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Immunochemical methods in biochemistry Jana Novotna Characteristics of antigens • Antigen - macromolecular substance of natural or synthetic origin, that the immune system recognizes as foreign. The presence in the organism - stimulation of antibody production (to induce an immune response). • Immunogenicity – property of substance (immunogens or antigens) to induce a detectable immune response. • Antigenicity – given by a surface structure of immunogen antigenic determinants. The organism responds only to those that are foreign to it. Characteristics of antigens • Antigenic specificity – property of antigen molecule (or its part) to react with the specific antibody. • The number of antigenic determinants of the antigen depends on its size and chemical complexity of macromolecule – egg ovalbumin, MW 42 000 - 5 antigenic determinants – thyroglobulin, MW 700 000 - many than 40 antigenic determinants • Hapten – small molecule that can elicit immune response only when attached to a large carrier such as a protein. • In general, only large molecules, infectious agents, or insoluble foreign matter can elicit an immune response in the body. Characteristics of antigens • Chemical nature of antigens: – – – – – – – – – proteins polysacchrides lipopolysaccharides nucleoproteins glycoproteins steroid hormones bacterial cells, viruses synthetic polypeptides synthetic polymers Characteristics of antigens • The antibody recognizes an epitope - defined segment of the antigen on the macromolecular carrier (about 5-8 amino acids) • Immunoreactivity of epitope depends on – primary, secondary and tertiary stricture – variability of epitope Antibodies (immunoglobulins) and their composition • Immunoglobulins produce plasma cells, which differentiate from B cells. • Proteins with specific binding activity to the antigen (antigenic determinant), formed after it´s stimulation. • Immunoglobulins account for ~ 20% of the total plasma proteins. • The chemical nature glycoproteins • The antibody binding site occupies 5% of the antibody molecule, distinguishes very fine differences in the antigenic determinants. • The structure of the binding site is exactly complementary to the antigenic determinant (lock and key). • The variety of specific antibodies in the body is more than 108. Characteristics of antibodies (immunoglobulins) • Variability of antibodies is subject to 5-classes of Ig: G, A, M, D, E • Heavy chains – g, a, m, d, e • Light chains – k, l • Subclasses of immunoglobulins: – IgG – g1, g2, g3, g4 – IgA – a1, a2 – IgM - m1, m2 Characteristics of antibodies (immunoglobulins) Flexibility and motion of imunoglibulins The forces binding antigen to antibody • Electrostatic : between attraction oppositely charged ionic group – (NH3-) of lysine and (-COO-) of aspartate. • Hydrogen bonding – relatively weak and reversible hydrogen bridges between hydrophilic group (OH, -NH2, COOH). • Hydrophobic– non-polar, hydrophobic side chains of Val, Leu, Ile (hydrophobic groups come close together and exclude water molecules between them. The force of attraction increases. • Van der Waals – forces which depend upon interaction between the external „electron clouds“. Nonspecific attractive forces. Polyclonal antibodies • Host animals ca be used to raise antibodies against a given antigen • Each epitope in the antigen molecule stimulates the production of antibodies to a single clone of B lymphocytes (complete antigens having multiple epitopes, some clones of activated B-cells). • Polyclonal antibody consists of mixture of monoclonal antibodies having specificity for complex antigens. • They are conventional produced by immunization of animals with antigen. Monoclonal antibody • Products of single clone of plasma cells by B lymphocytes; • Mostly prepared in laboratory. They are directed against single epitope – identical copies with same structure and antigen specificity. • They have excellent specificity but poor ability to precipitate antigen. Antibody affinity antibody + antigen k2 immunokomplex [AbAg] k1 [AbAg] Kekv = [Ab] [Ag] = k1 k2 equilibrium constant Affinity of antibody the equilibrium constant that describes the antigenantibody reaction. Affinity is the strength of the reaction between a single antigenic determinant and a single combining site on the antibody (the sum of the attractive and repulsive forces operating between the antigenic determinant and the combining site of the antibody). Antibody avidity • Avidity is a measure of the overall strength of binding of an antigen with many antigenic determinants and multivalent antibodies. • Avidity refers to the overall strength of binding between multivalent antigens and antibodies. Specificity and cross reactivity