Antigen-Antibody Reactions Lecture Notes PDF

ANTIGEN - ANTIBODY REACTIONS By: Haidy Samir Mohamed khalil Professor of Medical Microbiology & Immunology Faculty of Medicine Helwan University  Objectives Recognize the proerities of Ag-Ab reactions Enumerate different types of Ag-Ab reactions...

ANTIGEN - ANTIBODY REACTIONS By: Haidy Samir Mohamed khalil Professor of Medical Microbiology & Immunology Faculty of Medicine Helwan University  Objectives Recognize the proerities of Ag-Ab reactions Enumerate different types of Ag-Ab reactions Explain the principles of different Ag—Ab reactions Identify the uses of different Ag- Ab reactions  Introduction Reactions of antigens and antibodies are highly specific in the sense that an antibody can react only with the antigen which induced its formation.  These interactions are widely used in vitro for diagnostic purposes i.e. for the detection and identification of either antigen or antibody and are termed serologic reactions. In these tests one of the reactants should be known while the other is unknown.  The interactions of antigen with antibody may result in a variety of consequences, including agglutination if the antigen is particulate, precipitation if the antigen is soluble, or activation and fixation of complement. The extent of the reaction depends on the proportions of the interacting antigen and antibody.  At high antibody levels, i.e. antibody excess, the reaction may not occur. This is referred to as a "prozone" effect. Prozone effect  Types of. Ag-Ab reaction 1- Agglutination 2- Precipitation 3- Complement Fixation 4- Immunoflouresence tests 5- ELIZA 6- Radioimmunoassay  AGGLUTINATION The antigen in the agglutination reaction is in the form of particles, e.g. suspensions of microorganisms, cells (e.g. red blood cells), or latex particles coated with antigens. When mixed with the specific antiserum, these particles become clumped, i.e. agglutinated.  If one of the reactants (antigen or antibody) is known, the reaction can be used for the identification of the other. So known antisera are used to identify microorganisms isolated from clinical specimens. On the other hand, known organisms can be used to detect antibodies in sera of patients. There are several types of the agglutination reaction:- Agglutination: 1-Direct : Slide & tube 2- Coomb’s 3- Latex agglutination 4- Coagglutination 5- Virus haemagglutination inhibition test 6- Heterophil antibody agglutination test  1. Direct Agglutination: can be done in: A- slide B- Tube  A- The slide method It is rapid and useful in identification and typing of isolated organisms by mixing a drop of the bacterial suspension with known antisera. Clumping occurs if the serum is specific to the organism under test. The slide method is also used in blood grouping.  B- The tube method it is a quantitative test, which is used to determine the amount of antibodies in the serum of patients. It is done by mixing serial dilutions of the patient's serum (1/10, 1/20, 1/40, 1/80, etc.) with a constant amount of a known bacterial suspension, suspected to be the cause of the disease. The last tube showing visible agglutination will reflect the serum antibody titer of the patient.  The classical application of the tube method is the Widal test used to detect antibodies to salmonella in the serum of patients suspected to have enteric fever. The agglutination tests are also used in diagnosis of brucellosis, and other diseases.  In diagnosis of such infectious diseases, two serum samples separated by 7-10 days interval should be tested. A rising antibody titer of two folds or more is diagnostic.  The titer in this test is 1/160 = Highest dilution that shows agglutination  The titer in this test is 1/40 = Highest dilution that shows agglutination 2. Antiglobulin Agglutination test = Coomb's test: This test is used to determine the presence of Rh incompatibility which causes "erythroblastosis foetalis".  Most anti-Rh antibodies are incomplete IgG (unagglutinable) antibodies which can only coat the Rh positive RBCs, but can not bridge between two RBCs to cause agglutination. These antibodies can be detected by the antiglobulin Coomb's test which is performed in two ways:  Indirect Coomb's test: The mother's serum -containing incomplete anti-Rh antibodies- is mixed with Rh-positive RBCs (group O). After incubation, the mixture is centrifuged. The deposit containing red cells coated with incomplete antibodies is washed, and antihuman globulin is added, and the tubes incubated. The antihuman globulin causes agglutination by linking together the incomplete antibody molecules.  Direct Coomb's test: This test can detect incomplete Rh antibodies coating the RBCs of the newborn in erythroblastosis foetalis. The antihuman globulin is added directly to a washed suspension of the newborn RBCs, agglutination occurs. Both the direct and indirect Coomb's tests are also used to detect incomplete antibodies in autoimmune haemolytic anaemias. 