# Diseases of the Immune System

## Organ Transplantation and Rejection

### Learning Objectives

By the end of this section, you will be able to:
1. Explain why human leukocyte antigens (HLAs) are important in tissue transplantation
2. Explain the types of grafts possible and their potential for interaction with the immune system
3. Describe what occurs during graft-versus-host disease (GVHD)
A graft is the transplantation of an organ or tissue to a different location, with the goal of replacing a missing or damaged organ or tissue. Grafts are typically moved without their attachments to the circulatory system and must reestablish these, in addition to the other connections and interactions with their new surrounding tissues. There are different types of grafts depending on the source of the new tissue or organ. Tissues that are transplanted from one genetically distinct individual to another within the same species are called allografts. An interesting variant of the allograft is an isograft, in which tissue from one twin is transplanted to another. As long as the twins are monozygotic (therefore, essentially genetically identical), the transplanted tissue is virtually never rejected. If tissues are transplanted from one area on an individual to another area on the same individual (e.g., a skin graft on a burn patient), it is known as an autograft. If tissues from an animal are transplanted into a human, this is called a xenograft.

### Transplant Rejection

The different types of grafts described above have varying risks for rejection (). Rejection occurs when the recipient’s immune system recognizes the donor tissue as foreign (non-self), triggering an immune response. The major histocompatibility complex markers MHC I and MHC II, more specifically identified as human leukocyte antigens (HLAs), play a role in transplant rejection. The HLAs expressed in tissue transplanted from a genetically different individual or species may be recognized as non-self molecules by the host’s dendritic cells. If this occurs, the dendritic cells will process and present the foreign HLAs to the host’s helper T cells and cytotoxic T cells, thereby activating them. Cytotoxic T cells then target and kill the grafted cells through the same mechanism they use to kill virus-infected cells; helper T cells may also release cytokines that activate macrophages to kill graft cells.
With the three highly polymorphic MHC I genes in humans (HLA-A, HLA-B, and HLA-C) determining compatibility, each with many alleles segregating in a population, odds are extremely low that a randomly chosen donor will match a recipient's six-allele genotype (the two alleles at each locus are expressed codominantly). This is why a parent or a sibling may be the best donor in many situations—a genetic match between the MHC genes is much more likely and the organ is much less likely to be rejected.
Although matching all of the MHC genes can lower the risk for rejection, there are a number of additional gene products that also play a role in stimulating responses against grafted tissue. Because of this, no non-self grafted tissue is likely to completely avoid rejection. However, the more similar the MHC gene match, the more likely the graft is to be tolerated for a longer time. Most transplant recipients, even those with tissues well matched to their MHC genes, require treatment with immunosuppressant drugs for the rest of their lives. This can make them more vulnerable than the general population to complications from infectious diseases. It can also result in transplant-related malignancies because the body’s normal defenses against cancer cells are being suppressed.

### Graft-versus-Host Disease

A form of rejection called graft-versus-host disease (GVHD) primarily occurs in recipients of bone marrow transplants and peripheral blood stem cells. GHVD presents a unique situation because the transplanted tissue is capable of producing immune cells; APCs in the donated bone marrow may recognize the host cells as non-self, leading to activation of the donor cytotoxic T cells. Once activated, the donor’s T cells attack the recipient cells, causing acute GVHD.
Acute GVHD typically develops within weeks after a bone marrow transplant, causing tissue damage affecting the skin, gastrointestinal tract, liver, and eyes. In addition, acute GVHD may also lead to a cytokine storm, an unregulated secretion of cytokines that may be fatal. In addition to acute GVHD, there is also the risk for chronic GVHD developing months after the bone marrow transplant. The mechanisms responsible for chronic GVHD are not well understood.
To minimize the risk of GVHD, it is critically important to match the HLAs of the host and donor as closely as possible in bone marrow transplants. In addition, the donated bone marrow is processed before grafting to remove as many donor APCs and T cells as possible, leaving mostly hematopoietic stem cells.

### The Future of Transplantation

Historically speaking, the practice of transplanting tissues—and the complications that can accompany such procedures—is a relatively recent development. It was not until 1954 that the first successful organ transplantation between two humans was achieved. Yet the field of organ transplantation has progressed rapidly since that time.
Nonetheless, the practice of transplanting non-self tissues may soon become obsolete. Scientists are now attempting to develop methods by which new organs may be grown in vitro from an individual’s own harvested cells to replace damaged or abnormal ones. Because organs produced in this way would contain the individual’s own cells, they could be transplanted into the individual without risk for rejection.
An alternative approach that is gaining renewed research interest is genetic modification of donor animals, such as pigs, to provide transplantable organs that do not elicit an immune response in the recipient. The approach involves excising the genes in the pig (in the embryo) that are most responsible for the rejection reaction after transplantation. Finding these genes and effectively removing them is a challenge, however. So too is identifying and neutralizing risks from viral sequences that might be embedded in the pig genome, posing a risk for infection in the human recipient.

### Key Concepts and Summary

1. Grafts and transplants can be classified as autografts, isografts, allografts, or xenografts based on the genetic differences between the donor’s and recipient’s tissues.
2. Genetic differences, especially among the MHC (HLA) genes, will dictate the likelihood that rejection of the transplanted tissue will occur.
3. Transplant recipients usually require immunosuppressive therapy to avoid rejection, even with good genetic matching. This can create additional problems when immune responses are needed to fight off infectious agents and prevent cancer.
4. Graft-versus-host disease can occur in bone marrow transplants, as the mature T cells in the transplant itself recognize the recipient’s tissues as foreign.
5. Transplantation methods and technology have improved greatly in recent decades and may move into new areas with the use of stem cell technology to avoid the need for genetic matching of MHC molecules.

