INTRODUCTION

Immunology, the study of the body’s immune system. It was originally the branch of medical science dealing with defence or resistance against infections, but the term has broadened over the past four decades to encompass all processes and mechanisms which discriminate between “self”—that is, the body’s own innate mechanisms, molecules, cells, and tissues, and everything that belongs to it—and “non-self”—anything which comes from outside the body; that which is alien to it. The latter term includes infectious micro-organisms (protozoa, fungi, bacteria, mycoplasmas, and viruses); parasites; toxins and poisons of sufficient size and appropriate composition, tumours and neoplastic cells; transplants; and transfused cells or molecules from genetically non-identical animals.

THE IMMUNE RESPONSE

Most animals are capable of mounting a defensive response against non-self substances; this is known as the immune response. The study of the natural development of mechanisms involved in the immune response is the main feature of immunology and immunological research. Immune responses can be classified as innate (meaning those which occur without prior exposure to the foreign substance, organism, or tissue) or acquired (meaning those which require exposure to the non-self material).

Hybridoma Technology

Hybridoma are cells that have been engineered to produce a desired antibody in large amounts. To produce monoclonal antibodies, B-cells are removed from the spleen of an animal that has been challenged with the relevant antigen. These B-cells are then fused with myeloma tumor cells that can grow indefinitely in culture (myeloma is a B-cell cancer). This fusion is performed by making the cell membranes more permeable. The fused hybrid cells (called hybridomas), being cancer cells, will multiply rapidly and indefinitely and will produce large amounts of the desired antibodies. They have to be selected and subsequently cloned by limiting dilution. Supplemental media containing Interleukin-6 (such as briclone) are essential for this step.
Selection occurs via culturing the newly fused primary hybridoma cells in selective-media, specifically media containing 1x concentration HAT for roughly 10-14 days. After using HAT it is often desirable to use HT containing media. Cloning occurs after identification of positive primary hybridoma cells. Clone by limited dilution. While some may believe that IL-6 is essential for this step, it is not necessary to add that expensive supplement, rather use 50% heat-inactivated FBS for the first week. Add 10% FBS DMEM to the clone culture plate after screening for single colony wells.

Method
HAT medium is used for preparation of monoclonal antibodies.Laboratory animals (eg. mice) are first exposed to an antigen to which we are interested in isolating anantibody against. Once splenocytes are isolated from the mammal, the B cells are fused with HGPRT negative, immortalized myeloma cells using polyethylene glycol or the Sendai virus. Fused cells are incubated in the HAT medium. Aminopterin in the medium blocks the de novo pathway. Hence, unfused myeloma cells die, as they cannot produce nucleotides by de novo or salvage pathway. Unfused B cells die as they have a short life span. In this way, only the B cell-myeloma hybrids survive. These cells produce antibodies (a property of B cells) and are immortal (a property of myeloma cells). The incubated medium is then diluted into multiwell plates to such an extent that each well contains only 1 cell. Then the supernatant in each well can be checked for desired antibody. Since the antibodies in a well are produced by the same B cell, they will be directed towards the same epitope, and are known as monoclonal antibodies.
The production of monoclonal anti-bodies was first invented by Cesar Milstein, Georges J. F. Köhler and Niels Kaj Jerne in 1975.

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DISCRIMINATION BETWEEN SELF AND NON-SELF:

The discrimination between self and non-self is fundamental to immunology. Clones of cells which have the capacity to recognize self antigens are eliminated early (usually neonatally) in an animal’s development. This “clonal deletion”, which occurs possibly by processes involving programmed cell death (apoptosis), is not yet fully understood, but is known to produce tolerance to self antigens. Tumour cells can be destroyed by immune processes if they express antigens that are not present on normal cells (the so-called “tumour associated antigens”). The magnitude of the immune response against an antigen is often considerably affected by the difference between its structure and that of host antigens, and by the extent of recruitment of “help” (largely mediated by T-cells) for the antigen.

Tumor associated transplantation antigens (TATAs)

Tumor specific transplantation antigens are unique to tumor
cells and do not occur on normal cells in the body. They may result from
mutations in tumor cells that generate altered cellular proteins; cytosolic
processing of these proteins are presented with class I MHC molecules inducing a
cell mediated response by tumor specific CTLs.

