Precipitation Reactions PDF

Section 2-Part 2 Precipitation reactions LM 335 Dr. Hassan Kofahi ANTIGEN–ANTIBODY RACTIONS Antigen-antibody reactions serve as the basis of a large number of immunological assays A known reagent antibody could be used to detect or quantify an unknown antigen and vice...

Section 2-Part 2 Precipitation reactions LM 335 Dr. Hassan Kofahi ANTIGEN–ANTIBODY RACTIONS Antigen-antibody reactions serve as the basis of a large number of immunological assays A known reagent antibody could be used to detect or quantify an unknown antigen and vice versa. Immunological assays play an important role in the diagnosis of Infectious diseases (bacterial, viral, fungal and parasitic), Autoimmune diseases and many other clinical conditions. Lattice formation Under certain conditions, Antigen-antibody reactions may result in the formation of a lattice. The lattice is a network formed by cross- linking the antigens by the antibodies. Formation of an Ag-Ab lattice depends on the valency of both the antibody and antigen, Lattice formation requires that: Formation of the lattice Each antibody molecule contains at least two is responsible for two antigen-binding sites (bivalent). monovalent Fab types of Ag-Ab fragments can not form a lattice. reactions: The antigen is bivalent or multivalent (the number of epitopes on the surface is 2 or Precipitation reactions more). Agglutination reactions Precipitation reactions This type of reaction takes place when antibodies react with soluble antigens. In this reaction, antigens and antibodies combine to form multimolecular lattice that increases in size until it precipitates out of solution to produce insoluble complexes that are visible. The amount of precipitation depends largely on the ratio of antibodies to the antigens. Optimal precipitation occurs when the number of multivalent sites of antigen and antibody are approximately equal. This ratio is achieved in the zone of equivalence of the precipitation curve.  Precipitation curve Precipitation curve was used to study the effect of antigen:antibody ratio on the precipitation reactions. Precipitation curve can be generated by adding increasing concentrations of antigen to a set of tubes, all contain a constant concentration of an antibody. As a result, variable amounts of precipitations will form. Plotting the amount of precipitation against the amount of antigen added generates a precipitation curve. Amount of precipitation Three zones can be seen in the precipitation curve: Zone of equivalence: antibody ≈ Antigen Prozone: zone of antibody excess Postzone: Zone of antigen excess Prozone and postzone Excess antibody is responsible for the prozone phenomenon. Due to the antibody excess, each antigen molecule binds to one or two antibodies and only one antigen-binding site is used of each antibody molecule (cross-linking and lattice formation do not occur). In diagnostic immunology lab, prozone phenomenon may be responsible for false-negative reactions due to high antibody concentration in the sample. If a false-negative reaction is suspected, diluting the sample and performing the test again may produce a positive result. Prozone Postzone phenomenon occur when the antigen is present in excess Too many antigens block the lattice formation. The presence of a small amount of antibody in the patient serum may be obscured (due to postzone phenomenon), causing false-negative results Test is repeated about a week later with a new specimen to give time for further production of antibody If the test is negative again, it is unlikely that the patient has the antibody Postzone Uses of the precipitation reactions in the immunology/serology Lab Measurement of precipitation by light scattering Turbidimetry Nephelometry Passive immunodiffusion techniques Radial immunodiffusion (RID) Ouchterlony Double Diffusion Electrophoretic techniques Rocket Immunoelectrophoresis Immunoelectrophoresis Immunofixation Electrophoresis Turbidimetry In the fluids, reaction of a soluble multivalent antigens with the antibodies forms As the light passes through the precipitates that causes an initial solution, the intensity of turbidity or cloudiness of the transmitted light will be reduced solution. due to the scattering. The turbidity can be measured Scattering of light occurs in by placing a detection device in proportion to the size, shape, and direct line with a source of light, concentration of molecules to collect the light after it has present in solution. passed through the vial that contains the solution Higher turbidity  higher (transmitted light). scattering  lower light intensity detected Nephelometry Measures the light that is scattered at a particular angle as it passes through a solution. The detection device is placed Many automated instruments uses at an angle (10°- 90°) to the the principles of turbidimetry and original light path. nephelometry for executing a large number of diagnostic tests such as: The amount of scattered light Quantification of different classes of detected correlates with the immunoglobulins in the serum. amount of Ag-Ab complexes. Quantifications of complement proteins in the serum. Quantification of C-reactive protein in the serum (the high sensitivity method). Passive immunodiffusion techniques In these techniques, precipitation reactions occur in a semisolid support media (gel-like). Agarose is used as the support media for these reactions. When antigen and antibody diffuse toward one another