Development Team. Department of Zoology, University of Delhi. Department of Zoology, University of Delhi. Hindu College, University of Delhi

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1 Paper No.: 10: Module : 24: Immunity in health and diseases: Principles and applications of Development Team Principal Investigator: Co-Principal Investigator: Paper Coordinator: Content Writer: Content Reviewer: Prof. Neeta Sehgal Head, Department of Zoology, University of Delhi Prof. D.K. Singh Department of Zoology, University of Delhi Prof. Anju Srivastava Department of Zoology, University of Delhi Dr. Divya Bajaj Hindu College, University of Delhi Prof. Sukhmahendra Singh Banaras Hindu University 1

2 Description of Module Subject Name Paper Name Module Name/Title Module ID Keywords Zool 010: Immunity in Health and Diseases M24: Principles and applications of cross-reactivity- precipitation, agglutination, SPR Cross reactivity, precipitation, agglutination, affinity, avidity, immunodiffusion, SPR, specificity Contents 1. Learning Objectives 2. Introduction 2.1. Nature of Antigen-Antibody Reactions 2.2. Affinity and Avidity 2.3. Specificity and Cross Reactivity 3. Detecting Antigen-Antibody Reactions 3.1. Factors Affecting Measurement of Antigen-Antibody Reactions 4. Antigen-Antibody Interactions 4.1. Agglutination 4.2. Precipitation 4.3. SPR 5. Summary 2

3 1. Learning Objectives After reading this unit, you will be able to: 1. Understand nature of Antigen-antibody interactions. 2. Define the basic characteristics of an immune reaction. 3. Identify and define factors important for detection of Antigen-antibody reactions. 4. Define the methods and principles of agglutination, precipitation and SPR. 5. Describe the various applications of these techniques. 6. Describe the principles used in clinical applications of these basic tests. 2. Introduction 2.1. Nature of Antigen-Antibody Reactions Lock and Key Concept An antibody molecule comprises of the Fab portion where the active site consisting the hyper variable regions of the heavy and light chains is located. The antigenic determinant resides in a cleft formed by the active site of the immunoglobulin molecule as indicated by X-ray crystallography studies. Thus, the antigen-antibody interactions can be simulated by a key (the antigen) which fits into a lock (the antibody). Non-covalent Bonds The binding of an antibody and antigen is highly specific and involves weak and reversible noncovalent interactions comprising mainly of van der Waals forces, electrostatic forces, H-bonding and hydrophobic forces. The antigen combines to the antibody at the active site by non-covalent bonds. Multiple bond formation ensures that the antigen will be bound tightly to the antibody. Reversible Nature Antigen-antibody complexes are strengthened by non-covalent bonds, thus making their nature reversible Affinity and Avidity Affinity The intensity of the reaction between an antigenic determinant and one active site on the antibody molecule defines the affinity of that antibody. Affinity is the equilibrium constant characteristic of a Ag-Ab interaction. It is the sum of the attractive and repulsive forces effective between the antigenic determinant and the active site of a specific antibody. Most antibodies have extremely high affinity and specificity for their antigens. Avidity Avidity amounts to the total strength by which an antigen binds to multiple antigenic determinants and multivalent antibodies. Avidity is affected by the valence of the antibody as well as that of the antigen and is therefore more than the sum of individual affinities. 3

4 Hence, affinity is the strength of binding between a single antigenic determinant and its corresponding individual antibody combining site whereas avidity refers to the overall strength of binding between multivalent antigens and antibodies Specificity and Cross Reactivity Specificity Specificity is defined as the ability of a particular antibody active site to recognize and interact with only a single antigenic determinant or the ability of a population of antibody molecules to react with only a single antigen. Antigen-antibody reactions possess extremely high degree of specificity. Antibodies are capable of discriminating between- a. the primary structure of an antigen b. isomeric forms of an antigen c. secondary and tertiary structure of an antigen. Cross Reactivity Cross reactivity is the capability of a particular antibody active site to react with more than a single antigenic determinant or that of a population of antibody molecules to react with multiple antigens. Cross reactions are multispecific interactions arising due to sharing of an epitope by a cross reacting antigen and the immunizing antigen or because the epitope is structurally similar to one on the immunizing antigen. Cross-reactivity usually occurs among polysaccharide antigens containing similar oligosaccharide residues. For instance, the ABO blood-group antigens are glycoproteins expressed on the surface of erythrocytes. Factors distinguishing the blood-group antigens A and B include fine variations in the terminal sugar residues of these surface proteins. A person with type A blood has anti-b antibodies; a type B person has anti-a; and a type Operson therefore has both anti-a and anti-b antibodies. An individual lacking one or both of these antigens would generate serum antibodies to the missing antigen (s). The serum antibody response is not induced by exposure to erythrocyte antigens but by cross-reacting microbial antigens present on common intestinal bacteria. These antigens intern induce the production of antibodies in individuals lacking the similar blood-group antigens on their erythrocyte surfaces. These antibodies would cross-react with the oligosaccharides on foreign erythrocytes, forming the basis for blood typing and accounting for the essential compatible nature of blood types during blood transfusions. Table 1: ABO blood type- Antigens present on surface of RBCs act as epitopes for generation of serum antibodies. Blood type Antigens on RBCs Serum antibodies A A Anti-B B B Anti-A AB A and B Neither O Neither Anti-A and Anti-B 4

