Use of Whole Sheep Red Blood Cells in ELISA to Assess ImmunosuppressionIn Vivo

Abstract

An enzyme-linked immunosorbent assay (ELISA) using whole sheep red blood cells (SRBC) has been reported as one of the methods for detecting a T-lymphocyte-dependent antibody response. However, it has not been widely used because of SRBC problems such as the weak attachment to ELISA plates, specificity and shortterm stability. The objectives of this study were to address these issues and to validate the SRBC-specific antibody response assay. Male Sprague-Dawley rats were bled after 6 days of SRBC immunization. In our new procedure, glutaraldehyde was added before discarding the supernatant of inoculated SRBC suspension to attach SRBC firmly to the plate, while in the original method it was added after discarding. As a result, the attached SRBC was maintained throughout the ELISA procedures. No interference was observed in the titration curve of IgM and IgG antibodies in rats and IgM-antibody in mice when control sera were analyzed to evaluate specificity of this method. The short-term stability of SRBC was overcome by using the different lots of SRBC. They provided antibody titers, which were consistent with those measured using the same lot for immunization. In addition, cyclophosphamide, cyclosporine, prednisolone and methotrexate, well-known immunosuppressive agents, were tested to confirm the applicability of the improved ELISA method to detect the T-lymphocyte-dependent antibody response. All four compounds inhibited the IgM antibody responses dose-dependently. These results demonstrate that the improved whole SRBC-ELISA method provides reproducible and reliable results in the T-lymphocyte-dependent antibody response assay.

Interaction of Plasmodium vivax Tryptophan-rich Antigen PvTRAg38 with Band 3 on Human Erythrocyte Surface Facilitates Parasite Growth

“…Erythrocyte Binding Assay by Cell-ELISA-ELISA-based erythrocyte binding assay was performed as described earlier (9,17). Briefly, ϳ1 million human erythrocytes were added to each well of a 96-well microtiter plate.…”

Augmentation of expression of immunocytes’ functions by seed extract of Ziziphus mauritiana (Lamk.)

“…Hence, comparative study of levamisole and ZMS was planned where effect on Th-1, Th-2 cytokines, B cells and macrophages was studied in SRBC immunized Swiss albino mice. SRBC mediated immune response is a highly sensitive indicator of immunological integrity and requires coordinated interaction of various immune system cells (Koganei et al, 2007) and our study is a mirror of this coordination between the various immune system cells.…”

Modulation of in vivo immunoglobulin production by endogenous histamine and H1R and H2R agonists and antagonists

“…In the present study, we investigated the production of total serum Igs, total serum IgM and total serum IgG to SRBC, a T lymphocyte-dependent test antigen [7,10] modulated by endogenous histamine released from effector cells. Our investigation consisted of control (untreated), histamine, H1R agonist (HTMT), H2R agonist (amthamine), H1R antagonist (pheniramine) and H2R antagonist (ranitidine) treated groups.…”

“…To determine the SRBC-Igs, SRBC-IgM and SRBCIgG response, the whole SRBC enzyme linked immunosorbent assay (ELISA) [7] was also carried out on polystyrene plates with some modifications. Briefly, polystyrene MaxiSorp immunoplates were coated with an SRBC suspension (5 × 10 6 cells/100 µl PBS).…”