Zbl. Bakt. 282, 303-314 (1995) © Gustav Fischer Verlag, Stuttgart· Jena . New York
Intrathecal Immune Response in Neuroborreliosis: Importance of Cross-reactive Antibodies R. KAISER Department of Neurology, University of Freiburg, Germany Received July 25,1994· Revision received November 9,1994· Accepted December 8,1994
Summary The intrathecal IgG response to Borrelia burgdorferi was evaluated in 35 patients with neuroborreliosis (NB). Samples were tested with and without preabsorption with Treponema phagedenis. Specific antibody concentrations in the CSF and serum were determined by ELISA. The antibody index (AIBb = QBJ!QIgG) was calculated from the ratio between the CSF/serum quotients for specific antibodies (QBb) and total IgG (QIgG)' Intrathecal synthesis of B. burgdorferi antibodies was demonstrated in 31 samples before and in 24 samples after preabsorption with T. phagedenis. The clonal distribution of intrathecally produced IgG antibodies was determined by isoelectric focusing combined with affinity blotting. B. burgdorferi-specific oligoclonal IgG bands occurring predominantly in the CSF were demonstrated in 32/35 patients. In 29/32 samples, the major proportion of these bands also reacted with T. phagedenis. Preabsorption of samples with T. phagedenis removed a considerable share of bands reacting with B. burgdorferi. In patients with neurosyphilis, intrathecal synthesis of antibodies with specificity for B. burgdorferi was demonstrated in 711 0 samples before, and in no sample, after preabsorption of cross-reactive antibodies. Due to the lower sensitivity in determining the AIBb (-20%), preabsorption of cross-reactive antibodies cannot be generally recommended. In all patients with suspected neuroborreliosis, but uncommon neurological symptoms and missing anamnestic data concerning a tick bite or erythema migrans, neurosyphilis should be excluded by a negative TPHA test. Introduction Lyme disease is a systemic disease caused by infection with the tick-transmitted spirochaete, Borrelia burgdorferi. Neurological symptoms have been reported in about 20% of North American and as many as 50% of European patients, with meningitis, cranial neuritis and painful radiculoneuritis (Bannwarth's syndrome) occurring most frequently (16). In the last ten years, an increasing spectrum of neurologic disorders has been associated with B. burgdorferi infection, but in some syndrome, causality has never been established (3, 10). In hyperendemic areas, 5% to 25% of asymptomatic individuals may exhibit serologic evidence of exposure (18), Consequently, seropositivity alone cannot be con-
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sidered as a proof that a patient's problem are caused by B. burgdorferi infection. False-positive reactions can also occur among individuals with syphilis and in areas where closely related Borrelia species coexist with B. burgdorferi (5). Attempts to increase the specificity of antibody tests have employed preabsorption of serum with Treponema phagedenis, Biotype Reiter (2), or Escherichia coli (12). This procedure, however, reduces not only titres of cross-reactive antibodies, but may also decrease homologous titres. Infections of the central nervous system (CNS) frequently give rise to an autochthonous immune response to the etiologic agent (1). In several studies, this experience has been successfully applied to investigate the intrathecal immune response in patients with suspected neuroborreliosis (3, 19, 20). In a recent study, however, Hansen et al. demonstrated the reactivity of CSF antibodies from patients with neurosyphilis with B. burgdorferi (4). The aim of this study was to investigate the effect of preabsorption of CSF and serum samples with T. phagedenis on calculation of the intrathecal antibody synthesis in patients with neuroborreliosis and neurosyphilis. Results from ELISA studies were supplemented by the demonstration of specificity and cross-reactivity of oligo clonal IgG bands in the CSF of these patients.
