Background: Two enzyme immunoassays, ELISPOT and sandwich ELISA, were compared in order to evaluate the production of interferon gamma (IFN-γ) in peripheral blood mononuclear cell (PBMC) cultures from Hypoderma (Diptera: Oestridae) infested cattle.
Methods: Cell cultures from Hypoderma-infested (sensitized) and uninfested cattle (non-sensitized) were stimulated with the mitogen phytohemaglutinin A (PHA) and with different H. lineatum antigens, crude larval extract (CLE) and its purified fractions (hypodermin A, B and C). IFN-γ secreting cells (SC) were detected using an ELISPOT test, whereas theIFN-γ levels presented in supernatants from parallel cell cultures were measured by a sandwich ELISA; the same bovine specific IFN-γ antibodies were employed in both tests.
Results: The addition of H. lineatum antigens had an immunomodulatory effect on PBMC cells from both infested and uninfested cattle, characterized by suppression in the production of IFN-γ. ELISPOT results showed that hypodermin B was the antigen with major immunosuppressive effect on non-sensitized cultures, while CLE had the strongest impact on previously sensitized cultures. Our results revealed that the ELISPOT showed a high sensitivity allowing the determination of IFN-γ-SC frequencies in non-stimulated cultures; in contrast, the sandwich ELISA was not useful for detecting IFN-γ levels in parallel culture supernatants.
Conclusion: The ELISPOT test allows an accurately determination of the frequency of IFN-γ-SC in ex vivo PBMCs without the need for extensive re-stimulation in vitro with antigen or mitogen over long periods of time.
Keywords: Cattle-arthropoda, hypoderma, IFN-γ, ELISPOT, ELISA, cellular responses