Principles of Immunoassays Part 1 PDF

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Summary

- Immunoassay, an analytical test that uses antibodies or antigens as the reagent to measure small amounts of specific analytes. - Most commonly an antibody is used as a reagent and an antigen is the analyte. IgG is the most common reagent antibody. - These assays measure the formation of Ab-Ag comp...

Description

MLSC-3111, Clinical Biochemistry II -

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Immunoassay, an analytical test that uses antibodies or antigens as the reagent to measure small amounts of specific analytes. Most commonly an antibody is used as a reagent and an antigen is the analyte. IgG is the most common reagent antibody. These assays measure the formation of Ab-Ag complexes. Immunoassays are highly sensitive (amplifies measured product) and specific (uses unique binding characteristics of AB and Ag). Antibodies are made of two light chains and heavy chains. The heavy chain (constant region) determines the immunoglobulin isotype (IgA, IgD, IgM, etc.). The variable regions allow for binding specificity. The hypervariable regions causes increased variation through the variable region and is responsible for antibody combining site specificity.

Immunoassay (IA) Principles and Definitions -

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Antigen, high molecular weight foreign substance that induces an immune response. In immunoassay anything that binds to an Ab, regardless of immune response, is the analyte of interest. Hapten, a low molecular weight substance that induces an immune response when coupled to a high molecular weight carrier molecule (incomplete antigen). These are measured using IA to detect drugs and non-peptide hormones. Binding Forces, several forces act cooperatively to produce Ag-Ab reactions. o Complementarity of Fit, occurs in the hypervariable region to cause electrostatic attraction and is based on the conformation of Ab and Ag. o Lock and Key Concept, Ab combining site constructed from the hypervariable regions of the heavy and light chains. The Ab-Ag bond is non-covalent and reversible. Affinity, the strength of any given bond between a single antigenic determinant and a single combing site on the antibody. Reflects closeness of fit at a sing binding site. This is a property of antigens. Avidity, overall strength of Ab-Ag binding and includes the sum of the binding affinities of all combining sites on the Ab. Also known as functional affinity and is a property of antibodies. Specificity, the degree to which Ab bonds to its corresponding Ag while not reacting with other similar substances. Cross Reactivity, the ability of an Ab to bond with additional substances that are similar to its specific antigen. Causes a lowered specificity and is caused by antigens with similar structure or shared epitopes. The Ab-Ag complex allows for quantitation of the analyte when the IA measures complexes only. Ligand, the Ag in the IA test. Binding Agent, the Ab in the IA test. Law of Mass Action, in a given time, free reactants will achieve equilibrium with bound reactants in proportion to concentration. Ab-Ags take time to form in IAs.

MLSC-3111, Clinical Biochemistry II -

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Binding Factors, include… o Ion Species, Cationic Salts inhibit binding of cationic haptens. Anionic Salts inhibit binding of anionic haptens. o Polymers, Addition of polymers increases the rate of immune complex formation and enhances precipitation of immune complex. o Non-Specific Binding (NSB), the binding of Ags substances other than the intended assay Ab. Ag can bind to other “non-assay” Abs. Leads to inaccuracy in final Ag measurement. NSB is suspected when results do not make sense and are not linear upon dilution (NSB error acts in an unpredictable pattern). Ab increases can be polyclonal or monoclonal. A diffuse increase in Ab is a result of a mixture of increased Ab and a discrete increase is due to an increase of a specific antibody.

Polyclonal Abs used as Reagents -

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Production involves specific human Ag being injected into animals. The animals will then produce antibodies against the human antigen. A diverse range of Abs are produced by the animal to the differing antigenic sites on the Ag. The animals are given two doses spaced apart since the second sensitization will elicit a much stronger immune response. Polyclonal reagents are prone to cross reactivity since they can react with multiple substances, causing unwanted recognition of other antigens. These Abs are produced in relatively small amounts compared to the world market. The animals also die as time goes on meaning that they must be replaced.

