Assay

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An assay is an investigative (analytic) procedure in

According to Etymology Online,[3] the verb assay means "to try, endeavor, strive, test the quality of"; from Anglo-French assaier, from assai (noun), from Old French essai, "trial". Thus the noun assay means "trial, test of quality, test of character" (from mid-14th century), from Anglo-French assai; and its meaning "analysis" is from the late 14th century.

For assay of currency coins this literally meant analysis of the purity of the gold or silver (or whatever the precious component) that represented the true value of the coin. This might have translated later (possibly after the 14th century) into a broader usage of "analysis",^{[citation needed]} e.g., in pharmacology, analysis for an important component of a target inside a mixture—such as the active ingredient of a drug inside the inert excipients in a formulation that previously was measured only grossly by its observable action on an organism (e.g., a lethal dose or inhibitory dose).

General steps

An assay (analysis) is never an isolated process, as it must be accompanied with pre- and post-analytic procedures. Both the communication order (the request to perform an assay plus related information) and the handling of the specimen itself (the collecting, documenting, transporting, and processing done before beginning the assay) are pre-analytic steps. Similarly, after the assay is completed the results must be documented, verified and communicated—the post-analytic steps. As with any multi-step

While the analytic steps of the assay itself get much attention,^[4] it is those that get less attention of the chain of users—the pre-analytic and post-analytic procedures—that typically accumulate the most errors; e.g., pre-analytic steps in medical laboratory assays may contribute 32–75% of all lab errors.^[5]

Assays can be very diverse, but generally involve the following general steps:

1. Sample processing and manipulation in order to selectively present the target in a discernible or measurable form to a discrimination/identification/detection system. It might involve a simple centrifugal separation or washing or filtration or capture by some form of selective binding or it may even involve modifying the target e.g. epitope retrieval in immunological assays or cutting down the target into pieces e.g. in
   Mass Spectrometry
   . Generally there are multiple separate steps done before an assay and are called preanalytic processing. But some of the manipulations may be inseparable part of the assay itself and will not thus be considered pre-analytic.
2. Target-specific discrimination/identification principle: to discriminate from background (noise) of similar components and specifically identify a particular target component ("analyte") in a biological material by its specific attributes. (e.g. in a
   base pairing
   based on the specific nucleotide sequence unique to the target).
3. Signal (or target) amplification system: The presence and quantity of that analyte is converted into a detectable signal generally involving some method of signal amplification, so that it can be easily discriminated from noise and measured - e.g. in a PCR assay among a mixture of DNA sequences only the specific target is amplified into millions of copies by a DNA polymerase enzyme so that it can be discerned as a more prominent component compared to any other potential components. Sometimes the concentration of the analyte is too large and in that case the assay may involve sample dilution or some sort of signal diminution system which is a negative amplification.
4. Signal detection (and interpretation) system: A system of deciphering the amplified signal into an interpretable output that can be quantitative or qualitative. It can be visual or manual very crude methods or can be very sophisticated electronic digital or analog detectors.
5. Signal enhancement and noise filtering may be done at any or all of the steps above. Since the more downstream a step/process during an assay, the higher the chance of carrying over noise from the previous process and amplifying it, multiple steps in a sophisticated assay might involve various means of signal-specific sharpening/enhancement arrangements and noise reduction or filtering arrangements. These may simply be in the form of a narrow
   quenching reagent in a fluorescence detection system that prevents "autofluorescence" of background objects. ^{[citation needed}
   ]

1. An end point assay, in which a single measurement is performed after a fixed incubation period; or
2. A kinetic assay, in which measurements are performed multiple times over a fixed time interval. Kinetic assay results may be visualized numerically (for example, as a slope parameter representing the rate of signal change over time), or graphically (for example, as a plot of the signal measured at each time point). For kinetic assays, both the magnitude and shape of the measured response over time provide important information.
3. A high throughput assay can be either an endpoint or a kinetic assay usually done on an automated platform in 96-, 384- or 1536-well microplate formats (High Throughput Screening). Such assays are able to test large number of compounds or analytes or make functional biological readouts in response to a stimuli and/or compounds being tested.[6]

Number of analytes detected

Depending on how many targets or analytes are being measured:

