Serodiagnosis and Immunotherapy in Infectious Disease (1988) 2, 375-386

Characterization of antibody response in patients with Borrefia meningitis

Mats Karlsson, Ingrid Miilleghd, G&an Stiernstedt, Anne Marie Henriksson and Bengt Wretlind

Departments of Infectious Diseases and Clinical Bacteriology, Danderyd Hospital, S-18288 Danderyd, and Department of Neurology, Regionsjukhuset in Linkiiping. Linkiiping,

Sweden

Western blot analysis was used to characterize the antibody response of 38 patients with Borrelia meningitis to different strains of Borrelia spirochetes. Eight strains of Borrelia spirochetes were analysed by SDS-PAGE which showed major proteins of 60,41, 3 I.534 and 22-23 kD. Immunoblots of all sera, and all except one CSF from patients with clinically active disease showed IgG and/or IgM antibodies to at least one Borrefia protein. Antibodies to a 41 kD protein was the first to appear and patients with a longer duration of neurological disease had antibodies to as many as 19 different proteins. Some of the 40 controls showed weak bands, in some cases with the same location as in the Borrelia infected patients. In comparison with an ELBA assay based on a whole cell sonicate as antigen, Western blot was more sensitive in detecting an early antibody response, especially in serum. We conclude that Western blot might be used as a complement for immunological diagnosis of Borrelia infection in selected cases with low or border-line ELISA titers. However, a more sensitive/ specific ELISA assay might be developed with a single protein as antigen.

Keywords: Borrelia, meningitis, serodiagnosis, Western blot, ELISA.

Introduction

Lyme disease in the U.S.A. and related diseases in Europe are caused by spirochetes belonging to the genus Borrelia. The species has been named Borrelia burgdorferi after Willy Burgdorfer who in 1982’ was the first to isolate these spirochetes from the most important vector of Lyme disease, the tick Ixodes dammini. It is still unclear whether the European strains of Borrelia belong to the species Borrelia burgdorferi or to another species. Nevertheless, both in the U.S.A. and Europe the diagnosis has relied on epidemiological, clinical and serological data. The serological assays used so far have mainly been indirect immunofluorescence assay (IFA) and enzyme-linked immunosor- bent assay (ELISA)2-5. The aim of the present study was to identify important major antigens of different strains of Borrelia spirochetes, to analyse the antibody response in

Correspondence should be addressed to: Mats Karlsson, Dept. of Infectious Diseases, Danderyd Hospital. S-l 82 88 Danderyd, Sweden.

375 0888-0786/88/050375 + 12 $03.00/O Q 1988 Academic Press Limited

376 M. Karlsson cf al.

patients with Borreliu meningitis, and to investigate the usefulness of Western blot for the serological diagnosis of Borreliu infections.

Material and methods

Strains

Eight strains of Borreliu spirochetes were used. Strains STI, ST3, STl5, STG25, STG74 and STG152 were isolated from six different Swedish Zxodes ricinus ticks6: strain B31 from an American Zxodes dummini tick’ and strain SL20 from cerebrospinal fluid (CSF) of a Swedish patient with meningitis.

ELZSA

The enzyme-linked immunosorbent assay (ELISA) used has been described in detail previously4. Briefly, microtiterplates (Dynatech) were coated with 10 ug/ml protein from a whole cell sonicate of Borrelia spirochetes. Antibodies were detected with alkaline- phosphatase-conjugated swine antihuman IgG (Orion) diluted l/250, or with goat antihuman IgM (Sigma) diluted l/500. p-Nitrophenyl phosphate (Sigma) was used as substrate. Test sera were diluted l/l000 and CSF l/l0 andl/lOO. The ELISA titer was defined as the A,, value multiplied by the serum or CSF dilution factor. The cut-off level in serum was defined as the 95th percentile of 120 healthy controls4, which corresponded to serum titers of 450 in IgG and 580 in IgM4. In CSF, a titer of 10 was used as cut-off level in both IgG and IgM4.

