Microbiology: Detecting Antigen-Antibody Complexes PDF

20.2 Detecting Antigen-Antibody Complexes 787 FIGURE 20.8 The Ouchterlony test places antigen (well A) and antisera (wells 1 through 5) in a gel. The antibodies and antigen diffuse through the gel, causing a precipitin arc to form at the zone of equivalence. In this example, only the a...

20.2 Detecting Antigen-Antibody Complexes 787 FIGURE 20.8 The Ouchterlony test places antigen (well A) and antisera (wells 1 through 5) in a gel. The antibodies and antigen diffuse through the gel, causing a precipitin arc to form at the zone of equivalence. In this example, only the antiserum in well 1 contains antibodies to the antigen. The resulting precipitin arc is stable because the lattice is too large to diffuse through the gel. (credit left: modification of work by Higgins PJ, Tong C, Borenfreund E, Okin RS, Bendich A) Radial Immunodiffusion Assay The radial immunodiffusion (RID) assay is similar to the Ouchterlony assay but is used to precisely quantify antigen concentration rather than to compare different antigens. In this assay, the antiserum is added to tempered agar (liquid agar at slightly above 45 °C), which is poured into a small petri dish or onto a glass slide and allowed to cool. Wells are cut in the cooled agar, and antigen is then added to the wells and allowed to diffuse. As the antigen and antibody interact, they form a zone of precipitation. The square of the diameter of the zone of precipitation is directly proportional to the concentration of antigen. By measuring the zones of precipitation produced by samples of known concentration (see the outer ring of samples in Figure 20.9), we can prepare a standard curve for determining the concentration of an unknown solution. The RID assay is a also useful test for determining the concentration of many serum proteins such as the C3 and C4 complement proteins, among others. FIGURE 20.9 In this radial immunodiffusion (RID) assay, an antiserum is mixed with the agar before it is cooled, and solutions containing antigen are added to each well in increasing concentrations (wells 1–4). An antigen solution of an unknown concentration is added to well 5. The zones of precipitation are measured and plotted against a standard curve to determine the antigen concentration of the unknown sample. (credit circles: modification of work by Kangwa M, Yelemane V, Polat AN, Gorrepati KD, Grasselli M, Fernández-Lahore M) 788 20 Laboratory Analysis of the Immune Response CHECK YOUR UNDERSTANDING Why does a precipitin ring form in a precipitin ring test, and what are some reasons why a ring might not form? Compare and contrast the techniques used in an Ouchterlony assay and a radial immunodiffusion assay. Flocculation Assays A flocculation assay is similar to a precipitin reaction except that it involves insoluble antigens such as lipids. A flocculant is similar to a precipitin in that there is a visible lattice of antigen and antibody, but because lipids are insoluble in aqueous solution, they cannot precipitate. Instead of precipitation, flocculation (foaming) is observed in the test tube fluid. MICRO CONNECTIONS Using Flocculation to Test for Syphilis Syphilis is a sexually transmitted infection that can cause severe, chronic disease in adults. In addition, it is readily passed from infected people to their newborns during pregnancy and childbirth, often resulting in stillbirth or serious long-term health problems for the infant. Unfortunately, syphilis can also be difficult to diagnose in people, because it is often asymptomatic, especially in females. In addition, the causative agent, the bacterium Treponema pallidum, is both difficult to grow on conventional lab media and too small to see using routine microcopy. For these reasons, presumptive diagnoses of syphilis are generally confirmed indirectly in the laboratory using tests that detect antibodies to treponemal antigens. In 1906, German scientist August von Wassermann (1866–1925) introduced the first test for syphilis that relied on detecting anti-treponemal antibodies in the patient’s blood. The antibodies detected in the Wassermann test were antiphospholipid antibodies that are nonspecific to T. pallidum. Their presence can assist in the diagnosis of syphilis, but because they are nonspecific, they can also lead to false-positive results in patients with other diseases and autoimmune conditions. The original Wasserman test has been modified over the years to minimize false-positives and is now known as the Venereal Disease Research Lab test, better known by its acronym, the VDRL test. To perform the VDRL test, patient serum or cerebral spinal fluid is placed on a slide with a mixture of cardiolipin (an antigenic phospholipid found in the mitochondrial membrane of various pathogens), lecithin, and cholesterol. The lecithin and cholesterol stabilize the reaction and diminish false positives. Anti-treponemal antibodies from an infected patient’s serum will bind cardiolipin and form a flocculant. Although the VDRL test is more specific than the original Wassermann assay, false positives may still occur in patients with autoimmune diseases that cause extensive cell damage (e.g., systemic lupus erythematosus). Neutralization Assay To cause infection, viruses must bind to receptors on host cells. Antiviral antibodies can neutralize viral infections by coating the virions, blocking the binding (Figure 18.7). This activity neutralizes virions and can result in the formation of large antibody-virus complexes (which are readily removed by phagocytosis) or by antibody binding to the virus and blocking its binding to host cell receptors. This neutralization activity is the basis of neutralization assays, sensitive assays used for diagnoses of viral infections. When viruses infect cells, they often cause damage (cytopathic effects) that may include lysis of the host cells. Cytopathic effects can be visualized by growing host cells in a petri dish, covering the cells with a thin layer of agar, and then adding virus (see Isolation, Culture, and Identification of Viruses). The virus will diffuse very slowly through the agar. A virus will enter a host cell, proliferate (causing cell damage), be released from the dead host cell, and then move to neighboring cells. As more and more cells die, plaques of dead cells will form (Figure 20.10). During the course of a viral infection, the patient will mount an antibody response to the virus, and we can quantify those antibodies using a plaque reduction assay. To perform the assay, a serial dilution is carried out on a serum sample. Each dilution is then mixed with a standardized amount of the suspect virus. Any virus-specific antibodies in the serum will neutralize some of the virus. The suspensions are then added to host cells in culture to allow any Access for free at openstax.org  20.2 Detecting Antigen-Antibody Complexes 789 nonneutralized virus to infect the cells and form plaques after several days. The titer is defined as the reciprocal of the highest dilution showing a 50% reduction in plaques. Titer is always expressed as a whole number. For example, if a 1/64 dilution was the highest dilution to show 50% plaque reduction, then the titer is 64. The presence of antibodies in the patient’s serum does not tell us whether the patient is currently infected or was infected in the past. Current infections can be identified by waiting two weeks and testing another serum sample. A four-fold increase in neutralizing titer in this second sample indicates a new infection. FIGURE 20.10 In a neutralization assay, antibodies in patient serum neutralize viruses added to the wells, preventing the formation of plaques. In the assay pictured, the wells with numerous plaques (white patches) contain a low concentration of antibodies. The wells with relatively few plaques have a high concentration of antibodies. (credit: modification of work by Centers for Disease Control and Prevention) CHECK YOUR UNDERSTANDING In a neutralization assay, if a patient’s serum has high numbers of antiviral antibodies, would you expect to see more or fewer plaques? Immunoelectrophoresis When a patient has elevated protein levels in the blood or is losing protein in the urine, a clinician will often order a polyacrylamide gel electrophoresis (PAGE) assay (see Visualizing and Characterizing DNA, RNA, and Protein). This assay compares the relative abundance of the various types of serum proteins. Abnormal protein electrophoresis patterns can be further studied using immunoelectrophoresis (IEP). The IEP begins by running a PAGE. Antisera against selected serum proteins are added to troughs running parallel to the electrophoresis track, forming precipitin arcs similar to those seen in an Ouchterlony assay (Figure 20.11). This allows the identification of abnormal immunoglobulin proteins in the sample. IEP is particularly useful in the diagnosis of multiple myeloma, a cancer of antibody-secreting cells. Patients with multiple myeloma cannot produce healthy antibodies; instead they produce abnormal antibodies that are monoclonal proteins (M proteins). Thus, patients with multiple myeloma will present with elevated serum protein levels that show a distinct band in the gamma globulin region of a protein electrophoresis gel and a sharp spike (in M protein) on the densitometer scan rather than the normal broad smear (Figure 20.12). When antibodies against the various types of antibody heavy and light chains are used to form precipitin arcs, the M protein will cause distinctly skewed arcs against one class of heavy chain and one class of light chain as seen in Figure 20.11. 