Antigen–Antibody Reactions

Antigen–Antibody 14 Reactions

■ High-affinity binding is believed to result from a very close Introduction fit between the antigen-binding sites and the corresponding antigenic determinants that facilitates development of strong The interactions between antigens and antibodies are known as noncovalent interactions between antigen and antibody. antigen–antibody reactions. The reactions are highly specific, and an antigen reacts only with antibodies produced by itself or with closely related antigens. Since these reactions are essentially spe- Avidity cific, they have been used in many diagnostic tests for the detec- Avidity is a measure of the overall strength of binding of tion of either the antigen or the antibody in vitro. The antigen an antigen with many antigenic determinants and multiva- and antibody reactions also form the basis of immunity against lent antibodies. Avidity is a better indicator of the strength microbial diseases in vivo. In the host, it may cause tissue injury of interactions in real biological systems than affinity. in hypersensitivity reactions and in autoimmune diseases. Therefore, the avidity of an antigen–antibody reaction is dependent on the valencies of both antigens and antibodies and is greater than the sum total of individual affinities. General Features of Antigen–Antibody Reactions Specificity

Antigen and antibody bind through noncovalent bonds in a The term specificity refers to the ability of an individual antibody- manner similar to that in which proteins bind to their cellular combining site to react with only one antigenic determinant or receptors, or enzymes bind to their substrates. But antigen– the ability of a population of antibody molecules to react with antibody reactions differ from the latter as there is no irreversible only one antigen. Antigen–antibody reactions usually show a chemical alteration in either of the participants, i.e., antigen or the high degree of specificity. antibody. The antigen and antibody binding is reversible and can be prevented or dissociated by high ionic strength or extreme pH. Key Points Following are some of the general features of these interactions: Antibodies can specifically recognize differences in:

■ primary structure of an antigen, Physicochemical Properties ■ isomeric forms of an antigen, and ■ secondary and tertiary structure of an antigen. Electrostatic bonds, hydrogen bonding, van der Waals bonds, and hydrophobic interactions are the intermolecular forces involved in antigen–antibody reactions. All these types of intermolecular Despite this, cross-reactions between antigens and antibodies, forces depend on the close proximity of the antigen and antibody however, do occur and are sometimes responsible for causing molecules. For that reason, the “good fit” between an antigenic diseases in hosts and for causing false results in diagnostic tests. determinant and an antibody-combining site determines the stability of the antigen–antibody reaction. Multiple bonding Cross-Reactivity between the antigen and the antibody ensures that the antigen Although antigen–antibody reactions are highly specific, in will be bound tightly to the antibodies. some cases antibody elicited by one antigen can cross-react with an unrelated antigen. Such cross-reactivity occurs if two Affinity different antigens share an identical or very similar epitope. In the latter case, the antibody’s affinity for the cross-reacting epi- Affinity denotes the intensity of attraction between antigen tope is usually less than that for the original epitope. Antisera and antibody. containing polyclonal antibodies can often be found to cross- ■ Low-affinity antibodies bind antigen weakly and tend to dis- react with immunogens partially related to those used for sociate readily, whereas high-affinity antibodies bind antigen immunization, due to the existence of common epitopes or of more tightly and remain bound longer. epitopes with similar configurations. 102 IMMUNOLOGY

test. Higher titer means greater level of antibodies in serum. For Stages of Antigen–Antibody Reactions example, a serum with a titer of 1/128 contains more antibodies than a serum with a titer of 1/8. The antigen–antibody reaction occurs in two stages: primary and secondary.

Primary Stage Types of Antigen–Antibody Reactions Primary stage is the initial interaction between antigen and Serological tests are widely used for detection of either serum antibody. It is rapid and reversible, but without any visible antibodies or antigens for diagnosis of a wide variety of effects. The ionic bonds, hydrogen bonds, van der Waals infectious diseases (Table 14-1). These serological tests are also forces, and hydrophobic interactions are the weaker intermo- used for diagnosis of autoimmune diseases and in typing of lecular forces that bind antigen and antibodies together in this blood and tissues before transplantation. The following are the primary stage. examples of antigen–antibody reactions: (a) precipitation, (b) Covalent binding, which is a stronger intermolecular force agglutination, (c) complement-dependent serological tests, (d) Section II Section between antigen and antibody, however, does not occur in this neutralization test, (e) opsonization, (f ) immunofluorescence, stage. (g) enzyme immunoassay, (h) radioimmunoassay, (i) western blotting, ( j) chemiluminescence assay, and (k) immunoelec- Secondary Stage tronmicroscopic tests. Secondary stage is an irreversible interaction between anti-

Chapter 14 gen and antibody, with visible effects, such as agglutination, Precipitation precipitation, neutralization, complement fixation, and Precipitation test shows the following features: immobilization of motile organisms. The binding between antigen and antibody during this stage occurs by covalent ■ It is a type of antigen–antibody reaction, in which the antigen binding. occurs in a soluble form. A single antibody is capable of causing different types of ■ It is a test in which antibody interacts with the soluble antigen–antibody reactions, and a single antigen is capable of antigen in the presence of electrolyte at a specified pH and inducing production of different classes of immunoglobulins, temperature to produce a precipitate. A lattice is formed which differ in their biological properties. between the antigens and antibodies; in certain cases, it is The results of agglutination, precipitation, neutralization, visible as an insoluble precipitate. and other tests are usually expressed as a titer. Titer is defined ■ Antibodies that aggregate soluble antigens are called as the highest dilution of serum that gives a positive reaction in precipitins.

