Diagnostic Immunology

Immunoassays for Disease

Immunoassays are laboratory techniques based on the detection of antibody production in response to foreign antigens.

Learning Objectives

Describe how immunoassays aid in the diagnosis of disease

Key Takeaways

Key Points

  • When microbial agents penetrate the body, they elicit an immune response that involves cellular and humoral components.
  • An immune response is usually characterized by antibody secretions. These can be measured in the laboratory through various biochemical and serological techniques.
  • Most immunoassays rely on the formation of antibody- antigen complexes that can be identified using an indicator molecule.

Key Terms

  • humoral: Of or relating to the body fluids or humours.
  • antibody: A protein produced by B-lymphocytes that binds to a specific antigen.

The Immune System

Immunology is the study of molecules, cells, and organs that make up the immune system. The function of the immune system is to recognize self antigens from non-self antigens and defend the body against non-self (foreign) agents. Through specific and non-specific defense mechanisms, the body’s immune system is able to react to microbial pathogens and protect against disease. The first line of defense against infection is intact skin, mucosal membrane surfaces, and secretions that prevent pathogens from penetrating into the body.

When a foreign agent penetrates the first line of resistance, an immune reaction is elicited and immune cells are recruited into the site of infection to clear microorganisms and damaged cells by phagocytosis. If the inflammation remains aggravated, antibody-mediated immune reaction is activated and different types of immune cells are engaged to resolve the disease. The immune system is composed of cellular and humoral elements. The cellular component includes mast cells, neutrophils, macrophages, T and B lymphocytes, and plasma cells. The humoral component includes complement, lyzozyme, interferon, antibodies, and cytokines. All work cooperatively to eliminate immunogenic foreign substances from the body.

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Immune complex: This is the complex formation of a specific antibody-antigen.

Immunoassays

To aid in the diagnosis of disease caused by infectious microorganisms, immunoassays have been developed. These biochemical and serological techniques are based on the detection and quantitation of antibodies generated against an infectious agent, a microbe, or non-microbial antigen.

Because antibodies can be produced against any type of macromolecule, antibody-based techniques are useful in identifying molecules in solution or in cells. A blood sample is collected from the patient during the acute phase of the disease when antibody levels are high. Serum is then isolated and the concentration of antibodies is measured through various methods. Most assays rely on the formation of large immune complexes when an antibody binds to a specific antigen which can be detected in solution or in gels. Recent methods employ pure antibodies or antigens that have been immobilized on a platform and that can be measured using an indicator molecule. These methods provide high sensitivity and specificity and have become standard techniques in diagnostic immunology.

Antibody Functions

Antibodies, part of the humoral immune response, are involved in pathogen detection and neutralization.

Learning Objectives

Differentiate among affinity, avidity, and cross-reactivity in antibodies

Key Takeaways

Key Points

  • Antibodies are produced by plasma cells, but, once secreted, can act independently against extracellular pathogen and toxins.
  • Antibodies bind to specific antigens on pathogens; this binding can inhibit pathogen infectivity by blocking key extracellular sites, such as receptors involved in host cell entry.
  • Antibodies can also induce the innate immune response to destroy a pathogen, by activating phagocytes such as macrophages or neutrophils, which are attracted to antibody-bound cells.
  • Affinity describes how strongly a single antibody binds a given antigen, while avidity describes the binding of a multimeric antibody to multiple antigens.
  • A multimeric antibody may have individual arms with low affinity, but have high overall avidity due to synergistic effects between binding sites.
  • Cross reactivity occurs when an antibody binds to a different-but-similar antigen than the one for which it was raised; this can increase pathogen resistance or result in an autoimmune reaction.

Key Terms

  • avidity: the measure of the synergism of the strength individual interactions between proteins
  • affinity: the attraction between an antibody and an antigen

Antibody Functions

Differentiated plasma cells are crucial players in the humoral immunity response. The antibodies they secrete are particularly significant against extracellular pathogens and toxins. Once secreted, antibodies circulate freely and act independently of plasma cells. Sometimes, antibodies can be transferred from one individual to another. For instance, a person who has recently produced a successful immune response against a particular disease agent can donate blood to a non-immune recipient, confering temporary immunity through antibodies in the donor’s blood serum. This phenomenon, called passive immunity, also occurs naturally during breastfeeding, which makes breastfed infants highly resistant to infections during the first few months of life.

