Basic Immunoassay Principles and Guidelines

Defining Immunoassays

The immunoassay is a technique which incorporates the binding reaction of a target substance (antigen) with an antibody. Antibodies are basically immunoglobins that bind to different natural and synthetic antigens in the body such as carbohydrates, lipids, proteins and nucleic acids. The antibodies have a common structure but have different special components that help them identify and bind to specific antigens. This results in the flexibility needed for effective immunity in a heterogenic environment. The specificity of antibodies which enable them to bind to different antigens is shown in Figure 2.1 below:

Immunogens, also known as antigens are substances commonly proteins which are coupled to hapten (a carrier) that can cause the formation of antibodies when it is introduced in a host body. The mechanism exhibited by antigen and antibody is like a ‘lock and key’ one which determines the affinity and binding pattern of these substances in a solution. The core concept of immunoassay is the ‘antigen-antibody’ binding reaction which has a great impact on assay effectiveness and development. These reaction are however, quite variable.

In a normal host, after immunization, poly colonal anti sera is generated. This anti sera is a heterogeneous mixture of antibodies which have different binding specificities and affinities. The assays which have polyclonal antibodies would have a broader range of cross reactivity, which depends on their application. It is quite easy and cheap to produce polyclonal antisera.

Isolated cloned cells produce mono colonal antibodies. In 1975, George Kohler and Cesar Milstein produced hybrid cells which were formed by the fusing of B lymphocytes and immortal myeloma cells. These hybrid cells, also known as hybridomas, were able to generate very specific antibodies which can differentiate between a variety of target molecules. These antibodies were invaluable to the field of therapeutics and diagnosing. When compared to poly colonal antibodies, the mono colonal antibodies were much more specific for immunoassays and provided continuous antibodies. Recombination of DNA technology is now used to further improve the flexibility and specificity of this technology.

Principles Regarding The Methodology Of Immunoassays

Immunoassays have been classified as being:

  • Competitive and Non competitive
  • Heterogeneous and Homogenous

The immunoassays basically identify a label which is detected to measure the amount of antigen or antibody which is present in a sample. These ‘labels’ can either be radioactive isotopes or enzymes that cause changes in color or produce light. Depending on the type of assays, labels either exist on the antigen or the antigen.

Limited reagent or competitive immunoassays are related to the phenomenon of the sample’s analyte and the labeled analyte’s competition for the antibody. (See Figure 2.2) The signal’s measurement would indicate the amount of target compound that is present. Mostly, primary assays types are the competitive assays that are being utilized in the drug testing programs of workplaces.

While doing the noncompetitive immunoassays, the antibody is adsorbed on the solid phase’s surface and the sample interacts with the solid phase. This system has excessive labeled antibodies in it which bind to the whole target analyte. Then, another labeled antibody is inserted which results in the sandwiching of the target analyte. The sample is then measured to know the quantity of the analyte which is there in the sample. That is why, non competitive assays are also known as ‘2-site’ or ‘sandwich’ immunoassays.

Heterogeneous assays are those which require the separation of bound and free labels. In heterogeneous assays, the portion of antigen bound with the antibody is disconnected from the remaining unbound part after the reaction takes place. This separation of assays is done via solid phase adsorption, liquid phase adsorption or precipitation.

However, homogenous assays are those where the label is changed by the binding process, thereby allowing the binding to be observed without separation step. These systems are marked by their speed and ease and can be easily automated. These assays are commonly used to monitor therapeutic drug measurement and also, illicit drug testing programs for workplaces where low detection limits are not needed.

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