### Matching

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### Short Answer


# Diseases of the Immune System

## Immunodeficiency

### Learning Objectives

By the end of this section, you will be able to:
1. Compare the causes of primary and secondary immunodeficiencies
2. Describe treatments for primary and secondary immunodeficiencies
Immunodeficiencies are inherited (primary) or acquired (secondary) disorders in which elements of host immune defenses are either absent or functionally defective. In developed countries, most immunodeficiencies are inherited, and they are usually first seen in the clinic as recurrent or overwhelming infections in infants. However, on a global scale, malnutrition is the most common cause of immunodeficiency and would be categorized as an acquired immunodeficiency. Acquired immunodeficiencies are more likely to develop later in life, and the pathogenic mechanisms of many remain obscure.

### Primary Immunodeficiency

Primary immunodeficiencies, which number more than 250, are caused by inherited defects of either nonspecific innate or specific adaptive immune defenses. In general, patients born with primary immunodeficiency (PI) commonly have an increased susceptibility to infection. This susceptibility can become apparent shortly after birth or in early childhood for some individuals, whereas other patients develop symptoms later in life. Some primary immunodeficiencies are due to a defect of a single cellular or humoral component of the immune system; others may result from defects of more than one component. Examples of primary immunodeficiencies include chronic granulomatous disease, X-linked agammaglobulinemia, selective IgA deficiency, and severe combined immunodeficiency disease.

### Chronic Granulomatous Disease

The causes of chronic granulomatous disease (CGD) are defects in the NADPH oxidase system of phagocytic cells, including neutrophils and macrophages, that prevent the production of superoxide radicals in phagolysosomes. The inability to produce superoxide radicals impairs the antibacterial activity of phagocytes. As a result, infections in patients with CGD persist longer, leading to a chronic local inflammation called a granuloma. Microorganisms that are the most common causes of infections in patients with CGD include  spp., , , , and .

### X-Linked Agammaglobulinemia

Deficiencies in B cells due to defective differentiation lead to a lack of specific antibody production known as X-linked agammaglobulinemia. In 1952, Ogden C. Bruton (1908–2003) described the first immunodeficiency in a boy whose immune system failed to produce antibodies. This defect is inherited on the X chromosome and is characterized by the absence of immunoglobulin in the serum; it is called Bruton X-linked agammaglobulinemia (XLA). The defective gene, BTK, in XLA is now known to encode a tyrosine kinase called Bruton tyrosine kinase (Btk). In patients whose B cells are unable to produce sufficient amounts of Btk, the B-cell maturation and differentiation halts at the pre-B-cell stage of growth. B-cell maturation and differentiation beyond the pre-B-cell stage of growth is required for immunoglobulin production. Patients who lack antibody production suffer from recurrent infections almost exclusively due to extracellular pathogens that cause pyogenic infections: , , S. pyogenes, and S. aureus. Because cell-mediated immunity is not impaired, these patients are not particularly vulnerable to infections caused by viruses or intracellular pathogens.

### Selective IgA Deficiency

The most common inherited form of immunoglobulin deficiency is selective IgA deficiency, affecting about one in 800 people. Individuals with selective IgA deficiency produce normal levels of IgG and IgM, but are not able to produce secretory IgA. IgA deficiency predisposes these individuals to lung and gastrointestinal infections for which secretory IgA is normally an important defense mechanism. Infections in the lungs and gastrointestinal tract can involve a variety of pathogens, including H. influenzae, S. pneumoniae, , S. aureus, , or pathogenic strains of .

### Severe Combined Immunodeficiency

Patients who suffer from severe combined immunodeficiency (SCID) have B-cell and T-cell defects that impair T-cell dependent antibody responses as well as cell-mediated immune responses. Patients with SCID also cannot develop immunological memory, so vaccines provide them no protection, and live attenuated vaccines (e.g., for varicella-zoster, measles virus, rotavirus, poliovirus) can actually cause the infection they are intended to prevent. The most common form is X-linked SCID, which accounts for nearly 50% of all cases and occurs primarily in males. Patients with SCID are typically diagnosed within the first few months of life after developing severe, often life-threatening, opportunistic infection by  spp., , or pathogenic strains of .
Without treatment, babies with SCID do not typically survive infancy. In some cases, a bone marrow transplant may successfully correct the defects in lymphocyte development that lead to the SCID phenotype, by replacing the defective component. However, this treatment approach is not without risks, as demonstrated by the famous case of David Vetter (1971–1984), better known as “Bubble Boy” (). Vetter, a patient with SCID who lived in a protective plastic bubble to prevent exposure to opportunistic microbes, received a bone marrow transplant from his sister. Because of a latent Epstein-Barr virus infection in her bone marrow, however, he developed mononucleosis and died of Burkitt lymphoma at the age of 12 years.

