Hemagglutination assay

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 quantitating the relative concentration of viruses, bacteria, or antibodies.

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 RBC’s and virus particles. The agglutinated lattice maintains the RBC’s in a suspended distribution, typically viewed as a diffuse reddish solution. The formation of the lattice depends on the concentrations of the virus and RBC’s, and when the relative virus concentration is too low, the RBC’s are not constrained by the lattice and settle to the bottom of the well. 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 RBC’s 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.

A 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. 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. Each row of the plate typically has a different virus and the same pattern of dilutions. After serial dilutions, a standardized concentration of RBCs is added to each well and mixed gently. The plate is incubated for 30 minutes at room temperature. Following the incubation period, the assay can be analyzed to distinguish between agglutinated and non-agglutinated wells. The images across a row will typically progress from agglutinated wells with high virus concentration and a diffuse reddish appearance to a series of wells with low virus concentrations containing a dark red pellet, or button, in the center of the well. The low concentration wells appear nearly identical to the no-virus negative control well. The button appearance occurs because the RBC’s are not held in the agglutinated lattice structure and settle into the low point of the U or V-bottom well. The transition from agglutinated to non-agglutinated wells occurs distinctively, within 1 to 2 wells.

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