SDS-PAGE

Spirochetes were grown in modified Kelly’s medium’ for eight days, pelleted and washed four times in phosphate buffered saline (PBS) containing 5 mM MgCl, (pH 7.4). The pellet was resuspended in PBS without MgCl,, sonicated and dialysed against distilled water. The protein content was determined by Biorad protein assay (Biorad, Richmond, California). Antigen was dissolved and boiled for 3 min in 40 mM Tris-Hcl buffer, pH 7.0, containing 1.6% sodium dodecyl sulphate (SDS), 21 mM dl-dithiothreitol, 20% (v/v) glycerol and 0.004% bromphenol blue. Samples (10 ug) were subjected to SDS- polyacrylamid gel electrophoresis (SDS-PAGE) in 7.5-15% gradient gels, which were stained with Coomassie brilliant blue (modified after Laemmli’). Molecular weight standards (Biorad), l&250 kD, were included in each gel.

Western blot

Antigen from unstained SDS-PAGE gels were transferred electrophoretically to nitro- cellulose paper (Millipore HA 0.45 um)8. The paper was stained with Amido black and immersed for 30 min in 50 mM Tris-HCL buffer containing 150 mM NaCl, 1 mM EDTA, 0.05% Tween 20 (v/v) and 2% bovine serum albumine (BSA) (pH 7.4). The paper was then cut into strips which were incubated overnight at room temperature with serum or CSF samples diluted l/l00 in Tris-HCL buffer without BSA. Antibodies were detected with alkaline phosphatase-conjugated swine antihuman IgG (Orion) diluted l/250 or with goat antihuman IgM (Sigma) diluted l/500. Naphtol-AS-TR-phosphate (Sigma) was used as substrate.

Antibody response to Borelia spirochetes 377

Patients and controls

Thirty-eight patients with Borrelia meningitis and 40 controls were studied. The patients ( 15 men and 23 women) were between 11 and 81 years old (median 53 years). All patients had a pleocytosis in CSF and specific antibodies to Borrelia in at least one serum and/or CSF sample determined by ELISA4. Ten patients had a recent history of a tick bite and nine of erythema migrans. The most common neurological symptoms were headache, radicular pain, peripheral nerve paresis, ataxia, sensory nerve disturbances and distur- bances of sight and hearing. Twenty-three patients received 9-12 g penicillin G, two patients received 200 mg doxycyclin, and one patient 9 g cefuroxime i.v. daily for l@--14 days. One patient was given trimethoprim-sulfametoxazole and pyrimethamine for two weeks against suspected cerebral toxoplasmosis. This patient and the 11 untreated patients had recovered, some with residual symptoms, when the diagnosis of Borrelia infection was established retrospectively.

At least one paired serum and CSF sample from 34 patients were analysed by Western blot and ELISA. From two patients, only serum samples, and from another two patients only CSF samples, were analysed.

Pretreatment samples from 17/27 antibiotic treated patients were drawn between 2.5 and 84 weeks after onset of neurological symptoms, and post-treatment samples from 20127 patients were drawn between two and 277 weeks after start of antibiotic therapy. Samples from the 11 untreated patients were drawn between two weeks and six years after onset of neurological symptoms.

CSF samples from two patients with multiple sclerosis, from one patient with cerebral infarction and from seven patients with meningitis/encephalitis of verified non Borrelia etiology served as CSF controls. Sera from these patients, from 10 patients with syphilis, from four patients in convalescence after bacterial pneumonia and from 16 healthy individuals served as serum controls.

Serum and CSF samples were analysed by Western blot against strain SL20 in all patients and controls, against strain STG152 in 19 patients and against strain B31 in 3 patients. Serum and CSF samples were analysed by ELISA against strain STG152 in all patients and controls, against strain SL20 in 911 patients and against strain B31 in three patients.

Results

ELISA

Patients. ELISA titers against strain STG152 are shown in Figures 1 and 2. Of 15 patients with a duration of neurological symptoms for eight weeks or less, 7/14 (50%) had an elevated IgG titer and 4/14 (29%) had an elevated IgM titer in serum. Twelve of 13 (92%) CSF samples from these patients showed an elevated IgG titer and 8/l 3 (62%) showed an elevated IgM titer. Of 13 patients with neurological symptoms for more than eight weeks, 13/l 3 (100%) had an elevated IgG titer and l/l 1 (9%) had an elevated IgM titer in serum. Twelve of 13 (92%) CSF samples from these patients showed an elevated IgG titer and 8/l 1 (73%) showed an elevated IgM titer. From lo/38 patients only post- treatment samples were analysed. Six of nine (67%) patients had an elevated IgG titer, but O/7 had an elevated IgM titer in serum. CSF samples showed elevated IgG titers in 7/ 10 (70%) and elevated IgM titers in 3/7 (67%).