790 20 Laboratory Analysis of the Immune Response FIGURE 20.11 (a) This graph shows normal measurements of serum proteins. (b) This photograph shows an immunoelectrophoresis of urine. After electrophoresis, antisera were added to the troughs and the precipitin arcs formed, illustrating the distribution of specific proteins. The skewed arcs (arrows) help to diagnose multiple myeloma. (credit a, b: modification of work by Izawa S, Akimoto T, Ikeuchi H, Kusano E, Nagata D) MICRO CONNECTIONS Protein Electrophoresis and the Characterization of Immunoglobulin Structure The advent of electrophoresis ultimately led to researching and understanding the structure of antibodies. When 7 Swedish biochemist Arne Tiselius (1902–1971) published the first protein electrophoresis results in 1937, he could identify the protein albumin (the smallest and most abundant serum protein) by the sharp band it produced in the gel. The other serum proteins could not be resolved in a simple protein electrophoresis, so he named the three broad bands, with many proteins in each band, alpha, beta, and gamma globulins. Two years later, American immunologist Elvin Kabat (1914–2000) traveled to Sweden to work with Tiselius using this new technique and 8 showed that antibodies migrated as gamma globulins. With this new understanding in hand, researchers soon learned that multiple myeloma, because it is a cancer of antibody-secreting cells, could be tentatively diagnosed by the presence of a large M spike in the gamma-globulin region by protein electrophoresis. Prior to this discovery, studies on immunoglobulin structure had been minimal, because of the difficulty of obtaining pure samples to study. Sera from multiple myeloma patients proved to be an excellent source of highly enriched monoclonal immunoglobulin, providing the raw material for studies over the next 20-plus years that resulted in the elucidation of the structure of immunoglobulin. FIGURE 20.12 Electrophoresis patterns of myeloma (right) and normal sera (left). The proteins have been stained; when the density of each band is quantified by densitometry, the data produce the bar graph on the right. Both gels show the expected dense band of albumin at the bottom and an abnormal spike in the gamma-globulin region. (credit: modification of work by Soodgupta D, Hurchla MA, Jiang M, 7 Tiselius, Arne, “Electrophoresis of Serum Globulin: Electrophoretic Analysis of Normal and Immune Sera,” Biochemical Journal 31, no. 9 (1937): 1464. 8 Tiselius, Arne and Elvin A. Kabat. “An Electrophoretic Study of Immune Sera and Purified Antibody Preparations,” The Journal of Experimental Medicine 69, no. 1 (1939): 119-31. Access for free at openstax.org  20.2 Detecting Antigen-Antibody Complexes 791 Zheleznyak A, Weilbaecher KN, Anderson CJ, Tomasson MH, Shokeen M) CHECK YOUR UNDERSTANDING In general, what does an immunoelectrophoresis assay accomplish? Immunoblot Assay: The Western Blot After performing protein gel electrophoresis, specific proteins can be identified in the gel using antibodies. This technique is known as the western blot. Following separation of proteins by PAGE, the protein antigens in the gel are transferred to and immobilized on a nitrocellulose membrane. This membrane can then be exposed to a primary antibody produced to specifically bind to the protein of interest. A second antibody equipped with a molecular beacon will then bind to the first. These secondary antibodies are coupled to another molecule such as an enzyme or a fluorophore (a molecule that fluoresces when excited by light). When using antibodies coupled to enzymes, a chromogenic substrate for the enzyme is added. This substrate is usually colorless but will develop color in the presence of the antibody. The fluorescence or substrate coloring identifies the location of the specific protein in the membrane to which the antibodies are bound (Figure 20.13). Typically, polyclonal antibodies are used for western blot assays. They are more sensitive than mAbs because of their ability to bind to various epitopes of the primary antigen, and the signal from polyclonal antibodies is typically stronger than that from mAbs. Monoclonal antibodies can also be used; however, they are much more expensive to produce and are less sensitive, since they are only able to recognize one specific epitope. Several variations of the western blot are useful in research. In a southwestern blot, proteins are separated by SDS- PAGE, blotted onto a nitrocellulose membrane, allowed to renature, and then probed with a fluorescently or radioactively labeled DNA probe; the purpose of the southwestern is to identify specific DNA-protein interactions. Far-western blots are carried out to determine protein-protein interactions between immobilized proteins (separated by SDS-PAGE, blotted onto a nitrocellulose membrane, and allowed to renature) and non-antibody protein probes. The bound non-antibody proteins that interact with the immobilized proteins in a far-western blot may be detected by radiolabeling, fluorescence, or the use of an antibody with an enzymatic molecular beacon. 792 20 Laboratory Analysis of the Immune Response FIGURE 20.13 (a) This diagram summarizes the process of western blotting. Antibodies are used to identify specific bands on the protein gel. (b) A western blot test for antibodies against HIV. The top strip is the negative control; the next strip is the positive control. The bottom two strips are patient serum samples containing antibodies. (credit a: modification of work by “Bensaccount”/Wikimedia Commons) CHECK YOUR UNDERSTANDING What is the function of the enzyme in the immunoblot assay? Access for free at openstax.org  20.2 Detecting Antigen-Antibody Complexes 793 Complement-Mediated Immunoassay One of the key functions of antibodies is the activation (fixation) of complement. When antibody binds to bacteria, for example, certain complement proteins recognize the bound antibody and activate the complement cascade. In response, other complement proteins bind to the bacteria where some serve as opsonins to increase the efficiency of phagocytosis and others create holes in gram-negative bacterial cell membranes, causing lysis. This lytic activity can be used to detect the presence of antibodies against specific antigens in the serum. Red blood cells are good indicator cells to use when evaluating complement-mediated cytolysis. Hemolysis of red blood cells releases hemoglobin, which is a brightly colored pigment, and hemolysis of even a small number of red cells will cause the solution to become noticeably pink (Figure 20.14). This characteristic plays a role in the complement fixation test, which allows the detection of antibodies against specific pathogens. The complement fixation test can be used to check for antibodies against pathogens that are difficult to culture in the lab such as fungi, viruses, or the bacteria Chlamydia. To perform the complement fixation test, antigen from a pathogen is added to patient serum. If antibodies to the antigen are present, the antibody will bind the antigen and fix all the available complement. When red blood cells and antibodies against red blood cells are subsequently added to the mix, there will be no complement left to lyse the red cells. Thus, if the solution remains clear, the test is positive. If there are no antipathogen antibodies in the patient’s serum, the added antibodies will activate the complement to lyse the red cells, yielding a negative test (Figure 20.14). 794 20 Laboratory Analysis of the Immune Response FIGURE 20.14 The complement fixation test is used to determine whether a patient’s serum contains antibodies to a specific antigen. If it does, complement fixation will occur, and there will be no complement available to lyse the antibody-bound sheep red blood cells that are added to the solution in the next step. If the sample does not contain antibodies to the antigen, hemolysis of the sheep blood cells will be observed. Access for free at openstax.org  20.3 Agglutination Assays 795 LINK TO LEARNING View this video (https://openstax.org/l/22complfixatst) to see an outline of the steps of the complement fixation test. CHECK YOUR UNDERSTANDING In a complement fixation test, if the serum turns pink, does the patient have antibodies to the antigen or not? Explain. Table 20.2 summarizes the various types of antibody-antigen assays discussed in this section. Mechanisms of Select Antibody-Antigen Assays Type of Mechanism Examples Assay Precipitation Antibody binds to soluble antigen, forming a Precipitin ring test to visualize lattice visible precipitin formation in solution Immunoelectrophoresis to examine distribution of antigens following electrophoresis Ouchterlony assay to compare diverse antigens Radial immunodiffusion assay to quantify antigens Flocculation Antibody binds to insoluble molecules in VDRL test for syphilis suspension, forming visible aggregates Neutralization Antibody binds to virus, blocking viral entry into Plaque reduction assay for detecting target cells and preventing formation of plaques presence of neutralizing antibodies in patient sera Complement Antibody binds to antigen, inducing complement Complement fixation test for patient activation activation and leaving no complement to lyse antibodies against hard-to-culture bacteria red blood cells such as Chlamydia TABLE 20.2 20.3 Agglutination Assays LEARNING OBJECTIVES By the end of this section, you will be able to: Compare direct and indirect agglutination Identify various uses of hemagglutination in the diagnosis of disease Explain how blood types are determined Explain the steps used to cross-match blood to be used in a transfusion In addition to causing precipitation of soluble molecules and flocculation of molecules in suspension, antibodies can also clump together cells or particles (e.g., antigen-coated latex beads) in a process called agglutination (Figure 796 20 Laboratory Analysis of the Immune Response 18.9). Agglutination can be used as an indicator of the presence of antibodies against bacteria or red blood cells. Agglutination assays are usually quick and easy to perform on a glass slide or microtiter plate (Figure 20.15). Microtiter plates have an array of wells to hold small volumes of reagents and to observe reactions (e.g., agglutination) either visually or using a specially designed spectrophotometer. The wells come in many different sizes for assays involving different volumes of reagents. FIGURE 20.15 Microtiter plates are used for conducting numerous reactions simultaneously in an array of wells. (credit: modification of work by “Microrao”/Wikimedia) Agglutination of Bacteria and Viruses The use of agglutination tests to identify streptococcal bacteria was developed in the 1920s by Rebecca Lancefield 9 working with her colleagues A.R. Dochez and Oswald Avery. She used antibodies to identify M protein, a virulence factor on streptococci that is necessary for the bacteria’s ability to cause strep throat. Production of antibodies against M protein is crucial in mounting a protective response against the bacteria. Lancefield used antisera to show that different strains of the same species of streptococci express different versions of M protein, which explains why children can come down with strep throat repeatedly. Lancefield classified beta- hemolytic streptococci into many groups based on antigenic differences in group-specific polysaccharides located in the bacterial cell wall. The strains are called serovars because they are differentiated using antisera. Identifying the serovars present in a disease outbreak is important because some serovars may cause more severe disease than others. The method developed by Lancefield is a direct agglutination assay, since the bacterial cells themselves agglutinate. A similar strategy is more commonly used today when identifying serovars of bacteria and viruses; however, to improve visualization of the agglutination, the antibodies may be attached to inert latex beads. This technique is called an indirect agglutination assay (or latex fixation assay), because the agglutination of the beads is a marker for antibody binding to some other antigen (Figure 20.16). Indirect assays can be used to detect the presence of either antibodies or specific antigens. 