TABLE 14-1 Commonly used tests in clinical microbiology Test Uses Flocculation test Detection of reaginic antibodies in syphilis by VDRL test Radial immunodiffusion Detection of fungal antigen and antibodies Counter-current immunoelectrophoresis Detection of both antigen and antibodies in bacterial, viral, fungal, and parasitic diseases Slide agglutination test Identification of bacterial isolates, such as Salmonella, Shigella, Vibrio, etc. Tube agglutination test Detection of antibodies in bacterial infections, e.g., Widal test for enteric fever Latex agglutination test Quantitation and detection of antigen and antibodies Hemagglutination test Detection of both antigens and antibodies in viral and parasitic infections Coagglutination test Detection of microbial antigens Complement fixation test Quantitation and detection of antibodies Direct immunofluorescence test Detection and localization of antigen in a cell or tissue Indirect immunofluorescence test Detection of specific antibodies in the serum Sandwich ELISA Detection of antigens and antibodies Indirect ELISA Quantitation and detection of antibodies Radioimmunoassay Quantitation of hormones, drugs, etc. Western blot Detection of antigen-specific antibody Section II Chapter 14 Highly diluted antibody Antigen 103 Post zone excess antigen no aggreagation Diluted antibody Antigen Dilution proper ratio aggregation Equivalence zone Antibody Antigen Dilution cient lattice formation. This is because antigen combines cient lattice formation. Marrack’s lattice hypothesis. ANTIGEN–ANTIBODY REACTIONS ANTIGEN–ANTIBODY with only few antibodies and no cross-linkage is formed. with only few In postzone phenomenon, small aggregates are surrounded is formed. by excess antigen and again no lattice network precipitation Thus, for reactions to be detectable, they must (Fig. 14-2). be run in the zone of equivalence Key Points antibody for In the prozone phenomenon, there is too much efﬁ Prozone Precipitation in solution Precipitation in agar Precipitation in agar with an electric field no aggregation excess antibody The prozone and postzone phenomena are takenThe prozone and postzone phenomena into con- Types of precipitation reactions Types ◗ FIG. 14-2. Precipitation reactions can be broadly of three types: 1. 2. 3. Precipitation in solution Ring test and flocculation of precipitation test are examples in solution. sideration in the interpretation tests, because of serological in either of these conditions. reactions can occur false negative reaction suspected to be due to prozone phe- A false negative nomenon can be rectified by diluting out the antibody and performing the test. In the postzone phenomenon, excess anti- obscure the presence of small amount of antibodies. gen may test is repeated with an additional patient such a Typically, time for give This would later. specimen taken about a week the further on production of antibodies. If the test is negative that the patient has that particularthis occasion, it is unlikely antibody. . prozone prozone to explain to explain excess flocculation (Fig. 14-1). On (Fig. 14-1). On Zone of antigen lattice hypothesis Precipitate formed (zone of equivalence) Zone of equivalence (optimal antigen and . antibody concentration) and the zone of antigen excess is known as as known and the zone of antigen excess is excess Prozone phenomenon. Prozone Zone of antibody Antigen Antibody The antigen must be either bivalent or polyvalent;The antigen must that or copies of the same epitope, least two at is, it must have different with different epitopes that react have antibodies antisera. present in polyclonal The antibody must be bivalent;The antibody must a precipitate form will not fragments. Fab with monovalent When instead of sedimenting, the precipitate of sedimenting, When instead remains sus- floccules,pended as as reaction is known the on the lattice depends of an antigen–antibody Formation and antigen. the antibody both valency of Marrack in 1934 proposed the Prozone phenomenon ■ ■ ■ ■ ◗ Antigen and antibody reaction occurs optimally only when only when reaction occurs optimally Antigen and antibody the proportion in the reaction of the antigen and antibody mixture is equivalent FIG. 14-1. either side of the equivalence zone, precipitation is actually zone, precipitationeither side of the equivalence is actually anti- because of an excess of either antigen or prevented as the The zone of antibody excess is known body. phenomenon postzone phenomenon is based on hypothesis the prozone phenomenon. Marrack’s at must have that each antibody molecule the assumptions sites, and antigen must be multivalent. In binding least two precipitation where optimum occurs, the zone of equivalence and antibody are the number of multivalent sites of antigen In this zone, precipitation equal. as a occurs approximately anti- reactions whereby each result of random, reversible form- and vice versa, body binds to more than one antigen or lattice. As they combine, it results in a stable network ing a in size until it precipi- multimolecular lattice that increases tates out of solution. 104 IMMUNOLOGY

