Assay - Assay Types Based On The Nature of The Assay Process

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 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 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 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 (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 absorbance 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 plates, 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 e.g. 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 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 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 e.g. viscometry, elastography (e.g. 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 e.g. 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