### Secondary Immunodeficiency

A secondary immunodeficiency occurs as a result an acquired impairment of function of B cells, T cells, or both. Secondary immunodeficiencies can be caused by:
1. Systemic disorders such as diabetes mellitus, malnutrition, hepatitis, or HIV infection
2. Immunosuppressive treatments such as cytotoxic chemotherapy, bone marrow ablation before transplantation, or radiation therapy
3. Prolonged critical illness due to infection, surgery, or trauma in the very young, elderly, or hospitalized patients
Unlike primary immunodeficiencies, which have a genetic basis, secondary immunodeficiencies are often reversible if the underlying cause is resolved. Patients with secondary immunodeficiencies develop an increased susceptibility to an otherwise benign infection by opportunistic pathogens such as  spp., P. jirovecii, and .
HIV infection and the associated acquired immunodeficiency syndrome (AIDS) are the best-known secondary immunodeficiencies. AIDS is characterized by profound CD4 T-cell lymphopenia (decrease in lymphocytes). The decrease in CD4 T cells is the result of various mechanisms, including HIV-induced pyroptosis (a type of apoptosis that stimulates an inflammatory response), viral cytopathic effect, and cytotoxicity to HIV-infected cells.
The most common cause of secondary immunodeficiency worldwide is severe malnutrition, which affects both innate and adaptive immunity. More research and information are needed for the more common causes of secondary immunodeficiency; however, the number of new discoveries in AIDS research far exceeds that of any other single cause of secondary immunodeficiency. AIDS research has paid off extremely well in terms of discoveries and treatments; increased research into the most common cause of immunodeficiency, malnutrition, would likely be as beneficial.
 summarizes primary and secondary immunodeficiencies, their effects on immune function, and typical outcomes.

### Key Concepts and Summary

1. Primary immunodeficiencies are caused by genetic abnormalities; secondary immunodeficiencies are acquired through disease, diet, or environmental exposures
2. Primary immunodeficiencies may result from flaws in phagocyte killing of innate immunity, or impairment of T cells and B cells.
3. Primary immunodeficiencies include chronic granulomatous disease, X-linked agammaglobulinemia, selective IgA deficiency, and severe combined immunodeficiency disease.
4. Secondary immunodeficiencies result from environmentally induced defects in B cells and/or T cells.
5. Causes for secondary immunodeficiencies include malnutrition, viral infection, diabetes, prolonged infections, and chemical or radiation exposure.

### Multiple Choice

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### Short Answer


# Diseases of the Immune System

## Cancer Immunobiology and Immunotherapy

### Learning Objectives

By the end of this section, you will be able to:
1. Explain how the adaptive specific immune response responds to tumors
2. Discuss the risks and benefits of tumor vaccines
Cancer involves a loss of the ability of cells to control their cell cycle, the stages each eukaryotic cell goes through as it grows and then divides. When this control is lost, the affected cells rapidly divide and often lose the ability to differentiate into the cell type appropriate for their location in the body. In addition, they lose contact inhibition and can start to grow on top of each other. This can result in formation of a tumor. It is important to make a distinction here: The term “cancer” is used to describe the diseases resulting from loss of cell-cycle regulation and subsequent cell proliferation. But the term “tumor” is more general. A “tumor” is an abnormal mass of cells, and a tumor can be benign (not cancerous) or malignant (cancerous).
Traditional cancer treatment uses radiation and/or chemotherapy to destroy cancer cells; however, these treatments can have unwanted side effects because they harm normal cells as well as cancer cells. Newer, promising therapies attempt to enlist the patient’s immune system to target cancer cells specifically. It is known that the immune system can recognize and destroy cancerous cells, and some researchers and immunologists also believe, based on the results of their experiments, that many cancers are eliminated by the body’s own defenses before they can become a health problem. This idea is not universally accepted by researchers, however, and needs further investigation for verification.

### Cell-Mediated Response to Tumors

Cell-mediated immune responses can be directed against cancer cells, many of which do not have the normal complement of self-proteins, making them a target for elimination. Abnormal cancer cells may also present tumor antigens. These tumor antigens are not a part of the screening process used to eliminate lymphocytes during development; thus, even though they are self-antigens, they can stimulate and drive adaptive immune responses against abnormal cells.
Presentation of tumor antigens can stimulate naïve helper T cells to become activated by cytokines such as IL-12 and differentiate into TH1cells. TH1 cells release cytokines that can activate natural killer (NK) cells and enhance the killing of activated cytotoxic T cells. Both NK cells and cytotoxic T cells can recognize and target cancer cells, and induce apoptosis through the action of perforins and granzymes. In addition, activated cytotoxic T cells can bind to cell-surface proteins on abnormal cells and induce apoptosis by a second killing mechanism called the CD95 (Fas) cytotoxic pathway.
Despite these mechanisms for removing cancerous cells from the body, cancer remains a common cause of death. Unfortunately, malignant tumors tend to actively suppress the immune response in various ways. In some cancers, the immune cells themselves are cancerous. In leukemia, lymphocytes that would normally facilitate the immune response become abnormal. In other cancers, the cancerous cells can become resistant to induction of apoptosis. This may occur through the expression of membrane proteins that shut off cytotoxic T cells or that induce regulatory T cells that can shut down immune responses.
The mechanisms by which cancer cells alter immune responses are still not yet fully understood, and this is a very active area of research. As scientists’ understanding of adaptive immunity improves, cancer therapies that harness the body’s immune defenses may someday be more successful in treating and eliminating cancer.

