Immunoassays-1 PDF

IMMUNOLOGY & SEROLOGY IMMUNOASSAY ONE: AN and autologous antigens INTRODUCTION Primary union of binding sites on antibody with...

IMMUNOLOGY & SEROLOGY IMMUNOASSAY ONE: AN and autologous antigens INTRODUCTION Primary union of binding sites on antibody with specific epitopes on an antigen depends on two characteristics of antibody:  Affinity  Avidity Such characteristics are important because they relate to the sensitivity and specificity of testing in the clinical laboratory Both affinity and avidity contribute to the stability of the antigen-antibody complexes, which is essential to detecting the presence of an unknown, whether it is antigen or antibody AFFINITY Affinity is the initial force of attraction that exists between a single Fab site on an antibody molecule and a single epitope or determinant site on the ANTIGEN- ANTIBODY BINDING corresponding antigen As epitope and binding site come into close Antibodies combine in vitro reversibly with proximity to each other, several types of antigenic determinants to form immune complexes noncovalent interactions hold them together: (antigen-antibody complexes)  Ionic bonds Detection of these reactions form the basis of serology  Hydrogen bonds Serology is defined as a subdivision of immunology  Hydrophobic bonds concerned with in vitro antigen-antibody (Ag-Ab) reactions  Van der Waals forces In general, these reactions make use of serum but These are all rather weak bonds that can occur only other body fluids can also be tested over a short distance of approximately 1x10–7 mm Tests in serology are also called immunologic Therefore, there must be a very close fit between assays or immunoassays antigen and antibody Immunoassays can be used for the detection of The strength of attraction then depends on the either antigen (Ag) or antibody (Ab) specificity of antibody for a particular antigen  For antigen detection, the corresponding One antibody molecule may initially attract specific antibody should be prepared as one numerous different antigens, but it is the epitope’s of the reagents shape and the way it fits together with the binding sites on an antibody molecule that determines  The reverse is true for antibody detection whether the bonding will be stable Immunoassays can be applied for the detection of: Antibodies are capable of reacting with antigens that are structurally similar to the original antigen  Haptens as small molecules that induced antibody production, a characteristic  Proteins and protein complexes as known as cross-reactivity macromolecules  Any antibody to allergens, infectious agents, Degones, C.  IMMUNOLOGY & SEROLOGY The more the cross-reacting antigen resembles the the antigen-antibody complexes, which is essential original antigen, the stronger the bond will be to detecting the presence of an unknown, whether between the antigen and the binding site it is antigen or antibody However, if the epitope and the binding site have a perfect lock-and-key relationship, as is the case with the original antigen, the affinity will be maximal, because there is a very close fit When the affinity is higher, the assay reaction is more sensitive because:  More antigen-antibody complexes will be formed Avidity is the sum of the forces binding multivalent  More antigen-antibody complexes will be antigens to multivalent antibodies. In a comparison visualized more easily between IgG and IgM, IgM has the most potential binding sites for antigen and thus the higher avidity. Note that the monomers in IgM can swing up or down in order to bind more effectively. LAW OF MASS ACTION All antigen-antibody binding is reversible and is governed by the law of mass action Affinity is determined by the three-dimensional fit The law states that free reactants are in equilibrium and molecular attractions between one antigenic with bound reactants determinant and one antibody-binding site. The The equilibrium constant represents the difference antigenic determinant on the left has a better fit in the rates of the forward and reverse reactions and charge distribution than the epitope on the according to this equation: right and hence will have a higher affinity. AVIDITY Avidity represents the sum of all the attractive forces between an antigen and an antibody It involves the strength with which a multivalent antibody binds a multivalent antigen, and it is a measure of the overall stability of an antigen- antibody complex Once binding has occurred, avidity is the force that Therefore, the equilibrium constant Keq is: keeps the molecules together A high avidity can actually compensate for a low affinity Different classes of antibodies actually differ in avidities The more bonds that form between antigen and antibody, the higher the avidity is IgM has a higher avidity than IgG because it has the potential to bind 10 different antigens Both affinity and avidity contribute to the stability of Degones, C.  IMMUNOLOGY & SEROLOGY This constant can be seen as a measure of the as labels goodness of fit  Fluorescent immunoassay (FIA) uses Keq depends on the strength of binding between fluorescent dyes or fluorophores as antibody and antigen markers As the strength of binding (or avidity) increases, the  Chemiluminescent immunoassay (CLIA) tendency of the antigen-antibody complexes to uses chemiluminescent or dissociate decreases, and the value of K2 electrochemiluminescent compounds as decreases – i.e., it increases the value of K1 markers The higher the value of Keq, the larger the amount CATEGORIES OF ANTIGEN-ANTIBODY of antigen-antibody complex and the more visible REACTION (SECONDARY REACTION) or easily detectable the reaction is Secondary reactions are consequences of antigen- Therefore, the ideal conditions in the clinical antibody interactions which involve building of laboratory would be to have: lattice to form visible precipitates or agglutinates  An antibody with a high affinity, or initial Secondary reactions take a longer time to occur, force of attraction electrolytes are required, but involve very little  A high