■ Ring test: In this test, antigen solution is layered over antiserum in a test tube. Precipitation between antigen and antibodies in antiserum solution is marked by the appearance of a ring of precipitation at the junction of two liquid layers. C-reactive protein (CRP) and streptococcal grouping by the Lancefield methods are the examples of the ring test. ■ Flocculation test: Flocculation test may be performed in a slide or tube. VDRL test for detection of reaginic antibodies in syphilis is an example of a slide flocculation test. In this test, a drop of VDRL antigen suspension is added to a drop of patients’ serum on a cavity slide, and the result is recorded after shaking the slide on a VDRL shaker. In a positive test, the floccules appear, which can be demonstrated well under a microscope. Kahn test for syphilis is an example of tube FIG. 14-3. Radial immunodiffusion. flocculation test. The tube flocculation test for standardiza- Section II Section the surface of gel. The antigen is then applied to a well cut tion of toxins and toxoids is another example. into the gel. When antibody already present in the gel reacts with the antigen, which diffuses out of the well, a ring of pre- Precipitation in agar cipitation is formed around the wells. The diameter of the The precipitation test in agar gel is termed as immunodiffusion ring is directly proportional to th=e concentration of antigen. test. In this test, reactants are added to the gel and antigen– The greater the amount of antigen in the well, the farther the antibody combination occurs by means of diffusion. The rate ring will be from the well (Fig. 14-3, Color Photo 6). Chapter 14 of diffusion is affected by the size of the particles, temperature, gel viscosity, amount of hydration, and interactions between the matrix and reactants. Key Points An agar concentration of 0.3–1.5% allows for diffusion of Radial immunodiffusion has been used for the quantitative most reactants. Agarose is often preferred to agar because agar estimation of antibodies and antigens in the serum. It is used has a strong negative charge, while agarose has almost none, so to measure: that interactions between the gel and reactants are minimized. ■ IgG, IgM, IgA, and complement components in the serum, ■ antibodies to influenza virus in sera, and ■ serum transferrin and ␣-fetoprotein. Key Points Immunodiffusion reactions have the following advantages: However, the test has recently been replaced by more sen- ■ In this test, the line of precipitation is visible as a band, sitive and automated methods, such as nephelometry and which can also be stained for preservation. enzyme-linked immunosorbent assays (ELISAs). ■ The test can be used to detect identity, cross-reaction, and nonidentity between different antigens in a reacting mixture. ■ Double diffusion in one dimension: This method is also called Oakley–Fulthrope procedure. In this method, the antibody is incorporated in agar gel in a test tube, above which a layer Types of immunodiffusion reactions: Immunodiffusion reactions of plain agar is placed. The antigen is then layered on top of are classified based on the (a) number of reactants diffusing and this plain agar. During the course of time, the antigen and (b) direction of diffusion, as follows: antibody move toward each other through the intervening ■ Single diffusion in one dimension: Single diffusion in one layer of plain agar. In this zone of plain agar, both antigen dimension, as the name suggests, is the single diffusion of and antibody react with each other to form a band of pre- antigen in agar in one dimension. It is otherwise called Oudin cipitation at their optimum concentration. procedure because this technique was pioneered by Oudin ■ Double diffusion in two dimensions: This method is also who for the first time used gels for precipitation reactions. called the Ouchterlony procedure. In this method, both the In this method, antibody is incorporated into agar gel in a antigen and antibody diffuse independently through agar test tube and the antigen solution is poured over it. During gel in two dimensions, horizontally and vertically. The test is the course of time, the antigen diffuses downward toward performed by cutting wells in the agar gel poured on a glass the antibody in agar gel and a line of precipitation is formed. slide or in a Petri dish. The antiserum consisting of anti- The number of precipitate bands shows the number of dif- bodies is placed in the central well, and different antigens ferent antigens present in the antigen solution. are added to the wells surrounding the center well. After an ■ Single diffusion in two dimensions: Single diffusion in two incubation period of 12–48 hours in a moist chamber, the dimensions is also called radial immunodiffusion. In this lines of precipitins are formed at the sites of combination method, antiserum solution containing antibody is incorpo- of antigens and antibodies (Color Photo 7). Three types of rated in agar gel on a slide or Petri dish. The wells are cut on reactions can be demonstrated as follows (Fig. 14.4): Section II Chapter 14 ve ve ve + − + 105 Counter-current immu- Counter-current is an adaptation This technique -fetoprotein, hydatid and amoebic antigens in hydatid -fetoprotein, Increasing antigen concentration ␣ Ag Ab the serum, and cryptococcal antigen in the CSF. It is commonly used for Hepatitis B surfaceIt is commonly used for antigen (HBsAg), It is a rapid and a highly specific of detection for method in the serum, cerebrospinal antigen and antibodies both fluid, body fluids and other infectious in diagnosis of many diseases including bacterial, viral, fungal, and parasitic. ■ ■ Rocket electrophoresis. Rocket Counter-current immunoelectrophoresis. Counter-current ANTIGEN–ANTIBODY REACTIONS ANTIGEN–ANTIBODY ve Key Points uses: has many immunoelectrophoresis The counter-current Antigen wells − Electrophoretic current Electrophoretic Antibody in agarose gel Precipitation band FIG. 14-7. Counter-current immunoelectrophoresis: Counter-current of antigen towards depends on movement noelectrophoresis the cathode through the the anode and of antibody towards agar under electric field. The test is performed on a glass slide is punched out. One well with agarose in which a pair of wells Electric is filled with antibody. with antigen and the other gel. The migration of current is then passed through the antigen and antibody is greatly facilitated under electric field, and the line of precipitation is made visible in 30–60 minutes (Fig. 14-6). Rocket electrophoresis: of radial immunodiffusion by Laurell. It is called developed so due to the appearance of the precipitin bands in the appearance) at the end structures (rocket shape of cone-like 8). In this method, of the reaction (Fig. 14-7, Color Photo antibody is incorporated antigen is placed in in the gel and FIG. 14-6. b ab α . b ab α Nonidentical identical Partially Corynebacterium diphtheriae aa aa Immunoelectrophoresis is a process a α Identical a demonstration of antibodies in serodiagnosis of small pox, demonstration of antibodies in serodiagnosis identification of fungal antigens, and of antibodies to extractabledetection nuclear antigens. ■ ■ ■ Ouchterlony procedure. Ouchterlony Immunoelectrophoresis. Elek’s precipitation demon- test in gel is a special test used for Elek’s of stration of toxigenicity Double diffusion has been used for: dimension in two Key Points −+ A pattern of crossed lines demonstrates two separate separate lines demonstrates two A pattern of crossed antigens are and indicates that the compared reactions unrelated and share no common epitopes. lines with a spur indicates cross-reaction Fusion of two antigens share In this last case, the two or partial identity. Line of precipitation at their junction forming an arc Line of precipitation an arc junction forming at their the presence of a common represents serologic identity or epitope in antigens. a common epitope, but some antibody molecules are not molecules are not a common epitope, but some antibody through the initial and traverse by the antigen captured epitopes found precipitin line to combine with additional antigen. in the more complex Antigen Antibody 2. 3. 1. Precipitation in agar with an electric field Precipitation in agar with an electric Immunoelectrophoresis: of combination of immunodiffusion and electrophoresis. It is in which differenta method antigens in serum are separated according to their charge under an electric field. In this method, glass slide. in agar on a a drop of antigen is placed into a well During An electric current is then passed through the agar. in the electric fieldelectrophoresis, antigens move according electrophoresis, a trough is to their charge and size. Following cut into the agar and is filled with the antibody and diffusion As the antigen and antibody diffuse toward to occur. is allowed a series of lines of precipitation they form (Fig. 14-5). each other, The main advantage of immunoelectrophoresis is that a number of antigens can be identified is used to in serum. The method such as myeloma as abnormal proteins, normal as well detect in human serum. proteins FIG. 14-4. FIG. 14-5. 106 IMMUNOLOGY

phenomenon of light scattering by precipitates in a solution. Turbidimetry is a measurement of turbidity or cloudiness of precipitate in a solution. In this method, a detection device is placed in direct line with the incident light that collects the light after it has passed through the solution. It thus measures the reduction in the intensity of light due to reflection, absorption, or scatter. Scattering of light occurs in proportion to the size, shape, and concentration of precipitates present in solution. Nephelometry is an improvement on this technique in that it measures the light that is scattered at a particular angle from the incident beam as it passes through a suspension containing the antigen–antibody precipitate. The amount of light scattered is an index of the concentration of the solution. Beginning with a constant amount of antibody, an increasing amount of antigen would result in an increase in antigen–antibody complexes. Section II Section Thus the relationship between antigen concentrations, as indicated by the antigen–antibody complex formation, and light scattering approaches linearity. By using a computer, the exact values of the antigen or antibody in the serum can be estimated through this system. To improve the sensitivity of this system, FIG. 14-8. Photograph of rocket electrophoresis. laser beams have been used as the source of incident light. Chapter 14 wells cut in the gel. Electric current is then passed through the gel, which facilitates the migration of antigen into the agar. This Key Points results in formation of a precipitin line that is conical in shape, Nephelometry is now becoming the method of choice for use resembling a rocket. The height of the rocket, measured from in various laboratories for the measurement of plasma proteins the well to the apex, is directly in proportion to the amount of including IgG, IgM, and IgA, complement components, RA antigen in the sample (Fig. 14-8). Rocket electrophoresis is used (rheumatoid arthritis) factor, ASLO (anti-streptolysin O), etc. mainly for quantitative estimation of antigen in the serum. Two-dimensional immunoelectrophoresis: Two-dimensional immunoelectrophoresis is a variant of rocket electrophoresis. Agglutination It is a double diffusion technique used for qualitative as well Agglutination is an antigen–antibody reaction in which a par- as quantitative analysis of sera for a wide range of antigens. ticulate antigen combines with its antibody in the presence of This test is a two-stage procedure. In the first stage, antigens in electrolytes at a specified temperature and pH resulting in for- solution are separated by electrophoresis. In the second stage, mation of visible clumping of particles. Agglutination occurs electrophoresis is carried out again, but perpendicular to that optimally when antigens and antibodies react in equivalent of first stage to obtain rocket-like precipitation. proportions. In this test, first, a small trough is cut in agar gel on a glass plate and is filled with the antigen solution. Electric current is ■ Agglutination reactions are mostly similar to precipitation then passed through the gel, and the antigens migrate into the reactions in their fundamentals and share similar features. gel at a rate proportional to their net electric charge. In the sec- This reaction is analogous to the precipitation reaction in ond stage, after electrophoresis, the gel piece containing the that antibodies act as a bridge to form a lattice network of separated antigens is placed on a second glass plate and the agar antibodies and the cells that carry the antigen on their sur- containing antibody is poured around the gel piece. A second face. Because cells are so much larger than a soluble anti- electric potential is applied at right angles to the first direction of gen, the result is more visible when the cells aggregate into migration. The preseparated antigens then migrate into the gel clumps. containing antibodies at a rate proportional to their net charge ■ Agglutination differs from precipitation reaction in that and precipitate with antibodies in the gel, forming precipitates. since the former reaction takes place at the surface of the This method is both qualitative, in that it identifies different particle involved, the antigen must be exposed and be able to antigens that are present in the serum solution, and quantita- bind with the antibody to produce visible clumps. tive, in that it detects the amount of different antigens present In agglutination reactions, serial dilutions of the antibody in the solution. solution are made and a constant amount of particulate Turbidimetry and nephelometry: Turbidimetry and nephelo- antigen is added to serially diluted antibody solutions. After metry are the two methods used to detect and quantitate several hours of incubation at 37°C, clumping is recorded precipitation reactions in serum and are based on the by visual inspection. The titer of the antiserum is recorded Section II Chapter 14

