Recombinant immunoblot in the serodiagnosis of Lyme borreliosis

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.     

Antibody-mediated disease remission in the mouse model of lyme borreliosis

In the mouse model of Lyme borreliosis, the host immune response during infection with Borrelia burgdorferi results in the remission of carditis and arthritis, as well as global reduction of spirochete numbers in tissues, without elimination of infection. These events were recapitulated by passive transfer of immune serum from infected immunocompetent mice or T-cell-deficient mice to severe combined immunodeficient (SCID) mice. Previous studies have shown that immune serum is reactive against arthritis-related protein (Arp) and that Arp antiserum induces arthritis remission. However, although immune serum from T-cell-deficient mice induced disease remission, it was not reactive against Arp, suggesting that antibody to another antigen may be responsible. T-cell-deficient mouse immune serum was reactive to decorin binding protein A (DbpA). Therefore, DbpA antiserum was tested to determine its ability to induce disease remission in SCID mice. Antisera to Arp or DbpA induced both carditis and arthritis remission but did not significantly reduce spirochete numbers in tissues, based upon quantitative flaB DNA analysis, nor did treatment affect RNA levels of several genes, including arp and dbpA. Immunohistochemical labeling of spirochetes in hearts and joints during disease remission induced by adoptive transfer of lymphocytes, passive transfer of immune serum, or passive transfer of DbpA antiserum revealed that such treatment resulted in elimination of spirochetes from heart base and synovium but not vascular walls, tendons, or ligaments. These results suggest that Arp and DbpA antibodies may be active as disease-resolving components in immune serum but antibody against other antigens may be involved in reductions of spirochetes in tissues.     

Seroprevalence of lyme borreliosis in forestry workers from Brandenburg,Germany

In 1992 blood samples were taken from 630 forestry workers in the state of Brandenburg, Germany, and an inquiry about tick bites and possible symptoms of Lyme borreliosis carried out in order to determine the seroprevalence of the disease. To estimate the rate of seroconversion within six months, 406 of the individuals were investigated a second time. IgG and IgM antibodies againstBorrelia burgdorferi were detected in serum using an indirect immunofluorescence assay (IFA) and an immunoblot assay (IBA). Fifty-three percent of the forestry workers reported suffering a tick bite, 8% of whom recalled an erythema after the bite. Positive results were found more frequently in the forestry workers than in a control group of 200 healthy blood donors in both the IgG-IFA (8% vs. 4%, p<0.05) and the IgG-IBA (18% vs. 5%, p<0.05). The detection of IgG antibodies correlated with a tick bite and erythema history. There was a tendency of lower seropositivity by the IgG-IBA in individuals who treated the ticks before removal with chemicals or other agents compared to those without such treatment (16.8% vs. 23.9%, 0.05 >p<0.1). Likewise, there was a tendency of lower seropositivity by the IgG-IFA in individuals being treated with antibiotics for other reasons compared to untreated individuals (3.15% vs. 8.9%, p<0.05), although the two groups did not differ in the IgG-IBA (13.8% vs. 18.5%, p>0.1). The rate of seroconversion within six months ranged from 5 to 7%. It is concluded that forestry workers in Brandenburg, Germany, are at risk for infection withBorrelia burgdorferi, but clinical signs of infection are rare.     

Humoral immune response associated with lyme borreliosis in nonhuman primates: analysis by immunoblotting and enzyme-linked immunosorbent assay with sonicates or recombinant proteins

The immune response to Borrelia burgdorferi, the causative agent of Lyme disease, is complex. We studied the immunoglobulin M (IgM) and IgG antibody response to N40Br, a sensu stricto strain, in the rhesus macaque(nonhuman primate [NHP]) model of infection to identify the spirochetal protein targets of specific antibody. Antigens used in enzyme-linked immunosorbent assays were whole-cell sonicates of the spirochete and recombinant proteins of B. burgdorferi. Immunoblotting with a commercially available strip and subsequent quantitative densitometry of the bands were also used. Sera from four different groups of NHPs were used: immunocompetent, transiently immunosuppressed, extended immunosuppressed, and uninfected. In immunocompetent and transiently immunosuppressed NHPs, there was a strong IgM and IgG response. Major proteins for the early IgM response were P39 and P41 and recombinant BmpA and OspC. Major proteins for the later IgG response were P39, P41, P18, P60, P66, and recombinant BmpA and DbpA. There was no significant response in the NHPs to recombinant OspA or to Arp, a 37-kDa protein that elicits an antibody response during infection in mice. Most antibody responses, except for that to DbpA, were markedly diminished by prolonged dexamethasone treatment. This study supports the hypothesis that recombinant proteins may provide a useful adjunct to current diagnostic testing for Lyme borreliosis.     

