Explanation on immunological assays

Transcription

1 Explanation on immunological assays Single Radial Diffusion Single radial difussion (SRD) is an immunodiffusion technique used in immunology to determine the quantity of an antigen. Typically, wells are made in an agar gel that holds an antibody suspension. The antibody is directed against the antigen to be quantified. When antigen is added to the wells, complexes with the antibody will be formed. These complexes are soluble when the antigen is in excess, but as the antigen diffuses in the agar gel and its concentration decreases, insoluble precipitin complexes will form, which can be seen as circles. By measuring the diameter of these circles and using a standard curve with known antigen concentrations, the quantity of the antigen can be calculated. Many factors influence this process, such as time, temperature, incubation process,... Often the washing steps are crucial for good imaging! Relationship between concentration of the antigen and the diameter of the resulting precipitin complex (source: immuneweb.xxmu.edu.cn) Image of an SRD plate (source: GSK doc)

2 Colony counting Bacteria grow in colonies. Typically, a single bacterium growing on a culture medium, will replicate and form a colony overnight. Colonies can be counted to estimate the number of bacteria in the original sample. Image of an agar plate with bacterial colonies (source: clinx.cn) Seroneutralization Viruses need cells to grow, whereas antibodies can inhibit this growth, rendering the virus inactive (= neutralization). Antibodies are found in serum (a blood component) of patients who are infected with the virus or have been vaccinated. By incubating virus with serum, a researcher can determine the neutralizing properties of the serum. An example of a seroneutralization test is the Plaque Reduction Neutralization Assay. Viruses are typically grown on monolayers of confluent cells (meaning that the cells are grown in a single layer and have fully covered the well of the plate in which they are grown). Virus growth can be observed under the microscope as plaques). When performing a seroneutralisation test, the researcher can measure the number of remaining plaques Example of virus plaques in a plaque reduction neutralization test (source: Plaque+Reduction+Neutr alization+test.html)

3 ELISpot The Enzyme-Linked ImmunoSpot (ELISpot) assay is a widely-used method for monitoring cellular immune responses. It is based on the principle of an ELISA, but works with cells. Rather than measuring the presence of antibodies or antigens in a suspension (as is done in ELISA), an ELISpot assay will measure the presence of antibody-secreting cells (or cytokine-secreting cells). In the end, the secreting cells are washed away but the antibodies or cytokine they have secreted can be visualised. They can be observed under a microscope as spots (hence the name), which can be counted. Example of spots in an ELISpot assay. (source: Hemagglutination inhibition Some viruses can bind to molecules present on red blood cells, a process called hemagglutination. As each virus molecule binds to multiple RBCs, a lattice- structure will form. This process can be inhibited by antibodies directed against the virus, blocking the virus from binding to the red blood cells. This is called hemagglutination inhibition. This test can be used to determine quantities of virus present in a sample. Image of a HAI 96-well plate. (source: /05/27/influenza- hemagglutination- inhibition-assay/)

4 Macro-analysis Any type of object can be placed under a microscope to measure its characteristics in real-time. Interesting for industrial applications. MTX Applications Macrolab SRD application Single radial immunodiffusion assays determine the quantity of an antigen by measuring the diameters of circles of precipitin complexes.the MacroLab is a reader based on a high-end lens with color camera that can capture the complete plate in one go without losing information. Once the image is acquired the software will process the image and present the ring diameters, height and width to a spreadsheet format that can be read by most LIMS systems. Description: high resolution camera with telecentric lens, motorized drawer with adaptable plate holder (now: SRD glass plate, possible for petri dishes, 96 well plate,...) Capacity: Manual loading Productivity: 20sec./plate + visual check Microtechnix benefits: Quality, Reproducibility (variation <5%), time-saving (20sec vs 20 minutes), objective, allowing archival of results, traceability Scanlab HAI application Some viruses can bind to molecules present on red blood cells, a process called hemagglutination. As each virus molecule binds to multiple RBCs, a lattice-structure will form. This process can be inhibited by antibodies directed against the virus, blocking the virus from binding to the red blood cells. This is called hemagglutination inhibition. This test can be used to determine quantities of virus present in a sample. Virus, red blood cells and serum containing anti-virus antibodies are incubated together. From a control wells with known virus quantities, the amount of virus in the test samples can be deducted. The Scanlab will detect the absence/presence of the lattice.

5 The system is equipped with fully motorized optomechanical components to ensure the highest traceability towards the image analysis conditions. With a total load capacity of 90 plates, the system can run unattended for several hours. Specific to this system is the robot that has a fifth axis, to turn the microtiter plates to a vertical position for imaging (standing up). Description: Camera and lens, robotic arm with customized tilt axis Used for: HAI testing in 96 well plate Capacity: 90 or 135 plates (extended capacity) Productivity: 50 sec./plate Microtechnix benefits: Quality, Reproducibility, Traceability