ϩ A, and strains causing 107 Proteus Proteus ) to detect the ) to detect H S. paratyphi S. paratyphi , Agglutination of RBCs , and B H erythrocytes in saline, ϩ , A H strains OXK, OX19, and OX2). OX2). and OX19, strains OXK, , T Mycoplasma pneumoniae Mycoplasma O Salmonella typhi Coombs’ test was devised This test depends on demons- Proteus Proteus (Coombs reagent) antigens (T MG agglutination test is a similar test used for for MG agglutination test is a similar test used B in patient’s serum. + Antihuman immunoglobulin Salmonella OXK, OX19, and OX2. OX19, OXK, Weil–Felix test is an example of heterophile agglutina- of heterophile test is an example Weil–Felix In infections. serodiagnosis of rickettsial tion reaction for against this test, the cross-reacting antibodies produced by using cross-reacting pathogen are detected rickettsial related antigens (e.g., agglutination heterophile Paul–Bunnell test is another mono- antibodies in infectious test, which is used to detect using sheep erythrocytesnucleosis by antigens. as Streptococcus Widal test is used to diagnose enteric fever and uses differ- fever used to diagnose enteric Widal test is ent The standard agglutination test is a commonly used test of brucellosis. The tube agglutination serodiagnosis for by the prozone complicated is however, brucellosis, test for due to high concentration of bru- phenomenon. This is serum, resulting in false nega- cella antibodies in patient’s of several reactions. This problem is obviated by use tive reactions. The false positive prevent dilutions of serum to in the serum antibodies incomplete presence of blocking or antiglobulin by using This is avoided problem. is another these antibodies. (Coombs’ test) to detect of antibodies to detection primary atypical pneumonia. Although the antibodies are produced against rickettsial rickettsial Although the antibodies are produced against organisms, they cross-react with antigens of presence of antibodies to presence of antibodies S. paratyphi S. paratyphi ■ ■ ■ ■ ■ Principle of the direct Coombs’ test. ANTIGEN–ANTIBODY REACTIONS ANTIGEN–ANTIBODY Key Points Key Points patient’s RBCs patient’s Antibody bound incomplete antibody antiglobulin coats the surface of incomplete erythrocytes agglutination. When such cause any but does not erythrocytes are treated with antiglobulin or Coombs’ serum erythrocytes in saline. When serum containing incomplete with Rh anti-Rh antibodies is mixed FIG. 14-9. tration of heterophilic antibodies in serum present in certaintration of heterophilic bacterial infections: Antiglobulin (Coombs’) test: of detection originally by Coombs’, Mourant, and Race for agglutinate Rh anti-Rh antibodies that do not incomplete Heterophile agglutination test: blocking blocking ) heterophile c , etc., isolated from clinical , etc., Vibrio ) tube agglutination, ( , b It is a basic type of agglutination Shigella agglutination test, as the name Tube , ) antiglobulin (Coombs’) test. d Salmonella specimens. For blood grouping and cross-matching. For As a routine procedure to identify bacterial strains, such as . Anti-Rh antibodies and anti-brucella antibodies are . Anti-Rh antibodies ■ ■ ) slide agglutination, ( Key Points Slide agglutination is used: a Occasionally, antibodies are formed that react with the that react with the are formed antibodies Occasionally, Agglutination reactions have a wide variety of applications a wide variety have Agglutination reactions Types of agglutination reactions of agglutination Types ◗ agglutination, and ( reaction that is performed on a slide. Identification of bacterial of a direct slide agglutinationtypes represents a classic example In this test, a suspension of bacteria is that is still used today. standardizedprepared and is added to a drop of antiserum. and of bacteria by clumping reaction is indicated A positive occurs instantly solution. Clumping clearing of the background control consisting of antigen test. A or within seconds in a positive antiserum is included onsuspension in saline without adding results and also to detectthe same slide. It is used to validate the due to autoagglutination of the antigen.possible false positives Slide agglutination test: Agglutination reactions where the antigens are found naturally are found Agglutination reactions where the antigens different as direct agglutination. This is on a particle are known particles are that which employs agglutination, from passive on their surfaces. normally found coated with antigens not Tube agglutination test: agglutination Tube suggests, is performed in glass tubes. Typically, in these tests, suggests, is performed in glass tubes. Typically, patient’s serum is diluted in a series of tubes and bacterial antigens specific the suspected disease are added to it. Antigen for and antibody reactions are demonstrated by demonstration of of agglutination. It is a standard used visible clumps method estimation of antibodies in the serum. Tube quantitative for demonstration of agglutination tests are routinely used for and serodiagnosis of enteric fever antibodies in the serum for brucellosis, as follows: Direct agglutination reactions can broadly be of the followingDirect agglutination reactions can broadly types: ( Direct agglutination as the reciprocal of the highest dilution that causes clump- dilution that of the highest as the reciprocal on determinants antigenic many the cells have ing. Since their surface, is rarely of antibody excess phenomenon the encountered. agglu- cause any of a cell but does not antigenic determinants anti- the agglutination by the complete tination. They inhibit antibodies are called Such bodies added subsequently. antibodies few examples of such blocking antibodies. of such examples few in serum and antigens and antibodies of both in the detection fluids. body other They are very of the and the result sensitive with ease. test can be read visually 108 IMMUNOLOGY

Latex agglutination test: It is a test that employs latex particles + as carrier of antigen or antibodies. In 1955, Singer and Plotz accidentally found that IgG was naturally adsorbed to the surface of polystyrene latex particles. Antibodies in Target RBCs Antibody bound RBCs Latex particles are inexpensive, relatively stable and are patient’s serum not subject to cross-reactivity with other antibodies. These particles can be coated with antibodies to detect antigen in the serum and other body fluids. Use of monoclonal antibodies has reduced the cross-reactions resulting in reduction of false positive reactions. Additionally, the large particle size of the latex facilitates better visualization of antigen–antibody reactions by the Agglutination of RBCs Antihuman immunoglobulin naked eye observation. The tests are usually performed on (Coombs reagent) cardboard cards or glass slides and positive reactions are graded on a scale of 1ϩ to 4ϩ. Section II Section FIG. 14-10. Principle of the indirect Coombs’ test.

