Chemiluminescence in Analytical Chemistry

vance of environmental factors, life style, East-West gap, etc., on health, fitness for work and life-span)

Possible industrial applications include screening of substances with antiradical activity, quality testing of raw materials, pharmaceuticals, cosmetic products, fruit juices, wines, beers, edible oils, detection of food irradiation, and many more.

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Figure 2 Structure of acridinium ester.

of acridinium ester chemiluminescence is in the order of 10 amol. This is considerably better than the sensitivity of radioactive labels like 3H and 125I. Even greater sensitivities can be reached by using an enzyme as the label: in this case, because of enzyme turnover, many chemiluminescent molecules can be produced from one enzyme molecule. Examples include the luminol-enhanced chemiluminescence [9–11] catalyzed by horseradish peroxidase (HRP) and certain luminol analogs [12] reported to be more efficient than luminol.

Recently, it was found that certain acridan esters could be oxidized to acridinium esters in a reaction catalyzed by HRP [13, 14]. Like the luminol case it was found that the light yield of the reaction could be increased when certain additives (enhancers) were added.

1.1 Immunoassay

Immunoassays are based on the ability of the immune system to produce a virtually unlimited variety of antibodies each with a high affinity for foreign compounds (immunogens like viruses, bacteria, proteins, and haptens). Analytically, this phenomenon can be exploited by detection of this immunoreaction using labeled antibodies or antigens (i.e., compounds that can be bound by antibodies). The equilibrium between antibody (Ab), antigen (Ag), and immune complex (AbAg) may be expressed as:

kd

where ka and kd represent the association and dissociation rate constants, respectively. The equilibrium (affinity) constant K is defined as:

this type of assay increases since at low concentrations a variation in the amount of competing antigen has a larger influence on the change of signal that is ultimately measured. When using low concentrations of antibody and tracer, the rate of immunocomplex formation is reduced, resulting in relatively long incubation times. It can be shown that in this case the ultimate sensitivity of the assay (when the [Ab] approaches zero) is dependent on the equilibrium constant K and the reliability of the signal measurement of the bound fraction at zero dose (B0) [15]. For example, an antibody-antigen system with a K of 1010 M 1 and a relative error of 5% can be detected at 5* 10 12 M. With a low-molecular-weight compound (mw 200) this corresponds to a maximal attainable sensitivity of 10 9 g/L or 1 ppt.

1.2 Enzyme Immunoassay

1.2.1Introduction

Using enzymes as labels in immunoassays potentially affords much more sensitive tracer (i.e., enzyme-labeled antigen or antibody) detection in comparison with radiolabels because of the great catalytic power resulting in generation of many product molecules from one enzyme molecule through turnover. A major advantage of radiolabels is that their measurement is not prone to chemical interference. This is not the case with enzymes. Measurement of enzyme activity is strongly influenced by suboptimal temperature, pH, salt concentration, etc. However, the ease and low price of enzyme detection, together with the increased costs of using radiolabels, has made enzymes popular labels in immunoassay. The most widely used enzyme for immunoassay purposes is horseradish peroxidase (HRP)

1.2.2Enzyme Detection

Although HRP is often detected and quantified using a chromogenic substrate (actually, hydrogen peroxide is the substrate for HRP, generating an oxidized form of HRP, which is subsequently reduced in two steps converting a hydrogen donor DH to an oxidized donor D; in this case the chromogenic substrate is colorless DH, which is transformed to colored product D), there are other possibilities to measure HRP. One of the most sensitive and convenient is use of chemiluminogenic substrates.

1.3 Practical Aspects of Enzyme Immunoassay

An enzyme-linked immunosorbent assay (ELISA) has two major components. The first is the immunological reaction that occurs between an antigen and antibody. This reaction is crucial and needs careful optimization. The second compo-

nent is the surface to which antigens or antibodies are immobilized. This aspect too needs consideration with respect to the effect of the solid phase on, e.g., reproducibility of antigen or antibody coating, nonspecific binding, sensitivity of the assay, etc. Furthermore, parameters like temperature, pH, composition of coating and assay buffer, incubation times, etc., play important roles with respect to assay sensitivity and reliability. In the next sections some of these factors are discussed in detail, taking as an example the assay format described in Figure 3.

