Many problems concerned with the production and the purification of recombinant proteins must be addressed prior to launching an industrial production process. Among these problems, attention is focused on low-level expression that complicates the purification step and can jeopardise the process. The expression of a membrane protein, rP30, of Toxoplasma gondii in the yeast Schizosaccharomyces pombe led to a secretion of only 0.5 microg ml(-1). In order to obtain a sufficient quantity for biochemical characterization and evaluation in vitro diagnostic test development, strategies for both production and purification had to be optimized. First, the influence of four nitrogen sources (three peptones and yeast extract) on the growth rate, but also on the separation between the protein and the components of the fermentation broth was assessed. Second, batch and fed-batch fermentations were compared in terms of final biomass and rP30 concentrations. Third, three different protocols that included fixed and expanded bed ion exchange chromatography were compared for processing a large volume of feedstock. By using the most appropriate strategies, i.e. fed-batch fermentation, capture on EBA cation exchanger and affinity chromatography polishing, a purification factor of 1778 and a yield of 49% were achieved. These performances allowed a 12.5-fold increase for the overall rP30 process productivity.

Toxoplasma gondii, the intracellular parasite responsible for toxoplasmosis infects more than one-third of the world population and can be life-threatening for fetuses and immunocompromised patients. The surface protein SAG1 is an important immune target, which provides a strong immune response against the invasive tachyzoite while the other forms of the parasite, devoid of SAG1 at their surface, are multiplying. In addition to this role as a "hot spot" decoy, SAG1 is predicted to act as an adhesin during host-cell attachment through its binding to proteoglycans. To begin to understand the relationships between SAG1 epitopes and the ligand-binding site, we have solved the crystal structure of the monomeric form of T.gondii SAG1 complexed to a Fab derived from a monoclonal antibody raised against tachyzoite particles. This antibody competes strongly with human Toxoplasma-specific sera, suggesting that its epitope is part of an immunodominant region present on the surface of SAG1. The structure reveals that this conformational epitope, located within the SAG1 N-terminal domain, does not overlap with the proposed ligand-binding pocket. This study provides the first structural description of the monomeric form of SAG1, and significant insights into its dual role of adhesin and immune target during parasite infection.

A new serological test, Vidas Toxo IgG IV, has been developed with antigens obtained from tachyzoites cultured on cells. Vidas Toxo IgG IV replaces Vidas Toxo IgG II by offering a more standardized antigenic production and a lower number of indeterminate results while retaining equivalent sensitivity and specificity.

The hepatitis C virus (HCV) nonstructural 3 (NS3) protein is composed of an amino terminal protease and a carboxyl terminal RNA helicase. NS3 contains major antigenic epitopes. The antibody response to NS3 appears early in the course of infection and is focused on the helicase region. However, this response cannot be defined by short synthetic peptides indicating the recognition of conformation-dependent epitopes. In this study, we have screened a dodecapeptide library displayed on phage with anti-NS3 mouse monoclonal antibodies (mAbs) that compete with each other and human anti-HCV NS3 positive sera. Two peptides (mimotopes) were selected that appeared to mimic an immunodominant epitope since they were recognized specifically by the different anti-NS3 mAbs of the study and by human sera from HCV infected patients. Homology search between the two mimotopes and the NS3 sequence showed that one of the two peptides shared amino acid similarities with NS3 at residues 1396-1398 on a very accessible loop as visualized on the three-dimensional structure of the helicase domain whereas the other one had two amino acids similar to nearby residues 1376 and 1378. Reproduced as synthetic dodecapeptides, the two mimotopes were recognized specifically by 19 and 22, respectively, out of 49 sera from HCV infected patients. These mimotopes allowed also the detection of anti-NS3 antibodies in sera of HCV patients at the seroconversion stage. These results suggest that the two NS3 mimotopes are potential tools for the diagnosis of HCV infection.

