Antibody-forming cell assay

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. 

Lakew M, Nordstrom I, Czerkinsky C, Quiding-Jarbrink M. Combined immunomag-netic cell sorting and ELISPOT assay for the phenotypic characterization of specific antibody-forming cells. J Immunol Methods 1997 203 193-198. 

The splenic foci technique was developed independently in two laboratories (Kennedy et al., 1965 Playfair et al., 1965), with the same goal to obtain an assay for the precursors of antibody-forming cells. In principle, a small inoculum of donor spleen cells and antigen (red cells) is injected into lethally irradiated syngeneic mice. After 5 or more days the host spleens are cut into many slices, each of which is further cut into separate pieces. These are placed on petri dishes containing a layer of agar with embedded red cells. Antibody is allowed to diffuse out and complement-dependent lysis is observed around some pieces. In this way, it is possible to construct a three-dimensional map of foci of antibody-producing cells. 

Several immune function assays measuring lymphocyte proliferation and antibody production to experimental antigens have been developed as surrogates for measuring protective immune responses. Mice exposed to carbon dust inhalation (2-4 mg/m ) had a decreased number of antibody-forming cells in the spleen that was proportional to exposure time (64). In local mediastinal lymph nodes... 

Modifications are made also in order to use this reaction to conjugate nucleosides to erythrocyte surfaces, allowing use of the coated cells as targets for assays of antibody-forming splenic lymphocytes. Nucleoside, 10-20 mg, is oxidized in 1.5 ml of 0.1 M sodium periodate in 0.15M NaHCOa for 20 min at room temperature the reaction is stopped by the addition of 15 /zl of ethylene glycol. Sheep erythrocytes are washed twice with 0.15 M NaHCOs, and 0.5 ml of packed cells is then suspended in 2.0 ml of the bicarbonate solution in a40-ml centrifuge tube. The oxidized nucleoside is added dropwise to the cell suspension and the mixture is kept at room temperature for 15 min. fert-Butylamine borane (Aldrich Chemical Co.), 100 mg in 5 ml of 0.15 M NaHCOs, is added. The suspension is kept at room temperature for 3 min, and the tube is then quickly filled with bicarbonate solution and centrifuged at 1500 rpm for 10 min. 

Antibodies are a powerful and essential tool in scientific laboratories being used in an array of applications such as immuno-histochemistry, immunobloting, immunoprecipitation and enzyme-linked immunosorbent assays (ELISA). The different sources for antibodies include polyclonal antisera from immunized animals and monoclonal antibodies from cells in culture or from ascites in animals. Both polyclonal and monoclonal antibodies have their advantages, and or disadvantages, but in general the production of monoclonal antibodies is more time consuming and requires tissue culture facilities and skills. The use of either monoclonal or polyclonal antibodies in some of the applications may require that the antibody is in a purified form. They can be purified by a variety of methods described in the next few chapters. The availability of commercially available kits primarily designed for the purification of IgG and IgM classes of antibodies derived from all common animal species should also be mentioned. 

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. 

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. 

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