### Cancer Vaccines

There are two types of cancer vaccines: preventive and therapeutic. Preventive vaccines are used to prevent cancer from occurring, whereas therapeutic vaccines are used to treat patients with cancer. Most preventive cancer vaccines target viral infections that are known to lead to cancer. These include vaccines against human papillomavirus (HPV) and hepatitis B, which help prevent cervical and liver cancer, respectively.
Most therapeutic cancer vaccines are in the experimental stage. They exploit tumor-specific antigens to stimulate the immune system to selectively attack cancer cells. Specifically, they aim to enhance TH1 function and interaction with cytotoxic T cells, which, in turn, results in more effective attack on abnormal tumor cells. In some cases, researchers have used genetic engineering to develop antitumor vaccines in an approach similar to that used for DNA vaccines (see Micro Connections: DNA vaccines). The vaccine contains a recombinant plasmid with genes for tumor antigens; theoretically, the tumor gene would not induce new cancer because it is not functional, but it could trick the immune system into targeting the tumor gene product as a foreign invader.
The first FDA-approved therapeutic cancer vaccine was sipuleucel-T (Provenge), approved in 2010 to treat certain cases of prostate cancer.National Institutes of Health, National Cancer Institute. "Cancer Vaccines." http://www.cancer.gov/about-cancer/causes-prevention/vaccines-fact-sheet#q8. Accessed on May 20, 2016. This unconventional vaccine is custom designed using the patient’s own cells. APCs are removed from the patient and cultured with a tumor-specific molecule; the cells are then returned to the patient. This approach appears to enhance the patient’s immune response against the cancer cells. Another therapeutic cancer vaccine (talimogene laherparepvec, also called T-VEC or Imlygic) was approved by the FDA in 2015 for treatment of melanoma, a form of skin cancer. This vaccine contains a virus that is injected into tumors, where it infects and lyses the tumor cells. The virus also induces a response in lesions or tumors besides those into which the vaccine is injected, indicating that it is stimulating a more general (as opposed to local) antitumor immune response in the patient.

### Key Concepts and Summary

1. Cancer results from a loss of control of the cell cycle, resulting in uncontrolled cell proliferation and a loss of the ability to differentiate.
2. Adaptive and innate immune responses are engaged by tumor antigens, self-molecules only found on abnormal cells. These adaptive responses stimulate helper T cells to activate cytotoxic T cells and NK cells of innate immunity that will seek and destroy cancer cells.
3. New anticancer therapies are in development that will exploit natural adaptive immunity anticancer responses. These include external stimulation of cytotoxic T cells and therapeutic vaccines that assist or enhance the immune response.

### Multiple Choice

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### Short Answer


# Laboratory Analysis of the Immune Response

## Introduction

Many laboratory tests are designed to confirm a presumptive diagnosis by detecting antibodies specific to a suspected pathogen. Unfortunately, many such tests are time-consuming and expensive. That is now changing, however, with the development of new, miniaturized technologies that are fast and inexpensive. For example, researchers at Columbia University are developing a “lab-on-a-chip” technology that will test a single drop of blood for 15 different infectious diseases, including HIV and syphilis, in a matter of minutes.Chin, Curtis D. et al., “Mobile Device for Disease Diagnosis and Data Tracking in Resource-Limited Settings,”  The blood is pulled through tiny capillaries into reaction chambers where the patient’s antibodies mix with reagents. A chip reader that attaches to a cell phone analyzes the results and sends them to the patient’s healthcare provider. Currently the device is being field tested in Rwanda to check pregnant women for chronic diseases. Researchers estimate that the chip readers will sell for about $100 and individual chips for $1.Evarts, H., “Fast, Low-Cost Device Uses the Cloud to Speed Up Testing for HIV and More,” January 24, 2013. Accessed July 14, 2016. http://engineering.columbia.edu/fast-low-cost-device-uses-cloud-speed-diagnostic-testing-hiv-and-more.


# Laboratory Analysis of the Immune Response

## Polyclonal and Monoclonal Antibody Production

### Learning Objectives

By the end of this section, you will be able to:
1. Compare the method of development, use, and characteristics of monoclonal and polyclonal antibodies
2. Explain the nature of antibody cross-reactivity and why this is less of a problem with monoclonal antibodies
In addition to being crucial for our normal immune response, antibodies provide powerful tools for research and diagnostic purposes. The high specificity of antibodies makes them an excellent tool for detecting and quantifying a broad array of targets, from drugs to serum proteins to microorganisms. With , antibodies can be used to precipitate soluble antigens, agglutinate (clump) cells, opsonize and kill bacteria with the assistance of complement, and neutralize drugs, toxins, and viruses.
An antibody’s specificity results from the antigen-binding site formed within the variable regions—regions of the antibody that have unique patterns of amino acids that can only bind to target antigens with a molecular sequence that provides complementary charges and noncovalent bonds. There are limitations to antibody specificity, however. Some antigens are so chemically similar that cross-reactivity occurs; in other words, antibodies raised against one antigen bind to a chemically similar but different antigen. Consider an antigen that consists of a single protein with multiple epitopes (). This single protein may stimulate the production of many different antibodies, some of which may bind to chemically identical epitopes on other proteins.
Cross-reactivity is more likely to occur between antibodies and antigens that have low affinity or avidity. Affinity, which can be determined experimentally, is a measure of the binding strength between an antibody's binding site and an epitope, whereas avidity is the total strength of all the interactions in an antibody-antigen complex (which may have more than one bonding site). Avidity is influenced by affinity as well as the structural arrangements of the epitope and the variable regions of the antibody.  If an antibody has a high affinity/avidity for a specific antigen, it is less likely to cross-react with an antigen for which it has a lower affinity/avidity.

