Neutralization assay to resolve discrepancies between positive results in new highly sensitive anti-Toxoplasma gondii IgG assays and negative results in reference tests.

High sensitivity of IgG assays is required in order to diagnose Toxoplasma infection in immunocompromised patients and women before and during early pregnancy. Positive results obtained in new highly sensitive Toxoplasma IgG assays have been observed in sera that gave negative results in reference assays. To allow the resolution of these discrepancies, we developed a neutralization assay using 80 microg/ml of a soluble membrane fraction of Toxoplasma in the automated Elecsys IgG assay. We were able to block the detection of Toxoplasma IgG antibodies in 283 sera with IgG titers ranging from below the limit of detection to >2,300 IU/ml. The mean percentage of neutralization was 88%, with a minimum neutralization of 77%. In summary, the neutralization assay represents a valuable tool to confirm the specificity of Toxoplasma IgG antibodies in the sera of immunocompromised patients and women before and during early pregnancy. Discrepancies between positive results in highly sensitive Toxoplasma IgG assays and negative results in reference assays can, thereby, be resolved and may allow a decision to be made on the management of patients.

Identification of somatostatin receptor subtypes 1, 2A, 3, and 5 in neuroendocrine tumours with subtype specific antibodies.

BACKGROUND AND AIMS: Recently, novel somatostatin receptor (sstr) subtype specific ligand analogues have been developed for medical treatment of neuroendocrine tumours expressing different sstrs (sstr1-5). At present, individual expression patterns of sstr subtypes are based on methods such as in situ hybridisation and polymerase chain reaction at the transcriptional level. Therefore, we generated subtype specific antibodies against sstr1, 2A, 3, and 5 and analysed their presence, cellular localisation, distribution, and expression pattern in 33 gastrinomas, 36 insulinomas, and 35 tumours associated with a carcinoid syndrome by immunohistochemistry at the translational level. METHODS: Western blotting experiments were performed in the normal human pancreas used as a reference organ and in tumour tissues; at the cellular level, sstrs were localised by immunohistochemistry in tissue paraffin sections. RESULTS: In western blot analyses, the antibodies identified the respective receptors in their correct molecular range in extracts of the pancreas and neuroendocrine tumours. Using immunohistochemistry and immunofluorescence, the antibodies specifically detected the receptors in islet cells of the normal pancreas. Immunohistochemistry in the tumours revealed that all investigated sstr subtypes were highly expressed in the different tumour types. The frequency and expression pattern of the individual sstr subtypes varied considerably not only between the different tumour types but also in each patient. CONCLUSIONS: We conclude that immunohistochemistry with subtype specific antibodies can be used in clinical routine work to analyse sstr expression patterns for each patient before treatment and to facilitate well directed individual medical therapy by administering subtype specific somatostatin analogues.

[Critical view of the health care of tomorrow]. / Kritischer Blick auf die Medizin von morgen.

There are two issues in question concerning future developments in medicine: progress and reform. In so much as the "Scientific Paradigm" will be shifted from causality to the idea of a feed-back control system progress in medical sciences might lead treatment to enhancing the patient's biological individuality but it will not lead to better knowledge of the patient's person. Therefore the physicians functions will have to include communicational skills on a higher level. The health care system's reform primarily in Germany has to face the inevitability of rationing medical services. For alteration two examples will have to be considered: health care systems in the UK and in the US. Is health care a public or a private task? Answering this question leads to a new assignment to the medical profession.

Mobilization and retardation of uranium DOC species at three mine piles in Schlema/Alberoda, Saxony, Germany.

