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  • 1
    Electronic Resource
    Electronic Resource
    Recombinant immunoblot in the serodiagnosis of Lyme borreliosis (1993)
    Springer
    Medical microbiology and immunology 182 (1993), S. 255-270 
    Details
    ISSN: 1432-1831
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract A recombinant immunoblot was developed for detection of IgM and IgG antibodies in patients with Lyme borreliosis. The recombinant antigens were the chromosomal-encodedBorrelia burgdorferi proteins p100, the flagellin and an internal flagellin fragment thereof as well as the plasmid-encoded outer surface proteins A (OspA) and C (OspC). A panel of 144 sera from patients with Lyme borreliosis (erythema migrans,n = 31; neuroborreliosis state II,n = 60; Lyme arthritis,n = 24 and acrodermatitis chronica atrophicans,n =19) have been investigated and the results have been compared to the immunofluorescence absorption test (IFA-ABS) and to two different enzyme-linked immunosorbent assays [the flagellin ELISA and a newly developed ELISA (OGP-ELISA)]. The two ELISAs were comparable in sensitivity, whereas the IFA-ABS was less sensitive for IgM antibody but equally sensitive for IgG antibody detection. Immunoblot analysis revealed that IgG antibodies are mainly reactive with p 100 and the internal flagellin fragment (sensitivity 51% and 32%, respectively) and rarely with OspC (14%). All patients with late Lyme borreliosis had IgG antibodies against the p100. IgM antibodies were predominantly directed against OspC (43%) and in a lower extent against the internal flagellin fragment and p100 (15% and 13%, respectively). The complete flagellin was not useful due to a high number of unspecific reactions with control sera and the OspA was only exceptionally reactive in Lyme borreliosis patients. The sensitivity of IgM antibody detection could be increased in cases with early Lyme borreliosis from 46% to 65% when the OspC blot was performed in addition to the flagellin ELISA, or from 56% to 65% when performed in addition to the OGP-ELISA. The recombinant blot is, therefore, a valuable diagnostic test to increase sensitivity of early antibody detection and is regarded as a valuable confirmatory test also in late disease.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00579624
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  • 2
    Electronic Resource
    Electronic Resource
    Genetic heterogenity of the genes coding for the outer surface protein C (OspC) and the flagellin of Borrelia burgdorferi (1993)
    Springer
    Medical microbiology and immunology 182 (1993), S. 37-50 
    Details
    ISSN: 1432-1831
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The ospC gene coding for the outer surface protein OspC and the fla gene coding for the flagellin have been investigated in three different Borrelia burgdorferi sensu lato strains. These strains (the North American strain B31 and the European strains PKo and PBi) derive from various biological sources (lxodes dammini, human skin and human CSF) and belong to three different B. burgdorferi OspA serotypes and genospecies (OspA serotype 1, B. burgdorferi sensu stricto; OspA serotype 2, group VS461 and OspA serotype 4, B. garinii, respectively). The ospC and fla genes of the respective strains have been amplified by polymerase chain reaction, cloned in pUC8 and sequenced. The fla as well as the ospC genes were different among the three strains investigated. In general the fla genes are more conserved than the ospC genes. The fla genes have the same length of 1008 nucleotides coding for proteins of 336 amino acids, whereas the ospC genes differ in length. The ospC genes of strains B31, PKo and PBi have 630, 636 and 621 nucleotides encoding proteins of 210, 212 and 207 amino acids, respecctively. The ospC genes exhibit sequence identities between 70% and 74% among each other, sequence identities of the fla genes are in the range 96–97%. The ospC genes could be expressed in Escherichia coli to obtain proteins with and without leader peptides. The expression of the fla gene and an internal gene fragment resulted in the complete flagellin protein and a truncated protein (amino acids 129–251). The different ospC and fla gene products were immunoreactive with monoclonal antibodies and human sera and, thus, enlarge the spectrum of recombinant antigens to improve antibody detection in patients with Lyme borreliosis.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00195949
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  • 3
    Electronic Resource
    Electronic Resource
    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 (1995)
    Springer
    Medical microbiology and immunology 184 (1995), S. 73-80 
    Details
    ISSN: 1432-1831
    Keywords: Borrelia burgdorferi, afzelii, garinii ; Outer surface protein A ; ospA genes ; ospA sequence analysis ; Outer surface protein A serotypes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract 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 heterogenous 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. The latter group has not been described previously and is specifically recognized by an OspA-specific monoclonal antibody L32 1F7.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00221390