5 3. Detecting Antigen-Antibody Reactions 3.1. Factors Affecting Measurement of Antigen-Antibody Reactions Antigen-antibody complexes are formed under specific conditions of temperature and ph. In order to determine whether an antigen-antibody reaction has occurred, the Ag-Ab complexes formed have to be detected by direct or indirect means. A number of factors influence the detection of these complexes. a. Affinity - Higher affinity of the antibody for the antigen ensures a stable reaction between the two thus facilitating the detection of the complex formed. b. Avidity - Interactions between multivalent antigens and multivalent antibodies are very stable aiding in their detection. c. Antigen to antibody ratio - The formation of Ag-Ab complexes is related to the concentration of the antigen and antibody, therefore its detection is directly dependent on the ratio in which they are present at a particular instance. d. Physical form of the antigen - The physical form of the antigen plays an important role in detection of its interaction with an antibody. Particulate antigens result in agglutination on reaction with an antibody. However, precipitation of the antigen would occur in case it is soluble in nature when large insoluble Ag-Ab complexes are formed. 4. Antigen-Antibody Interactions 4.1. Agglutination The word Agglutination comes from the Latin agglutinare, meaning "to glue, referring to clumping of substances. Agglutination is defined as the visible clumping of a particulate antigen when mixed with antibodies specific for it, in the presence of electrolytes at an appropriate temperature and ph. Antibodies are capable of binding multiple antigen molecules, linking them to create a large lattice like complex which is visible to the naked eye. Antibodies that generate such reactions are called agglutinins. All antibodies can theoretically agglutinate particulate antigens but IgM, due to its high valence, is particularly a good agglutinin. Large antigens with multiple epitopes easily adhere to particles such as animal cells or bacteria when combined with specific antibodies resulting in crosslinking. The process of agglutination involves two steps. First step is sensitization and second is lattice formation. Sensitization is the recognition and attachment of specific antibody to corresponding antigen. Temperature, ph and time of incubation influence the reaction. A Lattice is formed by cross linking between sensitized particles. Agglutination reaction used for diagnosis of diseases in lab either uses the particulate or soluble antigens. Example of agglutination reaction using particulate antigens is Salmonella typhi bacteria to detect specific antibody in serum from patient suffering from typhoid fever (Widal test). Agglutination is a serological reaction similar to precipitation; with the exception of the antigen being large and particulate in case of agglutination. Both reactions are inhibited by antibody excess and this phenomenon is called the prozone effect, whereas in case of antigen excess postzone effect occurs. If the antigen is an integral part of the surface of a cell or other insoluble particle, the agglutination 5

6 reaction is known as direct agglutination. However, a cell or insoluble particle can be coated with a soluble antigen such as a viral antigen, a polysaccharide or a hapten and the coated cells can be used in an agglutination test for antibody to the soluble antigen in a reaction called passive agglutination. IgM Epitopes Y Y Bacterium Figure 1: Agglutination Pentavalent Immunoglobulin IgM is shown to interact and bind with multiple antigenic epitopes on the surface of bacterial cells causing clumping or agglutination reaction. Agglutination Inhibition In agglutination inhibition, absence of agglutination is diagnostic of an antigen. Agglutination inhibition, a modified agglutination reactionis a highly sensitive assay for detecting small quantities of an antigen. Its applications include a simple type of home pregnancy test kit where latex particles coated with human chorionic gonadotropin (HCG) and antibody to HCG. On reaction with urine from a pregnant woman, which would contain the HCG antigen, agglutination of the latex particles is inhibited when the anti-hcg antibody is added; thus absence of agglutination preliminarily confirmed pregnancy. Another instance where agglutination inhibition assay can be used is to determine whether an individual has consumed certain types of illegal drugs, such as cocaine or heroin. A urine or blood sample from the suspect is first incubated with antibody specific for a particular drug. Then erythrocytes (or other particles) coated with the drug are added. If the erythrocytes are not agglutinated by the antibody, the sample contains an antigen that binds to the antibody, indicating that the individual had consumed the illicit drug. However, some legal drugs have chemical structures like those of illicit drugs, and may cross-react with the antibody, giving a false-positive reaction. Therefore a positive reaction is a preliminary test followed by a non immunologic method for confirmation. 6