9 Lancefield, Rebecca C., “The Antigenic Complex of Streptococcus haemoliticus. I. Demonstration of a Type-Specific Substance in Extracts of Streptococcus haemolyticus,” The Journal of Experimental Medicine 47, no. 1 (1928): 91-103. Access for free at openstax.org  20.3 Agglutination Assays 797 FIGURE 20.16 Antibodies against six different serovars of Group A strep were attached to latex beads. Each of the six antibody preparations was mixed with bacteria isolated from a patient. The tiny clumps seen in well 4 are indicative of agglutination, which is absent from all other wells. This indicates that the serovar associated with well 4 is present in the patient sample. (credit: modification of work by American Society for Microbiology) To identify antibodies in a patient’s serum, the antigen of interest is attached to latex beads. When mixed with patient serum, the antibodies will bind the antigen, cross-linking the latex beads and causing the beads to agglutinate indirectly; this indicates the presence of the antibody (Figure 20.17). This technique is most often used when looking for IgM antibodies, because their structure provides maximum cross-linking. One widely used example of this assay is a test for rheumatoid factor (RF) to confirm a diagnosis of rheumatoid arthritis. RF is, in fact, the presence of IgM antibodies that bind to the patient’s own IgG. RF will agglutinate IgG-coated latex beads. In the reverse test, soluble antigens can be detected in a patient’s serum by attaching specific antibodies (commonly mAbs) to the latex beads and mixing this complex with the serum (Figure 20.17). Agglutination tests are widely used in underdeveloped countries that may lack appropriate facilities for culturing bacteria. For example, the Widal test, used for the diagnosis of typhoid fever, looks for agglutination of Salmonella enterica subspecies typhi in patient sera. The Widal test is rapid, inexpensive, and useful for monitoring the extent of an outbreak; however, it is not as accurate as tests that involve culturing of the bacteria. The Widal test frequently produces false positives in patients with previous infections with other subspecies of Salmonella, as well as false negatives in patients with hyperproteinemia or immune deficiencies. In addition, agglutination tests are limited by the fact that patients generally do not produce detectable levels of antibody during the first week (or longer) of an infection. A patient is said to have undergone seroconversion when antibody levels reach the threshold for detection. Typically, seroconversion coincides with the onset of signs and symptoms of disease. However, in an HIV infection, for example, it generally takes 3 weeks for seroconversion to take place, and in some instances, it may take much longer. Similar to techniques for the precipitin ring test and plaque assays, it is routine to prepare serial two-fold dilutions of the patient’s serum and determine the titer of agglutinating antibody present. Since antibody levels change over time in both primary and secondary immune responses, by checking samples over time, changes in antibody titer can be detected. For example, a comparison of the titer during the acute phase of an infection versus the titer from the convalescent phase will distinguish whether an infection is current or has occurred in the past. It is also possible to monitor how well the patient’s immune system is responding to the pathogen. 798 20 Laboratory Analysis of the Immune Response FIGURE 20.17 (a) Latex beads coated with an antigen will agglutinate when mixed with patient serum if the serum contains IgM antibodies against the antigen. (b) Latex beads coated with antibodies will agglutinate when mixed with patient serum if the serum contains antigens specific to the antibodies. LINK TO LEARNING Watch this video (https://openstax.org/l/22agglrealatbe) that demonstrates agglutination reactions with latex beads. CHECK YOUR UNDERSTANDING How is agglutination used to distinguish serovars from each other? In a latex bead assay to test for antibodies in a patient's serum, with what are the beads coated? What has happened when a patient has undergone seroconversion? Hemagglutination Agglutination of red blood cells is called hemagglutination. One common assay that uses hemagglutination is the direct Coombs’ test, also called the direct antihuman globulin test (DAT), which generally looks for nonagglutinating antibodies. The test can also detect complement attached to red blood cells. The Coombs’ test is often employed when a newborn has jaundice, yellowing of the skin caused by high blood concentrations of bilirubin, a product of the breakdown of hemoglobin in the blood. The Coombs’ test is used to determine whether the child’s red blood cells have been bound by antibodies during pregnancy. These antibodies would activate complement, leading to red blood cell lysis and the subsequent jaundice. Other conditions that can cause positive direct Coombs’ tests include hemolytic transfusion reactions, autoimmune hemolytic anemia, infectious mononucleosis (caused by Epstein-Barr virus), syphilis, and Mycoplasma pneumonia. A positive direct Coombs’ test may also be seen in some cancers and as an allergic reaction to some drugs (e.g., penicillin). The antibodies bound to red blood cells in these conditions are most often IgG, and because of the orientation of the antigen-binding sites on IgG and the comparatively large size of a red blood cell, it is unlikely that any visible agglutination will occur. However, the presence of IgG bound to red blood cells can be detected by adding Coombs’ reagent, an antiserum containing antihuman IgG antibodies (that may be combined with anti-complement) (Figure 20.18). The Coombs’ reagent links the IgG attached to neighboring red blood cells and thus promotes agglutination. There is also an indirect Coombs’ test known as the indirect antiglobulin test (IAT). This screens an individual for Access for free at openstax.org  20.3 Agglutination Assays 799 antibodies against red blood cell antigens (other than the A and B antigens) that are unbound in a patient’s serum (Figure 20.18). IAT can be used to screen pregnant people for antibodies that may cause hemolytic disease of the newborn. It can also be used prior to giving blood transfusions. More detail on how the IAT is performed is discussed below. FIGURE 20.18 The steps in direct and indirect Coombs’ tests are shown in the illustration. Antibodies that bind to red blood cells are not the only cause of hemagglutination. Some viruses also bind to red blood cells, and this binding can cause agglutination when the viruses cross-link the red blood cells. For example, influenza viruses have two different types of viral spikes called neuraminidase (N) and hemagglutinin (H), the latter 800 20 Laboratory Analysis of the Immune Response named for its ability to agglutinate red blood cells (see Viruses). Thus, we can use red blood cells to detect the presence of influenza virus by direct hemagglutination assays (HA), in which the virus causes visible agglutination of red blood cells. The mumps and rubella viruses can also be detected using HA. Most frequently, a serial dilution viral agglutination assay is used to measure the titer or estimate the amount of virus produced in cell culture or for vaccine production. A viral titer can be determined using a direct HA by making a serial dilution of the sample containing the virus, starting with a high concentration of sample that is then diluted in a series of wells. The highest dilution producing visible agglutination is the titer. The assay is carried out in a microtiter plate with V- or round-bottomed wells. In the presence of agglutinating viruses, the red blood cells and virus clump together and produce a diffuse mat over the bottom of the well. In the absence of virus, the red blood cells roll or sediment to the bottom of the well and form a dense pellet, which is why flat-bottomed wells cannot be used (Figure 20.19). A modification of the HA assay can be used to determine the titer of antiviral antibodies. The presence of these antibodies in a patient’s serum or in a lab-produced antiserum will neutralize the virus and block it from agglutinating the red cells, making this a viral hemagglutination inhibition assay (HIA). In this assay, patient serum is mixed with a standardized amount of virus. After a short incubation, a standardized amount of red blood cells is added and hemagglutination is observed. The titer of the patient’s serum is the highest dilution that blocks agglutination (Figure 20.20). FIGURE 20.19 A viral suspension is mixed with a standardized amount of red blood cells. No agglutination of red blood cells is visible when the virus is absent, and the cells form a compact pellet at the bottom of the well. In the presence of virus, a diffuse pink precipitate forms in the well. (credit bottom: modification of work by American Society for Microbiology) Access for free at openstax.org  20.3 Agglutination Assays 801 FIGURE 20.20 In this HIA, serum containing antibodies to influenzavirus underwent serial two-fold dilutions in a microtiter plate. Red blood cells were then added to the wells. Agglutination only occurred in those wells where the antibodies were too dilute to neutralize the virus. The highest dilution of patient serum that blocks agglutination is the titer of antibody in the patient’s serum. In the case of this test, Sample A shows a titer of 64, and Sample