of antibody • Specificity of antibody - the ability of an individual antibody combining site to react with only one antigenic determinant or the ability of a population of antibody molecules to react with only one antigen. • Antibodies can distinguish differences in: – the primary structure of an antigen – isomeric forms of an antigen – secondary and tertiary structure of an antigen • Cross reactivity - the ability of an individual antibody combining site to react with more than one antigenic determinant or the ability of a population of antibody molecules to react with more than one antigen. Imunoprecipitation reaction • Used for qualitative and quantitative detection of antigens and antibodies: – phase 1– formation of primary complexes with low MW – phase 2 – interconnection of Ag and Ab to the three dimensional network (formation of insoluble aggregates ) Quantitative (Heidelbergova) immunoprecipitation curve Immunochemical tests for detection of antigen-antibody reaction • Use of reaction antibody - antigen in vitro is the basis of immunochemical methods. • Factors affecting reaction: – the affinity of antibody, – the ratio of Ab/Ag – the physical form of the antigen (particulates or antigen in solubilized form) Agglutination tests • Agglutination (from Latin, agglutino – to glue/ attach) - specific antibody reacts with the corpuscular antigen. • Agglutination reaction is based on the formation of bridges between bivalent (IgG) or multivalent (IgM) antibodies and antigenic particles with multiple epitopes. • Among all other antibodies, IgM is a good agglutinin, since it has high affinity to different antigens. • Hemagglutination is a variant of agglutination technique in which red blood cells are used as the antigen bearing particles. Agglutination tests • Agglutination reactions are performed on slides, in test tubes or microtiter plates. • They are more sensitive in comparison with immunoprecipitation methods. • The agglutination methods produce qualitative or semiquantitative results. • Agglutination may be either: – Qualitative test – to identify the presence of an antigen or an antibody. – Quantitative test – to measure the level of antibodies to particulate antigens; • Serial dilutions of sample is used. • Agglutination assays may be classified as direct or indirect tests. Microtitration plates - variety of materials (polystyrene, polypropylene, polycarbonate) Direct agglutination • The antigen is an integral part of the cell surface (red blood cells, bacteria). • A suspension of particles is directly agglutinated by specific antibodies present in the examined sample. • This assay is frequently used in the hematology for the – determination of blood group – in the immunological diagnostics for detection of specific antibodies directed against naturally occurring antigens on the surface of some microbes (for example against Salmonella typhi – the Widal test). Indirect agglutination • • Indirect agglutination assay utilizes particles with the antigens that have been passively attached to their surface. Particles already coated with antigens (antibodies) to determine the antibody (antigen) in given sample. Eg: – – Pregnancy test – using hormone secretion (human chorionic gonadotropin), diagnosis of rheumatoid arthritis (rheumatoid factors are anti-immunoglobulin antibodies directed against Fc-fragment of IgG usually class IgM) Immunoassays Highly sensitive techniques High sensitivity is achieve by labeling of one reacting component (antibody or antigen) with a substance which detection is more sensitive 1) radioisotope – radio immunoassay (RIA) 2) enzyme – enzyme immunoassay (EIA) 3) fluorescent dye (e.g. fluorescein) • Detection limits of can be as low as 10−15 – 10−20 mol/l • Enzyme immunoassays utilize enzymes, usually peroxidase or alkaline phosphatase, to detect and quantify immunochemical reactions. Heterogeneous enzyme immunoassay • Enzyme-linked immunosorbent assay (ELISA) • One of the immunochemical reactants (antigen or antibody) is first non-specifically adsorbed to the surface of a solid phase (tubes, wells of microtiter plates or magnetic particles) Competitive enzyme immunoassay • Always performed under condition of antigen excess • A limited quantity of specific antibodies bound to the solid phase. • The enzyme-labeled antigen (conjugate) is mixed with serum sample containing the unknown amount of antigen. • The serum antigen and enzyme-labeled antigen compete for binding sites of antibody. • Labeled and non-labeled antigen bind to the antibody in the same proportion the more non-labeled antigen is contained in the mixture the less labeled antigen is bound Competitive enzyme immunoassay The probability of the antibody binding the labeled antigen is inversely proportional to the concentration of unlabelled antigen. Non-competitive enzyme immunoassay (sandwich methods) • Heterogeneous immunoassay - Used for the determination of antigens or antibodies. Antibody (or antigen) is bound to the surface of solid phase (plastic microwell surface) which must always be in excess over the analyte being measured. • Suitable for the determination of large antigens having multiple binding sites for the antibody (e.g. human chorionic gonadotropin, α1-fetoprotein, total IgE). 