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.     

Importance of sample preparation for molecular diagnosis of lyme borreliosis from urine

Urine PCR has been used for the diagnosis of Borrelia burgdorferi infection in recent years but has been abandoned because of its low sensitivity and the irreproducibility of the results. Our study aimed to analyze technical details related to sample preparation and detection methods. Crucial for a successful urine PCR were (i) avoidance of the first morning urine sample; (ii) centrifugation at 36,000 x g; and (iii) the extraction method, with only DNAzol of the seven different extraction methods used yielding positive results with patient urine specimens. Furthermore, storage of frozen urine samples at -80 degrees C reduced the sensitivity of a positive urine PCR result obtained with samples from 72 untreated erythema migrans (EM) patients from 85% in the first 3 months to <30% after more than 3 months. Bands were detected at 276 bp on ethidium bromide-stained agarose gels after amplification by a nested PCR. The specificity of bands for 32 of 33 samples was proven by hybridization with a GEN-ETI-K-DEIA kit and for a 10 further positive amplicons by sequencing. By using all of these steps to optimize the urine PCR technique, B. burgdorferi infection could be diagnosed by using urine samples from EM patients with a sensitivity (85%) substantially better than that of serological methods (50%). This improved method could be of future importance as an additional laboratory technique for the diagnosis of unclear, unrecognized borrelia infections and diseases possibly related to Lyme borreliosis.     

Recombinant BBK32 protein in serodiagnosis of early and late Lyme borreliosis

Borrelial protein BBK32 was evaluated as an antigen in the serodiagnosis of early and disseminated Lyme borreliosis (LB). bbk32 was cloned and sequenced from eight isolates of the three pathogenic Borrelia species. The identities between the amino acid sequences of the BBK32 proteins from Borrelia burgdorferi sensu stricto, B. garinii, and B. afzelii isolates were 71 to 100%. By immunoglobulin G (IgG) Western blotting (WB) or enzyme-linked immunosorbent assay (ELISA), up to 74 and 100% of acute- and convalescent-phase samples, respectively, from 23 patients with erythema migrans (EM) were positive for recombinant BBK32 protein from B. afzelii. In the serology of disseminated LB, the three variant BBK32 antigens cross-reacted. In total, 14 of 14 samples from patients with neuroborreliosis and 15 of 15 samples from patients with Lyme arthritis were positive. The specificities of the IgG ELISA with the variant BBK32 antigens for EM and disseminated borreliosis were 81 to 92% and 89 to 95%, respectively. Our findings indicate that the BBK32 proteins are promising serodiagnostic antigens for the detection of early and disseminated LB but that variant BBK32 proteins may be needed either in parallel or in combination with an immunoassay for LB to cover all the relevant borrelial species that cause the disease.     

Use of Bartonella adhesin A (BadA) immunoblotting in the serodiagnosis of Bartonella henselae infections

Bartonella henselae causes a variety of human diseases (e.g. cat scratch disease and the vasculoproliferative disorders, bacillary angiomatosis and peliosis hepatis). The laboratory diagnosis of B. henselae infections is usually based on the detection of anti-B. henselae antibodies by an indirect immunofluorescence assay (IFA) which, unfortunately, suffers from a significant amount of cross-reactivity and hence is prone to deliver false-positive results. In this pilot study, we evaluated the use of a potential two-step serodiagnosis of B. henselae infections by combining IFA and anti-Bartonella adhesin A (BadA) immunoblotting. Our data revealed that approximately 75% of the IFA-positive sera of patients with a suspected B. henselae infection reacted specifically with BadA but only approximately 25% of the IFA-negative sera of healthy blood donors. Although Yersinia adhesin A (YadA) is structurally closely related to BadA, no cross-reactivity of sera from patients suffering from a Yersinia enterocolitica or Y. pseudotuberculosis infection with BadA was detected in immunoblotting. Unfortunately, recombinantly expressed BadA domains (head, connector, stalk fragment) were not suitable for immunoblotting. Finally, the best resolution for full-length BadA immunoblotting was obtained when whole cell lysates of B. henselae were separated using continuous 4-15% sodium dodecyl sulfate polyacrylamide gels. In summary, our results show that BadA antibodies are detectable in the sera of B. henselae-infected patients and, therefore, this pilot study suggests to include BadA immunoblotting in the laboratory diagnosis of B. henselae infections.     

