Assay

This article is about assays associated with biological applications. For assays of various metals, seemetallurgical assay.For the French commune in the Indre-et-Loire department, seeAssay, Indre-et-Loire.

Anassayis an investigative (analytic) procedure inlaboratory medicine,mining,pharmacology,environmental biologyandmolecular biologyfor qualitatively assessing or quantitatively measuring the presence, amount, or functional activity of a target entity. The measured entity is often called theanalyte,themeasurand,or thetargetof the assay. The analyte can be adrug,biochemical substance,chemical elementorcompound,orcellin anorganismor organicsample.An assay usually aims to measure an analyte'sintensive propertyand express it in the relevantmeasurement unit(e.g.molarity,density,functional activity in enzyme international units, degree of effect in comparison to a standard, etc.).

If the assay involvesexogenousreactants (thereagents), then their quantities are kept fixed (or in excess) so that the quantity and quality of the target are the only limiting factors. The difference in the assay outcome is used todeducethe unknown quality or quantity of the target in question. Some assays (e.g., biochemical assays) may be similar tochemical analysisandtitration.However, assays typically involvebiological materialor phenomena that are intrinsically more complex in composition or behavior, or both. Thus, reading of an assay may benoisyand involve greater difficulties in interpretation than an accurate chemical titration. On the other hand, older generation qualitative assays, especiallybioassays,may be much more gross and less quantitative (e.g., counting death or dysfunction of an organism or cells in a population, or some descriptive change in some body part of a group of animals).

Assays have become a routine part of modernmedical,environmental,pharmaceutical,andforensic technology.Other businesses may also employ them at theindustrial,curbside, or field levels. Assays in high commercial demand have been well investigated inresearch and developmentsectors of professional industries. They have also undergone generations of development and sophistication. In some cases, they are protected by intellectual property regulations such aspatentsgranted for inventions. Such industrial-scale assays are often performed in well-equippedlaboratoriesand with automated organization of the procedure, from ordering an assay to pre-analytic sample processing (sample collection, necessary manipulations e.g.spinning for separation,aliquotingif necessary, storage, retrieval,pipetting,aspiration,etc.). Analytes are generally tested in high-throughputautoanalyzers,and the results are verified and automatically returned to ordering service providers andend-users.These are made possible through the use of an advancedlaboratory informatics systemthatinterfaceswith multiplecomputer terminalswith end-users, centralservers,the physical autoanalyzer instruments, and other automata.

Etymology

According to Etymology Online,the verbassaymeans "to try, endeavor, strive, test the quality of"; from Anglo-Frenchassaier,fromassai(noun), from Old Frenchessai,"trial". Thus the nounassaymeans "trial, test of quality, test of character" (from mid-14th century), from Anglo-Frenchassai;and its meaning "analysis" is from the late 14th century.

Forassay of currency coinsthis 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",e.g., in pharmacology, analysis for an important component of a target inside a mixture—such as theactive ingredientof a drug inside the inertexcipientsin 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)andthe 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-stepinformationhandling andtransmissionsystem, the variation and errors in reporting final results entail not only those intrinsic to the assay itself but also those occurring in the pre-analytic and post-analytic procedures.

While the analytic steps of the assay itself get much attention,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.

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

1. Sample processing and manipulationin 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. inMass 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 aPCRassay a specific oligonucleotide primer identifies the target bybase pairingbased 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 aPCRassay among a mixture of DNA sequences only the specific target is amplified into millions of copies by aDNA polymeraseenzyme 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 filteringmay 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 narrowband-passoptical filter, or a blocking reagent in a binding reaction that prevents nonspecific binding or aquenchingreagent in a fluorescence detection system that prevents "autofluorescence" of background objects.

Assay types based on the nature of the assay process

Time and number of measurements taken

Depending on whether an assay just looks at a single time point or timed readings taken at multiple time points, an assay may be:

1. Anend point assay,in which a single measurement is performed after a fixed incubation period; or
2. Akinetic 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. Ahigh throughput assaycan 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.