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  • 4
    Electronic Resource
    Electronic Resource
    Molecular analysis and expression of a Borrelia burgdorferi gene encoding a 22kDa protein (pC) in Escherichia coli (1992)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 6 (1992), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: We describe the cloning and expression of the pc gene which encodes a major immunodominant protein of Borrelia burgdorferi, the causative agent of Lyme borreliosis. The pC protein was purified from lysates of B. burgdorferi strain PKo. After tryptic digestion of the pC protein the resulting oligopeptides were applied to a gas-phase sequenator. Thus partial amino acid sequences were obtained. The deduced oligonucleotides were used as hybridization probes. After Southern blotting a reactive band in the 3 kb range of PstI-digested genomic DNA was detected. The insertion of these fragments into pUC vectors finally resulted in pc-positive Escherichia coli clones. The gene (encoding a protein with 212 amino acids) was expressed in E. coli with varying deletions at the 5′ end. A sequence comparison with other outer membrane proteins of B. burgdorferi indicates a processing of pC that is similar to that of lipoproteins.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.1992.tb01495.x
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  • 5
    Electronic Resource
    Electronic Resource
    Purification of a recombinantly produced transmembrane protein (gp41) of HIV I
    Amsterdam : Elsevier
    Journal of Chromatography A 521 (1990), S. 267-277 
    Details
    ISSN: 0021-9673
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Chemistry and Pharmacology
    Type of Medium: Electronic Resource
    URL: http://linkinghub.elsevier.com/retrieve/pii/0021-9673(90)85051-V
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  • 6
    Electronic Resource
    Electronic Resource
    Immunodominant proteins of Borrelia burgdorferi, the etiological agent of lyme borreliosis (1991)
    Springer
    World journal of microbiology and biotechnology 7 (1991), S. 130-136 
    Details
    ISSN: 1573-0972
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Lyme borreliosis, a multisystem disorder involving the skin, the nervous system, the heart, the joints and many other organs, is a worldwide infectious disease which is transmitted by ticks of the Ixodes complex. Most frequently diagnosis is accomplished by detection of antibodies because the Borrelia are difficult to cultivate. Present serodiagnostic methods, however, are impaired by low sensitivity and unspecific reactions. The selection of immunodominant antigens with low cross-reactivity to other bacteria should improve antibody detection. Borrelia burgdorferi proteins have been analysed for cross-reactivity with immune sera from unrelated bacteria, and sera from patients with different stages of the disease. Suitable antigens for improving serodiagnosis have been detected and are reported here. In view of the immunological heterogeneity of Borrelia proteins, sensitivity of antibody detection may possibly be increased by using recombinant antigens derived from different strains. Immunization with recombinant OspA (a flagellum-associated protein) from a North American isolate protected mice from the challenge with three North American isolates. However, for development of an effective vaccine (especially in Europe), the heterogeneity of OspA has to be considered.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00328982
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  • 7
    Electronic Resource
    Electronic Resource
    Active immunization with pC protein ofBorrelia burgdorferi protects gerbils againstB. burgdorferi infection (1992)
    Springer
    Infection 20 (1992), S. 342-349 
    Details
    ISSN: 1439-0973
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Description / Table of Contents: Zusammenfassung Weite Verbreitung und zunehmende Inzidenz der Lyme Borreliose sowie Therapieprobleme bei schweren Erkrankungsformen besonders im Spätstdadium der Infektion sind Gründe für die Entwicklung einer möglichen Alternative zur Antiobitikatherapie wie zum Beispiel Schutzimpfung. Die Schutzwirkung einer aktiven Immunisierung mit rekombinanten OspA und pC gegen die Infektion mitBorrelia burgdorferi wurde bei Gerbils geprüft. Die Tiere wurden mit rekombinanten OspA und pC vomB. burgdorferi-Stamm PKo immunisiert; die Infektion erfolgte vier Wochen nach der Immunisierung mit dem PKo-Stamm (schwache OspA, gute pC Expression). Als Kontrollgruppe dienten infizierte, nicht immunisierte Gerbils. Die immunisierten Tiere bildeten Antikörper gegen rekombinante Vakzine. Die mit pC immunisierten Tiere waren vor der Infektion geschützt, die Kontrollgruppe zeigte eine generalisierte Infektion. Die Immunisierung mit OspA schützte nicht, die Tiere zeigten, wie die Kontrollgruppe Merkmale der Infektion. Die Ergebnisse dieser Studie zeigen, daß pC für eine Immunisierung in Frage kommt. Eine Kombination von OspA und pC scheint für eine effektive Vakzine gegenBorrelia burgdorferi nötig zu sein.