C6 peptide ELISA test in the serodiagnosis of Lyme borreliosis in Sweden

The aim of this study was to evaluate the synthetic C6 peptide test as a first-line test in a two-tiered scheme for Borrelia serology in a clinically well-characterized population of patients with Lyme borreliosis in Kalmar County, Sweden. The study population consisted of a prospective group (n = 200), a control group (n = 255), and a retrospective group (n = 29). The test panel consisted of the Immunetics Quick ELISA C6 Borrelia assay kit (Immunetics, Cambridge, MA, USA), the Virotech Borrelia burgdorferi ELISA (Genzyme Virotech, Rüsselsheim, Germany), and the Liaison Borrelia CLIA (DiaSorin, Saluggia, Vercelli, Italy). Seroprevalence among 200 healthy blood donors was significantly lower in the C6 test (8%) compared to the Virotech ELISA (14%) and the Liaison CLIA (12%). In convalescent sera (2–3 months and 6 months post infection) from 158 patients with erythema migrans, the seropositivity in the C6 test was also significantly lower compared to both the Virotech ELISA and the Liaison CLIA. Serosensitivity in the acute phase of erythema migrans and other clinical manifestations of borreliosis did not differ significantly between the C6 test and the Virotech ELISA or the Liaison CLIA. Overall, a positive C6 test seems to correlate well with acute borreliosis. Cross-reactivity was lower in the C6 test in sera positive for Epstein-Barr virus infection as compared to the Virotech ELISA. This study supports the use of the C6 test as a screening test for borreliosis, in endemic areas.     

Validity of western immunoblot band patterns in the serodiagnosis of Lyme borreliosis.

Serodiagnosis of Lyme disease is hampered by low specificity of the standard assays currently used. The Western immunoblot has therefore been proposed as a potential confirmatory test. For the present report, the method was evaluated by testing sera from patients with clinically defined early- and late-stage borreliosis. In early-stage borreliosis, the 41,000-molecular-weight flagellin protein (41K) of Borrelia burgdorferi was the major antigen detected by antibodies in sera, but the specificity of the reaction pattern was dependent on the intensity of the band. The evaluation of different interpretation rules based on a semiquantitative record of band intensities showed the highest specificity (96%) and a corresponding sensitivity of 78% if there was at least one distinct (optical density range, 0.2 to 0.4) immunoglobulin G and immunoglobulin M reaction with the 41K band. Blots of B. burgdorferi proteins were also probed with sera from patients who were diagnosed by clinical criteria as having stage III Lyme borreliosis and with a control group of sera from asymptomatic persons with positive antibody titers against B. burgdorferi in the standard assays. Reaction patterns were recorded densitometrically. Statistical analysis and graphical marker analysis revealed significant discriminating capacities and relatively high specificities, respectively, for the 94K, 30K, and 21K bands, whereas the 41K and 60K bands were not discriminative between the symptomatic and asymptomatic groups and were specific only at high intensity values. Different multiple-band rules were evaluated, revealing a low specificity for positivity definitions of the type "four or five bands present" if the rules were not confined to known major bands.     

Use of the immunoblotting method in serodiagnosis of M. kansasii mycobacteriosis]

The indirect enzyme test ELISA with soluble complex antigen of M. kansasii, used for assessment of the titre of IgG serum antibodies in a group of patients suffering from mycobacteriosis M. kansasii and in a control group of patients suffering from tuberculosis did not reveal statistically significant differences between serological responses of these two groups. On the other hand, the immunoblot analysis revealed in sera selected at random from these two groups differences at the level of subprotein units against the complex antigen of M. kansasii. In a group of 15 sera of patients with M. kansasii the immunodominant area was 42-45 kD protein subunits, in the control group of 10 sera with M. tuberculosis the area was 35-39 kD. The western blot technique is more perspective for improvement of the serum diagnosis of M. kansasii, in particular where a specific antigen is not available.     

