSAYS Lymphocyte function can be compromised in certain diseases or can occur as a result of a genetic abnormality. A diagnosis can be confirmed in many cases if it is known whether or not the B or T cells are normal, if the existing B cells can make antibodies, or if the T cells can produce the correct cytokines. Mitogen Activation Lipopolysaccharides can cause polyclonal stimulation of B cells in vitro. This activation is accurately measured by incorporation of radioactive nucleosides. Several lectins, including concanavalin A and phytohemagglutinin are effective T cell mitogens. Pokeweed mitogen stimulates polyclonal activation of both B and T cells. 21

22 Numerous assays can measure antibody production by stimulated B cells (ie ELISA). Cytotoxicity assays measure the ability of cytotoxic T cells or NK cells to kill radioactive target cells that express a specific antigen for which the cytotoxic T cells may be sensitive. MONOCLONAL ANTIBODIES AND T CELL HYBRIDOMAS Due to cross reactivity of antibodies and the need for more controllable assays it is sometimes of great advantage to have a homogeneous antibody preparation that is specific for only a single epitope and with high affinity. Since polyclonal antibody mixtures consist of a multitude of antibodies specific for different epitopes on even simple antigens like tetanus toxoid, and the fact that there are an array of different subpopulations of antibodies with different affinities even in the subset specific for a single epitope, significant cross reactions can occur when using polyclonal antibodies for analytical assays. This can lead to misinterpretation of results occasionally. Kohler and Milstein developed a method for making murine antibodies that are monoclonal, that is, all antibodies are derived from a single precursor plasma cell so that all the antibodies in the preparation are identical and derived from the same original clone. T CELL HYBRIDOMAS The general method is also used for making T cell hybridomas. STUDY PROBLEMS: Review the animations provided below and answer the questions that follow to test your understanding

23 F) Study questions for class discussion 1. From memory, draw the classical, lectin, and alternative pathways of complement. Understand how each is activated. 2. Name 5 biological functions mediated by complement activation. 3. Name the complement activation products that mediate each function. 4. What are the three complement anaphylatoxin peptides? 5. How does complement clear immune complexes from the circulation? 6. What complement activation products bind covalently to cell surfaces? 7. Know how the complement activation pathways are affected if a certain component is missing. 8. If a patient is missing a particular complement protein, what disease(s) are they predisposed? 9. On the figure shown below, assign the activation fragments responsible for that function. 23

24 Study questions for Antigen-Antibody Interactions 10. What is the classic example where a Coomb s type agglutination assay would be more sensitive than the direct agglutination method. 11. Describe the steps in setting up a quantitative precipitation reaction. What does the experiment tell you? 12. Make a list of all the immunoassays in this chapter and categorize them as a) Quantitative or as b) Qualitative. 24

25 13. Describe a situation where you would order an Ig class quantitation measurement done on a patient s serum. What instrument would the lab use to do this? 14. Describe the steps in developing an enzyme linked immunosorbent assay. How would you make it quantitative? 15. Write the two equations that together define antigen-antibody Affinity. 25