| ink"> | | | | protocols are necessary for measurement. The |
| | | | amount of analyte recovered is divided by the |
| The exact definition of immunoassay can vary | | | | amount of analyte in the pre-extraction sample. |
| based on where and in what environment it is | | | | This number is usually expressed as a percentage. |
| being used. For example, Wikipedia defines it | | | | Fluorimetric Detection: An assay detection method |
| as
a biochemical test that measures the | | | | based on the measurement of emitted light given |
| concentration of a substance in a biological liquid, | | | | off by an excited molecule. In a Fluorescent EIA, |
| typically serum or urine, using the reaction of an | | | | the enzyme converts the substrate resulting in |
| antibody or antibodies to its antigen. A | | | | the production of fluorescent molecules that emit |
| dentist may define it differently from a veterinary | | | | light at one wavelength when excited by the |
| though. The terms typically associated with | | | | presence of light at a different wavelength. The |
| immunoassays stay relatively consistent though, | | | | light is measured in Relative Fluorescent Units |
| as seen in the glossary below. | | | | (RFU) and is proportional to the amount of |
| Antibody: An immunoglobulin that recognizes some | | | | substrate converted. Assuming that all of the |
| portion of an antigen molecule. The antibody | | | | enzyme molecules have the same activity, the |
| contains a species-specific Fc region and | | | | RFU values become a relative measure of how |
| antigen-specific Fab portion. | | | | many conjugate molecules were bound. |
| Analyte: The molecule being quantified or analyzed. | | | | IgG: A specific immunoglobulin class that binds an |
| Antigen: A molecule that is specifically bound by a | | | | antigen. IgG is the major immunoglobulin of the |
| given antibody. | | | | blood, lymph, cerebrospinal and peritoneal fluids. |
| Bo: The binding maximum in competitive | | | | Interference: A condition that prevents the |
| immunoassays. The Bo typically contains antibody, | | | | completion of an unrestricted competitive binding |
| enzyme-linked conjugate and buffer. The | | | | reaction and its subsequent detection. |
| conjugate and antigen in the standard or sample | | | | Interference can be caused by the presence of |
| normally compete for the available antibody | | | | certain antibodies (species interference), an |
| binding sites but in the absence of antigen, only | | | | overwhelming amount of sample constituents |
| conjugate molecules are bound resulting in the | | | | (matrix effect) or inappropriate chemicals in a |
| maximum detection for the assay conditions used. | | | | sample. |
| Chemiluminescent Detection: An assay detection | | | | Linearity: The ability to consistently detect the |
| method based on the measurement of light given | | | | same amount of antigen through multiple serial |
| off by a molecule. In a chemiluminescent EIA, the | | | | dilutions. When the sample cannot be detected |
| enzyme converts the substrate resulting in the | | | | linearly, interference is usually involved. |
| emission of light that is detectable at a specific | | | | Matrix: The environment in which something is |
| wavelength. The light is measured in Relative Light | | | | found. This is a multifunctional term that can be |
| Units (RLU) and is proportional to the amount of | | | | used to refer to the solid matrix that is used |
| substrate converted. Assuming that all of the | | | | during the assay (eg: tubes or microtiter wells) or |
| enzyme molecules have the same activity, the | | | | the source of the sample (eg serum, plasma, |
| RLU values become a relative measure of how | | | | saliva, urine, media, etc). |
| many conjugate molecules were bound. | | | | Matrix Effect: A type of interference caused by a |
| Colorimetric Detection: An assay detection | | | | constituent of the sample itself. This usually |
| method based on the optical density evaluation of | | | | relates to the pH, osmolarity or composition of |
| a colored sample. In a colorimetric EIA, the | | | | the sample. If the sample characteristics exceed |
| enzyme converts the substrate resulting in a | | | | the limitations tolerated by the assay, a matrix |
| colored product whose optical density is | | | | effect will result and sample detection becomes |
| proportional to the amount converted. Assuming | | | | non-linear. |
| that all of the enzyme molecules have the same | | | | Monoclonal Antibody: A type of antibody derived |
| activity, the optical density becomes a relative | | | | from hybridoma cells. These antibodies are of |
| measure of how many conjugate molecules were | | | | higher purity and specificity than polyclonals. |