Monoclonal Abs used as Reagents -

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A single B lymphocyte is used to generate antibodies to one specific epitope. Complex process that creates monoclonal antibodies for testing. Hybrid cells are created by fusing normal cells with tumour cells to cause the continual cloning of the target lymphocyte. Hybridoma, fused cells that possess immortal characteristics of myeloma cells plus the Ab production capabilities of the original lymphocyte. A culture of Ab secreting cells from a single spleen cell produces reagent volumes of monoclonal antibodies. Recombinant Monoclonal Abs, Monoclonal Abs generated in vitro using synthetic genes via cloning. These can produce reagent quantities of monoclonal Abs. Advantages, the hybridoma cell line is potentially immortal and can produce the same antibody consistently and indefinitely without replacement. Provides reproducible reagent of known specificity and affinity Disadvantages, the antibodies can be hyper-specific and may not recognize slightly different variations of its specific antigen. The changes are usually not physiologically different, but the antibody becomes too specific and will not detect those variants (seen in LH hormones variants). Results in falsely low/negative results.

MLSC-3111, Clinical Biochemistry II

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o HAMA, human anti-mouse antibodies can interfere with the monoclonal Ab since the hybridoma came from a mouse. o Heterophile Abs, other human anti-“animal” Abs may also interfere if the cell comes from a different animal. Dilution of tests with inconsistent results is a sign of HAMA or heterophile interference. The physician can also be called after the dilution to confirm based on patient exposure history. Preincubation of the sample with non-immune globulin blocking agents can help omit these interferences.

Other Types of Receptors in IAs -

Transport Proteins, acts as binding agents like TBG which binds thyroxine. However, the body also makes TBG, causing a decreased specificity. Cellular Receptors, membrane bound receptors can be used to bind analyte. Directed toward biologically actives sites like thyroid cell membranes and can be used to measure TSH.

Calibration of Immunoassays -

Dose-Response Curve, same as preparation of a standard curve in non-IAs. A series of standards/calibrators are assayed with known analyte concentrations. The curve is often not linear and requires data manipulation.

Immune Precipitation Techniques -

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Precipitation, soluble antigen binds to soluble Ab and forms network large enough to settle out/become insoluble (precipitate) on its own. Ag is placed into a gel containing Ab. As Ag diffuses into the gel it reacts with the Ab and the complex then forms precipitates. The Lattice Theory, the reaction of Ags possessing multiple antigenic sites to bivalent Ab sites produces a lattice of interlocking molecules. As the size and complexity of the lattice increases, the lattice becomes insoluble and precipitates out of solution. Precipitation Curve, the plot of the % of antibody precipitated vs. increasing antigen concentration. o Prozone, the phase of the curve at which the antibody is in excess. Results in no cross-linking being formed because there is not enough AB in solution. o Equivalence Zone, contains no free Ab or Ag and is the concentration at which both Ab and Ag can react at their fullest. o Postzone, the phase of the curve at which the antigen is in excess. As antigen concentration increases, precipitation decreases. High Dose Hook (HDH) Effect, phenomenon of IAs that occurs in the postzone. The very high concentration of Ag causes all Abs to be saturated. Results in the lattice formation

MLSC-3111, Clinical Biochemistry II

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not being formed to decrease precipitation proportionally to the antigen concentration. Causes falsely low/negative results. Sample should be diluted if the antigen concentration is too high and causing HDH effect. Passive Gel Diffusion, Ag and Ab solutions are allowed to diffuse toward each other through a semi-solid gel. Precipitation occurs at the line where they meet in solution. A positive result is seen as a line of precipitation where the antibody and antigen concentrations are the most ideal. Double Immunodiffusion (DID), wells cut into agar and solutions of antigens and antibodies placed into the gels. The wells contain control Ags and test Ags, with a central well containing the Ab. If there is a corresponding Ag is the sample it will perform a line of precipitation between its Ag well and the Ab well. Most commonly used to detect Auto-Abs in patient samples. Radial Immunodiffusion, a quantitative variation of DID. Ab is added into the agar gel and evenly distributed throughout the gel plate and sample is added to wells. Presence of Ag in samples produces precipitation ring around sample well at zone of equivalence. Therefore, detects presence of Ag in patient serum. Quantitation is done through standards and standard curves. Advantages o Easy to perform o Can be very sensitive under appropriate conditions o Reliable for detection of Abs or Ags Disadvantages, o Time consuming and can require 24-72h incubation time to allow all of the Ag/Ab to diffuse completely into the gel. Nephelometry and Turbidimetry assays can be used as IA’s that measure immune complex formation. Ag + Ab produce large complex seen as particles in solution. Rate assays occur in the prozone phase. Endpoint assays occur in the zone of equivalence....