Serially diluted serum which contain antibody is loaded to each well of the microtiter plate, after that antigen coated red blood cells is applied to each well. The characteristic pattern of agglutinated red blood cells on the wells is used as a tool for assaying the agglutination reactions. If the antigen is particulate, then the antigen can react with the antibody in the serum and results in the clumping of antigen which shows a positive result. Over the past several years, there has been a shift away from red blood cells to synthetic particles, such as latex beads.
The preparation can either be used immediately or stored for later use. The use of synthetic beads offers the advantages of consistency, uniformity, and stability.
Furthermore, agglutination reactions employing synthetic beads can be read rapidly, often within 3 to 5 minutes of mixing the beads with the test sample. Whether based on red blood cells or the more convenient and versatile synthetic beads, agglutination reactions are simple to perform, do not require expensive equipment, and detect small amount of antibody concentrations as low as nanograms per millilitre.
The initial step in the test is the linking together of the latex particle by the antibody molecules that specifically attach to the antigenic determinants on the surface of the particles. There is a formation of large lattices through these cross links and these large lattices sediment readily due to the large size of clumps and are visible to the unaided eye within minutes.
The degree of agglutination can be determined by plotting the aggutinant concentration which gives a bell shaped curve. The antigen-antibody complexes can be magnified using the latex particles.
Many of the latex agglutination tests are performed manually and detected by visual observation. To determine agglutination there must contain about clumps, and these clumps must be of about 50 micrometer in size to be seen by eye. If the antibody is incubated with antigen prior to mixing with latex, agglutination is inhibited; this is because free antibodies are not available for agglutination.
In agglutination inhibition, the absence of agglutination is diagnostic of antigen, provides a high sensitive assay for small quantities of antigen. For example home pregnancy kits contain human chorionic gonadotropin HCG hormone coated latex particle and antibody to HCG.
A pregnant woman urine contain HCG which is secreted by the developing placenta after fertilization. The addition of urine containing HCG, inhibits agglutination of latex particles when the anti-HCG antibody is added; and thus the pregnancy is indicated by the absence of agglutination. Emulsion polymerization is the procedure that is applied for the preparation of latex particles. Firstly the styrene is mixed with the surfactant sodium dodecyl sulfate solution, forms a billions of emulsified micelles which are in uniform diameter.
Then a little amount of potassium persulfate is added to it which is a water soluble polymerization initiator. When the polymerization process is finished, the polystyrene chains are arranged into the micelles.
The hydrocarbon part of the polystyrene chain is attached to the center and the terminal sulfate ion to the spheres surface which is exposed to the water phase. Other hydrocarbons and its derivatives are also used for the production of the uniform latex particles, some of the examples are stryrene-dlvinylbenzene, polymethyl methacrylate, styrene vinyl toluene, polyvinyl toluene etc.
The process of latex particle production is evolved from synthetic rubber production and also the emulsion have a milky appearance, the term latex is given to it.
The desired diameter of latex particle can be made by modifying the process of preparation, hydrocarbons, the surfactants and the initiator. The particle size of latexes are usually ranges between 0. How to Prepare for the Test. Why the Test is Performed. What Abnormal Results Mean. If there is an antigen-antibody match, agglutination will occur. The risk level depends on the type of test.
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Immune System and Disorders Read more. Infectious Diseases Read more. Antibodies that produce such reactions are known as agglutinins. The principle of Agglutination reactions are similar to precipitation reactions ; they depend on the cross linking of polyvalent antigens.
When the antigen is an erythrocyte it is called hemagglutination. Theoretically all antibodies can agglutinate particulate antigens but IgM, due to its high specificity is a particularly good agglutinin.
There is no agglutination can be observed when the concentration of antibody is high, lower dilutions , and then the sample is diluted, agglutination occurs.
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