Key Points (rabbit antiserum against human ␥ globulin), then the cells are agglutinated. Coombs’ test is of two types: (a) direct Coombs’ The latex agglutination tests have following uses: test and (b) indirect Coombs’ test. ■ The tests are used for rapid identification of antigens of Chapter 14 ■ Direct Coombs’ test: In this test, the sensitization of red group B Streptococcus, Staphylococcus aureus, Neisseria menin- gitidis, Cryptococcus neoformans, etc. blood cells (RBCs) with incomplete antibodies takes place in ■ The tests have also been found to be useful for detection of vivo. The cell-bound antibodies can be detected by this test soluble microbial antigens in urine, spinal fluid, and serum in which antiserum against human immunoglobulin is used for diagnosis of a variety of infectious diseases. to agglutinate patient’s red cells (Fig. 14-9). ■ These tests are being used to detect RA factor, ASLO, CRP, ■ Indirect Coombs’ test: In this test, the sensitization of etc., in serum specimens. RBCs with incomplete antibodies takes place in vitro. In this test, the patient’s serum is mixed with normal red cells and antiserum to human immunoglobulin is added. Agglutina- Hemagglutination test: RBCs are used as carrier particles tion occurs if antibodies are present in the patient’s serum in hemagglutination tests. RBCs of sheep, human, chick, (Fig. 14-10). etc. are commonly used in the test. When RBCs are coated Coombs’ tests are used for detection of (a) anti-Rh with antigen to detect antibodies in the serum, the test antibodies and (b) incomplete antibodies in brucellosis and is called indirect hemagglutination (IHA) test. The IHA is other diseases. a most commonly used test for serodiagnosis of many parasitic diseases including amoebiasis, hydatid disease, and toxoplasmosis. Passive agglutination When antibodies are attached to the RBCs to detect micro- Passive agglutination employs carrier particles that are bial antigen, it is known as reverse passive hemagglutination coated with soluble antigens. This is usually done to convert (RPHA). The RPHA has been used extensively in the past precipitation reactions into agglutination reactions, since to detect viral antigens, such as in HBsAg in the serum for the latter are easier to perform and interpret and are more diagnosis of hepatitis B infection. The test has also been used sensitive than precipitation reactions for detection of anti- for detection of antigens in many other viral and parasitic bodies. When the antibody instead of antigens is adsorbed on infections. the carrier particle for detection of antigens, it is called reverse Viral hemagglutination: Many viruses including influenza, passive agglutination. mumps, and measles have the ability to agglutinate RBCs with- Until the 1970s, erythrocytes were the major carrier particles out antigen–antibody reactions. This process is called viral used for coating of antigens. Recently, however, a variety of other hemagglutination. This hemagglutination can be inhibited particles including polystyrene latex, bentonite, and charcoal are by antibody specifically directed against the virus, and this used for this purpose. Particle size vary from 7 ␮ for RBCs to 0.05 ␮ phenomenon is called hemagglutination inhibition. This forms for very fine latex particles. The use of synthetic beads or particles the basis of the viral hemagglutination inhibition test, which provides the advantage of consistency, uniformity, and stability. is used to detect antibodies in patient’s sera that neutralize the Reactions are also easy to read visually. Passive agglutination reac- agglutinating viruses. To perform this test, patient’s serum is tion, depending on the carrier particles used, can be of the follow- first incubated with a viral preparation. Then RBCs that the ing types: (i) latex agglutination test, (ii) hemagglutination test, virus is known to agglutinate are added to the mixture. If anti- and (iii) coagglutination test. body is present, this will combine with viral particles and prevent Section II Chapter 14 109 test ) an indicator a Negative + Antigen Complement antigen available (unused Hemolysis sheep RBC Sheep RBC Antibody to Serum without complement No complement fixation reaction antibody against for hemolysis) test Positive Complement + ++ been preincu- It consists of RBCs that have ) a test system. In the test system, patient’s serum is first heated Antigen b antigen Complement sheep RBC Sheep RBC Antibody to No hemolysis Complement fixation test. fixation reaction up in antigen– antibody against ANTIGEN–ANTIBODY REACTIONS ANTIGEN–ANTIBODY Serum containing antibody reaction) (complement used Complement fixationComplement test Immune adherence test Immobilization test Cytolytic or cytocidal reactions Complement fixation test ◗ promoting phagocytosis, and in immune adherence. The com- phagocytosis, and promoting following be of the may plement-dependent serological tests types: 1. 2. 3. 4. to 56°C to inactivate the native complement. Then the inac- complement. to 56°C to inactivate the native tivated serum is adsorbed with washed sheep RBC to elimi- anti-RBC antibodies (also known nate broadly cross-reactive antibodies), which could interfere with the as Forssman-type purified with is then mixed The serum antigen and assay. of with a dilution of fresh guinea pig serum, used as source Indicator system: bated with a specific in concentrations that anti-RBC antibody, cause agglutination, and no hemolysis of RBCs occurs do not Such RBCs are designated as in the absence of complement. “sensitized” red cells. system: Test system and ( The principle of the complement fixationThe principle of the complement test is that when anti- com- gen and antibodies of the IgM or the IgG classes are mixed, plement is “fixed” If this occurs to the antigen–antibody complex. on the surface cascade will be activated of RBCs, the complement fixation The complement and hemolysis will occur. test consists systems: ( antigen–antibody complement of two FIG. 14-12. is ) coated S. aureus S. aureus Antigen Precipitate visible S. aureus Antibody-coated particles ( + Coagglutination is a type of reaction in which Cowan I strain of reaction in which Cowan Neisseria gonorrhoeae. Neisseria Detection of amoebic and hydatid antigens in the serum antigens of amoebic and hydatid Detection diagnosis of amoebiasis and cystic echinococcosis, for respectively; and typing and for and mycobacteria Grouping of streptococci of Detection of cryptococcalDetection diagnosis antigen in the CSF for of cryptococcal meningitis; Principle of the coagglutination. ■ ■ ■ contains protein A, an anti-antibody, that combines contains A, protein an anti-antibody, Key Points Coagglutination test has been used for: FIG. 14-11. Complement-Dependent Serological Tests The complement system is a group of serum proteins that is a group of serum proteins system is The complement present in normal serum. The system consists of 20 or more and with cell that interact with one another serum proteins membrane. It is a biochemical cascade that helps to clear patho- bacteria, It aids the antibodies in lysing gens from the body. agglutination Coagglutination test: agglutination, and a lack of or reduction in agglutination indi- agglutination, and a lack of or reduction serum. cates presence of antibody in patient’s used as carrier particle to coat antibodies. Cowan I strain of used as carrier particle to coat antibodies. Cowan S. aureus with the Fc portion the Fab of immunoglobulin, IgG, leaving present in the specimens region free to react with the antigen A bearing test, protein (Fig. 14-11). In a positive with specific if mixed with antibodies will be agglutinated antigen. The advantage is that these particles of the test show greater stability than latex particlesare more refractory and to changes in ionic strength. 110 IMMUNOLOGY

