Immunization and Immune Testing

Immunization and ImmuneTesting Rights versus Responsibilities susceptible people are too rarely in contact with infected individuals to allow disease transmission. This effect is called herd immunity. The currently recommended vaccine against measles is MMR, which protects against measles, mumps, and rubella. One dose is to be given at age 12–15 months, followed by a booster at 4–6 years, and a second booster between 19 and 40. There is no controversy in the scientific community on MMR efficacy: the vaccine is highly effective. However, there is an ethical dilemma between the individual rights of people to refuse to be vaccinated as compared to their responsibility towards public health, since lack of vaccination can allow the virus to spread, resulting in outbreaks. If you were on a panel advising the CDC, how would you answer these questions? 1. Should we force people to be vaccinated? ­ http://www.cdc.gov/measles/cases-outbreaks.html 2. What procedures should be used to ensure the highest vaccination rates? Go to the study area to solve the problem. Immunization ­ In this chapter, we will discuss three applications of immunology: active immunization (vaccination), passive immunotherapy using immunoglobulins (antibodies), and immune testing. Vaccination has proven the most efficient and costeffective method of controlling infectious diseases. Without the use of effective vaccines, millions more people worldwide would suffer each year from potentially fatal infectious diseases, including measles, mumps, and polio. The administration of immunoglobulins has further reduced morbidity and mortality from certain infectious diseases, such as hepatitis A, in unvaccinated individuals. Medical personnel also make practical use of immune responses as a diagnostic procedure. For example, the detection of antibodies to HIV in a person’s blood indicates that the individual has been exposed to that virus and may develop AIDS. The remarkable specificity of antibodies also enables the detection of drugs in urine, recognition of pregnancy at early stages, and the identification or characterization of other biological material. The many tests developed for these purposes are the focus of the discipline of serology and are discussed in the second half of this chapter. M17_BAUM7206_05_SE_C17.indd 500 3. What other facts would help make decisions related to vaccination? ­ In the early 20th century, almost all children contracted measles, with four million new infections per year, resulting in approximately 50,000 hospitalizations and 6,000 deaths. But, in 2000, the Centers for Disease Control and Prevention (CDC) declared measles eliminated (defined by lack of disease transmission for a year) in the United States. This medical advance capped a campaign of vaccination that began in 1963. However, multiple outbreaks in 2014 resulted in the greatest number of cases of measles in the United States since 1995. How did measles reemerge? Several factors contributed. Measles is still common in other parts of the world, and travelers can bring the measles virus here. A measles outbreak occurs when there are significant numbers of unvaccinated people in a community. When the vaccination rate in a community is above 95 percent, then even unvaccinated individuals are protected because An individual may be made immune to an infectious disease by two artificial methods: active immunization, which involves administering antigens to a patient so that the patient actively mounts an adaptive immune response, and passive immunother­ apy, in which a patient acquires temporary immunity through the transfer of antibodies formed by other individuals or animals (see Table 16.5 on p. 493). In the following sections, we will review the history of immunization before examining immunization and immunotherapy in more detail. Brief History of Immunization LE A R N I N G O UTC O M E 17.1 Discuss the history of vaccination from the 12th century through the present. As early as the 12th century, the Chinese noticed that children who recovered from smallpox never contracted the disease a 11/23/16 3:35 AM Immunization      501 1From Latin vacca, meaning “cow.” M17_BAUM7206_05_SE_C17.indd 501 Number of reported cases (thousands) 50 Introduction of two different vaccines Polio 40 30 20 10 0 1950 Number of reported cases (thousands) second time. They therefore adopted a policy of deliberately infecting young children with particles of ground smallpox scabs from other children who had survived mild cases. By doing so, they succeeded in significantly reducing the population’s overall morbidity and mortality from the disease. News of this procedure, called variolation (var’ē-ō-lā’shŭn), spread westward through central Asia, and the technique was widely adopted. Lady Mary Montagu (1689–1762), the wife of the English ambassador to the Ottoman Empire, learned of the procedure, had it performed on her own children, and told others about it upon her return to England in 1721. As a result, variolation came into use in England and in the American colonies. Although effective and usually successful, variolation caused death from smallpox in 1% to 2% of recipients and in people exposed to recipients, so in time variolation was outlawed. The English physician Edward Jenner demonstrated in 1796 that protection against smallpox could be conferred by inoculation with crusts from a person infected with cowpox—a related but very mild disease. His new technique became widely adopted. Because cowpox was also called vaccinia1 (vak-sin’ē-ă), Jenner called the new technique vaccination (vak’-si-nā’shŭn), and the protective inoculum a vaccine (vak-sēn’). Today, we use the term immunization to refer to the administration of any antigenic inoculum, which are all called vaccines. For many years thereafter, vaccination against smallpox was widely practiced, even though no one understood how it worked or whether similar techniques could protect against other diseases. In 1879, Louis Pasteur conducted experiments on the bacterium Pasteurella multocida (pas-ter-el’ă mul-tŏ’si-da) and demonstrated that he could make an effective vaccine against this organism (which causes a disease in birds called fowl cholera). Once he understood the basic principle of vaccine manufacture, vaccines against anthrax and rabies rapidly followed. In 1890, researchers in Germany discovered that vaccines provide protection through the actions of antibodies. They developed the technique of transferring protective antibodies to susceptible individuals—that is, passive immunotherapy (im’yū-nō-thār’ă-pē). By the late 1900s, immunologists and health care providers had formulated vaccines that significantly reduced the number of cases of many infectious diseases (FIGURE 17.1). We also have successful vaccines against some types of cancer. Health care providers, governments, and international organizations working together have rid the world of naturally occurring smallpox, and we hope for the worldwide eradication of polio, measles, mumps, and rubella. Even though immunologists have produced vaccines that protect people against many deadly diseases, a variety of political, social, economic, and scientific problems prevent vaccines from reaching all those who need them. In developing nations worldwide, over 3 million children still die each year from vaccine-preventable infectious diseases, primarily because of political obstacles. Additionally, some pathogens, such as the protozoa of malaria and the virus of AIDS, still frustrate attempts to develop effective vaccines against them. 800 1960 1970 1980 Year 1990 2000 Introduction of vaccine 2010 Measles 600 400 200 0 1950 1960 1970 1980 Year 1990 2000 2010 ▲ FIGURE 17.1 The effect of immunization in reducing the prevalence of two infectious diseases in the United States. Polio is no longer endemic in the United States. Measles is nearly eradicated. Furthermore, the existence of vaccine-associated risks—both medical risks (the low but persistent incidence of vaccine-caused diseases) and financial risks (the high costs of developing and producing vaccines and the risk of lawsuits by vaccine recipients who have adverse reactions)—has in recent years discouraged investment in new vaccines. Thus, although the history of immunization is marked by stunning advancements in public health, the future of immunization poses challenges. Next, we take a closer look at active immunization, commonly known as vaccination. Active Immunization LE A R N I N G O UTC O M E S 17.2 Describe the advantages and disadvantages of five types of vaccines. 17.3 Describe three methods by which recombinant genetic techniques can be used to develop improved vaccines. 17.4 Delineate the risks and benefits of routine vaccination in healthy populations, mentioning contact immunity and herd immunity. 11/23/16 3:35 AM e 17    Immunization and Immune Testing r hapt       C In the following subsections, we examine types of vaccines, the roles of technology in producing modern vaccines, and issues concerning vaccine safety. Vaccine Types Scientists are constantly striving to develop vaccines of maximal efficacy and safety. In each case, a pathogen is altered or inactivated so that it is less likely to cause illness; however, not all types of vaccines are equally safe or effective. Effectiveness can be checked by measuring the antibody level—called the titer (tī’ter)— in the blood. When a titer is low, antibody production can be bolstered by administration of more antigen—a booster immunization. The general types of vaccines, each of which has its own combination of strengths and weaknesses, are attenuated (mod­ ified live) vaccines, inactivated (or killed) vaccines, toxoid vaccines, combination vaccines, and recombinant gene vaccines. Each of these is named for the type of antigen used in the inoculum. ­ Attenuated (Modified Live) Vaccines Virulent microbes are not used in vaccines because they cause disease. Immunologists can reduce virulence so that, although still active, a pathogen is less likely to cause disease. The process of reducing virulence is called attenuation (ă-ten-yū-ā’shŭn). A common method for attenuating viruses involves raising them for numerous generations in cells in which they normally don’t replicate well. As the viruses adapt to this inhospitable environment, they can lose the ability to produce disease in humans. Scientists can reduce virulence in bacteria by culturing them under unusual conditions or by using genetic manipulation. At tenuated vaccines—those