Assay Development

The use of light-producing chemical reactions for quantitative detection in ligand-binder assays is a rapidly growing field. Automated immunoassay analyzers from several different manufacturers are in commercial use and still others are in development. The technical demands of these assay markets requires detection technologies which are highly sensitive, not overly prone to interferences, robust and simple to use. Chemiluminescent processes have sufficient sensitivity and so are well suited for these applications. Most assays are heterogeneous and require separation of bound from unbound label. These assays are simpler to develop but somewhat more complicated to perform. A homogeneous assay, in contrast, requires the light-producing reaction to be affected in some way by binding of the ligand to its receptor.

One reason accounting for the growing popularity of chemiluminescent assays is their exquisite detection sensitivity. Unlike absorbance (colorimetric) or fluorescent measurements, assay samples typically contribute little or no native background chemiluminescence. The most serious source of background signal comes from the label being nonspecifically associated with the analyte through nonspecific binding events. This problem occurs in all detection systems. Measurement of light intensity is relatively simple, requiring only a photomultiplier or photodiode and the associated electronics to convert and record signals. The lack of inherent background and the ability to easily measure very low and very high light intensities with simple instrumentation provide a large potential dynamic range of measurement. Linear measurement over a dynamic range of 106 or 107 using purified compounds and standards is routine.

Factors in Choosing a Detection Reaction

Enzyme Label or Direct Label?
A common means for generating chemiluminescence is to use a label enzyme to catalyze a chemiluminescent reaction. Each label can initiate multiple chemiluminescent reactions, so it is usually only necessary to incorporate one or a few enzyme labels/analyte. The chemiluminescent compound is supplied as the enzyme substrate in excess to assure saturation kinetics.

Light intensity is a linear function of the amount of label enzyme. The intensity, which is the rate of emission of photons/sec, is the product of the catalytic turnover of substrate E + S → E + S' (steps 1-3 below) and the lifetime of the light-producing compound S' → P* (step 4). The latter process is usually first order with a rate constant k and can be characterized by its half life

t1/2 = (ln 2)/k.

The lifetime of the excited state product P* → P (step 5) is extremely short in comparison to the other steps and has no effect on the observed reaction kinetics.

reaction kinetics

The chemiluminescence intensity/time profile consists of an initial rise period up to a prolonged emission at a plateau or pseudo-plateau level. When the first order reaction of S' → P* is slow, the rise time to plateau will be extended as the steady state concentration of S' is reached. When the reaction of S' → P* is fast, there will be a rapid rise and prolonged emission. The absence of a steady plateau value indicates either substrate depletion or inactivation of the enzyme. Detection of enzyme-generated chemiluminescence provides great flexibility in the measurement process. Light intensity at any time point through the plateau can be related to the amount of enzyme. Measurement can be made during the rising portion if speed is an issue as either a single point or a multi-point slope type measurement. For maximal sensitivity, measurement can be performed on the plateau.

enzymatic chemiluminescence

Another way to use a chemiluminescent reaction for detecting an analyte in an immunoassay is to covalently label one of the complementary binding partners directly with a chemiluminescent compound. Triggering the chemiluminescent label to undergo the light-emitting reaction produces a signal for detecting the analyte. This approach has the advantage of being more straightforward and avoids the need to use large, relatively "sticky" labels. However, not many suitable chemiluminescent compounds exist. A suitable candidate must first have some functionality to allow for attachment to other molecules. More importantly, the label, upon triggering, should emit all of the light in the briefest possible period of time. When the chemiluminescence is emitted gradually over a period of time, signal intensity (photons/sec) is diminished and, in the worst case, can be low enough to impair detection sensitivity.

binding molecule

The optimum number of chemiluminescent tags on the species to be detected is best determined empirically. Even though more labels should theoretically provide more photons, quenching effects can occur if labels are too closely spaced. The surface area and the number of derivatizable groups, typically amino or sulfhydryl groups, provide further limitations. In practice 10-20 labels, at most, can be incorporated onto a large molecule. Moreover, the more labels on the surface of a binding molecule, the more likely that the label will interfere with its binding properties.

Factors in Choosing Assay Format

Homogeneous or Heterogeneous?
Homogeneous or nonseparation assays are desirable because of their simple mix-and-measure nature. This can be achieved when the key binding event (mix) modulates signal generation (measure) without the need for separating bound and unbound ligands. Signal modulation can, in principle, be any of several types. For example, the complement to the chemiluinescent labeled partner can be labeled with a quencher or a wavelength-shifting agent or a substance which affects the time course of emission. In practice, such chemiluminescent assays are very difficult to design. It is necessary that two critical components for the reaction be separately linked to the respective binding partners and that they interact to modulate the production of chemiluminescence only when the binding event occurs and not when the two labeled components are both physically present in the assay mixture in the unbound state. The discrimination between the two signal states must be at least 3-4 orders of magnitude.

It is far simpler to design heterogeneous assays in which labeled binding pair complexes are separated from unbound labeled reactants. Most chemiluminescent reactions can be adapted to this assay format, by labeling either with a chemiluminescent compound or with an enzyme and using a chemiluminescent substrate. Most commercially developed immunoassays are of this type.

The Chemiluminescent Reaction

There are many known chemiluminescent reactions to choose from in designing a detection scheme. However, only a fraction of the known reactions have found their way into high volume clinical diagnostic testing methods. Several factors influence whether a particular chemiluminescent technology is suitable for use in automated immunoassays.

  1. The reaction, of course, should demonstrate an adequate chemiluminescence quantum yield.
  2. All aspects of the detection chemistry, including labeling methods, labeling reagents, triggering agents and chemiluminescent reagents, must be robust and reproducibly prepared. Methods using enzyme labels face the additional requirement that the enzyme must be reasonably stable and easy to conjugate without diminishing its catalytic activity.
  3. Ideal chemiluminescent reagents possess an easily measured signal by virtue of an efficient chemiluminescent reaction with a predictable time course of light emission.;
  4. Analytical performance must be highly reproducible. Since inter- and intra-assay CVs must often be on the order of 3-5%, reagent performance must not contribute more than 1-2% to the CV. Of course, the reagent not be overly prone to interferences and must produce a stable and reproducible signal under laboratory use conditions. Constant reagent performance over a period of 1-2 weeks is important so that on-board calibration curves can be stored.
  5. Processing and shipping considerations mandate that the detection reagent be capable of bulk manufacture. The reagent must show extended storage stability at refrigeration temperatures or ideally at ambient temperature.