Cunningham Plaque Assay

The Cunningham Plaque Assay (hemolytic assays in general) was used as a method of directly detecting and evaluating the IgM response. Ig secreted by mouse plasma cells, to sheep red blood cells (sRBCs) and assess potential modulation of the humoral immune response. The assay is simple. If the mouse produced a humoral immune response to the injected foreign sRBCs, than it would have produced activated B cells, plasma cells, which would be secreting IgM antibody, primary immune response, specific for antigens found only on the sRBC. Therefore splenic B cells are isolated and mixed with guinea pig complement, sRBC (termed indicator cells) and culture media (usually isotonic solution of glucose, salts and amino acids, to prevent any spontaneous cell lysis). This solution is placed into a special slide with a rectangular sized chamber that allows a liquid monolayer of cells to form. If the splenic B cells are producing and secreting anti-sRBC IgM, the sRBCs in the chamber will be lysed by antibody activation of the guinea pig complement. The anti-sRBCs antibodies Fab (variable region) bind to the sRBCs surface antigens, while the Fc region binds to the complement component, C1q, that initiates the terminal complement components to form a membrance attack complex on the sRBCs and lead to their lysis. If there was no immune response (humoral) to the sRBCs, then no plaques will be formed, as there are no plasma cells specific to the sRBCs. Antibody-secreting (anti-sRBC) B cells, plasma cells, create a clear region, or plaque, around themselves from their peripheral secretion of antibody. The surrounding are of sRBC 'ghosts' and are referred to as a hemolytic plaque. Each plaque corresponds to an individual plasma B cell *when counting". Quantification of the plaques will give the number of antibody producing B cells against sRBCs (2,3).

The Cunningham Plaque Assay is a very fast and sensitive method for evaluating the humoral response. Moreover, this method can only detect plasma cells secreting IgM against sRBCs, because IgM is pentameric and can therefore synergize its' ability to activate the complement pathway and lyse the sRBCs. IgM plaques are usually referred to as direct plaques. To quantify a mixture of IgM and IgG plaques, antibodies against the mouse IgG would have to be utilized to crosslink the IgG antibodies, forming multimeric-like structures like IgM on the sRBCs surface, thereby enabling complement activation to pursue, with the subsequent formation of plaques. These anti-mouse IgG antibodies are usually of rabbit origin.

Cunningham - Szenberg assay

Since its discovery years ago, the plaque forming cell (PFC) response of mice immunized with foreign, heterologous erythrocytes is one of the most studied phenomena within immunology. This system, the hemolytic plaque assay, allows for the quantification of the number of antibody secreting plasma cells through the ability of antibodies to form hemolytic plaques in vitro with the subsequent addition of complement. The initial finding of this methodology was first described by Jerne and Nordin (1963), followed by many modified versions of the PFC assay appearing later on, such as the Cunningham - Szenberg assay. The Cunningham - Szenberg assay differs only in its' use of special chambers, Cunningham chambers, that allow for the development of plaques in a liquid monolayer of cells, while the initial methodology used agarose slides (2,3,4,5).

Experiment

Visualization of IgM and IgG Plaques separately against Chimpanzee IgG formed after Immunizing Mice with Chimpanzee IgG.

Prepare splenocytes from a chimpanzee IgG immunized mouse, as mentioned above, however this time we can quantify the amount of plasma cells secreting IgM and IgG antibodies against the chimpanzee IgG. In order to obtain hemolytic plaques, chimpanzee IgG will be attached to erythrocytes (e.g. sheep), which now act as the indicator cells. The RBCs used must never have been introduced into the mouse previously, to prevent the formation of non-chimp IgG plaques. In addition, in order to obtain IgG secreting plasma cells, an incubation time within the immunized mouse greater than 7 days is necessary. Using the Cunningham Plaque Assay (also do a control, in addition to the immunized mouse), mix culture media, splenocytes, indicator cells (sRBCs conjugated with chimp IgG) and goat anti0IgM mouse antibodies, followed by the addition of rabbit anti-IgG mouse, into a test tube. Guinea pig complement would then be added after a short incubation period. The goat anti-IgM mouse antibodies will inhibit the formation of IgM plaques by binding to and blocking IgM from carrying out its functions, noth of which are binding to the chimp IgG and activating complement. Addition of rabbit anti-IgG mouse is required in order to 'multimerize' the IgG against the chimp IgG, so IgG can become more efficient in activating complement (similar to pentameric IgM). The solution would be pipetted into a chamber slide, incubated and viewed. The quantified plaques would be derived from IgG secreting plasma cells only. IgM functions were blocked and could not form plaques. To quantify the IgM plaques, a method identical to the one used in the methodology would be used, except that the chimp IgG would be conjugated to the sRBC.