C shows a titer of 32. (credit: modification of work by Evan Burkala) CHECK YOUR UNDERSTANDING What is the mechanism by which viruses are detected in a hemagglutination assay? Which hemagglutination result tells us the titer of virus in a sample? EYE ON ETHICS Animals in the Laboratory Much of what we know today about the human immune system has been learned through research conducted using animals—primarily, mammals—as models. Besides research, mammals are also used for the production of most of the antibodies and other immune system components needed for immunodiagnostics. Vaccines, diagnostics, therapies, and translational medicine in general have all been developed through research with animal models. Consider some of the common uses of laboratory animals for producing immune system components. Guinea pigs are used as a source of complement, and mice are the primary source of cells for making mAbs. These mAbs can be used in research and for therapeutic purposes. Antisera are raised in a variety of species, including horses, sheep, goats, and rabbits. When producing an antiserum, the animal will usually be injected at least twice, and adjuvants may be used to boost the antibody response. The larger animals used for making antisera will have blood harvested repeatedly over long periods of time, with little harm to the animals, but that is not usually the case for rabbits. Although we can obtain a few milliliters of blood from the ear veins of rabbits, we usually need larger volumes, which results in the deaths of the animals. We also use animals for the study of disease. The only way to grow Treponema pallidum for the study of syphilis is in living animals. Many viruses can be grown in cell culture, but growth in cell culture tells us very little about 802 20 Laboratory Analysis of the Immune Response how the immune system will respond to the virus. When working on a newly discovered disease, we still employ Koch’s postulates, which require causing disease in lab animals using pathogens from pure culture as a crucial step in proving that a particular microorganism is the cause of a disease. Studying the proliferation of bacteria and viruses in animal hosts, and how the host immune system responds, has been central to microbiological research for well over 100 years. While the practice of using laboratory animals is essential to scientific research and medical diagnostics, many people strongly object to the exploitation of animals for human benefit. This ethical argument is not a new one—indeed, one of Charles Darwin's daughters was an active antivivisectionist (vivisection is the practice of cutting or dissecting a live animal to study it). Most scientists acknowledge that there should be limits on the extent to which animals can be exploited for research purposes. Ethical considerations have led the National Institutes of Health (NIH) to develop strict regulations on the types of research that may be performed. These regulations also include guidelines for the humane treatment of lab animals, setting standards for their housing, care, and euthanization. The NIH document “Guide for the Care and Use of Laboratory Animals” makes it clear that the use of animals in research is a privilege granted by society to researchers. The NIH guidelines are based on the principle of the three R’s: replace, refine, and reduce. Researchers should strive to replace animal models with nonliving models, replace vertebrates with invertebrates whenever possible, or use computer-models when applicable. They should refine husbandry and experimental procedures to reduce pain and suffering, and use experimental designs and procedures that reduce the number of animals needed to obtain the desired information. To obtain funding, researchers must satisfy NIH reviewers that the research justifies the use of animals and that their use is in accordance with the guidelines. At the local level, any facility that uses animals and receives federal funding must have an Institutional Animal Care and Use Committee (IACUC) that ensures that the NIH guidelines are being followed. The IACUC must include researchers, administrators, a veterinarian, and at least one person with no ties to the institution, that is, a concerned citizen. This committee also performs inspections of laboratories and protocols. For research involving human subjects, an Institutional Review Board (IRB) ensures that proper guidelines are followed. LINK TO LEARNING Visit this site (https://openstax.org/l/22NIHcareuseani) to view the NIH Guide for the Care and Use of Laboratory Animals. Blood Typing and Cross-Matching In addition to antibodies against bacteria and viruses to which they have previously been exposed, most individuals also carry antibodies against blood types other than their own. There are presently 33 immunologically important blood-type systems, many of which are restricted within various ethnic groups or rarely result in the production of antibodies. The most important and perhaps best known are the ABO and Rh blood groups (see Figure 19.4). When units of blood are being considered for transfusion, pretransfusion blood testing must be performed. For the blood unit, commercially prepared antibodies against the A, B, and Rh antigens are mixed with red blood cells from the units to initially confirm that the blood type on the unit is accurate. Once a unit of blood has been requested for transfusion, it is vitally important to make sure the donor (unit of blood) and recipient (patient) are compatible for these crucial antigens. In addition to confirming the blood type of the unit, the patient’s blood type is also confirmed using the same commercially prepared antibodies to A, B, and Rh. For example, as shown in Figure 20.21, if the donor blood is A-positive, it will agglutinate with the anti-A antiserum and with the anti-Rh antiserum. If no agglutination is observed with any of the sera, then the blood type would be O-negative. Following determination of the blood type, immediately prior to releasing the blood for transfusion, a cross-match is performed in which a small aliquot of the donor red blood cells are mixed with serum from the patient awaiting transfusion. If the patient does have antibodies against the donor red blood cells, hemagglutination will occur. To confirm any negative test results and check for sensitized red blood cells, Coombs’ reagent may be added to the mix Access for free at openstax.org  20.3 Agglutination Assays 803 to facilitate visualization of the antibody-red blood cell interaction. Under some circumstances, a minor cross-match may be performed as well. In this assay, a small aliquot of donor serum is mixed with patient red blood cells. This allows the detection of agglutinizing antibodies in the donor serum. This test is rarely necessary because transfusions generally use packed red blood cells with most of the plasma removed by centrifugation. Red blood cells have many other antigens in addition to ABO and Rh. While most people are unlikely to have antibodies against these antigens, people who have had multiple pregnancies or patients who have had multiple transfusions may have them because of repeated exposure. For this reason, an antibody screen test is used to determine if such antibodies are present. Patient serum is checked against commercially prepared, pooled, type O red blood cells that express these antigens. If agglutination occurs, the antigen to which the patient is responding must be identified and determined not to be present in the donor unit. FIGURE 20.21 This sample of a commercially produced “bedside” card enables quick typing of both a recipient’s and donor’s blood before transfusion. The card contains three reaction sites or wells. One is coated with an anti-A antibody, one with an anti-B antibody, and one with an anti-Rh antibody. Agglutination of red blood cells in a given site indicates a positive identification of the blood antigens: in this case, A and Rh antigens for blood type A-positive. CHECK YOUR UNDERSTANDING If a patient's blood agglutinates with anti-B serum, what is the patient’s blood type? What is a cross-match assay, and why is it performed? Table 20.3 summarizes the various kinds of agglutination assays discussed in this section. Mechanisms of Select Antibody-Antigen Assays Type of Assay Mechanism Example Agglutination Direct: Antibody is used to clump bacterial cells or other Serotyping bacteria large structures Indirect: Latex beads are coupled with antigen or antibody Confirming the presence of to look for antibody or antigen, respectively, in patient rheumatoid factor (IgM- serum binding Ig) in patient serum Hemagglutination Direct: Some bacteria and viruses cross-link red blood cells Diagnosing influenza, and clump them together mumps, and measles Direct Coombs’ test (DAT): Detects nonagglutinating Checking for maternal antibodies or complement proteins on red blood cells in antibodies binding to vivo neonatal red blood cells TABLE 20.3 804 20 Laboratory Analysis of the Immune Response Mechanisms of Select Antibody-Antigen Assays Type of Assay Mechanism Example Indirect Coombs’ test (IAT): Screens an individual for Performing pretransfusion antibodies against red blood cell antigens (other than the A blood testing and B antigens) that are unbound in a patient’s serum in vitro Viral hemagglutination inhibition: Uses antibodies from a Diagnosing various viral patient to inhibit viral agglutination diseases by the presence of patient antibodies against the virus Blood typing and cross-matching: Detects ABO, Rh, and Matches donor blood to minor antigens in the blood recipient immune requirements TABLE 20.3 20.4 EIAs and ELISAs LEARNING OBJECTIVES By the end of this section, you will be able to: Explain the differences and similarities between EIA, FEIA, and ELISA Describe the difference and similarities between immunohistochemistry and immunocytochemistry Describe the different purposes of direct and indirect ELISA Similar to the western blot, enzyme immunoassays (EIAs) use antibodies to detect the presence of antigens. However, EIAs differ from western blots in that the assays are conducted in microtiter plates or in vivo rather than on an absorbent membrane. There are many different types of EIAs, but they all involve an antibody molecule whose constant region binds an enzyme, leaving the variable region free to bind its specific antigen. The addition of a substrate for the enzyme allows the antigen to be