Tumor associated transplantation antigens (TATAs) are not unique
to tumor cells; may be proteins that are expressed on normal cells during fetal
development when the immune system in immature. Reactivation of the embryonic
genes that encode these proteins in tumor cells results in their expression on
the fully differentiated tumor cells.

Transformed African Green MonkeyKidney Fibroblast Cells (COS-7 Line)

Innate Immunity:

Animals have natural barriers and substances which help to prevent infections. Animals have natural barriers and substances which help to prevent infections with micro-organisms and parasites. The skin and mucus-containing secretions act as barriers, and proteolytic enzymes (digestive enzymes which break down protein) present in body fluids have the ability to destroy some invading organisms. In addition to this, cells with specific innate immune functions rapidly respond to invading organisms to destroy them. These cells are primarily of two types: monocytes (especially macrophages) and polymorphonuclear leucocytes. Both of these can ingest micro-organisms by a process known as “phagocytosis” and destroy them. They also synthesize and secrete many substances, including cytokines and enzymes, which protect against infections and promote the development of the immune response.

protection against parasites. The function of circulating IgD is unclear. Immunoglobulins are produced and secreted by B-lymphocytes.
Antibodies bind specifically to non-self organisms and substances. This often results in inactivation of undesired properties. Antigen-antibody complexes are removed from the body by various processes and antibody-coated micro-organisms are particularly susceptible to phagocytosis by macrophages and other cells. After interacting with an antigen, antibodies trigger a range of immunobiological mechanisms which protect against infections and other undesirable effects.

Cell-mediated immunity is mediated by T-lymphocytes. Antigen-specific T-cells are produced, which interact with antigens to mediate a number of immunobiological functions. One example of this is the production of cytotoxic cells that specifically “kill” unwanted micro-organisms or cells. A third class of lymphocyte, known as large granular lymphocytes or natural killer (NK) cells, also destroy non-self cells and micro-organisms. The acquired immune response complements the innate response to provide a specific, very efficient defence system.

CONTROLLING THE DEVELOPMENT OF ACQUIRED IMMUNITY:

The acquired immune system is tightly controlled. B-lymphocytes that secrete antibodies are strongly influenced by T-cells which can either “help” the response or suppress it. Such T-cells secrete cytokines and other potent, biologically active molecules which enhance or inhibit B-cell activation, maturation, and ability to secrete appropriate immunoglobulin. B-cells (and other cell types) also secrete cytokines which modulate immunity. In particular, Interleukins 1,2,4,6,10,12,13,14,15, and 16, •-interferon, and transforming growth factor β influence the development and modulation of the acquired immune response. IL-12 is a potent stimulator of NK cells.

IMMUNITY AND DISEASE:

Immunological processes are normally beneficial. However, development of inappropriate immunity can cause disease or at least adverse clinical effects. The breakdown of tolerance to self can result in autoimmune diseases, such as rheumatoid arthritis, primary biliary cirrhosis, systemic lupus erythematosus, Hashimoto’s thyroiditis, myasthenia gravis, and insulin-dependent diabetes mellitus. Production of antibodies against sperm can result in a woman’s inability to conceive. Excessive aggressive immune reactivity (hypersensitivity) can result in conditions like anaphylaxis, allergy, and asthma. An impaired immune response occurs in a group of pathological conditions known as immunodeficiencies. Such conditions can affect innate or acquired immunity and may involve humoral or cellular responses. They vary in severity. Bruton’s congenital agammaglobulinaemia results in a grossly impaired ability to produce immunoglobulins. Common variable immunodeficiency is the most frequently occurring immunodeficiency disease and involves a defect in B-cell function such that the cells cannot secrete antibodies. DiGeorge and Nezelof syndromes involve defects in T-cell development. Severe combined immunodeficiency results in total lack of ability to mount humoral and cellular immune responses. The underlying basis for such conditions is often genetic, but in several cases is unclear. Other immunodeficiencies are caused by viruses. The acquired immune deficiency syndrome (AIDS) results from infection with the human immunodeficiency virus (HIV); this virus destroys CD4-positive T-cells, causing dramatic immunosuppression. Immunosuppressive disorders result in an inability to cope with infections and, in some cases, the spontaneous growth of tumours. Severe illness or death often results.

