assay

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An assay is an investigative (analytic) procedure in laboratory medicine, pharmacology, environmental biology, and molecular biology for qualitatively assessing or quantitatively measuring the presence or amount or the functional activity of a target entity (the analyte) which can be a drug or biochemical substance or a cell in an organism or organic sample.^[1][2]

Generally in an assay the quantity of the exogenous materials (the reagents) are kept fixed (or in excess) so that the quantity (and quality) of the target is the only limiting factor for the reaction/assay process, and the difference in the assay outcome is used to deduce the unknown quality or quantity of the target in question. Some assays (e.g., biochemical assays) may be similar to or have overlap with chemical analysis and titration. But generally assays involve biological material or phenomena which tend to be intrinsically more complex either in composition or in behavior or both. Thus reading of an assay may be quite noisy and may involve greater difficulties in interpretation than an accurate chemical titration. On the other hand older generation qualitative assays especially bioassays 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).

An assay (analysis) is never an isolated process and needs to be preceded by certain necessary procedures which are the preanalytic steps and must be followed by certain necessary post analytic steps. The information communication (eg request to perform an assay and further information processing) or specimen handling (eg collection transport and processing) done before and till the point of beginning of an assay are the preanalytic steps. Similarly after the actual assay is done the result may be documented, verified and transmitted/communicated in steps which are called post-analytic steps related to an assay. Like any multistep information handling and transmission systems, variation and errors in the communicated final results of an assay involves corresponding parts in every such step ie not only analytic variations and errors intrinsic to the assay itself but also variations and errors involved in preanalytic and post analytic steps. Since the assay itself (the analytic step) gets a lot of attention^[3], steps that get less attention by the chain of users ie the preanalytic and the post analytic steps are often less stringently regulated and generally more prone to errors- eg preanalytic steps in medical laboratory assays may contribute to 32-75% of all lab errors^[4].

In modern practice Assays have become routine part of medical, environmental, pharmaceutical, forensic and many other business at various scales from industrial to curbside or field level. Those assays that have very high commercial demand have been well investigated in research and development sectors of professional industries and have gone through various generations of development of better and sophisticated assays that have become copyrighted intellectual properties through highly competitive process patenting. These kind of industrial scale assays are often done in well equipped laboratories and use high level of automation in organizing the procedure, right from ordering of an assay, to pre-analytic sample processing (sample collection, necessary manipulations eg spinning for separation or other processes, aliquoting if necessary, storage, retrieval, pipetting/aspiration etc) and are generally tested in high thruput AutoAnalyzers and the results are verified, and transmitted automatically back to the ordering service providers and the end users. These are made possible through use of advanced Laboratory informatics system that interfaces with multiple computer terminals with end users as well as central servers and the physical autoanalyser instruments and other automata.

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 eg principles of measurement (including identification, amplification and detection), dynamic range of detection (usually the range of linearity of the standard curve), analytic sensitivity, functional sensitivity, analytic specificity, positive, negative predictive values, turn around time ie time taken to finish a whole cycle from the preanalytic steps till the end of the last post analytic step (report dispatch/transmission), thruput ie number of assays done per unit time (usually expressed as per hour) etc. Organizations or laboratories that perform Assays for professional purposes eg medical diagnosis and prognostics, environmental analysis, forensic proceeding, pharmaceutical research and development must undergo well regulated quality assurance procedures including method validation, regular callibration, Analytical quality control, Proficiency testing, test accreditation, test licensing and 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.

Etymology

According to Etymology Online^[5] the verb assay, at least since the 13th century meant "to try, endeavor, strive; test the quality of," from Anglo-Fr. assaier, from assai (n.), from O.Fr. essai "trial". And the noun assay thus means "trial, test of quality, test of character," mid-14c., from Anglo-Fr. assai and Meaning "analysis" is from late 14c. For assay of currency coins this litterally meant analysis of the purity of the gold or silver or whatever precious component used to represent the true value of the coin. This might have translated later (possibly after 14th century) into a generalized meaning of analysis eg of important/principle component of a target inside a mixture such as active ingradient of a drug inside the inert excepients in a pharmacological formulation which originally used to be measured by its actual action on an organism (eg lethal dose or inhibitory dose).

General steps of any assay

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

1. Sample processing/manipulation in order to selectively present that target in a discernible/measurable form to a discrimination/identification/detection system. It might involve a simple centrifugal separation or washing or flitration or capture by some form of selective binding or it may even involve modifying the target eg epitope retrieval in immunological assays or cutting down the target into pieces eg 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. (eg in a PCR assay a specific oligonucleotide primer identifies the target by 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 - eg 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 a interpretable output that can be quantitative or qualitative. It can be visual or manual very crude methods or can be vary sophisticaed electronic digital or analog detectors.
5. Signal enhancement and noise filtering: may be done at any/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 band-pass optical filer, or a blocking reagent in a binding reaction that prevents nonspecific binding or a quenching reagent in a fluorescence detection system that prevents "autofluorescence" of background objects.