Number of analytes detected

Depending on how many targets or analytes are being measured:

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

Result type

Depending on the quality of the result produced, assays may be classified into:

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 RBCagglutinationin 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 -Von Willebrand diseaseisVWFantigen 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 isRistocetinCofactor assay, which measures the functional activity of the VWF present in a patient's plasma by adding exogenousformalin-fixedplateletsand gradually increasing quantities of drug named ristocetin while measuring agglutination of the fixed platelets. A similar assay but used for a different purpose is calledRistocetin Induced Platelet Aggregationor 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 vivobody part (e.g. leg of a frog)
   3. ex vivoorgan (e.g. heart of a dog)
   4. ex vivopart of an organ (e.g. a segment of an intestine).
   5. tissue (e.g. limulus lysate)
   6. cell (e.g. platelets)
2. Ligand binding assaywhen a ligand (usually a small molecule) binds a receptor (usually a large protein).
3. Immunoassaywhen 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 orabsorbanceat a particular wavelength or light orElectrochemiluminescenceor electrical/redox activity.
2. Light detection systems that may use amplification e.g. by aphotodiodeor aphotomultiplier tubeor a cooledcharge-coupled device.
3. Radioisotopelabeled substrates as used inradioimmunoassaysand equilibrium dialysis assays and can be detected by the amplification inGamma countersorX-ray plates,orphosphorimager
4. Polymerase Chain ReactionAssays that amplify a DNA (or RNA) target rather than the signal
5. Combination MethodsAssays may utilize a combination of the above and other amplification methods to improve sensitivity. e.g.Enzyme-linked immunoassayor EIA,enzyme linked immunosorbent assay.

Detection method or technology

Depending on the nature of the Detection system assays can be based on:

1. Colony formingorvirtual colony count:e.g. by multiplying bacteria or proliferating cells.
2. Photometry/spectrophotometryWhen the absorbance of a specific wavelength of light while passing through a fixed path-length through a cuvette of liquid test sample is measured and the absorbance is compared with a blank and standards with graded amounts of the target compound. If the emitted light is of a specific visible wavelength it may be calledcolorimetry,or it may involve specific wavelength of light e.g. by use oflaserand emission offluorescentsignals of another specific wavelength which is detected via very specific wavelength optical filters.
3. Transmittanceof 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 plateletagglutinationreaction.
4. Turbidimetrywhen the opacity of straight-transmitted light passing through a liquid sample is measured by detectors placed straight across the light source.
5. Nephelometrywhere 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.
6. ReflectometryWhen 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. opticFlow cytometriccell or particle counters, orcoulter/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. involvingamperometry,Voltammetry,coulometrymay 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

Assay types based on the targets being measured

DNA

Assays for studyinginteractionsofproteinswithDNAinclude:

• DNase footprinting assay
• Filter binding assay
• Gel shift assay

Protein

• Bicinchoninic acid assay(BCA assay)
• Bradford protein assay
• Lowry protein assay
• Secretion assay

RNA

• Nuclear run-on
• Ribosome profiling

Cell counting, viability, proliferation or cytotoxicity assays

A cell-counting assay may determine the number of living cells, the number of dead cells, or the ratio of one cell type to another, such as enumerating and typingredversus different types ofwhiteblood cells. This is measured by different physical methods (light transmission, electric current change). But other methods use biochemical probing cell structure or physiology (stains). Another application is to monitorcell culture(assaysof cell proliferation orcytotoxicity). A cytotoxicity assay measures how toxic a chemical compound is to cells.

• MTT assay
• Cell Counting Kit-8 (WST-8 based cell viability assay)
• SRB (Sulforhodamine B) assay
• CellTiter-Glo® Luminescent Cell Viability Assay
• Cell counting instruments and methods:CASY cell counting technology,Coulter counter,Electric cell-substrate impedance sensing
• Cellviability assays:resazurin method,ATP test,Ethidium homodimer assay(detect dead or dying cells),Bacteriological water analysis,Clonogenic assays,...

Environmental or food contaminants

• Bisphenol F
• Aquatic toxicity tests

Surfactants

• AnMBAS assayindicatesanionicsurfactantsin water with a bluing reaction.

