Today's requirement for reducing the cost to produce a reportable patient result is driving the laboratory to consolidate as much testing as possible onto the fewest instruments. Manufacturers must respond by developing systems that provide a large menu of assays. he Access immunoassay system (Sanofi Diagnostics Pasteur, Inc., Chaska, MN) provides a tool for developing assays for a wide array of molecules. The instrument design has been described previously.1,2 This article focuses on the two key chemistry parameters, paramagnetic particles (PMPs) and chemiluminescence, that provide the basis for developing high-sensitivity assays with large dynamic ranges.
Access Solid Phase
Like most available immunoassay systems, the Access system uses heterogeneous immunochemistry. The solid phase is a submicron-size paramagnetic particle. The particles are polystyrene with incorporated iron particles and carboxyl functional groups on the surface. Antibodies, antigens, or other ligands are coupled covalently to the particle during reagent manufacturing. Standard sandwich or competitive assay formats are used; unbound enzyme label is washed from the particles before initiation of the chemiluminescent substrate reaction.
Why choose paramagnetic particles? Particles are commercially available in a variety of sizes and with a variety of functional groups for covalent coupling of biologicals. Covalent attachment of antibodies and antigens to the surfaces of PMPs yields reagents with shelf lives of a year or more at 2°10°C. Since PMPs can be suspended in solution, coating them is an easier manufacturing process than coating classic solid phases, such as microplates or 1/4-in. beads. We use a fully automated liquid-handling process to couple ligands to particles. Particle concentrates are easily stored in bottles until they are needed for production. One liter of coupled PMP concentrate is enough to manufacture 200,000 tests. By comparison, over 50 L of 1/4-in. beads plus additional coating solution are required to manufacture the same number of tests.
Convenience and stability aside, PMPs provide exceptional assay performance and ease of integration into an immunoassay analyzer. The high surface area provided by submicron particles and the ability to suspend them near the target analyte enable fast reaction times. Our test results show that reactions come to equilibrium approximately four times faster with PMPs than with a standard 1/4-in. bead.
Processing the PMPs as a liquid reagent allows the assay developer to optimize the mass of solid phase for each analyte. Assays requiring a large dynamic range, such as hCG, employ 100 mg of PMPs, whereas other assays may use only 25 mg.
The Luminescence Phenomenon
Close observation of the glowing firefly is fascinating, whether the observer understands the phenomenon of luminescence or not. The firefly's glow is just one example of luminescence. Thousands of species, including insects, bacteria, fungi, worms, and countless sea animals, use light emission as a tool.
In the broad sense, luminescence is simply the conversion of energy into light. The energy is stored in chemical bonds and released by a chemical reaction. Just as the chemical reaction of burning wood releases chemical bond energy to produce heat, enzymes in the firefly help to release chemical bond energy from adenosine triphosphate (ATP) to produce light.
In living organisms, this luminescence is referred to as bioluminescence. The reactions in such species require enzyme action. Through the study of bioluminescence, researchers were eventually able to synthesize molecules that could emit light in a test tube without an enzyme catalyst. This phenomenon has been termed chemiluminescence.
In the last 15 years, the utility of luminescent labels has steadily increased. The phenomenon has been applied to biomedical science in immunoassays, DNA probe assays, and measurement of important enzymes and metabolites.
Immunoassays based on chemiluminescence have substantially greater sensitivity and dynamic range than those based on earlier-generation detection techniques. Efficient light emission with low background is coupled with the high sensitivity and broad range of the photomultiplier detector. For every photon of light striking the surface of the photomultiplier, there is a 106-fold electronic amplification of the signal. Photomultipliers have very low background noise and inherent dynamic ranges of 5 to 6 orders of magnitude.
Many excellent review articles and reference books cover performance characteristics.37 The various luminescent reactions do not all result in the same sensitivity or the same ease of use. Their utility is governed by several key variables.