1. Usual assays are simple or single target assays which is usually the default unless it is called multiplex.
2. Multiplex assays are used to simultaneously measure the presence, concentration, activity, or quality of multiple analytes in a single test. The advent of multiplexing enabled rapid, efficient sample testing in many fields, including immunology, cytochemistry, genetics/genomics, pharmacokinetics, and toxicology.^[7]

1. Qualitative assays, i.e. assays which generally give just a pass or fail, or positive or negative or some such sort of only small number of qualitative gradation rather than an exact quantity.
2. Semi-quantitative assays, i.e. assays that give the read-out in an approximate fashion rather than an exact number for the quantity of the substance. Generally they have a few more gradations than just two outcomes, positive or negative, e.g. scoring on a scale of 1+ to 4+ as used for blood grouping tests based on RBC agglutination in response to grouping reagents (antibody against blood group antigens).
3. Quantitative assays, i.e. assays that give accurate and exact numeric quantitative measure of the amount of a substance in a sample. An example of such an assay used in coagulation testing laboratories for the most common inherited bleeding disease -
   VWF
   antigen assay where the amount of VWF present in a blood sample is measured by an immunoassay.
4. Functional assays, i.e. an assay that tries to quantify functioning of an active substance rather than just its quantity. The functional counterpart of the VWF antigen assay is
   Ristocetin Induced Platelet Aggregation
   or RIPA, which tests response of endogenous live platelets from a patient in response to Ristocetin (exogenous) & VWF (usually endogenous).

Sample type and method

Depending on the general substrate on which the assay principle is applied:

1. Bioassay: when the response is biological activity of live objects. Examples include
   1. in vivo, whole organism (e.g. mouse or other subject injected with a drug)
   2. ex vivo body part (e.g. leg of a frog)
   3. ex vivo organ (e.g. heart of a dog)
   4. ex vivo part of an organ (e.g. a segment of an intestine).
   5. tissue (e.g. limulus lysate)
   6. cell (e.g. platelets)
2. Ligand binding assay when a ligand (usually a small molecule) binds a receptor (usually a large protein).
3. Immunoassay when the response is an antigen antibody binding type reaction.

Signal amplification

Depending on the nature of the signal amplification system assays may be of numerous types, to name a few:

1. Enzyme assay: Enzymes may be tested by their highly repeating activity on a large number of substrates when loss of a substrate or the making of a product may have a measurable attribute like color or absorbance at a particular wavelength or light or Electrochemiluminescence or electrical/redox activity.
2. Light detection systems that may use amplification e.g. by a
   charge coupled device
   .
3. Radioisotope labeled substrates as used in radioimmunoassays and equilibrium dialysis assays and can be detected by the amplification in Gamma counters or X-ray plates, or phosphorimager
4. Polymerase Chain Reaction
   Assays that amplify a DNA (or RNA) target rather than the signal
5. Combination Methods Assays may utilize a combination of the above and other amplification methods to improve sensitivity. e.g.
   enzyme linked immunosorbent assay
   .

1. Colony forming or virtual colony count: e.g. by multiplying bacteria or proliferating cells.
2. fluorescent
   signals of another specific wavelength which is detected via very specific wavelength optical filters.
3. Transmittance of light may be used to measure e.g. clearing of opacity of a liquid created by suspended particles due to decrease in number of clumps during a platelet agglutination reaction.
4. Turbidimetry when the opacity of straight-transmitted light passing through a liquid sample is measured by detectors placed straight across the light source.
5. Nephelometry where a measurement of the amount of light scattering that occurs when a beam of light is passed through the solution is used to determine size and/or concentration and/or size distribution of particles in the sample.^[8]
6. Reflectometry When color of light reflected from a (usually dry) sample or reactant is assessed e.g. the automated readings of the strip urine dipstick assays.
7. Viscoelastic measurements e.g. viscometry, elastography (e.g. thromboelastography)
8. Counting assays: e.g. optic
   coulter
   /impedance principle based cell counters
9. Imaging assays, that involve image analysis manually or by software:
   1. Cytometry: When the size statistics of cells is assessed by an image processor.
10. Electric detection e.g. involving amperometry, Voltammetry, coulometry may be used directly or indirectly for many types of quantitative measurements.
11. Other physical property based assays may use
    1. Osmometer
    2. Viscometer
    3. Ion Selective electrodes
    4. Syndromic testing