Antigen-Antibody Reaction

NATURE OF ANTIGEN-ANTIBODY REACTIONS
A. Lock and Key Concept The combining site of an antibody is located in the Fab portion of the molecule and is constructed from the hypervariable regions of the heavy and light chains. X-Ray crystallography studies of antigen-antibody interactions show that the antigenic determinant nestles in a cleft formed by the combining site of the antibody as illustrated in Figure 1. Thus, our concept of antigen-antibody reactions is one of a key (i.e. the antigen) which fits into a lock (i.e. the antibody).
B. Non-covalent Bonds The bonds that hold the antigen to the antibody combining site are all non-covalent in nature. These include hydrogen bonds, electrostatic bonds, Van der Waals forces and hydrophobic bonds. Multiple bonding between the antigen and the antibody ensures that the antigen will be bound tightly to the antibody.
C. Reversibility Since antigen-antibody reactions occur via non-covalent bonds, they are by their nature reversible.

KEY WORDSAffinityAviditySpecificityCross reactivityAgglutinationHemagglutinationAgglutininTiterProzonePassive hemagglutinationDirect Coomb's testIndirect Coomb's testHemagglutination inhibitionEquivalence pointAntibody excessAntigen excessRadial immunodiffusionImmunoelectrophoresisCountercurrent immunoelectrophoresisRadioimmunoassayEnzyme linked immunosorbent assayCompetitive RIA/ELISANoncompetitive RIA/ELISAImmunofluorescenceFlow cytometryComplement fixation
II. AFFINITY AND AVIDITY
A. Affinity Antibody affinity is the strength of the reaction between a single antigenic determinant and a single combining site on the antibody. It is the sum of the attractive and repulsive forces operating between the antigenic determinant and the combining site of the antibody as illustrated in Figure 2.
Affinity is the equilibrium constant that describes the antigen-antibody reaction as illustrated in Figure 3. Most antibodies have a high affinity for their antigens.
B. AvidityAvidity is a measure of the overall strength of binding of an antigen with many antigenic determinants and multivalent antibodies. Avidity is influenced by both the valence of the antibody and the valence of the antigen. Avidity is more than the sum of the individual affinities. This is illustrated in Figure 4.
To repeat, affinity refers to the strength of binding between a single antigenic determinant and an individual antibody combining site whereas avidity refers to the overall strength of binding between multivalent antigens and antibodies.

III. SPECIFICITY AND CROSS REACTIVITY
A. Specificity Specificity refers to the ability of an individual antibody combining site to react with only one antigenic determinant or the ability of a population of antibody molecules to react with only one antigen. In general, there is a high degree of specificity in antigen-antibody reactions. Antibodies can distinguish differences in 1) the primary structure of an antigen, 2) isomeric forms of an antigen, and 3) secondary and tertiary structure of an antigen.
B. Cross reactivity Cross reactivity refers to the ability of an individual antibody combining site to react with more than one antigenic determinant or the ability of a population of antibody molecules to react with more than one antigen. Figure 5 illustrates how cross reactions can arise. Cross reactions arise because the cross reacting antigen shares an epitope in common with the immunizing antigen or because it has an epitope which is structurally similar to one on the immunizing antigen (multispecificity).
IV. TESTS FOR ANTIGEN-ANTIBODY REACTIONS
A. Factors affecting measurement of antigen-antibody reactions The only way that one knows that an antigen-antibody reaction has occurred is to have some means of directly or indirectly detecting the complexes formed between the antigen and antibody. The ease with which one can detect antigen-antibody reactions will depend on a number of factors.
1. Affinity The higher the affinity of the antibody for the antigen, the more stable will be the interaction. Thus, the ease with which one can detect the interaction is enhanced.
2. Avidity Reactions between multivalent antigens and multivalent antibodies are more stable and thus easier to detect.
Figure 6
3. Antigen to antibody ratio The ratio between the antigen and antibody influences the detection of antigen-antibody complexes because the size of the complexes formed is related to the concentration of the antigen and antibody. This is depicted in Figure 6.
4. Physical form of the antigen The physical form of the antigen influences how one detects its reaction with an antibody. If the antigen is a particulate, one generally looks for agglutination of the antigen by the antibody. If the antigen is soluble one generally looks for the precipitation of the antigen after the production of large insoluble antigen-antibody complexes.
Figure 7
B. Agglutination Tests
1. Agglutination/Hemagglutination When the antigen is particulate, the reaction of an antibody with the antigen can be detected by agglutination (clumping) of the antigen. The general term agglutinin is used to describe antibodies that agglutinate particulate antigens. When the antigen is an erythrocyte the term hemagglutination is used. All antibodies can theoretically agglutinate particulate antigens but IgM, due to its high valence, is particularly good agglutinin and one sometimes infers that an antibody may be of the IgM class if it is a good agglutinating antibody.
a. Qualitative agglutination test Agglutination tests can be used in a qualitative manner to assay for the presence of an antigen or an antibody. The antibody is mixed with the particulate antigen and a positive test is indicated by the agglutination of the particulate antigen. (Figure 7).
For example, a patient's red blood cells can be mixed with antibody to a blood group antigen to determine a person's blood type. In a second example, a patient's serum is mixed with red blood cells of a known blood type to assay for the presence of antibodies to that blood type in the patient's serum.
Figure 8
b. Quantitative agglutination test Agglutination tests can also be used to measure the level of antibodies to particulate antigens. In this test, serial dilutions are made of a sample to be tested for antibody and then a fixed number of red blood cells or bacteria or other such particulate antigen is added. Then the maximum dilution that gives agglutination is determined. The maximum dilution that gives visible agglutination is called the titer. The results are reported as the reciprocal of the maximal dilution that gives visible agglutination. Figure 8 illustrates a quantitative hemagglutination test.
Prozone effect - Occasionally, it is observed that when the concentration of antibody is high (i.e. lower dilutions), there is no agglutination and then, as the sample is diluted, agglutination occurs (See Patient 6 in Figure 8). The lack of agglutination at high concentrations of antibodies is called the prozone effect. Lack of agglutination in the prozone is due to antibody excess resulting in very small complexes that do not clump to form visible agglutination.

