Plaque-forming assay

Humoral immunity Plaque forming assay Plaque forming assays... 

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. 

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. 

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... 

Luster et al., 1992a, b). Specifically, the use of either a humoral response assay for plaque-forming colonies (PFC response) or determination of surface marker expression in combination with almost any other parameter significantly increased the ability to predict immunotoxicity when compared to the predictivity of any assay alone. 

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)... 

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. 

CPE). At 50-75% CPE, decant medium, add fetal calf serum to 20% and freeze at -70°C. After freezing, prepare tenfold dilution series down to 10-5 and assay plaque numbers as noted below. Note plaque forming unit titer and dilution to be used for assay. 

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. 

The influenza virus host resistance model has been characterized in mice and rats, and has been widely used to evaluate the potential immunotoxicity of therapeutics. Influenza virus is used as the infectious challenge agent and administered intranasally in a 28-day repeat-dose study. Mice or rats are dosed for 7 days, infected and then dosed for an additional 21 days. Viral clearance is quantified by measuring infectious virus (plaque-forming units) at various times following infection. Dexamethasone may be used as a positive immunomodulatory control. This host resistance assay has been used in Balb/c, C57BL/6, and B6C3F1 mice and Fischer 344 (CDF), Brown Norway, and... 

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. 

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