378 M. Karlsson cr al.

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Figure I. Serum IgG and IgM antibody titers measured by ELISA in patients with Borrelia meningitis and in controls. A titer level of 450 and 580 (indicated as horizontal lines) was used as upper limit of normal values for IgG and IgM respectively. Borrelia strain STG152 was used as antigen.

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Figure 2. CSF IgG and IgM antibody titers measured by ELISA in patients with Eorrelia meningitis and in controls. A titer level of 10 (indicated as horizontal lines) was used as upper limit of normal values for both IgG and IgM. Borreliu strain STG152 was used as antigen.

Antibody response to Borefh spirochetes 379

Controls. Four of 10 (40%) syphilis patients and two of the other 30 controls (6%) had slightly elevated IgG titers in serum. One of the latter suffered from a Mycoplasma pneumonia and the other had a tick-borne encephalitis. Two of 40 (4%) controls had elevated IgM titers in serum. Both were healthy individuals with no history of Borrelia infection. None of the 10 CSF controls had elevated IgG or IgM titers.

SDS- PAGE

The SDS-PAGE of the eight Borrelia strains examined are shown in Figure 3. All strains had major proteins with a molecular weight of 60 and 41 kD. Seven of eight strains had two major proteins in the 3 l-34 kD region and 4/8 strains had one or two major proteins in the 22 kD region. Differences were seen mainly in the 32 and 20 kD regions. The American strain B31, derived from a tick, had two major proteins with a molecular weight of about 3 1.5 and 34 kD. All Swedish strains, except strain ST3, derived from a tick, had two major proteins with a molecular weight of about 32 and 34 kD. Strain SL20, isolated from a human CSF, had another major protein of 23 kD, also found in strain ST15 and strain STl, both derived from ticks. Strain ST1 had another major

A BCDEFGH

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Figure 3. Eight different Borreliu strains separated by SDS-PAGE. A, strain B3 1 isolated from an American Ixodes dunmini tick; E, strain SL 20 isolated from the CSF of a Swedish patient with meningitis; BH, strains STGl52, STG74, STG25. ST15, ST3 and STl, respectively, isolated from Swedish Ixodes ricks ticks. The gel is stained with Coomassie brilliant blue. Arrows to the left indicate major proteins of the strains with a molecular weight of 60.41, 33-31.5 and 23-22 kD. Molecular weight markers, 144200 kD. are shown to the left and to the right.

380 M. Ksrlsaon et al.

protein of 22 kD, also found in strain ST3. In contrast, strains STG25, STG74, STG152 and B31 (U.S.A), all derived from ticks, had only small amounts of proteins in the 22 kD region.

Western blot

Patients. Thirty-three of 36 (92%) patients had at least one IgG band in serum demonstrated by Western blot (WB). From l/3 patients with no visible serum IgG bands, the only serum examined was drawn after four weeks of neurological disease. This patient had one serum IgM band corresponding to a 41 kD protein. From the other two patients, the only sera examined were drawn 110 and 157 weeks after penicillin G treatment. These patients had neither IgG bands in CSF, nor any IgM bands in serum or CSF. Thirty-three of 36 (92%) patients had IgG bands in CSF. The only CSF samples analysed from three patients with no visible IgG bands were sampled 110, 157 and 7 weeks after onset of antibiotic treatment. The last patient had neurological symptoms for only one week before treatment.

Twenty-eight of 36 (78%) patients had IgM bands in serum. From 5/8 patients with no IgM bands, sera were drawn before antibiotic treatment, after 14-69 weeks of neurological disease. Sera from the other three patients were drawn between 13 and 157 weeks post-treatment. These patients had had neurological symptoms for 2-52 weeks before treatment. Twenty-two of 36 (61%) patients had IgM bands in CSF. From 8/14 patients with no IgM bands, CSF were sampled before antibiotic treatment, after 8869 weeks of neurological disease. CSF from the other six patients were sampled between 7 and 277 weeks after antibiotic treatment. These patients had had neurological symptoms for 1-8 weeks before antibiotic treatment.