Patients and Methods Subjects. Paired CSF and serum samples from 35 patients were tested. All patients had a positive antibody response to B. burgdorferi in CSF and serum. Patients were negative in TPHA screening. Of them, 26 (ages: 22-80 years) had early Lyme neuroborreliosis (ELN), defined as neurologic symptoms lasting more than one week but not more than six months. These patients had meningitis mostly accompanied by N. VII palsy (n = 14), painful radiculitis (n = 10), myelitis (n = 1), and papillitis (n = 1). Nine patients (ages: 24-61 years) presented themselves with late Lyme neuroborreliosis (LLN), defined as neurological symptoms lasting for at least seven months (range 7 months - 4 years), that could not be attributed to another cause. Of these, eight had spastic hemi- or paraparesis, and one had motor neuron disease. Examinations, including neurophysiological studies, computed tomography, magnetic resonance imaging, and, immunological and serological studies were not indicative of other etiologic causes, i.e. neurosyphilis, stroke or cerebral vasculitis, tumour, multiple sclerosis (MS) or neurosarcoidosis. A preceding erythema migrans (EM) had been reported in 14 Patients (ELN: 12, LLN: 2), 15 individuals (ELN: 14, LLN: 1) remembered a tick bite. Patients were treated with ceftriaxone 1 X 2 g intravenously for 14 days. All measurements were done on the first pretreatment samples taken one week to four years after onset of neurologic symptoms. CSF and sera were drawn at the same time. For controls, paired CSF/serum samples from 30 patients with MS and from 10 patients with neurosyphilis were analyzed. The latter patients had been admitted to hospital because of paranoid syndrome (n = 4), depression (n = 1), dementia (n = 1) and lumbar pain (n = 4). The TPHA was highly positive in serum in 10 patients, and in CSF in 7 patients. The fluorescent treponemal antibody absorption (FTA-abs) test was positive in all patients. Standard CSF determination. CSF cells were counted by phase contrast microscopy within 30 min of lumbar puncture. Albumin, IgG, IgM and IgA were measured in CSF and corresponding serum by laser nephelometry. With this apparatus, detection limit for IgM is 6.9 mlll and for IgA 11.1 mg/1. In the case of IgM and IgA concentrations in the CSF below the detection limit, levels in paired CSF and sera were determined by ELISA (8). Detection sensitivity with this assay was 10 !lgl for IgM and IgA. The blood-CSF barrier was evaluated by determination of the CSF/serum albumin ratio. Intrathecal synthesis of IgG, IgM and 19A was calculated according to Reiber. Fractions of locally produced immunoglobulins are
Intrathecal Immune Response in Neuroborreliosis
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given as percentage (%) of the total IgGIMIA content in the CSF (14). Findings from routine CSF studies are shown in Table 1. Routine Lyme borreliosis serology
Screening for B. burgdorferi-specific antibodies in sera and CSF was done by the indirect immunofluorescence assay (IFA) according to standard techniques (17). Samples were tested at the Department of Medical Microbiology of the University of Freiburg. For IFA, the same B. burgdorferi strain was used as in ELISA (Z-136). For IgG-antibodies, specimens were tested with and without preabsorption with T. phagedenis. In this laboratory serum IgG titres after absorption 2: 1: 32 were considered as positive. For the IgM-specific test, the samples were pretreated with anti-human IgG (RF-Adsorbents, Behringwerke AG, Marburg, F.R.G). Serum IgM titres 2: 1: 48 were considered as positive. Antigen preparation
For ELISA, western blotting and affinity blotting, sedimented spirochaetes (B. burgdorferi, T. phagedenis, T. pallidum) were supplemented with 6M ureaJPBS and sonicated for 5 min at 60 percent of maximum setting on a Branson B-12 Sonifier. By comploying this procedure, cells were completely dissolved and then diluted in 6M ureaJPBS to a final protein concentration of 2.0 mg/mL. For absorption procedures, T. phagedenis was sonicated in PBS without urea and used as a crude cell suspension. ELISA procedure
Determination of antigen-specific intrathecal antibody synthesis by ELISA was done by a concentration ELISA as described previously (9). This ELISA technique allows the quantification of relative concentrations of specific antibodies in CSF and serum instead of antibody titres: ODs of the reference samples were related to arbitrary concentrations units. The OD (1.8) of the greatest measurable standard concentration (serum dilution 1: 1000) was defined to be 1000 arbitrary concentration units and the lowest OD (0.10) to be 15.625 concentration units (1000/2 6 ). The standard values (reference sample) were evaluated in a logllog diagram (OD/relative concentrations). Final antibody concentrations in CSF and serum were calculated by multiplication of relative concentrations units with the dilution factor. Samples to be tested for IgG antibodies against B. burgdorferi