complement. The mixture is incubated for 30 minutes at 37°C ◗ Immobilization test to allow antibody in the patient’s serum to form complexes Immobilization test is a complement-dependent test in which with the antigen and to fix complement (Fig. 14-12). certain live bacteria, such as T. pallidum, are immobilized when In complement fixation test, “sensitized” red cells are then mixed with patient’s serum in the presence of complement. added to the mixture. If the red cells are lysed, it indicates that This forms the basis of T. pallidum immobilization test. A posi- there were no antibodies specific to the antigen in the serum tive test shows serum to contain treponemal antibodies. of the patient. The complement therefore was not consumed in the test system and was available to be used by the anti-RBC ◗ Cytolytic or cytocidal reactions antibodies, resulting in hemolysis. This reaction is considered negative. The test is considered positive if the red cells are not When a live bacterium, such as V. cholerae, is mixed with its lysed. Nonlysis of the cells indicates that patient’s serum had specific antibody in the presence of complement, the bacterium antibodies specific to the antigen, which have “fixed” comple- is killed and lysed. This forms the basis of test used to measure ment. Hence, no complement was available to be activated by anti-cholera antibodies in the serum. the indicator system. Neutralization Tests Section II Section Key Points Neutralization is an antigen–antibody reaction in which the biological effects of viruses and toxins are neutralized by The complement ﬁ xation reactions were used earlier for diagnosis homologous antibodies known as neutralizing antibodies. of many infections, such as: These tests are broadly of two types: (a) virus neutralization ■ Wassermann test for syphilis and tests and (b) toxin neutralization tests. ■ Tests for demonstration of antibodies to M. pneumoniae, Chapter 14 Bordetella pertussis, many different viruses, and to fungi (such as ◗ Cryptococcus spp., Histoplasma, and Coccidioides immitis). Virus neutralization tests Since this test is technically very cumbersome, and often Neutralization of viruses by their specific antibodies are called difﬁ cult, it is no longer used now-a-days. virus neutralization tests. Inoculation of viruses in cell cul- tures, eggs, and animals results in the replication and growth of viruses. When virus-specific neutralizing antibodies are Indirect complement fixation test: Indirect complement injected into these systems, replication and growth of viruses fixation test is carried out to test the sera that cannot fix guinea is inhibited. This forms the basis of virus neutralization test. pig complement. These include avian sera (e.g., parrot, duck) Viral hemagglutination inhibition test is an example of virus and mammalian sera (e.g., cat, horse). The test is carried out neutralization test frequently used in the diagnosis of viral in duplicate and after the first test, the standard antiserum infections, such as influenza, mumps, and measles. If patient’s known to fix the complement is added to one set. Hemolysis serum contains antibodies against certain viruses that have the indicates a positive test. In a positive test, if the serum contains property of agglutinating the red blood cells, these antibodies antibody, the antigen would have been used up in the first test, react with the viruses and inhibit the agglutination of the red standard antiserum added subsequently would fail to fix the blood cells. complement, therefore causing hemolysis. Conglutinating complement adsorption test: It is an alter- ◗ Toxin neutralization tests native method for systems that do not fix guinea pig complement. Sheep erythrocytes sensitized with bovine serum Toxin neutralization tests are based on the principle that bio- are used as the indicator system. The bovine serum con- logical action of toxin is neutralized on reacting with specific tains conglutinin, a ␤ globulin that acts as antibody to the neutralizing antibodies called antitoxins. Examples of neutral- complement. Therefore, conglutinin causes agglutination ization tests include: of sensitized sheep erythrocytes if these are combined with ■ In vivo—(a) Schick test to demonstrate immunity against complement, which is known as conglutination. If the horse diphtheria and (b) Clostridium welchii toxin neutralization complement had been used up by the antigen–antibody reac- test in guinea pig or mice. tion in the first step, the agglutination of the sensitized cells ■ In vitro—(a) antistreptolysin O test and (b) Nagler reaction does not occur. used for rapid detection of C. welchii.

◗ Immune adherence test Opsonization Immune adherence test is a test in which certain pathogens (e.g., Vibrio cholerae, Treponema pallidum, etc.) react with specific Opsonization is a process by which a particulate antigen antibodies in the presence of complement and adhere to eryth- becomes more susceptible to phagocytosis when it combines rocytes or platelets. The adherence of cells to bacteria is known with opsonin. The opsonin is a heat-labile substance present in as immune adherence, which facilitates phagocytosis of the fresh normal sera. Unlike opsonin, bacteriotropin is heat-stable bacteria. substance present in the serum but with similar activities. Section II Chapter 14 , 111 C. diphtheriae , N. gonorrhoeae , etc. directly in appropriate clinical specimens. directly , etc. T. pallidum T. Detect specificDetect serodiagnosis of syphilis, antibodies for other and many amoebiasis, toxoplasmosis, leptospirosis, diseases; infectious antibody by using fluorescentIdentify the class of a given antibodies specific different for immunoglobulin isotypes; subpopulations byIdentify and enumerate lymphocyte monoclonal antibodies and cytofluorographs;employing and autoantibodies, such as antinuclear antibodies in Detect autoimmune diseases. Direct immunofluorescence antemortem test for diagnosis of rabies virus anti- detection for of rabies: The test is used from the nape of the neck in gen in the skin smear collected of dogs. humans and in the saliva of detection Also used for ■ ■ ■ ■ ■ ■ ANTIGEN–ANTIBODY REACTIONS ANTIGEN–ANTIBODY Indirect immunoﬂ used widely to: uorescence test is Indirect immunoﬂ Key Points Key Points Indirect immunofluorescence is a two-stage process. In The first step in the indirect immunofluorescencetest is advantage has the The indirect method of using a single labeled Indirect immunofluorescence test Indirect immunofluorescence The indirect immunofluorescence of detection for test is used specific body fluids antibodies in the serum and other sero- for diseases. infectious diagnosis of many the first antigen is fixed stage, a known on a slide. Then the to the slide, followed patient’s serum to be tested is applied by careful washing. If the patient’s serum contains antibody antigen on the slide. against the antigen, it will combine with In the second stage, with anti- the combination of antibody by addition of a fluorescentgen can be detected dye-labeled antibody to human IgG, which is examined by a fluorescence microscope. the incubation of a fixed in a cell or tissue) with antigen (e.g., antigen. which becomes associated with the unlabeled antibody, afterNext, careful washing, a fluorescent antibody (e.g., fluores- antibody This second cent labeled anti-IgG) is added to the smear. will become associated to the first, and the antigen–antibody can be visualized on the fluorescencecomplex microscope. reagent” to detectantiglobulin (antibody to IgG) as a “universal different specificmany antigen–antibody reactions. The test is often direct immunofluorescence than the more sensitive test. microscope (Fig. 14-13). Direct immunofluorescence (Fig. 14-13). Direct microscope test is fungi, parasites, viruses, of bacteria, detection for widely used and other stool, urine, tissues, blood, in CSF, antigens or other include: examples Few specimens. each preparation of separate labeled antibody for The need for pathogen is the major disadvantage of the direct immunofluorescence test. ◗ . Antigen on cell surface Fluorochrome fluorescence Antibodies fluorochrome combined with Antibodies Bacterial cell fluorochrome combined with bound antibodies Bacterial cell with Direct fluorescent antibody test. Indirect immunofluorescence test Direct immunofluorescence test The term “opsonic index” is defined “opsonic index” The term of the as the ratio Direct immunofluorescence test Direct immunofluorescence unknown detect test is used to labeled anti- a known antigen in a cell or tissue by employing antigen. If antigen body that interacts directly with unknown and the antibody- is present, it reacts with labeled antibody coated antigen is observed under UV light of the fluorescence FIG. 14-13. ◗ 2. 1. Immunofluorescence The property certain of parti- at one light rays absorbing dyes them at a light) and emitting (ultraviolet cular wavelength as light) is known (visible different wavelength phagocytic activity of patient’s blood for a particular blood for activity of patient’s phagocytic bacterium individual. It blood from a normal activity of to the phagocytic of resistance study the progress is used to of during the course with by incubating fresh citrated blood disease. It is measured 15 minutes and estimat- at 37°C for the suspension of bacteria from the stained number of phagocytic bacteria ing the average blood films. Fluorescent dyes, such as fluoresceinFluorescent dyes, and lissa- isothiocyanate mine rhodamine, can be tagged antibody molecules. They with emit blue-green and orange-red fluorescence, under respectively in the fluorescence (UV) rays ultraviolet microscope. This forms the basis of the immunological test. Immunofluorescence tests and diagnostics. These tests applications in research wide have types: are broadly of two 112 IMMUNOLOGY

