A sensitive two-site enzyme immunoassay for the detection of rat interferon-gamma in biological fluids.

Two noncompeting monoclonal antibodies (mAbs) directed to rat interferon-gamma (IFN-gamma) were used in a sensitive two-site enzyme-linked immunosorbent assay (ELISA). This permitted quantitation of rat IFN-gamma in culture medium, and serum and was quicker and more sensitive than the conventional antiviral bioassay. A standard curve was linear between 0.1 (15 pg) and 10 (1,500 pg) U/ml. There was no reaction with rat IFN-alpha or -beta or human IFN-gamma, but mouse IFN-gamma was detected with a lower limit of sensitivity of 300 U/ml. Complement-dependent inactivation of rat IFN-gamma reduced the sensitivity of the ELISA approximately seven-fold but this could be overcome by adding ethylenediamine tetraacetic acid (EDTA, 10 mM) or other anticomplementary substances to the serum samples. IFN-gamma activity determined by ELISA and bioassay decreased in parallel upon heat denaturation and pH 2.5 treatment, but proteases caused a relatively greater reduction in biological activity.

Antiviral activity of recombinant rat interferon gamma in immunologically impaired and immunosuppressed rats.

Treatment of Wag/Rij rats with recombinant rat interferon gamma (rRIF-gamma) resulted in complete protection against a lethal pseudorabies virus (PRV) infection. To investigate whether the protection resulted from direct inhibition of virus replication or from a stimulation of immune mechanisms, we tested rRIF-gamma activity in naturally immunocompromised and artificially immunosuppressed rats. The antiviral effect of rRIF-gamma was not abolished in silica- and carrageenan-treated, phagocyte-depleted rats. Immunologically immature newborn and T cell-deficient nude rats were also protected under a regime of rRIF-gamma treatment as well as whole body gamma-irradiated rats. Sera of the protected rats were devoid of PRV-neutralizing antibodies. Our results indicate that the protective activity of rRIF-gamma is based on direct inhibition of virus replication; stimulation of the immune system is not required but may be responsible for protection upon challenge several weeks after infection.

Elongation factor Tu isolated from Escherichia coli mutants altered in TufA and tufB.

In a previous paper we described a number of Escherichia coli mutants resistant to the antibiotic kirromycin. These mutants are altered in both tufA and tufB, the genes coding for elongation factor Tu (EF-Tu). We have now isolated EF-Tu in a homogeneous form from the mutant strains and have studied its function in polypeptide synthesis. These EF-Tu preparations were examined in renaturation studies of Qbeta RNA replicase, described in another paper. In order to characterize the factor we have inactivated the tufB gene by insertion of bacteriophage Mu or by an amber mutation. This enabled us to isolate EF-Tu as a single gene product derived from tufA (designated EF-TuA in contrast to the tufB product, which is called EF-TuB). Kirromycin-resistant EF-TuA did not respond to addition of the antibiotic in three assays: [(3)H]GDP exchange with EF-Tu-GDP at 0 degrees C, in vitro translation of poly(U), and kirromycin-induced GTPase activity of EF-Tu. In contrast, wild-type EF-TuA responded normally to the antibiotic in these assays. One of our mutants (LBE 2012) harbors the kirromycin-resistant EF-TuA and an EF-TuB that is able to bind kirromycin. This binding does not cause inhibition of protein synthesis, indicating that EF-TuB from LBE 2012 is unable to reach the ribosome under these conditions. The two types of EF-Tu from this mutant are equal in size but differ by 0.1 pH unit in isoelectric point. In the soluble fractions of LBE 2012 cells they are present in approximately equal amounts. Our results also show that the tufB gene is not necessary for bacterial growth.