3. Latex or Passive Agglutination: It is an agglutination reaction in which inert particles, e.g. latex or RBCs are coated with various antigens or antibodies. These particles are aggregated in the presence of specific antibody or antigen, respectively. Examples of passive agglutination include the following:- a-Immunologic pregnancy test: It depends on the appearance of human chorionic gonadotrophic hormones (HCG) in the urine of pregnant females. The test is done by adding a drop of latex particles coated with anti-HCG to a drop of urine; agglutination occurs if HCG is present in urine. b- Rheumatoid factor c-C-reactive protein (CRP) d-anti-streptolysin 'O' (ASO) tests, used for diagnosis of acute rheumatic fever  4- Co-agglutination - Staphylococcus aureus (Cowan I strain) has specific receptors for the antibody ( attached from fc portion, leaving the fab fragment free to bind to the antigen. - The same idea of the latex 5. Haemagglutination Inhibition assays. They are used to determine whether an individual has been exposed to certain types of viruses that cause agglutination of red blood cells.  The patient serum containing specific antiviral antibodies is mixed with a known virus, then RBCs are added, the antibodies will bind to the virus and interfere with haemagglutination of RBCs by the virus i.e. virus haemagglutination inhibition. 6- Heterophil antibodies agglutination tests 1- Paul Bunnell test Diagnose infectious mononucleosis (Epstein Barr) Virus II- Cold agglutinins Patients with mycoplasma pneumonia III -VDRL and RPR test: In sera of syphilitic patients  Types of. Ag-Ab reaction 1- Agglutination 2- Precipitation 3- Complement Fixation 4- Immunoflouresence tests 5- ELIZA 6- Radioimmunoassay  PRECIPITATION This is an antigen antibody reaction in which the antigen is soluble. Precipitation reactions can be done in solution or in agar gel A- Precipitation in solution: This reaction can be made quantitative It is used primarily in research. B- Precipitation in agar: In this technique, diffusion of antigen and antibody is allowed to occur in agar gel. It can also be done in presence of an electric field. 1- Double diffusion (Ouchterlony): The antigen and antibody are placed in different wells punched in the agar. Both will diffuse in the agar and where they meet at optimal proportions, precipitation bands will appear. This method can show whether antigens are identical, related but not identical, or not related.  A clinical application for this method is the Elek's test which is used to prove the toxigenicity of diphtheria bacilli A strip of filter paper, saturated with diphtheria antitoxin, is embedded in serum agar.  A heavy inoculum of the diphtheria bacillus to be tested is inoculated at right angle to the strip of filter paper. Plates are examined after 2 days incubation at 37°C.  If the organism is toxigenic, the toxin will diffuse sideways from the inoculum and the antitoxin diffuses from the filter paper, and where they meet at optimal concentrations, a precipitate is formed which will appear as fine white lines. 2- Single diffusion: The antibody is incorporated in the agar before pouring it in the plates. The antigen is placed in a well punched in this agar. The antigen diffuses in all directions, and where its concentration is optimal in relation to the antibody, a precipitation ring will form around the well.  The diameter of the ring depends on the antigen concentration. This technique is used to estimate the quantity of the various immunoglobulin classes, complement components and other substances in human serum. 3- Precipitation in agar with an electric field: Immunoelectrophoresis: No details Western Blots (immunoblots): No Details  Types of. Ag-Ab reaction 1- Agglutination 2- Precipitation 3- Complement Fixation 4- Immunoflouresence tests 5- ELIZA 6- Radioimmunoassay  COMPLEMENT FIXATION (CF) This is an antigen antibody reaction that occurs in the presence of a third component known as the complement. The antigen unites with its specific antibody and the resulting complex fixes the complement.  The CF test is used for diagnosis of many diseases by detecting complement fixing antibodies in the serum of patients, as in syphilis, whooping cough, chronic gonorrhoea, typhus, small pox and other diseases. N.B. This test is infrequently used nowadays and is replaced by more sensitive and faster tests.  