containing attenuated microbes—are also called modified live vaccines. Because they contain active but generally avirulent microbes, these vaccines cause very mild infections but no serious disease under normal conditions. Attenuated viruses in such a vaccine infect host cells and replicate; the infected cells then process endogenous viral antigens. Because modified live vaccines contain active microbes, a large number of antigen molecules are available. These tend to elicit vigorous antibody and cell-mediated immune responses. Further, vaccinated individuals can infect those around them, providing contact immunity—that is, immunity beyond the individual receiving the vaccine. Although usually very effective, attenuated vaccines can be hazardous because modified microbes may retain enough residual virulence to cause disease in immunosuppressed people. Pregnant women should not receive live vaccines because of the danger that the attenuated pathogen will cross the placenta and harm the developing baby. Occasionally, modified viruses actually revert to wild type or mutate to a form that causes persistent infection or disease. For example, in 2000, a polio epidemic in the Dominican Republic and Haiti resulted from the reversion of an attenuated virus in oral polio vaccine to a virulent poliovirus. For this reason, we no longer use oral polio vaccine to immunize children in the United States. Inactivated (Killed) Vaccines For some diseases, live vaccines have been replaced by inactivated vaccines, which are of two types: • Whole agent vaccines are produced with inactive but whole microbes, such as rabies vaccine and hepatitis A vaccine. M17_BAUM7206_05_SE_C17.indd 502 • Subunit vaccines are produced with antigenic fragments of microbes. Because neither whole agent nor subunit vaccines can replicate, revert, mutate, or retain residual virulence, they are safer than live vaccines. However, because they cannot replicate, booster doses must be administered to achieve full immunity, and immunized individuals do not stimulate immunity in their contacts. Also, with whole agent vaccines, nonantigenic portions of the microbe occasionally stimulate a painful inflammatory response in some individuals. As a result, whole agent pertussis vaccine has been replaced with a subunit pertussis vaccine called acellular pertussis vaccine, which is a mixture of four proteins from the pertussis bacterium. When microbes are killed for use in vaccines, it is important that their antigens remain as similar to those of living organisms as possible. If chemicals are used for killing, they must not alter the antigens responsible for stimulating protective immunity. A commonly used inactivating agent is formalde­ hyde, which denatures proteins and nucleic acids. Because the microbes of inactivated vaccines cannot reproduce, they do not present as many antigenic molecules to the body as do live vaccines; therefore, inactivated vaccines are antigenically weak. They are administered in high doses or in multiple doses, or they are incorporated with materials called adjuvants2 (ad’joo-văntz), substances that increase the effective antigenicity of the vaccine by stimulating immune cell receptors and their actions. Unfortunately, high individual doses and multiple dosing increase the risk of producing allergies, and the use of adjuvants to increase antigenicity may stimulate local inflammation. Because all types of killed vaccines are recognized by the immune system as exogenous antigens, they stimulate an antibody immune response. Toxoid Vaccines For some bacterial diseases, notably tetanus and diphtheria, it is more efficient to induce an immune response against toxins than against cellular antigens. Toxoid vaccines (tok’soyd) are chemically or thermally modified toxins that are used in vaccines to stimulate active immunity. As with killed vaccines, toxoids stimulate antibody-mediated immunity. Because toxoids have few antigenic determinants, effective immunization requires multiple childhood doses as well as reinoculations every 10 years for life.▶ ANIMATIONS:Vaccines: Function,Types Combination Vaccines The Centers for Disease Control and Prevention (CDC) has approved several combination vaccines for routine use. These vaccines combine antigens from several attenuated and inactivated pathogens and toxoids that are administered simultaneously. Examples include MMR—a vaccine against measles, mumps, and rubella—and Pentacel, which is a vaccine against diphtheria, tetanus, pertussis (whooping cough), polio, and diseases of Haemophilus influenzae (hē-mof’i-lŭs in-flu-en’zī). Vaccines Using Recombinant Gene Technology Although live attenuated, inactivated, and toxoid vaccines have been highly successful in controlling infectious diseases, researchers continue to seek ways to make vaccines more effective, cheaper, 2From latin adjuvo, meaning “to help.” 11/23/16 3:35 AM       Immunization and safer and to make new vaccines against pathogens that have been difficult to protect against. For example, scientists have developed a recombinant DNA vaccine against Blastomy­ ces (blas-tō-mī’sēz) infection in dogs—the first vaccine against a fungal pathogen. Scientists can also use a variety of genetic recombinant techniques to improve vaccines. For example, they can selectively delete virulence genes from a pathogen, producing an irreversibly attenuated microbe, one that cannot revert to a virulent pathogen (FIGURE 17.2a). Scientists also use recombinant techniques to produce large quantities of very pure antigens for use in vaccines. In Production of vaccine this process, scientists isolate the gene that codes for an antigen and insert it into a bacterium, yeast, or other cell, which then expresses and releases the antigen (FIGURE 17.2b). Vaccine manufacturers produce a hepatitis B subunit vaccine in this manner using recombinant yeast cells. Alternatively, a genetically altered microbial cell or virus may express the antigen and act as a live vaccine (FIGURE 17.2c). Experimental recombinant vaccines of this type have used genetically modified adenoviruses, herpesviruses, poxviruses, or bacteria such as Salmonella (sal’mŏ-nel’ă). Vaccinia virus (cowpox virus) is often used because it is easy to administer orally or Components of vaccine Virulence gene is deleted Virulent pathogen with antigens Harmless, attenuated pathogen (a) Antigen Cell Insertion of Isolated DNA DNA into a cell coding for antigenic protein Cell releases antigenic protein ◀ FIGURE 17.2 Some uses of recombinant DNA technology for making improved vaccines. (a) Deletion of virulence gene(s) to create an attenuated pathogen for use in a vaccine. (b) Insertion of a gene that codes for a selected antigenic protein into a cell, which then produces large quantities of the antigen for use in a vaccine. (c) Insertion of a gene that codes for a selected antigenic protein into a cell or virus, which displays the antigen. The entire recombinant is used in a vaccine. (d) Injection of DNA containing a selected gene (in this case, as part of a plasmid) into an individual. Once some of this DNA is incorporated into the genome of a patient’s cells, those cells synthesize and process the antigen, which stimulates an immune response. Antigenic protein molecules (b) Antigen Isolated DNA coding for antigen Insertion of DNA into virus or cell Virus or cell presenting pathogen’s antigen (c) Antigen Isolated DNA coding for antigen Insertion of DNA into plasmid vector Recombinant plasmids produced Plasmid in body’s cell, which then synthesizes pathogen’s antigen (d) M17_BAUM7206_05_SE_C17.indd 503 11/23/16 3:35 AM r e 17    Immunization and Immune Testing hapt       C by dermal scratching. Further, its large genome makes inserting a new gene into it relatively easy. Another method results in the body’s own cells expressing the antigen. In this type of vaccein, DNA coding for a pathogen’s antigen is inserted into the body in a vector, perhaps inside a plasmid (FIGURE 17.2d). The body’s cells take up the DNA and then transcribe and translate the gene to produce antigen molecules, which trigger an immune response. No recombinant DNA vaccine of this type has yet been approved for use in humans. Vaccine Manufacture Manufacturers mass-produce many vaccines by growing bacteria in laboratory culture vessels, but because viruses require a host cell to reproduce, they are cultured inside chicken eggs. Availability of sterile eggs is thus critical for manufacturing viral vaccines such as flu vaccines. Because the vaccines are produced in eggs, physicians must withhold such immunizations from patients with egg allergies. Research on gene-based vaccines and development of vaccines in genetically modified plants may result in safer vaccines. Recommended Immunizations The CDC and medical associations publish recommended immunization schedules for children, adults, and special populations, such as health care workers and HIV-positive individuals. The recommendations are frequently modified to reflect changes in the relationships between pathogens and the human population. FIGURE 17.3 highlights the CDC-Recommended Immunization Schedule—United States, 2016 Childhood Vaccine Birth Hepatitis B (Hep B) Dose 1 1 mo 2 mos 4 mos 6 mos 12 mos Adolescent 15 mos 18 mos 19–23 mos 2–3 yrs Dose 3 Dose 2 Rotavirus 1 2 Diphtheria, tetanus, pertussis 1 2 3 Haemophilus influenzae type b (Hib) 1 2 3 4 Pneumococcal (PCV) 1 2 3 4 Inactivated polio (IPV) 1 2 4–6 yrs 7–10 11–12 13–15 yrs yrs yrs 16 yrs 17–18 19–49 50–64 yrs yrs yrs Ú65 yrs Catch-up immunization 4 5 Tdap once, and Td every 10 yrsa 6 5 3 4 Annually Influenza Measles, mumps, rubella (MMR) 1 2 Varicella-zoster 1 2 Hepatitis A 1 or 2 1 2 2 Doses Meningococcal 1 Human papillomavirus (HPV) 1 2 3 Range of recommended ages for immunization Adult 2 3 Doses a Tdap and Td, used for adult boosters, are slightly different vaccines than the childhood vaccine, DTaP. Range for catch-up immunization ▲ FIGURE 17.3 The CDC’s recommended immunization schedule for the general population. Adult meningococcal vaccine is recommended for college students who live in dormitories. M17_BAUM7206_05_SE_C17.indd 504 11/23/16 3:35 AM       Immunization general 2016 immunization schedules recommended by the CDC. TABLE 17.1 lists facts concerning the types of vaccines available for each of the diseases in the vaccination schedule, as well as some other available vaccines. Vaccines against anthrax, cholera, plague, tuberculosis, and other diseases are available, but the CDC does not recommend them for the general