1. Non-competitive enzyme immunoassay for determination of antigen. 2. Non-competitive enzyme immunoassay for determination of antibody Antigen determination Two different molecules of antibodies directed against various epitopes are necessary. The first antibody is in excess adsorbed to a solid phase. The serum sample or calibrators containing the desired antigen are added to the well with immobilized antibody incubation all the non-reacting material is washed away a second enzyme-labeled antibody (different from the first antibody) is added in excess (another antigen epitope binds to the second labeled antibody). “Sandwich complex“ consisting of solidphase antibody – antigen – enzyme labeled antibody complex is formed. After washing of all the unreacted enzyme-labeled antibody, the substrate is added. The intensity of the finally measured colored product of the enzyme reaction is directly proportional to the amount of antigen. Antibody determination Especially useful for detection of specific serum antibodies against viruses and parasites, antibodies and specific IgE antibodies to individual allergens. The antigen must be first immobilized on the solid phase. After adding test samples or calibrators the antibodies in the sample react with the immobilized antigen and forms immunocomplexes. The next steps of the assay procedure are similar to that described for the measurement of antigens. Immunochemical methods in fast diagnostics • Fast and tentative “point-of-care” or “bed-side” tests based on “dry chemistry” • Reagents are anchored in a porous substrate that is attached on a plastic pad or inserted in a frame with windows for sample application and result reading. • Examples of application: – estimation of human chorionic gonadotropin (hCG) for diagnostics of pregnancy, – troponine T for acute heart infarction, – tests for narcotics Detection of narcotics in urine or saliva • • • • • • Test is based on competition of a drug in the sample with the same drug immobilized in the detection area of the test for a limited amount of specific antibody. The sample is mixed with antibodies in the reaction zone. The antibodies are labeled with stained micro-particles. Both the sample and antibodies move by capillary action through the reaction zone to the detection area (contains the immobilized drug). If the sample contains molecules of the drug it fully saturates the binding sites of color-labeled antibodies. The antibody molecules cannot then react with the immobilized drug and color of the detection area stays unchanged. Detection of human chorionic gonadotropin, hCG (pregnancy test) • • • • • Three different antibodies are used. The first sample area contains specific anti-hCG antibody labeled with micro particles of colloid gold or blue latex (Ab1). Urine sample is applied, molecules of Ab1 flow to the second, detection area where another specific antibody against hCG (Ab2) is anchored. If hCG is contained in the sample a combined immunocomplex is formed with both labeled and anchored antibody (Ab2hCG-Ab1), a colored band displays in the detection area. Excess of the labeled antibody is caught in the third (control), with immobilized antibodies against labeled anti-hCG two bands are interpreted as a positive result. Western blotting • is a widely used analytical technique used to detect specific proteins in a sample of tissue homogenate or extract. • It uses gel electrophoresis to separate native or denatured proteins. • The proteins are then transferred to a membrane (typically nitrocellulose), where they are stained with antibodies specific to the target protein. Electrophoretic profile of separated collagen fraction isolated from peripheral pulmonary arteries of rats exposed to hypoxia and its Western blot with antibodies to collagen type I References • http://www.nios.ac.in/media/documents/dmlt/Biochemistry/Lesson24.pdf • https://www.google.cz/search?q=imunoglobulin+struktura&biw=1280 &bih=862&source=lnms&sa=X&ei=igfzVOD4K4HsUM7Hgng&ved=0 CAYQ_AUoAA&dpr=1#q=immunochemical+techniques • http://elte.prompt.hu/sites/default/files/tananyagok/practical_biochem istry/ch07s05.html • http://www.microbiologybook.org/mayer/ab-ag-rx.htm • http://ulbld.lf1.cuni.cz/file/1610/immunochemical-methods.pdf