Antibodies coat extracellular pathogens and neutralize them by blocking key sites on the pathogen that enhance their infectivity, such as receptors that “dock” pathogens on host cells. Antibody neutralization can prevent pathogens from entering and infecting host cells, as opposed to the cytotoxic T-cell-mediated approach of killing cells that are already infected to prevent progression of an established infection. The neutralized antibody-coated pathogens can then be filtered by the spleen and eliminated in urine or feces.

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Mechanisms of antibody action: Antibodies may inhibit infection by (a) preventing the antigen from binding to its target, (b) tagging a pathogen for destruction by macrophages or neutrophils, or (c) activating the complement cascade.

Antibodies also mark pathogens for destruction by phagocytic cells, such as macrophages or neutrophils, because they are highly attracted to macromolecules complexed with antibodies. Phagocytic enhancement by antibodies is called opsonization. In another process, complement fixation, IgM and IgG in serum bind to antigens, providing docking sites onto which sequential complement proteins can bind. The combination of antibodies and complement enhances opsonization even further, promoting rapid clearing of pathogens.

Affinity, avidity, and cross reactivity

Not all antibodies bind with the same strength, specificity, and stability. In fact, antibodies exhibit different affinities (attraction) depending on the molecular complementarity between antigen and antibody molecules. An antibody with a higher affinity for a particular antigen would bind more strongly and stably. It would be expected to present a more challenging defense against the pathogen corresponding to the specific antigen.

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Antibody affinity, avidity, and cross reactivity: (a) Affinity refers to the strength of single interactions between antigen and antibody, while avidity refers to the strength of all interactions combined. (b) An antibody may cross-react with different epitopes.

The term avidity describes binding by antibody classes that are secreted as joined, multivalent structures (such as IgM and IgA). Although avidity measures the strength of binding, just as affinity does, the avidity is not simply the sum of the affinities of the antibodies in a multimeric structure. The avidity depends on the number of identical binding sites on the antigen being detected, as well as other physical and chemical factors. Typically, multimeric antibodies, such as pentameric IgM, are classified as having lower affinity than monomeric antibodies, but high avidity. Essentially, the fact that multimeric antibodies can bind many antigens simultaneously balances their slightly-lower-binding strength for each antibody/antigen interaction.

Antibodies secreted after binding to one epitope on an antigen may exhibit cross reactivity for the same or similar epitopes on different antigens. Cross reactivity occurs when an antibody binds not to the antigen that elicited its synthesis and secretion, but to a different antigen. Because an epitope corresponds to such a small region (the surface area of about four to six amino acids), it is possible for different macromolecules to exhibit the same molecular identities and orientations over short regions.

Cross reactivity can be beneficial if an individual develops immunity to several related pathogens despite having been exposed to or vaccinated against only one of them. For instance, antibody cross reactivity may occur against the similar surface structures of various Gram-negative bacteria. Conversely, antibodies raised against pathogenic molecular components that resemble self molecules may incorrectly mark host cells for destruction, causing autoimmune damage. Patients who develop systemic lupus erythematosus (SLE) commonly exhibit antibodies that react with their own DNA. These antibodies may have been initially raised against the nucleic acid of microorganisms, but later cross-reacted with self-antigens. This phenomenon is also called molecular mimicry.

Serology

Serology is the study of blood serum and other bodily fluids for the identification of antibodies.

Learning Objectives

Describe how serology can be used to identify antibodies in blood serum and other bodily fluids

Key Takeaways

Key Points

  • Serology is based on detecting immunoglobulin levels during the course of an infection.
  • Serological techniques can differentiate between IgM and IgG antibodies, thus determining the stage of the infection.
  • Serological techniques are important for the diagnosis of immunological diseases.

Key Terms

  • immunoglobulin G: Most abundant antibody isotype secreted by plasma B cells.
  • immunoglobulin M: largest antibody produced by B cells and the first to appear in response to initial exposure to antigen.
  • serology: the scientific study of blood serum and other bodily fluids.

Serology is the scientific study of blood serum and other bodily fluids. In practical immunological terms, serology is the diagnostic identification of antibodies in the serum. Serological tests are performed on blood serum, and body fluids such as semen and saliva. In practice, the term usually refers to the diagnostic identification of antibodies in the serum or the detection of antigens of infectious agents in serum. Such antibodies are typically formed in response to an infection (against a given microorganism), against other foreign proteins (in response, for example, to a mismatched blood transfusion), or to one’s own proteins (in instances of autoimmune disease).