U.S. population. It is important that patients follow the recommended immunization schedule not only to protect themselves but also to provide society with herd immunity. Herd immunity is the protection provided all individuals in a population due to the inability of a pathogen to effectively spread when a large proportion of individuals (typically more than 75%) are resistant. When immunization compliance in a population has fallen, local epidemics have resulted. Vaccine Safety Health care providers must carefully weigh the risks associated with all medical procedures, including vaccines, against their benefits. A common vaccine-associated problem is mild toxicity. Some vaccines—especially whole agent vaccines that contain adjuvant—may cause pain at the injection site for several TABLE 17.1 Principal Vaccines to Prevent Human Diseases Vaccine Method of Administration Disease Agent Disease Vaccine Type Hepatitis B Hepatitis B virus Hepatitis B Inactive subunit from recombinant yeast Intramuscular Rotavirus Rotavirus Gastroenteritis Attenuated, recombinant Oral Diphtheria/tetanus/ acellular pertussis (DTaP) Diphtheria toxin Diphtheria Tetanus Toxoid Intramuscular Tetanus toxin Whooping cough Toxoid Recommended by the CDC Bordetella pertussis Inactivated subunit (inactivated whole also available) Haemophilus influenzae type b (Hib) Haemophilus influenzae Meningitis, pneumonia, epiglottitis Inactivated subunit Intramuscular Pneumococcal (PCV) Streptococcus pneumoniae Pneumonia Inactivated subunit Intramuscular Polio Poliovirus Poliomyelitis Inactivated (attenuated also available) Subcutaneous or intramuscular (attenuated: oral) Influenza Influenzaviruses Flu Inactivated subunit Intramuscular or oral Measles/mumps/rubella Measles virus Measles Attenuated Subcutaneous (MMR) Mumps virus Mumps Attenuated Rubella virus Rubella (German measles) Attenuated Varicella-zoster Chicken pox virus Chicken pox, shingles Attenuated Subcutaneous Hepatitis A Hepatitis A virus Hepatitis A Inactivated whole Intramuscular Human papillomavirus (HPV) Human papillomaviruses Genital warts, cervical cancer Inactive recombinant Intramuscular Meningococcal Neisseria meningiditis Meningitis Inactive Subcutaneous or intramuscular Available But Not Recommended for General Population in the United States Bacillus anthracis Anthrax Inactivated whole Subcutaneous BCG (bacillus of Calmette and Guérin) Mycobacterium tuberculosis, M. leprae Tuberculosis, leprosy Attenuated Intradermal Japanese encephalitis vaccine Japanese encephalitis virus Inactive Subcutaneous Rabies Rabies virus Rabies Inactivated whole Intramuscular or intradermal Typhoid fever vaccine Salmonella enterica Typhoid fever Attenuated (inactive also available) Oral (inactive: subcutaneous or intramuscular) Vaccinia (cowpox) Smallpox virus, monkeypox virus Smallpox, monkey pox Attenuated Subcutaneous Yellow fever Yellow fever virus Yellow fever Attenuated Subcutaneous M17_BAUM7206_05_SE_C17.indd 505 E Anthrax ncephalitis 11/23/16 3:35 AM r e 17    Immunization and Immune Testing hapt       C hours or days after injection. In rare cases, toxicity may result in general malaise and possibly a fever high enough to induce seizures. Although not usually life threatening, the potential for these symptoms may be sufficient to discourage people from being immunized or having their infants immunized. A much more severe problem associated with immunization is the risk of anaphylactic shock, an allergic reaction that may develop to some component of the vaccine, such as egg proteins, adjuvants, or preservatives. Because people are rarely aware of such allergies ahead of time, recipients should remain for several minutes in the health care setting, where epinephrine is readily available to counter any signs of an allergic reaction. A third major problem associated with immunization is that of residual virulence, which we previously discussed. Attenuated viruses occasionally cause disease not only in fetuses and immunosuppressed patients but also in healthy children and adults. A good example is the attenuated oral poliovirus vaccine (OPV) mentioned previously. Though a very effective vaccine, it causes clinical poliomyelitis in one of every 2 million recipients or their close contacts. Medical personnel in the United States eliminated this problem by switching to inactivated polio vaccine (IPV). Over the past two decades, lawsuits in the United States and Europe have alleged that certain vaccines against childhood diseases cause or trigger disorders such as autism, diabetes, and asthma. Extensive research has failed to substantiate these allegations. Vaccine manufacturing methods have improved tremendously in recent years, ensuring that modern vaccines are much safer than those in use even a decade ago. The U.S. Food and Drug Administration (FDA) has established a Vaccine Adverse Event Reporting System for monitoring vaccine safety. The CDC and FDA have determined that the problems associated with immunization are far less serious than the suffering and death that would result if we stopped immunizing people. Beneficial Microbes: Cowpox: To Vaccinate or Not to Vaccinate? discusses issues surrounding the administration of smallpox vaccinations to the general public. Passive Immunotherapy LE A R N I N G O UTC O M E S 17.5 Identify two sources of antibodies for use in passive immunotherapy. 17.6 Compare the relative advantages and disadvantages of active immunization and passive immunotherapy. Passive immunotherapy (sometimes called passive immuniza­ tion) involves the administration of antibodies to a patient. Physicians use passive immunotherapy when protection against a recent infection or an ongoing disease is needed quickly. Rapid protection is achieved because passive immunotherapy does not require the body to mount a response; instead, preformed antibodies are immediately available to bind to antigen, enabling neutralization and opsonization to proceed without delay. For example, in a case of botulism poisoning (caused by the toxin of Clostridium botulinum [klos-trid’ē-ŭm bo-tū-lī’num]), passive immunotherapy with preformed antibodies against the toxin can prevent death. Antibodies directed against toxins are also called antitox­ ins (an-tē-tok’sinz); antivenom (antivenin) used to treat venomous bites or stings is an example of an antitoxin. In some cases, E E E B N FICIAL MICROB S Cowpox: To Vaccinate or Not to Vaccinate? M17_BAUM7206_05_SE_C17.indd 506 E Dr. dward Jenner developed an early use for a beneficial microbe in medicine—administering cowpox virus as the smallpox vaccine. Medical personnel in the United States followed Jenner’s example by regularly immunizing the general public until 1971, at which time the risk of contracting smallpox was deemed too low to justify required vaccinations. Indeed, in 1980, the World Health Assembly declared smallpox successfully eradicated from the natural world, though the virus still exists in research laboratories. However, recent concerns about the potential use of smallpox virus as an agent of bioterrorism has sparked debate about whether citizens should once again be vaccinated with cowpox to protect against smallpox. Although safe and effective for most healthy adults, for others the attenuated cowpox virus can result in serious side effects— even death. Individuals with compromised immune systems (such 10 mm Vaccinia necrosum as AIDS patients or cancer patients undergoing chemotherapy) are considered to be at particularly high risk for developing adverse reactions. Pregnant women, infants, and individuals with a history of the skin condition eczema are also considered poor candidates for the vaccine. Though rare, adverse reactions to the vaccine may also develop in certain otherwise healthy individuals. The more serious side effects include vaccinia necrosum (characterized by progressive cell death in the area of vaccination) and encephalitis (inflammation of the brain). Approximately one in every 1 million individuals receiving cowpox virus as a vaccine for the first time develops a fatal reaction to it. Is the risk of a bioterrorist smallpox attack great enough to warrant the exposure to the known risks of administering cowpox virus as a vaccine to the general population? If you were a public health official, what would you decide? 11/23/16 3:35 AM       Immunization infections with certain viruses—hepatitis A and B, measles, rabies, Ebola, chicken pox, and shingles—are treated with antibodies directed against the causative viruses. To acquire antibodies for passive immunotherapy, clinicians remove blood cells and clotting factors from the blood of donors; the result is serum, which contains a variety of antibodies—particularly gamma globulins. When used for passive immunotherapy, such serum is called antiserum (an-tē-sē’rŭm) or sometimes immune serum. Antisera are typically collected from human blood plasma donors or from large animals intentionally exposed to a pathogen of interest, because the large blood volume of the animals contains more antibodies than can be obtained from smaller animals. Pooled antisera from a group of human donors can be administered intravenously (intravenous immunoglobulins, IVIg) to treat immunodeficiencies and some autoimmune and inflammatory diseases. Passive immunotherapy has the following limitations: • Repeated injections of animal-derived antisera can trigger an allergic response called serum sickness, in which the recipient mounts an immune response against animal antigens found in the antisera. • The patient may degrade the antibodies relatively quickly; therefore, protection is not long lasting. • The body does not produce memory B cells in response to passive immunotherapy, so the patient is not protected against subsequent infections. ­ ­ ­ M17_BAUM7206_05_SE_C17.indd 507 Long-lived myeloma cell lines are grown in culture. 2 Plasma cells, which secrete antibodies, are removed. Antibodies 3 Hybridomas are formed by mixing and fusing plasma cells and myeloma cells; hybridomas are long lived and produce antibodies. Hybridoma 4 Hybridomas are placed individually in small wells, and their antibodies are tested for reactivity against the antigen. 