The modeling of geo-chemical processes needs the detailed and comprehensive knowledge of all chemical interactions existing in the flow path of waste dumps, subsoil and aquifer. This includes the adsorption, displacement and transport of heavy metal species of fulvic and humic acids, which represent the main amount of DOC in the liquid/solid system of the flow path. Comparative measurements of DOC concentrations in the input and output flow at the three waste dumps in the district Schlema/Alberoda indicated that DOC is produced and/or supplied within the waste dumps. The speciation of heavy metal compounds of fulvic and humic acids was achieved by means of sequential chromatographic analysis SCA and isoelectric focusing IEF. Between 5% and 20% of uranium exist in the flow path in the form of stable fulvic and humic acid species. Their distribution coefficients are strongly correlated with the pH values of the geo-chemical system.

Genetic analysis of Borrelia garinii OspA serotype 4 strains associated with neuroborreliosis: evidence for extensive genetic homogeneity.

Infection with Borrelia garinii outer surface protein (Osp) A serotype 4 strains has been correlated with the development of neuroborreliosis in Lyme borreliosis patients in Europe. OspA serotype 4 isolates have been recovered primarily from human cerebrospinal fluid, suggesting a tropism for this environment. Previous studies with monoclonal antibodies directed against OspA and OspC demonstrated that OspA serotype 4 strains are antigenically closely related. In view of the pronounced antigenic and genetic variability that has been noted in the Osps of other Borrelia isolates, we sought to determine if OspA serotype 4 strains represent a recently emerged clonal lineage of B. garinii. Toward this goal, a representative group of OspA serotype 4 strains was analyzed for traits that typically exhibit hypervariability among isolates that cause Lyme borreliosis. The following criteria were assessed: (i) ospC sequences, (ii) plasmid composition, (iii) genomic restriction fragment length polymorphism (RFLP) patterns, and (iv) the RFLP patterns of the upstream homology box (UHB) element which flanks members of the UHB gene family at their 5' end. Collectively, these analyses demonstrate genetic homogeneity, suggesting that OspA serotype 4 strains are a recently emerged clonal lineage with an apparent tropism for the central nervous system.

Osp17, a novel immunodominant outer surface protein of Borrelia afzelii: recombinant expression in Escherichia coli and its use as a diagnostic antigen for serodiagnosis of Lyme borreliosis.

Western blot analyses of the human humoral response of patients with Lyme borreliosis have shown that a 17-kDa protein is an immunodominant protein in late disease. Immune electron microscopy with a monoclonal antibody against this protein revealed that the 17-kDa protein is abundantly expressed on the surface of Borrelia afzelii strain PKo. Therefore, the protein has been renamed outer surface protein (Osp) 17. Recombinant Osp 17 of strain PKo was expressed in Escherichia coli and purified by chromatography. Immunoblot analysis of human sera showed a comparable sensitivity with recombinant and natural proteins. The DNA sequences of the osp17 genes from different B. afzelii strains were determined. The DNA sequences of the different osp 17 homologues (six isolates from skin, three isolates from CSF and one isolate from synovial fluid) had high sequence identities of at least 94%. Using a polyclonal antibody against recombinant Osp 17, it was shown that Osp 17 expression varied considerably among the investigated B. afzelii strains. As previously also observed for OspA- and OspC-encoding genes, the osp 17 gene is present in strains not expressing the respective protein. It has been shown that OspA and OspC expression varies in different environments such as tick and vertebrate host. Studies are underway to examine whether this is also true for Osp 17. For diagnostic purposes the use of recombinant Osp 17 has the advantage that the amount of Osp 17 antigen can be easily standardized for immunoblotting, and that this antigen can be used in a protein-specific enzyme-linked immunosorbent assay.

An improved recombinant IgG immunoblot for serodiagnosis of Lyme borreliosis.