1.3.1Coating of the Solid-Phase with Antibody

Although other assay containers can be used (e.g., coated tubes or beads), the most popular one is the 96-well microtiter plate. ELISAs that are detected in a microplate luminometer must be performed in opaque white or black plates.

Biomolecules like antibodies attach to surfaces via a variety of mechanisms. This attachment phenomenon is controlled by the chemical properties of the surface, but can be influenced by factors such as pH and temperature. In the case of antibody coating to a solid support the use of so-called medium-binding plates is to be recommended. Coating conditions can be optimized by performing a checkerboard titration (in the following example the optimal coating antibody concentration is determined):

A.Antibody is serially diluted across the plate (starting concentration of approximately 10 g/mL in coating buffer (PBS or carbonate buffer), allowed to coat to the surface (overnight at 4°C), and excess is washed away.

B.The surface is then blocked (PBS containing 0.02% Tween 20 and 1% BSA).

C.The antigen is serially diluted down the plate, allowed to react with the immobilized antibody, and excess is washed away (PBS containg 0.1% Tween 20, PBST).

D.Enzyme-labeled antigen is used at a constant (and excess) concentration of approximately 10 g/mL (such that it is not the limiting factor in the assay), allowed to react, and excess is washed away (PBST).

E.Signal reagent is added to each well.

F.Following signal detection, select the optimal reading (this reading is normally chosen based on the following: readings will increase from the bottom right corner to the upper left corner of the plate; the optimal reading lies just prior to the set of wells exhibiting the highest readings).

G.From the well corresponding to the optimal reading, derive the optimal concentration of coating antibody.

This procedure can be repeated for other coating conditions (time, temperature, pH, buffer compositions, blocking steps, wash buffers, etc.).

1.3.2Determining the Optimal Concentration of Labeled Antigen

The optimal concentration of labeled antigen can also be determined via a checkerboard titration protocol:

A.The antigen is serially diluted down the plate (optimized with respect to antibody concentration as described in Sec. 1.3.1.), allowed to react with the immobilized antibody, and excess is washed away (PBS containing 0.1% Tween 20, PBST).

B.Enzyme-labeled antigen is serially diluted across the plate, allowed to react, and excess is washed away (PBST).

C.Signal reagent is added to each well.

D.Following signal detection, select the optimal set of column wells (this will be dependent on the goal of the assay: if, for instance, a screening assay is required with a certain cutoff concentration, e.g., 0.1 ng/mL for pesticides in water, then the labeled antigen concentration that yields a B/B0 of 50% at 0.1 ng/mL will be ideal).

E.From the wells corresponding to the optimal reading, derive the optimal concentration of labeled antigen.

2. CHEMILUMINOGENIC SIGNAL REAGENTS FOR HRP

2.1Enhanced Chemiluminescence Using Luminol and Analogs

In the early 1980s a serendipitous discovery was made when to a mixture of HRP, luminol, and hydrogen peroxide in buffer, firefly luciferin was accidentally added. The light yield of the HRP-catalyzed peroxidation of luminol was greatly enhanced. This remarkable discovery marked the beginning of a very successful analytical tool for immunoassay and all kinds of blotting (protein, DNA, and RNA) applications [16–18].

2.2 Enhanced Chemiluminescence Using Acridan Esters

In 1994 at the 8th International Symposium on Bioluminescence and Chemiluminescence, Schaap and Akhavan-Tafti presented a new technology for the detection of HRP [13, 19, 20]. They proposed the use of certain aromatic acridan esters (see Fig. 4 for the structures) as part of a signal reagent for very sensitive detection