c. Applications of agglutination tests
i. Determination of blood types or antibodies to blood group antigens.
ii. To assess bacterial infections
e.g. A rise in titer of an antibody to a particular bacterium indicates an infection with that bacterial type. N.B. a fourfold rise in titer is generally taken as a significant rise in antibody titer.
d. Practical considerations Although the test is easy to perform, it is only semi-quantitative.
Figure 9
2. Passive hemagglutination The agglutination test only works with particulate antigens. However, it is possible to coat erythrocytes with a soluble antigen (e.g. viral antigen, a polysaccharide or a hapten) and use the coated red blood cells in an agglutination test for antibody to the soluble antigen (Figure 9). This is called passive hemagglutination. The test is performed just like the agglutination test. Applications include detection of antibodies to soluble antigens and detection of antibodies to viral antigens.
Figure 10
3. Coomb's Test (Antiglobulin Test)
a. Direct Coomb's Test When antibodies bind to erythrocytes, they do not always result in agglutination. This can result from the antigen/antibody ratio being in antigen excess or antibody excess or in some cases electrical charges on the red blood cells preventing the effective cross linking of the cells. These antibodies that bind to but do not cause agglutination of red blood cells are sometimes referred to as incomplete antibodies. In no way is this meant to indicate that the antibodies are different in their structure, although this was once thought to be the case. Rather, it is a functional definition only. In order to detect the presence of non-agglutinating antibodies on red blood cells, one simply adds a second antibody directed against the immunoglobulin (antibody) coating the red cells. This anti-immunoglobulin can now cross link the red blood cells and result in agglutination. This test is illustrated in Figure 10 and is known as the Direct Coomb's test.
Figure 11
b. Indirect Coomb's Test If it is necessary to know whether a serum sample has antibodies directed against a particular red blood cell and you want to be sure that you also detect potential non- agglutinating antibodies in the sample, an Indirect Coomb's test is performed (Figure 11). This test is done by incubating the red blood cells with the serum sample, washing out any unbound antibodies and then adding a second anti-immunoglobulin reagent to cross link the cells.
c. Applications These include detection of anti-rhesus factor (Rh) antibodies. Antibodies to the Rh factor generally do not agglutinate red blood cells. Thus, red cells from Rh+ children born to Rh- mothers, who have anti-Rh antibodies, may be coated with these antibodies. To check for this, a direct Coombs test is performed. To see if the mother has anti-Rh antibodies in her serum an Indirect Coombs test is performed.
Figure 12
4. Hemagglutination Inhibition The agglutination test can be modified to be used for the measurement of soluble antigens. This test is called hemagglutination inhibition. It is called hemagglutination inhibition because one measures the ability of soluble antigen to inhibit the agglutination of antigen-coated red blood cells by antibodies. In this test, a fixed amount of antibodies to the antigen in question is mixed with a fixed amount of red blood cells coated with the antigen (see passive hemagglutination above). Also included in the mixture are different amounts of the sample to be analyzed for the presence of the antigen. If the sample contains the antigen, the soluble antigen will compete with the antigen coated on the red blood cells for binding to the antibodies, thereby inhibiting the agglutination of the red blood cells. as illustrated in Figure 12.
By serially diluting the sample, you can quantitate the amount of antigen in your unknown sample by its titer. This test is generally used to quantitate soluble antigens and is subject to the same practical considerations as the agglutination test.