We have previously described the use of the following recombinant antigens for serodiagnostic immunoblots: p83/100, p39, OspC and p41 (flagellin) internal fragment [Wilske et al. (1993) Med Microbiol Immunol 182:255-270; Rossler et al. (1997) J Clin Microbiol 35:2752-2758]. In our currently used immunoblot p83/100 is derived from strain PKo (Borrelia afzelii), p39 (BmpA) and OspC from strains PKa2 (B. hurgdorferi sensu stricto), PKo and PBi (B. garinii), respectively; the p41 (flagellin) internal fragments were cloned from strains PKo and PBi. In this study we describe the use of two additional recombinantly expressed highly immunogenic proteins Osp 7 (derived from PKo) and p58 (derived from PBi). A clinically well-defined panel of sera from 147 Lyme borreliosis patients and 139 controls previously tested by a standardized whole cell lysate immunoblot [Hauser et al. (1997) J Clin Microbiol 35:1433-1444] was investigated in the recombinant immunoblot without (old recombinant immunoblot) and with Ospl7 and p58 (new recombinant immunoblot) for IgG antibodies. The sensitivity of the recombinant IgG immunoblot for diagnosis of stage II and stage III could be significantly improved by addition of Osp17 and p58 without loss of specificity. With the exception of sera from patients with erythema migrans the diagnostic sensitivity is comparable to the whole cell lysate IgG immunoblot. The main advantage of the recombinant immunoblot is the easy identification of diagnostic bands, whereas the identification of bands in the whole cell lysate immunoblot is difficult. The recombinant immunoblot is especially suitable where large series of sera need to be investigated.

Heterogeneity of Borrelia burgdorferi sensu lato demonstrated by an ospA-type-specific PCR in synovial fluid from patients with Lyme arthritis.

Borrelia burgdorferi sensu lato, the etiological agent of Lyme borreliosis, has been divided into three genospecies: B. burgdorferi sensu stricto (OspA-type 1), B. afzelii (OspA-type 2) and B. garinii (OspA-type 3-7). Whereas in Europe B. afzelii (OspA-type 2) is predominant among human skin isolates and B. garinii (OspA-type 3-7) among human CSF isolates, some previous serological studies suggested that Lyme arthritis is also associated with B. burgdorferi sensu stricto in Europe. In the present study we designed ospA type-specific PCRs and identified four different ospA types associated with Lyme arthritis. Our study group consisted of 20 patients with positive serology (ELISA and immunoblotting) and clinical criteria for Lyme arthritis. B. burgdorferi DNA was detected in 13 patients and in none of 10 control patients from synovial fluid. We identified ospA-type 1 (26.6%), ospA-type 2 (33.3%), ospA-type 4 (6.6%) and ospA-type 5 (33.3%). Our conclusion is that in Europe B. burgdorferi sensu lato strains causing Lyme arthritis are considerably heterogeneous and that there is no prevalence of certain genospecies or OspA-types among this strains.

Development of enzyme-linked immunosorbent assays using recombinant borrelial antigens for serodiagnosis of Borrelia burgdorferi infection.

To improve IgG antibody detection in the serodiagnosis of Borrelia burgdorferi infection, indirect enzyme-linked immunosorbent assays (ELISAs) were developed utilizing purified recombinant antigens of B. burgdorferi sensu lato: the chromosomally encoded proteins p100 of strain PKo (B. afzelii) and p4li (internal flagellin fragments) derived from strains PKo, PBi (B. garinii), and B31 (B. burgdorferi sensu stricto). In Western blot analysis, these proteins have proved to be highly specific and sensitive for IgG antibody detection, especially in late manifestations of Lyme borreliosis. Sera from 464 forest workers, a high-risk group for infections with B. burgdorferi, were investigated and the results compared with those obtained by a commercial ELISA using an octyl-beta-D-glucopyranoside (OGP) extract from B. afzelii (PKo) cells as the antigenic substrate. Sera derived from 200 blood donors served for determination of the 95% specific cut-off level. The number of positive test results using OGP-ELISA (23.9%) was only slightly higher than that using p100-ELISA (19.8%); corresponding results were observed in 84.7% of all sera (14.2% positive, 70.5% negative). The frequency and interassay correspondence of positive results increased with the age of the forest workers, as most markedly demonstrated by p100 and OGP assays (P < 0.0001). Using the p41i-ELISAs, generally no significant strain-dependent differences in sensitivity were found (PKo, 13.8%; PBi, 14.2%; B31, 12.9%). Compared with the p100-ELISA, the p41i-assays showed significantly lower detection rates for IgG antibodies (P < 0.03) and a reduced capacity for quantitative discrimination between seropositive individuals and negative controls (P < 0.0007). At a 95% specificity, the IgG antibody detection rate could be increased to 23.1% when the p100-ELISA was evaluated in combination with the assays using p41i/PBi and P41i/B31. Due to its high sensitivity, the specific recombinant p100-ELISA might be a suitable test for detection of late immune responses to B. burgdorferi, thus being especially useful for epidemiological investigations.

