Immunoassay Classifications Essay PDF

**Write an essay on each of the following Immunoassay Classifications with an example and diagram of each:** **[Label Free ]** - Category of immunoassay that don't require a label to detect the Ab-Ag complex. - Based on principle that when Ab binds to it's Ag; they form a complex that...

**Write an essay on each of the following Immunoassay Classifications with an example and diagram of each:** **[Label Free ]** - Category of immunoassay that don't require a label to detect the Ab-Ag complex. - Based on principle that when Ab binds to it's Ag; they form a complex that can be detected w/o label being needed. - Rely on physical properties of complex, such as size or ability to to scatter light. - Can visualise the complexes via precipitation or Agglutination. - When Ab or Ag coated onto cells, the binding of the Ab to it's target Ag will cause the cells to clump together forming the precipitation or agglutination. - One of the **advantages** = Simplicity -\> they don't require complex labelling procedures - Another **advantage** = Cost-effectiveness -\> They're less expensive than labelled assays due to the lack of need for labelling procedures - One **Disadvantage** = Sensitivity -\> They have a lower sensitivity compared to labelled assays - Another **Disadvantage** = Hook Effect -\> highly concentration of analyte can inhibit complex formation; leads to inaccurate results - To overcome Hook Effect; appropriate dilutions should be conducted to determine the yield. - **Example: Latex Agglutination Assays (LAAs)** - Faster alternative to traditional single radial immunodiffusion (sRID) - Known concentrations of analyte (Ag) are used to create a standard curve. - Serial dilutions are done on bother the test sample and standards to ensure they are within the detectable range - Latex beads coated with a specific amount of purified antigen are mixed in wells with sample containing the antibody. - Mixture is incubated. - If antibody present -- they will bind to the antigen in the beads - Causes agglutination (clumping together) - An equivalence curve can be used to calculate the point at which the ab and ag are equal in conc. - Represents relationship between Ag con and degree of agglutination observed - The degree of agglutination can then be measure with a spectrophotometer. **[Reagent Excess]** - Non competitive immunoassay - Use an excess of reagents, usually Abs to ensure the target analyte in the sample is captured and detected. - Maximizes sensitivity and allows for quant of analyses over wide concentration range. - Example: - 2-Site/Sandwich Assay - Analyta captured between 2 Abs that bind to different epitopes on target molecule. - 1^st^ Ab is immobilised on solid phase -Microtitre plate/magnetic beads- - Sample containing analyte then added; allows for capture Ab to bind specifically to target. - 2^nd^ Ab -Detection Ab- introduced. - Ab is conjugated to a detectable label such as Enzyme or fluorescent molecule. - Detection Ab binds to different epitope on analyte; sandwiching target between 2 Abs - Following wash step to remove unbound material; signal generated by label is measured. - Signal is directly proportional to conc of analyte in sample. - Advantages of Sandwich Assay: - High specificity and sensitivity - Due to the use of 2 Abs that recognise distinct epitopes on the analyte - Excellent Detection in complex small samples - Such as serum and plasma - Due to dual Ab binding minizing interference from other components - Disadvantages of Sandwich Assay: - Requires carefully matched Ab pairs - due to the need for them to bind to different epitopes on analyte without interfering with each other - Difficult to test small Ags - Finding 2 distinct epitopes is challenging - Applications: - Detecting and quant of Protes in serum/blood sample - Diagnosis and monitoring disease **[Reagent Limited]** - Competitive Assay - Principle = Competition between labelled and unlabelled Ags for a limited number of binding sites. - Utilizes fixed and limited amount of Ab - Can be heterogeneous or homogenous - Uses a labelled Ag or labelled Ab - Both labelled Ag and unlabelled analyte in sample compete for binding sites - Amount of labelled Ag bound to the Ab is inversley proportional to the conc of unlabelled analyte in sample. - Higher signal = lower conc of analyte in sample - Example: - Enzyme Multiplied Immunoassay Technique (EMIT) - Free analyte analog molecules labelled with enzyme -Glucose-6-Phosphate Dehydrogenase- competes with analyte of interest in test solution - Active Enzyme reduces NAD to NADH which is measurable spectrophotometrically - When bound, no reduction, therefore, more target bound -\>more free enzyme -\> more signal - Signal intensity = directly proportional to analyte conc