5 A hybridoma that makes antibodies that react with the antigen is cloned. ­ Scientists have overcome the limitations of antisera by developing hybridomas (hī-brid-ō’măz), which are tumor cells created by fusing antibody-secreting plasma cells with cancerous plasma cells called myelomas (mī-ě-lō’măz) (FIGURE 17.4). Each hybridoma divides continuously (because of the cancerous plasma cell component) to produce clones of itself, and each clone secretes large amounts of a single antibody molecule. These identical antibodies are called monoclonal antibodies (mon-ō-klō’năl) because all of them are secreted by clones originating from a single plasma cell. Once scientists have identified the hybridoma that secretes antibodies complementary to the antigen of interest, they maintain it in tissue culture to produce the antibodies needed for passive immunotherapy. For example, physicians use such a monoclonal antibody to treat newborns infected with respiratory syncytial virus. The FDA has approved over 30 monoclonal antibodies for medical usage in America. Repeated injection of antibodies made in lab animals, such as rabbits or mice, into humans results in immune rejection of those foreign proteins, so scientists have generated transgenic mice in which the mouse immunoglobulin genes have been replaced with human immunoglobulin genes. Hybridomas made with cells from these mice secrete human antibodies, eliminating rejection. Active immunization and passive immunotherapy are used in different circumstances because they provide protection with different characteristics (FIGURE 17.5). As just noted, passive immunotherapy with preformed antibodies is used whenever immediate protection is required. However, because preformed antibodies are removed rapidly from the 1 Mouse is injected with antigen. Monoclonal antibodies Hybridoma clone ▲ FIGURE 17.4 The production of hybridomas. After a laboratory animal is injected with the antigen of interest 1 , plasma cells are removed from the animal and isolated 2 . When these plasma cells are fused with cultured cancer cells called myelomas, hybridomas result 3 . Once the hybridomas are cultured individually and the hybridoma that produces antibodies against the antigen of interest is identified 4 , it is cloned to produce a large number of hybridomas, all of which secrete identical antibodies called monoclonal antibodies 5 . blood and no memory B cells are produced, protection is temporary, and the recipient becomes susceptible again. Active immunization provides long-term protection that is capable of restimulation. TELL ME WHY Vaccines have drastically reduced the number of cases of many diseases, such as measles and whooping cough. Why should parents have their children vaccinated given that there are so few cases? 11/23/16 3:35 AM r e 17    Immunization and Immune Testing hapt       Antibody (IgG, IgM) concentration (titer) C Passive immunotherapy Injection Boosters Active immunization agglutination, neutralization, and labeled antibody tests. Some of these procedures are presented for historical reasons—more accurate and faster modern tests have replaced them. For example, one modern method—polymerase chain reaction (PCR)—can amplify copies of genes. Thus, PCR enables testing for the presence of viral genetic material rather than the presence of antibodies against the viruses, allowing infection to be detected before the body produces antibodies. Precipitation Tests LE A R N I N G O UTC O M E S 17.10 Describe the general principles of precipitation testing. 17.11 Describe the technique of immunodiffusion. Initial inoculation Time ▲ FIGURE 17.5 The characteristics of immunity produced by active immunization (red) and passive immunotherapy (green). Passive immunotherapy provides strong and immediate protection, but it disappears relatively quickly. Active immunity takes some time and may require additional booster inoculations to reach protective levels, but it is long lasting and capable of restimulation. Serological Tests That Use Antigens and Corresponding Antibodies LE A R N I N G O U TC O M E S 17.7 Define serology. 17.8 Describe several uses of serological tests. 17.9 In general terms, compare and contrast precipitation, agglutination, neutralization, complement fixation, and labeled-antibody testing methods. ­ The analysis of blood to determine the presence of particular antigens or specific antibodies is called serology (sĕ-rol’ō-jē). Scientists have developed a variety of serological tests to identify antigens or antibodies in serum. Serological methods range from simple manual procedures to complex and automated ones. Serological tests have many uses. Epidemiologists review serological test results to monitor the spread of infection through a population. Physicians order relevant tests, which are conducted by medical laboratory scientists or lab technologists, to establish diagnoses. For example, when a physician suspects a patient might be infected by both HIV and hepatitis B virus, the doctor orders both an anti-HIV test and a hepatitis B virus surface antigen test. The first determines the presence of antibodies against HIV in the serum—strong evidence that the patient is infected with HIV. A surface antigen test indicates infection with hepatitis B virus. In the following sections, we examine various types of serological methods: precipitations, turbidimetry, nephelometry, M17_BAUM7206_05_SE_C17.indd 508 One of the simplest of serological tests relies on the fact that when antigens and antibody are mixed in proper proportions, they form large, insoluble, lattice-like complexes called precipitates. For example, when a solution of a soluble antigen, such as that of the fungus Coccidioides immitis (kok-sid-ē-oy’dēz im’mi-tis), is mixed with an antiserum containing antibodies against the fungal antigen, the mixture quickly becomes cloudy because of the formation of a precipitate consisting of antigen-antibody complexes, also called immune complexes. When a given amount of antibody is added to each of a series of test tubes containing increasing amounts of antigen, the amount of precipitate increases gradually until it reaches a maximum (FIGURE 17.6a). In test tubes containing still more antigen molecules, the amount of precipitate declines; in fact, in test tubes containing antigen in great excess over antibody, no precipitate at all develops. Thus, a graph of the amount of precipitate versus the amount of antigen has a maximum in the middle values. The reasons behind this pattern of precipitation reactions are simple. Complex antigens are generally multivalent—each possesses many epitopes—and antibodies have pairs of active sites and therefore can simultaneously cross-link the same epitope on two antigen molecules (review the agglutination function of antibodies in Figure 16.14c). When there is excess antibody, each antigen molecule is covered with many antibody molecules, preventing extensive cross-linkage and thus precipitation (FIGURE 17.6b). Since there is no precipitation, an observer might conclude that there is no antigen in the solution—a negative test result. This is untrue; antigen is present. Such a false negative interpretation is called a prozone phenomenon. When the reactants are in optimal proportions, the ratio of antigen to antibody is such that cross-linking and lattice formation are extensive. As this immune complex grows, it precipitates. In mixtures in which antigen is in excess, little or no precipitation occurs because there are few cross-linkages. Single antibodies bound to antigen are small and soluble, so no precipitation occurs, which would result in a false negative interpretation. Because precipitation requires the mixing of antigen and antibody in optimal proportions, it is not possible to perform a precipitation test by combining just any two solutions containing these reagents. To ensure that the optimal concentrations of antibody and antigen come together, scientists historically 11/23/16 3:35 AM       Serological Tests That Use Antigens and Corresponding Antibodies ◀ FIGURE 17.6 Characteristics of precipitation reactions. (a) When increasing amounts of antigen are Amount of antibody precipitated Precipitate placed in tubes containing a constant amount of antibody, precipitate forms only in tubes having moderate amounts of antigen. The overlay graph shows the amount of precipitate formed versus the amount of antigen present. (b) In the presence of excess antigen or antibody, immune complexes are small and soluble; only when antigen and antibody are in optimal proportions do large complexes form and precipitate. Increasing amount of antigen (a) Antibody Antigen (b) Antibody excess Optimal proportions Antigen excess have used a technique involving movement of the molecules through an agar gel: immunodiffusion. Well containing antigen molecules Line of immune precipitation Well containing antibodies against the antigen Immunodiffusion Turbidimetric and Nephelometric Tests Turbidimetry and nephelometry are automated methods that measure the cloudiness of a solution, as occurs when antibodies and antigens are mixed together. As noted previously, when the concentrations of antibodies and antigens are optimal, the initial cloudiness is followed by precipitation. In turbidimetry, a light detector measures the amount of light passing through a solution, whereas in nephelometry, a machine measures the amount of light reflected from the antigen-antibody complexes within the solution. Medical laboratory scientists use these methods to quantify the amounts of proteins, such as antibodies and complement, in serum. M17_BAUM7206_05_SE_C17.indd 509 Agar (a) Well containing four different antigens Zone of antigen excess Zone of optimal precipitation Zone of antibody excess Lines of immune precipitation Well containing a mixture of antibodies, each reacting to a different antigen (b) ▲ FIGURE 17.7 Immunodiffusion, a type of precipitation reaction. (a) Antigen and antibody placed in wells diffuse out and through the agar; where they meet in optimal proportions, a line of precipitation forms. The artist has used color for the antigen, antibodies, and line of precipitation to clarify what happens, but in reality antigens and antibodies are colorless, and lines of precipitation are off-white. (b) When multiple antigens and antibodies are placed in the wells, multiple lines of precipitation mark the sites where different antigen-antibody combinations occurred in optimal proportions. Why did only three lines of precipitation occur when the antigen well contained four antigens? Figure 17.7a-b None of the antibodies used in the test was complementary to the fourth antigen. In the precipitation technique called immunodiffusion (im’yūnō-di-fyū’zhŭn), a researcher cuts cylindrical holes called wells in an agar plate. If the researcher is examining a serum for the presence of antibodies, then one well is filled with a solution of antigen and the other with the serum. Antigen and antibody molecules (if present) diffuse in all directions out of the wells and into the surrounding agar, and where they meet in optimal proportions, a line of precipitation appears (FIGURE 17.7a). If the solutions contain many different antigens and antibodies, each complementary pair of reactants reaches optimal proportions at different positions, and numerous lines of precipitation are produced—one for each interacting antigen-antibody pair (FIGURE 17.7b). Such an immunodiffusion test has been used to indicate exposure to complex mixtures of antigens from pathogens such as fungi. Only patients exposed to the fungi have serum antibodies against the fungal antigens, so only these patients’ sera show precipitation. Physicians can monitor and treat such patients. 