Immunological and molecular variability of OspA and OspC. Implications for Borrelia vaccine development.

Borrelia burgdorferi sensu lato, the etiological agent of Lyme borreliosis is considerably heterogeneous in Europe. Since the outer surface proteins OspA and OspC are the most promising candidates for a Borrelia vaccine the immunological heterogeneity of these proteins was investigated. By immunological analysis with monoclonal antibodies and sequence analysis of PCR amplified OspA and OspC at least seven and 16 different types, respectively, were found. Whereas skin isolates (n = 68) were quite homogeneous (84% belonged to OspA-serotype 2 or Borrelia afzelii), isolates from human cerebrospinal fluid and from ticks (n = 43 and n = 90 respectively) were highly heterogeneous in their OspA-serotypes with prevalence of the Borrelia garinii associated types (about 70%). OspA-type 4 was often found among isolates from cerebrospinal fluid (28%). In ticks type 4 OspA has not been detected by culture so far. However, as reported in a previous study, type 4 OspA could be detected in ticks by the highly sensitive PCR technique.

Diversity of OspA and OspC among cerebrospinal fluid isolates of Borrelia burgdorferi sensu lato from patients with neuroborreliosis in Germany.

Neuroborreliosis is the most frequent manifestation of the second stage of Lyme borreliosis in Europe. However, only few isolates from the cerebrospinal fluid (CSF) have been characterized with controversial results. A large panel of 36 CSF isolates isolated over a 10-year period in Munich has now been analyzed for their OspA and OspC type, resulting in at least eight different types, respectively. Representatives of the different types cultivated from CSF in Munich have also been isolated from other geographical regions in Europe from CSF or ticks, suggesting a widespread distribution of pathogenic strains. A certain OspA type (type 4) was frequently observed in adults but rarely in children or ticks. Since OspA and OspC are the most promising candidates for a Borrelia vaccine, the considerable heterogeneity found among CSF isolates has important implications for development of a vaccine in Europe.

Borrelia burgdorferi sensu lato strains isolated from cutaneous Lyme borreliosis biopsies differentiated by pulsed-field gel electrophoresis.

25 skin biopsy isolates of Borrelia burgdorferi sensu lato, mainly from German patients with erythema migrans [EM], borrelial lymphocytoma [BL] and acrodermatitis chronica atrophicans [ACA], were species-differentiated using pulsed-field gel electrophoresis (PFGE). The isolates revealed 15 B. afzelii, 8 B. garinii and 2 B. burgdorferi sensu stricto species according to Miu I digestion. All 6 ACA isolates were identified as B. afzelii species, whereas the 2 borrelial lymphocytoma isolates were grouped into B. garinii species. 12 B. afzelii strains, including 4 ACA isolates, were further investigated by digestion with 5 restriction enzymes. Most of the strains revealed an individual pattern. No specific characteristically large restriction fragment pattern (LRFP) could be detected within the ACA and/or EM group. The ACA isolates could not be clearly differentiated from the EM isolates according to the LRFP patterns. PFGE is thus a highly effective method for detecting differences among B. afzelii isolates.