7 Table 2: Aggutination reaction in ABO blood typing- Serum of individuals would contain different types of antigens on surface of RBCs and corresponding antibodies to these antigens. Red blood cells from individuals of type Serum from individuals of type O A B AB Y O Y Anti-A & Anti- B antibodies A Y Anti-B antibodies B Y Anti-A antibodies A B No antibodies to A or B No agglutination Agglutination Agglutination Agglutination No agglutination No agglutination Agglutination Agglutination No agglutination Agglutination No agglutination Agglutination No agglutination No agglutination No agglutination No agglutination Qualitative Agglutination Test Agglutination tests could be performed in a qualitative manner to assay for the presence of an antigen or an antibody. When the antibody is combined with a particulate antigen, the agglutination of this antigen indicates a positive test. For eg., an individual s erythrocytes can be mixed with antibody specific to different blood group antigens to determine his blood type. Alternately, an individual s serum is mixed with erythrocytes of a known blood type to assay for the presence of antibodies to that blood type in the person s serum. Quantitative Agglutination Test Agglutination tests could also be quantitative where the level of antibodies to particulate antigens can be measured. This test is performed by making serial dilutions of the sample to be tested for antibody and then a fixed number of erythrocytes or bacteria or other such particulate antigen is added. The maximum dilution that gives visible agglutination is then determined and is known as the titer. The results are reported as the reciprocal of the maximal dilution that gives visible agglutination. Hemagglutination Hemagglutination reactions involve agglutination reactions using erythrocytes. These reactions are used in blood typing, diagnosis of certain diseases, and identification of certain viruses. Blood typing tests with the ABO antigens involve mixing of the RBCs on a slide with antisera to the antigens A or B. If the corresponding antigen is present on the cells, agglutination results in the form of visible clumping on the 7

8 slide. Identification of the antigens present on donor and recipient RBCs is the fundamentalprinciple for matching blood types for transfusions. Figure 2: Hemagglutination- Red blood cells interact with viruses to produce a clumping reaction. Kuby, Sixth edition, WH Freeman and Company. Applications of Agglutination Tests i. Determination of blood group types or antibodies to blood group antigens. ii. Assessment of bacterial infections. eg. infection with a particular bacterium is indicated by the rise in titer of an antibody to this bacterium. Coombs Test When erythrocytes interact with antibodies, agglutination may not always result. This could be due to deviation from antigen-antibody ratio from its optimal concentration, or zeta potential on the erythrocytes could be preventing cross-linking of cells. In order to detect the non-agglutinating antibodies on erythrocytes, a second antibody directed against the antibodies attached to their respective epitopes on erythrocytes is added. This anti-immunoglobulin can now cross-link the erythrocytes and result in agglutination. This test is known as the Coombs test or anti-immunoglobulin test. The Coomb s test is based on two important facts: i. Antibodies of one species are immunogenic when injected into another species leading to production of anti-immunoglobulins. ii. Many anti-immunoglobulins bind with antigenic determinants present on the Fc portion of the antibody and leave the Fab portions free to react with antigens Precipitation The smallest unit of an antigen molecule that can bind with an antibody is known as antigenic determinant or epitope. The corresponding region on the antibody molecule that interacts with the epitope is called paratope. The number of epitopes on the surface of an antigen is known as its valence and it determines the number of antibody molecules that can combine with the antigen at one time. Monovalent antigens are those having a single epitope, however, more than one copy of the same epitope is present on most antigens called polyvalent antigens. Immunoprecipitation involves interaction of a soluble antibody with a soluble antigen resulting inthe formation of an insoluble product, the precipitate. These reactions consist of lattice (cross-links) formation when the corresponding antigen and antibody combine in optimal ratios. Lattice formation relies upon the valency of both antibody and antigen. Crosslinked complexes result when bi- or polyvalent antigens interact with more than one multivalent antibodies. If the Ag-Ab complexes formed are too large to stay in solution, visible precipitation results. Excess of either component reduces lattice formation and subsequent precipitation 8