The major limitation of immunofluorescence is that the tech- been formed and will not get washed away. On the other hand, nique requires (a) expensive fluorescence microscope and if the specific antibody was not present in the specimen, there reagents, (b) trained personnel, and (c) have a factor of subjec- would not be any complex formation. Next, an anti-isotype tivity that may result in erroneous results. antibody conjugated with an enzyme is added and incubated. After another washing step, a substrate for the enzyme is added. If there was complex formation in the initial step, the second- Enzyme Immunoassays ary anti-isotype antibody would have bound to the primary antibody, and there would be a chromogenic reaction between Enzyme immunoassays (EIAs) can be used for detection of the enzyme and substrate. By measuring the optical density val- either antigens or antibodies in serum and other body fluids of ues of the wells, after a stop solution has been added to arrest the patient. In EIA techniques, antigen or antibody labeled with the chromogenic reaction, one can determine the amount of enzymes are used. Alkaline phosphatase, horseradish peroxi- antigen–antibody complex formed in the first step (Fig. 14-14). dase, and galactosidase are the enzymes used in the EIA tests. The commonly used EIAs are enzyme-linked immunosorbent assays (ELISAs). The ELISA technique was first conceptualized Key Points Section II Section and developed by Peter Perlmann and Eva Engvall at Stockholm University, Sweden. The test is extensively used for determination of serum antibodies for diagnosis of human immunodeﬁ ciency virus (HIV) These assays involve the use of an immunosorbent specific to infection, Japanese encephalitis, dengue, and many other viral either the antigen or antibody. Following the antigen– antibody infections. reaction, chromogenic substrate specific to the enzyme (o-phenyl- diamine dihydrochloride for peroxidase, p-nitrophenyl phosphate

Chapter 14 for alkaline phosphatase, etc.) is added. The reaction is detected ◗ Sandwich ELISA by reading the optical density. Usually, a standard curve based on known concentrations of antigen or antibody is prepared from The sandwich ELISA is used for the detection of antigen. In which the unknown quantities are calculated. There are different this test, the known antibody is coated and immobilized onto types of ELISAs available for the detection and quantitation of the wells of microtiter plates. The test sample containing the either the antigen or antibodies in serum and other body fluids. suspected antigen is added to the wells and is allowed to react These include: (a) indirect ELISA, (b) sandwich ELISA, (c) competi- with the antibodies in the wells. After the step of washing the tive ELISA, and (d) ELISPOT assay. well, a second enzyme-conjugated antibody specific for a different epitope of the antigen is added and allowed to incubate. After removing any free secondary antibody by rewashing, the ◗ Indirect ELISA specific substrate is added, and the ensuing chromogenic reac- The indirect ELISA is used for the quantitative estimation of tion is measured. The chromogenic reaction is then compared antibodies in the serum and other body fluids. In this method, with a standard curve to determine the exact amount of the specimens are added to microtiter plate wells coated with antigen present in the test sample. In a positive test, an enzyme antigen to which specific antibodies are to be detected. After acts on the substrate to produce a color, and its intensity can be a period of incubation, the wells are washed. If antibody was measured by spectrophotometer or ELISA reader. The change present in the sample, antigen–antibody complex would have of color can also be observed by the naked eye (Fig. 14-15).

Antigen Antibody Enzyme conjugated antibody Enzyme substrate Excess unbound enzyme Excess unbound antibody is conjugated antibody is Product washed off during washing washed off during washing

Antigen is adsorbed to Clinical specimen is added: Enzyme conjugated Enzyme substrate ( ) is added well surface antibody binds to antigen anti-immunoglobulin and substrate is converted to antibody binds product ( ) that causes a to antibody visible color