sonicate were diluted 200, 1.000 and 10.000 times (serum) and 10,50 and 500 times (CSF). For absorption studies, 100 ilL of 100-fold prediluted serum and 100 ilL of 5-fold prediluted CSF were incubated with 100 IlL of T. phagedenis (1.0 mg/mL). After incubation at 37°C for 1 h, samples were pelleted at 13.000 U/min for 20 min. The supernatant was used for further dilution steps. Control samples were treated in the same way. Microtiter plates were coated overnight at 4°C with 50 IlL of B. burgdorferi or T. phagedenis (20 Ilg/ml) in ureaJPBS. Antibodies to B. burgdorferi were tested on the same plate with and without preabsorption with T. phagedenis. ODs, which were 3 SD above the mean OD of 30 negative controls (2: 0.10), were defined as a positive antibody response to B. burgdorferi. Samples were tested in duplicate, and the mean value was calculated. If the two values differed by more than 10% from the mean, the samples were retested. Negative controls were included on every plate. Evaluation of specific antibody synthesis in the CNS. Specific antibody synthesis in the CNS was calculated by the antibody index (AIBb ), which is defined as the ratio between the CSF/serum quotient for specific antibodies (~) and the quotient of total IgG in CSF and serum (
IgG esE IgG serum
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R. Kaiser
As proposed by Reiber and Lange (15), in the case of local synthesis of polyclonal IgG in the CNS (Ig~ocaJ > 0), QIgG was replaced by Qlim with QJim = 0.8
VQ3\lb + 15.10-6
-
10-3
In this procedure, Qlim includes only immunoglobulins which statistically enter the CSF by diffusion at a certain blood-CSF-barrier permeability represented by the CSF/serumratios of albumin (QAlb). The normal reference range for the AI was determined to be between 0.7 and 1.3. As shown by Reiber and Lange (15) and confirmed by our own studies, quotients ~ 1.5 were considered indicative of an intrathecal antibody response to B. burgdorferi (AIBb ) or T. phagedenis (AITp ) antigens. Isoelectric focusing and affinity blotting. Analysis of oligoclonal IgG bands was performed as described previously (7). Briefly, samples were focused on thin agarose gels and afterwards blotted onto nitrocellulose strips loaded with goat antibodies to human IgG. Bound antibodies were detected by incubating the sheets with peroxidase-conjugated goat anti-human IgG (Dianova, Hamburg, F.R.G.) diluted 1: 1000 in PBSlTween. Oligoclonal IgG bands related to B. burgdorferi, T. phagedenis, and T. pallidum were detected by the same procedure, except that NC strips had been coated with the corresponding antigens (50 1J.g/10 cm2/mL) and the concentration of immunoglobulins increased to 20 mgIL. Intrathecal synthesis of IgG was assumed to be present when two or more bands were detected in CSF exclusively or bands were stained more intensively in CSF. Specific transfer of antibodies was confirmed using native NC strips as a negative control. For absorption of cross-reactive antibodies, CSF and serum samples were added each to a microcentrifuge tube containing an equivalent volume of T. phagedenis (200 mgIL) and incubated at 37°C for 1 hour (final IgG concentration in CSF and serum: 20 mgIL). SDS gel and immunoblotting. SDS-PAGE, transfer of electrophoretically separated proteins (B. burgdorferi, T. phagedenis, and T. pallidum lysates) to nitrocellulose, and subsequent blotting was done according to the previously described method (9). After blocking for 1 h at room temperature with dilution buffer, the strips were incubated with 0.5 mL of CSF and sera, each diluted to an IgG concentration of 10 mgIL. After 2 h, the strips were washed three times with TweenlPBS and incubated for 1 h at room temperature with peroxidase-labelled goat anti-human IgG (1: 1000). The NC strips were developed by addition of a freshly prepared solution of 3 mg/mL 4-chloro-1-naphthol (Sigma) and 3 IJ.UmL hydrogen peroxide in PBS for 10 min. The reaction was stopped by washing the strips with distilled water. Intrathecal synthesis of antibodies to the individual spirochaetal proteins was assessed either from the isolated presence or more intense staining of antibody bands in the CSF. Results Indirect immunofluorescence assay (IFA) All patients with neuroborreliosis had IgG-antibodies to B. burgdorferi in serum (1: 64 - 1: 4096) and CSF (1: 2 - 1: 1024). After preabsorption of cross-reactive antibodies, 30/35 neuroborreliosis patients were IgG antibody-positive in serum (1: 32 - 1: 1024) and 29/35, in the CSF (1: 2 - 1: 1024). Elevated IgM antibody to B. burgdorferi in serum was demonstrated in 16 patients and in CSF, in 17/35 patients. In patients with neurosyphilis, IgG antibody to B. burgdorferi in serum (1: 32 - 1: 2048) was demonstrated in seven subjects and in the CSF, in four patients (1: 2 - 1: 256). All of them became negative after preabsorption with T. phagedenis. IgM-antibody to B. burgdorferi was not detected in patients with neurosyphilis either in serum or CSF.
