A new vine snake (Reptilia, Colubridae, Oxybelis) from Peru and redescription of O. acuminatus

The Brown Vine Snake, Oxybelis aeneus, was until recently considered a single species, distributed from southern Arizona through the Neotropics into southeastern Brazil. However, newly conducted research restructured the species with a substantial taxonomic revision, recognizing five additional taxa (i.e. O. koehleri, O. microphthalmus, O. potosiensis, O. rutherfordi, O. vittatus) in this species complex. This revision focused on populations in North America, Central America, and northern South America while neglecting the southern portion of its distribution. Here, we examine the taxonomic history of the complex and use it along with specimen data to resurrect O. acuminatus from southeastern Brazil. Finally, we describe a new species from the Peruvian Amazon based on morphological characters. This work increases the species diversity of the O. aeneus complex to eight, and we expect further increases in biodiversity discoveries with continued exploration of the New World vine snakes

Antigen-binding properties of monoclonal antibodies reactive with EBNA1 and use in immunoaffinity chromatography.

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) was overexpressed and purified from Escherichia coli. Mouse monoclonal antibodies (mAbs) were prepared that react with EBNA1. Eleven high affinity mAbs were recovered. Nine mAbs are isotype IgG (all subisotype IgG(1)) and two mAbs are isotype IgM. All mAbs react strongly with EBNA1 in an ELISA assay while only one mAb (designated 1EB6) fails to react in a Western blot assay. The epitopes for these mAbs were mapped to seven different regions, providing good coverage of the entire EBNA1 protein. The mAbs had differing affinity for an EBNA1/DNA complex with four mAbs able to supershift the complex completely. All mAbs can immunoprecipitate EBNA1 from E. coli overexpressing EBNA1. A modified ELISA assay, termed ELISA-elution assay, was used to screen for mAbs that release EBNA1 in the presence of a low molecular weight polyhydroxylated compound (polyol) and a nonchaotropic salt. MAbs with this property, termed polyol-responsive (PR)-mAbs, allow gentle elution of labile proteins and protein complexes. Four mAbs are polyol-responsive with two showing usefulness in gentle immunoaffinity chromatography. Purification with these PR-mAbs may be useful in purifying EBNA1 complexes and elucidating EBNA1-associated proteins. This panel of anti-EBNA1 mAbs will advance the study of EBV by providing new tools to detect and purify EBNA1

Immunoaffinity chromatography using PR-mAbs.

<p>The soluble fraction of whole cell extract from <i>E. coli</i> overexpressing EBNA1 was added to A) mAb 1EB2-Sepharose column and B) mAb 3EB7-Sepharose column, washed, and eluted with TE+0.75 M AS+40% PG. Coomassie blue stained gels are shown.</p

Ability of mAbs to bind EBNA1/DNA complex.

<p>MAbs were tested by EMSA to determine binding efficiency to a pre-formed EBNA1/DNA complex. Purified EBNA1 protein was bound to a ³²P-labelled oligonucleotide encoding one palindromic EBNA1 binding site. Equal concentrations of purified mAb were added to each reaction and allowed to incubate. The ability of the mAbs to shift the EBNA1/DNA complex was analyzed by gel electrophoresis. The first lane does not include any protein. The second lane contains only DNA and EBNA1. MAbs are listed by the last number in their name.</p

Summary of anti-EBNA1 murine mAbs.

<p>WB: Western blot, WB': Western blot signal with endogenous EBNA1, ELISA: the average ELISA signal when adding equal concentrations of purified mAb to an EBNA1-coated well, PR-mAb: polyol-responsive mAb, IP: immunoprecipitation, Supershift: ability to bind to EBNA1/DNA complex and change its mobility during non-denaturing gel electrophoresis, NYT: not yet tested. Epitope was determined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004614#pone-0004614-g001" target="_blank">Fig. 1</a>.</p>*<p>particularly high affinity for endogenous EBNA1.</p>a<p>responsive to salt alone.</p

Immunoprecipitation of EBNA1.

<p><i>E. coli</i>-expressed EBNA1 (∼50 kDa) was isolated from whole cell extract by mixing with equal quantities of mAb-rProteinA-agarose resin. Reactions were allowed to incubate for 1 h, washed to remove nonspecifically bound proteins, and eluted with SDS sample buffer. MW is molecular weight. The Western blot was probed with rat mAb 2B4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004614#pone.0004614-Grasser1" target="_blank">[23]</a>.</p

Polyol-responsiveness of anti-EBNA1 mAbs.

<p>A) ELISA-elution assay results to determine polyol-responsiveness. A standard ELISA protocol was performed with the addition of an intermediate step. After the anti-EBNA1 mAbs are bound to the EBNA1-coated well, either a control buffer (TE: 50 mM Tris-HCl, pH 7.9+0.1 mM EDTA) or a salt-polyol buffer (AS/PG: TE buffer+1 M ammonium sulfate+40% propylene glycol or NaCl/PG: TE buffer+1 M NaCl+40% propylene glycol) was added to the wells. MAbs that are polyol-responsive will have lower affinity for their antigen in the salt-polyol buffer which will result in a decrease in signal after the addition of a secondary antibody and the ELISA substrate. The experiment was done in triplicate. B) Detailed analysis of mAb 3EB7 response to various combinations of ammonium sulfate (AS) and propylene glycol.</p

A new vine snake (Reptilia, Colubridae, Oxybelis) from Peru and redescription of O. acuminatus

The Brown Vine Snake, Oxybelis aeneus, was until recently considered a single species, distributed from southern Arizona through the Neotropics into southeastern Brazil. However, newly conducted research restructured the species with a substantial taxonomic revision, recognizing five additional taxa (i.e. O. koehleri, O. microphthalmus, O. potosiensis, O. rutherfordi, O. vittatus) in this species complex. This revision focused on populations in North America, Central America, and northern South America while neglecting the southern portion of its distribution. Here, we examine the taxonomic history of the complex and use it along with specimen data to resurrect O. acuminatus from southeastern Brazil. Finally, we describe a new species from the Peruvian Amazon based on morphological characters. This work increases the species diversity of the O. aeneus complex to eight, and we expect further increases in biodiversity discoveries with continued exploration of the New World vine snakes