Detection Methods for Immunoassays

The immunological part of an immunoassay ends typically with either a wash or separation step. In these steps, any unbound labeled materials in addition to any unbound components from the sample are discarded and the primary antibody or antibodies are left. In a competitive immunoassay, a mixture of both labeled and sample antigen is bound to the primary antibodies. In an immunometric assay, the capture antibody has the antigen and the labeled detection antibody bound to it. In an indirect immunoassay, the immobilized antigen has antibody bound to it and a labeled secondary antibody to the antigen-bound antibody is detected. In all of these assays, the assay quantification is achieved by measuring the amount of the labeled material isolated. The detection systems most commonly used are radioactivity, colorimetry, fluorescence, and chemiluminescence. The first immunoassays used 125I, 14C, or 3H-labeled antigens as the conjugates, or so-called “tracers”. Radioactivity has some distinct advantages, such as very minor changes to the structure of the labeled antigen. Moreover, a tritiated molecule has the same molecular size as the non-radioactive antigen and this makes the use of these materials very convenient to study binding reactions of small molecules. However, low specific activity coupled with the labile nature of some radioactively modified molecules and the regulatory pressures and constraints in their use, makes radioactivity a poor choice for convenient immunoassay applications.

The move toward non-radioactive approaches for detection in immunoassays in the 1970’s, 80’s and 90’s lead to the use of other detection vehicles. The first of these was colorimetry, normally achieved by attachment of an enzyme to an antigen for the competitive methods, attachment to a specific antibody for an immunometric or “sandwich” assay and to a generic secondary antibody for an ELISA approach.

The most commonly used enzymes are horseradish peroxidase (HRP) or alkaline phosphatase (AP). HRP is a relatively small enzyme with a high turnover and is derived from non-mammalian sources. It has been used very extensively for detection. When used with a variety of substrates, it generates high signals by the production of colored products in the presence of hydrogen peroxide. However, the first substrates used for HRP were carcinogenic. HRP is susceptible to inactivation from routine buffer constituents, such as azide, and peroxide forms a complex with the heme group of HRP, which leads to rapid loss of enzymatic activity. AP is a 140,000 Da bovine intestinal enzyme, which is stable and compatible with most buffers. As an enzyme that uses zinc and magnesium for activity, it is effected by very high concentrations of chelators such as EDTA. It has high turnover, is not inactivated by common buffer constituents, and is stable in solution at 4°C for years. Unlike HRP, AP does have a high temperature coefficient for the incubation with substrate. Both AP and HRP have been used for diverse applications using a variety of solid phase supports, separations and assay systems. Both enzyme systems liberate a soluble or insoluble colored product when incubated with suitable substrate. The amount of color generated is measured at a specific wavelength after a fixed incubation time. The optical density obtained is then related back to the concentration of the antigen in the sample. Another enzyme less commonly used is acetylcholine esterase (AChE). AChE is a multi-subunit enzyme that tends to aggregate into dimers, tetramers, and higher molecular weight species in solution. Although it has a high turnover number, its use has been limited by instability in solution. Once reconstituted, the lyophilized enzyme loses all of its activity in a week or two, limiting its desirability for robust and convenient immunoassay use.

Most colorimetric enzyme detection systems equal or beat the best radioactive assays, thus offering a convenient and safe alternative to radioisotopes. To reach lower concentrations or to obtain a result faster, fluorometric or chemiluminescent detections have been used. In general, fluorescent detection methods allow more sensitive or faster detection than colorimetric, but chemiluminescence has been used most successfully for both rapid and sensitive assay detection.

 

In general, the use of a more sensitive detection system, such as chemiluminescence, may allow for a faster assay or the use of a smaller sample size, which could result in a better detection system overall. Chemiluminescence is the chemical generation of visible light by a reaction and, as such, does not use any light source. This eliminates complicated and inefficient optical wavelength filtering systems. Chemiluminescent systems fall into two classes. The first and easiest to develop, uses enzymes to produce the chemiluminescent signal. Typically either HRP or AP is used with substrates that give rise to visible emission. This type of signal enhancement has allowed the development of immunoassays that are faster and more sensitive than any traditional colorimetric or radioactive assay. Other chemiluminescent systems use a direct, non-enzymatic chemiluminescent label. The direct labels tend to produce lower background signals than the enzyme systems and will typically elicit a signal very quickly. With the Acridinium ester system, the signal takes only two seconds to develop, as compared with 30 minutes or longer for an enzyme-generated system. One question occasionally asked is how chemiluminescence improves an immunoassay system. They may be accustomed to RIA systems, where the maximum signal bound is perhaps 1,500 cpm, or an EIA system where only 0.2 OD units are bound. They look at these assay system “statistics” and realize that between the 80% and 100% binding there may only be a few hundred cpm, or 0.05 or less OD units. They then look at one of our CLIA assays and may have hard time realizing that there may be 50,000 RLU’s bound between 100% and 80% binding. With precision of 1-2%, you should be able to make accurate concentration determinations with 90-95% binding. Even considering all of the sophisticated technology available for increasing sensitivity, the advances may be for naught if the immunological system, especially the efficient coating and preparation of the solid phase is not optimal. High non-specific binding (NSB) or variable antigen or antibody coating may lead to lower sensitivities.