The objective of the study was to determine the diagnostic value for rheumatoid arthritis (RA) of anti-filaggrin autoantibodies (autoAb) recognizing citrullinated recombinant rat filaggrin (ACRF) in community cases of very early arthritis. To evaluate the diagnostic value of ACRF, were studied sera from patients with different classified rheumatic diseases and healthy subjects (group 1, n= 422) and 314 community cases of very early arthritis (group 2) that were classified as RA (n = 176), non-RA (n = 63) and undifferentiated (n = 75) arthritides after 1 years of follow-up. ACRF were measured using a new ELISA, with results expressed as the difference between the OD value obtained on citrullinated minus that on noncitrullinated rat filaggrin (differential ACRF; dACRF). For both groups, rheumatoid factors (RF), anti-keratin autoAb (AKA) and anti-perinuclear factor (APF) were tested; for group 2, anti-CCP autoAb were also tested. Different reactivity patterns against citrullinated and noncitrullinated filaggrin were observed. Almost all sera reacting with citrullinated but not noncitrullinated filaggrin were from RA patients. Among RA and non-RA sera that recognized both forms of filaggrin, a positive result was obtained only with RA sera. For groups 1 and 2, dACRF sensitivity was 58.4% and 30.7%, and specificity for RA was 99.5% and 98.4%, respectively. In group 2, dACRF specificity for RA was better than that of RF (92.1%), APF (95.2%), AKA (96.8%) and anti-CCP (95.2%). dACRF positive predictive value was high (98.2) and close to that given by the concomitant positivity of RF and anti-CCP autoAb. Despite a high positive correlation between AKA, APF, anti-CCP and dACRF test results, they were complementary since some sera were positive for only one test. Thus, in a community setting, anti-citrullinated rat filaggrin reactivity detected by a new ELISA, whose originality is based on the difference between serum's reactivities on the citrullinated and native forms of filaggrin, had a higher diagnostic value for RA than other autoAb.

MATI spectra of NH3 have been recorded using two-color (2 + 1') multiphoton excitation via the (B) over tilde and (C) over tilde states and have been simulated using multichannel quantum defect theory. New vibrational bands in the MATI spectra are reported for intermediate levels up to nu (2) = 6 in the (B) over tilde state. The spectra obtained-should in principle be identical to ZEKE: spectra recorded under the same resolution. A good agreement:,between experiment and theory-is obtained by using quantum defects determined previously from optical spectra and optimization of unknown parameters; The effects of p pi -d delta mixing in the core region are included to account for "forbidden" transitions, and the optimization of quantum defect parameters is used to quantify the strength of this mixing. Molecular symmetry group arguments are used to define which channels must be included in the quantum-defect matrix. The paper demonstrates that in favorable cases a complete theory of ZEKE/MATI line intensities is achievable including the effects of bound-state couplings.

Background Objectives To evaluate the predictive value of autoantibodies (Ab) directed against citrullinated recombinant rat filaggrin (ACRFA) detected by a new ELISA for prognosis in a cohort of community cases of RA. Methods 127 patients (mean age: 50.5 yr, F/M: 2.9) with early RA (median disease duration: 2 yr) were enrolled prospectively in 1996 (T1), primarily from a population based-recruitment (80%), and were followed until 1999 (T2). At T1 were tested rheumatoid factors determined by the latex fixation test (LFT) and anti-filaggrin Abs identified by different assays, i.e. anti-keratin Ab (AKA), and anti-perinuclear factor (APF) by indirect immunofluorescence, ACRFA by an ELISA test whose results are expressed as the difference of OD values given by sera tested against the citrullinated and non-citrullinated forms of filaggrin. At T1 and T2: radiographs of the hands and wrists. Prognosis was defined by (1) radiological damage (van der Heijde modified Sharp method) at T2; (2) progression of radiological damage between T1 and T2. Results At T1: RA had mild activity (Ritchie articular index: 11/78, mean CRP: 15 mg/l), mild functional disability (HAQ: 0.8/3), mild X-ray destruction (Sharp: 9.2/280); 96% of patients were treated at T1 (DMARDs: 95%; prednisone: 72%); frequency of LFT, AKA, APF and ACRFA were respectively: 50%, 33%, 45% and 45%. At T2: total Sharp score: 22.8/280. The only parameter at T1 associated with a higher Sharp score at T2 (p = 0.03) and particularly with a more important radiological progression (p = 0.03) was the LFT. Conclusion Whatever the method used, indirect immunofluorescence or ELISA, anti-filaggrin do not allow to predict radiological damage in community cases of RA of limited duration.

