Polyclonal and Monoclonal Antibody Production Quiz

PDF page 869: 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). Table 20.1 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 (Figure 20.4).

PDF page 867: 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 (Figure 20.3). 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 antigenbinding sites. On re-exposure to the antigen, those B cells capable of producing antibody with higher affinity antigenbinding 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.

PDF page 869: 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). Table 20.1 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 (Figure 20.4).

PDF page 869: 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). Table 20.1 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 (Figure 20.4).

PDF page 870: Characteristics of Polyclonal and Monoclonal Antibodies Monoclonal Antibodies Polyclonal Antibodies Expensive production Inexpensive production Long production time Rapid production Large quantities of specific antibodies Large quantities of nonspecific antibodies Recognize a single epitope on an antigen Recognize multiple epitopes on an antigen Production is continuous and uniform once the hybridoma is made Different batches vary in composition

PDF page 870: Characteristics of Polyclonal and Monoclonal Antibodies Monoclonal Antibodies Polyclonal Antibodies Expensive production Inexpensive production Long production time Rapid production Large quantities of specific antibodies Large quantities of nonspecific antibodies Recognize a single epitope on an antigen Recognize multiple epitopes on an antigen Production is continuous and uniform once the hybridoma is made Different batches vary in composition

PDF page 870: Characteristics of Polyclonal and Monoclonal Antibodies Monoclonal Antibodies Polyclonal Antibodies Expensive production Inexpensive production Long production time Rapid production Large quantities of specific antibodies Large quantities of nonspecific antibodies Recognize a single epitope on an antigen Recognize multiple epitopes on an antigen Production is continuous and uniform once the hybridoma is made Different batches vary in composition

PDF page 866: 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 in vitro assays, 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 (Figure 20.2). 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.

PDF page 868: 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 crossreact 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

PDF page 868: 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 crossreact 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

PDF page 871: 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

PDF page 871: 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