Assay


An assay is an investigative procedure in laboratory medicine, pharmacology, environmental biology and molecular biology for qualitatively assessing or quantitatively measuring the presence, amount, or functional activity of a target entity. The analyte can be a drug, biochemical substance, or cell in an organism or organic sample. The measured entity is often called the analyte, the measurand, or the target of the assay. An assay usually aims to measure an analyte's intensive property and express it in the relevant measurement unit.
If the assay involves exogenous reactants, then their quantities are kept fixed so that the quantity and quality of the target are the only limiting factors. The difference in the assay outcome is used to deduce the unknown quality or quantity of the target in question. Some assays may be similar to chemical analysis and titration. However, assays typically involve biological material or phenomena that are intrinsically more complex in composition or behavior, or both. Thus, reading of an assay may be noisy and 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.
Assays have become a routine part of modern medical, environmental, pharmaceutical, and forensic technology. Other businesses may also employ them at the industrial, curbside, or field levels. Assays in high commercial demand have been well investigated in research and development sectors of professional industries. They have also undergone generations of development and sophistication. In some cases, they are protected by intellectual property regulations such as patents granted for inventions. Such industrial scale assays are often performed in well equipped laboratories and with automated organization of the procedure, from ordering an assay to pre-analytic sample processing. Analytes are generally tested in high throughput autoanalyzers, and the results are verified and automatically returned to ordering service providers and end users. These are made possible through the use of an advanced laboratory informatics system that interfaces with multiple computer terminals with end users, central servers, the physical autoanalyzer instruments, and other automata.

Etymology

According to Etymology Online, the verb assay means "to try, endeavor, strive; test the quality of", from Anglo-French assaier, from assai, from Old French essai "trial", and the noun assay thus means "trial, test of quality, test of character", mid-14th century, from Anglo-French assai and the 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 precious component was used to represent the true value of the coin. This might have translated later into a generalized meaning of analysis, e.g. of an important or principal component of a target inside a mixture such as an active ingredient of a drug inside the inert excipients in a pharmacological formulation which originally used to be measured by its actual action on an organism.

General steps

An assay is never an isolated process. It must be combined with pre- and post-analytic procedures. The information communication or specimen handling that are done until the beginning of an assay are the preanalytic steps. Similarly, after the assay, the result may be documented, verified and transmitted/communicated in steps that are called post-analytic steps. Like any multi-step information handling and transmission systems, variation and errors in the communicated final results of an assay involve corresponding parts in every such step; i.e. 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 gets much attention, steps that get less attention by the chain of users, i.e. the preanalytic and the post analytic steps, are often less stringently regulated and generally more prone to errors – e.g. preanalytic steps in medical laboratory assays may contribute to 32–75% of all lab errors.
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 of similar components and specifically identify a particular target component in a biological material by its specific attributes..
  3. Signal 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 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 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

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. 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, or graphically. 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. 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 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.

    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 RBC agglutination in response to grouping reagents.
  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 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 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 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 & VWF.

    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
  2. #in vivo, whole organism
  3. #ex vivo body part
  4. #ex vivo organ
  5. #ex vivo part of an organ.
  6. #tissue
  7. #cell
  8. Ligand binding assay when a ligand binds a receptor.
  9. 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 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 Gamma counters or X-ray plates, or phosphorimager
  4. Polymerase Chain Reaction Assays that amplify a DNA 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 immunoassay or EIA, enzyme linked immunosorbent assay.

    Detection method or technology

Depending on the nature of the Detection system assays can be based on:
  1. Colony forming or virtual colony count: e.g. by multiplying bacteria or proliferating cells.
  2. Photometry / spectrophotometry When 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 called colorimetry, or it may involve specific wavelength of light e.g. 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 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.
  6. Reflectometry When color of light reflected from a sample or reactant is assessed e.g. the automated readings of the strip urine dipstick assays.
  7. Viscoelastic measurements e.g. viscometry, elastography
  8. Counting assays: e.g. optic Flow cytometric cell or particle counters, or coulter/impedance principle based cell counters
  9. Imaging assays, that involve image analysis manually or by software:
  10. #Cytometry: When the size statistics of cells is assessed by an image processor.
  11. Electric detection e.g. involving amperometry, Voltammetry, coulometry may be used directly or indirectly for many types of quantitative measurements.
  12. Other physical property based assays may use
  13. #Osmometer
  14. #Viscometer
  15. #Ion Selective electrodes

    Assay types based on the targets being measured

DNA

Assays for studying interactions of proteins with DNA include:
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. But other methods use biochemical probing cell structure or physiology. Another application is to monitor cell culture.
A cytotoxicity assay measures how toxic a chemical compound is to cells.
Many cell assays have been developed to assess specific parameters or response of cells. Techniques used to study_cells include :
Metastasis Assay

Petrochemistry

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 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

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, dynamic range of detection, 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, throughput i.e. number of assays done per unit time 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 regulated quality assurance procedures including method validation, regular calibration, 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.

List of BioAssay databases

[Bioactivity databases]

Bioactivity databases correlate structures or other chemical information to bioactivity results taken from bioassays in literature, patents, and screening programs.
NameDeveloperInitial release
ScrubChemJason Bret Harris2016
PubChem-BioAssayNIH 2004
ChEMBLEMBL-EBI2009

[Protocol databases]

Protocol databases correlate results from bioassays to their metadata about experimental conditions and protocol designs.
NameDeveloperInitial release
BioMetaData or BioAssay ExpressCollaborative Drug Discovery2016
PubChem-BioAssayNIH 2004