    Notes: Summary Serious infection due toBorrelia burgdorferi and the disseminated infection characteristic of the disease possess unique treatment problems. The wide and still increasing incidence of Lyme borreliosis as well as the problems in treatment call for effective prevention strategies by active immunization. Vaccination experiments were done to determine if active immunization of gerbils with recombinant OspA and pC protects against infection with strains ofB. burgdorferi. Gerbils were vaccinated with recombinant OspA and pC (20 kDa protein) and challenged four weeks later with a clone (derived fromB. burgdorferi strain PKo) which expresses an abundant amount of pC but only little OspA. Non-immunized gerbils challenged with the sameB. burgdorferi strain were used as controls. Both groups of immunized gerbils developed antibodies against the recombinant vaccines. The pC vaccinated group was protected against infection, whereas the OspA vaccinated group showed signs of infection. The non-vaccinated group developed generalised infection. These results show that pC should be considered as a further vaccine candidate and probably needs to be combined with OspA for an efficient vaccine againstB. burgdorferi.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01710681
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  • 8
    Electronic Resource
    Electronic Resource
    Immunoblot using recombinant antigens derived from different genospecies of Borrelia burgdorferi sensu lato (1994)
    Springer
    Medical microbiology and immunology 183 (1994), S. 43-59 
    Details
    ISSN: 1432-1831
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract 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 p 100, 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 acrodermatitits 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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00193630
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  • 9
    Electronic Resource
    Electronic Resource
    Genetic polymorphism of the gene encoding the outer surface protein A (OspA) of Borrelia burgdorferi (1992)
    Springer
    Medical microbiology and immunology 181 (1992), S. 57-70 
    Details
    ISSN: 1432-1831
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The genes coding for the outer surface protein (OspA) of Borrelia burgdorferi, the causative agent of Lyme borreliosis have been cloned and sequenced. Two German strains (skin isolate PKo and cerebrospinal fluid isolate PBi) have been analyzed. Using an OspA-specific monoclonal antibody (L32 2E7) for immunological screening of a genomic pUC 18 library of B. burgdorferi strain PKo, an OspA-producing clone was detected and subclones containing the open reading frame were constructed. The gene coding for the OspA protein of B. burgdorferi strain PBi was amplified using polymerase chain reaction (PCR) and cloned in pUC8. The open reading frame of both ospA genes consists of 822 nucleotides corresponding to a protein of 273 amino acids. Both proteins have a calculated molecular mass of 29.6 kDa. Molecular analysis revealed significant differences between each other and to already-published sequences of ospA of B. burgdorferi strains B31, ZS7 and N40 (the ospA genes of B31, ZS7 and N40 are nearly identical). The deduced amino acid sequences of the OspA protein of strains PKo and PBi showed a homology of 83% to each other and 77% and 80%, respectively, to OspA protein of strain B 31. The three proteins contain a variable middle region, whereas the N and the C terminus are conserved. This unexpected high dissimilarity of the ospA genes may be important in respect to vaccination studies and diagnostic procedures (i.e., development of PCR primers or serodiagnostic antigens). Moreover, the molecular heterogeneity of OspA confirms three out of seven immunologically defined OspA serotypes of a recently proposed OspA serotyping system.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00189424
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  • 10
    Electronic Resource
    Electronic Resource
    Molecular characterization of the p83/100 proteins of variousBorrelia burgdorferi sensu lato strains (1996)
    Springer
    Cellular and molecular life sciences 52 (1996), S. 305-306 
    Details
    ISSN: 1420-9071
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Conclusions As in OspA-serotyping experiments, theB garinii group (OspA-sterotype 3–7) showed highest diversity within this internal fragment of p83/100, whereas theB. afzelii group (OspA-type 2) and theB. burgdorferi sensu stricto group (OspA-type 1) were nearly identical. Determination of the size of the PCR products as well as restriction fragment length polymorphism analysis (DraI) can be used for classification into the three species ofB. burgdorferi sensu lato. Since p83/100 is chromosomally encoded, this protein might be a more stable marker for classification than the plasmid-encoded OspA. In contrast to the flagellin gene a subclassification of theB. garinii group is possible due to the diversity of the p83/100 internal fragment.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01919522
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