The results of such an experiment would vary, as the number of IgM and IgG plasma cells and therefore, secreted IgM and IgG vary according to time after immunization. Isolation of splenocytes only a few days after immunization (7 days) would display a far greater majority of IgM plaques compared to IgG plaques. However, splenocytes isolated 25 days after immunization, there would be more IgG secreting plasma cells, as the immune response is in its' later stages. In the later stages, there is more IgG present due to its' longer half-life and persistence, T cells would have had a longer time to influence isotype switching with secreted cytokines and we would be measuring subsequent immune responses, as antigen becomes trapped by APCs which further stimulate lymphocytes. Thus, there would be more IgG PFCs (however, far more after a secondary immunization with the same antigen), but there would still be a sufficient and visible level of IgM PFCs. Ig serum levels in the mouse would consist of a large amount of IgG after 20 days, compared to IgM, which would be prominent before day 20 (inclusion of 2-mercaptoethanol in the assay mixture can prevent the formation of direct, IgM-mediated plaques also (1,4,5,6,8).

Direct Detection of Plasma Cells Secreting IgM versus Sheep Erythrocytes

As mentioned previously in the Cunningham Plaque Assay, sRBC (sheep) injected into the mouse produce an immune response, as these are foreign to the mouse. Therefore, immunized mice form an adaptive immune response and become primed to the sRBC. Sheep RBCs are T-dependent antigens that require T cells to help stimulate the B cells to differentiate and proliferate. However, the plasma cells were able to secrete antibody specific to sRBCs and form plaques upon in vitro confrontation with the sRBCs, because these plasma cells had been previously stimulated and activated to clear sRBC from the blood of the mouse and were thus secreting antibodies. Therefore, the immunized mouse contained plasma cells against the sRBCs, and therefore its' splenocyte suspension formed plaques upon the sRBC monolayer. However, the control mouse, un-primed, and un-stimulated previously with the sRBCs, did not have the opportunity to mount an immune response, humoral, against the sRBCs and form plasma cells specific to the sRBCs. Lymphocytes in the control animal had never seen sRBCs before and levels of T cells and B cells would be far too low, as no clonal expansion could have occurred. In addition, the lymphocytes would have to differentiate into effector cells. Such processes did not occur within the control mouse. Our value of 480/million splenocytes, is far below the 1200 PFC/millions spleen cells usually obtained, as seen in the paper by Kissinger and Myl, 1983, that used a similar protocol.

Pitfalls to the Cunningham Plaque Assay

The size of the hemolytic plaques is directly in relation to the affinity and secretory rate by plasma cells. Most plaques are seen as fairly small. The actual sensitivity of this type of assay is decreased, because antibodies are lost in the 3-Dimensional assay space in the protocol used above., sRBC monolayer. Highly secreting cells, and high in affinity can probably be only detected. However, due to the incubators being a distance away from where most of the work is being done, the danger of accidental resuspension of the assay chamber could obscure highly the round lytic zones, refilling the plaques up and hiding the results. In addition, usaing a 1 hour incubation period, it is possible that the protocol time could have only allowed for the development of small, highly indistinguishable plaques. Moreover, 1 week immunizations could have only produced low affinity antibodies (not enough time for somatic mutations and affinity maturation) decreasing binding to sRBCs. Therefore, these low binders could have not been easily detected, as less sRBCs were lysed around them.

The difficulty of detecting and distinguishing plaques may be a problem within the above assay. The chambers contain after the method, numerous air bubbles trapped, within the solution. These are similar to plaques in appearance, and which to a novice in immunology become mistaken easily as plaques. Some plaques, the larger ones, may have been formed by more than one plasma cell, thereby decreasing PFC (plaque forming colony) number count. Well mixing can benefit somewhat. High affinity antibodies can have more efficiency in binding to the sRBC. More sRBC binding can result in higher complement activation and sRBC lysis. The chamber slides, which are supposed to produce uniform a volume, seemed to not fill-up in certain regions, mainly the corners, thereby weakening any plaque-counting reproducibilty. The use of an automated counter can greatly increase the accuracy of assaying the large numbers of PFCs. In addition, too little addition of a molecule within the complement pathway can result in a decreased level of sRBC lysis, thereby creating a difficulty in visualizing and quantifying plaques. Lacking a complement pathway mediator can result in no plaques, as the pentameric IgM can agglutinate the sRBCs.

References:

1. Janeway, C.A., Travers P., Walport M., and Capra J.D. 1999. Immunobiology: The immune system in health and disease. Garland Publishing, 4th ed., New York, USA, pages 1-40, 2.5-2.22, 3.1-3.12

2. Kissinger R, and Myl A.D. 1984. Improvements to the Plaque Assay for Antibody Secreting Cells. J. of Immun. Meth. 66:377-382

3. Wilson S., Munson A., and Meade J. 1999. Assessment of the Functional Integrity of the Humoral Immune Response: The Plaque-Forming Cell Assay and the Enzyme-Linked Immunosorbent Assay. Methods 19:2-7

4. Cunningham, A.J. 1965. Nature (London) 207, 1106

5. Cunningham A.J. and Szenberg A. 1968. Immuniligy 14: 599

6. Jerne N. and Nordin A. 1963. Science 140: 405

7. Delves P. and Roitt I. 1999. Encyclopedia of Immunology, Academic Press Inc., 2nd ed., San Diego, USA page 238.

8. Cruse, J and Lewis, R. 1995. Illustrated Dictionary of Immunology. CRC Press Inc., USA pages 1960-1965