IgG bands to the CSF strain SL20 in sera drawn before antibiotic treatment from 27 patients are listed in Table 1, together with the corresponding ELISA titers. There was great interindividual variation between the patients, but a clear tendency toward more intense and more numerous bands in patients with a longer disease duration. In 5/35 (14%) patients the IgG bands in CSF were more intense than in the corresponding serum, judged visually, in 15/35 (43%) patients the bands were equally intense and in 15/ 35 (43%) patients the bands in CSF were weaker and less numerous than in the corresponding serum. Examples of IgG and IgM bands in paired serum and CSF samples are shown in Figures 4 and 5.

The most common IgG bands in WB corresponded to proteins with molecular weights of 84, 72, 59, 58,56,41, 39, 38, 31.5, 22, 18 and 16 kD. IgM bands were generally in the same position as the IgG bands. A 41 kD IgG or IgM band was present in the first pretreatment serum sample in 25/27 (93%) patients, and was the only band in the sera from two patients with neurological symptoms for less than four weeks. One patient with neurological symptoms for eight weeks lacked the 41 kD band, but had one 18 kD band in serum and one 18 and one 16 kD band in CSF. One or both of these two bands were seen in the first pretreatment serum sample from 12/27 (44%) patients.

In four untreated patients, followed with several pairs of serum and CSF samples, additional IgG and IgM bands were seen in later samples, drawn during clinically active disease. IgM bands in serum and CSF disappeared between 15 and 25 weeks after onset of neurological symptoms. In all four patients, the IgG bands in CSF had disappeared in samples drawn four years after the onset of disease, but weak bands could still be seen in

Antibody response to Borelia spirochetes 381

Table 1. Molecular weights of Borrelia proteins in strain SL20 recognized by immunoblotting (IgG) in sera from 27 patients with Borrelia meningitis. ELBA IgG titer in each serum is shown to

the right. Titers regarded as significantly elevated are in italics

Patient No. Duration of neurological symptoms (weeks)

I 2 3 4 5 6 7 8 9

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I7 14

IX I8 I9 I9 20 20 21 22 22 29 23 37 24 37

25 47

26 69 21 84

Molecular weight of proteins (kD) ELBA antigen SL20

58, 41, 39, 18 41 58, 56,4l 84, 58, 41, 39, 31.5

72, 58, 41 58,41, 39, 18 84, 58, 56,43,41, 31.5, 30, 18 60, 58, 47, 41, 39, 32, 22, 18 84, 59, 58, 56, 47, 43,41, 39, 31.5, 30 18 58, 56, 41, 31.5 84, 58, 41, 31.5, 23, I8 58, 56 84, 60, 59, 58, 56. 55,42,41, 39, 38, 31.5, 23, 18, I3 84, 72, 60, 59, 58, 56. 55, 47. 42, 41, 39, 31.5, 30, 24. 22, I8 84, 72. 60, 58, 56, 55,49, 46.43. 41, 39. 38, 31.5, 29, 22, 21, I8 58, 56,44, 41, 31, 26, 23 72, 58, 57, 42, 41 41 58, 57, 47,41 72, 63, 58, 56, 54,47,42,41, 39, 32, I3 72, 63, 60, 58, 56, 41, 38, 31.5, 25, 20, I6 84, 72, 68, 64, 60, 59, 58, 56, 55,41, 39, 38, 36, 32, 30, 29, 26, 18, 13 84, 72, 70, 68, 65, 63, 60, 59, 51, 53,41, 39, 35. 31, 22,21, I3 63, 60, 59, 58, 56,47, 39, 38 84, 79, 72, 63, 60, 59, 58, 56, 55, 45, 43, 41. 37, 34, 31, 20, 18, 13

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the corresponding serum samples. At this time the patients had recovered or had minor residual symptoms. In nine patients, followed with several posttreatment serum and CSF samples, the bands which were seen initially gradually became weaker. No additional bands appeared, except in one patient. This patient had a history of erythema migrans two years after treatment for Borrelia meningitis, and subsequently new IgG and IgM bands appeared in serum.

Controls. IgG and IgM bands in some of the controls are shown in Figure 6. Sera from two patients with herpes encephalitis showed one 35 kD IgG band in one patient and one

382 M. Karlsson et al.

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Figure 4. Immunoblot of IgG (G) and IgM (M) antibodies to Borrelia strain SL20 in serum (S) and/or CSF (C) from six patients with Borreliu meningitis (Nos IA) and a healthy control (No. 7). Patients Nos 16 had had neurological symptoms for 2.5, 5.5, 3, 4, 4, and 4 weeks, respectively, when the samples were drawn. Molecular weight markers, 1492 kD, are indicated to the left.