Assay types based on the nature of the assay process

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. end point assay: when the only reading that matters is the end result after a fixed assay incubation period.
2. kinetic assay: when readings are taken multiple times at fixed time intervals during an assay and a kinetic graph of the readings is important.

Depending on how many targets or analytes are being measured:

1. Usual assay are simple or single target assays which is usually the default unless it is called multiplex.
2. Multiplex assays are assays that in a same reaction detect multiple analytes simultaneously.

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

1. Qualitative assay, 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 gives 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 gives 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 commonest inherited bleeding disease - Von Willebrand disease is VWF antigen assay where the amount of VWF present in a blood sample is measured by an immunoassay.
4. Functional assay, i.e. an assay that tries to quantitative functioning of an active substance rather than just its quantity. The functional counterpart of the VWF antigen assay is Ristocetin Cofactor assay, which measures the functional activity of the VWF present in a patients plasma by adding exogenous formalin-fixed platelets and gradually increasing quantities of drug named ristocetin while measuring agglutination of the fixed platelets. A similar assay but used for a different purpose is called Ristocetin Induced Platelet Aggregation or RIPA, which tests response of endogenous live platelets from a patient in response to Ristocetin (exogenous) & VWF (usually endogenous).

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

1. Bioassay: when the response is biological activity of live objects e.g.
   1. Organism (e.g. mouse 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.

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

1. Enzyme activity 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 absorbence at a particular wavelength or light or chemiluminiscence or electrical/redox activity.
2. Light detection systems that may use amplification e.g. by a photodiode or a photomultiplier tube or a cooled charge coupled device.
3. Radioisotope labeled substrates as used in radioimmunoassays and equilibrium dialysis assays and can be detected by the amplification in GM counters or [X-ray plate]]s, or phosphorimager
4. Polymerase Chain Reaction Assays that amplifies a DNA (or RNA) target itself rather than the signal

Assays may be a combination of the above eg Enzyme Immuno assay or EIA, enzyme linked immunosorbent assay.

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

1. colony forming: e.g. by multiplying bacteria or proliferating cells.
2. Photometry /Spectrophotometry When the absorbence 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 called colorimetry, or it may involve specific wavelength of light eg by use of laser and emission of fluorescent signals of another specific wavelength which is detected via very specific wavelength optical filters.
3. Transmittance of light may be used to measure eg 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 are measured by detectors placed straight across the light source.
5. Nephelometry when the scattered lights are measured by detectors placed at fixed angles to the path of light.
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 eg viscometry, elastography (eg thromboelastography)
8. Counting assays: e.g. optic Flowcytometric cell or particle counters, or 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 eg involving amperometry, voltametry, 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

Assay types based on the targets being measured

This unreferenced section requires citations to ensure verifiability.

DNA

Assays for studying interactions of proteins with DNA include:

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

Protein

• Bicinchoninic acid assay (BCA assay)
• Bradford protein assay
• Lowry protein assay^[6][7]
• Secretion assay

RNA

• Nuclear run-on

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 numerating and typing red versus different types of white blood 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 monitor cell culture (assays of cell proliferation or cytotoxicity). A cytotoxicity assay measures how toxic a chemical compound is to cells.

• MTT assay
• SRB (Sulforhodamine B) assay

• Cell counting instruments and methods: CASY cell counting technology, Coulter counter, Electric cell-substrate impedance sensing
• Cell viability assays: resazurin method, ATP test, Ethidium homodimer assay (detect dead or dying cells), Bacteriological water analysis, Clonogenic assays, ...

Surfactants

• An MBAS assay indicates anionic surfactants in 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_cells include :

• reporter assays using i.e. Luciferase, calcium signaling assays using Coelenterazine, CFSE or Calcein
• Immunostaining of cells on slides by Microscopy (ImmunoHistoChemistry or Fluorescence), on microplates by photometry including the ELISPOT (and its variant FluoroSpot) to enumerate B-Cells or antigen-specific cells, in solution by Flow cytometry
• Immunostaining of cells on slides by Microscopy, on microplates by photometry
• Molecular biology techniques such as DNA microarrays, in situ hybridization, combined to PCR, Computational genomics, and Transfection; Cell fractionation or Immunoprecipitation
• Migration assays, Chemotaxis assay
• Secretion assays
• Apoptosis assays such as the DNA laddering assay, the Nicoletti assay, caspase activity assays, and Annexin V staining
• Chemosensitivity assay measures the number of tumor cells that are killed by a cancer drug
• Tetramer assay detect the presence of antigen specific T-cells
• Gentamicin protection assay or survival assay or invasion assay to asses ability of pathogens (bacteria) to invade eukaryotic cells

Petrochemistry

• Crude oil assay

Virology

The HPCE-based viral titer assay uses a proprietary, high-performance capillary electrophoresis system to determine baculovirus titer.

The Trofile assay is used to determine HIV tropism.

The viral plaque assay is to calculate the number of viruses present in a sample. In this technique the number of viral plaques formed 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 specific antibody or cytokine) can be detected using the ELISA technique. The number of cells which secrete those particular substances can be determined using a related technique, the ELISPOT assay.

Drugs

• Testing for Illegal Drugs
• Radioligand binding assay

See also

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

References