Precipitation tests

Precipitation tests
1. Radial Immunodiffusion (Mancini) In radial immunodiffusion antibody is incorporated into the agar gel as it is poured and different dilutions of the antigen are placed in holes punched into the agar. As the antigen diffuses into the gel, it reacts with the antibody and when the equivalence point is reached a ring of precipitation is formed as illustrated in Figure 13.
The diameter of the ring is proportional to the log of the concentration of antigen since the amount of antibody is constant. Thus, by running different concentrations of a standard antigen one can generate a standard cure from which one can quantitate the amount of an antigen in an unknown sample. Thus, this is a quantitative test. If more than one ring appears in the test, more than one antigen/antibody reaction has occurred. This could be due to a mixture of antigens or antibodies. This test is commonly used in the clinical laboratory for the determination of immunoglobulin levels in patient samples.
Figure 14
2. Immunoelectrophoresis In immunoelectrophoresis, a complex mixture of antigens is placed in a well punched out of an agar gel and the antigens are electrophoresed so that the antigen are separated according to their charge. After electrophoresis, a trough is cut in the gel and antibodies are added. As the antibodies diffuse into the agar, precipitin lines are produced in the equivalence zone when an antigen/antibody reaction occurs as illustrated in Figure 14.
This tests is used for the qualitative analysis of complex mixtures of antigens, although a crude measure of quantity (thickness of the line) can be obtained. This test is commonly used for the analysis of components in a patient' serum. Serum is placed in the well and antibody to whole serum in the trough. By comparisons to normal serum, one can determine whether there are deficiencies on one or more serum components or whether there is an overabundance of some serum component (thickness of the line). This test can also be used to evaluate purity of isolated serum proteins.
Figure 15
3. Countercurrent electrophoresis In this test the antigen and antibody are placed in wells punched out of an agar gel and the antigen and antibody are electrophoresed into each other where they form a precipitation line as illustrated in Figure 15. This test only works if conditions can be found where the antigen and antibody have opposite charges. This test is primarily qualitative, although from the thickness of the band you can get some measure of quantity. Its major advantage is its speed.

Figure 16

Figure 17
D. Radioimmunoassay (RIA)/Enzyme Linked Immunosorbent Assay (ELISA)
Radioimmunoassays (RIA) are assays that are based on the measurement of radioactivity associated with immune complexes. In any particular test, the label may be on either the antigen or the antibody. Enzyme Linked Immunosorbent Assays (ELISA) are those that are based on the measurement of an enzymatic reaction associated with immune complexes. In any particular assay, the enzyme may be linked to either the antigen or the antibody.
1. Competitive RIA/ELISA for Ag Detection The method and principle of RIA and ELISA for the measurement of antigen is shown in Figure 16. By using known amounts of a standard unlabeled antigen, one can generate a standard curve relating radioactivity (cpm) (Enzyme) bound versus amount of antigen. From this standard curve, one can determine the amount of an antigen in an unknown sample.
The key to the assay is the separation of the immune complexes from the remainder of the components. This has been accomplished in many different ways and serves as the basis for the names given to the assay:
a. Precipitation with ammonium sulphate Ammonium sulphate (33 - 50% final concentration) will precipitate immunoglobulins but not many antigens. Thus, this can be used to separate the immune complexes from free antigen. This has been called the Farr Technique
b. Anti-immunoglobulin antibody The addition of a second antibody directed against the first antibody can result in the precipitation of the immune complexes and thus the separation of the complexes from free antigen.
c. Immobilization of the Antibody The antibody can be immobilized onto the surface of a plastic bead or coated onto the surface of a plastic plate and thus the immune complexes can easily be separated from the other components by simply washing the beads or plate (Figure 17). This is the most common method used today and is referred to as Solid phase RIA or ELISA. In the clinical laboratory, competitive RIA and ELISA are commonly used to quantitate serum proteins, hormones, drugs metabolites.
TUTORIAL ELIZA ASSAY HHMIRequires Flash
Figure 18

