Following the incubation period, the assay can be analyzed to distinguish between agglutinated and non-agglutinated wells. The plate is incubated for 30 minutes at room temperature. After serial dilutions, a standardized concentration of RBCs is added to each well and mixed gently. Each row of the plate typically has a different virus and the same pattern of dilutions. The most concentrated sample in the first well is often diluted to be 1/5x of the stock, and subsequent wells are typically two-fold dilutions (1/10, 1/20, 1/40, etc.).The final well serves as a negative control with no virus. The RBCs used in HA and HI assays are typically from chickens, turkeys, horses, guinea pigs, or humans depending on the selectivity of the targeted virus or bacterium and the associated surface receptors on the RBC.Ī general procedure for HA is as follows, a serial dilution of virus is prepared across the rows in a U or V- bottom shaped 96-well microtiter plate. Hemagglutination is observed in the presence of staphylococci, vibrios, and other bacterial species, similar to the mechanism viruses use to cause agglutination of erythrocytes. The formation of the lattice depends on the concentrations of the virus and RBCs, and when the relative virus concentration is too low, the RBCs are not constrained by the lattice and settle to the bottom of the well. The agglutinated lattice maintains the RBCs in a suspended distribution, typically viewed as a diffuse reddish solution. HA and HI apply the process of hemagglutination, in which sialic acid receptors on the surface of red blood cells (RBCs) bind to the hemagglutinin glycoprotein found on the surface of influenza virus (and several other viruses) and create a network, or lattice structure, of interconnected RBCs and virus particles. The hemagglutination assay or haemagglutination assay ( HA) and the hemagglutination inhibition assay ( HI or HAI) were developed in 1941–42 by American virologist George Hirst as methods for quantifying the relative concentration of viruses, bacteria, or antibodies. Seropositivity was suspected in Sample 179 Different serum samples diluted from the left to the right. Indirect hemagglutination assay for human echinococcosis.
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