### Producing Polyclonal Antibodies

Antibodies used for research and diagnostic purposes are often obtained by injecting a lab animal such as a rabbit or a goat with a specific antigen. Within a few weeks, the animal’s immune system will produce high levels of antibodies specific for the antigen. These antibodies can be harvested in an antiserum, which is whole serum collected from an animal following exposure to an antigen. Because most antigens are complex structures with multiple epitopes, they result in the production of multiple antibodies in the lab animal. This so-called polyclonal antibody response is also typical of the response to infection by the human immune system. Antiserum drawn from an animal will thus contain antibodies from multiple clones of B cells, with each B cell responding to a specific epitope on the antigen ().
Lab animals are usually injected at least twice with antigen when being used to produce antiserum. The second injection will activate memory cells that make class IgG antibodies against the antigen. The memory cells also undergo affinity maturation, resulting in a pool of antibodies with higher average affinity. Affinity maturation occurs because of mutations in the immunoglobulin gene variable regions, resulting in B cells with slightly altered antigen-binding sites. On re-exposure to the antigen, those B cells capable of producing antibody with higher affinity antigen-binding sites will be stimulated to proliferate and produce more antibody than their lower-affinity peers. An adjuvant, which is a chemical that provokes a generalized activation of the immune system that stimulates greater antibody production, is often mixed with the antigen prior to injection.
Antiserum obtained from animals will not only contain antibodies against the antigen artificially introduced in the laboratory, but it will also contain antibodies to any other antigens to which the animal has been exposed during its lifetime. For this reason, antisera must first be “purified” to remove other antibodies before using the antibodies for research or diagnostic assays.

### Clinical Uses of Polyclonal Antisera

Polyclonal antisera are used in many clinical tests that are designed to determine whether a patient is producing antibodies in response to a particular pathogen. While these tests are certainly powerful diagnostic tools, they have their limitations, because they are an indirect means of determining whether a particular pathogen is present. Tests based on a polyclonal response can sometimes lead to a false-positive result—in other words, a test that confirms the presence of an antigen that is, in fact, not present. Antibody-based tests can also result in a false-negative result, which occurs when the test fails to detect an antibody that is, in fact, present.
The accuracy of antibody tests can be described in terms of test sensitivity and test specificity. Test sensitivity is the probability of getting a positive test result when the patient is indeed infected. If a test has high sensitivity, the probability of a false negative is low. Test specificity, on the other hand, is the probability of getting a negative test result when the patient is not infected. If a test has high specificity, the probability of a false positive is low.
False positives often occur due to cross-reactivity, which can occur when epitopes from a different pathogen are similar to those found on the pathogen being tested for. For this reason, antibody-based tests are often used only as screening tests; if the results are positive, other confirmatory tests are used to make sure that the results were not a false positive.
For example, a blood sample from a patient suspected of having hepatitis C can be screened for the virus using antibodies that bind to antigens on hepatitis C virus. If the patient is indeed infected with hepatitis C virus, the antibodies will bind to the antigens, yielding a positive test result. If the patient is not infected with hepatitic C virus, the antibodies will generally not bind to anything and the test should be negative; however, a false positive may occur if the patient has been previously infected by any of a variety of pathogens that elicit antibodies that cross-react with the hepatitis C virus antigens. Antibody tests for hepatitis C have high sensitivity (a low probability of a false negative) but low specificity (a high probability of a false positive). Thus, patients who test positive must have a second, confirmatory test to rule out the possibility of a false positive. The confirmatory test is a more expensive and time-consuming test that directly tests for the presence of hepatitis C viral RNA in the blood. Only after the confirmatory test comes back positive can the patient be definitively diagnosed with a hepatitis C infection. Antibody-based tests can result in a false negative if, for any reason, the patient’s immune system has not produced detectable levels of antibodies. For some diseases, it may take several weeks following infection before the immune system produces enough antibodies to cross the detection threshold of the assay. In immunocompromised patients, the immune system may not be capable of producing a detectable level of antibodies.
Another limitation of using antibody production as an indicator of disease is that antibodies in the blood will persist long after the infection has been cleared. Depending on the type of infection, antibodies will be present for many months; sometimes, they may be present for the remainder of the patient’s life. Thus, a positive antibody-based test only means that the patient was infected at some point in time; it does not prove that the infection is active.
In addition to their role in diagnosis, polyclonal antisera can activate complement, detect the presence of bacteria in clinical and food industry settings, and perform a wide array of precipitation reactions that can detect and quantify serum proteins, viruses, or other antigens. However, with the many specificities of antibody present in a polyclonal antiserum, there is a significant likelihood that the antiserum will cross-react with antigens to which the individual was never exposed. Therefore, we must always account for the possibility of false-positive results when working with a polyclonal antiserum.

### Producing Monoclonal Antibodies

Some types of assays require better antibody specificity and affinity than can be obtained using a polyclonal antiserum. To attain this high specificity, all of the antibodies must bind with high affinity to a single epitope. This high specificity can be provided by monoclonal antibodies (mAbs).  compares some of the important characteristics of monoclonal and polyclonal antibodies.
Unlike polyclonal antibodies, which are produced in live animals, monoclonal antibodies are produced in vitro using tissue-culture techniques. mAbs are produced by immunizing an animal, often a mouse, multiple times with a specific antigen. B cells from the spleen of the immunized animal are then removed. Since normal B cells are unable to proliferate forever, they are fused with immortal, cancerous B cells called myeloma cells, to yield hybridoma cells. All of the cells are then placed in a selective medium that allows only the hybridomas to grow; unfused myeloma cells cannot grow, and any unfused B cells die off. The hybridomas, which are capable of growing continuously in culture while producing antibodies, are then screened for the desired mAb. Those producing the desired mAb are grown in tissue culture; the culture medium is harvested periodically and mAbs are purified from the medium. This is a very expensive and time-consuming process. It may take weeks of culturing and many liters of media to provide enough mAbs for an experiment or to treat a single patient. mAbs are expensive ().