9 Antibody in precipitate hence, these should occur at optimal concentrations. Antibodies that aggregate soluble antigens are called precipitins. Precipitation and agglutination reactions differ in size, solubility of the antigen and sensitivity. Antigens are soluble molecules and larger in size in precipitation reactions. Antigen-Antibody lattice formation is governed by the valency of both the antibody and antigen: The antibody should be polyvalent (Fab fragments) in order to form a precipitate. The antigen should be bi- or polyvalent; ie. it should possess at least two copies of an epitope, or have different epitopes that are capable of reacting with various antibodies in a polyclonal antisera. Antigen Antibody Zone of antibody excess Zone of antigen excess Zone of equivalence Precipitate formed Antigen added Figure 3: Precipitation curve Interaction of Antigens with specific antibodies generates the precipitate. Different zones are highlighted according to the concentration of differently distributed components. Precipitation Reactions in Fluids Yield a Precipitin Curve When a constant amount of antibody is taken in a series of tubes and increasing amounts of antigen is added to these, variable amounts of precipitates form resulting in a quantitative reaction. Initially, this method was used to determine the amount of antigen or antibody present in a particular sample. After precipitation, the tubes are centrifuged to pellet the precipitate and the amount of precipitate is measured on removing the supernatant. The amount of precipitate when plotted against increasing antigen concentrations yields a precipitin curve. Excess of either antibody or antigen interferes with maximal 9

10 precipitation. Hence, formation of an insoluble antigen-antibody complex occurs within a narrow optimal concentration range known as the zone of equivalence. This zone represents the conditions under which antigen-antibody complexes formed are sufficiently large to be precipitated. At equivalence, a large multi-molecular complex is formed which increases in size and precipitates out of solution. On the other hand, outside this zone antigen or antibody excess occurs resulting in the formation of small soluble complexes. Figure 4: Test tube reaction for antigen-antibody combination. Both reactants migrate towards each other and a precipitation band is formed in the zone of equivalence. Immunoprecipitation Reactions in Gels Yield Visible Precipitin Lines Immune precipitates obtained on an agar matrix are referred to as immunodiffusion reactions. When antigen and antibody diffuse toward each other on a solid matrix, or when antibody is incorporated into the gel and antigen diffuses into the antibody-containing matrix, a visible line of precipitation, precipitin line would form. Visible precipitation reaction occurs in the zone of equivalence, whereas no reaction is visible in antibody or antigen excess regions. Different types of immunodiffusion reactions are used to determine relative concentrations of antibodies or antigens, to compare antigens, or to estimate the relative purity of an antigen such as: 1. Radial Immunodiffusion (Mancini method) Radial immunodiffusion is a simple quantitative technique where a serially diluted antigen present in wells diffuses into the gel matrix containing uniformly distributed antibody. When the antigen diffuses into the gel it reacts with the antibody as soon as it encounters the latter. This is followed by attainment of the equivalence point visualized in the form of a ring of precipitation around the well. The diameter of the precipitation ring is directly proportional to log concentration of antigen since the amount of 10

11 antibody is constant. Thus, the amount of an antigen in an unknown sample can be quantitated from a standard curve obtained from different concentrations of a standard antigen. If more than one antigenantibody reaction has occurred, corresponding number of multiple rings would form indicating that the sample contains a mixture of antigens or antibodies. This test is commonly used for the determination of immunoglobulin levels in clinical samples. Antigen diffusion Antibody incorporated in agar Antigen Y Figure 5: Radial immunodiffusion The precipitin ring formed as a result of antigen-antibody interaction is visible in the agar matrix in the form of a radial sphere. 2. Immunoelectrophoresis Immunoelectrophoresis involves placing a complex mixture of antigens in a well and electrophoresing it on an agar gel so that the antigens are separated according to their charge. Following electrophoresis, a trough is cut in the gel and antibodies added. The antibodies diffuse into the gel resulting information of precipitin lines in the equivalence zone as a consequence of the antigen-antibody reaction. This test provides qualitative analysis of complex mixtures of antigens along with a crude measure of quantity represented by the thickness of the line. The components in a patient's serum are commonly analyzed using this test, where the serum is placed in the well, while antibody to whole serum in the trough. Comparison with normal serum can determine whether there are deficiencies or excess of one or more serum components on visualizing the thickness of the precipitin line. 3. Countercurrent Electrophoresis/ Double Immunodiffusion (Ouchterlony) Precipitate forms ring Ouchterlony test named after the Swedish physician who invented it one of the most commonly used serologic precipitation reactions and is based on double immunodiffusion. Also known as countercurrent electrophoresis, it involves placing the antigen and antibody in wells punched in a gel matrix and allowing the two to radially diffuse into each other hence producing a concentration gradient. Different geometrical patterns have been observed in the precipitin lines formed between the antigen and antiserum wells. The patterns of these lines are indicative of the amount of similarity or dissimilarity between the antigens. Rates of diffusion of these molecules increase in proportion to their concentration in the wells, but decrease in proportion to their sizes. Only antigen and antibody having opposite charges 11

