The concentrations of analytes of clinical interest in serum span a wide range. Hemoglobin is the most abundant protein in blood at 150 milligrams per deciliter. But how does that relate to the concentrations of parathyroid hormone (PTH) at 10 picograms per mL or human chorionic gonadotropin (hCG) at 20 milliInternational Units per milliliter(mIU/mL) and what is an international unit anyway? This is far from an academic question if an assay or detection technology for a new system is being considered for these and other analytes. To get analyte concentrations into a form where they can be compared, it is best to convert them into concentrations expressed in moles/liter. All that is needed is a molecular weight and the ability to convert milligrams and picograms into grams and to convert milliliters and deciliters into liters. In the hemoglobin example above, multiplying both the milligrams and the deciliters by ten does not change anything but yields 1500 milligrams per liter. Converting milligrams to grams by dividing by 1000 gives 1.5 grams per liter (g/L). Dividing by the molecular weight of hemoglobin (64500 grams/mole) yields a concentration of 2.3e-5 moles/liter. Note that keeping track of the units involved is a good internal quality control measure to ensure that the calculation is being done correctly.
International units (IU) pose a special problem. They were defined decades ago somewhat arbitrarily for some protein analytes. They are commercially available as lyophilized materials in a sealed glass ampule. An approximate weight of the material is given in an enclosed package insert. If one assumes that the material is essentially pure and it dissolved in a measured amount of liquid, an approximate concentration can be estimated. Antibody titrations, using solution phase capture or unlabeled detector antibody, can be used to more accurately derive IU to molar concentration conversions.
Antibody affinities also vary enormously from association constants (Ka) of 10e3 to 10e14. Lower affinity antibodies are easier to find than ones with higher affinities. Antibody affinities determine the concentration range where they can be used successfully. Competitive assays have their midpoints at approximately 1/Ka. The useable analytical range of a competitive assay is approximately a factor of ten around the midpoint. For sandwich assays the useful analytical range is about an order of magnitude more than for a competitive assay. The analytical sensitivity of a competitive assay is approximately 0.1/Ka. For sandwich assays, it is approximately 0.01/Ka.
Both types of assays have calibration curves that flatten out at the top and the bottom of their ranges. Competitive assays flatten out at the top end, or lower concentrations because at the concentrations of antibodies used, the affinity of the antibody is no longer capable of binding enough analyte to displace the tracer molecule which is often an analog of the analyte. Competitive assays flatten out at the bottom of the calibration curve, or at higher concentrations, because the amount of antibody used in the assay is saturated and cannot bind anymore analyte. Note that the range of a competitive assay can be shifted to the left by decreasing the amount of antibody used. The assay will be a little more sensitive, but at a cost in the higher concentration upper limit of detection and in the maximum amount of binding of the label that can be achieved. Sandwich assays flatten out at the bottom end of the calibration curve because non-specific binding begins to approach the amount of binding of the label at the most sensitive portion of the calibration curve. Indeed, sandwich assay sensitivity can be significantly improved by switching to a solid phase with lower non-specific binding. Sandwich assays flatten out at the lower end of the calibration curve, or higher concentration region, because they reach the limits of what the capture and/or detection antibodies can bind.
For the average user of immunoassay, the above discussion doesn’t matter very much because the assay manufacturer builds all of this into the formulation of the assay. The problem for the user comes when they wish to measure an analyte that is outside the range of a given technology. The range of an immunoassay technology is determined by several factors such as the the sensitivity of the detection technology, the affinity of the antibodies used in the assay and the size of the sample.
In the 1980s a new detection technology called fluorescence polarization was commercialized and a wide range of assays for drugs were commercialized. These assays worked very well and quickly displaced older technologies such as radioimmunoassay (RIA) for most drug assays such as phenobarbital and gentamicin. They reached their limit for assays such as digoxin and thyroxine because their physiological concentration ranges were several orders of magnitude lower than the higher concentration drugs such as phenobarbital and gentamicin. Fluorescence assays depend on getting light into a molecule so that it can be re-emitted as fluorescence. Light absorption is in turn dependent on the innate capacity of molecules to absorb light. This property is often expressed as the extinction coefficient of a molecule at a given wavelength and extinction coefficients in nature are almost never above 10e6 M-1cm-1. Combined with practical limits on optical pathlengths in the cm and below range, this translates into lower levels of detection in the 10-9 M range which is just about where digoxin and thyroxine occur in human plasma.
Two revolutions happened in the late 1980s that made more sensitive assays possible. They were the availability of monoclonal antibodies and the emergence of chemiluminescence as a practical detection technology. Polyclonal antibodies from rabbits, sheep and goats had been used as capture antibodies on solid phases such as beads and test tubes since the early 1970s and functioned quite well. The fact that these specific antibodies were only available as a minor component (usually much less than 0.1%) in partially purified antibody fractions did not matter when they were used in competitive assays or even as capture antibodies in sandwich assays. The problem came when they were pressed into service as labelled detector antibodies. When partially purified polyclonal antibody preparations were labelled, a thousand times more irrelevant antibodies than specific antibodies were present. Non-specific binding is driven by the total amount of labelled antibody and backgrounds with these formulations were too high to make practical assays. Afinity purification could be used to overcome this but it is time consuming, technically demanding and expensive. Monoclonals overcame these difficulties in one bound.
Chemiluminescence provided an exquisitely sensitive detection technology that overcame the barriers encountered by RIA, fluorescence and even enzyme immunoassays. Detecting emitted light, produced not by incoming light, but chemical means proved to be the key for making ultra-sensensitive assays such as Parathyroid Hormone (PTH) and so called third generation Thyroid Stimulating Hormone (TSH) practical. These assays typically have detection limits in the 10e-13 M range. Enzyme immunoassays can be made this sensitive by using substrates that are detected with fluorescence measurements or with long colorimetric substrate generation times, but this comes at the expense of complex instrumentation and lower assay throughput.
Modern clinical laboratories often have instruments that can measure more than 100 different analytes on a single platform. There is never enough space in a lab and having this capability on one instrument is critical for the productivity needed to be competitive. It is common to have 20 or so assays on the instrument at one time and to rotate the other 80 or so through on an as needed basis.
An analysis of one of the most popular immunoassay instrument’s menu of about 75 different assays shows that 42 percent of the assays have analytical sensitivities of less than 10e-9 M, 27 percent are more sensitive than 10e-10 M, 22 percent are more sensitive than 10e-12 M and 4 assays or 2.3% are at about the limit of current assay technology at about 10e-13 M. Even more sensitive immunoassays can and have been developed, but none have gained traction in the commercial marketplace probably because it has not been shown as yet that they can provide medically useful information.
DCN Dx is an international leader in the contract development and commercialization of rapid diagnostic tests at its ISO 9001:2015 and EN 13485:2016 certified facility in Carlsbad, Calif. The company’s team of in-house scientists and engineers develop and integrate all aspects of assay systems, including cassettes, sample handling devices, and reader systems. Since its founding more than 12 years ago, DCN Dx has been committed to furthering the rapid diagnostic test market through the continued evolution of technologies and applications related to lateral flow assays.
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