A primary immune response occurs when a B cell sees an antigen for the first time. Antigen binding to the surface of the B cell stimulates the production of antibodies that are capable of binding directly to the antigen. Because this first recognition process takes time for antibody development, there is an initial delay for the body to fight the invading antigens. Immunoglobulin M (IgM) is an antibody produced during the primary immune response and plays a significant role fighting infection. When an antigen is introduced into the body for the first time, large quantities of IgM are produced. Meanwhile, the B cells are producing highly specific Immunoglobulin G (IgG) more slowly. Once IgG is produced in quantity, the IgG plays a greater role in the removal of antigens from the body due to its ability to bind to the antigen molecules more tightly. Through the course of an infection, a rapid spike of circulating IgM can be seen in the bloodstream. This is followed by a decrease of IgM as the amount of IgG increases. Medical laboratory personnel can identify the course and duration of an infection by measuring the ratio of IgM to IgG in the bloodstream. A ratio high in IgM indicates that an infection is in its early stages, while a ratio high in IgG indicates that the infection is in its later stage.

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Immune response: When B and T cells begin to replicate, some of the offspring that they produce will end up becoming long-lived memory cells. These memory cells will remember all specific pathogens encountered during the animal’s lifetime and can thus call forth a strong response if the pathogen ever invades the body again.

Precipitation Reactions

Precipitation reactions are serological assays for the detection of immunoglobulin levels from the serum of a patient with infection.

Learning Objectives

Describe how precipitation reactions can be used for the detection of immunoglobulin levels in the serum of a patient

Key Takeaways

Key Points

  • Precipitation assays are performed in semi-solid media such as agar or agarose where antibodies and antigens can diffuse toward one another and form a visible line of precipitation.
  • There are several precipitation methods applied in the diagnostic laboratory. These include single, double, and electroimmunodiffusion.
  • The most widely used gold standard precipitation methods are Ouchterlony test and Mancini test.

Key Terms

  • precipitin: Any antibody which reacts with an antigen to form a precipitate.

Precipitation reactions are based on the interaction of antibodies and antigens. They are based on two soluble reactants that come together to make one insoluble product, the precipitate. These reactions depend on the formation of lattices (cross-links) when antigen and antibody exist in optimal proportions. Excess of either component reduces lattice formation and subsequent precipitation. Precipitation reactions differ from agglutination reactions in the size and solubility of the antigen and sensitivity. Antigens are soluble molecules and larger in size in precipitation reactions. There are several precipitation methods applied in clinical laboratory for the diagnosis of disease. These can be performed in semisolid media such as agar or agarose, or non-gel support media such as cellulose acetate.

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Precipitation reaction: Difference in the visual appearance of an aggregate and a precipitate.

Precipitation methods include double immunodiffusion (qualitative gel technique that determines the relationship between antigen and antibody), radial immunodiffusion (semi-quantitation of proteins by gel diffusion using antibody incorporated in agar), and electroimmunodiffusion (variation of the double immunodiffusion method reaction that uses an electric current to enhance the mobility of the reactants toward each other). Precipitation reactions are less sensitive than agglutination reactions but remain gold standard serological techniques. The most commonly used serologic precipitation reactions are the Ouchterlony test (based on double immunodiffusion and named after the Swedish physician who invented it), and the Mancini method (based on single radial immunodiffusion). In the double immunodiffusion technique, three basic reaction patterns result from the relationship of antigens and antibodies. These patterns are identity, non-identity, and partial identity. The Mancini method results in precipitin ring formation on a thin agarose layer. The diameter of the ring correlates with the concentration of proteins in the precipitin.

Agglutination Reactions

Agglutination reactions are used to assess the presence of antibodies in a specimen by mixing it with particulate antigens.

Learning Objectives

Describe how agglutination reactions can be used to assess the presence of antibodies in a specimen

Key Takeaways

Key Points

  • Agglutination reactions produce visible aggregates of antibody – antigen complexes when antibodies or antigens are conjugated to a carrier.
  • Carriers used in agglutination methods could be artificial (e.g., latex or charcoal) or biological (e.g., erythrocytes ).
  • There are various methods of agglutination reactions that follow the same principle, but they differ in the elements they employ based on the desired endpoint and the main purpose of the test.