Figure 5. Immunoblot of IgG antibodies to Borreliu strain SL20 in serum and CSF from 10 patients with Borrelia meningitis. Serum is shown to the left in each pair of strips. Patients Nos l-10 had had neurological symptoms for 4, 8, 8, 11, 14, 19, 37, 47, 69, and 84 weeks, respectively, when the samples were drawn. Strips No. 11 shows serum and CSF drawn from patient No. 10, three years after antibiotic treatment with penicillin G. Strips No. 12 shows the serum from a healthy control. Some strips are from different gradient gels which explains differences in the position of bands measured to be identical. Serum and CSF were sampled simultaneously in each patient. Molecular weight markers, 14-92 kD, are indicated to the left.

Antibody response to Bore& spirochetes 383

Figure 6. Immunoblot of IgG and IgM antibodies to Born& strain SL20 in controls. Patients Nos 14 show IgG antibodies in serum (the left strip in each pair) and CSF from No. 1. a patient with Borrelia meningitis (shown as a positive control), No. 2, a patient with Herpes simplex encephalitis, No. 3, a patient with Influensa A encephalitis and No. 4. a patient with multiple sclerosis. Patients >I 1 show IgG (the left strip in each pair) and IgM (the right strip in each pair) antibodies in sera from patients with viral tick-borne encephalitis (No. 5). EpsteinBarr virus encephalitis (No. 6), cerebral infarction (No. 7), syphilis (No. 8). and three healthy individuals (Nos 9911). Molecular weight markers, 14-92 kD, are indicated to the left.

58 kD IgG band in the other. Serum from one patient with Epstein-Barr virus encephalitis showed a few weak IgG bands between 60 and 56 kd, and several IgM bands, most intense between 63-56 kD. Serum from one patient with influensa A encephalitis showed one 54 kD IgG band and weak IgM bands of 61,55 and 54 kD. One of two patients with multiple sclerosis had one weak 54 kD IgG band, and one patient with a cerebral infarction had one weak 56 kD band and three weak IgM bands of 58,56 and 55 kD. Three patients with viral tick-borne encephalitis had several IgG bands and weak IgM bands of 60,59,58,56,55,41,39,30,20 and 13 kD. There were no bands in the CSF samples from these 10 patients.

Sera from 10 syphilis patients with Treponemapallidum haemagglutinin titers between l/60 and 1 /S 120 showed several weak IgG and IgM bands, including 58,56,41,30 and in two patients 18 kD bands. Sera from four patients in convalescence after bacterial pneumonia and 16 healthy individuals showed weak 58-56 kD IgG bands in seven sera, one weak 35 kD IgG band in three sera, and one weak 41 kd IgM band in five sera. Four sera had weak IgM bands in the range 6456 kD.

Antibodies to d#erent Borrelia strains. The IgG band patterns of serum and CSF samples from three patients tested against strains STG152, B3 1 and SL20 differed somewhat in location and intensity of individual bands, but were generally very similar. Figure 7 shows immunoblots of serum samples from two patients tested against these three strains. One patient with bands against the Swedish strains STG152 and SL20 had no bands against the American strain B31 in the first serum and CSF samples. This patient had erythema migrans two years after treatment for meningitis, and subse- quently showed a similar band pattern in serum against strain B31 as against strains STG152 and SL20. Serum and CSF from another 16 patients tested against strain

M. Karlsson et ol.

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Figure 7. Immunoblot of IgG antibodies in serum from two patients with Borreh meningitis to three different strains of Borreliu spirochetes. A. strain STG 152, isolated from a Swedish Ixodes ricinus tick; B, strain SL20, isolated from the CSF of a Swedish patient with meningitis; C. strain B3 1, isolated from an American h-odes dummini tick. The last strip to the right shows the immunoblot of IgG antibodies to strain SL20 in serum from a healthy control. Molecular weight markers, 1492 kD, are indicated to the left.

STG152 and SL20 also showed a similar pattern against the two strains, although 13 of 16 (8 1%) patients had more bands toward strain SL20, especially low molecular bands.