### Clinical Uses of Monoclonal Antibodies

Since the most common methods for producing monoclonal antibodies use mouse cells, it is necessary to create humanized monoclonal antibodies for human clinical use. Mouse antibodies cannot be injected repeatedly into humans, because the immune system will recognize them as being foreign and will respond to them with neutralizing antibodies. This problem can be minimized by genetically engineering the antibody in the mouse B cell. The variable regions of the mouse light and heavy chain genes are ligated to human constant regions, and the chimeric gene is then transferred into a host cell. This allows production of a mAb that is mostly “human” with only the antigen-binding site being of mouse origin.
Humanized mAbs have been successfully used to treat cancer with minimal side effects. For example, the humanized monoclonal antibody drug Herceptin has been helpful for the treatment of some types of breast cancer. There have also been a few preliminary trials of humanized mAb for the treatment of infectious diseases, but none of these treatments are currently in use. In some cases, mAbs have proven too specific to treat infectious diseases, because they recognize some serovars of a pathogen but not others. Using a cocktail of multiple mAbs that target different strains of the pathogen can address this problem. However, the great cost associated with mAb production is another challenge that has prevented mAbs from becoming practical for use in treating microbial infections.Saylor, Carolyn, Ekaterina Dadachova and Arturo Casadevall, “Monoclonal Antibody-Based Therapies for Microbial Diseases,”
One promising technology for inexpensive mAbs is the use of genetically engineered plants to produce antibodies (or plantibodies). This technology transforms plant cells into antibody factories rather than relying on tissue culture cells, which are expensive and technically demanding. In some cases, it may even be possible to deliver these antibodies by having patients eat the plants rather than by extracting and injecting the antibodies. For example, in 2013, a research group cloned antibody genes into plants that had the ability to neutralize an important toxin from bacteria that can cause severe gastrointestinal disease.Nakanishi, Katsuhiro et al., “Production of Hybrid-IgG/IgA Plantibodies with Neutralizing Activity against Shiga Toxin 1,”  Eating the plants could potentially deliver the antibodies directly to the toxin.

### Key Concepts and Summary

1. Antibodies bind with high specificity to antigens used to challenge the immune system, but they may also show cross-reactivity by binding to other antigens that share chemical properties with the original antigen.
2. Injection of an antigen into an animal will result in a polyclonal antibody response in which different antibodies are produced that react with the various epitopes on the antigen.
3. Polyclonal antisera are useful for some types of laboratory assays, but other assays require more specificity. Diagnostic tests that use polyclonal antisera are typically only used for screening because of the possibility of false-positive and false-negative results.
4. Monoclonal antibodies provide higher specificity than polyclonal antisera because they bind to a single epitope and usually have high affinity.
5. Monoclonal antibodies are typically produced by culturing antibody-secreting hybridomas derived from mice. mAbs are currently used to treat cancer, but their exorbitant cost has prevented them from being used more widely to treat infectious diseases. Still, their potential for laboratory and clinical use is driving the development of new, cost-effective solutions such as plantibodies.

### Multiple Choice

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### Short Answer

### Critical Thinking


# Laboratory Analysis of the Immune Response

## Detecting Antigen-Antibody Complexes

### Learning Objectives

By the end of this section, you will be able to:
1. Describe various types of assays used to find antigen-antibody complexes
2. Describe the circumstances under which antigen-antibody complexes precipitate out of solution
3. Explain how antibodies in patient serum can be used to diagnose disease
Laboratory tests to detect antibodies and antigens outside of the body (e.g., in a test tube) are called in vitro assays. When both antibodies and their corresponding antigens are present in a solution, we can often observe a precipitation reaction in which large complexes (lattices) form and settle out of solution. In the next several sections, we will discuss several common in vitro assays.

### Precipitin Reactions

A visible antigen-antibody complex is called a precipitin, and in vitro assays that produce a precipitin are called precipitin reactions. A precipitin reaction typically involves adding soluble antigens to a test tube containing a solution of antibodies. Each antibody has two arms, each of which can bind to an epitope. When an antibody binds to two antigens, the two antigens become bound together by the antibody. A lattice can form as antibodies bind more and more antigens together, resulting in a precipitin (). Most precipitin tests use a polyclonal antiserum rather than monoclonal antibodies because polyclonal antibodies can bind to multiple epitopes, making lattice formation more likely. Although mAbs may bind some antigens, the binding will occur less often, making it much less likely that a visible precipitin will form.
The amount of precipitation also depends on several other factors. For example, precipitation is enhanced when the antibodies have a high affinity for the antigen. While most antibodies bind antigen with high affinity, even high-affinity binding uses relatively weak noncovalent bonds, so that individual interactions will often break and new interactions will occur.
In addition, for precipitin formation to be visible, there must be an optimal ratio of antibody to antigen. The optimal ratio is not likely to be a 1:1 antigen-to-antibody ratio; it can vary dramatically, depending on the number of epitopes on the antigen and the class of antibody. Some antigens may have only one or two epitopes recognized by the antiserum, whereas other antigens may have many different epitopes and/or multiple instances of the same epitope on a single antigen molecule.
 illustrates how the ratio of antigen and antibody affects the amount of precipitation. To achieve the optimal ratio, antigen is slowly added to a solution containing antibodies, and the amount of precipitin is determined qualitatively. Initially, there is not enough antigen to produce visible lattice formation; this is called the zone of antibody excess. As more antigen is added, the reaction enters the equivalence zone (or zone of equivalence), where both the optimal antigen-antibody interaction and maximal precipitation occur. If even more antigen were added, the amount of antigen would become excessive and actually cause the amount of precipitation to decline.

