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Glossary of Terms

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  • Antibody
    Antibody
    

    An antibody is a protein synthesized by the immune system of an animal in response to the presence of a foreign substance, called an antigen or immunogen. An antibody is a large protein called immunoglobulin and consists of heavy and light chains in a Y-shaped structure. Within the body, antibodies are modified to provide the optimal binding fit for a region on the antigen known as the epitope. Once bound, the foreign antigens are targeted for removal by the body.

    Two types of antibodies are used in immunodiagnostics, polyclonal and monoclonal antibodies. Both types of antibodies are engineered by creating an immunogen of the target to be measured which is then injected into host animals. For monoclonal production, the immunogen is injected into a mouse and after incubation the host’s spleen cells are removed and fused with a mouse myeloma cell line to form hybridoma cells. The myeloma cell line is a cancer tumor cell line that can make a large number of cells of the same kind and therefore, when fused with the mouse spleen cells, the same antibody clone. The hybridoma that produces the antibody with the optimal characteristics for specificity is chosen and then amplified to produce significant levels of that antibody. In this case only one “clone” is chosen for amplification hence the term monoclonal. For polyclonal production, the immunogen can be injected into different host animals, but the most popular are rabbit or goat. After incubation, the host animal’s blood is drawn and the antibody of choice can be purified from the serum. Here, many different cells have contributed to antibody production and numerous antibody clones are produced, hence the term polyclonal.

  • Antigen
    Antigen
    

    An antigen is a substance, such as a small molecule, protein, or virus, that causes an antigenic response within the body to which an antibody is produced. The antigen is the “target” for the antibody to bind. An antigen is also called an immunogen when it is used for engineered antibody production.

  • AP (Alkaline Phosphatase)
    AP (Alkaline Phosphatase)
    

    AP (Alkaline Phosphatase) is a phosphatase enzyme that catalyzed the removal of a phosphate group from the substrate. The enzymatic activity can be measured using a chromogenic substrate such as PNPS. The color produced by the reaction is directly proportional to concentration. This is a common enzyme used for the development of enzyme linked immunosorbent assays (ELISA).

  • Array
    Array
    

    Array refers to the pattern of a biomolecule (typically DNA or antibody) spotted on a surface, usually glass or plastic. Arrays are often used to enable the measurement of multiple species in one sample.

  • Assay diluent
    Assay diluent
    

    An assay diluent is a buffer formulation used dilute biological reagents and samples in an immunoassay format. It is important to dilute both standards and samples in the same assay diluent to ensure accurate quantitation. Often assay diluent formulations are designed to reduce interfering matrix effects while still maintaining assay integrity.

  • Assay sensitivity
    Assay sensitivity
    

    Assay sensitivity (not the same as Clinical Sensitivity) is the change in response (e.g., absorbance) per unit of concentration of the analyte, i.e. the more sensitive an assay the more of a response is observed to slight changes in concentration.

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  • Background
    Background
    

    Background is the level of signal in an assay that is not due to the specific or desired measurement.

  • BCIP
    BCIP
    

    A colorimetric substrate (5-Bromo-4-chloro-3-indolyl phosphate) used in immunoassays, that forms a dark blue or purple product when allowed to react with enzymes such as alkaline phosphatase.

  • Blocking
    Blocking
    

    Blocking is the prevention of unwanted interactions. Often blocking is used for the prevention of non-specific binding to a surface. However, the term can also be used to describe unwanted interactions between proteins in solution.

  • Blotting
    Blotting
    

    Blotting is the method of transferring biomolecules onto a membrane. Typical examples are western (protein), northern (RNA) or southern (DNA) blots.

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  • Chemiluminescent substrate
    Chemiluminescent substrate
    

    A chemiluminescent substrate is a chemical reagent that enables an immuno-enzymatic reaction to produce a measurable, light emitting end-product.

  • Clinical sensitivity
    Clinical sensitivity
    

    Clinical sensitivity is the proportion of actual positives which are correctly identified as such, e.g. identifying a patient correctly with a certain disease or condition.

  • Clinical specificity
    Clinical specificity
    

    Clinical specificity is the proportion of negatives which are correctly identified, e.g. identifying a patient that does not have a certain disease or condition.

  • Colorimetric substrate
    Colorimetric substrate
    

    A colorimetric substrate is a chemical reagent (chromogen) that enables an immuno-enzymatic reaction to produce a measurable, colored end-product.

  • Conjugate
    Conjugate
    

    A conjugate is a protein to which another molecule is attached or conjugated, hence the term conjugate. This term usually refers to an antibody to which a detection enzyme such as HRP or AP is conjugated. Conjugation is usually a covalent attachment through the lysine molecules of the antibody or enzyme, although other types of conjugation can be used. It is also possible to conjugate small molecules to detection enzymes. These are often used in competitive ELISA.