in a gel matrix, visible lines of precipitation will form. Agarose help in stabilizing the diffusion process and allow visualization of the precipitin lines. “Passive” because the reactants migrate in the gel by diffusion (no electrical current is used). Immunodiffusion reactions can be classified according to the number of reactants diffusing and the direction of diffusion. Radial Immunodiffusion (RID) Is a passive immunodiffusion technique, in which: The antibody is incorporated and uniformly distributed in the support gel. The antigen is applied to a well cut into the gel. After applying the antigen, it starts to diffuse in all directions. As a result, antigen-antibody combinations at different proportions occur at different distances from the well At a certain point, the zone of equivalence proportions are achieved. At the zone of equivalence, a ring of visible precipitation forms around the well.  The area of the precipitation ring is proportional to the concentration of the antigen. Several standards, of known Ag concentrations, are used to generate a standard curve. The diameter of the precipitation rings for each standard is measured and this value is used for generating the standard curve. The concentration of the Ag in the patient sample can be obtained from the standard curve. RID: interpretation of the results End point method (Mancini method): In this technique, antigen is allowed to diffuse to completion, and when equivalence is reached, there is no further change in the ring diameter. The diameter of the precipitation ring is measured after 24-72 hours. The squares of the precipitation rings- diameters is plotted against the concentrations of the standards to generate the standard graph. The concentration of the Ag in the unknown sample is obtained by measuring Disadvantages: takes long time to the diameter and using the value of the obtain the result (72 hours) squared diameter to obtain the concentration value from the graph. RID: interpretation of the results Kinetic method (Fahey Method): The measurement is taken before the end-point. The diameter is plotted against the log of the concentration. Results can be read after 18 hours. Clinical uses of RID RID technique is used frequently for performing the following tests: Quantitative measurement of the different classes of immunoglobulins (IgG, IgM, IgA, IgD) Quantitative measurement of the complement proteins (C3, C4). Currently, RID is being replaced by the more sensitive and automated methods such as nephelometry. RID: Sources of error Overfilling or underfilling the wells. Nicking the side of the wells when filling. Spilling sample outside the wells. Improper incubation time and temperature. Incorrect measurement of the diameter. Ouchterlony Double Diffusion Both the antigen and the antibody are placed in two different adjacent wells in a semi-solid media. Double diffusion: both the antigen and the antibody diffuse radially and toward each other. Precipitin lines form where the moving front of antigen meets that of antibody. Semi-quantitative technique the density of the lines reflects the amount of immune complex formed. Used to identify fungal antigens. Preparation of the Ouchterlony plates Ouchterlony double diffusion test is used to test whether different antigens share the same epitopes. The Ouchterlony plates are prepared by cutting a central well in the gel surrounded by 4-6 outer wells. Polyspecific antibody is placed in the central well and the antigens to be tested are placed in the outer wells. In 12-48 hours, precipitation lines form. Depending on the pattern of the precipitation lines, antigens can be compared with one another. Patterns of precipitation lines: Fusion of the lines at the junction to form an arc indicates a serological identity (the two Ags are identical and share the same epitope). Formation of crossed lines at the junction indicates non-identity (the two Ags share no epitopes). Fusion of the two lines with a spur indicates partial identity (the two Ags share an epitope but one of them contains an additional epitope that can react with some antibodies in the polyspecific antisera) Electrophoretic techniques Precipitation reactions can be combined with electrophoresis to speed up the reaction. Electrophoresis is the separation of molecules, including proteins, based on differences in their net charge when they are placed in an electric field. Negatively charged molecules migrate toward the anode. The speed of migration depends on the charge of the molecule. Rocket Immunoelectrophoresis This technique is a modification of the RID. Like RID, the antibodies are incorporated into the gel and the antigen is placed in wells cut into the gel. Electrophoresis is used to speed up the migration of antigens in the gel. The pH of the reaction is kept at 8.6. At this pH the antigen is negatively charged and will migrate but not the antibody. As the antigens move away from the well, there is dissolution and reformation of the precipitate at increasing distances from the well. The end result is a rocket-shaped precipitation. The height of the rocket is directly proportional to the concentration of the antigen in the solution. If standards are run, a standard curve can be generated, from which the antigen concentration in an unknown sample can be obtained.  