Intrathecal Immune Response in Neuroborreliosis
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Antibody index (AI) determination by ELISA The completeness of absorption procedures was examined by testing individual CSF specimens and sera, with and without preabsorption with T. phagedenis, on the same plate in T. phagedenis ELISA. Mter preabsorption, the OD of samples was reduced to about 10% of the initial OD before absorption. No further reduction in OD could be achieved by higher concentrations of T. phagedenis. Intrathecal synthesis of antibodies to B. burgdorferi (AIBb ;::: 1.5) was demonstrated in 31/35 samples (Tab. 1). Mter preabsorption with T. phagedenis, 24/35 samples (ELN: 17, LLN: 7) were still positive. AIBb values were compared in samples with and without preabsorption. After preabsorption, the AIBb values increased in 16 samples (ELN: 11, LLN: 5) and decreased in 15 samples (ELN: 11, LLN: 4). In 7/31 samples, the AIBb fell below 1.5. Individual changes of AIBb values are shown in Fig. 1. Employment of T. phagedenis in ELISA revealed elevated AITp values in 26/35 samples (ELN: 18, LLN: 8),25/26 patients also had elevated AIBb values in B. burgdor-
Table 1. Findings from standard CSF determinations in patients with neuroborreliosis Normal values
Early Lyme Neuroborreliosis N = 26
Late Lyme Neuroborreliosis N = 9
<5
25 234± 150
8 76±93
abnormal values m±s (x 10-3 )
<7.5
25 23.5 ± 11.1
7 42.2±41
IgG Synthesis abnormal values m±a (%)
0
20 22±17
8 46±26
IgM Synthesis abnormal values m±s (%)
0
23 60±30
3 10± 16
IgA Synthesis abnormal values m±s(%)
0
5 8±20
6 28±29
OCB-IgG
0
25
9
OCB-Bb
0
23
9
AI-Bb
<1.5
22
9
Cell abnormal values m±s (filL)
Q Albumin
OCB-IgG: Oligo clonal bands of total IgG OCB-Bb: B. burgdorferi-specific oligo clonal bands AI-Bb: B. burgdorferi-specific antibody index
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"wn_"" " before absorption
after absorption
0
I
100
• ?
"'\LLL
AI 10
"j;
-"" 5
10
15
.""I 1 20
j-'a I
61
25
~"
""
ll!ll 30
35
Fig. 1. Significance of pre absorption of cross-reactive antibodies in CSF and serum on determination of the antibody index (AI). Samples were ranked by decreasing AI values. AIBb values 2:: 1.5 were considered positive. The last four samples were negative.
feri ELISA. Comparison of the AI values calculated from B. burgdorferi and T. phagedenis ELISA without preabsorption of cross-reactive antibodies, provided similar values for both agents in 10 samples (ELN: 9, LLN: 1), higher values in B. burgdorferi ELISA in 16 samples (ELN: 12, LLN: 4), and higher values in T. phagedenis ELISA in 9 samples (ELN: 5, LLN: 4). No correlation between the duration of disease and the level of AIBb or AITp could be derived from these findings Elevated AI values (7.1-72.2) in B. burgdorferi ELISA were also found in 7110 patients with neurosyphilis, all of whom became negative after preabsorption with T. phagedenis. Retesting in T. phagedenis ELISA provided elevated AIs (3.1-38.0) in 8110 samples. Samples from patients with MS were negative in B. burgdorferi and T. phagedenis ELISA. Specificity of oligoclonal IgG bands in the CSF Oligoclonal bands of total IgG were found exclusively or more intensely in the CSF in 34/35 patients with neuroborreliosis and in 8110 patients with neurosyphilis. In one patient with facial palsy occurring one week after EM, affinity blotting did not show either oligoclonal bands of total IgG nor of B. burgdorferi-specific IgG. In patients with neuroborreliosis, affinity blotting demonstrated reactivity of electrofocused IgG bands with B. burgdorferi in 32/35 CSF samples. In 3/32 patients, however, a similar IEF pattern of agent-specific oligoclonal IgG bands was detectable in CSF and serum. Even though these patients had elevated concentrations of B. IJUrgdorferi-specific antibodies in the CSF and serum, calculation of the AIBb did not indicate intrathecal synthesis of these antibodies. However, this criterion could be established from the finding of additional agent-specific bands in the CSF (Fig. 2).