avidity, or strength of binding change in free energy The higher the values are for both of these and the These reactions do not occur when monovalent more antigen-antibody complexes that are formed, antibodies (still bivalent but the other Fab arm the more sensitive the test will be cannot interact on another antigenic site of the same Ag) or haptens are involved CATEGORIES OF ANTIGEN-ANTIBODY Types of secondary reactions include: REACTION (PRIMARY REACTION)  Precipitation of soluble antigen is also known as initial interaction, or sensitization  Agglutination (“clumping”) of particulate It is the basic event and consists of the binding of antigen antigen with an antibody  Neutralization of bacteria, viruses, or toxins It occurs almost instantaneously and is rarely directly visible  Activation of complement enzymes Antigen and antibody can combine in the absence Secondary binding tests or secondary of electrolytes immunoassays are the techniques used to detect secondary reactions Major change in free energy occurs during antigen- antibody interaction These immunoassays are less sensitive than primary binding tests, since more antigens and Labeled immunoassays or primary binding tests antibodies are required for lattice formation are the techniques used to detect primary antigen- antibody interactions However, secondary immunoassays do not usually require sophisticated techniques Primary binding assays are the most sensitive tests in terms of the amount of antigen or antibody Examples include precipitation immunoassays, detectable particle agglutination immunoassays, neutralization tests, and complement fixation tests Visualization is usually accomplished by labelling antibodies or antigens with markers (or labels): CATEGORIES OF ANTIGEN-ANTIBODY  Radioimmunoassay (RIA) uses REACTION (TERTIARY REACTION) radioisotopes as labels Tertiary reactions are biologic expressions of  Enzyme immunoassay (EIA) uses enzymes antigen-antibody interaction which may either be Degones, C.  IMMUNOLOGY & SEROLOGY beneficial or harmful; e.g., pollens interact with IgE, Insulin, for example, was quantified by RIA, which which may lead to an asthma attack subsequently replaced the insulin bioassay These are consequences of immune response in RIA may be formatted in a solid-phase procedure vivo for easy separation of bound and free labels These reactions are measured by tertiary binding Since the development of RIA, the search for tests which are more complex than the other two alternative labels to hazardous radioisotopes has intensified, with the aim of developing nonisotopic Tertiary binding tests are performed to determine immunoassays using enzymes, fluorescent labels, the protective effects of the antibody and other reporter groups CLASSES OF IMMUNOASSAYS CLASSES OF IMMUNOASSAYS (ENZYME (PRECIPITATION IMMUNOASSAY) IMMUNOASSAY EIA) Precipitation immunoassay involves combining EIA uses enzymes as labels soluble antigen with soluble antibody to produce visible insoluble complexes This assay was developed in the early 1970s, and rapidly gained wide popularity These immunoassays provide the simplest method for antigens and antibodies to react with each other Enzymes can amplify signals, depending on the without involving the detection of any labels turnover of enzyme catalytic activity The resulting antigen-antibody complex in gel or Efforts to improve substrates and to increase liquid phase may be observed qualitatively as a sensitivity have led to the introduction of precipitant by the naked eye and quantitatively with chromophore, fluorophore, and later a detector chemiluminescent compounds CLASSES OF IMMUNOASSAYS (PARTICLE Depending on the substrate chosen, the assay AGGLUTINATION IMMUNOASSAY) method can be defined as a fluorescent enzyme immunoassay or as a chemiluminescent enzyme Agglutination is the process by which particulate immunoassay antigens (e.g. cells) aggregate to form larger CLASSES OF IMMUNOASSAYS complexes when a specific antibody is present (FLUORESCENT IMMUNOASSAY FIA) This technique uses inert particles as carriers, as opposed to direct precipitation of the Ag-Ab FIA uses fluorophores as labels complexes Fluorophores require optimal wavelength light Antigens or antibodies, attached to particles, such energy for their excitation to produce detectable as erythrocytes, latex, or metals, react with the emission light analyte in the specimen FIA sensitivity is likely to decrease because of the As a result of this immune reaction, large particles nonspecific background fluorescence present in show significant agglutination patterns that may be biological specimens seen by the naked eye Introduction of a new class of fluorescent CLASSES OF IMMUNOASSAYS compounds has resulted in improvements in FIA such as the elimination of background noise (RADIOIMMUNOASSAY RIA) Sophisticated instrumentation has been introduced Yalow and Berson in 1959 reported on the that can detect low concentrations (10-15 M) of development of RIA using radioisotopes as labels analytes using FIAs This breakthrough allowed for the quantitative detection of a trace level of analytes and contributed to the advancement of basic research and clinical medicine Degones, C.  IMMUNOLOGY & SEROLOGY CLASSES OF IMMUNOASSAYS (CHEMILUMINESCENT IMMUNOASSAY CLIA uses chemiluminescent compounds as labels Chemiluminescent compounds include chemically synthesized molecules as well as natural products such as aequorin Unlike fluorophores, most chemiluminescent compounds require chemical rather than light energy to generate emission light Reduction-oxidation (redox) reaction is a process common to all chemiluminescent assays Signal amplification is not expected of chemiluminescent labels because chemiluminescent molecules generate just one photon through molecular decomposition A series of new and innovative compounds for electrochemiluminescence have proven suitable for application on immunoassays Metal chelate with tribiphenyls emits light through a continuous reduction-oxidation reaction on the surface of electrodes Degones, C.