A biosynthetic antibody binding site, which incorporated the variable domains of anti-digoxin monoclonal antibody 26-10 in a single polypeptide chain (Mr = 26,354), was produced in Escherichia cofi by protein engineering. This variable region fragment (Fv) analogue comprised the 26-10 heavy-and light-chain variable regions (VH and VL) connected by a 15-amino acid linker to form a single-chain Fv (sFv). The sFv was designed as a prolyl-VH-(linker)-VL sequence of 248 amino acids. A 744-base-pair DNA sequence corresponding to this sFv protein was derived by using an E. colt codon preference, and the sFv gene was assembled starting from synthetic oligonucleotides. The sFv polypeptide was expressed as a fusion protein in E. colt, using a leader derived from the trp LE sequence. The sFv protein was obtained by acid cleavage of the unique Asp-Pro peptide bond engineered at the junction of leader and sFv in the fusion protein [(leader)-Asp-Pro-VH-(linker)-VL]. After isolation and renaturation, folded sFv displayed specificity for digoxin and related cardiac glycosides similar to that of natural 26-10 Fab fragments. Binding between afirmity-purified sFv and digoxin exhibited an association constant [Ka = (3.2 ± 0.9) x 107 M -1] that was about a factor of 6 smaller than that found for 26-10 Fab fragments [K. = (1.9 @ 0.2) x 108 M 'I under the same buffer conditions, consisting of 0.01 M sodium acetate, pH 5.5/0.25 M urea.It is known that antigen binding fragments of antibodies (1,2) can be refolded from denatured states with recovery of their specific binding activity (3)(4)(5)(6). The smallest such fragment that contains a complete binding site is termed Fv, consisting of an Mr 25,000 heterodimer of the VH and VL domains (2, 5-11). Givol and coworkers were the first to prepare an Fv by peptic digestion of murine IgA myeloma MOPC 315 (2). However, subsequent development of general cleavage procedures for Fv isolation has met with limited success (7-11). As a result, the Mr 50,000 Fab (1) has remained the only monovalent binding fragment used routinely in biomedical applications.An Fv analogue was constructed in which both heavy-and light-chain variable domains (VH and VL) were part of a single polypeptide chain. Synthetic genes for the 26-10 anti-digoxin VH and VL regions were designed to permit their connection through a linker segment, as well as other manipulations (12,13 MATERIALS AND METHODSModel Antibody. The digoxin binding site of the IgG2a,K monoclonal antibody 26-10 has been analyzed by MudgettHunter and colleagues (14-16). The 26-10 V region sequences were determined from both protein sequencing (17) (14) and has a well-defined specificity profile (15) (Fig. 1).Gene Synthesis. Design of the 744-base sequence for the synthetic sFv gene was derived from the sFv protein sequence by choosing codons preferred by E. coli (25). Synthetic genes encoding the trp promoter-operator, the modified trp LE leader peptide (MLE), and VH were prepared largely as described (26). The gene encoding VH was assembled from 46...

The Glucatell (1r3)-b-D-glucan (BG) detection assay (Associates of Cape Cod) was studied as a diagnostic adjunct for invasive fungal infections (IFIs). On the basis of findings from a preliminary study of 30 candidemic subjects and 30 healthy adults, a serum BG level of у60 pg/mL was chosen as the cutoff. Testing was performed with serial serum samples obtained from 283 subjects with acute myeloid leukemia or myelodysplastic syndrome who were receiving antifungal prophylaxis. At least 1 serum sample was positive for BG at a median of 10 days before the clinical diagnosis in 100% of subjects with a proven or probable IFI. IFIs included candidiasis, fusariosis, trichosporonosis, and aspergillosis. Absence of a positive BG finding had a 100% negative predictive value, and the specificity of the test was 90% for a single positive test result and у96% for у2 sequential positive results. The Glucatell serum BG detection assay is highly sensitive and specific as a diagnostic adjunct for IFI.The mortality rate for invasive fungal infections in neutropenic subjects is 50% for subjects with Candida infection [1,2] and may approach 100% for those with invasive aspergillosis [3,4], fusariosis [5], or trichosporonosis [6]. Early diagnosis of invasive fungal infection in neutropenic subjects has the potential to increase antifungal therapeutic response, but meaningful diagnostic tests have proven to be elusive. Histopathologic demonstration of organisms in tissue specimens or growth of fungal agents in culture media is still the

