Plaque-forming cell assay

Temple, L. et al., Comparison of ELISA and plaque-forming cell assays for measuring the humoral immune response to SRBC in rats and mice treated with benzo[a]pyrene or cyclophosphamide, Fundam. Appl. Toxicol., 21, 412, 1993. 

In rodents, sheep red blood cells (SRBC) are routinely used for immunization. The antibody response is determined using the plaque forming cells assay (PFC) or by plasma SRBC-specific antibody titer [21, 22], As an alternative to SRBC, other T-cell-dependent antigen may be used, including keyhole limpet hemocyanin (KLH), tetanus toxoid (TT), or pneumococcal antigen. 

Wilson, S.D., Munson, A.E. and Meade, B.J., Assessment of the functional integrity of the humoral immune response the plaque-forming cell assay and the enzyme-linked immunosorbent assay, Methods, 19, 3, 1999. 

Humoral immunity Plaque forming cell assay Rosette formation, AMP poreforming activity... 

Chien, C.C., Lieberman, R., and Inman, J.K., Preparation of functionalized derivatives of inulin conjugation of erythrocytes for hemagglutination and plaque-forming cell assays, J. Immunol. Methods, 26, 39-46, 1979. 

Organ weights (spleen, thymus, all animals) Immunotoxicity tests (1) functional tests (either a splenic plaque-forming cell (PEC) assay or an Enzyme-Linked Immunosorbent Assay (ELISA) to determine the response to antigen administration) (2) enumeration of splenic or peripheral blood total B cells, total T cells, and T-cell subpopulations Detailed clinical observations Functional observations (sensory reactivity to stimuli of different types, grip strength, motor activity, more specialized tests on indication)... 

Preclinical studies have shown that impaired immune function determined using a variety of assays, e.g., the plaque-forming cell (PFC) assay, lymphocyte proliferation, delayed-type hypersensitivity, and NK cell activity, is associated with decreased resistance toward experimental infections (Luster et al., 1994). When a drug candidate has been shown to impair immune function in animal studies, the question arises whether similarly negative effects can also be seen in treated human subjects. 

No adverse effects on immune function were observed in the plaque-forming cell and host-resistance assays in mice orally administered coriander oil at doses up to 1250 mg/kg daily for 5 days (Gaworski et al. 1994). 

Antibody producing cells to sheep erythrocytes (SRBC) were measured by the hemolytic plaque assay as modified by Cunningham and Szenberg (1968). Mice were inoculated i.p. with 5 x 10 SRBC on day 0, and with 25 mg/kg pyran (NSC 46051) i.v. on days — 1, 0, 1 or 2 with respect to SRBC and the splenic plaque forming cells determined on day 3,4, 5 and 6 post SRBC inoculation. 

Kennedy, J. C. and Axelrad, M. A. An Improved Assay for Haemolytic Plaque-Forming Cells , Immunology 20, 253-257 (1971). 

The communication box was used to provide psychological stress. The box consisted of small compartments (10 x 10 cm) equipped with either an electric foot-shock floor or a non-shock floor, and the electric shock and the non-shock floor were placed reciprocally. A mouse (sender) placed on an electric shock floor made emotional responses when charged with electricity to the floor for 10 sec at intervals of 50 sec and a mouse (responder) placed in a non-shock floor were exposed only to psychological stress. The electric current for the shock was increased stepwise from 1.6 mA to 2.0 mA at the rate of 0.2 mA per 1 h over 3 h. Sender mice were changed daily to naive mice in order to avoid reduced emotional responses to the electric shock due to adaptation to repeated exposures. Responder mice were daily administered with either AGE (10 ml/kg) or water (control) one hr prior to the 3 h emotional stress for 4 days. On day 1, responder mice were immunized with sheep red blood cells (SRBC, 1 X10 ) after the 3 h stress. After the last day of stress exposure, the spleen weight and number of spleen cells were measured and the number of anti-SRBC plaque-forming-cells (PEC) were assayed. 

The study of the immune response at cellular level was therefore undertaken by two methods the haemolysin plaque forming cells (PFC) assay using the modified Jerne technique" and the rosette forming cells (RFC) test using the method described previously The PFC are the antibody secreting cells (mature or immature plasmocytes) derived from antigen-induced differentiation of B lymphocytes. 

Fig. 11.7 A, diagrammatic representation of plaque assay for evaluating virucidal activity and B, monolayers of baby hamster ki(hiey (BHK) cells C, virus tte untreated virus (o represents a plaque-forming unit, pfu, in BHK cells) D, virus titre disinfectant-treated virus (before plating onto BHK, die disinfectant must be neuh alized in an appropriate manner). Note the greatly reduced nimiber of pfu in D, indicative of fewer iminactivated virus particles than in C. 

For assaying herpes virus, monolayers of baby hamster kidney (BHK) cells are used. Virus titre is expressed as the number of plaque-forming units (pfu) per millilitre before and after exposure to a disinfectant, so that the virucidal efficacy of the test agent can be determined. A diagrammatic representation is given in Fig. 11.7. 

Currently there are few methods for specific investigation of immunotoxic effects, which are regarded as sufficiently validated for routine use (EC 2003). The plaque forming assay or the equivalent using the ELISA method (Enzyme-linked Immunosorbent Assay) are recommended to identify altered T-cell-dependent humoral responses. Of particular value for hazard assessment are the so-called host resistance models, in which the clinical relevance of immunotoxicity can be evaluated. Other methods may also be of value to provide information on the mode of immunotoxic action, e.g., mitogen stimulation tests and leucocyte phenotyping. However, further work is needed on standardization and validation of these test methods. 

Assessment of the virulence of Listeria monocytogenes Agreement between a plaque-forming assay with HT-29 cells and infection of immunocompetent mice. Int. ]. Food Microbiol. 68, 33-44. 

Jordan and Seet studied the antiviral effects of Amphotericin B Methyl Ester (AME), an antimicrobial agent, on several vimses including VACV in a plaque reduction assay [64], The concentration of AME resulting in a 50% inactivation of the plaque forming units, after a 60 min exposure at 35°C, was reported to be 5.0 pg/mL. The authors suggested lipid components in the host cell membrane which incorporate into the viral envelope may serve as a susceptible site for AME. 

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