11/23/16 3:35 AM r e 17    Immunization and Immune Testing hapt       C Agglutination Tests Serum added in increasing dilutions LE A R N I N G O U TC O M E S 1:1 1:10 1:100 1:1,000 Control (no 1:10,000 specimen added) 17.12 Contrast agglutination and precipitation tests. 17.13 Describe how agglutination is used in immunological testing, including titration. Not all antigens are soluble proteins that can be precipitated by antibody. Because of their multiple antigen-binding sites, antibodies can also cross-link particles, such as whole bacteria or antigen-coated latex beads, causing agglutination (ă-glooti-nā’shŭn; clumping). The difference between agglutination and precipitation is that agglutination involves the clumping of insoluble particles, whereas precipitation involves the aggregation of soluble molecules. Agglutination reactions are sometimes easier to see and interpret with the unaided eye. When the particles agglutinated are red blood cells, the reaction is called hemagglutination (hē-mă-gloo’ti-nā’shŭn). One use of hemagglutination is to determine blood type in humans. Blood is considered type A if the red blood cells possess surface antigens called A antigen, type B if they possess B antigens, type AB if they possess both antigens, and type O if they have neither antigen. In a hemagglutination reaction to determine blood type (FIGURE 17.8), two portions of a given blood sample are placed on a slide. Anti-A antibodies are added to Anti-A antibody added Anti-B antibody added Blood sample A A (a) Negative result: no agglutination of blood cells B B Positive result: agglutination of blood cells Antigen (identical in each well) ++++ Very strong agglutination +++ ++ + – Control No agglutination ▲ FIGURE 17.9 Titration, the use of agglutination to quantify the amount of antibody in a serum sample. Serial dilutions of serum are added to wells containing a constant amount of antigen. At lower serum dilutions (higher concentrations of antibody), agglutination occurs; at higher serum dilutions, antibody concentration is too low to produce agglutination. The serum’s titer is the highest dilution at which agglutination can be detected—in this case, 1:1000. one portion and anti-B antibodies to the other; the antibodies agglutinate those blood cells that possess corresponding antigens. Another use of agglutination is in a type of test that determines the concentration of antibodies in a clinical sample. Although the simple detection of antibodies is sufficient for many purposes, it is often more desirable to measure the amount of antibodies in serum. By doing so, clinicians can determine whether a patient’s antibody levels are rising, as occurs in response to the presence of active infectious disease, or falling, as occurs during the successful conclusion of a fight against an infection. One way of measuring antibody levels in blood sera is by titration (tī-tra’shŭn). In titration, the serum being tested undergoes a regular series of dilutions, and each dilution is then tested for agglutinating activity (FIGURE 17.9). Eventually, the antibodies in the serum become so dilute that they can no longer cause agglutination. The highest dilution of serum giving a positive reaction is a titer, which is expressed as a ratio reflecting the dilution. Thus, a serum that must be ◀ FIGURE 17.8 The use of hemagglutination to determine blood types in humans. (a) Antibodies with active sites that bind to either of two surface antigens (antigen A or antigen B) of red blood cells are added to portions of a given blood sample. Where the antibodies react with the surface antigens, the blood cells can be seen to agglutinate, or clump together. (b) Photo of actual test, which also includes a test for Rh antigen, to be denoted positive (+) or negative (-). What is the blood type of the person whose blood was used in this hemagglutination reaction? M17_BAUM7206_05_SE_C17.indd 510 Figure 17.8 The individual who donated the blood sample has type B+ blood. (b) 11/23/16 3:35 AM       Serological Tests That Use Antigens and Corresponding Antibodies greatly diluted before agglutination ceases (for example, has been diluted a thousandfold; that is, has a titer of 1:1000) contains more antibodies than a serum that no longer agglutinates after minimal dilution (has a titer of 1:10). The Complement Fixation Test Neutralization Tests LE A R N I N G O UTC O M E LE A R N I N G O U TC O M E S 17.14 Explain the purpose of neutralization tests. 17.15 Contrast a viral hemagglutination inhibition test with a hemagglutination test. Neutralization tests work because antibodies can neutralize the biological activity of many pathogens and their toxins. For example, combining antibodies against tetanus toxin with a sample of tetanus toxin renders the sample harmless to mice because the antibodies have reacted with and neutralized the toxin. Next we briefly consider two neutralization tests that, although not simple to perform, effectively reveal the biological activity of antibodies. Viral Neutralization ­ One neutralization test is viral neutralization, which is based on the fact that many viruses introduced into appropriate cell cultures will invade and kill the cells, a phenomenon called a cytopathic effect (seen in plaque formation; see Chapter 13, Figure 13.17). However, if the viruses are first mixed with specific antibodies against them, their ability to kill cultured cells is neutralized. In a viral neutralization test, a mixture containing serum and a known pathogenic virus is introduced into a cell culture; if there are no cytopathic effects, antibodies against that virus are present in the serum. For example, if a mixture containing an individual’s serum and a sample of hantavirus produces no cytopathic effect in a culture of susceptible cells, then it can be concluded that the individual’s serum contains antibodies to hantavirus, and these antibodies neutralized the virus. Viral neutralization tests are sufficiently sensitive and specific to ascertain whether an individual has been exposed to a particular virus or viral strain, which may lead a physician to a diagnosis or treatment or to recommendations to prevent future infection or disease. Viral Hemagglutination Inhibition Test ­ ­ ­ Because not all viruses are cytopathic—they do not kill their host cell—a neutralization test cannot be used to identify all viruses. However, many viruses (including influenzaviruses) have surface proteins that naturally clump red blood cells. (This natural process, called viral hemagglutination, must not be confused with the hemagglutination test we discussed previously—viral hemagglutination is not an antibody-antigen reaction.) Antibodies against influenzavirus inhibit viral hemagglutination; therefore, if serum from an individual stops viral hemagglutination, we know that the individual’s serum contains antibodies to that particular strain of influenzavirus. Such viral hemagglutination inhibition tests can be used to M17_BAUM7206_05_SE_C17.indd 511 detect antibodies against influenza, measles, mumps, and other viruses that naturally agglutinate red blood cells, thus revealing that a patient has been infected or immunized with the virus in question. 17.16 Briefly explain the phenomenon that is the basis for a complement fixation test. Activation of the classical complement system by antibody leads to the generation of membrane attack complexes (MACs) that disrupt cytoplasmic membranes (see Figure 15.9). This phenomenon is the basis for the complement fixation test (kom’plěment fik-sā’shŭn), which is a complex assay used to detect the presence of specific antibodies in an individual’s serum. The test can detect the presence of small amounts of antibody— amounts too small to detect by agglutination—though complement fixation tests have been replaced by other serological methods such as ELISA (discussed shortly) or genetic analysis using polymerase chain reaction (PCR) (see Figure 8.5). Labeled Antibody Tests LE A R N I N G O UTC O M E S 17.17 List three tests that use labeled antibodies to detect either antigen or antibodies. 17.18 Compare and contrast the direct and indirect fluores- cent antibody tests, and identify at least three uses for these tests. 17.19 Compare and contrast the methods, purposes, and advantages of ELISA and immunoblotting tests. A different form of serological testing involves labeled (or tagged) antibody tests, so named because these tests use antibody molecules that are linked to some molecular “label” that enables them to be detected easily. Labels include radioactive chemicals, fluorescent dyes, and enzymes. Automated machines can detect and quantify labels. For example, gamma radiation detectors can count radioactive chemicals, and fluorescence microscopes can measure fluorescent labels. Labeled antibody tests using radioactive or fluorescent labels can be used to detect either antigens or antibodies. Currently, radioactivity is rarely used, given the regulatory challenges of using and disposing of it. In the following sections, we will consider fluorescent antibody tests, ELISA, and immunoblotting tests. Fluorescent Antibody Tests Fluorescent dyes are used as labels in several serological tests. Some fluorescent dyes can be chemically linked to an antibody without affecting the antibody’s ability to bind antigen. When exposed to a specific wavelength of light (as in a fluorescence microscope), the fluorescent dye glows. Fluorescently labeled antibodies are used in direct and indirect fluorescent antibody tests. 