Intrathecal Immune Response in Neuroborreliosis
Detection
Sample
Total IgG
Serum
Borrelia burglfulfeli
Serum
Treponema pbagedenis
Serum
309
IEF-Pattem
CSF
CSF
CSF 8
pH-gradient
10
Fig. 2. Oligoclonal IgG bands of similar intensity in CSF and serum. Only the presence of two additional bands in CSF indicates intrathecal synthesis of agent-specific antibodies. Calculation of the AI was negative « 1.5) in this patient.
Detection
Sample
Total IgG
Serum
Borrelia bUlogdolferi
Serum
Treponema phagedenis
Serum
pH-gradient
IEF-Pattem
CSF
CSF
CSF 8
10
Fig. 3. IEF and affinity blotting of CSF and serum in a patient with late Lyme neuroborreliosis (AI: 9.5). A considerable proportion of agent-specific bands is cross-reactive with B. burgdorferi and T. phagedenis.
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Reactivity of oligoclonal IgG bands in the CSF with T. phagedenis was shown in 29/ 32 samples (ELN: 20, LLN: 9). In 21129 CSF samples, IEF disclosed a very similar pattern of bands reacting with B. burgdorferi and T. phagedenis (Fig.3). In ten patients with neuroborreliosis, oligoclonal bands in the CSF Were tested for reactivity with T. pallidum. In nine patients, individual IgG bands not only bound to B. burgdorferi and T. phagedenis but also to T. pallidum. After preabsorption with T. phagedenis, a considerable portion of bands have been prior reacting with B. burgdorferi, was eliminated. In 7110 patients with neurosyphilis, most of the oligoclonal IgG bands in the CSF reacted with T. pallidum. The CSF of these patients was also positive in the TPHA test. However, a considerable portion of these bands bound to B. burgdorferi and T. phagedenis, too. In general, affinity blotting disclosed a very similar pattern of both antigen preparations (T. phagedenis and B. burgdorferi). The typical finding in patients with neurosyphilis is shown in Fig.4. Preabsorption with T. phagedenis eliminated most of the B. burgdorferi bands but only a minor portion of the T. pallidum-specific bands. No reactivity of oligo clonal IgG-bands with B. burgdorferi, T. phagedenis or T. pallidum could be shown in any patient with MS. CSF and sera were diluted to the same IgG concentration and tested in western blotting for antibodies to B. burgdorferi and T. phagedenis. B. burgdorferi bands, showing the highest frequency of occurrence in the CSF, appeared to be in the range of 41 kDa (ELN: 16, LLN: 7), 45 kDa (ELN: 11, LLN: 6), and 60-65 kDa (ELN: 11, LLN: 6). Intrathecally produced IgG antibodies to T. phagedenis were detected less
Detection
Sample
Total IgG
Serum
Treponema pallidum
Serum
IEF-Pattem
CSF
CSF
Treponema pbagedenis Borrelia burgdorferi 10 Fig. 4. IEF pattern of a patient with chronic neurosyphilis showing several IgG bands specific for T. pallidum. Identical and additional IgG bands in the CSF are reacting with T. phagedenis and B. burgdorferi indicating cross-reactivity of intrathecally produced antibodies.
Intrathecal Immune Response in Neuroborreliosis
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frequently. Mostly, these antibodies reacted with p45 (ELN: 9, LLN: 7) and p60 (ELN: 8, LLN: 2). In several patients, western blotting of B. burgdorferi and T. phagedenis revealed CSF antibodies to proteins of similar molecular weight in both antigen preparations. These antibody reactions were related to p45 (n = 12), p60 (n = 7), and p70 (n = 5). In a similar manner, samples from patients with neurosyphilis were tested for IgG antibodies to T. pallidum, T. phagedenis, and B. burgdorferi. In 8/10 patients, this procedure revealed an intrathecal synthesis of IgG antibodies to various proteins of these agents. These antibodies were related to p47 (8/8), p18 (6/8), and p33 (5/8) of T. pallidum, to p45 (8/8) and to p33 (3/8) of T. phagedenis, and to p41 (5/8) and to p60 (4/8) of B. burgdorferi. Fig. 5 illustrates the immunoreactivity of CSF IgG antibodies to proteins of T. pallidum, T. phagedenis, and B. burgdorferi in a patient with neuroborreliosis and neurosyphilis, respectively. In general, in patients with neuroborreliosis, intrathecally produced IgG-antibodies reacted with a higher number of borrelial antigens than of treponemal antigens.