### Precipitin Ring Test

A variety of techniques allow us to use precipitin formation to quantify either antigen concentration or the amount of antibody present in an antiserum. One such technique is the precipitin ring test (), which is used to determine the relative amount of antigen-specific antibody in a sample of serum. To perform this test, a set of test tubes is prepared by adding an antigen solution to the bottom of each tube. Each tube receives the same volume of solution, and the concentration of antigens is constant (e.g., 1 mg/mL). Next, glycerol is added to the antigen solution in each test tube, followed by a serial dilution of the antiserum. The glycerol prevents mixing of the antiserum with the antigen solution, allowing antigen-antibody binding to take place only at the interface of the two solutions. The result is a visible ring of precipitin in the tubes that have an antigen-antibody ratio within the equivalence zone. This highest dilution with a visible ring is used to determine the titer of the antibodies. The titer is the reciprocal of the highest dilution showing a positive result, expressed as a whole number. In , the titer is 16.
While a measurement of titer does not tell us in absolute terms how much antibody is present, it does give a measure of biological activity, which is often more important than absolute amount. In this example, it would not be useful to know what mass of IgG were present in the antiserum, because there are many different specificities of antibody present; but it is important for us to know how much of the antibody activity in a patient’s serum is directed against the antigen of interest (e.g., a particular pathogen or allergen).

### Ouchterlony Assay

While the precipitin ring test provides insights into antibody-antigen interactions, it also has some drawbacks. It requires the use of large amounts of serum, and great care must be taken to avoid mixing the solutions and disrupting the ring. Performing a similar test in an agar gel matrix can minimize these problems. This type of assay is variously called double immunodiffusion or the Ouchterlony assay for Orjan Ouchterlony,Ouchterlony, Örjan, “In Vitro Method for Testing the Toxin-Producing Capacity of Diphtheria Bacteria,”  who first described the technique in 1948.
When agar is highly purified, it produces a clear, colorless gel. Holes are punched in the gel to form wells, and antigen and antisera are added to neighboring wells. Proteins are able to diffuse through the gel, and precipitin arcs form between the wells at the zone of equivalence. Because the precipitin lattice is too large to diffuse through the gel, the arcs are firmly locked in place and easy to see ().
Although there are now more sensitive and quantitative methods of detecting antibody-antigen interactions, the Ouchterlony test provides a rapid and qualitative way of determining whether an antiserum has antibodies against a particular antigen. The Ouchterlony test is particularly useful when looking for cross-reactivity. We can check an antiserum against a group of closely related antigens and see which combinations form precipitin arcs.

### 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 ), 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.

### 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.

### 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 (). 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 ().
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 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.

### 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 (). 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 (). 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 .

### 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 ().
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.

### 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 (). 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 ().
 summarizes the various types of antibody-antigen assays discussed in this section.

### Key Concepts and Summary

1. When present in the correct ratio, antibody and antigen will form a precipitin, or lattice that precipitates out of solution.
2. A precipitin ring test can be used to visualize lattice formation in solution. The Ouchterlony assay demonstrates lattice formation in a gel. The radial immunodiffusion assay is used to quantify antigen by measuring the size of a precipitation zone in a gel infused with antibodies.
3. Insoluble antigens in suspension will form flocculants when bound by antibodies. This is the basis of the VDRL test for syphilis in which anti-treponemal antibodies bind to cardiolipin in suspension.
4. Viral infections can be detected by quantifying virus-neutralizing antibodies in a patient’s serum.
5. Different antibody classes in plasma or serum are identified by using immunoelectrophoresis.
6. The presence of specific antigens (e.g., bacterial or viral proteins) in serum can be demonstrated by western blot assays, in which the proteins are transferred to a nitrocellulose membrane and identified using labeled antibodies.
7. In the complement fixation test, complement is used to detect antibodies against various pathogens.

### Multiple Choice

### Fill in the Blank

### Short Answer

### Critical Thinking


# Laboratory Analysis of the Immune Response

## Agglutination Assays

### Learning Objectives

By the end of this section, you will be able to:
1. Compare direct and indirect agglutination
2. Identify various uses of hemagglutination in the diagnosis of disease
3. Explain how blood types are determined
4. 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 (). 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 (). 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.

### Agglutination of Bacteria and Viruses

The use of agglutination tests to identify streptococcal bacteria was developed in the 1920s by Rebecca Lancefield working with her colleagues A.R. Dochez and Oswald Avery.Lancefield, Rebecca C., “The Antigenic Complex of  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 (). Indirect assays can be used to detect the presence of either antibodies or specific antigens.
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 (). 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 ().
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  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.

### 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 the mother’s antibodies. 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 . 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) (). 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 antibodies against red blood cell antigens (other than the A and B antigens) that are unbound in a patient’s serum (). IAT can be used to screen pregnant women 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.
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 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 ().
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 ().

### 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 ).
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 , 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 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, women 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.
 summarizes the various kinds of agglutination assays discussed in this section.