11/23/16 3:35 AM e 17    Immunization and Immune Testing r hapt       C Direct fluorescent antibody tests identify the presence of antigen in a tissue. The test is straightforward: A scientist floods a tissue sample suspected of containing the antigen with labeled antibody, waits a short time to allow the antibody to bind to the antigen, washes the preparation to remove any unbound antibody, and examines it with a fluorescence microscope. If the suspected antigen is present, labeled antibody will adhere to it, and the scientist will see fluorescence. This is not a quantitative test—the amount of fluorescence observed is not directly related to the amount of antigen present. Scientists use direct fluorescent antibody tests to identify small numbers of bacteria in patient tissues. This technique has been used to detect Mycobacterium tuberculosis in sputum and rabies viruses infecting a brain. In one use, medical laboratory scientists employ a direct fluorescent antibody test to detect the presence of fungi in the lungs of a patient, corroborating a diagnosis of fungal pneumonia (FIGURE 17.10). Indirect fluorescent antibody tests are used to detect the presence of specific antibodies in an individual’s serum via a threestep process (FIGURE 17.11a): An antigen of interest is fixed to a microscope slide. 2 The individual’s serum is added and remains long enough to allow serum antibodies, if present, to bind to the antigen. The serum is then washed off, leaving the antibodies bound to the antigen (but not yet visible). 3 Fluorescently labeled antibodies against human antibodies (anti–human antibody antibodies) are added to the slide and bind to the antibodies already bound to the antigen. After the slide is washed to remove unbound antiantibodies, it is examined with a fluorescence microscope. Antigen on cell bound to slide 2 Slide is flooded with patient’s serum. IgG from patient’s serum 3 Fluorescent-labeled anti-Ig antiglobulin is added. Fluorescent label Anti-IgG (antiglobulin, anti-antibody) ­ (a) ­ ­    1 1 Cells with antigen are attached to slide. The presence of fluorescence indicates the presence of the labeled anti-antibodies, which are bound to serum antibodies bound to the fixed antigen; thus, fluorescence indicates that the individual has serum antibodies against the antigen of interest. Scientists can use indirect fluorescent antibody tests to detect antibodies against many viral, protozoan, or bacterial pathogens, including Neisseria gonorrhoeae (nī-se’rē-ă go-norrē’ī), the causative agent of gonorrhea (FIGURE 17.11b). The presence of antibodies indicates that the patients have been exposed to the pathogen and may need treatment or counseling on steps to take to lower their risk of future infection. LM 25 µm ▲ FIGURE 17.10 Here rabies proteins (green) are revealed in tissue. M17_BAUM7206_05_SE_C17.indd 512 (b) LM 10 µm ▲ FIGURE 17.11a-b The indirect fluorescent antibody test. This test detects the presence of a specific antibody in a patient’s serum. (a) The test procedure. 1 Antigen is attached to the slide. 2 Slide is flooded with an individual’s serum to allow specific antibodies in the serum to bind to the antigen. 3 After anti-antibodies labeled with fluorescent chemical are added and then washed off, the slide is examined with a fluorescence microscope. (b) A positive indirect antibody test, in which fluorescence indicates the presence of antibodies in an individual’s serum against a particular antigen (here, the gonorrhea bacterium). Scientists routinely identify and separate types of white blood cells, such as lymphocytes, by using specific monoclonal antibodies produced against each cell type. The researchers can attach differently colored fluorescent dyes to the antibodies, allowing them to differentiate types of lymphocytes by the color of the dye attached to each type of antibody. Such identification tests can quantify the numbers and ratios of lymphocyte subsets, information critical in diagnosing and monitoring 11/23/16 3:35 AM       Serological Tests That Use Antigens and Corresponding Antibodies disease progression and effectiveness of treatment in patients with AIDS, other immunodeficiency diseases, leukemias, and lymphomas. ELISAs (EIAs) In another type of labeled antibody test, called an enzymelinked immunosorbent assay (ELISA) (im’yū-nō-sōr’bent as’sā), or simply an enzyme immunoassay (EIA), the label is not a dye but instead an enzyme that reacts with its substrate to produce a colored product that indicates a positive test. One form of ELISA is used to detect the presence and quantify the abundance of antibodies in serum—an example of indirect testing—as might be used in diagnosis of Lyme disease, legionellosis (a type of pneumonia), or cat scratch disease. An ELISA, which can take place in wells in commercially produced plates, has five basic steps with washes to remove excess chemicals between steps (FIGURE 17.12): 1 Each of the wells in the plate is coated with antigen molecules in solution. 2 Excess antigen molecules are washed off, and another protein (such as gelatin) is added to the wells to completely coat any of the surface not coated with antigen. 3 One well is assigned to each serum being tested, and a sample of the serum is added to that well. Whenever a serum sample contains antibodies against the antigen, the antibodies bind to the antigen affixed to the plate. 4 Anti-antibodies labeled with an enzyme are added to each well. 5 The enzyme’s substrate is added to each well. The enzyme and substrate are chosen because their reaction results in products that cause a visible color change. 1 Antigen is attached to well in plate.    2 A protein such as gelatin is added to block the uncoated surface.   3 Patient serum is added; complementary antibody binds to antigen. A positive reaction in a well, indicated by the development of color, can occur only if the labeled anti-antibody has bound to antibodies attached to the antigen of interest. The intensity of the color, which can be estimated visually or measured accurately using a spectrophotometer, is proportional to the amount of antibody present in the serum. Enzyme Anti-antibody 4 Enzyme-linked anti-antibody is added and binds to bound antibody. ▶ FIGURE 17.12 Enzyme-linked immunosorbent assay (ELISA), also known as enzyme immunoassay (EIA). Shown is one well in a plate. The well is washed between o E utor id steps. 1 Antigen added to the well attaches to it irreversibly. 2 After excess antigen is removed by washing, gelatin is added to cover any V e T portion of the well not covered by antigen. 3 Test serum is added to the well; any specific antibodies in it bind to the antigen. 4 nzymelabeled anti-antibodies are added to the well and bind to any bound antibody. 5 The enzyme’s substrate is added to the well, and the enzyme converts the substrate into a colored product; the amount of color, which can be measured via spectrophotometry, is directly proportional to the amount of antibody bound to the antigen. M17_BAUM7206_05_SE_C17.indd 513 Substrate Colored product 5 Enzyme’s substrate is added, and reaction produces a visible color change. 11/23/16 3:35 AM e 17    Immunization and Immune Testing r A modification of the ELISA technique, called an antibody sand­ wich ELISA, can be used to detect antigen (FIGURE 17.13)—an example of direct testing. In testing for the presence of HIV in blood serum, for example, the plates are first coated with Colored product Gelatin An immunoblot (also called a western blot) is a technique used to detect a specific protein, such as an antibody, in a complex mixture. Immunoblotting tests are used to confirm the presence of proteins, including antibodies against pathogens. Physicians use immunoblots to verify the presence of HIV proteins or antibodies against the bacterium of Lyme disease in the blood serum of patients. Immunoblotting involves three steps (FIGURE 17.14a): 1 Electrophoresis. Antigens in a solution (in this example, HIV proteins) are placed into wells and separated by gel electrophoresis. Each of the proteins in the solution is resolved into a single band, producing invisible protein bands. (In the figure, the artist has colored the invisible bands to show you where they are.) 2 Blotting. The protein bands are transferred to an overlying nitrocellulose membrane. This can be done by absorbing the solution into absorbent paper—a process called blotting. The nitrocellulose membrane is then cut into strips. 3 Antibody testing. Each nitrocellulose strip is incubated with a test solution— in this example, samples from each of six individuals who are being tested for antibodies against HIV. After the strips are washed, an enzyme-labeled antiantibody solution is added for a time; then the strips are washed again and exposed to the enzyme’s substrate. Color develops wherever antibodies against the HIV proteins in the test solutions have bound to their substrates, as shown in the positive control. In this example, the individual tested in strip 3 is positive for antibodies against HIV, whereas the other five individuals are negative for antibodies against HIV (FIGURE 17.14b). Immunoblots are sensitive and can detect many types of proteins simultaneously. Colored bands common to all patients are normal serum proteins. Enyme-linked antibody Substrate Antigen in patient’s serum Antibody bound to microwell ­ (a) Immunoblots  • ELISAs are sensitive, able to detect very small amounts of antibody (or antigen). • Unlike some diffusion and fluorescent tests, ELISA can quantify amounts of antigen or antibody. Knowing the amount of antigen or antibody in a patient’s serum can provide information concerning the course of an infection or the effectiveness of a treatment. • ELISAs are easy to perform. • ELISAs are relatively inexpensive. • ELISAs can simultaneously test many samples quickly at once. • ELISAs lend themselves to efficient automation and can be read easily, either by direct observation or by machine. • Plates coated with antigen and gelatin can be stored for testing whenever they are needed. antibody against HIV (instead of antigen). Then the sera from individuals being tested for HIV are added to the wells, and any HIV in the sera will bind to the antibody attached to the well. Finally, each well is flooded with enzyme-labeled antibodies specific to the antigen. The name “antibody sandwich ELISA” refers to the fact that the antigen being tested for is “sandwiched” between two antibody molecules. Such tests can also be used to quantify the amount of antigen in a given sample, such as with tests for illegal drugs.  