MWM A
B
c
A
B
- -------
fJ7
67 45
30
A
B
c
----- ..
-
-•
21
Total protein stain
Neuroborreliosis
Neurosyphilis
Fig. 5. Western blotting of CSF IgG antibodies. Lane A: T. pallidum, lane B: T. phagedenis, lane C: B. burgdorferi. Left side: Total protein stain, centre: patient with neuroborreliosis, right side: patient with neurosyphilis.
Discussion Previous studies have indicated that determination of the intrathecal synthesis of B. burgdorferi-specific antibodies is useful in suspected cases of neuroborreliosis (6, 19, 20). Most of these examinations were done by B. burgdorferi sonicate ELISA. In general, samples were tested without preabsorption with T. phagedenis. For these
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studies, sensitivity in detecting an intrathecal synthesis of B. burgdorferi-specific IgG antibodies is reported to range from 87% in solid-phase ELISA (3) to 92% in antibody capture ELISA (4). Demonstration of an intrathecal antibody response to an infectious agent is not only dependent on technical equipment but also on the duration of disease: In this study, the B. burgdorferi antibody index (AIBb ) was elevated in 84.6% of samples from patients with an acute course and in all samples from patients with a chronic course of Lyme neuroborreliosis. In 4/26 patients (15.4%) with a one-week history of neurological symptoms, no intrathecal synthesis of B. burgdorferi-specific IgG could be demonstrated in the CSF. In these patients, the suspicion of neuroborreliosis was supported by the occcurrence of an EM in three of four subjects, and by pathological findings in the CSF (pleocytosis, impairment of the blood-CSF barrier, intrathecal synthesis of IgM), and elevated IgM-antibodies to B. burgdorferi in the CSF and serum. Even though antibody determination without preabsorption is very sensitive, this procedure is quite unspecific with regard to the intrathecal immune response in patients with neurosyphilis: In this study, 7110 patients with neurosyphilis had elevated antibody indices in B. burgdorferi sonicate ELISA; all of them were negative after preabsorption with T. phagedenis. This procedure, however, resulted in a significant loss of sensitivity concerning the AIBb determination in patients with neuroborreliosis. In those seven patients with AIBb values < 1.5 after absorption procedures, the clinical diagnosis of neuroborreliosis was supported by pathological findings in the CSF (pleocytosis: 7/7, blood-CSF-barrier impairment: 6/7, IgG synthesis: 3/7, IgM synthesis: 5/7), and by elevated IgM ( n = 4) or IgG (n = 7) antibodies to B. burgdorferi in serum and/or CSF determined by IFA. In two of these patients, duration of disease was more than one year indicating that even in chronic courses cross-reactive antibodies might represent the major portion of locally produced IgG-antibodies. Besides the calculation of the B. burgdorferi-specific antibody index, the determination of B. burgdorferi-specific oligoclonal IgG bands in the CSF represents a suitable method to demonstrate a specific intrathecal immune response in patients with neuroborreliosis (5, 13). In some patients, however, affinity blotting may even be superior to the calculation of indices. This was particularly shown in three patients in whom the intrathecal synthesis of agent-specific antibodies could only be assessed from the different pattern of specific oligo clonal bands in CSF and serum (Fig.2). In this study, B. burgdorferi-specific oligoclonal IgG bands occurring predominantly in the CSF, were detected in 32/35 patients with neuroborreliosis (91%). In 90% of these specimens, the major portion of bands also reacted with T. phagedenis. In a preceding study, 35/45 patients with neuroborreliosis (78%) exhibited B. burgdorferi-specific oligo clonal IgG bands in the CSF (5). No cross-reactivity of oligoclonal bands with T. phagedenis could be detected in four samples tested. Similarly as in neuroborreliosis, in patients with neurosyphilis, oligoclonal IgG bands in the CSF have been shown to react specifically with Treponema pallidum (22). In the present study, however, both in patients with neuroborreliosis and neurosyphilis, oligoclonal IgG bands were demonstrated to react with B. burgdorferi and T. pallidum as well. In individual patients IEF disclosed a considerable number of identical oligoclonal bands reacting with B. burgdorferi and T. pallidum, while western blotting of the same CSF sample demonstrated antibody reactions to five or more proteins of B. burgdorferi but only to one protein of T. pallidum. The molecular masses of this single protein, however, differed between the patients tested. In conclusion, results from ELISA, western blotting, and IEF along with affinity
