Overexpression and purification of a recombinant chimeric HIV type 2/HIV type 1 envelope peptide and application in an accelerated immunobased HIV type 1/2 antibody detection system (AIBS): a new rapid serological screening assay.

A chimeric HIV-2/HIV-1 envelope sequence containing an immunodominant region of HIV-2 gp36 and the corresponding region of HIV-1 gp41 was constructed and overexpressed in Escherichia coli. The recombinant product (rp21/18) was purified and applied in a novel antibody-screening assay. Characteristics in the design of this new principle are as follows: (1) the overall assay time is about 30 min; (2) the assay procedure includes three manipulation steps; and (3) the test shows a reliable performance with respect to sensitivity and specificity. The diluted serum sample and the protein G-horseradish peroxidase conjugate are added simultaneously into a coated (hybrid antigen HIV-1/2) and blocked microtiter plate well. The in-batch incubation of serum sample with protein G-horseradish peroxidase saves two manipulation steps that are normally necessary in the five-step procedure of a classical ELISA. AIBS was evaluated with commercially available seroconversion panels and with random negative serum samples from a blood bank. Seroconversion results demonstrated that AIBS has equivalent sensitivity to ELISAs and the third generation assays. The specificity was determined on a total blood donor population of 5012 (Red Cross Vienna, Austria). The repeat reactive rates for donor population were 0.02%. AIBS represents a general immunometric system (not only HIV antibodies). The entire assay procedure of AIBS evaluated for HIV-1/2 screening, including result reporting, can be performed automatically by several commercially available systems. Depending on these systems AIBS is potentially useful in laboratories or blood banks that have both high- and low-volume testing.

Indications for the presence of antibodies cross-reactive with HTLV-I/II, but not HIV, in patients with myelodysplastic syndrome.

Serological evidence is presented for the fact that patients with the myelodysplastic syndrome exhibit a statistically significant reactivity in confirmatory assays for antibodies to human T-lymphotropic viruses types I and II (HTLV-I/II). This antibody reactivity, evident by indirect immunofluorescence and Western blot, was not confined to HTLV core antigens but extended to native and recombinant envelope glycoproteins. The effect was also observed in cases of acute myeloic leukemia, albeit to a lesser degree. It was essentially absent from patients with chronic myeloic leukemia or lymphocytic leukemias and healthy or multitransfused controls. No antibodies to human immunodeficiency viruses types 1 or 2 were detected in any of the specimens. The investigated clinical population had no known risk factor for retroviral infection other than a history of multiple platelet transfusions, and none of the specimens was seropositive for HTLV-I or HTLV-II according to recommended criteria. The cause of this cross-reactivity remains to be determined.

Purification and properties of an extreme thermostable glutamate dehydrogenase from the archaebacterium Sulfolobus solfataricus.

Glutamate dehydrogenase (L-glutamate:NAD(P)+ oxidoreductase, deaminating, EC 1.4.1.3.) of the extreme thermophilic archaebacterium Sulfolobus solfataricus was purified to homogeneity by (NH4)2SO4 fractionation, anion-exchange chromatography and affinity chromatography on 5'-AMP-Sepharose. The purified native enzyme had a Mr of about 270,000 and was shown to be a hexamer of subunit Mr of 44,000. It was active from 30 to 95 degrees C, with a maximum activity at 85 degrees C. No significant loss of enzyme activity could be detected, either after incubation of the purified enzyme at 90 degrees C for 60 min, or in the presence of 4 M urea or 0.1% SDS. The enzyme was catalytically active with both NADH and NADPH as coenzyme and was specific for 2-oxoglutarate and L-glutamate as substrates. With respect to coenzyme utilization the Sulfolobus solfataricus glutamate dehydrogenase resembled more closely the equivalent enzymes from eukaryotic organisms than those from eubacteria.