Homogenous and Heterogenous Immunoassays PDF

Homogeneous and heterogeneous enzyme immunoassays Dr. Rakesh Kumar Verma  Definition: Analytical methods combining antibody specificity with enzyme sensitivity Introduction Purpose: Detection and to Enzyme quan...

Homogeneous and heterogeneous enzyme immunoassays Dr. Rakesh Kumar Verma  Definition: Analytical methods combining antibody specificity with enzyme sensitivity Introduction Purpose: Detection and to Enzyme quantification of specific Immunoassa molecules in biological samples ys (EIAs) Key components: Antibodies, antigens, and enzyme labels Homogenous Enzyme Immunoassay Homogenous enzyme immunoassay is defined as an immunoassay system in which both antigen-antibody reaction and the measurement of the degree of immune reaction are performed in solution without separation of the free and antibody-bound components. The assay is based on the conformational change of the active site of the enzyme or the steric hindrance of the substrate by the binding of enzyme-labeled antigen to the antibody. Characteristics: 1. No separation of bound and free labels required 2. Faster and simpler to perform 3. Generally less sensitive 4. Often used for small molecule detection Work Principle When the antibody binds to the analyte conjugated to the reporter enzyme, a physical blockage and/or a conformational change of the enzyme's active site occurs, hindering the substrate from accessing the active site or reducing intrinsic catalytic activity. As a result, the reporter enzyme activity is repressed. Because the number of antibody sites available to inhibit the enzyme depends on the free analyte concentration, the measurable reporter enzyme activity is related to the free analyte concentration. Enzyme-multiplied Immunoassay Technique (EMIT)  EMIT: https://www.lino-biotech.com/heterogeneous-vs-homogeneous-assays/ The main component of the assay is a complex made of an enzyme (“reporter enzyme” in figure below), attached to the analyte of interest. The enzyme’s active site (the part that is specific for binding) is specific for a substrate that is fluorescent after its binding event with the enzyme. In short, when this substrate and the enzyme-analyte complex bind, the substrate changes color (yielding a detectable product). These complexes are added to a sample solution, usually issued from a patient; when the color change of the substrate occurs, the whole sample solution will change color. This however still does not help us explain how we can determine the presence of the analyte of interest in the sample. To this end, we add an antibody specific for the analyte of interest to the sample (anti-analyte antibody). If the sample does not contain the analyte of interest, the antibody will only be able to bind it whilst attached to the enzyme (in the enzyme-analyte complex), because there is no free analyte in the present in the sample solution. When this happens, the antibody blocks the active site of the enzyme. This means that no other molecule can bind this site, because there is no access possible. The color-substrate that would usually bind there and become fluorescent can therefore no longer bind; it never becomes fluorescent and there is no color change in the sample. If, however, the solution does contain the analyte of interest, the added antibodies have many more molecules to bind: they will primarily bind the free analyte in solution (because of its easy access), as well as some of the enzyme-analyte complexes. Heterogeneous assays heterogeneous assays require one or more steps of separation, where unbound antibodies and/or unbound analyte must be washed away. This makes heterogeneous assays often longer and more complex to conduct; however, they are often more precise than homogeneous assays, and can be very helpful to detect more complex analytes. Homogeneous assays are usually reserved for the detection of small, simple molecules. Characteristics: 1. Involve washing steps to remove unbound reagents 2. Generally more sensitive 3. Can detect a wider range of analytes, including large proteins 4. More time-consuming and complex Principle of operation: 1. Immobilization of one assay component (usually antibody or antigen) 2. Binding of analyte 3. Addition of enzyme-labeled reagent 4. Washing to remove unbound materials 5. Addition of enzyme - substrate for signal generation Enzyme-Linked Immunosorbent Assay (ELISA) Comparison: Sensitivity: Heterogeneous assays are generally more sensitive due to the washing steps that reduce background noise. Complexity: Homogeneous assays are simpler and faster to perform. Application: Homogeneous assays are often used for small molecules, while heterogeneous assays can detect a wider range of analytes. Automation: Homogeneous assays are easier to automate due to fewer steps. Cost: Homogeneous assays can be less expensive due to fewer reagents and simpler equipment requirements.