Intrathecal Immune Response in Neuroborreliosis
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blotting demonstrate that in patients with neuroborreliosis, a considerable proportion of intrathecally produced IgG-antibodies is related to cross-reactive antigens of B. burgdorferi. Even though absorption procedures may improve specificity of antibody reactions to B. burgdorferi in serum and CSF (2, 21), this procedure cannot be recommended generally for the determination of the intrathecal synthesis of agent-specific antibodies in neuroborreliosis. In 20% of patients with an autochthonous immune response to B. burgdorferi, locally produced antibodies were related mainly to crossreactive antigens of the spirochaete. In patients with uncommon neurological symptoms and missing anamnestic data concerning a tick bite or erythema migrans, neither elevated antibody indices nor B. burgdorferi-reactive oligoclonal IgG bands in CSF can be considered as a proof for neuroborreliosis, as both parameters were also positive in patients with neurosyphilis. The latter diagnosis may be better excluded by a negative TPH-assay than by absorption studies. Negative results after preabsorption of cross-reactive antibodies in ELISA and IEF and affinity blotting were demonstrated both in patients with neurosyphilis and neuroborreliosis. In doubtful cases, western blotting represents a valuable method showing different magnitudes of antibody reactions to both agents. Acknowledgments. This study was supported by grant no 01KI9001/0 from the German Federal Ministry for Research and Technology (Bundesministerium fur Forschung und Technologie). Prof. Vogt, Institute of Medical Microbiology, University of Freiburg is gratefully acknowledged for donating sonicates of B. burgdorferi and T. phagedenis, Prof. Wellensiek, Institute of Medical Microbiology, University of GieBen for donating T. pallidum, Gisela Fleig for her expert and skilful technical assistance, and Prof. Batsford for help in editing the manuscript.
References 1. Dorries, R., R. Kaiser, S. Schwender, H. Imrich, A. Pohl-Koppe, and K. Dorries: Recent aspects on the diagnosis of viral infections in the central nervous system. Lab. Med. 15 (1991) 99-102 2. Fawcett, P. T., A. E. O'Brien, and R. A. Doughty: An adsorption procedure to increase the specificity of enzyme-linked immunosorbent assays for Lyme disease without decreasing sensitivity. Arthritis and Rheumatism 32 (1989) 1041-1044 3. Halperin, ]. ]., D. ]. Volkman, and P. Wu: Central nervous system abnormalities in Lyme neuroborreliosis. Neurology 41 (1991) 1571-1582 4. Hansen, K., P. Hindersson, and N. Strandberg-Pedersen: Measurement of antibodies to Borrelia burgdorferi flagellum improves serodiagnosis in Lyme disease. ]. Clin. MicrobioI. 26 (1988) 338-346 5. Hansen, K., M. Cruz, and H. Link: Oligoclonal Borrelia burgdorferi-specific IgG antibodies in cerebrospinal fluid in Lyme neuroborreliosis. J. Infect. Dis. 161 (1990) 1194-1202 6. Hansen, K. and A. M. Lebech: Lyme neuroborreliosis: a new sensitive diagnostic assay for intrathecal synthesis of Borrelia burgdorferi-specific immunoglobulin G, A, and M. Ann. Neurol. 30 (1991) 197-205 7. Kaiser, R.: Affinity immunoblotting: rapid and sensitive detection of oligoclonal IgG, IgA and IgM in unconcentrated CSF by agarose isoelectric focusing. J. Neurol. Sci. 101 (1991) 216-225 8. Kaiser, R. and C. H. Lucking: Intrathecal synthesis of IgM and IgA in neurological diseases: Comparison of two formulae with the isoelectric focusing. Clin. Chim. Acta 216 (1993)39-51
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