ELISA has become a test of choice for many diagnostic procedures, such as determination of HIV infection, because of its many advantages:  hapt       C Point-of-Care Testing LE A R N I N G O UTC O M E S 17.20 Discuss the benefits of using immunofiltration assays rather than an ELISA. (b) graphic assays. E ▲ FIGURE 17.13 An antibody sandwich ELISA. Because this variation of LISA is used to test for the presence of antigen, antibody is attached to the well in the initial step. A second antibody sandwiches the antigen. (a) Artist’s rendition. (b) Actual results. How many wells are positive? 17.21 Contrast immunofiltration and immunochromato- Figure 17.13 Seventeen wells are positive. M17_BAUM7206_05_SE_C17.indd 514 Recent years have seen the development of simple immunoassays that give clinicians useful results within minutes. These assays allow point­of­care testing; that is, health care 11/23/16 3:35 AM       Serological Tests That Use Antigens and Corresponding Antibodies Wells containing proteins Polyacrylamide gel 1 Electrophoresis (not shown) Polyacrylamide gel 2 Blotting Filter paper Nitrocellulose membrane Polyacrylamide gel Absorbent paper Nitrocellulose is removed from blotting stack. 1 2 3 Nitrocellulose is incubated with a test solution. 3 4 5 6 Polypeptides (a) Patient 1 2 3 4 5 6 Positive control providers do not have to send specimens to a laboratory for testing but can perform the test at the patient’s bedside or in a doctor’s office. Common point-of-care tests include immu­ nofiltration and immunochromatography assays. These tests are not quantitative, but they do rapidly give a positive or negative result, making them very useful in arriving at a quick diagnosis. Immunofiltration assays (im’yū-nō-fil-trā’shŭn) are rapid ELISAs based on the use of antibodies bound to a membrane filter rather than to plates. Because of the large surface area of a membrane filter, reactions proceed faster, and assay times are significantly shorter than a traditional ELISA. Immunochromatographic assays (im’yū-nō-krō’mat-ōgraf’ik) are faster and easier-to-read immunoassays. In these systems, an antigen solution (such as diluted blood or sputum) flowing through a porous material encounters antibody labeled with either pink colloidal3 gold or blue colloidal selenium. Where antigen and antibody bind, colored immune complexes form in the fluid, which then flows through a region where the complexes encounter antibody against them, resulting in a clearly visible pink or blue line, depending on the label used. These assays are used for pregnancy testing, which tests for human chorionic gonadotropin—a hormone produced only by an embryo or fetus—and for rapid identification of infectious agents such as HIV, Escherichia coli (esh-ě-rik’ê-ă kō’lī) O157:H7, group A Streptococcus, respiratory syncytial virus (RSV), and influenzaviruses. In one adaptation, the antibodies are coated on membrane strips, which serve as dipsticks. At one end, anti-antibodies are fixed in a line so that they cannot move in the membrane. The lower portion of the membrane is coated with antibodies against the antigen in question. These antibodies are linked to a color indicator in the form of a colloidal metal and are free to move in the membrane by capillary action. FIGURE 17.15 illustrates the procedure used to test for the presence of group A Streptococcus in the nasal secretion of a patient. A laboratory scientist prepares a solution with a nasal swab from the patient so as to release Streptococcus antigens if they are present. The scientist then dips the membrane into the solution. The membrane’s antibodies bind to streptococcal antigens, forming complexes. The complexes move up the membrane by capillary action until they reach the line of anti-antibodies, where they bind and must stop because the anti-antibodies are chemically bound to the strip. Previously, the complexes were invisible because they were dilute; now they are concentrated at the line of anti-antibodies and become visible, indicating that this patient has group A Streptococcus in the nose. Knowing that the infection is bacterial and not viral, the physician can prescribe antibacterial drugs. The Negative control (b) 3Colloidal refers to small particles suspended in a liquid or gas. ▲ FIGURE 17.14 Immunoblotting (western blotting). This technique detects specific proteins in a complex mixture, taking advantage of the high affinity and specificity of antibodies against the proteins. (a) Steps in immunoblotting test. Proteins are separated by gel electrophoresis. The artist has shown the positions of the proteins, but in reality, they would be invisible 1 . Separated proteins are transferred to a nitrocellulose membrane. This is the “blotting” 2 . Test solutions, enzyme-labeled anti-antibody, and the enzyme’s substrate are added; color changes are detected wherever antibody in the test solutions has bound to proteins 3 . (b) Real results of an immunoblot. Patient 3 has the same proteins as the positive control. M17_BAUM7206_05_SE_C17.indd 515 06/03/2018 09:58 e 17    Immunization and Immune Testing r hapt       C Line of fixed anti-antibody Zone of antibodies linked to colloidal metal, color too diffuse to see TABLE 17.2 Antibody-Antigen Immunological Tests and Some of Their Uses Strep A Dip Strep A Anti-antibodies stop movement of antibodyantigen complexes. Color becomes visible because of density of complexes. Test Use Immunodiffusion (precipitation) Diagnosis of syphilis, pneumococcal pneumonia Agglutination Blood typing; pregnancy testing; diagnosis of salmonellosis, brucellosis, gonorrhea, rickettsial infection, mycoplasma infection, yeast infection, typhoid fever, meningitis caused by Haemophilus Viral neutralization Diagnosis of infections by specific strains of viruses Viral hemagglutination inhibition Diagnosis of viral infections including influenza, measles, mumps, rubella, mononucleosis Complement fixation In the past, diagnosis of measles, influenza A, syphilis, rubella, rickettsial infections, scarlet fever, rheumatic fever, infections of respiratory syncytial virus and Coxiella Direct fluorescent antibody Diagnosis of rabies, infections of group A Streptococcus, identification of lymphocyte subsets Indirect fluorescent antibody Diagnosis of syphilis, mononucleosis Movement of fluid containing complexes of antibodies bound to antigen Prepared antigen extract from patient’s nasal sample ▲ FIGURE 17.15 Immunochromatographic dipstick. The dipstick is impregnated with colloidal metal particles linked to movable antibodies against particular antigens at one end and anti-antibodies fixed in a line closer to the other end of the membrane. E Dip LISA Immunoblot (western blot) Pregnancy testing; presence of drugs in urine; diagnosis of hepatitis A, hepatitis B, rubella; initial diagnosis of HIV infection Confirmation of infection with HIV; diagnosis of Lyme disease TELL ME WHY ­ ­ procedure from antigen preparation to diagnosis takes less than 10 minutes. TABLE 17.2 lists some antibody-antigen immune tests that can be used to diagnose selected bacterial and viral diseases. Complications of Premature Birth? Dr. Connors examines Ryan and collects a nasal wash (saline squirted into and immediately withdrawn from the nostril) to send for lab analysis. Noting Ryan’s labored breath sounds and his previous lung issues, Dr. Connors admits Ryan to the hospital so he can be monitored until the lab results return. The analysis of the nasal wash reveals that Ryan has respiratory syncytial virus (RSV). Doug and Stacy are scared by the diagnosis— Ryan’s been through so much already. Dr. Connors tells them that premature babies like Ryan are at risk for acquiring RSV infection; it is actually very common. The Centers for Disease Control and Prevention (CDC) estimates that almost all children become infected with RSV before their second birthday. There is no specific treatment for RSV, but because of Ryan’s medical history, Dr. Connors keeps Ryan in the hospital. Over the next couple of days, Ryan develops M17_BAUM7206_05_SE_C17.indd 516 A diagnostician used an ELISA to show that a newborn had antibodies against HIV in her blood. However, six months later, the same test was negative. How can this be? bronchiolitis (inflammation of the bronchioles). He is treated with supplemental oxygen and suctioning of mucus to help him breathe more easily. During the next 10 days, Ryan’s condition progressively improves, and he returns home, fully recovered. 1. The laboratory test used to identify RSV in Ryan’s nasal wash is based on detecting RSV antigen in the sample. Describe one approach to identifying antigens in a sample from a patient. 2. Even though RSV infection in infants is common, a vaccine does not currently exist. Imagine you are designing a recombinant vaccine for RSV—what viral components would you use in your vaccine? Justify your choice. 11/23/16 3:35 AM       Chapter Summary CHAPTER SUMMARY 1. The first vaccine was developed by Edward Jenner against smallpox. He called the technique vaccination. Immunization is a more general term referring to the use of vaccines against rabies, anthrax, measles, mumps, rubella, polio, and other diseases. 2. Individuals can be protected against many infections by either active immunization or passive immunotherapy. ▶ ANIMATION: Vaccines: Function 3. Active immunization involves giving antigen in the form of either attenuated vaccines, inactivated vaccines (killed vaccines), toxoid vaccines, or recombinant gene vaccines. Antibody titer refers to the amount of antibody produced. ▶ ANIMATION: Vaccines: Types 4. Immunologists can reduce the virulence of microbes used in vaccines so that they are less likely to cause disease, a practice known as attenuation. Pathogens in attenuated vaccines are weakened so that they no longer cause disease, though they are still alive or active and can provide contact immunity in unimmunized individuals who associate with immunized people. 5. Inactivated vaccines are either whole agent or subunit vaccines and often contain adjuvants, which are chemicals added to increase their ability to stimulate active immunity. ­ 6. Toxoid vaccines use modified toxins to stimulate antibodymediated immunity. 7. A combination vaccine is composed of antigens from several pathogens so they can be administered to a patient at once. 8. Vaccine developers can use recombinant gene technology to remove virulence genes from microbes to create attenuated vaccines, to produce large quantities of antigens for use in immunizations, or to alter cells, viruses, or plasmids by introducing genes for antigens into them. 9. Having a large proportion of immunized individuals (>75%) in a population interrupts disease transmission, providing protection to unimmunized individuals. Such protection is called herd immunity. 