Quantum efficiency is perhaps the most important variable. It is defined as the percentage of those molecules undergoing the chemical reaction that emit photons. The maximum obtainable luminescence quantum efficiency would be 100%, although in practice this level has never been obtained. Some bioluminescent reactions have efficiencies of up to approximately 90% in vivo. Most chemiluminescent reactions have well below 15% quantum efficiency. The percentage of chemical reaction energy that is not dissipated by light emission is dissipated as heat or as kinetic collisional energy with other molecules in solution.
The impact of luminescence quantum efficiency on the sensitivity of the immunoassay depends on how the luminescent molecule is used for detection. For chemiluminescent labels that are coupled directly to protein (e.g., acridinium esters), the sensitivity is directly proportional to the quantum yield. For reactions that are amplified by an enzyme, sensitivity depends on both the quantum efficiency and enzyme turnover. Enzyme amplification can turn a reaction system with moderate quantum efficiency into an ultrasensitive reaction.
Background luminescence is another key variable. The background comes from two major sources:
One is simply nonspecific binding of the label. Nonspecific binding is not directly related to the chemiluminescent reaction. Its effect on performance characteristics depends on how well a given immunoassay procedure has succeeded in washing away nonspecifically bound proteins or in inhibiting nonspecific binding in the first place.
The second important source of background is nonspecific light emission of the chemiluminescent molecule. This phenomenon can occur before or during the initiation of the specific reaction and is caused by unwanted oxidants, metal catalysts, pH differences, enzymatic activity, and other variables. A review of various chemiluminescent compounds and their potential interferences is outside the scope of this article. Washing unbound material from a solid phase before triggering the specific luminescent reaction helps keep these interferences to a minimum.
Thermal degradation is another mode of unwanted light emission. The level of thermal-initiated background is specific for the chemiluminescent label or substrate in question and for the particular temperature.
For a sandwich immunoassay format, the detection limit is a function of signal-to-noise ratio. The considerable disparity in the quantum efficiency of different luminescent reactions makes the choice of a label particularly important. Once the label is chosen, however, proper control of background can make the difference between moderate and optimal sensitivity.
The Access system uses LumiPhos 530 (Lumigen, Inc., Southfield, MI), a chemiluminescent enzyme substrate for alkaline phosphatase. LumiPhos has a one-year shelf life when stored at 4°C and a one-week shelf life once a bottle of it is plumbed into the instrument. The LumiPhos reaction combines the inherent sensitivity of chemiluminescent light detection with the signal amplification of an enzyme label. As little as 1021 M of alkaline phosphatase can be detected with this substrate.8 Figure 1 depicts the detection reaction for this system.
Detection of the low levels of light produced by chemiluminescence generally requires the use of a photomultiplier tube (PMT). A PMT consists of an evacuated glass tube containing a photocathode, a number of dynodes, and an anode. As photons strike the photocathode, electrons are ejected and accelerate toward the first dynode. Each electron striking the dynode then ejects several more electrons. This process is repeated for each dynode in the chain. Thus, each impact of a photon upon the photocathode results in production of an electron current pulse at the anode. Typical amplification throughout the dynode chain is 106 to 107. If the incident light is intense enough, the individual pulses overlap and merge. The resulting signal then becomes a fluctuating dc current.
The output from a photomultiplier may be processed in either digital or analog mode. In digital (or photon-counting) mode, discrete photoelectron pulses are counted. Photon counting is the natural choice for the low light levels produced by chemiluminescence. It affords greater immunity to noise, drift, and variations in supply voltage and components. Its output is inherently digital, so there is no extra analog-to-digital conversion step (see box below). In analog mode, the fluctuating photocurrent is amplified, filtered, and read as dc signal. This detection mode is appropriate for higher light levels.
High Voltage and Discriminator Threshold
Even in the absence of light, PMTs produce some noise pulses (or dark pulses) stemming from a variety of sources.9,14 The number and size of both dark and signal pulses increase with increasing temperature and high voltage. On average, the noise pulses are smaller than the signal pulses. The system may be programmed to reject them by counting only pulses greater than a fixed threshold. This ability to reject noise is a chief advantage of photon counting over analog mode.