| bound. | | | | Neat Sample: Undiluted or unaltered sample. |
| Conditioned Media: Tissue Culture Media that has | | | | Non-conditioned Media: Tissue Culture Media that |
| been exposed to cells. The media would include all | | | | contains all supplements, but has not been |
| added supplements in addition to the molecules | | | | exposed to cells. Non-conditioned media should be |
| secreted by the cells themselves. Conditioned | | | | used for the standard diluent when samples are |
| media can be used as a sample matrix. (See | | | | conditioned media. |
| Non-Conditioned media) | | | | NSB: Non-specific binding. Wells run as NSBs |
| Conjugate: Two molecules that are covalently | | | | typically contain only buffer and the enzyme-linked |
| linked, one being detectable by some method. (eg: | | | | conjugate. Because no intermediate molecules are |
| enzyme activity) | | | | present to specifically retain the conjugate in the |
| Cross Reactivity: Although antibodies are | | | | well, any enzyme activity that is detected after |
| antigen-specific, they can frequently bind other | | | | the washing procedure is there due to non-specific |
| related molecules. The cross reactivity | | | | binding. |
| measurement quantifies how efficiently the | | | | Polyclonal Antibody: An antibody that is produced |
| antibody can bind other molecules. | | | | by more than one type of cell. |
| CV: Coefficient of Variation. The CV is a statistical | | | | Precision: A statistical evaluation of the ability to |
| expression of precision based on the standard | | | | detect the same value over multiple |
| deviation and average of multiple measurements. | | | | measurements. Intra-assay Precision looks at the |
| %CV = (Std. Dev/Mean) * 100. | | | | statistical repeatability within a single assay while |
| Diluent: Buffer or liquid medium used to dilute a | | | | Inter-Assay Precision looks at the statistical |
| standard or sample. | | | | repeatability over a number of different assay |
| Drift: The difference in signal seen from one side | | | | runs. |
| of the plate to the other. This is often due to a | | | | Sample Recovery: A statistical expression of the |
| delay in the addition of reagents. The left side of | | | | ability to measure antigen that has been added to |
| the plate (where reagents were added first) will | | | | the sample. Sample recovery experiments are |
| have longer for the binding reaction to occur than | | | | conducted to determine the presence and extent |
| the right side of the plate. This can generally be | | | | of a matrix effect for a typical type of sample. |
| avoided by adding reagents in a timely manner | | | | The dilution factor required for an approximate |
| without interruption. | | | | 100% recovery of the added antigen is the |
| DynamicRange: The continuous span of high to | | | | recommended minimum dilution to avoid a matrix |
| low analyte concentrations that can be reliably | | | | effect. |
| detected in the assay. | | | | Serial dilution: A set of successive dilutions where |
| ED50: The concentration of the analyte when the | | | | the prior dilution step serves as the sample |
| %B/Bo is at 50%. It is commonly used as a | | | | source for the next dilution step. This type of |
| reference of sensitivity in competitive | | | | format is used to dilute non-discrete standards to |
| immunoassays. | | | | make up the standard curve. Samples are also |
| Edge Effect: The difference in signal between the | | | | serially diluted when examining linearity or when |
| exterior and interior wells of a microtiter plate. | | | | large final dilutions are required. |
| This is usually due to uneven incubation | | | | Sensitivity: The smallest increment that can be |
| temperature or incomplete plate sealing at | | | | reliably detected. This is the calculated value based |
| non-ambient temperatures. | | | | on optical density statistical data from the Bo and |
| EIA: Enzyme Immunoassay. A quantitative | | | | lowest concentration standard. |
| analysis involving an antigen bound by an antibody. | | | | Spiked Sample: A sample into which a known |
| The detection step is driven by an enzyme that is | | | | concentration of analyte has been added. |
| covalently linked to some portion of the antigen | | | | TA: Total Activity: The measurement of the |
| antibody complex acting upon a substrate to | | | | maximum enzymatic activity expected for the |
| create either a colorimetric, chemiluminescent or | | | | assay. Total Activity wells typically contain a |
| fluorescent signal. | | | | specific amount of the detection enzyme molecule |
| Extraction Efficiency: Calculation used to determine | | | | (conjugate) and substrate. |
| the true analyte concentration when extraction | | | | |