visualized or quantified (Figure 20.22). In EIAs, the substrate for the enzyme is most often a chromogen, a colorless molecule that is converted into a colored end product. The most widely used enzymes are alkaline phosphatase and horseradish peroxidase for which appropriate substrates are readily available. In some EIAs, the substrate is a fluorogen, a nonfluorescent molecule that the enzyme converts into a fluorescent form. EIAs that utilize a fluorogen are called fluorescent enzyme immunoassays (FEIAs). Fluorescence can be detected by either a fluorescence microscope or a spectrophotometer. FIGURE 20.22 Enzyme immunoassays, such as the direct ELISA shown here, use an enzyme-antibody conjugate to deliver a detectable substrate to the site of an antigen. The substrate may be a colorless molecule that is converted into a colored end product or an inactive Access for free at openstax.org  20.4 EIAs and ELISAs 805 fluorescent molecule that fluoresces after enzyme activation. (credit: modification of work by “Cavitri”/Wikimedia Commons) MICRO CONNECTIONS The MMR Titer The MMR vaccine is a combination vaccine that provides protection against measles, mumps, and rubella (German measles). Most people receive the MMR vaccine as children and thus have antibodies against these diseases. However, for various reasons, even vaccinated individuals may become susceptible to these diseases again later in life. For example, some children may receive only one round of the MMR vaccine instead of the recommended two. In addition, the titer of protective antibodies in an individual’s body may begin to decline with age or as the result of some medical conditions. To determine whether the titer of antibody in an individual’s bloodstream is sufficient to provide protection, an MMR titer test can be performed. The test is a simple immunoassay that can be done quickly with a blood sample. The results of the test will indicate whether the individual still has immunity or needs another dose of the MMR vaccine. Submitting to an MMR titer is often a pre-employment requirement for healthcare workers, especially those who will frequently be in contact with young children or immunocompromised patients. Were a healthcare worker to become infected with measles, mumps, or rubella, the individual could easily pass these diseases on to susceptible patients, leading to an outbreak. Depending on the results of the MMR titer, healthcare workers might need to be revaccinated prior to beginning work. Immunostaining One powerful use of EIA is immunostaining, in which antibody-enzyme conjugates enhance microscopy. Immunohistochemistry (IHC) is used for examining whole tissues. As seen in Figure 20.23, a section of tissue can be stained to visualize the various cell types. In this example, a mAb against CD8 was used to stain CD8 cells in a section of tonsil tissue. It is now possible to count the number of CD8 cells, determine their relative numbers versus the other cell types present, and determine the location of these cells within this tissue. Such data would be useful for studying diseases such as AIDS, in which the normal function of CD8 cells is crucial for slowing disease progression. Immunocytochemistry (ICC) is another valuable form of immunostaining. While similar to IHC, in ICC, extracellular matrix material is stripped away, and the cell membrane is etched with alcohol to make it permeable to antibodies. This allows antibodies to pass through the cell membrane and bind to specific targets inside the cell. Organelles, cytoskeletal components, and other intracellular structures can be visualized in this way. While some ICC techniques use EIA, the enzyme can be replaced with a fluorescent molecule, making it a fluorescent immunoassay. 806 20 Laboratory Analysis of the Immune Response FIGURE 20.23 Enzyme-linked antibodies against CD8 were used to stain the CD8 cells in this preparation of bone marrow using a chromogen. (credit: modification of work by Yamashita M, Fujii Y, Ozaki K, Urano Y, Iwasa M, Nakamura S, Fujii S, Abe M, Sato Y, Yoshino T) CHECK YOUR UNDERSTANDING What is the difference between immunohistochemistry and immunocytochemistry? What must be true of the product of the enzymatic reaction used in immunohistochemistry? Enzyme-linked Immunosorbent Assays (ELISAs) The enzyme-linked immunosorbent assays (ELISAs) are widely used EIAs. In the direct ELISA, antigens are immobilized in the well of a microtiter plate. An antibody that is specific for a particular antigen and is conjugated to an enzyme is added to each well. If the antigen is present, then the antibody will bind. After washing to remove any unbound antibodies, a colorless substrate (chromogen) is added. The presence of the enzyme converts the substrate into a colored end product (Figure 20.22). While this technique is faster because it only requires the use of one antibody, it has the disadvantage that the signal from a direct ELISA is lower (lower sensitivity). In a sandwich ELISA, the goal is to use antibodies to precisely quantify specific antigen present in a solution, such as antigen from a pathogen, a serum protein, or a hormone from the blood or urine to list just a few examples. The first step of a sandwich ELISA is to add the primary antibody to all the wells of a microtiter plate (Figure 20.24). The antibody sticks to the plastic by hydrophobic interactions. After an appropriate incubation time, any unbound antibody is washed away. Comparable washes are used between each of the subsequent steps to ensure that only specifically bound molecules remain attached to the plate. A blocking protein is then added (e.g., albumin or the milk protein casein) to bind the remaining nonspecific protein-binding sites in the well. Some of the wells will receive known amounts of antigen to allow the construction of a standard curve, and unknown antigen solutions are added to the other wells. The primary antibody captures the antigen and, following a wash, the secondary antibody is added, which is a polyclonal antibody that is conjugated to an enzyme. After a final wash, a colorless substrate (chromogen) is added, and the enzyme converts it into a colored end product. The color intensity of the sample caused by the end product is measured with a spectrophotometer. The amount of color produced (measured as absorbance) is directly proportional to the amount of enzyme, which in turn is directly proportional to the captured antigen. ELISAs are extremely sensitive, allowing antigen to be quantified in the nanogram (10–9 g) per mL range. In an indirect ELISA, we quantify antigen-specific antibody rather than antigen. We can use indirect ELISA to detect antibodies against many types of pathogens, including Borrelia burgdorferi (Lyme disease) and HIV. There are three important differences between indirect and direct ELISAs as shown in Figure 20.25. Rather than using antibody to capture antigen, the indirect ELISA starts with attaching known antigen (e.g., peptides from HIV) to the bottom of the microtiter plate wells. After blocking the unbound sites on the plate, patient serum is added; if antibodies are present (primary antibody), they will bind the antigen. After washing away any unbound proteins, the secondary Access for free at openstax.org  20.4 EIAs and ELISAs 807 antibody with its conjugated enzyme is directed against the primary antibody (e.g., antihuman immunoglobulin). The secondary antibody allows us to quantify how much antigen-specific antibody is present in the patient’s serum by the intensity of the color produced from the conjugated enzyme-chromogen reaction. As with several other tests for antibodies discussed in this chapter, there is always concern about cross-reactivity with antibodies directed against some other antigen, which can lead to false-positive results. Thus, we cannot definitively diagnose an HIV infection (or any other type of infection) based on a single indirect ELISA assay. We must confirm any suspected positive test, which is most often done using either an immunoblot that actually identifies the presence of specific peptides from the pathogen or a test to identify the nucleic acids associated with the pathogen, such as reverse transcriptase PCR (RT-PCR) or a nucleic acid antigen test. 808 20 Laboratory Analysis of the Immune Response FIGURE 20.24 (a) In a sandwich ELISA, a primary antibody is used to first capture an antigen with the primary antibody. A secondary antibody conjugated to an enzyme that also recognizes epitopes on the antigen is added. After the addition of the chromogen, a spectrophotometer measures the absorbance of end product, which is directly proportional to the amount of captured antigen. (b) An ELISA plate shows dilutions of antibodies (left) and antigens (bottom). Higher concentrations result in a darker final color. (credit b: modification of work by U.S. Fish and Wildlife Service Pacific Region) Access for free at openstax.org  20.4 EIAs and ELISAs 809 FIGURE 20.25 The indirect ELISA is used to quantify antigen-specific antibodies in patient serum for disease diagnosis. Antigen from the suspected disease agent is attached to microtiter plates. The primary antibody comes from the patient’s serum, which is subsequently bound by the enzyme-conjugated secondary antibody. Measuring the production of end product allows us to detect or quantify the amount of antigen-specific antibody present in the patient’s serum. CHECK YOUR UNDERSTANDING What is the purpose of the secondary antibody in a direct ELISA? 