Amino acid sequences of two tryptic peptides derived from enriched bovine osteogenic protein preparations revealed considerable homology to two members of the TGF‐beta (transforming growth factor beta) supergene family, DPP (decapentaplegic protein) of Drosophila and Vg‐1 (vegetal protein) of Xenopus. Building upon this information we constructed a synthetic consensus gene to use as a probe to screen human genomic libraries. This resulted in the isolation of three interrelated genes. Among these were BMP‐2b and BMP‐3 which have recently been described by others. The third gene, termed OP‐1 (osteogenic protein one), is new and was subsequently shown to encode the human homolog of a major component of bovine osteogenic protein. The genomic clones were used to isolate the corresponding complementary DNA (cDNA) clones. Sequence analysis indicates that OP‐1 is a relative of the murine Vgr‐1 (Vg‐1 related gene). This report describes the cDNA structure and putative amino acid sequence of OP‐1.

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Endotoxin-neutralizing protein (ENP) of the horseshoe crab is one of the most potent neutralizers of endotoxins [bacterial lipopolysaccharide (LPS)]. Here, we report on the interaction of LPS with recombinant ENP using a variety of physical and biological techniques. In biological assays (Limulus amebocyte lysate and tumour necrosis factor-a induction in human mononuclear cells), ENP causes a strong reduction of the immunostimulatory ability of LPS in a dose-dependent manner. Concomitantly, the accessible negative surface charges of LPS and lipid A (zeta potential) are neutralized and even converted into positive values. The gel to liquid crystalline phase transitions of LPS and lipid A shift to higher temperatures indicative of a rigidification of the acyl chains, however, the only slight enhancement of the transition enthalpy indicates that the hydrophobic moiety is not strongly disturbed. The aggregate structure of lipid A is converted from a cubic into a multilamellar phase upon ENP binding, whereas the secondary structure of ENP does not change due to the interaction with LPS. ENP contains a hydrophobic binding site to which the dye 1-anilino-8-sulfonic acid binds at a K d of 19 lM, which is displaced by LPS. Because lipopolysaccharide-binding protein (LBP) is not able to bind to LPS when ENP and LPS are preincubated, tight binding of ENP to LPS can be deduced with a K d in the low nonomolar range. Importantly, ENP is able to incorporate by itself into target phospholipid liposomes, and is also able to mediate the intercalation of LPS into the liposomes thus acting as a transport protein in a manner similar to LBP. Thus, LPS-ENP complexes might enter target membranes of immunocompetent cells, but are not able to activate due to the ability of ENP to change LPS aggregates from an active into an inactive form.Keywords: endotoxins; IR spectroscopy; LALF; LPS; tumor necrosis factor. Lipopolysaccharides (LPS) represent the main amphiphilic component of the outer leaflet of the outer membrane of Gram-negative bacteria. They are often referred to as endotoxins due to their ability to induce a variety of biological effects in mammals, in particular the production of proinflammatory cytokines [1]. At low endotoxin concentrations, the biological effects may be beneficial, as some cytokines such as tumour necrosis factor-a (TNFa) have been shown to possess antitumour activity. At higher endotoxin concentrations, however, the release of cytokines leads to an inflammatory response, eventually resulting in the septic shock syndrome [2].LPS consists of a sugar moiety comprising the O-specific chain, outer core and inner core covalently linked to the hydrophobic moiety of LPS, lipid A, which anchors the LPS molecule to the membrane [2]. The sugar moiety type and length depends on the strain of bacteria. Smooth strains have varying length O-specific chains, while rough mutants have no O-specific chain (Ra) and can also lack parts of the core regions (Rb-Re). Deep rough mutant LPS (LPS Re) comprises the inner core, which co...