Figure 19
2. Noncompetitive RIA/ELISA for Ag or Ab Noncompetitive RIA and ELISAs are also used for the measurement of antigens and antibodies. In Figure 18, the bead is coated with the antigen and is used for the detection of antibody in the unknown sample. The amount of labeled second antibody bound is related to the amount of antibody in the unknown sample. This assay is commonly employed for the measurement of antibodies of the IgE class directed against particular allergens by using a known allergen as antigen and anti-IgE antibodies as the labeled reagent. It is called the RAST test (radioallergosorbent test). In Figure 19, the bead is coated with antibody and is used to measure an unknown antigen. The amount of labeled second antibody that binds is proportional to the amount of antigen that bound to the first antibody.
Figure 20
E. Tests for Cell Associated Antigens
1. Immunofluorescence Immunofluorescence is a technique whereby an antibody labeled with a fluorescent molecule (fluorescein or rhodamine or one of many other fluorescent dyes) is used to detect the presence of an antigen in or on a cell or tissue by the fluorescence emitted by the bound antibody.
a. Direct Immunofluorescence In direct immunofluorescence, the antibody specific to the antigen is directly tagged with the fluorochrome (Figure 20).
Figure 21
b. Indirect Immunofluorescence In indirect immunofluorescence, the antibody specific for the antigen is unlabeled and a second anti-immunoglobulin antibody directed toward the first antibody is tagged with the fluorochrome (Figure 21). Indirect fluorescence is more sensitive than direct immunofluorescence since there is amplification of the signal.
Figure 22
c. Flow CytometryFlow cytometry is commonly used in the clinical laboratory to identify and enumerate cells bearing a particular antigen. Cells in suspension are labeled with a fluorescent tag by either direct or indirect immunofluorescence. The cells are then analyzed on the flow cytometer.
Figure 22 illustrates the principle of flow cytometry. In a flow cytometer, the cells exit a flow cell and are illuminated with a laser beam. The amount of laser light that is scattered off the cells as they passes through the laser can be measured, which gives information concerning the size of the cells. In addition, the laser can excite the fluorochrome on the cells and the fluorescent light emitted by the cells can be measured by one or more detectors.
Figure 23
The type of data that is obtained from the flow cytometer is shown in Figure 23. In a one parameter histogram, increasing amount of fluorescence (e.g. green fluorescence) is plotted on the x axis and the number of cells exhibiting that amount of fluorescence is plotted on the y axis. The fraction of cells that are fluorescent can be determined by integrating the area under the curve. In a two parameter histogram, the x axis is one parameter (e.g. red fluorescence) and the y axis is the second parameter (e.g. green fluorescence). The number of cells is indicated by the contour and the intensity of the color.
Figure 24
PowerPoint animation of figure 24 of this figure
F. Complement Fixation Antigen/antibody complexes can also be measured by their ability to fix complement because an antigen/antibody complex will "consume" complement if it is present, whereas free antigens or antibodies do not. Tests for antigen/antibody complexes that rely on the consumption of complement are termed complement fixation tests and are used to quantitate antigen/antibody reactions. This test will only work with complement fixing antibodies (IgG and IgM are best).
The principle of the complement fixation test is illustrated in Figure 24. Antigen is mixed with the test serum to be assayed for antibody and antigen/antibody complexes are allowed to form. A control tube in which no antigen is added is also prepared. If no antigen/antibody complexes are present in the tube, none of the complement will be fixed. However, if antigen/antibody complexes are present, they will fix complement and thereby reduce the amount of complement in the tube. After allowing complement fixation by any antigen/antibody complexes, a standard amount of red blood cells, which have been pre-coated with anti-erythrocyte antibodies is added. The amount of antibody-coated red blood cells is predetermined to be just enough to completely use up all the complement initially added, if it were still there. If all the complement was still present (i.e. no antigen/antibody complexes formed between the antigen and antibody in question), all the red cells will be lysed. If antigen/antibody complexes are formed between the antigen and antibody in question, some of the complement will be consumed and, thus, when the antibody-coated red cells are added not all of them will lyse. By simply measuring the amount of red cell lysis by measuring the release of hemoglobin into the medium, one can indirectly quantitate antigen/antibody complexes in the tube. Complement fixation tests are most commonly used to assay for antibody in a test sample but they can be modified to measure antigen.