Sequence analysis of ospA genes shows homogeneity within Borrelia burgdorferi sensu stricto and Borrelia afzelii strains but reveals major subgroups within the Borrelia garinii species.

The genes coding for the outer surface protein A (OspA) of 19 different Borrelia burgdorferi strains belonging to the seven OspA-serotypes 1-7, previously described [Wilske et al. (1993) J Clin Microbiol, 31: 340-350], have been investigated. B. burgdorferi sensu lato strains were chosen from various biological sources (ticks, human skin and cerebrospinal fluid) as well as different geographical origins (Germany, Slovenia, Austria, United States). The open reading frames of all ospA genes consist of 819-825 nucleotides corresponding to proteins of approximately 30 kDa. The ospA sequences obtained in this study and previous published studies were compared with the results from OspA serotyping with monoclonal antibodies. The classification into the seven OspA serotypes could be confirmed on a genetic basis (ospA genotypes 1-7) for all strains analyzed so far (n = 29). In addition, one strain without OspA expression could be assigned to ospA genotype 2. Genetic stability could be proven for the ospA gene of B. burgdorferi strain PWudI after inocculation and reisolation from a gerbil. However, we found evidence for intragenic recombination by cluster analysis of ospA sequence data. Accordance of ospA genotype 1 strains with B. burgdorferi sensu stricto and ospA genotype 2 strains with B. afzelii, as well as the ospA genotype strains 3-7 with B. garinii was confirmed by pulsed-field gel electrophoresis of MluI-digested genomic DNA. B. garinii is not only more heterogeneous in respect to the OspA-encoding genes, but shows moreover major subgroups formed by genotypes 4, 5 and 6 and genotypes 3 and 7, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular analysis of genes encoding outer surface protein C (OspC) of Borrelia burgdorferi sensu lato: relationship to ospA genotype and evidence of lateral gene exchange of ospC.

It has been shown by analysis with monoclonal and polyclonal antibodies that outer surface protein C (OspC) of Borrelia burgdorferi sensu lato is highly heterogeneous. To determine if the heterogeneity has a genetic basis, the genes of 18 different B. burgdorferi sensu lato strains have been amplified by PCR, cloned, and sequenced. The ospC genes could be amplified from all strains tested, even from two strains which did not express OspC in detectable amounts. Among the 18 strains, 16 significantly different types of ospC sequences have been found. The sequence identities of the deduced amino acid sequences of different ospC genotypes range between 62 and 80% (determined without the leader peptide). The sequences range between 62 and 80% (determined without the leader peptide). The sequences correspond to one of the 13 OspC types distinguishable by analysis with monoclonal antibodies (B. Wilske, S. Jauris-Heipke, R. Lobentanzer, I. Pradel, V. Preac-Mursic, D. Roessler, E. Soutschek, and R. C. Johnson, J. Clin. Microbiol. 33:103-109, 1995) or represent additional types. Two completely new types were found, and OspC type 8 (which was found in Borrelia afzelii and Borrelia garinii) could be divided into two groups with different sequences but the same antibody pattern. Thus, strains belonging to different species or OspA serotypes were always significantly different in their ospC sequences. This was also confirmed by ospA sequence analysis. Interestingly, some strains of the same OspA serotype or genotype were very heterogeneous with respect to OspC, while others had nearly identical OspC proteins. Such groups of strains were found among B. burgdorferi sensu stricto, B. afzelii, and B. garinii strains. Cluster analysis of 5'-terminal and 3'-terminal stretches of ospC suggested recent intragenic recombination events in the ospC gene at least one B. afzelii strain. In addition, other recombination events between ancestors of strains belonging to the same or different species were evidenced by this type of analysis.

Molecular and immunological characterization of the p83/100 protein of various Borrelia burgdorferi sensu lato strains.