10. Vaccine developers and administrators monitor vaccine safety by checking for adverse reactions to vaccines, including mild irritation at the site of inoculation, anaphylactic shock, and the possibility of an attenuated vaccine reverting to virulence. 13. Health providers weigh the advantages and disadvantages of active versus passive immunization. Active immunization, in which patients synthesize their own antibodies and develop immunological memory, is longer lasting but relatively slow to develop. Passive immunity, in which preformed antibodies are administered, is fast acting but does not produce long-term effects. Serological Tests That Use Antigens and Corresponding Antibodies (pp. 508–516) 1. Serology is the study and use of immunological assays on blood serum to diagnose disease or identify antibodies or antigens. Scientists use antibodies to find an antigen in a specimen and use antigen to find antibodies. 2. The simplest of the serological tests is a precipitation test, in which antigen and antibody meet in optimal proportions to form immune complexes, which are often insoluble. Often, this test is performed in clear gels, where it is called immunodiffusion. 3. Automated light detectors can measure the cloudiness of a solution—an indication of the quantity of protein in the solution. Turbidimetry measures the passage of light through the solution, whereas nephelometry measures the amount of light reflected by protein in the solution. ­ (pp. 500–508) ­ Immunization 4. Agglutination tests involve the clumping of antigenic particles by antibodies. The amount of these antibodies, called a titer is measured by diluting the serum in a process called titration. 5. Antibodies to viruses or toxins can be measured using a neutralization test, such as a viral neutralization test. Infection by viruses that naturally agglutinate red blood cells can be demonstrated using a viral hemagglutination inhibition test. 6. The complement fixation test is a complex assay used to determine the presence of specific antibodies. 7. Fluorescently labeled antibodies—those chemically linked to a fluorescent dye—can be used in a variety of direct fluorescent antibody tests and indirect fluorescent antibody tests. The presence of labeled antibodies is visible through a fluorescence microscope. 9. Immunofiltration assays and immunochromatographic assays are modifications of ELISA tests that can give much more rapid diagnostic results. M17_BAUM7206_05_SE_C17.indd 517 utor e o 12. The fusion of myelomas (cancerous plasma cells) with plasma cells results in hybridomas, the source of monoclonal antibodies, which can be used in passive immunization. V T id 11. Passive immunotherapy (a type of passive immunization) involves administration of an antiserum containing preformed antibodies. Serum sickness results when the patient makes antibodies against the antiserum. 8. Enzyme-linked immunosorbent assays (ELISAs), or enzyme immunoassays (EIAs), are a family of simple tests that can be readily automated and read by machine. These tests are among the more common serological tests used. A variation of the ELISA is an immunoblot (western blot), which is used to detect antibodies against multiple antigens in a mixture. 11/23/16 3:35 AM 518     Chapter 17    Immunization and Immune Testing QUESTIONS FOR REVIEW Answers to the Questions for Review (except Short Answer questions) begin on p. A-1. Multiple Choice 1. To obtain immediate immunity against tetanus, a patient should receive                               . a. an attenuated vaccine of Clostridium tetani b. a modified live vaccine of C. tetani c. tetanus toxoid d. immunoglobulin against tetanus toxin (antitoxin) 2. Which of the following vaccine types is commonly given with an adjuvant? a. attenuated vaccine b. modified live vaccine c. chemically killed vaccine d. immunoglobulin 3. Which of the following viruses was widely used in living vaccines? a. coronavirus c. influenzavirus b. poliovirus d. retrovirus 4. When antigen and antibodies combine, maximal precipitation occurs when                               . a. antigen is in excess b. antibody is in excess c. antigen and antibody are at equivalent concentrations d. antigen is added to the antibody 5. An anti-antibody is used when                               . a. an antigen is not precipitating b. an antibody is not agglutinating c. an antibody does not activate complement d. the antigen is an antibody 6. The many different proteins in serum can be analyzed by a(n)                               . a. anti-antibody test c. precipitation test b. complement fixation test d. immunodiffusion test 7. A direct fluorescent antibody test requires which of the following? a. heat-inactivated serum b. fluorescent serum c. immune complexes d. antibodies against the antigen 8. An ELISA uses which of the following reagents? a. enzyme-labeled anti-antibody b. radioactive anti-antibody c. source of complement d. enzyme-labeled antigen 9. A direct fluorescent antibody test can be used to detect the presence of                               . a. hemagglutination c. antibodies b. specific antigens d. complement 10. Which of the following is a good test to detect rabies virus in the brain of a dog? a. agglutination b. hemagglutination inhibition c. virus neutralization d. direct fluorescent antibody M17_BAUM7206_05_SE_C17.indd 518 11. Attenuation is                               . a. the process of reducing virulence b. a necessary step in vaccine manufacture c. a form of variolation d. similar to an adjuvant 12. An antiserum is                               . a. an anti-antibody b. an inactivated vaccine c. formed of monoclonal antibodies d. the liquid portion of blood used for immunization 13. Monoclonal antibodies                               . a. are produced by hybridomas b. are secreted by clone cells c. can be used for passive immunization d. all of the above 14. The study of antibody-antigen interaction in the blood is                               . a. attenuation c. precipitation b. agglutination d. serology 15. Anti–human antibody antibodies are                               . a. found in immunocompromised individuals b. used in direct fluorescent antibody tests c. formed by animals reacting to human immunoglobulins d. an alternative method in ELISA True/False 1.                              Passive immunotherapy provides more ­prolonged immunity than active immunization. 2.                              It is standard to attenuate killed virus vaccines. 3.                              One single serological test is inadequate for an accurate diagnosis of HIV infection. 4.                              ELISA is very easily automated. 5.                              ELISA has basically replaced immunoblotting. Matching Match the characteristic in the first column with the therapy it most closely describes in the second column. Some choices may be used more than once. 1.          Induces rapid onset of immunity 2.          Induces mainly an antibody response 3.          Induces good cell-mediated immunity A. Attenuated viral vaccine B. Adjuvant C. Subunit vaccine D. Immunoglobulin E. Residual virulence 4.          Increases antigenicity 5.          Uses antigen fragments 6.          Uses attenuated microbes 11/23/16 3:35 AM Questions for Review      519 Short Answer VISUALIZE IT! 1. Identify the chemicals represented by this artist’s conception of an antibody sandwich ELISA. 1. Compare and contrast the Chinese practice of variolation with Jenner’s vaccination procedure. 2. What are the advantages and disadvantages of attenuated vaccines? d. ____________ 4. How does precipitation differ from agglutination? a. ____________ e. ____________ b. ____________ 5. Explain how a pregnancy test works at the molecular level. 6. Compare and contrast herd immunity and contact immunity. f. ____________ c. ____________ Controls 2 3 4 5 6 7. How does nephelometry differ from turbidimetry? CRITICAL THINKING 2. The two columns on the left show negative and positive immunoblot results for a particular pathogen. The numbered columns are blots of samples from 11 patients. Which patients are most likely uninfected? (–) (+) 1 3. Compare the advantages and disadvantages of passive immunotherapy and active immunization. 7 8 9 10 11 1. Is it ethical to approve the use of a vaccine that causes significant illness in 1% of patients if it protects immunized survivors against a serious disease? 2. Which is worse: to use a diagnostic test for HIV that may falsely indicate that a patient is not infected (false negative) or to use one that sometimes falsely indicates that a patient is infected (false positive)? Defend your choice. 3. Discuss the importance of costs and technical skill in selecting a practical serological test. Under what circumstances does automation become important? 4. What bodily fluids, in addition to blood serum, might be usable for immune testing? 5. Why might a serological test give a false positive result? 6. Some researchers want to distinguish B cells from T cells in a mixture of lymphocytes. How could they do this without killing the cells? 7. Describe three ways by which genetic recombinant techniques could be used to develop safer, more effective vaccines. 8. How does a toxoid vaccine differ from an attenuated vaccine? 9. Explain why many health organizations promote breast-feeding of newborns. What risks are involved in such nursing? 10. Contrast a hemagglutination test with a viral hemagglutination inhibition test. 11. Sixty years ago, parents would have done almost anything to get a protective vaccine against polio for their children. Now parents fear the vaccine, not the disease. Why? 12. Draw a picture showing, at both the molecular and the cellular level, IgM agglutinating red blood cells. M17_BAUM7206_05_SE_C17.indd 519 11/23/16 3:35 AM 520     Chapter 17    Immunization and Immune Testing CONCEPT MAPPING Using the following terms, fill in the following concept map that describes vaccines. Subunit vaccines Tetanus toxoid Modified toxins Pertussis vaccine Polio Rabies vaccine Immunizations Inactivated vaccines Measles vaccine Microorganisms Active immunity Adjuvants Attenuated vaccines Hepatitis A vaccine Vaccines provide also known as 1 9 can be 2 also known as 6 use also known as use used with 5 Live vaccines 10 Killed vaccines Antigenic fragments Toxoids are 13 with 11 Reduced virulence such as 3 7 such as such as such as 8 12 14 4 M17_BAUM7206_05_SE_C17.indd 520 3/6/18 4:54 PM