Types of. Ag-Ab reaction 1- Agglutination 2- Precipitation 3- Complement Fixation 4- Immunoflouresence tests 5- ELIZA 6- Radioimmunoassay  IMMUNOFLUORESCENCE These are antigen antibody reactions in which we use fluorescein labelled antibodies. Fluorescein is a dye which emits greenish fluorescence under ultraviolet light (UV) by the fluorescent microscope , and it can be tagged to immunoglobulin molecules. There are two ways for doing the test: 1- Direct Immunofluorescence: In this test, fluorescein labelled antibodies are layered over the antigen fixed on a slide. They are left to react for some time then the excess unattached antibody is washed thoroughly. The slide is then examined by the fluorescent microscope under ultraviolet light. The site where the antibody adheres to its specific antigen will be seen as apple green fluorescence. T his method can be used to detect bacteria, viruses and antigens in tissues or in pathological samples. 2- Indirect Immunofluorescence: The test is used to detect antibodies in serum of patients. The serum is layered on the antigen preparation which is fixed on a slide; they are allowed to react for some time, then the excess is washed.  Whether there is antibody in the serum, that adhered to the antigen, or not is determined by adding a fluorescein labelled anti-globulin which will attach to the antibody if present in patient's serum and give positive fluorescence under UV light. This method is used in the serologic diagnosis of syphilis (caused by Treponema pallidum) to detect anti-treponemal antibodies. This is known as Fluorescent treponemal antibody (FTA) test.  Types of. Ag-Ab reaction 1- Agglutination 2- Precipitation 3- Complement Fixation 4- Immunoflouresence tests 5- ELIZA 6- Radioimmunoassay  ENZYME-LINKED IMMUNO SORBENT ASSAY (ELISA) This technique is very sensitive and does not require specialized equipment and avoids the hazards of radioactivity. The method depends on conjugation of an enzyme to either antigen or antibody, then the enzyme activity on a substrate is used as a quantitative measure.  To measure antibody, the indirect method is used. A known antigen is fixed to a solid phase (e.g. plastic cup or microplate), incubated with the test serum, then washed to remove excess unattached antibody and re-incubated with antiglobulin labelled with a suitable enzyme (e.g. horseradish peroxidase).  The labelled antiglobulin will attach to the antibody bound to the fixed antigen. After washing, the enzyme activity is measured by adding a specific substrate and measuring the degree of colour change.  To measure an antigen, the double antibody technique is used. A known antibody is fixed to the solid phase. The test material containing antigen is added and the excess washed. A specific known antibody labelled with enzyme is added. After washing, a substrate is added and the enzyme activity measured colourimetrically and related to antigen concentration.  Types of. Ag-Ab reaction 1- Agglutination 2- Precipitation 3- Complement Fixation 4- Immunoflouresence tests 5- ELIZA 6- Radioimmunoassay  RADIOIMMUNO ASSAY (RIA) This is a sensitive method used to measure antigens or antibodies that can be radioactively labelled. There are many variations of the test. However, the solid phase RIA is a popular method. The principle of the assay method is that radio-iodine (125I) labelled antigen (e.g. thyroid hormones T3 or T4, hepatitis B antigen) competes with a non-labelled antigen in a test sample, for a fixed amount of a specific antibody in a limited time.  Read only The test is done by adding the serum sample (test antigen) to antibody adsorbed to solid phase (tubes or beads), then the labelled antigen is added and incubated for a limited time. After decanting the supernatant, the remaining bound radioactivity is measured in a gamma counter and the percentage bound labelled antigen is calculated.  Read only The concentration of test antigen is deduced from a standard curve drawn between concentrations of known standards (run at the same time with the test) and the percentage bound radiolabeled antigen.  Read only N.B. The shelf life time of the reagents used in RIA is 2 weeks to 2 months as compared to that of the reagents used in the ELISA which is 6-12 months. In addition, the RIA reagents are hazardous while ELISA reagents are not.  Types of. Ag-Ab reaction 1- Agglutination 2- Precipitation 3- Complement Fixation 4- Immunoflouresence tests 5- ELIZA 6- Radioimmunoassay  FLOW CYTOMETRY Flow cytometers are instruments capable of analyzing properties of single cells, in a fluid sheath, as they pass through an orifice at high velocity (50,000 cells/minute). They can sort out and count cells with certain characteristics from the general population. Properties measured include physical characters such as size, volume, refractive index, viscosity and chemical features such as content of DNA or RNA, proteins and enzymes.  Read only These properties are detected by measuring light scatter, cell volume and fluorescence after staining the cells with fluorescein labelled monoclonal antibodies to any specific cell surface markers.  Personal contact data [email protected] [email protected] 01091584654 Haidy Khalil