Comparison of Western blot and ELBA. All except one serum and one CSF sample with a significant elevated ELISA IgG and/or IgM titer had IgG and/or IgM bands in WB. The serum was drawn from a patient with 18 weeks of neurological symptoms and had an elevated IgM titer in ELISA but no IgG or IgM bands in WB. The CSF was sampled from a patient 110 weeks after antibiotic treatment and showed an elevated IgG titer in ELISA but no bands in WB. In 15 patients with a duration of neurological symptoms for eight weeks or less, WB detected serum IgG bands in 6/7 ELISA-IgG negative patients, and serum IgM bands in lO/lO ELISA-IgM negative patients. In CSF from these patients, WB detected IgM bands in 3/5 ELISA-IgM negative samples. In 13 patients with neurological symptoms for more than eight weeks, WB detected serum IGM bands in 7/10 ELISA-IgM negative patients. In CSF from these patients, the only patient with a negative ELISA-IgG titer had IgG bands in WB. In l/3 posttreatment sera and in l/3 posttreatment CSF samples which were negative in IgG ELISA, bands were still seen in WB. The same was true for 4/7 posttreatment sera which were negative in IgM ELISA.

Antibody response to Borelia spirochetes 385

Discussion

The SDS-PAGE patterns of the eight Borrefiu strains in this study showed major proteins of molecular weight 60, 41, 33-34, 32 and 22 kD, along with several minor proteins. The patterns are similar to those previously described by Barbour et ~1.~ and Wilske et al.“, who have analysed strain differences in detail by use of monoclonal antibodies. The two major proteins in the 3 l-34 kD region are surface proteins and have been named OspA and OspB, respectively”. Wilske ef ~1.” described another major protein with a molecular weight of approximately 22 kD in 15 of 23 European Borrelia strains and named it protein C. We found this protein in four of eight strains examined. Schwan & Burgdorfer” have shown that some Borrelia strains might lose the expression of major proteins during subcultivation, and we have found that the CSF strain SL20 almost lost its 22 kD protein after subcultivation once a month for nine months (unpubl. data). This fact must be considered when comparing different strains.

With Western blot analysis we found that sera from most patients with Borrelia meningitis recognized 6&56 kD proteins, 41-39 kD proteins, and in many cases also low molecular weight proteins of 22-16 kD. These results are in accordance with the findings of Barbour et a1.13, Wilske et a1.‘o*‘4 and Craft et al.“. Barbour et ~1.~ also reported antibodies to the OspA and OspB proteins late in the disease. We could not confirm this finding, but many of our patients recognized a 31.5 kD protein, close to the OspA protein in the SDS-PAGE separation. The finding that sera from syphilis patients and some healthy individuals recognize single Borrelia proteins in immunoblotting has been reported by others 9~‘3~‘5 This fact, to some extent, reduces the usefulness of immunoblot- . ting for diagnostic purposes. However, the immunoblots of syphilis sera showed a much weaker and different bandpattern compared with sera from Borrelia infected patients. Furthermore, the bands found in our other controls were, with a few exceptions, very weak. Sera from three elderly patients with viral tick-borne encephalitis recognized several Borreliu proteins and one of the patients had an elevated serum IgG titer. The risk of contracting tick-borne encephalitis in Sweden is rather low and it is likely that patients with this disease are primarily individuals heavily exposed to tick bites. The antibody response to Borreliu spirochetes in these patients might be explained by previous exposure toBorrelia.

In comparison with an ELISA assay based on a whole-cell sonicate as antigen, Western blot was more sensitive in detecting an early antibody response in serum and CSF. This difference was not as marked in CSF, which might be explained by the lower unspecified background in CSF compared with serum. Western blot also detected antibodies in sera and CSF sampled long after antibiotic treatment, at a time when the ELISA assay had become negative. We conclude thatwestern blot is a sensitive method to detect exposure to Borrelia and might be used as a complement for the immunological diagnosis of Borrelia infection in patients with borderline values in ELISA. However, because immunoblotting is primarily a qualitative method, an analysis of a single sample does not state if the patient has an active infection or not. To answer this question, a quantitative test is necessary. ELISA fulfills this criterion and one possibility of improving the serodiagnosis of Borreliu infection would be to develop an ELISA assay based on purified antigens identified by the Western blot. In a recent study, Hansen et ai.16 reported highly sensitive and specific ELISA assay using a purified preparation of spirochetal flagellas as antigen. The flagellar antigen is most probably identicalI to the 41 kD protein found to be immunogenic in most patients in the present study.

386 M. Karlsson et al.

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(Manuscript accepted 31~1 May 1988)