Assay


An assay is an investigative procedure in laboratory medicine, mining, pharmacology, environmental biology and molecular biology for qualitatively assessing or quantitatively measuring the presence, amount, or functional activity of a target entity. The measured entity is often called the analyte, the measurand, or the target of the assay. The analyte can be a drug, biochemical substance, chemical element or compound, or cell in an organism or organic sample. 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". Thus the noun assay means "trial, test of quality, test of character", 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 that represented the true value of the coin. This might have translated later into a broader usage of "analysis", 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.

General steps

An assay is never an isolated process, as it must be accompanied with pre- and post-analytic procedures. Both the communication order and the handling of the specimen itself 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 information handling and transmission system, 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 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 filter, 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 most common 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 patient's 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.