810 20 Laboratory Analysis of the Immune Response What do the direct and indirect ELISAs quantify? CLINICAL FOCUS Part 2 Although contacting and testing the 1300 patients for HIV would be time consuming and expensive, administrators hoped to minimize the hospital’s liability by proactively seeking out and treating potential victims of the rogue employee’s crime. Early detection of HIV is important, and prompt treatment can slow the progression of the disease. There are a variety of screening tests for HIV, but the most widely used is the indirect ELISA. As with other indirect ELISAs, the test works by attaching antigen (in this case, HIV peptides) to a well in a 96-well plate. If the patient is HIV positive, anti-HIV antibodies will bind to the antigen and be identified by the second antibody- enzyme conjugate. How accurate is an indirect ELISA test for HIV, and what factors could impact the test’s accuracy? Should the hospital use any other tests to confirm the results of the indirect ELISA? Jump to the previous Clinical Focus box. Jump to the next Clinical Focus box. Immunofiltration and Immunochromatographic Assays For some situations, it may be necessary to detect or quantify antigens or antibodies that are present at very low concentration in solution. Immunofiltration techniques have been developed to make this possible. In immunofiltration, a large volume of fluid is passed through a porous membrane into an absorbent pad. An antigen attached to the porous membrane will capture antibody as it passes; alternatively, we can also attach an antibody to the membrane to capture antigen. The method of immunofiltration has been adapted in the development of immunochromatographic assays, commonly known as lateral flow tests or strip tests. These tests are quick and easy to perform, making them popular for point-of-care use (i.e., in the doctor’s office) or in-home use. One example is the TORCH test that allows doctors to screen pregnant people or newborns for infection by an array of viruses and other pathogens (Toxoplasma, other viruses, rubella, cytomegalovirus, herpes simplex). In-home pregnancy tests are another widely used example of a lateral flow test (Figure 20.26). Immunofiltration tests are also popular in developing countries, because they are inexpensive and do not require constant refrigeration of the dried reagents. However, the technology is also built into some sophisticated laboratory equipment. In lateral flow tests (Figure 20.27), fluids such as urine are applied to an absorbent pad on the test strip. The fluid flows by capillary action and moves through a stripe of beads with antibodies attached to their surfaces. The fluid in the sample actually hydrates the reagents, which are present in a dried state in the stripe. Antibody-coated beads made of latex or tiny gold particles will bind antigens in the test fluid. The antibody-antigen complexes then flow over a second stripe that has immobilized antibody against the antigen; this stripe will retain the beads that have bound antigen. A third control stripe binds any beads. A red color (from gold particles) or blue (from latex beads) developing at the test line indicates a positive test. If the color only develops at the control line, the test is negative. Like ELISA techniques, lateral flow tests take advantage of antibody sandwiches, providing sensitivity and specificity. While not as quantitative as ELISA, these tests have the advantage of being fast, inexpensive, and not dependent on special equipment. Thus, they can be performed anywhere by anyone. There are some concerns about putting such powerful diagnostic tests into the hands of people who may not understand the tests’ limitations, such as the possibility of false-positive results. While home pregnancy tests have become widely accepted, at-home antibody-detection tests for diseases like HIV have raised some concerns in the medical community. Some have questioned whether self-administration of such tests should be allowed in the absence of medical personnel who can explain the test results and order appropriate confirmatory tests. However, with growing numbers of lateral flow tests becoming available, and the rapid development of lab-on-a-chip technology (Figure 20.1), home medical tests are likely to become even more commonplace in the future. Access for free at openstax.org  20.4 EIAs and ELISAs 811 FIGURE 20.26 A lateral flow test detecting pregnancy-related hormones in urine. The control stripe verifies the validity of the test and the test line determines the presence of pregnancy-related hormones in the urine. (credit: modification of work by Klaus Hoffmeier) FIGURE 20.27 Immunochromatographic assays, or lateral flow tests, allow the testing of antigen in a dilute solution. As the fluid flows through the test strip, it rehydrates the reagents. Antibodies conjugated to small particles bind the antigen in the first stripe and then flow 812 20 Laboratory Analysis of the Immune Response onto the second stripe where they are bound by a second, fixed antibody. This produces a line of color, depending on the color of the beads. The third, control stripe binds beads as well to indicate that the test is working properly. (credit: modification of work by Yeh CH, Zhao ZQ, Shen PL, Lin YC) CHECK YOUR UNDERSTANDING What physical process does the lateral flow method require to function? Explain the purpose of the third strip in a lateral flow assay. Table 20.4 compares some of the key mechanisms and examples of some of the EIAs discussed in this section as well as immunoblots, which were discussed in Detecting Antigen-Antibody Complexes. Immunoblots & Enzyme Immunoassays Type of Assay Mechanism Specific Procedures Examples Immunoblots Uses enzyme-antibody Western blot: Detects Detecting the presence conjugates to identify specific the presence of a of HIV peptides (or proteins that have been particular protein peptides from other transferred to an absorbent infectious agents) in membrane patient sera Immunostaining Uses enzyme-antibody Immunohistochemistry: Stain for presence of conjugates to stain specific Used to stain specific CD8 cells in host tissue molecules on or in cells cells in a tissue Enzyme-linked Uses enzyme-antibody Direct ELISA: Uses a Detection of HIV immunosorbent assay conjugates to quantify target single antibody to antigen p24 up to one (ELISA) molecules detect the presence of month after being an antigen infected Indirect ELISA: Detection of HIV Measures the amount antibodies in serum of antibody produced against an antigen Immunochromatographic Techniques use the capture of Sandwich ELISA: Detection of antibodies (lateral flow) assays flowing, color-labeled Measures the amount for various pathogens antigen-antibody complexes of antigen bound by the in patient sera (e.g., by fixed antibody for disease antibody rapid strep, malaria diagnosis dipstick) Pregnancy test detecting human chorionic gonadotrophin in urine TABLE 20.4 CLINICAL FOCUS Part 3 Although the indirect ELISA for HIV is a sensitive assay, there are several complicating considerations. First, if an Access for free at openstax.org  20.5 Fluorescent Antibody Techniques 813 infected person is tested too soon after becoming infected, the test can yield false-negative results. The seroconversion window is generally about three weeks, but in some cases, it can be more than two months. In addition to false negatives, false positives can also occur, usually due to previous infections with other viruses that induce cross-reacting antibodies. The false-positive rate depends on the particular brand of test used, but 10 0.5% is not unusual. Because of the possibility of a false positive, all positive tests are followed up with a confirmatory test. This confirmatory test is often an immunoblot (western blot) in which HIV peptides from the patient’s blood are identified using an HIV-specific mAb-enzyme conjugate. A positive western blot would confirm an HIV infection and a negative blot would confirm the absence of HIV despite the positive ELISA. Unfortunately, western blots for HIV antigens often yield indeterminant results, in which case, they neither confirm nor invalidate the results of the indirect ELISA. In fact, the rate of indeterminants can be 10–49% (which is why, combined with their cost, western blots are not used for screening). Similar to the indirect ELISA, an indeterminant western blot can occur because of cross-reactivity or previous viral infections, vaccinations, or autoimmune diseases. Of the 1300 patients being tested, how many false-positive ELISA tests would be expected? Of the false positives, how many indeterminant western blots could be expected? How would the hospital address any cases in which a patient’s western blot was indeterminant? Jump to the previous Clinical Focus box. Jump to the next Clinical Focus box. 20.5 Fluorescent Antibody Techniques LEARNING OBJECTIVES By the end of this section, you will be able to: Describe the benefits of immunofluorescent antibody assays in comparison to nonfluorescent assays Compare direct and indirect fluorescent antibody assays Explain how a flow cytometer can be used to quantify specific subsets of cells present in a complex mixture of cell types Explain how a fluorescence-activated cell sorter can be used to separate unique types of cells Rapid visualization of bacteria from a clinical sample such as a throat swab or sputum can be achieved through fluorescent antibody (FA) techniques that attach a fluorescent marker (fluorogen) to the constant region of an antibody, resulting in a reporter molecule that is quick to use, easy to see or measure, and able to bind to target markers with high specificity. We can also label cells, allowing us to precisely quantify particular subsets of cells or even purify these subsets for further research. As with the enzyme assays, FA methods may be direct, in which a labeled mAb binds an antigen, or indirect, in which secondary polyclonal antibodies bind patient antibodies that react to a prepared antigen. Applications of these two methods were demonstrated in Figure 2.19. FA methods are also used in automated cell counting and sorting systems to enumerate or segregate labeled subpopulations of cells in a sample. Direct Fluorescent Antibody Techniques Direct fluorescent antibody (DFA) tests use a fluorescently labeled mAb to bind and illuminate a target antigen. DFA tests are particularly useful for the rapid diagnosis of bacterial diseases. For example, fluorescence-labeled antibodies against Streptococcus pyogenes (group A strep) can be used to obtain a diagnosis of strep throat from a throat swab. The diagnosis is ready in a matter of minutes, and the patient can be started on antibiotics before even leaving the clinic. DFA techniques may also be used to diagnose pneumonia caused by Mycoplasma pneumoniae or Legionella pneumophila from sputum samples (Figure 20.28). The fluorescent antibodies bind to the bacteria on a microscope slide, allowing ready detection of the bacteria using a fluorescence microscope. Thus, the DFA technique is valuable for visualizing certain bacteria that are difficult to isolate or culture from patient samples. 10 Thomas, Justin G., Victor Jaffe, Judith Shaffer, and Jose Abreu, “HIV Testing: US Recommendations 2014,” Osteopathic Family Physician 6, no. 6 (2014). 