A European multicenter association study of HTR2A receptor polymorphism in bipolar affective disorder

The available data on the role of 5-HT in a variety of behaviors support the hypothesis that a dysfunction in brain serotoninergic system activity contributes to vulnerability to major depression. The diversity in the electrophysiological actions of 5-HT in the central nervous system can now be categorized according to receptor subtypes and their respective effector mechanisms. In particular, the implication of central postsynaptic 5-HT2A receptor in affective disorders has been supported by findings consistent with the hypothesis of 5-HT2A receptor up-regulation in depression. For these reasons, the 5-HT2A receptor (HTR2A) gene can be considered as a candidate gene in bipolar affective disorder (BPAD). We tested the possible genetic contribution of the polymorphic DNA variation T102C in exon 1 of HTR2A (chromosome 13q14-21) gene in a large European multicentric case-control sample. Allele and genotype frequencies, as well as homo-heterozygote distributions were compared between the two groups of 309 bipolar affective disorder patients and 309 matched controls. No significant differences were observed in the allelic and genotypic (also for homo-heterozygote) distribution between BPAD and controls. These results indicate that, in our sample, the 5-HT2A receptor polymorphism studied is unlikely to play a major role in the genetic susceptibility to BPAD. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:136-140, 2000.     

Two-year survey of the incidence of lyme borreliosis and tick-borne encephalitis in a high-risk population in Sweden

A survey was made over a two-year period (September 1987 to August 1989) of a population living in an area endemic for Lyme borreliosis and tick-borne encephalitis in Sweden. For each patient a blood sample was collected and a questionnaire completed annually. All sera were tested for an antibody response toBorrelia burgdorferi in an EIA using sonicated antigen and for an antibody response to the tick-borne encephalitis virus using an EIA and a haemagglutination inhibition test. Antibodies toBorrelia burgdorferi and tick-borne encephalitis virus were detected in 89 (25.7 %) and 40 (11.6 %) respectively of 346 samples collected in August 1987. In the first year of the study 14 of 303 subjects (4.6 %) developed Lyme borreliosis and in the second year 9 of 277 subjects (3.2 %). A significant increase in the antibody titre forBorrelia burgdorferi was seen in 14 of 303 (4.6 %) subjects in the first year and 8 of 277 (2.9 %) subjects in the second year. An earlier episode of Lyme borreliosis or an elevated antibody titre did not seem to protect against reinfection. One case of tick-borne encephalitis was seen each year. Seroconversion for tick-borne encephalitis virus was found in 3 of 258 (1.2 %) subjects in the first year and 5 of 211 (2.4 %) in the second year, excluding subjects who had undergone successful immunisation or had earlier been hospitalised for tick-borne encephalitis. The study thus demonstrated a high yearly incidence of tick-borne infections in a population at risk.     

Improved serodiagnosis of early Lyme borreliosis: immunoblot with local Borrelia afzelii strain

To improve the serodiagnosis of Lyme borreliosis (LB) the performances of four tests were evaluated. An indirect immunofluorescent assay based on Borrelia burgdorferi s.s., enzyme-linked immunosorbent assay (ELISA) based on local isolates of Borrelia afzelii and B. burgdorferi s.s., and immunoblot (IB) of B. afzelii were prepared. The serum panels contained 214 serum samples: control group (n=120) and patients at different stages of LB (n=94). The specificity of IB was 96%, of in-house ELISA 93%, and of IFA 89%. In early LB the sensitivity of IFA was 36%, ELISA 67%, and IB 93%. In late-stage LB the sensitivity was: 72% for IFA, 80% for ELISA, and 94% for IB. Comparison of in-house and Behring ELISA showed that the sensitivity of the serological assay could be increased when the test was based on local Borrelia strains. IgM and IgG antibodies from sera of patients with early and late LB most frequently demonstrated reactivity to OspC. The other significant proteins in early LB were: p39, p41 in IgM IB, and p83/100, p39, Osp17 in IgG IB; in late LB: p39, p41 in IgM IB, and p83/100, Osp17, p21 and p43 in IgG IB. Using IB based on local B. afzelii isolates improves the serodiagnosis of early LB in our geographical region.     