The system designer may select a particular high voltage and adjust the discriminator threshold to maximize the signal-to-noise ratio. Another option is to select the discriminator threshold first and then adjust the high voltage. We used several mathematical techniques to visualize the functional dependence and locate the optimum operating point.
To ensure a robust design, system developers must take into account the wide variability among PMTs. We obtained 11 randomly selected PMTs from the manufacturer and located the optimum operating point for each. From these 11 optimums, we then chose the one that gave the best signal-to-noise ratio across all 11 PMTs.
Individual tubes can vary in gain by as much as a factor of 10. For ease in manufacturing and calibration we wanted the system to require only a single adjustment--not both high voltage and discriminator threshold. We chose a constant discriminator threshold with a variable high voltage. We found that varying the high voltage gives a wider range of gain adjustment than varying the discriminator.
Optimal system performance requires precise temperature control. Antibody binding, alkaline phosphatase activity, the LumiPhos reaction, PMT gain and dark counts, luminometer electronics, and reference LED output are all sensitive to temperature. We found it necessary to control the temperature of the PMT, luminometer electronics, reference LED, and antibody reactions at 37° ±1°C, and the temperature of the LumiPhos addition and reaction at 37° ±0.25°C.
Figure 3 shows the response of the luminometer to serial dilutions of alkaline phosphatase reacting with LumiPhos 530. The plot shows that the luminometer is linear up to about 10 million counts per second (cps), with a usable range extending to 30 million cps. The dark-pulse counts for the system are generally less than 100 cps. This gives a total dynamic range of 5.5 decades. This range is not fully realized for assays, however, because of the background signal from the substrate, which in this case is at about 6000 cps. The usable range for assays is therefore 3.7 decades. The lower limit of detection (signal equal to two standard deviations above the substrate background) is 0.032 attomoles of enzyme. The luminometer thus covers 5.7 orders of magnitude in enzyme concentration.
The response plateaus at about 60 million cps. Other experiments have shown that the response actually decreases with higher enzyme concentrations. This "hook" effect is due primarily to pulse overlap at high count rates.
Signal Processing and Calculations
As mentioned previously, samples may be located in every third carousel position. When these locations are presented to the PMT, the system takes 10 1-second readings and computes the median. If a sample is present, the median is used to compute the sample response; otherwise, it is stored as the dark-pulse count. The median gives better rejection of noise than the average does.
Sample readings are adjusted for dark pulses and luminometer drift in the following way: The reference LED is read periodically in positions adjacent to samples. A drift correction factor (DCF) is calculated by comparison of the current reading to a previously stored value set at the factory.
The DCF is used to normalize the sample reading after the dark pulses are subtracted.
Normalized net sample reading = k * DCF (reading dark-pulse count)
The constant k adjusts for the prescaler in the circuit.
Calibration and Luminous Standards
To aid in development and allow matching between instruments, we employed various luminous standards. Early on, we used standards containing from 1 to 500 µCi of tritium-labeled palmitic acid in a liquid scintillation cocktail. Its spectral output peaks at 440 nm. Later, we developed standards using a proprietary solid-phase tritium-labeled scintillation polymer with an emission maximum at 545 nm. The latter more closely matches the spectral output of LumiPhos 530. This match is important, since variability in spectral sensitivity among PMTs may cause calibration and mismatching errors.
We have succeeded in building an immunoassay system that makes possible the development of a wide range of immunoassays. By combination of paramagnetic-particle, solid-phase, enhanced chemiluminescence and a sensitive luminometer, the system delivers assays with rapid kinetics and high sensitivity.
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Richard Creager, PhD, is vice president, research and development; David Knoll, PhD, and Curtis Shellum, PhD, are scientists; and Peter Werness, PhD, is director of advanced technology for Sanofi Diagnostics Pasteur, Inc. (Chaska, MN). They work together in Access research and development. Dr. Creager is a member of the Editorial Advisory Board of IVD Technology.