814 20 Laboratory Analysis of the Immune Response FIGURE 20.28 A green fluorescent mAb against L. pneumophila is used here to visualize and identify bacteria from a smear of a sample from the respiratory tract of a pneumonia patient. (credit: modification of work by American Society for Microbiology) LINK TO LEARNING Watch the animation (https://openstax.org/l/22dirfluorant) on this page to review the procedures of the direct fluorescent antibody test. CHECK YOUR UNDERSTANDING In a direct fluorescent antibody test, what does the fluorescent antibody bind to? Indirect Fluorescent Antibody Techniques Indirect fluorescent antibody (IFA) tests (Figure 20.29) are used to look for antibodies in patient serum. For example, an IFA test for the diagnosis of syphilis uses T. pallidum cells isolated from a lab animal (the bacteria cannot be grown on lab media) and a smear prepared on a glass slide. Patient serum is spread over the smear and anti-treponemal antibodies, if present, are allowed to bind. The serum is washed off and a secondary antibody added. The secondary antibody is an antihuman immunoglobulin conjugated to a fluorogen. On examination, the T. pallidum bacteria will only be visible if they have been bound by the antibodies from the patient’s serum. The IFA test for syphilis provides an important complement to the VDRL test discussed in Detecting Antigen- Antibody Complexes. The VDRL is more likely to generate false-positive reactions than the IFA test; however, the VDRL is a better test for determining whether an infection is currently active. IFA tests are also useful for the diagnosis of autoimmune diseases. For example, systemic lupus erythematosus (SLE) (see Autoimmune Disorders) is characterized by elevated expression levels of antinuclear antibodies (ANA). These autoantibodies can be expressed against a variety of DNA-binding proteins and even against DNA itself. Because autoimmunity is often difficult to diagnose, especially early in disease progression, testing for ANA can be a valuable clue in making a diagnosis and starting appropriate treatment. The IFA for ANA begins by fixing cells grown in culture to a glass slide and making them permeable to antibody. The slides are then incubated with serial dilutions of serum from the patient. After incubation, the slide is washed to remove unbound proteins, and the fluorescent antibody (antihuman IgG conjugated to a fluorogen) added. After an incubation and wash, the cells can be examined for fluorescence evident around the nucleus (Figure 20.30). The titer of ANA in the serum is determined by the highest dilution showing fluorescence. Because many healthy people express ANA, the American College of Rheumatology recommends that the titer must be at least 1:40 in the 11 presence of symptoms involving two or more organ systems to be considered indicative of SLE. 11 Gill, James M., ANNA M. Quisel, PETER V. Rocca, and DENE T. Walters. “Diagnosis of systemic lupus erythematosus.” American family Access for free at openstax.org  20.5 Fluorescent Antibody Techniques 815 FIGURE 20.29 (a) The IFA test is used to detect antigen-specific antibodies by allowing them to bind to antigen fixed to a surface and then illuminating these complexes with a secondary antibody-fluorogen conjugate. (b) In this example of a micrograph of an indirect fluorescent antibody test, a patient’s antibodies to the measles virus bind to viral antigens present on inactivated measles-infected cells affixed to a slide. Secondary antibodies bind the patient’s antibodies and carry a fluorescent molecule. (credit b: modification of work by American Society for Microbiology) physician 68, no. 11 (2003): 2179-2186. 816 20 Laboratory Analysis of the Immune Response FIGURE 20.30 In this test for antinuclear antibodies (ANA), cells are exposed to serum from a patient suspected of making ANA and then to a fluorescent mAb specific for human immunoglobulin. As a control, serum from a healthy patient is also used. Visible fluorescence around the nucleus demonstrates the presence of ANA in the patient’s serum. In the healthy control where lower levels of ANA are produced, very faint green is detected. (credit left, right: modification of work by Al-Hussaini AA, Alzahrani MD, Alenizi AS, Suliman NM, Khan MA, Alharbi SA, Chentoufi AA) CHECK YOUR UNDERSTANDING In an indirect fluorescent antibody test, what does the fluorescent antibody bind to? What is the ANA test looking for? Flow Cytometry Fluorescently labeled antibodies can be used to quantify cells of a specific type in a complex mixture using flow cytometry (Figure 20.31), an automated, cell-counting system that detects fluorescing cells as they pass through a narrow tube one cell at a time. For example, in HIV infections, it is important to know the level of CD4 T cells in the patient’s blood; if the numbers fall below 500 per μL of blood, the patient becomes more likely to acquire opportunistic infections; below 200 per μL, the patient can no longer mount a useful adaptive immune response at all. The analysis begins by incubating a mixed-cell population (e.g., white blood cells from a donor) with a fluorescently labeled mAb specific for a subpopulation of cells (e.g., anti-CD4). Some experiments look at two cell markers simultaneously by adding a different fluorogen to the appropriate mAb. The cells are then introduced to the flow cytometer through a narrow capillary that forces the cells to pass in single file. A laser is used to activate the fluorogen. The fluorescent light radiates out in all directions, so the fluorescence detector can be positioned at an angle from the incident laser light. Figure 20.31 shows the obscuration bar in front of the forward-scatter detector that prevents laser light from hitting the detector. As a cell passes through the laser bar, the forward-scatter detector detects light scattered around the obscuration bar. The scattered light is transformed into a voltage pulse, and the cytometer counts a cell. The fluorescence from a labeled cell is detected by the side-scatter detectors. The light passes through various dichroic mirrors such that the light emitted from the fluorophore is received by the correct detector. Access for free at openstax.org  20.5 Fluorescent Antibody Techniques 817 FIGURE 20.31 In flow cytometry, a mixture of fluorescently labeled and unlabeled cells passes through a narrow capillary. A laser excites the fluorogen, and the fluorescence intensity of each cell is measured by a detector. (credit: modification of work by “Kierano”/Wikimedia Commons) Data are collected from both the forward- and side-scatter detectors. One way these data can be presented is in the form of a histogram. The forward scatter is placed on the y-axis (to represent the number of cells), and the side scatter is placed on the x-axis (to represent the fluoresence of each cell). The scaling for the x-axis is logarithmic, so fluorescence intensity increases by a factor of 10 with each unit increase along the axis. Figure 20.32 depicts an example in which a culture of cells is combined with an antibody attached to a fluorophore to detect CD8 cells and then analyzed by flow cytometry. The histogram has two peaks. The peak on the left has lower fluorescence readings, representing the subset of the cell population (approximately 30 cells) that does not fluoresce; hence, they are not bound by antibody and therefore do not express CD8. The peak on the right has higher fluorescence readings, representing the subset of the cell population (approximately 100 cells) that show fluorescence; hence, they are bound by the antibody and therefore do express CD8. FIGURE 20.32 Flow cytometry data are often compiled as a histogram. In the histogram, the area under each peak is proportional to the number of cells in each population. The x-axis is the relative fluorescence expressed by the cells (on a log scale), and the y-axis represents the number of cells at a particular level of fluorescence. 818 20 Laboratory Analysis of the Immune Response CHECK YOUR UNDERSTANDING What is the purpose of the laser in a flow cytometer? In the output from a flow cytometer, the area under the histogram is equivalent to what? CLINICAL FOCUS Resolution After notifying all 1300 patients, the hospital begins scheduling HIV screening. Appointments were scheduled a minimum of 3 weeks after the patient’s last hospital visit to minimize the risk of false negatives. Because some false positives were anticipated, the public health physician set up a counseling protocol for any patient whose indirect ELISA came back positive. Of the 1300 patients, eight tested positive using the ELISA. Five of these tests were invalidated by negative western blot tests, but one western blot came back positive, confirming that the patient had indeed contracted HIV. The two remaining western blots came back indeterminate. These individuals had to submit to a third test, a PCR, to confirm the presence or absence of HIV sequences. Luckily, both patients tested negative. As for the lone patient confirmed to have HIV, the tests cannot prove or disprove any connection to the syringes compromised by the former hospital employee. Even so, the hospital’s insurance will fully cover the patient’s treatment, which began immediately. Although we now have drugs that are typically effective at controlling the progression of HIV and AIDS, there is still no cure. If left untreated, or if the drug regimen fails, the patient will experience a gradual decline in the number of CD4 helper T cells, resulting in severe impairment of all adaptive immune functions. Even moderate declines of helper T cell numbers can result in immunodeficiency, leaving the patient susceptible to opportunistic infections. To monitor the status of the patient’s helper T cells, the hospital will use flow cytometry. This sensitive test allows physicians to precisely determine the number of helper T cells so they can adjust treatment if the number falls below 500 cells/µL. Jump to the previous Clinical Focus box. Cell Sorting Using Immunofluorescence The flow cytometer and immunofluorescence can also be modified to sort cells from a single sample into purified subpopulations of cells for research purposes. This modification of the flow cytometer is called a fluorescence- activated cell sorter (FACS). In a FACS, fluorescence by a cell induces the device to put a charge on a droplet of the transporting fluid containing that cell. The charge is specific to the wavelength of the fluorescent light, which allows for differential sorting by those different