Western immunoblot and flagellum enzyme-linked immunosorbent assay for serodiagnosis of Lyme borreliosis.

Western immunoblot with a whole-cell lysate was compared with an enzyme-linked immunosorbent assay with a purified flagellum antigen of Borrelia burgdorferi for serodiagnosis of Lyme borreliosis. The assays showed similar sensitivities and specificities in detecting immunoglobulin M and/or immunoglobulin G antibodies in sera from 68 patients with neuroborreliosis and 44 controls with meningitis and encephalitis or with multiple sclerosis. Flagellum enzyme-linked immunosorbent assay is more easily standardized and seems to be a more suitable diagnostic test in a routine laboratory.     

Evidence for vaccine synergy between Borrelia burgdorferi decorin binding protein A and outer surface protein A in the mouse model of lyme borreliosis

Mice immunized with either the predominantly vector-stage lipoprotein outer surface protein A (OspA) or the in vivo-expressed lipoprotein decorin binding protein A (DbpA) are protected against Borrelia burgdorferi challenge. DbpA-OspA combinations protected against 100-fold-higher challenge doses than did either single-antigen vaccine and conferred significant protection against heterologous B. burgdorferi, B. garinii, and B. afzelii isolates, suggesting that there is synergy between these two immunogens.     

A long-term follow-up study of hyposensitization with immunoblotting

The formation of specific IgE, IgG1, and IgG4 antibodies was investigated by immunoblotting during hyposensitization with timothy grass-pollen extract and 6 years later, Until the end of immunotherapy, specific IgG antibody levels increased. Also simultaneously, the number of allergenic components detected by IgG increased. However, this IgG response was similar in responding and nonresponding patients; thus, it did not correlate with the clinical outcome of the therapy. More allergenic compounds were also detected by IgE on immunoblots, but again without correlation to success of therapy. Six years after immunotherapy, the therapeutic effect was still present, although by now the observed immunoglobulin-binding patterns were similar to patterns observed in the same patients' sera collected before the initiation of hyposensitization. Thus, changes of antibody-binding patterns in immunoblot do not relate to the success or failure of immunotherapy.     

C6 test as an indicator of therapy outcome for patients with localized or disseminated lyme borreliosis

Management of Lyme disease would benefit from a test to assess therapy outcome. Such a test could be employed to ascertain if treatment of early Lyme disease was successful and would be helpful to clinicians assessing patients with lingering posttreatment symptoms. We reported recently that levels of the antibody to C(6), a Borrelia burgdorferi-derived peptide that is used as an antigen in the C(6)-Lyme diagnostic test, declined after successful antibiotic treatment of Lyme borreliosis patients. We assessed retrospectively the change in anti-C(6) antibody titers in 131 patients with either early localized disease (erythema migrans) or disseminated disease. All of these patients were treated with antibiotics and were free of the clinical signs shown at presentation within 12 weeks after the initiation of treatment. Decreases in reciprocal geometric mean titers (rGMT) of the anti-C(6) antibody were quantified for the subpopulation of 45 patients whose baseline rGMT were >/=80 and whose second serum specimens were obtained at least 6 months after the baseline specimen. Eighty percent of this patient group (36 of 45) experienced a >/=4-fold decrease in their rGMT (P < 0.0003). These results suggest that a change in the anti-C(6) antibody titer may serve as an indicator of therapy outcome for patients with localized or disseminated Lyme borreliosis.     

Designing and clinical testing of immune-enzyme and immunofluorescence test systems for serodiagnosis of ixodes borreliosis

The methods of immune enzyme assay (MIEA) and of lanthanide immunofluorescence analysis (LIFA) were used to work out the test systems for the detection (in blood serum of patients) of specific IgM IgG antibodies to the B. burgdorferi spirochete--a causative agent of ixodic borrelioses. The test system was clinically tested versus the indirect immunofluorescence reaction (IIFR) and commercial immune enzyme test system (CIET). The results of antibodies' detection were shown to correlate with the analysis data for the same sera in IIFR and to be in line with a real presence or absence of the disease. Test systems based on LIFA were proven to be most sensitive and specific.