### Key Concepts and Summary

1. Antibodies can agglutinate cells or large particles into a visible matrix. Agglutination tests are often done on cards or in microtiter plates that allow multiple reactions to take place side by side using small volumes of reagents.
2. Using antisera against certain proteins allows identification of serovars within species of bacteria.
3. Detecting antibodies against a pathogen can be a powerful tool for diagnosing disease, but there is a period of time before patients go through seroconversion and the level of antibodies becomes detectable.
4. Agglutination of latex beads in indirect agglutination assays can be used to detect the presence of specific antigens or specific antibodies in patient serum.
5. The presence of some antibacterial and antiviral antibodies can be confirmed by the use of the direct Coombs’ test, which uses Coombs’ reagent to cross-link antibodies bound to red blood cells and facilitate hemagglutination.
6. Some viruses and bacteria will bind and agglutinate red blood cells; this interaction is the basis of the direct hemagglutination assay, most often used to determine the titer of virus in solution.
7. Neutralization assays quantify the level of virus-specific antibody by measuring the decrease in hemagglutination observed after mixing patient serum with a standardized amount of virus.
8. Hemagglutination assays are also used to screen and cross-match donor and recipient blood to ensure that the transfusion recipient does not have antibodies to antigens in the donated blood.

### Multiple Choice

### Fill in the Blank

### Short Answer

### Critical Thinking


# Laboratory Analysis of the Immune Response

## EIAs and ELISAs

### Learning Objectives

By the end of this section, you will be able to:
1. Explain the differences and similarities between EIA, FEIA, and ELISA
2. Describe the difference and similarities between immunohistochemistry and immunocytochemistry
3. 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 ().
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.

### 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 , 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.

### 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 (). 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 (). 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  (Lyme disease) and HIV. There are three important differences between indirect and direct ELISAs as shown in . 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 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.

### 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 women 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 (). 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 (), 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 (), home medical tests are likely to become even more commonplace in the future.
 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.

### Key Concepts and Summary

1. Enzyme immunoassays (EIA) are used to visualize and quantify antigens. They use an antibody conjugated to an enzyme to bind the antigen, and the enzyme converts a substrate into an observable end product. The substrate may be either a chromogen or a fluorogen.
2. Immunostaining is an EIA technique for visualizing cells in a tissue (immunohistochemistry) or examining intracellular structures (immunocytochemistry).
3. Direct ELISA is used to quantify an antigen in solution. The primary antibody captures the antigen, and the secondary antibody delivers an enzyme. Production of end product from the chromogenic substrate is directly proportional to the amount of captured antigen.
4. Indirect ELISA is used to detect antibodies in patient serum by attaching antigen to the well of a microtiter plate, allowing the patient (primary) antibody to bind the antigen and an enzyme-conjugated secondary antibody to detect the primary antibody.
5. Immunofiltration and immunochromatographic assays are used in lateral flow tests, which can be used to diagnose pregnancy and various diseases by detecting color-labeled antigen-antibody complexes in urine or other fluid samples

### Multiple Choice

### Fill in the Blank

### Short Answer

### Critical Thinking


# Laboratory Analysis of the Immune Response

## Fluorescent Antibody Techniques

### Learning Objectives

By the end of this section, you will be able to:
1. Describe the benefits of immunofluorescent antibody assays in comparison to nonfluorescent assays
2. Compare direct and indirect fluorescent antibody assays
3. Explain how a flow cytometer can be used to quantify specific subsets of cells present in a complex mixture of cell types
4. 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 . 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  (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  or  from sputum samples (). 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.

### Indirect Fluorescent Antibody Techniques

Indirect fluorescent antibody (IFA) tests () 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 (). 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 presence of symptoms involving two or more organ systems to be considered indicative of SLE.Gill, James M., ANNA M. Quisel, PETER V. Rocca, and DENE T. Walters. “Diagnosis of systemic lupus erythematosus.”

### Flow Cytometry

Fluorescently labeled antibodies can be used to quantify cells of a specific type in a complex mixture using flow cytometry (), 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.
 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.
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.  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.

### 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.
 compares the mechanisms of the fluorescent antibody techniques discussed in this section.

### Key Concepts and Summary

1. Immunofluorescence assays use antibody-fluorogen conjugates to illuminate antigens for easy, rapid detection.
2. Direct immunofluorescence can be used to detect the presence of bacteria in clinical samples such as sputum.
3. Indirect immunofluorescence detects the presence of antigen-specific antibodies in patient sera. The fluorescent antibody binds to the antigen-specific antibody rather than the antigen.
4. The use of indirect immunofluorescence assays to detect antinuclear antibodies is an important tool in the diagnosis of several autoimmune diseases.
5. Flow cytometry uses fluorescent mAbs against cell-membrane proteins to quantify specific subsets of cells in complex mixtures.
6. Fluorescence-activated cell sorters are an extension of flow cytometry in which fluorescence intensity is used to physically separate cells into high and low fluorescence populations.

### Multiple Choice

### Fill in the Blank

### Critical Thinking


# Skin and Eye Infections

## Introduction

The human body is covered in skin, and like most coverings, skin is designed to protect what is underneath. One of its primary purposes is to prevent microbes in the surrounding environment from invading underlying tissues and organs. But in spite of its role as a protective covering, skin is not itself immune from infection. Certain pathogens and toxins can cause severe infections or reactions when they come in contact with the skin. Other pathogens are opportunistic, breaching the skin’s natural defenses through cuts, wounds, or a disruption of normal microbiota resulting in an infection in the surrounding skin and tissue. Still other pathogens enter the body via different routes—through the respiratory or digestive systems, for example—but cause reactions that manifest as skin rashes or lesions.
Nearly all humans experience skin infections to some degree. Many of these conditions are, as the name suggests, “skin deep,” with symptoms that are local and non-life-threatening. At some point, almost everyone must endure conditions like acne, athlete’s foot, and minor infections of cuts and abrasions, all of which result from infections of the skin. But not all skin infections are quite so innocuous. Some can become invasive, leading to systemic infection or spreading over large areas of skin, potentially becoming life-threatening.