FIG. 14-14. Indirect ELISA test. Section II Chapter 14 113 visible color product ( ) that causes a and substrate is converted to and substrate is converted Enzyme substrate ( ) is added conjugated antibody is Excess unbound enzyme Excess unbound washed off during washing ANTIGEN–ANTIBODY REACTIONS ANTIGEN–ANTIBODY sandwich The classical RIA methods are based on the principle The classical RIA methods variable amounts of cold In the test, mixtures of known ELISPOT Assay ELISPOT assay is a modificationELISPOT assay of ELISA. the quanti- It allows of number of cells in a population that are tative determination producing antibodies specific antigen or an antigen a given for found which one has a specific These tests have for antibody. of cytokines. application widely in the measurement Radioimmunoassay are used as labels to instead of enzymes When radioisotopes of the technique be conjugated with antigens or antibodies, is called as radio- complex of the antigen–antibody detection (RIA). RIA was first immunoassay mea- described in 1960 for by Solomon Berson surement of endogenous plasma insulin Administration Hospital of the Veterans and Rosalyn Yalow Prize for the 1977 Nobel was awarded Yalow York. in New hor- peptide of the RIA for the development Medicine for mones, but because of his untimely death in 1972, Berson share the award. could not unlabeled antigen method, binding. In this competitive of to antibody binding with radiolabeled antigen for competes with the appropriate specificity. Thus, when mixtures of radiolabeled and unlabeled antigen are incubated with the to bound the amount of free (not corresponding antibody, antibody) radiolabeled antigen is directly proportional to the of unlabeled antigen in the mixture. quantity antigen and fixed of labeled antigen and mixtures of amounts identi- concentrations of antigen with with unknown samples cal amounts of labeled antigen are prepared in the first step. Identical amounts of antibody are added to the mixtures. are precipitated either by cross- Antigen–antibody complexes linking with a second antibody or by means of the addition of the precipitationreagents that promote of antigen–antibody Counting radioactivity in the precipitates allows complexes. ◗ binds to antigen forming specific for the test antigen Enzyme conjugated antibody Excess unbound antigen is Excess unbound antigen washed off during washing antigen binds to antibody Clinical specimen is added: in feces. Antigen Antibody antibody Enzyme conjugated Enzyme substrate Product to well surface Escherichia coli Escherichia Antibody is adsorbed Sandwich ELISA test. Key Points detec- ELISA is the most commonly used test for Competitive tion of HIV antibodies in serum in patients with HIV. Key Points and entero- rotavirus The sandwich ELISA is used to detect of toxin In this test, the microtiter wells are coated with HIV anti- wells In this test, the microtiter Competitive ELISA Competitive ELISA is another technique used for the esti- used for technique ELISA is another Competitive such as serum. mation of antibodies present in a specimen, specificPrinciple of the test is that two antibodies, one present in test serum conjugated with enzyme and the other antibodies), are used. Competition for (if serum is positive the same antigen. antibodies for the two occurs between test (absence of anti- Appearance of color indicates a negative test indicates a positive bodies), while the absence of color (presence of antibodies). and incu- gen. The sera to be tested are added to these wells bated at 37°C and then washed. If antibodies are present in the test serum, antigen–antibody reaction occurs. The antigen– by adding enzyme-labeled-specificantibody reaction is detected test, no antigen is left these HIV antibodies. In a positive for antibodies to act. Hence, the antibodies remain free and are the process of washing. When substrate during washed away positive to act on it. Therefore, is added, no enzyme is available test, in which no result indicates no color reaction. In a negative antibodies are present in the serum, antigen in the coated wells and to combine with enzyme-conjugated antibodies is available the enzyme acts on the substrate to produce color. FIG. 14-15. ◗ 114 IMMUNOLOGY

the determination of the amount of radiolabeled antigen pre- Western blotting is usually done on a tissue homogenate cipitated with the antibody. A standard curve is constructed by or extract. It uses SDS-PAGE (sodium dodecyl sulphate- plotting the percentage of antibody-bound radiolabeled anti- polyacrylamide gel electrophoresis), a type of gel electro- gen against known concentrations of a standardized unlabeled phoresis to first separate various proteins in a mixture on antigen, and the concentrations of antigen in patient samples the basis of their shape and size. The protein bands thus are extrapolated from that curve. The extremely high sensitivity obtained are transferred onto a nitrocellulose or nylon of RIA is its major advantage: membrane where they are “probed” with antibodies specific to the protein to be detected. The antigen–antibody com- Key Points plexes that form on the band containing the protein recog- nized by the antibody can be visualized in a variety of ways. Uses of RIA: If the protein of interest was bound by a radioactive anti- ■ The test can be used to determine very small quantities body, its position on the blot can be determined by exposing (e.g., nanogram) of antigens and antibodies in the serum. the membrane to a sheet of X-ray film, a procedure called ■ The test is used for quantitation of hormones, drugs, autoradiography. However, the most generally used detec- HBsAg, and other viral antigens. tion procedures employ enzyme-linked antibodies against Section II Section the protein. After binding of the enzyme–antibody conju- The main drawbacks of the RIA include: (a) the cost of equip- gate, addition of a chromogenic substrate that produces ment and reagents, (b) short shelf-life of radiolabeled com- a highly colored and insoluble product causes the appear- pounds, and (c) the problems associated with the disposal of ance of a colored band at the site of the target antigen. The radioactive waste. site of the protein of interest can be determined with much higher sensitivity if a chemiluminescent compound along Chapter 14 Western Blotting with suitable enhancing agents is used to produce light at the antigen site (Fig. 14-16). Western blotting is called so because the procedure is similar to Southern blotting, which was developed by Edwin Southern for the detection of DNA. While Southern blotting is done to detect DNA, Western blotting is done for the detection of proteins. Key Points Western blot technique has many uses as follows:

Proteins are ■ It is used for identification of a specific protein in a com- separated through plex mixture of proteins. In this method, known antigens of SDS-PAGE well-defined molecular weight are separated by SDS-PAGE and blotted onto nitrocellulose. The separated bands of Proteins separated on gel were known antigens are then probed with the sample suspected blotted onto nitrocellulose paper of containing antibody specifi c for one or more of these antigens. Reaction of an antibody with a band is detected by using either radiolabeled or enzyme-linked secondary Nitrocellulose antibody that is specific for the species of the antibodies paper in the test sample. ■ It is also used for estimation of the size of the protein as well as the amount of protein present in the mixture. Incubation with patient’s ■ The Western blot test is most widely used as a confirma- antisera (first antibody) tory test for diagnosis of HIV, where this procedure is used Incubation with enzyme to determine whether the patient has antibodies that react conjugated antihuman with one or more viral proteins or not. serum (second antibody) ■ The Western blotting is also used for demonstration of specific antibodies in the serum for diagnosis of neurocysticer- Antibodies cosis and tubercular meningitis. attach to specific antigen bands on nitrocellulose paper

Enzyme conversion of substrate identifies the antigen Chemiluminescence Assay The chemiluminescence assay uses chemiluminescent com- Bands can be pounds that emit energy in the form of light during the anti- visually interpreted gen–antibody reactions. The emitted lights are measured and the concentration of the analyze is calculated. The assay is a fully automated method, which is used commonly for drug sensitiv- FIG. 14-16. Western blot test. ity testing of Mycobacterium tuberculosis. Section II Chapter 14 115 ANTIGEN–ANTIBODY REACTIONS ANTIGEN–ANTIBODY Immunoenzyme test Immunoenzyme test Immunoferritin ◗ This test is used to detect antigen directly in tissue specimens, in in tissue specimens, antigen directly used to detect This test is antisera with peroxidase-labeled sections are treated which tissue bound to the corresponding antigen. The peroxidase to detect under the electron microscope. antigen is visualized Electron-dense substances, with are conjugated such as ferritin can labeled antibodies reacting with antigen antibody and such the electron microscope. be visualized under ◗ Immunoelectronmicroscopy This is a test used to detect rotavirus and hepatitis A virus rotavirus to detect This is a test used this test, viral particles In with spe- directly in feces. mixed are cific of virion particlesantisera and are demonstrated as clumps microscope. under the electron ◗ Immunoelectronmicroscopic Tests Immunoelectronmicroscopic that are reactions the types of antigen–antibody These are are of the microscope. These directly by electron visualized following types: