Everything You Wanted To Know About Immunoassays

Wikipedia defines an immunoassay as “…a biochemical test that measures the

concentration of a substance in a biological liquid, typically serum or urine, using the
reaction of an antibody or antibodies to its antigen.” Depending on how and for what
purpose it is used, the definition of an immunoassay can vary. The terms associated with
immunoassays stay relatively consistent though.

Below is a list of terms commonly used in association with immunoassays:

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.

Dynamic Range: 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 well, 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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