Key Terms

  • avidity: The measure of the synergism of the strength of individual interactions between proteins.
  • erythrocytes: Red blood cells.
  • agglutination: the clumping together of red blood cells or bacteria, usually in response to a particular antibody

Agglutination is the visible expression of the aggregation of antigens and antibodies. Agglutination reactions apply to particulate test antigens that have been conjugated to a carrier. The carrier could be artificial (such as latex or charcoal particles) or biological (such as red blood cells). These conjugated particles are reacted with patient serum presumably containing antibodies. The endpoint of the test is the observation of clumps resulting from that antigen-antibody complex formation. The quality of the result is determined by the time of incubation with the antibody source, amount and avidity of the antigen conjugated to the carrier, and conditions of the test environment (e.g., pH and protein concentration). Various methods of agglutination are used in diagnostic immunology and these incude latex agglutination, flocculation tests, direct bacterial agglutination, and hemagglutination.

In latex agglutination, many antibody molecules are bound to latex beads (particles), which increases the number of antigen-binding sites. If an antigen is present in a test specimen, it will bind to the antibody and form visible, cross-linked aggregates. Latex agglutination can also be performed with the antigen conjugated to the beads for testing the presence of antibodies in a serum specimen.

Flocculation tests are designed for antibody detection and are based on the interaction of soluble antigens with antibodies, producing a precipitate of fine particles that can be seen with the naked eye.

Direct bacterial agglutination uses whole pathogens as a source of antigen. It measures the antibody level produced by a host infected with that pathogen. The binding of antibodies to surface antigens on the bacteria results in visible clumps.

Hemagglutination uses erythrocytes as the biological carriers of bacterial antigens, and purified polysaccharides or proteins for determining the presence of corresponding antibodies in a specimen.

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Hemagglutination assay: Red blood cells are used as carriers to detect antibodies from a patient’s serum.

Agglutination tests are easy to perform and in some cases are the most sensitive tests currently available. These tests have a wide range of applications in the clinical diagnosis of non- infectious immune disorders and infectious disease.

Neutralization Reaction

Neutralization reactions are used to inactivate viruses and evaluate neutralizing antibodies.

Learning Objectives

Describe how neutralizing antibodies serve to block viral attachment to cells thus inhibiting viral replication

Key Takeaways

Key Points

  • When a vertebrate is infected with a virus, antibodies are produced against it. Some of the antibodies can block viral infection by neutralization which is usually the result of a formation of a virus-antibody complex. This complex can prevent viral infections in many ways.
  • Neutralizing antibodies have shown potential in the treatment of retroviral infections such as HIV. Recently, potent and broadly neutralizing human antibodies against influenza have been reported.
  • In diagnostic immunology and virology laboratories, the evaluation of neutralizing antibodies, which destroy the infectivity of viruses, can be measured by the neutralization method.

Key Terms

  • neutralization: In the immunological sense refers to the ability of antibodies to block the site(s) on bacteria or viruses that they use to enter their target cell. One example of this within biology is a neutralizing antibody.
  • virion: A single individual particle of a virus (the viral equivalent of a cell).
  • endosomes: membrane-bound compartments inside eukaryotic cells.

A neutralizing antibody defends a cell from an antigen or infectious body by inhibiting or neutralizing any effect it has biologically. The antibody response is crucial for preventing many viral infections and may also contribute to the resolution of an infection. When a vertebrate is infected with a virus, antibodies are produced against many epitopes of multiple virus proteins. A subset of these antibodies can block viral infection by a process called neutralization. This usually involves the formation of a virus-antibody complex.

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Neutralizing antibody: Antibody neutralizing an antigen and preventing its biological effect.

This virus-antibody complex can prevent viral infections in many ways. It may interfere with virion binding to receptors, block uptake into cells, prevent uncoating of the genomes in endosomes, or cause aggregation of virus particles. Many enveloped viruses are lysed when antiviral antibodies and serum complement disrupt membranes. Antibodies can also neutralize viral infectivity by binding to cell surface receptors.

Neutralizing antibodies have shown potential in the treatment of retroviral infections. Medical professionals and researchers have shown how the encoding of genes which influence the production of this particular type of antibody could help in the treatment of infections that attack the immune system. Experts in the field have used HIV treatment as an example of infections these antibodies can treat. Recently, potent and broadly neutralizing human antibodies against influenza have been reported, and have suggested possible strategies to generate an improved vaccine that would confer long-lasting immunity. Another disease which has been linked to the production of neutralizing antibodies is multiple sclerosis.