IMMUNOCHEMISTRY

Immunochemistry is a branch of immunology which encompasses the use of biochemical and biophysical procedures for the study and use of antibodies. Immunochemical procedures, particularly those using monoclonal antibodies, are widely used for research in many branches of biology and medicine

Research in immunology has had a fundamental impact on the understanding of many important aspects of biology and medicine. This impact has been acknowledged by the award of the Nobel Prize to several immunologists. As early as 1901, von Behring received the Nobel Prize for his work on immunotherapy; this was followed by awards to Paul Ehrlich and Élie Metchnikoff in 1908. Other Nobel laureates include Karl Landsteiner for discovery of the blood groups; Sir Frank Macfarlane Burnet and Peter Medawar for work on tolerance; Rodney Porter and Gerald Edelman for the discovery of the biochemical structure of antibodies; Rosalyn Yalow for the development of radioimmunoassay technology; Georges Köhler and César Milstein for development of monoclonal antibody technology; Niels Jerne for work on immune system function; and Tonegawa Susumu for discovering the genetic basis of antibody diversity.

Current research is diverse but includes examination of the roles of cytokines and their receptors in modulating the immune response, and classification of the functions of the various leucocyte types in this process. Identification of subtypes of T-lymphocytes (for example, the so-called TH1 and TH2 populations of “helper” T-cells) which direct the humoral and cellular immune response to mature in different ways by secreting different cytokines and other substances, has initiated a greater understanding of control of the immune system. Refinement of procedures for production of monoclonal antibodies, including the use of recombinant DNA technology, has increased the potential of immunochemical methods. It is hoped that intense activity in the area of vaccine design and production, including the development of DNA vaccines, will lead to better and safer vaccines for the prevention of a wide range of infectious and other diseases

ANTIGEN PRESENTATION AND THE MAJOR HISTOCOMPATIBILITY COMPLEX ANTIGENS

Most animal cells (except mammalian erythrocytes) express molecules on their surfaces which are strongly recognized as non-self by other, genetically non-identical, individuals. These major histocompatibility antigens show considerable similarity in structure to the immunoglobulins and T-cell receptor proteins. They play an important (though not exclusive) role in transplant rejection and some transfusion reactions. They are also fundamentally involved in the “presentation” of processed antigens to T-lymphocytes, which is especially important in the development of immune responses. Antigen-presenting cells such as macrophages, dendritic cells, and B-lymphocytes process antigens by degradation (which is mediated by enzymes) and present the resulting peptides, bound in grooves on the major histocompatibility complex (MHC) molecules, to responding T-cells (see Histocompatibility Groups). The type of the MHC molecules and their ability to present antigen influences significantly the strength and nature of the resulting immune response.

IMMUNOLOGICAL DIVERSITY

Fluorescence Microscopy Digital Image Gallery
Transformed African Green Monkey Kidney Fibroblast Cells (COS-7 Line)

The acquired immune system is capable of producing antibodies and T-cells which recognize a very large number of different molecules with remarkable specificity. It has been estimated that mammals can produce about a million different antibodies, and the mechanism able to achieve this generation of diversity has been a primary field of immunological research. It is also known that antibodies can be produced against synthetic substances which do not occur in nature. Early theories seeking to explain this diversity considered that an “instructive” process, involving the “induced fit” of antibody with antigens was the basis of specificity.

This theory has been largely abandoned as no convincing biological basis for its action has been conceived or discovered. It is now known that immunoglobulins are produced by reorganization and joining of several genes, and that somatic mutation of genetic material occurs during the development of acquired immunity. This especially affects the highly variable portions of the immunoglobulin protein that make up the antigen recognition site.

Such processes play a significant role in the generation of diversity and specificity of the immune system. Development of humoral immunity involves interaction of antigens with IgM or IgD molecules present on the B-cell surface. This triggers B-cell activation which (if sufficient help from T-cells and other systems is available) results in maturation of the response and secretion of appropriate antibodies. This concept was put forward by the pioneering German immunologist Paul Ehrlich decades before it was proven by experimentation. T-cells interact with antigens via T-cell receptors, which show structural similarities with immunoglobulins.