Scientific Terms Associated With Immunoassays

By: Ian Mackie

Antibody: An immunoglobulin that recognizes some portion of an antigen molecule. The antibody contains a species-specific Fc region and antigen-specific Fab portion.

Analyte: The molecule being quantified or analyzed.

Antigen: A molecule that is specifically bound by a given antibody.

Bo: The binding maximum in competitive immunoassays. The Bo typically contains antibody, enzyme-linked conjugate and buffer. The conjugate and antigen in the standard or sample normally compete for the available antibody binding sites but in the absence of antigen, only conjugate molecules are bound resulting in the maximum detection for the assay conditions used.

Chemiluminescent Detection: An assay detection method based on the measurement of light given off by a molecule. In a chemiluminescent EIA, the enzyme converts the substrate resulting in the emission of light that is detectable at a specific wavelength. The light is measured in Relative Light Units (RLU) and is proportional to the amount of substrate converted. Assuming that all of the enzyme molecules have the same activity, the RLU values become a relative measure of how many conjugate molecules were bound.

Colorimetric Detection: An assay detection method based on the optical density evaluation of a colored sample. In a colorimetric EIA, the enzyme converts the substrate resulting in a colored product whose optical density is proportional to the amount converted. Assuming that all of the enzyme molecules have the same activity, the optical density becomes a relative measure of how many conjugate molecules were bound.

Conditioned Media: Tissue Culture Media that has been exposed to cells. The media would include all added supplements in addition to the molecules secreted by the cells themselves. Conditioned media can be used as a sample matrix. (See Non-Conditioned media)

Conjugate: Two molecules that are covalently linked, one being detectable by some method. (eg: enzyme activity)

Cross Reactivity: Although antibodies are antigen-specific, they can frequently bind other related molecules. The cross reactivity measurement quantifies how efficiently the antibody can bind other molecules.

CV: Coefficient of Variation. The CV is a statistical expression of precision based on the standard deviation and average of multiple measurements. %CV = (Std. Dev/Mean) * 100.

Diluent: Buffer or liquid medium used to dilute a standard or sample.

Drift: The difference in signal seen from one side of the plate to the other. This is often due to a delay in the addition of reagents. The left side of the plate (where reagents were added first) will have longer for the binding reaction to occur than the right side of the plate.

This can generally be avoided by adding reagents in a timely manner without interruption.

DynamicRange: The continuous span of high to low analyte concentrations that can be reliably detected in the assay.

ED50: The concentration of the analyte when the %B/Bo is at 50%. It is commonly used as a reference of sensitivity in competitive immunoassays.

Edge Effect: The difference in signal between the exterior and interior wells of a microtiter plate. This is usually due to uneven incubation temperature or incomplete plate sealing at non-ambient temperatures.

EIA: Enzyme Immunoassay. A quantitative analysis involving an antigen bound by an antibody. The detection step is driven by an enzyme that is covalently linked to some portion of the antigen/antibody complex acting upon a substrate to create either a colorimetric, chemiluminescent or fluorescent signal.

Extraction Efficiency: Calculation used to determine the true analyte concentration when extraction protocols are necessary for measurement. The amount of analyte recovered is divided by the amount of analyte in the pre-extraction sample. This number is usually expressed as a percentage.

Fluorimetric Detection: An assay detection method based on the measurement of emitted light given off by an excited molecule. In a Fluorescent EIA, the enzyme converts the substrate resulting in the production of fluorescent molecules that emit light at one wavelength when excited by the presence of light at a different wavelength. The light is measured in Relative Fluorescent Units (RFU) and is proportional to the amount of substrate converted. Assuming that all of the enzyme molecules have the same activity, the RFU values become a relative measure of how many conjugate molecules were bound.

IgG: A specific immunoglobulin class that binds an antigen. IgG is the major immunoglobulin of the blood, lymph, cerebrospinal and peritoneal fluids.

Interference: A condition that prevents the completion of an unrestricted competitive binding reaction and its subsequent detection. Interference can be caused by the presence of certain antibodies (species interference), an overwhelming amount of sample constituents (matrix effect) or inappropriate chemicals in a sample.

Linearity: The ability to consistently detect the same amount of antigen through multiple serial dilutions. When the sample cannot be detected linearly, interference is usually involved.

Matrix: The environment in which something is found. This is a multifunctional term that can be used to refer to the solid matrix that is used during the assay (eg: tubes or microtiter wells) or the source of the sample (eg serum, plasma, saliva, urine, media, etc).

Matrix Effect: A type of interference caused by a constituent of the sample itself. This usually relates to the pH, osmolarity or composition of the sample. If the sample characteristics exceed the limitations tolerated by the assay, a matrix effect will result and sample detection becomes non-linear.

Monoclonal Antibody: A type of antibody derived from hybridoma cells. These antibodies are of higher purity and specificity than polyclonals.

Neat Sample: Undiluted or unaltered sample.

Non-conditioned Media: Tissue Culture Media that contains all supplements, but has not been exposed to cells. Non-conditioned media should be used for the standard diluent when samples are conditioned media.

NSB: Non-specific binding. Wells run as NSBs typically contain only buffer and the enzyme-linked conjugate. Because no intermediate molecules are present to specifically retain the conjugate in the wellFree Articles, any enzyme activity that is detected after the washing procedure is there due to non-specific binding.

Polyclonal Antibody: An antibody that is produced by more than one type of cell.

Precision: A statistical evaluation of the ability to detect the same value over multiple measurements. Intra-assay Precision looks at the statistical repeatability within a single assay while Inter-Assay Precision looks at the statistical repeatability over a number of different assay runs.

Sample Recovery: A statistical expression of the ability to measure antigen that has been added to the sample. Sample recovery experiments are conducted to determine the presence and extent of a matrix effect for a typical type of sample. The dilution factor required for an approximate 100% recovery of the added antigen is the recommended minimum dilution to avoid a matrix effect.

Serial dilution: A set of successive dilutions where the prior dilution step serves as the sample source for the next dilution step. This type of format is used to dilute non-discrete standards to make up the standard curve. Samples are also serially diluted when examining linearity or when large final dilutions are required.

Sensitivity: The smallest increment that can be reliably detected. This is the calculated value based on optical density statistical data from the Bo and lowest concentration standard.

Spiked Sample: A sample into which a known concentration of analyte has been added.

TA: Total Activity: The measurement of the maximum enzymatic activity expected for the assay. Total Activity wells typically contain a specific amount of the detection enzyme molecule (conjugate) and substrate.

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