Enhancing the detection of Toxoplasma gondii via an anti-SAG1 scFv-alkaline phosphatase immunoconjugate.

The purpose of this study was to implement a fluorometric method for enhancing the detection sensitivity of Toxoplasma gondii in biological fluids. To address this challenge, we designed and produced a recombinant immunoconjugate tool based on a single-chain antibody fragment anti-T. gondii SAG1 antigen (scFvSG15) genetically fused to the bacterial alkaline phosphatase (AP) using 4-methyl-umbelliferyl-phosphate as fluorogenic substrate. The anti-SAG1 scFv-AP conjugate was fully bifunctional and was used successfully in different assays including immunoblot, fluorometric ELISA and direct immunofluorescence. The fluorometric immunoassay afforded an extremely low detection limit (1 tachyzoite/well), which was in agreement with the real-time PCR control test. The immunofluorescence imaging has provided captivating visual evidence of T. gondii detection. These results strongly suggest that the recombinant anti-SAG1-AP conjugate generated here might serve as useful and highly sensitive immunoassay probe to direct detect T. gondii in a one-step procedure, opening up new perspectives for diagnosis of toxoplasmosis.

Engineering of a recombinant trivalent single-chain variable fragment antibody directed against rabies virus glycoprotein G with improved neutralizing potency.

Human and equine rabies immunoglobulins are currently available for passive immunization against rabies. However, these are hampered by the limited supply and some drawbacks. Advances in antibody engineering have led to overcome issues of clinical applications and to improve the protective efficacy. In the present study, we report the generation of a trivalent single-chain Fv (scFv50AD1-Fd), that recognizes the rabies virus glycoprotein, genetically fused to the trimerization domain of the bacteriophage T4 fibritin, termed 'foldon' (Fd). scFv50AD1-Fd was expressed as soluble recombinant protein in bacterial periplasmic space and purified through affinity chromatography. The molecular integrity and stability were analyzed by polyacrylamide gradient-gel electrophoresis, size-exclusion chromatography and incubation in human sera. The antigen-binding properties of the trimeric scFv were analyzed by direct and competitive-ELISA. Its apparent affinity constant was estimated at 1.4 ± 0.25 × 10(9)M(-1) and was 75-fold higher than its monovalent scFv (1.9 ± 0.68 × 10(7)M(-1)). The scFv50AD1-Fd neutralized rabies virus in a standard in vitro and in vivo neutralization assay. We showed a high neutralization activity up to 75-fold compared with monovalent format and the WHO standard serum. The gain in avidity resulting from multivalency along with an improved biological activity makes the trivalent scFv50AD1-Fd construct an important reagent for rabies protection. The antibody engineering approach presented here may serve as a strategy for designing a new generation of anti-rabies for passive immunotherapy.

Design and characterization of a recombinant colorimetric SAG1-alkaline phosphatase conjugate to detect specific antibody responses against Toxoplasma gondii.

The purpose of this study was to design a novel in vitro tool by using recombinant protein technology to detect specific antibody responses against Toxoplasma gondii in a simple, rapid and highly sensitive reagent. The surface T. gondii SAG1 protein is an important immunodominant target, which provides a great interest as a diagnostic antigen. To further exploit its immunodetection capacity, in the present study, the full length sag1 gene was inserted into the pLIP6 prokaryotic expression vector so as to produce a SAG1 antigen genetically fused to the bacterial alkaline phosphatase (AP). After expression optimization, the recombinant fusion protein folded correctly in soluble form in the periplasmic space and preserved both the AP enzymatic activity and the SAG1 immunoreactivity. Subsequently, direct-ELISA and dot-blot immunoassays were designed, using crude preparation SAG1-AP conjugate, to explore its value in serodiagnosis of human toxoplasmosis. We demonstrate that the recombinant SAG1-AP can detect specific T. gondii antibodies in one-step procedure and can successfully discriminate between T. gondii immune and non-immune patients, in agreement with the standard gold test. In conclusion, the present study shows that the genetic fusion protein provides a new tool for one-step T. gondii immunodetection, which was easily, quickly and reproducibly produced as homogeneous bi-functional reagent. Thus, this recombinant immunoconjugate is a promising marker for Toxoplasma serodiagnosis, requiring further evaluation on a larger series and could provide the basis for direct antibody capture enzyme-immunoassay for specific immunoglobulin M and G detection.

