Colorimetric diagnosis of prolonged bluetongue viremia in sheep, using an enzyme-linked oligonucleotide sorbent assay of amplified viral nucleic acids.

Each of 5 US-origin serotypes of bluetongue virus (BTV) was inoculated into a separate pair of sheep. The duration of each animal's ensuing viremia was monitored, using a BTV serogroup-specific nested polymerase chain reaction (PCR) method and an embryonating chicken egg (ECE) inoculation procedure. Mean duration of viremia was 100 and 38 days for the PCR and ECE methods, respectively. This difference was significant (P < 0.001) and documents a more prolonged viremia in virus-exposed sheep than has been reported. A dual internal oligonucleotide solution hybridization procedure was developed for the rapid (2 hours) colorimetric detection and identification of BTV-specific PCR products. This enzyme-linked oligonucleotide sorbent assay (ELOSA) relied on annealing of separate biotinylated and fluoresceinated probes to the amplified BTV nucleic acid; these complexes were captured on streptavidin-coated microtitration wells and were detected, using a horseradish peroxidase-labeled antifluorescein antibody conjugate. End-point dilution analyses of PCR products indicated that the ELOSA was more sensitive than gel electrophoretic or comparable colorimetric slot-blot hybridization techniques. The BTV PCR-ELOSA system represents a more sensitive and expeditious means of diagnosing BTV-induced viremia than does the ECE procedure currently used. The combination of ELOSA with PCR should facilitate practical application of nucleic acid technology to diagnostic veterinary medicine.

Comparison of spot-blot and microtitre plate methods for the detection of HIV-1 PCR products.

We have compared spot-blot methodology with a recently developed rapid microtitre plate assay for the specific detection of HIV-1 PCR products. We have studied blood specimens isolated from HIV-1 infected individuals (48 asymptomatic and 56 symptomatic patients). Mononuclear cells were isolated, lysed and processed for PCR. Both PCR product detection methods were carried out in parallel on all amplified samples. HIV-1 sequences were detected by spot-blot or microtitre plate hybridization in samples taken from 42/48 asymptomatic and 53/56 symptomatic subjects. Concordant results between the two detection methods were observed for 90 samples, with 81 positive and nine negative assays. On repeat evaluation of the 14 discordant samples, nine showed concordant positive results, near the limit of detection of the assay. Serial dilutions of ACH-2 cells were amplified, and the PCR products were detected using the microtitre plate assay, yielding semi-quantitative results. The sensitivity of this simple, rapid assay compares with that of more laborious DNA detection systems. This may become a useful tool in HIV-1 research and in the clinical care of seropositive individuals.

Effect of Plasmodium berghei on membranes of murine erythrocytes.

Membranes from normal and Plasmodium berghei-infected mice were analyzed to determine: 1) if any antigenic changes were present; and 2) the nature of these changes. Polyacrylamide gel electrophoresis (PAGE), immunological and biochemical analyses were performed on whole ghosts and glycoprotein fractions extracted from whole ghosts by chloroform-methanol. PAGE profiles and biochemical analyses revealed quantitative, but not qualitative, differences between membrane proteins and glycoproteins of normal and infected membranes. No antigenic changes were detected. The data presented here suggest that productive infection with P. berghei brings about an imbalance in the protein composition of the erythrocyte membrane, but does not result in the insertion of new proteins or glycoproteins.

Blocking induction of human cytotoxic T lymphocytes with anti-Ia xenoantiserum.

A xenoantiserum to human Ia antigens has been described that is capable of blocking not only stimulation in the mixed lymphocyte reaction (MLR) but also the induction of cytotoxic T lymphocytes (CTLs). Data from immunofluorescence as well as complement-dependent cytolytic assays indicate that the anti-Ia xenoantiserum is directed against B cell surface antigens. Inhibition of complement-dependent cytolysis with column fractions of B cell antigens and autoradiography of immune precipitates electrophoresed on sodium dodecyl sulfate (SDS)-polyacrylamide gels have established that the antigen detected by the xenoantiserum has characteristics of the human two-polypeptide Ia molecular complex. Allogeneic stimulator cells pretreated with anti-Ia at very low doses were unable to stimulate in the MLR and failed to induce CTLs. Neither anti-beta 2-microglobulin nor a non-HLA-associated antilymphocyte serum in similar dose ranges inhibited MLR or cell-mediated lympholysis (CML) assays. Absorption of anti-Ia xenoantiserum with B lymphoblasts, but not T lymphoblasts, removed inhibitory activity for both MLR and CML. Untreated third-party stimulator cells cocultivated with anti-Ia-pretreated stimulator cells provided stimulation in the MLR that apparently allowed partial recovery of CML against targets from the same donor as the anti-Ia-treated stimulator cells. Elimination of the helper effect, normally provided by MLR stimulation, may be one mechanism by which anti-Ia xenoantiserum prevents induction of CTLs.