Other cell assays

Many cell assays have been developed to assess specific parameters or response of cells (biomarkers,cell physiology).Techniques used to study cellsinclude:

• reporter assays using i.e.Luciferase,calcium signaling assays usingCoelenterazine,CFSEorCalcein
• Immunostainingof cells on slides byMicroscopy(ImmunoHistoChemistry or Fluorescence), onmicroplatesbyphotometryincluding theELISpot(and its variantFluoroSpot) to enumerate B-Cells or antigen-specific cells, in solution byFlow cytometry
• Molecular biologytechniques such asDNA microarrays,in situ hybridization,combined toPCR,Computational genomics,andTransfection;Cell fractionationorImmunoprecipitation
• Migration assays,Chemotaxis assay
• Secretion assays
• Apoptosisassays such as theDNA ladderingassay, theNicoletti assay,caspaseactivity assays, andAnnexin Vstaining
• Chemosensitivity assaymeasures the number of tumor cells that are killed by a cancer drug
• Tetramer assaydetect the presence of antigen specific T-cells
• Gentamicin protection assayor survival assay or invasion assay to assess ability of pathogens (bacteria) to invade eukaryotic cells

Metastasis Assay

Petrochemistry

• Crude oil assay

Virology

TheHPCE-based viral titer assayuses a proprietary, high-performancecapillary electrophoresissystem to determinebaculovirustiter.

TheTrofile assayis used to determineHIV tropism.

The viralplaque assayis to calculate the number ofvirusespresent in a sample. In this technique the number ofviral plaquesformed by a viral inoculum is counted, from which the actual virus concentration can be determined.

Cellular secretions

A wide range of cellular secretions (say, a specificantibodyorcytokine) can be detected using theELISAtechnique. The number of cells which secrete those particular substances can be determined using a related technique, theELISPOTassay.

Drugs

• Testing for Illegal Drugs
• Radioligand binding assay

Quality

When multiple assays measure the same target their results and utility may or may not be comparable depending on the natures of the assay and their methodology, reliability etc. Such comparisons are possible through study of general quality attributes of the assays e.g. principles of measurement (including identification, amplification and detection), dynamic range of detection (usually the range of linearity of thestandard curve),analytic sensitivity,functional sensitivity,analytic specificity,positive,negative predictive values,turn around time i.e. time taken to finish a whole cycle from the preanalytic steps till the end of the last post analytic step (report dispatch/transmission),throughputi.e. number of assays done per unit time (usually expressed as per hour) etc. Organizations or laboratories that perform Assays for professional purposes e.g. medical diagnosis and prognostics, environmental analysis, forensic proceeding, pharmaceutical research and development must undergo well regulatedquality assuranceprocedures includingmethod validation,regularcalibration,analytical quality control,proficiency testing,testaccreditation,testlicensingand must document appropriate certifications from the relevant regulating bodies in order to establish the reliability of their assays, especially to remain legally acceptable and accountable for the quality of the assay results and also to convince customers to use their assay commercially/professionally.

List of BioAssay databases

Bioactivity databases

Bioactivity databases correlate structures or other chemical information to bioactivity results taken frombioassaysin literature, patents, and screening programs.

Name	Developer(s)	Initial release
ScrubChem	Jason Bret Harris	2016
PubChem-BioAssay	NIH	2004
ChEMBL	EMBL-EBI	2009

Protocol databases

Protocol databases correlate results frombioassaysto their metadata about experimental conditions and protocol designs.

Name	Developer(s)	Initial release
BioMetaDataorBioAssay Express	Collaborative Drug Discovery	2016
PubChem-BioAssay	NIH	2004

See also

• Analytical chemistry
• MELISA
• Multiplex (assay)
• Pharmaceutical chemistry
• Titration

External links

• Blair, Andrew Alexander (1911)."Assaying".InChisholm, Hugh(ed.).Encyclopædia Britannica.Vol. 2 (11th ed.). Cambridge University Press. pp. 776–778.This includes a detailed, technical explanation of contemporaneous metallic ore assay techniques.
• The dictionary definition ofassayat Wiktionary