In diagnostic immunology and virology laboratories, the evaluation of neutralizing antibodies, which destroy the infectivity of viruses, can be measured by the neutralization method. In this procedure, patient serum is mixed with a suspension of infectious virus particles of the same type as those suspected of causing disease in the patient. A control suspension of virus is mixed with normal serum and is then inoculated into an appropriate cell culture. If the patient serum contains antibody to the virus, the antibody will bind to the virus particles and prevent them from invading the cells in culture, thereby neutralizing the infectivity of the virus. This technique is labor-intensive, demanding, and time consuming. It application is restricted to laboratories that perform routine viral cultures and related diagnosis.

Complement Fixation

Complement fixation is a method that demonstrates antibody presence in patient serum.

Learning Objectives

Describe how the complement fixation assay can be used to test for the presence of a specific antibody in a patient’s serum

Key Takeaways

Key Points

  • Complement fixation method is more demanding than other systems used to detect antibodies and has been replaced by more sensitive techniques.
  • Complement fixation requires several elements mixed together in optimum concentrations.
  • The indicator system for the complement fixation assay is sheep red blood cells bound to anti-sheep immunoglobulin G.

Key Terms

  • immunoglobulin G: Most abundant antibody isotype secreted by plasma B cells.

Complement fixation is a classic method for demonstrating the presence of antibody in patient serum. The complement fixation test consists of two components. The first component is an indicator system that uses combination of sheep red blood cells, complement-fixing antibody such as immunoglobulin G produced against the sheep red blood cells and an exogenous source of complement usually guinea pig serum. When these elements are mixed in optimum conditions, the anti-sheep antibody binds on the surface of red blood cells. Complement subsequently binds to this antigen -antibody complex formed and will cause the red blood cells to lyse.

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The complement pathway: Complement binds to antigen-antibody complex and leads to cell lysis.

The second component is a known antigen and patient serum added to a suspension of sheep red blood cells in addition to complement. These two components of the complement fixation method are tested in sequence. Patient serum is first added to the known antigen, and complement is added to the solution. If the serum contains antibody to the antigen, the resulting antigen-antibody complexes will bind all of the complement. Sheep red blood cells and the anti-sheep antibody are then added. If complement has not been bound by an antigen-antibody complex formed from the patient serum and known antigens, it is available to bind to the indicator system of sheep cells and anti-sheep antibody. Lysis of the indicator sheep red blood cells signifies both a lack of antibody in patient serum and a negative complement fixation test. If the patient’s serum does contain a complement-fixing antibody, a positive result will be indicated by the lack of red blood cell lysis.

Fluorescent Antibodies

Fluorescent antibodies are antibodies that have been tagged with a fluorescent compound to facilitate their detection in the laboratory.

Learning Objectives

Describe how fluorescent antibody conjugates can be used in immunoassays for protein detection

Key Takeaways

Key Points

  • Fluorescent labeling of antibodies is used in place of radioisotopes and enzymes to enhance the sensitivity and specificity of immunological tests.
  • Fluorescent antibodies can be used to stain proteins from patient serum or tissue sections fixed on a slide or live cells in suspension.
  • Fluorescent antibodies can be detected with a fluorescent microscope or a flow cell sorter.

Key Terms

  • radioisotope: A radioactive isotope of an element.

Fluorescent labeling is another method of demonstrating the complexity of antigens and antibodies. Fluorescent molecules are used as substitutes for radioisotope or enzyme labels. The fluorescent antibody technique consists of labeling antibody with dyes such as fluorescein isothiocyanate (FITC). These compounds have high affinity for proteins with which they conjugate.

Fluorescent techniques are very specific and sensitive, so fluorescent antibody-based techniques require a fluorescent microscope. A fluorescent substance absorbs light of one wavelength and emits light of a longer wavelength. Fluorescein fluoresces an intense apple-green color when excited under fluorescent microscopy. The chemical manipulation in labeling antibodies with fluorescent dyes to permit detection by direct microscopy examination does not impair antibody activity.

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Flow cell sorter: This instrument is used to analyze live cells in suspension after staining them with fluorescent antibodies.