Hepatitis B virus core protein (HBc) is an important serology marker of hepatitis B infection and patient follow-up. It is an M, 21,000 protein, which has the intrinsic capacity to self-assemble as a capsid-like particle. The hepatitis B core protein has been expressed in Escherichia coli and Pichia pastoris (three different constructions) in order to select a HBc recombinant antigen suitable for serodiagnosis requirements with a cost effective downstream strategy. The expression and purification of the different forms of recombinant HBc have been described. For the last step, ultracentrifugation and size-exclusion chromatography were compared. The morphology of these capsids was observed using an electron microscope. Our data shows that HBc antigen is produced in large quantities in E. coli but some contaminants remained which were associated with the E. coli HBc protein after ultracentrifugation or size-exclusion chromatography. The ultracentrifugation enables a higher purity of HBc antigen to be obtained than size-exclusion chromatography but the latter enables a higher recovery rate. P. pastoris enables the expression and extraction of a highly purified HBc antigen suitable for diagnostic purposes.

For isolation of low soluble recombinant full-length (amino acids 1-191) core protein of hepatitis C virus (HCV) overexpressed in Escherichia coli, the advantage of combining two electrophoretic techniques, in comparison with chromatographic separation, is demonstrated. The protein extract was first solubilized in agents compatible with electrophoretic separation. Using preparative liquid phase isoelectric focusing (IEF) the protein of interest was first concentrated within a defined acidic pH range. These fractions were then submitted to preparative sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) to isolate the 22 kDa protein. The second-dimensional step allowed the isolation of 2 mg of the purified recombinant HCV core protein (rHCV-C191) from 1.5 g bacterial pellet. This quantity is sufficient to characterize the protein and to perform immunogenicity studies. This procedure of two-dimensional preparative electrophoresis is applicable to a wide range of biological samples and represents an alternative for purification of insoluble proteins.

The industrial production of recombinant proteins requires control of both fermentation and purification steps. For the serodiagnosis of toxoplasmosis, the main antigen is a membrane protein of 30 kDa (P30). The P30 gene was cloned and expressed in Schizosaccharomyces pombe at 0.7 microg/ml in culture medium. Batch fermentation was optimized by the specific choice of peptones, which enabled optimum growth and protein expression without reducing the efficacy of the purification step. Analytical purification was then carried out using cation-exchange chromatography. For larger volumes, scaling up was performed on expanded mode by using a Streamline system (Pharmacia). This purification step allowed us to obtain a 67.5% recovery with a purification factor greater than 27-fold. Expanded bed adsorption technology is a convenient and effective technique for protein capture directly from feedstock, and the eluted fraction is ready for a second affinity chromatography step. This second step is performed with a yield of 40% and provides a final purification factor of 2000-fold.

NH3+ ions have been prepared in unique vibration-rotation states v(2)(+), N+, K+, by pulsed-field ionization of high Rydberg states of NH3, populated by two-colour multiphoton excitation. The selected states, ranging from v(2)(+) = 0 to 7 and N+, K+ less than or equal to 4 are accessed via the B and C' states as intermediates. The application of multichannel quantum defect theory to the understanding of the rotational propensities in the mass-analysed threshold ionization spectra is discussed. It is demonstrated that the interactions of the Rydberg pseudo-continuum both with bound Rydberg states and with other continua can be modelled.

The pulsed-field ionization (PFI) spectra of CO and NO are recorded using stepwise two-colour multiphoton excitation with ion detection. These spectra demonstrate the production of CO+ and NO+, state-selected in unique vibration-rotation levels. For CO a (2 + 1′) excitation process is employed via the E1Π (δ′=0) state, whereas for NO a (1 + 1′) process is used via the B2II (δ′=4) state. The one-colour REMPI spectrum of NO via this same state is also reported for the first time. The NO spectrum involves a nominal two-electron excitation in the final step and the intensity is derived from configuration interaction with the C2II Rydberg state. The PFI spectrum of CO shows marked differences in rotational intensities compared to those determined by Fujii et al. in direct ionization [1]. The effects of final state interactions are abundant in both spectra, with the observation of window resonances and intensity enhancements, including some unexplained enhancements of positive N+ − J′ lines in CO.

An inhibition EIA using a monoclonal antibody against the major P30 Toxoplasma gondii surface protein was designed for detection of specific antibodies in human sera. The assay was based on the inhibition of binding of peroxidase labelled monoclonal antibody to Toxoplasma gondii crude antigen coated plates by the corresponding antibodies present in human sera. This rapid and simple assay was compared to indirect immunofluorescence, direct agglutination and an immunosorbent agglutination assay using 435 human sera. The specificity and sensitivity were 100% and 97% respectively. This test was found to be as sensitive as the dye test.