Enzyme-linked immunosorbent assay using recombinant SAG1 antigen to detect Toxoplasma gondii-specific immunoglobulin G antibodies in human sera and saliva.

Serologic detection of Toxoplasma gondii IgG 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%.

Evaluation of an enzyme-linked immunosorbent assay based on crude Leishmania histone proteins for serodiagnosis of human infantile visceral leishmaniasis.

Human visceral leishmaniasis (VL) is routinely diagnosed by detecting IgG that specifically binds to Leishmania antigens. 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 crude Leishmania histone (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 against Leishmania recombinant 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 soluble Leishmania antigen (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, crude Leishmania histone extract could be a valuable antigen for clinical use.

Identification of cryptosporidium species infecting humans in Tunisia.

Prevalence and species distribution of Cryptosporidium spp. were determined among 633 immunocompetent children less than five years of age and 75 patients hospitalized for immunodeficiency who lived in northern Tunisia. Microscopy was used for initial screening to detect positive samples and a nested polymerase chain reaction and restriction fragment length polymorphism analysis was used to determine the species. Cryptosporidium spp. was identified in 2.7% of cases (19 stool samples), and there was a significant difference between samples collected from immunocompromised patients and those collected from healthy children (10.7% versus 1.7%). Prevalence was also significantly higher in diarrheal specimens than in formed specimens (6.3% versus 1.6%). Cryptosporidium hominis and C. parvum were responsible for most Cryptosporidium spp. infections (78.9%). Cryptosporidium hominis was more prevalent in children from urban areas than in those from rural areas, and C. parvum was found with similar prevalence rates in the two populations. Cryptosporidium meleagridis was identified in four children on farms.

Recombinant single-chain Fv antibody fragment-alkaline phosphatase conjugate: a novel in vitro tool to estimate rabies viral glycoprotein antigen in vaccine manufacture.

The purpose of this study was to design a novel in vitro tool by using recombinant protein technology to qualify the whole reagent preparation procedure, to be used to quantify rabies viral antigen preparation in a simple and rapid format for potency control of rabies vaccines. 50AD1 is a neutralizing monoclonal antibody directed against the rabies virus glycoprotein that binds to native conformational antigenic site III. In the present study, the DNA fragments encoding the variable domains of 50AD1 were inserted into a prokaryotic expression vector so as to produce a single-chain Fv antibody fragment (scFv) genetically fused to the bacterial alkaline phosphatase (AP). The recombinant fusion protein preserved both the AP enzymatic activity and the antigen-binding activity against the rabies virus glycoprotein nearly identical to the parental antibody, and was used successfully in different assays including ELISA, dot-blot and cell culture tests. The present study shows that the genetic fusion protein provides a new tool for one-step rabies virus immunodetection, which can be produced in homogeneous bifunctional reagent, easily, quickly and reproducibly. In addition, this recombinant immunoconjugate is a promising alternative reagent for applications involving immunodetection, it presents a similar sensitivity and specificity to that obtained with classical reagents.

Stable trimerization of recombinant rabies virus glycoprotein ectodomain is required for interaction with the p75NTR receptor.

Native rabies virus glycoprotein (RVGvir) is a trimeric, membrane-anchored protein that has been shown to interact with the p75NTR neurotrophin receptor. In order to determine if the RVG trimeric oligomerization state is required for its binding with p75NTR, 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 p75NTR 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 p75NTR. However, the exact mode of trimerization appears unimportant, as trimeric recombinant RVGect (fused to the fibritin foldon) and RVGvir both recognize p75NTR 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 p75NTR binding domain of RVG.