The complete coding regions of the chromosomally encoded p83/100 protein of four Borrelia garinii strains and one Borrelia burgdorferi sensu stricto strain have been amplified by the polymerase chain reaction (PCR), cloned and sequenced. From alignment studies with the deduced amino acid sequences presented here, and five other published p83/100 sequences, the most heterologous region of the p83/100 molecule was identified to be located between amino acid position 390-540. To study the structure of this heterogeneous region, and internal fragment of the p83/100 genes from 11 additional B. burgdorferi sensu lato strains was amplified by PCR. The PCR products were analyzed by DNA sequencing and restriction enzyme analysis. These internal p83/100 fragments varied in size and sequence. Cluster analysis of internal p83/100 fragments, as well as restriction enzyme analysis, revealed three major groups in accordance with grouping into the three species causing Lyme disease. Strains within the same species (six B. burgdorferi sensu stricto and six B. afzelii strains) showed similar p83/100 partial structures. Nevertheless, nine B. garinii strains showed more sequence variations and could be further divided into two major subgroups. One group is represented by OspA serotype 4 strains, the other more heterogeneous group is represented by OspA serotypes 3, 5, 6 and 7 strains. Phenotypic analysis with four p83/100-specific monoclonal antibodies revealed four distinct reactivity patterns. Antibody L100 1B4 recognized a common epitope of B. burgdorferi sensu stricto and B. afzelii. Antibodies L100 17D3 and L100 18B4 were reactive with an epitope shared by strains of all three species. The broadest reactivity was shown by L100 18B4 which, in contrast to L100 17D3, additionally recognized the relapsing fever borreliae B. turicatae and B. hermsii. L100 8B8 detected a subgroup of the B. burgdorferi sensu stricto strains. Since comparison of the p83/100 molecule with sequences from protein databases showed similarities with characteristics of eukaryotic cell structures, the p83/100 might mimic these structures and may, therefore, be involved in the immune escape mechanism of the pathogenic agent of Lyme disease.

Phenotypic analysis of outer surface protein C (OspC) of Borrelia burgdorferi sensu lato by monoclonal antibodies: relationship to genospecies and OspA serotype.

Molecular analyses of the genes encoding OspC, a major immunodominant protein of Borrelia burgdorferi sensu lato, revealed a considerable degree of heterogeneity. In the present study, we investigated whether a similar heterogeneity of the OspC phenotype can be shown by analysis with monoclonal antibodies (MAbs). Thirteen OspC-specific MAbs (L22 MAbs) were produced by immunizing mice with either different combinations of whole-cell antigens or recombinantly expressed OspCs cloned from strains belonging to different Borrelia spp. Ten of them differed in their reactivities with various strains. Western blot (immunoblot) analyses of 38 B. burgdorferi sensu lato strains resulted in 13 different reactivity patterns. These 13 different patterns were observed among only six different OspA serotypes, indicating that OspC is more heterogeneous than OspA. Patterns 1 to 4 were present only in B. burgdorferi sensu stricto, patterns 5 to 7 were present only in Borrelia afzelii, and patterns 9 to 13 were present only in Borrelia garinii. Pattern 8 was observed among B. afzelii and B. garinii strains but not among B. burgdorferi sensu stricto strains. One L22 MAb (2B8) recognized a common OspC-specific epitope of all 38 B. burgdorferi sensu lato strains analyzed, and another one (22C11) recognized a common epitope of OspC from both B. afzelii and B. garinii and was not reactive with OspC from B. burgdorferi sensu stricto. Western blot and sequence analysis of truncated OspCs located the 22C11 epitope as well as a species-specific sequence motif between amino acids 20 and 35. Other broadly reactive L22 MAbs were 10D3, 1F8, and 7G5. Some L22 MAbs (1C3, 1C3, 12E5, 1B11, 1F10, and 6C8) bound to epitopes present only in a few strains. Relapsing fever borreliae (Borrelia hermsii, Borrelia turicatae, and Borrelia duttoni) were nonreactive, with the following exception: three L22 MAbs (2B8, 6C4, and 10C5) recognized an abundantly expressed 20-kDa-range protein of B. turicatae. Because OspC is an immunodominant protein during the early immune response in Lyme borreliosis and has been shown to be effective as a vaccine in an animal model, our findings have important implications for the development of diagnostic reagents as well as vaccine research.