After the labeling of a specific antibody with a fluorescent molecule, it can still be reacted with its antigen and identified microscopically. Fluorescent antibody conjugates are commonly used in immunoassays. The basic methods utilizing fluorescent antibodies include direct, inhibition, and indirect immunofluorescent assay.

In the direct technique, a fluorescent antibody is used to detect antigen-antibody reactions at a microscopic level. The inhibition immunofluorescent assay is a blocking test in which an antigen is first exposed to an unlabeled antibody, then to a fluorescent antibody, and is finally washed and examined. Indirect immunofluorescence assay is based on the ability of antibodies to react with antigens as well as act as antigens and react with anti-antibody (anti-immunoglobulin). This technique is used extensively for the detection of autoantibodies and antibodies to tissue and cellular antigens. The methods described are mostly performed on glass slides with patient serum or tissue sections. Immunofluorescence can also be performed to identify specific antigens on live cells in suspension. This method is known as flow cytometry and requires a flow cell sorter rather than a fluorescent microscope.

Enzyme-Linked Immunosorbent Assay (ELISA)

Enzyme-linked immunosorbent assay (ELISA) is a solid-phase enzyme immunoassay used to detect the presence of a substance in solution.

Learning Objectives

Describe how the Enzyme-linked immunosorbent assay (ELISA) can be used to detect and quantitate antigens, antibodies and allergens

Key Takeaways

Key Points

  • ELISA is a quantitative technique that measures serum concentration of antigens, antibodies, and allergens.
  • Standard ELISA uses antibody-antigen-antibody trapping principle with the second antibody coupled to an enzyme. If the complex is formed, the enzyme converts a clear solution into a colored one that can be measured with a spectrophotometer.
  • ELISA is performed in a muti-well microtiter plate. In addition to the test solution, standard solutions are added with known antigen concentration. These solutions will be used to infer the concentration of the antigen being tested.

Key Terms

  • spectrophotometrically: By using spectrophotometry.
  • epitope: That part of a biomolecule (such as a protein) that is the target of an immune response.

Enzyme-linked immunosorbent assay (ELISA) is a method of quantifying an antigen immobilized on a solid surface. ELISA uses a specific antibody with a covalently coupled enzyme. The amount of antibody that binds the antigen is proportional to the amount of antigen present, which is determined by spectrophotometrically measuring the conversion of a clear substance to a colored product by the coupled enzyme.

Several variations of ELISA, seen in, exist but the most commonly used method is the sandwich ELISA. The sandwich assay uses two different antibodies that are reactive with different epitopes on the antigen with a concentration that needs to be determined. A fixed quantity of one antibody is attached to a series of replicate solid supports, such as plastic microtiter multi-well plate. Test solutions containing antigen at an unknown concentration are added to the wells and allowed to bind. Unbound antigen is removed by washing, and a second antibody which is linked to an enzyme is allowed to bind. This second antibody-enzyme complex constitutes the indicator system of the test. The antigen serves as bridge, so the more antigen in the test solution, the more enzyme-linked antibody will bind. The test solution is used in parallel with a series of standard solutions with known concentrations of antigen that serve as control and reference. The results obtained from the standard solutions are used to construct a binding curve of the second antibody as a function of antigen concentration. The concentration of antigens can be inferred from absorbance readings of standard solutions.

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Sandwich ELISA: Two antibodies recognize different epitopes on same antigen.

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ELISA: Direct ELISA diagram using a viral antigen as an example.

Immunoblot Procedures

Immunoblot is a technique for analyzing proteins via antigen-antibody specific reactions.

Learning Objectives

Describe how Western blotting allows individuals to detect specific solubilized proteins from serum or cell or tissue extracts

Key Takeaways

Key Points

  • In immunoblot techniques such as Western blot analysis, proteins are separated by electrophoresis and transferred onto nitrocellulose sheets, then are identified by their reaction with labeled antibodies.
  • Electrophoresis uses an electric current to separate proteins based on their size. Big proteins migrate slower and are represented by the highest bands on the blot, while small proteins migrate faster and are indicated by the lowest bands on the blot.
  • Immunoblot assays are usually performed to confirm results obtained by other techniques such as ELISA.

Key Terms

  • sodium dodecyl sulfate: strong detergent agent used to reduce and unfold native protein.
  • blot: method of transferring protein, DNA, or RNA onto a carrier membrane.

Immunoblot procedures like protein blotting, or Western blotting, allow individuals to detect specific solubilized proteins from extracts made from cells or tissues, before or after any purification steps.

