Chemiluminescence immunoassay, the way forward

Chemiluminescent immunoassay (CLIA) is the most advanced immunoassay platform in the in-vitro diagnostics. The wide dynamic range, high sensitivity and specificity of the results, high degree of automation and reduction in turnaround time, and ability to run a large number of tests, in random access mode, make CLIA the most advanced IVD tool. Current improvements with the development of new analytical platforms in CLIA like flow-injection, two-dimensional resolution, magnetic nanoparticles, etc., have improved the clinical efficacy of immunoassay tests very much.

Technology
CLIA is an immunoassay technique where the label, or the true indicator of the analytic reaction, is a luminescent molecule. Luminescence is the emission of visible or near-visible (λ=300–800 nm) radiation, which is generated when an electron transitions from an excited state to a ground state. The light generation in CLIA is facilitated with the conjugation of chemical probes with the detector molecules in the reagent.

CLIA reaction is described below.

In the presence of complementary antigen and antibody, the paratope of the antibody binds to the epitope of the antigen to form an antigen-antibody/immune complex. Substrate is added after incubation, which generates light. The intensity is directly proportional to the amount of labelled complexes and quantifies the analyte of interest. The intensity of light is measured in terms of relative light units (RLU). The main advantage of this technology includes sensitivity and its ability to be unaffected by background signals. Depending on the immunoreaction mode, the CLIA analyzer mainly applies three measurement principles – sandwich assay, competitive assay, and capture assay.

Sandwich assay
The sandwich assay uses two antibodies – a capture antibody and a detection antibody. It is called sandwich because the antigen is bound between antibodies. In a sandwich assay, the target antigen is specifically bound between a capture antibody and a detection antibody. The capture antibody is demobilized on a surface, while the conjugated detection antibody is applied as the last step before quantification.

Competitive assay
Also known as inhibition immunoassay, competitive assays measure the concentration of an antigen by detection of signal interference.

The sample antigen competes with a reference antigen for binding to a specific amount of labelled antibody. The reference antigen is pre-coated and the sample is pre-incubated with labelled antibody and added to the wells. Depending on the amount of antigen in the sample, more or less free antibodies will be available to bind the reference antigen. This means the more antigen there is in the sample, the less reference antigen will be detected and the weaker the signal.

Capture assay
Indirect immunoassay is a technique that uses a two-step process for detection, whereby a primary antibody, specific for the antigen, binds to the target, and a labelled secondary antibody against the host species of the primary antibody binds to the primary antibody for detection.

The method can be used to detect specific antibodies in a serum sample by substituting the serum for the primary antibody.

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