PFC assay

NY) and kept at 4°C in the dark until developed. The time of exposure varied between 12 days and one month. The films were developed in Kodak X-ray Developer-Replenisher ( 146-5327) and fixed in Kodak Rapid Fix ( 146-4106). Selected areas on the TLC plates directly under the spots on the films were eluted from the silica gel and tested in the anti-Thy-1 PFC assay. 

DEAE cellulose chromatography was performed by application of a small volume of glycolipid in C M (1 1) to a 3 ml column of DEAE cellulose, acetate form, followed by elution with 5 column volumes of C M W (60 40 8). Bound glycolipids were eluted with 5 column columes of chloroform-methanol-ammonium acetate. The samples were dried and tested in the PFC assay as described below. Mild HC1 treatment was performed as previously described with 0.1 N HC1 at 80°C for 30 minutes (8). After hydrolysis the samples were neutralized with 0.1 N NaOH, dried and tested in the PFC assay. 

Anti-Thy-1 PFC Assay. Fuji and Milgrom (4) originally developed an in vitvo PFC assay which detected Thy-1 alloantigen on whole thymocytes. A modified version, used here, has previously been described in detail and found to be effective for measuring the immune response to isolated glycolipid and glycoprotein Thy-1 alloantigens (3). 

Gangliosides labeled by intracranial injection of l-t(C]ManNAc were extracted from 1 g of brain, applied with solvent 1 (vertically) and solvent 2 (horizontally). Assayed fractions indicated by numbers (and ganglioside abbreviations) correspond to the PFC assay results in Figure 4. Fraction 6 refers to the area surrounding all the assayed spots (15). 

Gangliosides assayed were eluted from the plate shown in Figure 3, diluted, and the amount derived from 0.1 g of brain was added to the PFC assay cultures. Values are the mean standard error of five cultures. Application of the Student t test to the standard errors for the samples gave p values less than 0.05 when compared with the Thy-l-active fraction. Positive control for the anti-Thy-1.2 PFC assay was a column GM1 fraction containing Thy-l glycolipid (15). 

Chromatography was done as described in Figure 3, using the gangliosides derived from 2 X 10s cells. Assayed fractions are labeled with numbers that correspond to the anti-Thy-1 PFC assay in Figure 6. Number 10 refers to the area surrounding the spots that were assayed (15,). 

Figure 8. Anti-Thy-I PFC assay for allogenic specificity of brain and lymphoma Thy -l...

Measurement of a TDAR in vitro is another variation of the PFC assay. This approach was originally developed by Mishell and Dutton (1967). Sple-nocytes are treated in vitro with test compound or vehicle and are sensitized with sRBC for approximately 4-5 days. Following the incubation period, the cultures are then resuspended in agar with sRBC and complement. As in the PFC assay, the number of antibody-producing cells is determined by counting the number of plaques. The Mishell-Dutton assay requires less test substance compared to in vivo approaches and thus lends to the possibility of testing multiple compounds concurrently. 

Johnson CW, Williams WC, Copeland CB, DeVito MJ, Smialowicz RJ. Sensitivity of the SRBC PFC assay versus ELISA for detection of immunosuppression by TCDD and TCDD-like congeners. Toxicology 2000 156 1-11. 

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. 

PFC assays depend on division of a monomeric reporter enzyme into two separate inactive components that can reconstitute function upon association [Fig. 12.2(a)]. When these reporter fragments are fused to interacting proteins, reporter activity is reconstituted upon association of the interacting proteins. PFC strategies based on several enzymes including /3-galactosidase,... 

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. 

The triphasic response to selenium is similar in fact to antigenic stimulation of tumor transplants. The dose response effect of Se when added to the diets of mice or guinea pigs for immune enhancement appears to be 1-2 ppm as indicated by the PFC assay performed with mice and the DNCB challenge of guinea pigs. When selenium is administered by injection, the immune response is measured by the hemagglutination titers and is dose related as well... 

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