Immunoblot using recombinant antigens derived from different genospecies of Borrelia burgdorferi sensu lato.

Immunodominant proteins are variable in molecular and antigenic structure among different genospecies of Borrelia burgdorferi sensu lato. We have recently developed an immunoblot using five recombinant antigens: the chromosomal-encoded B. burgdorferi proteins p100, the flagellin and an internal flagellin fragment thereof, and the plasmid-encoded outersurface proteins A (OspA) and C (OspC). In the present study the same antigens (derived from strain PKo, genospecies B. afzelii) were compared with the homologous recombinant proteins from strain B31 (genospecies B. burgdorferi sensu stricto) and with OspA, OspC and the internal flagellin fragment from strain PBi (genospecies B. garinii). Patients with neuroborreliosis (n = 28) and patients with acrodermatitis chronica atrophicans (n = 20) were investigated in the IgG immunoblot; the IgM immunoblot was performed only in patients with neuroborreliosis. There was a small increase in the detection rate of OspA-specific IgG or IgM antibodies using the different variants of recombinant OspA; however, OspA remained an insensitive antigen for antibody detection in Lyme borreliosis. The same was true to OspC-specific IgG antibodies. The sensitivity of OspC, which is the immunodominant antigen for IgM antibody detection, could not be increased using recombinant antigens derived from different strains. However, some sera which were negative in the recombinant immunoblot reacted with OspC in the conventional immunoblot using B. burgdorferi whole cell lysate as antigen. The most unexpected finding was the high degree of immunological heterogeneity of the internal flagellin fragments: IgG antibodies were detected in 18 of 48 patients using B31 fragments, in 25 of 48 using PKo fragments, in 23 of 48 using PBi fragments versus 33 of 48 when the three recombinant proteins were combined. PKo-derived fragments were more sensitive for antibody detection in patients with acrodermatitis chronica atrophicans, B31- and PBi-derived fragments for antibody detection in patients with neuroborreliosis. This is in agreement with the fact that isolates from patients with neuroborreliosis are predominantly belonging to the genospecies B. burgdorferi sensu stricto and B. garinii. For detection of IgM antibodies in sera from patients with neuroborreliosis, recombinant internal fragments derived from strains B31 and PBi were more sensitive than the PKo-derived fragment. The best discrimination between neuroborreliosis sera and control sera was achieved when the IgM blot was performed using recombinant internal flagellin fragments derived from strains PKo and PBi and OspC derived from B31 or PKo.

Cloning, expression and characterization of human kininogen domain 3.

The internal domain 3 of the heavy chain of human kininogen, a cysteine proteinase inhibitor, was amplified by a polymerase chain reaction from the kininogen cDNA clone phKG36. The DNA fragment was expressed in Escherichia coli using the ompA expression vector pASK40 and the resulting protein was isolated from periplasm, purified by S-carboxymethylpapain affinity- and ion-exchange chromatography. The recombinant human kininogen domain 3 is 92% pure, reacts with anti-kininogen antibodies and is actively inhibitory. The expected amino acid sequence of ANSM-[G253-S377] kininogen was confirmed; the inhibitor has a molecular mass of 14,396 Da and an isoelectric point of 6.0 (pH). The determined Ki values of the complexes with papain and cathepsin L are similar to those measured previously with proteolytically liberated kininogen domain 3, and those of single-domain cystatins, like chicken egg white cystatin. However, recombinant kininogen domain 3 is a weak inhibitor of cathepsin B (Ki = 63 nM) as it has been found for native L-kininogen (Ki = 340 nM).