Monoclonal antibody 4C1 specifically binds to and neutralizes the most potent neurotoxin (AahII) of the scorpion Androctonus australis. The cDNAs encoding the variable regions of this antibody were isolated by PCR-mediated cloning. A single-chain Fv gene was engineered and expressed in Escherichia coli. The recombinant protein had neutralizing activity similar to that of the intact antibody in vitro and in vivo. We have thus neutralized the pharmacological and biological properties of a scorpion neurotoxin with a single-chain Fv, which opens new perspectives for the treatment of envenomizations.z 1999 Federation of European Biochemical Societies.
Native rabies virus glycoprotein (RVGvir) is a trimeric, membrane-anchored protein that has been shown to interact with the p75 NTR neurotrophin receptor. In order to determine if the RVG trimeric oligomerization state is required for its binding with p75 NTR , different soluble recombinant molecules containing the entire RVG ectodomain (RVGect) were expressed alone or fused at its C terminus to the trimerization domain of the bacteriophage T4 fibritin, termed 'foldon'. The oligomerization status of recombinant RVG was investigated using sedimentation in sucrose gradient and p75 NTR binding assays. It was found that, in the absence of the fibritin foldon, recombinant RVGect forms unstable trimers that dissociate into monomers in a concentration-dependent manner. C-terminal fusion with the foldon induces stable RVG trimerization, which is concentration-independent. Furthermore, the fibritin foldon maintains the native antigenic structure of the carboxy part of RVGect. Cell binding experiments showed that RVG trimerization is required for efficient interaction with p75 NTR . However, the exact mode of trimerization appears unimportant, as trimeric recombinant RVGect (fused to the fibritin foldon) and RVGvir both recognize p75 NTR with similar nanomolar affinities, as shown by surface plasmon resonance experiments. Altogether, these results show that the C-terminal fusion of the RVG ectodomain with the fibritin foldon is a powerful way to obtain a recombinant trimeric native-like structure of the p75 NTR binding domain of RVG.
Serologic detection of Toxoplasma gondiiIgG antibodies is widely accepted as a means to determine immune status and susceptibility to Toxoplasma infection during pregnancy. However, current commercial kits present some drawbacks, such as a requirement for whole-parasite antigen preparation or interassay variability. To address these problems, the purpose of this study was to produce a whole sequence of the recombinant T. gondii SAG1 antigen (rSAG1) to assess its diagnostic performance in Toxoplasma IgG screening and to explore a saliva-based method as a noninvasive alternative to serum-based testing. rSAG1 was expressed in recombinant bacteria as inclusion bodies, purified through one-step affinity chromatography, and refolded in native form by dialysis. A large amount was obtained, and the specific antigen immunoreactivity was confirmed by immunoblotting. Two rSAG1-based enzyme-linked immunosorbent assays (ELISAs) applied to paired serum and saliva samples were designed. The rSAG1-based ELISA evaluation consisted of testing intrinsic sensitivity and specificity of 49 serum samples from patients immune to toxoplasmosis and 42 serum samples from nonimmune controls identified by routinely used kits. To assess agreement between serum-based and saliva-based tests, the positive percent agreement (PPA) and negative percent agreement (NPA) between the 2 tests were estimated. The rSAG1 serum-based ELISA detected specific IgG with 100% sensitivity and specificity. The PPA and NPA between the serum-based and saliva-based tests varied according to the selected optical density threshold in saliva. Thus, for a selected cutoff of 0.14, the PPA was 100% and the NPA was 88.1%, whereas for a selected cutoff of 0.29, the PPA was 67.3% and the NPA was 100%.
ABSTRACTHuman visceral leishmaniasis (VL) is routinely diagnosed by detecting IgG that specifically binds toLeishmaniaantigens. The enzyme-linked immunosorbent assay (ELISA) remains a widely used method. However, the biggest challenge remains the choice of antigen with the highest specificity and sensitivity. This study is aimed at assessing the diagnostic performances of crudeLeishmaniahistone (CLH) protein-based ELISAs in Mediterranean VL patients. The CLH proteins were biochemically purified from promastigote nuclear extracts. Their reactivities were analyzed by Western blotting (WB) using rabbit polyclonal antibodies againstLeishmaniarecombinant histones and sera from VL patients, respectively. Then, the diagnostic potential of CLH proteins was validated by the CLH-based ELISA using 42 infantile VL patients' sera and 70 control subjects. The CLH-based ELISA performance was compared to that of the solubleLeishmaniaantigen (SLA)- and the recombinant K39 (rK39)-based ELISAs. Analysis of the WB profile with the use of polyclonal antibodies confirmed the histone origin of low molecular mass proteins (12 to 16 kDa). All VL samples tested presented antibodies reacting against different antigen fractions; however, recognition patterns were different depending on the reactivity of each serum. CLH-based ELISA showed an excellent ability to discriminate between VL cases and healthy controls (97.6% sensitivity and 100% specificity). It had a diagnostic performance similar to that of rK39-based ELISA (97.6% sensitivity and 97.1% specificity,P= 0.5) and a better serodiagnosis accuracy than the SLA-based ELISA (85.7% sensitivity and 90% specificity,P< 0.05). Therefore, crudeLeishmaniahistone extract could be a valuable antigen for clinical use.
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