Immunoblotting Procedures

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Immunoblot: proteins separated by molecular weight and represented by a dark band on a blot.

This analytic technique proceeds in the following steps. Proteins are generally separated by size using sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. After this, they are transferred to a synthetic membrane via dry, semi-dry, or wet blotting methods. In the electric field generated by a power supply, the proteins coated with negatively charged SDS migrate toward the positive electrode. As the proteins migrate out of the gel, they are captured on a membrane. Protein binding to the membrane is an irreversible mechanism. Membranes can be of the nitrocellulose, polyvinylidene difluoride (PVDF), or nylon variety. The membrane can then be blocked with serum albumin or milk solution to prevent non-specific antibody binding. This is followed by probing with antibodies specific to the protein being studied on the membrane, a method that is similar to immunohistochemistry, but without a need for fixation. This technique exploits the specificity inherent in antigen-antibody recognition. Detection is typically performed using chromogen or peroxide-linked secondary antibodies to catalyze a chromogenic or chemiluminescent reaction.

Applications of Immunoblotting

Western blotting is a routine molecular biology method that can be used to semi-quantitatively compare protein levels between extracts. The size separation, prior to blotting, allows the protein molecular weight to be gauged, as compared with known molecular weight markers. Immunoblots are most often used in research settings and are usually performed to confirm results from ELISA or other immunoassays. In clinical diagnostic settings, immunoelectrophoresis is applied, which involves the electrophoresis of serum or urine followed by immunodiffusion. The size and position of precipitation bands provides the same type of information about antibody amount as the double immunodiffusion method.

Tests That Differentiate Between T Cells and B cells

Methods used to differentiate T cells and B cells include staining cell surface receptors and functional assays like the T lymphocyte cytotoxicity assay.

Learning Objectives

Describe how T cells and B cells can be differentiated using staining of cell surface receptors and functional assays like the T lymphocyte cytotoxicity assay

Key Takeaways

Key Points

  • There are two types of lymphocytes: B cells and T cells. These two components of the immune system have different functions but cooperate to fight infection.
  • T cells elicit cell-mediated immune response, while B cells elicit humoral immunity.
  • Lymphocytes are the only immunologically specific cellular components of the immune system.

Key Terms

  • cytotoxic: of, relating to, or being a cytotoxin
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SEM Lymphocyte: A scanning electron microscope (SEM) image of a single human lymphocyte.

Lymphocytes are the only immunologically specific cellular components of the immune system. They are divided into two types based on the pathogen recognition receptors they express on their surface. T cells or T lymphocytes belong to a group of white blood cells known as lymphocytes. They are called T cells because they mature in the thymus. They play a central role in cell-mediated immunity along with initiating rejection of foreign tissues following organ transplantation. B-cells are also white blood cells and are a vital part of the humoral immunity branch of the adaptive immune system. These two cell types can function independently or cooperatively to defend the body against pathogens. T-lymphocytes can be distinguished from other lymphocytes like B cells and natural killer cells (NK cells) by the presence of a T cell receptor (TCR) on the cell surface. Alternatively, B-cells can be distinguished from other lymphocytes like T cells and natural killer cells (NK cells) by the presence of a protein on the B-cell’s outer surface called a B-cell receptor (BCR).

Traditionally, T-lymphocytes were defined by their ability to form E-rosettes when they bind selectively to sheep erythrocytes. T-lymphocytes express CD3, CD4, CD8, or CD25 markers. B-lymphocytes express CD19 marker. The expression of different markers allows the separation/ differentiation of T and B cells. Another functional assay used to identify T-lymphocyte is the cytotoxic activity assay. This assay is based on measuring the killing ability that a determined number of T lymphocytes have for a certain number of target cells when both populations are placed together. B-lymphocytes have membrane-bound immunoglobulins that can be stained with anti-immunoglobulin labeled with fluorescent dyes and detected with a fluorescent microscope. More modern techniques like flow cytometry and immunohistochemistry are commonly used and rely on the use of fluorescent antibodies. These techniques are based on staining B and T cells for unique cell surface markers known as cluster of differentiation (CD).

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Cluster of differentiation: T and B lymphocytes express unique CD markers.

In Vivo Testing

In vivo testing using animal models of disease help discover new ways of solving complex health problems.

