Recombinant Leishmania eukaryotic elongation factor-1 beta protein: A potential diagnostic antigen to detect tegumentary and visceral leishmaniasis in dogs and humans.

The laboratorial diagnosis of leishmaniasis is based on parasitological methods, which are invasive, present high cost, require laboratorial infrastructure and/or trained professionals; as well as by immunological methods, which usually present variable sensitivity and/or specificity, such as when they are applied to identify asymptomatic cases and/or mammalian hosts presenting low levels of antileishmanial antibodies. As consequence, new studies aiming to identify more refined antigens to diagnose visceral (VL) and tegumentary (TL) leishmaniasis are urgently necessary. In the present work, the Leishmania eukaryotic elongation factor-1 beta (EF1b) protein, which was identified in L. infantum protein extracts by antibodies in VL patients' sera, was cloned and its recombinant version (rEF1b) was expressed, purified and tested as a diagnostic marker for VL and TL. The post-therapeutic serological follow-up was also evaluated in treated and untreated VL and TL patients, when anti-rEF1b antibody levels were measured before and after treatment. Results showed that rEF1b was highly sensitive and specific to diagnose symptomatic and asymptomatic canine VL, as well as human TL and VL. In addition, low cross-reactivity was observed when sera from healthy subjects or leishmaniasis-related diseases patients were tested. The serological follow-up showed also that rEF1b-specific antibodies declined significantly after treatment, suggesting that this protein could be also evaluated as a prognostic marker for human leishmaniasis.

A protein chip approach for high-throughput antigen identification and characterization.

Proteomics research in humans and other eukaryotes demands a large number of high-quality mAbs. Here, we report a new approach to produce high-quality mAbs against human liver proteins using a combined force of high-throughput mAb production and protein microarrays. After immunizing mice with live cells from human livers, we isolated 54 hybridomas with binding activities to human cells and identified the corresponding antigens for five mAbs via screening on a protein microarray of 1058 unique human liver proteins. Finally, we demonstrated that using the five mAbs we could characterize the expression profiles of their corresponding antigens by using tissue microarrays. Among them, we discovered that eIF1A expressed only in normal liver tissues, not in hepatocellular carcinoma in humans.

Assignment of the beta-subunit of wheat eIF2 by protein and DNA sequence analysis and immunoanalysis.

Wheat germ initiation factor 2 (eIF2), like mammalian and yeast eIF2, contains three nonidentical subunits. The estimated molecular weights for the wheat subunits are 38,000 (p38), 42,000 (p42), and 50,000 (p50). Peptide sequence was obtained for the p38 subunit of wheat eIF2 and the resulting amino acid sequence suggested that it was actually the equivalent of the mammalian beta-subunit. A wheat sprout cDNA expression library was screened with antibody affinity purified to the p38 subunit. The DNA sequence of the clones obtained also indicated that the p38 subunit was the equivalent to the mammalian beta-subunit. The wheat p38 subunit was then expressed in Escherichia coli and antibodies raised to the purified recombinant protein. Only the p38 subunit of purified wheat germ eIF2 reacted with the antisera. The p38 subunit of wheat eIF2 is therefore the equivalent of mammalian eIF2beta.