Unit 5- Immunoassays(1).docx

UNIT 5 Immunochemical Techniques What are they? Also known as immunoassays Involve principles of antibody-antigen interactions The specificity and high affinity allows for the identification and quantification of specific substances by a variety of methods Terms Affinity- refers to the strength of...

UNIT 5 Immunochemical Techniques What are they? Also known as immunoassays Involve principles of antibody-antigen interactions The specificity and high affinity allows for the identification and quantification of specific substances by a variety of methods Terms Affinity- refers to the strength of the bond between the antigen and the antibody Avidity- describes the overall stability of an antibody-antigen complex. This is based on affinity of the antibody for the antigen, the number of binding sites available, and the way the two molecules combine. Igg and igd only has two binding sites where igm has 10 therefore igm has stringer avidity Specificity- a property of T cell and B cell receptors that results in their ability to recognize one, and only one, epitope Property of t or b cells, they are able to recognize different things they see the antigen and they call on the antibodies to come around Lattice Formation One antigen molecule crosslinks more than one antibody, resulting in the formation of a lattice structure When the lattice reaches a certain size, it loses its solubility and precipitates Maximum precipitation occurs when the relative concentration of the antigen and antibody allows each antibody and each antigen molecule to participate in the lattice Precipitates out agglutination when we have enough of the lattice formation Precipitation curve Graphic representation of precipitation reactions that occur when the concentration of one reactant is constant for every test sample, while the concentration of the second reagent is increased serially in the test samples Zone of antibody excess (prozone) At all stages in the zone of the antibody excess, free antibody is present. Antigen/antibody interactions occur, and immune complexes are formed, but there is insufficient antigen to form large immune complexes, which requires extensive crosslinking As more antigen is added precipitation is observed Zone of equivalence As the antigen concentration is increased serially in the various test samples, more precipitation is observed until all the antigen molecules and all antibody molecules interact and form a lattice There are not free antigen and no free antibody molecules present in solution Zone of antigen excess (postzone) When an increasing amount of antigen is added beyond the zone of equivalence, there is a gradual decrease in the amount of precipitation that occurs until finally no precipitation is observed In this zone, there is free antigen present in the solution Cross linking ends up disintegrating free floating antibodies and agglutination dissipates Immunochemical Methods Heterogeneous vs. Homogeneous Immunoassays Immunoassays can be categorized according to whether or not it is necessary to separate the bound reactants from the free ones Heterogeneous- require a step to physically separate free from bound analyte. Homogenous- do not need a separation step. The activity of the label attached to the antigen is diminished when binding of antibody and antigen occurs Competitive Immunoassay Unlabeled analyte in the test sample is measured by its ability to compete with labelled antigen for a limited number of antibody binding sites Unlabeled antigen blocks the ability of the labelled antigen to bind because that binding site is already occupied Therefore, less label measured in the assay means more unlabeled antigen is present The amount of antigen in the test sample is inversely related to the amount of label measured High response at detector equals low concentration in sample Low response at detector equals high concentration in sample Non-competitive Immunoassay Also called sandwich immunoassays because the analyte is bound (sandwiched) between two highly specific antibody reactions Reaction mixture typically includes an excess of labelled antibody so that all analyte is bound. The amount of antibody-antigen complex is then measured to determine the amount of analyte present in sample The measurement of labeled analyte, usually an antibody, is directly proportional to the amount of antigen present in the sample. Antibody is attached to support the antigen will bind then labelled antibodies will attach to antigen and these labels will emit a colour or a product produced, soething we can measure therefor signal is proportionate to concentration Particle Methods Radial Immunodiffusion (RID) Is a passive diffusion method in which a concentration gradient is established for a single reactant, usually the antigen. The antibody is dispersed uniformly in the gel matrix Antigen is allowed to diffuse from a well into the gel until antibody excess exists and immune precipitation occurs A well-defined ring around the well indicates the presence of antigen Label Methods Radioimmunoassay (RIAs) Use radioactive isotopes of iodine and tritium as labels Competition between radiolabeled and unlabeled antigen or antibody in a reaction is used to determine the concentration of the unlabeled antigen or antibody Requires that the antigen be available in a pure form and be labeled with a radioactive isotope Once popular, use of RIAs has declined over concerns of safe handling and disposal of radioactive reagents and waste Immunoradiometric assay (IRMA) Alternative assay design to RIAs Uses labelled antibody and does not require purified antigen because the antigen need not be labeled Antibodies are more stable proteins and are easier to label without damage to the proteins function Basicslly sandwhich with a radio labelled antobody Enzyme-linked Immunosorbent assay (ELISA)- antibodies are put on a surface and add antigen and we let it combine then we add our labelled antidy, after its sitting we add a substrate the labelled antibody has an enzyme and when the subtstrate combines it produces a signal Antibodies are absorbed to the surface of a solid phase, usually a microtiter plate well. Samples containing the antigen are added to the wells and incubated After incubation, the wells are washed, leaving the Ag-Ab complexes in the well Enzymes are added, wells are washed again, and a substrate is added Absorbance of the wells is measured Sample absorbance is directly proportional to the analyte concentration Enzyme-Multiplied Immunoassay Technique (EMIT) In this technique, the antibody against the analyte (drug, hormone, or metabolite) is added together with substrate to the patient’s sample. Binding of the antibody and analyte occurs An enzyme with the attached analyte is then added and will bind with any excess antibody which will alter the enzyme activity Change is enzymatic activity is proportional to the analyte concentration in the patient’s sample Chemiluminescent microparticle immunoassay (CMIA) This technique uses a chemiluminescent compound to detect analytes These labels produce light when combined with a trigger reagent This is a non-competitive sandwich assay, so the amount of analyte present in the sample is directly proportional to the intensity of the measured signal Fluorescence Polarization Immunoassay (FPIA) know for exam A type of fluoroimmunoassay Labeled, antibody-bound antigen in the sample competes with labeled, free antigen for antibody binding sites. This method provides accurate and sensitive measurement of therapeutic drugs, DOA, toxic substances, and hormones. Fluorescin is the label of choice; when the molecule is activated, it produces fluorescent light at a wavelength of 520 nm. The Ab-Ag-fluorescein molecules are large and rotate slowly in solution when excited by polarized light, whereas the smaller Ag-fluorescein molecules in solution rotate rapidly. When the solution is exposed to polarized light, the smaller Ag-fluorescein molecules emit light in a different plane (520 nm) from that which was absorbed (490 nm). When the larger Ab-Ag-fluorescein molecule is exposed to polarized light, it emits light in the same plane as the absorbed light energy. There is an inverse relationship between the signal and the concentration of analyte in the sample. High reaction at detector low concentration patient sample Low detection at detector indicate high concentration in patient sample Sources of Error High-Dose Hook Effect Usually seen when sandwich immunoassays are performed Very high antigen concentrations in the patient sample bind to all available antibody binding sites, in both the antibody solid phase and the antibody-labeled conjugate Prevents formation of the sandwich complex and cause falsely decreased test results To fix form a dilution on patient sample Human anti-mouse antibody (HAMA) A problem for sandwich immunoassays Caused by circulating human antibodies that react with animal immunoglobulins, particularly HAMAs Can cause positive or negative interference in two-site antibody based sandwich assays False positive- by bridging between a mouse immunoglobulin antibody and mouse conjugate and thus mimicking the specific analyte False negative- thought to be caused by HAMA reacting with one of the assay reagents and preventing formation of the sandwich