charges. The sorting is accomplished by an electrostatic deflector that moves the charged droplet containing the cell into one collecting vessel or another. The process results in highly purified subpopulations of cells. One limitation of a FACS is that it only works on isolated cells. Thus, the method would work in sorting white blood cells, since they exist as isolated cells. But for cells in a tissue, flow cytometry can only be applied if we can excise the tissue and separate it into single cells (using proteases to cleave cell-cell adhesion molecules) without disrupting cell integrity. This method may be used on tumors, but more often, immunohistochemistry and immunocytochemistry are used to study cells in tissues. LINK TO LEARNING Watch videos to learn more about how flow cytometry (https://openstax.org/l/22flowcytometry) and a FACS (https://openstax.org/l/22FACSwork) work. Access for free at openstax.org  20.5 Fluorescent Antibody Techniques 819 CHECK YOUR UNDERSTANDING In fluorescence activated cell sorting, what characteristic of the target cells allows them to be separated? Table 20.5 compares the mechanisms of the fluorescent antibody techniques discussed in this section. Fluorescent Antibody Techniques Type of Mechanism Examples Assay Direct Uses fluorogen-antibody conjugates to label bacteria Visualizing Legionella pneumophila fluorescent from patient samples from a throat swab antibody (DFA) Indirect Detects disease-specific antibodies in patent serum Diagnosing syphilis; detecting fluorescent antinuclear antibodies (ANA) for antibody lupus and other autoimmune (IFA) diseases Flow Labels cell membranes with fluorogen-antibody Counting the number of fluorescently cytometry conjugate markers excited by a laser; machine counts labeled CD4 or CD8 cells in a sample the cell and records the relative fluorescence Fluorescence Form of flow cytometry that both counts cells and Sorting cancer cells activated cell physically separates them into pools of high and low sorter (FACS) fluorescence cells TABLE 20.5 820 20 Summary Summary 20.1 Polyclonal and Monoclonal Antibody The presence of specific antigens (e.g., bacterial Production or viral proteins) in serum can be demonstrated by western blot assays, in which the proteins are Antibodies bind with high specificity to antigens transferred to a nitrocellulose membrane and used to challenge the immune system, but they identified using labeled antibodies. may also show cross-reactivity by binding to In the complement fixation test, complement is other antigens that share chemical properties used to detect antibodies against various with the original antigen. pathogens. Injection of an antigen into an animal will result in a polyclonal antibody response in which 20.3 Agglutination Assays different antibodies are produced that react with Antibodies can agglutinate cells or large particles the various epitopes on the antigen. into a visible matrix. Agglutination tests are Polyclonal antisera are useful for some types of often done on cards or in microtiter plates that laboratory assays, but other assays require more allow multiple reactions to take place side by specificity. Diagnostic tests that use polyclonal side using small volumes of reagents. antisera are typically only used for screening Using antisera against certain proteins allows because of the possibility of false-positive and identification of serovars within species of false-negative results. bacteria. Monoclonal antibodies provide higher specificity Detecting antibodies against a pathogen can be a than polyclonal antisera because they bind to a powerful tool for diagnosing disease, but there is single epitope and usually have high affinity. a period of time before patients go through Monoclonal antibodies are typically produced by seroconversion and the level of antibodies culturing antibody-secreting hybridomas derived becomes detectable. from mice. mAbs are currently used to treat Agglutination of latex beads in indirect cancer, but their exorbitant cost has prevented agglutination assays can be used to detect the them from being used more widely to treat presence of specific antigens or specific infectious diseases. Still, their potential for antibodies in patient serum. laboratory and clinical use is driving the The presence of some antibacterial and antiviral development of new, cost-effective solutions antibodies can be confirmed by the use of the such as plantibodies. direct Coombs’ test, which uses Coombs’ 20.2 Detecting Antigen-Antibody reagent to cross-link antibodies bound to red Complexes blood cells and facilitate hemagglutination. Some viruses and bacteria will bind and When present in the correct ratio, antibody and agglutinate red blood cells; this interaction is the antigen will form a precipitin, or lattice that basis of the direct hemagglutination assay, precipitates out of solution. most often used to determine the titer of virus in A precipitin ring test can be used to visualize solution. lattice formation in solution. The Ouchterlony Neutralization assays quantify the level of virus- assay demonstrates lattice formation in a gel. specific antibody by measuring the decrease in The radial immunodiffusion assay is used to hemagglutination observed after mixing patient quantify antigen by measuring the size of a serum with a standardized amount of virus. precipitation zone in a gel infused with Hemagglutination assays are also used to screen antibodies. and cross-match donor and recipient blood to Insoluble antigens in suspension will form ensure that the transfusion recipient does not flocculants when bound by antibodies. This is have antibodies to antigens in the donated blood. the basis of the VDRL test for syphilis in which anti-treponemal antibodies bind to cardiolipin in 20.4 EIAs and ELISAs suspension. Enzyme immunoassays (EIA) are used to Viral infections can be detected by quantifying visualize and quantify antigens. They use an virus-neutralizing antibodies in a patient’s serum. antibody conjugated to an enzyme to bind the Different antibody classes in plasma or serum antigen, and the enzyme converts a substrate are identified by using immunoelectrophoresis. into an observable end product. The substrate Access for free at openstax.org  20 Review Questions 821 may be either a chromogen or a fluorogen. 20.5 Fluorescent Antibody Techniques Immunostaining is an EIA technique for Immunofluorescence assays use antibody- visualizing cells in a tissue fluorogen conjugates to illuminate antigens for (immunohistochemistry) or examining easy, rapid detection. intracellular structures (immunocytochemistry). Direct immunofluorescence can be used to Direct ELISA is used to quantify an antigen in detect the presence of bacteria in clinical solution. The primary antibody captures the samples such as sputum. antigen, and the secondary antibody delivers an Indirect immunofluorescence detects the enzyme. Production of end product from the presence of antigen-specific antibodies in patient chromogenic substrate is directly proportional to sera. The fluorescent antibody binds to the the amount of captured antigen. antigen-specific antibody rather than the antigen. Indirect ELISA is used to detect antibodies in The use of indirect immunofluorescence assays patient serum by attaching antigen to the well of to detect antinuclear antibodies is an important a microtiter plate, allowing the patient (primary) tool in the diagnosis of several autoimmune antibody to bind the antigen and an enzyme- diseases. conjugated secondary antibody to detect the Flow cytometry uses fluorescent mAbs against primary antibody. cell-membrane proteins to quantify specific Immunofiltration and subsets of cells in complex mixtures. immunochromatographic assays are used in Fluorescence-activated cell sorters are an lateral flow tests, which can be used to extension of flow cytometry in which diagnose pregnancy and various diseases by fluorescence intensity is used to physically detecting color-labeled antigen-antibody separate cells into high and low fluorescence complexes in urine or other fluid samples populations. Review Questions Multiple Choice 1. For many uses in the laboratory, polyclonal b. precipitin antibodies work well, but for some types of c. coagulation assays, they lack sufficient ________ because d. a bright pink color they cross-react with inappropriate antigens. a. specificity 4. The titer of a virus neutralization test is the b. sensitivity highest dilution of patient serum c. accuracy a. in which there is no detectable viral DNA. d. reactivity b. in which there is no detectable viral protein. 2. How are monoclonal antibodies produced? c. that completely blocks plaque formation. a. Antibody-producing B cells from a mouse d. that reduces plaque formation by at least are fused with myeloma cells and then the 50%. cells are grown in tissue culture. b. A mouse is injected with an antigen and 5. In the Ouchterlony assay, we see a sharp then antibodies are harvested from its precipitin arc form between antigen and serum. antiserum. Why does this arc remain visible for a c. They are produced by the human immune long time? system as a natural response to an a. The antibody molecules are too large to infection. diffuse through the agar. d. They are produced by a mouse’s immune b. The precipitin lattice is too large to diffuse system as a natural response to an through the agar. infection. c. Methanol, added once the arc forms, denatures the protein and blocks 3. The formation of ________ is a positive result in diffusion. the VDRL test. d. The antigen molecules are chemically a. flocculant coupled to the gel matrix. 822 20 Review Questions 6. We use antisera to distinguish between various 12. When using an EIA to study microtubules or ________ within a species of bacteria. other structures inside a cell, we first chemically a. isotypes fix the cell and then treat the cells with alcohol. b. serovars What is the purpose of this alcohol treatment? c. subspecies a. It makes holes in the cell membrane large d. lines enough for antibodies to pass. b. It makes the membrane sticky so 7. When using antisera to characterize bacteria, we antibodies will bind and be taken up by will often link the antibodies to ________ to receptor-mediated endocytosis. better visualize the agglutination. c. It removes negative charges from the a. latex beads membrane, which would otherwise b. red blood cells repulse the antibodies. c. other bacteria