12 can be used for this reaction. This test is primarily qualitative, although semi quantitative analysis can be performed by analyzing the thickness of the line. Its major advantage is its speed. Antibody Antigen Y Y Agar matrix Precipitate Figure 6: Double Immunodiffusion The antigen and antibody diffuse toward each other in gel matrix until they interact to form the precipitate in the zone of equivalence. Immunologic relationship between two antigens can be analyzed using this technique. i. Identity occurs when two antigens share similar epitopes. ii. Non-identity occurs when two antigens are discrete and unrelated. ie. share no common epitopes. The antisera form an independent precipitin line with each antigen, and the two lines cross. iii. Partial identity occurs when two antigens share some epitope (line of identity) but one of them also bears a unique epitope (curved spur). This technique can also be used to estimate the relative concentrations of antigens. The distance of formation of the precipitin ring from the antigen well is indicative of concentration of the antigen. Higher the concentration of antigen, greater is the distance between the equivalent zone and the well. Identical Non- Identical Partially Identical Antigen a a a b a ab Antibody α a α α ab α α ab α Figure 7: Ouchterlony test The precipitin lines form differentiable patterns in different antigen-antisera interactions. Relationships between the reacting components can be identified on the basis of the nature of precipitin line produced SPR: Surface Plasmon Resonance SPR is non-invasive optical technique that involves label-free means of real-time tracking of binding of an injected analyte to an immobilized biomolecule. Analyte binding leads to a subsequent increase in 12

13 mass and thus proportional increase in refractive index observed as a shift in the resonance angle. The SPR procedure involves a flow injection comprising the analyte of interest dissolved in a buffer, traversing the sensing surface to network with the immobilized biomolecule. SPR transducers operate by prism coupling incident light onto an optical substrate coated with a semitransparent noble metal Au. Total internal reflection (TIR) occurs at this surface such that all of the light is reflected while none of it refracted above a certain incidence angle. First, TIR should occur at the interface, until the incident angle drops to the critical angle, at which a fraction of the light is refracted across the interface. At the point of reflection at the interface, an evanescent field (standing wave) would penetrate the exit medium to the order of 1/4 of the incident light wavelength. Figure 8: SPR Surface Plasmon Resonance. The basic principle of the technique is highlighted. Coupling of the incident light with the surface plasmons effects the intensity of the reflected light and the patterns are analysed. When monochromatic, polarised light strikes the interface of two transparent media, in this case a glass prism and a buffer solution, from the side of the media with the highest refractive index (glass prism), the light is partly reflected and partly refracted towards the plane of the interface. The 'coupling' of the incident light to the surface plasmons leads to loss of energy and thus a reduction in the intensity of the reflected light. The technique uses a photo-detector array to measure very slight deviations in SPR. The output can be viewed on the BIAcore as 'dips'. The deviations in refractive index are recorded in real time, and plotted as resonance units (RUs) versus time (a sensorgram). A background response is usually produced if there is a difference in the refractive indices of the running and sample buffers. This response value must be deducted from the sensorgram to obtain the actual binding response. Advantage of SPR Ability to perform real-time measurement: Eliminate the need for labeled reactants Exceptional sensitivity: Small quantities of purified reagents are required Disadvantage of SPR: Lack of sensitivity when monitoring low molecular weight adsorbates Rate limiting factor of mass transport-affecting kinetic analysis Coupling to AFM Coupling with Mass-spectrometry 13

14 5. Summary 1. Antigen-antibody interactions depend on four types of noncovalent interactions: hydrogen bonds, ionic bonds, hydrophobic interactions, and van der Waals interactions. 2. Cross reactions are multispecific interactions arising due to sharing of an epitope by a cross reacting antigen and the immunizing antigen or because the epitope is structurally similar to one on the immunizing antigen. 3. The interaction between a particulate antigen and agglutinating antibody (agglutinin) produces visible clumping, or agglutination that forms the basis of simple, rapid, and sensitive immunoassays. 4. The interaction of a soluble antigen and precipitating antibody in a liquid or gel medium forms an Ag-Ab precipitate. Electrophoresis can be combined with precipitationin gels in a technique called immunoelectrophoresis. 5. Precipitation reactions are based on the interaction of antibodies and antigens, two soluble reactants that combine resulting into one insoluble product, the precipitate. These reactions involve formation of lattices (cross-links). 6. SPR is non-invasive optical phenomenon that allows label-free means of observing interactions occurring during binding of an injected analyte to an immobilized biomolecule in real time. 14