Hemolytic plaque assay procedures

Developed in the early 1960s, hemolytic plaque assays are among the most sensitive biological measurement procedures. In principle, these assays resemble plaque techniques for the detection of bacterial viruses. Typically, agar-embedded (see below for variations) erythrocyte suspensions are mixed with lymphocyte populations which contain relevant B lineage cells. If these cells secrete antibody to antigenic determinants present on the surface of target red blood cells, the antibody will bind first to the nearest cells and then to erythrocytes further away. In the presence of a source of complement (usually rabbit or guinea pig serum), antibody-coated red cells lyse. Thus, after -6-12 h incubation (1-3 h in the thin-/monolayer techniques, see below), the result is formation of clear zones where erythrocytes have been lysed, and in the center of the zone is the single antibody-producing cell.

To facilitate the lytic function of antibodies that are not able to or inefficient in complement fixation (e.g. immunoglobulin G, IgG), addition of a secondary (e.g. goat anti-lgG) antibody will 'develop' such 'indirect' plaque-forming cell (PFC) assays (Table 1). If only secondary (e.g. IgG/IgA) responses are to be evaluated, it is possible to prevent the formation of 'direct' (IgM-mediated) plaques by the inclusion of 2-mercaptoethanol in the assay mixture. An excellent and extensive review of PFC assay techniques used in rodent research has been written by its inventor. Niels Jerne, and updates have been published by colleagues in his institution (see Further reading).

Numerous antigens and haptens have been conjugated to various red cells to serve as specific antigen, and the assay lends itself to functional estimates of affinity through, for example, titration of the amount of free hapten required for blockade of 50% of PFCs. However, since such blocking procedures also titrate the amount of antibody, i.e. secretory rates, and

Table 1 Key words and terms

Term Definition

PFC Hemolytic plaque-forming cell: a B lineage cell which secretes antibody that binds to epitopes present on surrounding erythrocytes; if the antibody can fix complement added to the assay, a zone of hemolysis is formed around the secretory B cell.

Direct PFC If the antibody produced by the PFC is of the IgM class, the added complement directly mediates lysis of surrounding red blood cells. Theoretically, a single, bound IgM molecule is sufficient for the lysis of a red cell.

Indirect PFC If the antibody produced by the PFC is not of the IgM class (e.g. IgG, IgA) then a

'secondary' (or developing) antibody (e.g. goat anti-IgG, rabbit anti-lgA) is added late during the assay to achieve more efficient complement activation on target erythrocytes.

Fluid-phase PFC assays Reduction of plaque assay volumes by omission of the agarose gel. The first of these assays ('Cunningham assay') uses hemocytometer chambers and coverslips to suspend appropriate mixtures of lymphocytes (i.e. PFCs) and target red blood cells. To avoid physical disturbance of these mixtures, another procedure uses red blood cell monolayers fixed to the bottoms of 96-well microtiter plates with poly-L-lysine. Relevant lymphocyte/complement mixtures are added to monolayers and close red cell/white cell contact can be achieved by gentle centrifugation.

Reverse PFC assays (rPFC) Nonantigen-specific version of indirect plaque assays where the target red blood cells are conjugated with anti-lg antibody (e.g. anti-IgG, anti-lgA or polyvalent antibody). One variation uses protein A instead of anti-lg. A nearby B cell secreting an antibody of the relevant class becomes a plaque-forming cell as its Ig product is bound to the red blood cell surface. rPFC assays permit studies of polyclonal B cell activation. This assay can be modified to detect the secretion of molecules other than Ig.

ELISPOT assays Reminiscent of rPFC. Replacement of conjugated erythrocytes with an enzymatic detection system typical of ELISA procedures allows enumeration of cells secreting molecules of interest such as specific cytokines. Instead of red cells, the primary (capture) antibody is coated to plastic and cells are incubated on top. Following addition of enzyme-conjugated secondary antibody and enzyme substrate, secretory cells are enumerated as dark spots surrounding a single lymphocyte.

since secretory rates can vary by orders of magnitude within a given immune response induced, results of hapten inhibition studies must be interpreted with caution.

The size of hemolytic zones is related to antibody affinity and secretory rate, but accurate measurements are cumbersome and have not been rigorously examined with cloned cell sources. Given that a single IgM molecule can fix one Clq complement molecule to the red cell surface and that this is sufficient for the formation of a lytic site, there is little doubt that hemolytic plaque assays are of exquisite sensitivity. The actual sensitivity is somewhat lower, probably due to antibody loss in the three-dimensional assay space, but cells secreting in the order of a few thousand antibody molecules per hour are clearly detectable.

Methodological variations of PFC assays

A number of variations of the original hemolysis-in-gel (agarose) procedure have been described with the goals to improve cell survival in the assay system, reduce the cell number and materials requirements and increase practical assay throughput and, perhaps, sensitivity. Agarose is still used extensively as the plaquing medium but smaller assay vessels than standard Petri dishes (e.g. slides) are now being used more often. The development of efficient microcul-tures generated a need for micro-PFC assays. The first such modification was Cunningham's fluid phase assay that used red blood cell/lymphocyte mixtures suspended in hemocytometer chambers. In this procedure PFCs are read microscopically. It requires small cell numbers and eliminates the need for a solid carrier medium. One potential drawback of the procedure is the danger of accidental resuspension which, of course, obscures the round lytic zones.

Modified plaque assays were further developed under Cunningham's influence in Toronto, including a solid-phase procedure, and its micromodification which permitted fast (1 h) PFC assays in 96-well formats using very small numbers of cells (e.g. limiting dilution microcultures). In this procedure, monolayers of red blood cells, modified with haptens or unmodified, are immobilized on plastic surfaces with poly-L-lysine. To ensure intimate contact, lymphocytes are sedimented on to that monolayer by centrifugation. Small volumes of medium and complement are added with the lymphocyte preparation. Plaques in this system develop quickly but often are small and a specific role of the poly-L-lysine itself modifying antibody release was suggested. A majority of experiments dealing with antigen-specific in vitro responses of human lymphocytes have used this system. Although successful in several laboratories these modifications were overall 'tender' techniques which are rarely used today.

Another, rather important development was the introduction of 'reverse' hemolytic PFC assays. This procedure is in essence the reverse of an indirect PFC technique where the secondary (developing) antibody becomes the primary reagent covalently coupled on to the surface of target erythrocytes. Any secretory B cell releasing an Ig bound by these anti-Ig-coated target red blood cells will now become a PFC. This is a rapid and elegant procedure for studies of polyclonal B cell activation, in particular in the human system where B cell activation has been less easily achieved and manipulated.

Reverse PFC assays have been further modified for the detection of cells secreting proteins other than Ig, broadening applications for this sensitive procedure beyond B lymphocyte studies. The latest of these modifications is a departure from the use of indicator erythrocytes and their replacement with an ELISA-type enzymatic read-out system. These ELISPOT assays are now increasingly used for the enumeration of T cells secreting TH1- or TH2-type cytokines. Reminiscent of rPFC assays, plastic dishes or plates are coated with an antibody specific for the cytokine of choice, T cells (fresh or stimulated) are added in culture medium, and the secreted cytokine is captured by the plastic-bound antibody. After some hours of incubation, the assays are developed through addition of enzyme-coupled secondary antibody to the cytokine. In the presence of relevant enzyme substrate, round areas of enzymatic activity (usually dark spots) are counted microscopically, each corresponding to one T cell producing the particular cytokine of interest.

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