  • Cross-reactivity
    Cross-reactivity
    

    Cross-reactivity is antibody binding to antigens that were not the specific target but may be similar in structure. For example, an antibody engineered against a small molecule steroid could react at some level with other classes of steroids that have similar structures. Another example would be an antibody that is targeted for immunoglobulin from goat but also reacts with immunoglobulin from bovine. Here the similarity is because goat and bovine are close on the phylogenetic tree which means there can often be structural similarities between the two immunoglobulin proteins even though they are from different species.

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  • Detection limit
    Detection limit
    

    Detection limit is the lowest concentration of analyte measured by the assay. It is often determined by 2 SD (standard deviations) over the background.

  • Diagnostic assay
    Diagnostic assay
    

    Diagnostic assay examples include:  Immunoblotting / Western Blotting, ELISA, EIA, ELISPOT, Immunohistochemistry, Radioimmunoassays, Rapid/Lateral Flow Assays, Microarrays, Microfluidics, Multiplexing Instruments, Flow Cytometry.

  • Dilution
    Dilution
    

    Dilution means reducing the concentration of a species usually done by addition of a liquid such as water or a buffer.

  • Dynamic range
    Dynamic range
    

    Dynamic range is the range over which there is a linear relationship between the concentration of analyte and the observed measurement of the assay. An example would be concentration versus absorbance in an ELISA.

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  • ELISA (Enzyme-Linked ImmunoSorbant Assay)
    ELISA (Enzyme-Linked ImmunoSorbant Assay)
    

    ELISA (Enzyme-Linked ImmunoSorbant Assay) is a type of immunoassay that utilizes antigen/antibody interactions (Immunohistochemistry) to measure the presence or concentration of an antigen or analyte. The detection signal is generated by an enzyme that is covalently linked or conjugated to an antibody or antigen which specifically binds the target. The activity of the enzyme is typically measured via a colorimetric reaction with substrate where signal is proportional to concentration. Typical enzymes used are HRP and AP. Detection signal can also be generated by chemiluminescent substrates. ELISA has been the “gold standard” for many years in clinical immunodiagnostics. Several different ELISA formats are commonly used for immunodiagnostics such as sandwhich, direct, indirect, sandwich and competitive.

  • Enzyme
    Enzyme
    

    A classification of proteins that catalyze or increase the rates of chemical reactions.  Enzymes work by lowering the amount of energy needed for a reaction to occur, thus dramatically increasing the rate of the reaction. As a result, products are formed faster and reactions reach their equilibrium state more rapidly.

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  • Heterophilic interference
    Heterophilic interference
    

    Heterophilic interference refers to antibodies in biological samples that have affinity for the species of antibody used for detection of the desired antigen or analyte. The most common example is human anti-mouse antibodies or HAMA that occur in samples where a monoclonal sandwich assay is used for antigen detection. Patients that have developed antibodies to mouse proteins will have cross-reactivity to the mouse monoclonal antibodies used for detection in the sandwich ELISA. These antibodies interfere with assay measurements by causing a false positive result.

  • HRP (Horseradish Peroxidase)
    HRP (Horseradish Peroxidase)
    

    HRP (Horseradish Peroxidase) is a peroxidase enzyme that catalyzes the reduction of hydrogen peroxide to water. This enzymatic activity can be measured using chromogenic substrates such as TMB, DAB, or ABTS. The color produced by the reaction is directly proportional to concentration. This is a common enzyme used for the development of enzyme linked immunosorbent assays (ELISA).

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  • Immunoassay
    Immunoassay
    

    An immunoassay is an assay or test that utilizes antigen/antibody interactions (Immunohistochemistry) to measure the presence or concentration of an antigen. The most common type of assay is the ELISA.

  • Immunoblotting
    Immunoblotting
    

    A protein blotting technique, that is used to determine the molecular weight and relative amounts of a protein present in biological samples. Briefly, proteins are separated by gel electrophoresis, usually SDS-PAGE. The proteins are then transferred to a sheet of blotting paper (nitrocellulose, PVDF or other membranes). The blot is blocked (incubated with formulated blocking reagents) to bind any remaining sticky places on the membrane. The location of the antibody is then revealed using antibodies specific to the target protein, which are then coupled to colorimetric or chemiluminescent substrates for detection and visualization.

  • Immunohistochemistry
    Immunohistochemistry
    

    Immunohistochemistry is the interaction between an antibody and an antigen that is coupled to a detection system so that amounts can be observed and/or quantified. The term was traditionally used when referring to tissue section preparation and denoted as IHC.

  • In situ hybridization (ISH)
    In situ hybridization (ISH)
    

    In microarray applications, ISH uses a labeled complementary nucleic acid (RNA or DNA) strand or protein to capture a specific protein or nucleic acid from a given biological sample, so that the specific analyte of interest may be measured.