Rocket immunoelectrophoresis is used to quantify serum immunoglobulins. Serum protein electrophoresis (SPE) and immunofixation Serum contains more than 100 proteins. These proteins can be separated by electrophoresis into 5 fractions: Albumin (58-70 % of serum proteins) Alpha-1 globulins (2-5%) Alpha-2 globulins (6-11%) Beta globulins (8-14%) Gamma globulins (9-18%) Each fraction, except the albumin, contains multiple proteins. The gamma globulin fraction contains the different classes of immunoglobulins. SPE: principle Serum samples are loaded into a gel soaked in a buffer at pH 8.6-8.8. Serum proteins are negatively charged at pH 8.6-8.8. In the presence of the electrical field, serum proteins migrate in the gel toward the anode. During their migration, serum proteins are separated into the 5 major fractions. Albumin has the greatest negative charge at pH 8.6, and hence it will travel the farthest in a given time. Gamma globulins are the least charged and will remain in a diffused band near the origin. After the separation, the proteins are fixed in the gel and stained. SPE: Results Each of the 5 fractions appears as a distinct band. The intensity of the band represents the concentration of the proteins in that band. A densitometer can be used to measure the intensities of the bands and to generate a graph. Abnormalities in the gamma fraction Polyclonal gammopathy: An overall increase in the intensity of the gamma region. Seen in infections and autoimmune diseases Monoclonal gammopathy: Appears as a sharp peak (called M- protein or paraprotein) within the gamma region. Seen in multiple myeloma (The cancer of plasma cells) and Waldenström’s macroglobulinemia. Immunoelectrophoresis A two-step technique that combines electrophoresis and double-diffusion. Typically, antigen mixture (usually serum) is electrophoresed to separate out the main protein fractions. After the separation, troughs cut into the gel parallel to the lines of separation are filled with antiserum. The gel is incubated for 18 to 24 hours to allow double diffusion and precipitation reactions to occur. Immunoelectrophoresis Patient serum is run in multiple lanes in parallel to multiple lanes of normal serum as a control (see the figure). The troughs are filled with the following antisera: Anti-total immunoglobulins (reacts with any type of immunoglobulins) or anti-human proteins (reacts with the serum proteins) Anti-γ (reacts only with IgG) Anti-α (reacts only with IgA) Anti-μ (reacts only with IgM) Anti-κ (reacts only with κ light chain) Anti-λ (reacts only with λ light chain) The lines or arcs of precipitation can be compared in shape, intensity, and location to that of a normal serum control to detect abnormalities. Immunoelectrophoresis results of a C serum sample obtained from a patient with monoclonal gammopathy. P The M protein detected in this case is IgG with kappa light chain. Anti-γ A reduction in the other classes of C immunoglobulins can be also seen. Anti-α P Anti-μ C Anti-κ P Anti-λ C  This method was used to detect monoclonal gammopathies (such as multiple myeloma). Drawbacks The sensitivity of this technique is relatively low. The results require long time to be obtained. The interpretation of results is very complicated and require experience. This technique has been replaced largely with Immunofixation. Immunofixation electrophoresis Similar to immunoelectrophoresis except that the antiserum is applied directly to the gel’s surface rather than placed in a trough. This test is usually ordered if an M-protein was detected in SPE or when there is a strong indication that the patient has a monoclonal gammopathy. Used to confirm the presence of M-protein and to identify its type in the serum or urine of patients with monoclonal gammopathies. Require a shorter time and results in a higher resolution than immuoelectrophoresis. Because diffusion occurs only across the thickness of the gel, the reaction usually takes place in less than 1 hour. Immunofixation: procedure Patient serum sample is applied in sex lanes of the gel. After separation by electrophoreses, each of the lanes is covered by one of the following antibodies: Anti-γ (IgG) Anti-α (IgA) Anti-μ (IgM) Anti-κ light chain Anti-λ light chain An antisera to all serum proteins or a protein fixative is added to one of the lanes to serve as a reference lane. Immunoprecipitates form only where specific antigen–antibody reactions occur, and the immune complexes become trapped in the gel. The gel is washed to remove any nonprecipitating proteins and can then be stained for easier visibility. Note: IgG, IgA or IgM comprise 99% of M-proteins seen. IgE or IgD M-proteins are very rare, hence they are not usually screened. However, in certain cases IgE and IgD might be added. Immunofixation: results Immunofixation: results The M-protein in this case is IgG with a lambda The M-protein in this case is IgA with a kappa light chain. light chain.  Immunofixation: results In this case, the initial test showed a band only in the λ lane. There are two possible explanations for this result, either the M-protein is a free lambda light chain (most propably) or that the M-protein is either IgE or IgD. To solve this problem, the test must be repeated with the addition of anti δ and anti ε antibodies. In the case above, the M-protein was found to be IgD with lambda light chain  Immunofixation: results The M-protein in this case is free kappa light chains Immunofixation: results In rare cases, more than one type of M-proteins might be detected. In the example shown here, two types of M-protein were detected (biclonal): IgG lambda and IgM kappa.