Learning Objectives

Describe how animals can be used for diagnostic antibody production

Key Takeaways

Key Points

  • In vivo testing is necessary for medical and research purposes. The medical field benefits from animal models to test the safety of drugs before they are used on patients. The research field benefits from in vivo testing by validating in vitro findings in vertebrates closest to humans.
  • The most used animal models are mice, rats, and other rodents.
  • In vivo testing is useful for the production of polyclonal antibodies applied in immunoassays and diagnostic immunology.

Key Terms

  • in vitro: In an artificial environment outside the living organism.
  • antiserum: a serum prepared from human or animal sources containing antigens specific for combatting an infectious disease
  • in vivo: Within a living organism.

In Vivo Testing

In vivo methods refer to the use of animals as a conduit to generate purified polyclonal antibody solutions ( antiserum ) for research purposes. Polyclonal antibodies are applied in immunological assays to diagnose disease.

In vivo testing follows strict guidelines and humane animal use ethics. The protocol for diagnostic antibody production in animals follows multiple steps. Animals are injected with microbes or antigenic fragments that elicit an immune response; the immune response is allowed to develop for 1-2 weeks, after which blood is harvested. This blood now contains antibodies created from the antigens that were introduced into the animals. Antibodies are purified from the serum to make antiserum or a purified antibody solution for one particular antigen.

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A white laboratory rat: Animals are used in laboratory experiments to translate in vitro findings.

These preparations will produce multiple antibody types that recognize different epitopes on the antigen, hence the term polyclonal. Polyclonal antibodies have various applications in the clinic and in research laboratories. Animals are also used to model human diseases in the research field. They are useful vehicles to understand how our bodies work, find cures and treatments for diseases, test new drugs for safety, and evaluate medical procedures before they are used on patients.

Mice, and other rodents such as rats and hamsters, make up over 90% of the animals used in biomedical research. In addition to having bodies that work similar to humans and other animals, rodents are small in size, easy to handle, relatively inexpensive to buy and keep, and produce many offspring in a short period of time. In vivo testing remains a crucial step for the evaluation of in vitro experimental findings and the production of immunological solutions needed for the diagnosis of human diseases.

The Future of Diagnostic Immunology

The future of diagnostic immunology lies in the production of specific antibody-based assays and the development of improved vaccines.

Learning Objectives

Describe how immunologic methods are used in the treatment and prevention of infectious diseases and immune-mediated diseases

Key Takeaways

Key Points

  • Diagnostic immunology has considerably advanced due to the development of automated methods.
  • New technology takes into account saving samples, reagents, and reducing cost.
  • The future of diagnostic immunology faces challenges in the vaccination field for protection against HIV and as anti-cancer therapy.

Key Terms

  • ELISA: enzyme-linked immunosorbent assay; assay based on the principle of antibody-antigen interaction.

The Future of Diagnostic Immunology

Modern immunology relies heavily on the use of antibodies as highly specific laboratory reagents. The diagnosis of infectious diseases, the successful outcome of transfusions and transplantations, and the availability of biochemical and hematologic assays with extraordinary specificity and sensitivity capabilities all attest to the value of antibody detection.

Immunologic methods are used in the treatment and prevention of infectious diseases and in the large number of immune -mediated diseases. Advances in diagnostic immunology are largely driven by instrumentation, automation, and the implementation of less complex and more standardized procedures. Examples of such processes are as follows:

  • miniaturization (use of microtiter plates to save samples and reagents),
  • amplified immunoassays (chemiluminesent ELISA),
  • flow cytometry with monoclonal antibodies,
  • immunoglobulins,
  • molecular methods (polymerase chain reactions).

These methods have facilitated the performance of tests and have greatly expanded the information that can be developed by a clinical laboratory. The tests are now used for clinical diagnosis and the monitoring of therapies and patient responses. Immunology is a relatively young science and there is still so much to discover. Immunologists work in many different disease areas today that include allergy, autoimmunity, immunodeficiency, transplantation, and cancer.

Interestingly, no matter what the areas of expertise, vaccine development and understanding how vaccines work pose the greatest challenges. The vaccines currently used primarily generate an antibody response, which is able to attack free-moving pathogens, but is unable to fight bacteria and viruses, such as human immunodeficiency virus (HIV). In the cancer research field, vaccines that stimulate the immune system to attack tumor cells are undergoing clinical trials.

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Antibody sculpture: Angel of the West is a sculpture by Julian Voss-Andreae based on the antibody structure.