  • Interference
    Interference
    

    In immunoassays, interference refers to the unintended specific interactions that add to the variability in determining the accuracy of the assay, or measure of the true negative or positive.  False results have significant impact on the quality of the diagnostic assay, as well as on the lives of those individuals who have been falsely diagnosed.

  • IVD (In Vitro Diagnostics)
    IVD (In Vitro Diagnostics)
    

    IVD (In Vitro Diagnostics) tests are devices that are used in assays performed in a controlled environment outside a living organism to diagnose diseases.

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  • Matrix effect
    Matrix effect
    

    Matrix effect is the combined effect of all components of the sample, other than the analyte, on the measurement of the specific analyte. If a specific component can be identified as causing an effect then this is referred to as interference.

  • Membrane
    Membrane
    

    A synthetic, porous sheet-like material which is used for separation purposes, often of proteins, in the laboratory. Membranes are commonly used in western blotting and lateral flow and are typically made of nitrocellulose, PVDF or nylon materials.

  • Microwell
    Microwell
    

    Microwell is a term usually associated with ELISA applications as it denotes the small wells in a microtiter plate on which ELISA’s are typically performed. Standard ELISA microtiter plates contain 96 microwells.

  • Molecular diagnostics
    Molecular diagnostics
    

    Molecular diagnostics is the measurement of DNA or RNA (nucleic acids) to detect disease (e.g., cancer).

  • Multiplexing
    Multiplexing
    

    Multiplexing is the ability to measure multiple analytes within a single assay measurement. This can be done on different assay formats such as microarrays and Luminex®.

    Luminex is a registered trademark of Luminex Corporation

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  • Non-specific binding
    Non-specific binding
    

    Non-specific binding is any binding that occurs that is not the specific or intended antigen/antibody interaction. Non-specific binding can be an issue in many assay formats, especially those utilizing complex biological samples.

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  • Passivating surface chemistry
    Passivating surface chemistry
    

    Passivating surface chemistry is a coating on a surface that prevents penetration of impure compounds to undesired locations.

  • pNPP
    pNPP
    

    pNPP is a colorimetric substrate (para-Nitrophenylphosphate) used in immunoassays, that forms a yellow product when allowed to react with enzymes such as alkaline phosphatase.

  • Protein stabilizers
    Protein stabilizers
    

    Protein stabilizers are reagent formulations that preserve the conformation of proteins in their native folded state when dried down or in solution so that biological activity is maintained.

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  • Rheumatoid factor
    Rheumatoid factor
    

    Proteins produced by a body’s immune system that can attack healthy tissue, and are most often associated with autoimmune diseases.  Rheumatoid factor in a patient’s sample can be a source of interference in immunoassays, resulting in false positive results.

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  • Sample matrix interference
    Sample matrix interference
    

    Sample matrix interference refers to samples, usually biological samples, are complex in nature and containing varying levels of components that negatively impact diagnostic assays. These complexities can be affected by many factors such as: high levels of non-specific proteins, interfering enzymes, high salt levels, or non-optimal pH. These factors can influence the quantitation of the “true value” of the analyte measured by the diagnostic test. Often the matrices of samples are quite different from one another and therefore this variation contributes to the difficulty in measurement. Commonly, samples are diluted significantly to reduce these effects.

  • Signal
    Signal
    

    Signal is the measurable aspect of an immunoassay.

  • Substrate
    Substrate
    

    In biochemistry, a substrate is a molecule upon which an enzyme acts. Enzymes catalyze chemical reactions involving the substrate(s). In the case of a single substrate, the substrate binds with the enzyme active site, and an enzyme-substrate complex is formed. The substrate is transformed into one or more products, which are then released from the active site. The active site is now free to accept another substrate molecule.

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  • Test performance
    Test performance
    

    Protein concentration sensitivity:  Either the minimal detectable concentration or the slope of the dose–response standard curve.

  • TMB
    TMB
    

    TMB is a colorimetric substrate (3,3’,5,5’-Tetramethylbenzidine) used in immunoassays, that forms a blue product when allowed to react with peroxidase enzymes such as horseradish peroxidase.

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  • Western Blot
    Western Blot
    

    Western Blot is a protein immunoblot technique, that is used to determine the molecular weight and relative amounts of a protein present in biological samples. Briefly, proteins are separated by gel electrophoresis, usually SDS-PAGE. The proteins are then transferred to a sheet of blotting paper (nitrocellulose, PVDF or other membranes). The blot is blocked (incubated with formulated blocking reagents) to bind any remaining sticky places on the membrane. The location of the antibody is then revealed using antibodies specific to the target protein, which are then coupled to colorimetric or chemiluminescent substrates for detection and visualization.