Sensitive and specific serodiagnosis of Lyme disease by enzyme-linked immunosorbent assay with a peptide based on an immunodominant conserved region of Borrelia burgdorferi vlsE

VlsE, the variable surface antigen of Borrelia burgdorferi, contains an immunodominant conserved region named IR(6). In the present study, the diagnostic performance of a peptide enzyme-linked immunosorbent assay (ELISA) based on a 26-mer synthetic peptide (C(6)) with the IR(6) sequence was explored. Sensitivity was assessed with serum samples (n = 210) collected from patients with clinically defined Lyme disease at the acute (early localized or early disseminated disease), convalescent, or late disease phase. The sensitivities for acute-, convalescent-, and late-phase specimens were 74% (29 of 39), 85 to 90% (34 of 40 to 35 of 39), and 100% (59 of 59), respectively. Serum specimens from early neuroborreliosis patients were 95% positive (19 of 20), and those from an additional group of patients with posttreatment Lyme disease syndrome yielded a sensitivity of 62% (8 of 13). To assess the specificity of the peptide ELISA, 77 serum samples from patients with other spirochetal or chronic infections, autoimmune diseases, or neurologic diseases and 99 serum specimens from hospitalized patients in an area where Lyme disease is not endemic were examined. Only two potential false positives from the hospitalized patients were found, and the overall specificity was 99% (174 of 176). Precision, which was assessed with a panel of positive and negative serum specimens arranged in blinded duplicates, was 100%. Four serum samples with very high anti-OspA antibody titers obtained from four monkeys given the OspA vaccine did not react with the C(6) peptide. This simple, sensitive, specific, and precise ELISA may contribute to alleviate some of the remaining problems in Lyme disease serodiagnosis. Because of its synthetic peptide base, it will be inexpensive to manufacture. It also will be applicable to serum specimens from OspA-vaccinated subjects.     

Characterization of a Borrelia burgdorferi VlsE invariable region useful in canine Lyme disease serodiagnosis by enzyme-linked immunosorbent assay

Sera collected from dogs experimentally infected with Borrelia burgdorferi by tick inoculation were analyzed for an antibody response to each of the six invariable regions (IRs; i.e., IR(1) to IR(6)) of VlsE, the variable surface antigen of B. burgdorferi. Six synthetic peptides (C(1) to C(6)), which reproduced the six IR sequences were used as peptide-based, enzyme-linked immunosorbent assay (ELISA) antigens. Two IRs, IR(2) and IR(6), were found to be immunodominant. Studies with serially collected serum samples from experimentally infected dogs revealed that the antibody response to IR(6) appears earlier and is stronger than that to IR(2). Thus, the IR(6) sequence alone appeared to be sufficient for serodiagnosis. When C(6) alone was used as antigen, the peptide-based ELISA was positive in 7 of 23 dogs (30%) as early as 3 weeks postinfection. All dogs (n = 33) became strongly positive 1 or 2 weeks later, and this response persisted for the entire study, which lasted for 69 weeks. Of 55 sera submitted by veterinarians from dogs suspected of having Lyme disease, 19 were also positive by the C(6) ELISA, compared to 20 positives detected by immunoblot analysis using cultured B. burgdorferi lysates as antigen. The sensitivity of using C(2) and C(6) together for detecting specific antibody in both experimentally infected and clinically diagnosed dogs was not better than sensitivity with C(6) alone, confirming that C(6) suffices as a diagnostic probe. Moreover, the C(6) ELISA yielded 100% specificity with serum samples collected from 70 healthy dogs, 14 dogs with infections other than B. burgdorferi, and 15 animals vaccinated with either outer surface protein A, whole-spirochete vaccines, or the common puppy-vaccines. Therefore, this C(6) ELISA was both sensitive and specific for the serodiagnosis of canine Lyme disease and could be used with vaccinated dogs.     

Use of recombinant OspC from Borrelia burgdorferi for serodiagnosis of early Lyme disease.

Infection with Borrelia burgdorferi, the etiologic agent of Lyme disease, is associated with an early and dominant humoral response to the spirochete's 23-kDa outer surface protein C (OspC). We have cloned and expressed OspC as a fusion protein in Escherichia coli and have shown that patient serum samples react with it in an enzyme-linked immunosorbent assay (ELISA) (S. J. Padula, A. Sampieri, F. Dias, A. Szczepanski, and R. W. Ryan, Infect. Immun. 61:5097-5105, 1993). Now we have compared the detection of B. burgdorferi-specific immunoglobulin M antibodies in 74 individuals with culture-positive erythema migrans by a whole-cell ELISA, immunoblot, and the recombinant OspC (rOspC) ELISA. Seventy-six negative controls were also studied. With all of the tests, there was a statistically significant association between the duration of disease and the frequency of a positive result. With the rOspC ELISA, the predictive value of a positive test was 100% and the predictive value of a negative test was 74%. Similar results were obtained with the whole-cell ELISA and with the immunoblot using as the source of test antigen a strain of B. burgdorferi which expresses abundant levels of OspC. We conclude that the use of rOspC in an ELISA is a convenient, readily automated, and easily standardized test for the serodiagnosis of early Lyme disease.     

Rapid decline of OspC borreliacidal antibodies following treatment of patients with early Lyme disease

We determined whether the levels of OspC borreliacidal antibodies declined following treatment of early Lyme disease and whether the OspC7 peptide enzyme-linked immunosorbent assay (ELISA) could be used as an alternative test for detecting the response. Serum samples were collected from 37 subjects at the onset of illness and 2 and 6 months after treatment with doxycycline. The ELISA detected IgM and IgG OspC7 antibodies within 2 months in 18 (49%) and 5 (14%) sera, respectively. Moreover, the sera from 12 subjects who tested positive by the ELISA also showed borreliacidal activity which was completely abrogated when the antibodies to OspC7 were removed. The borreliacidal activity decreased greater than 4-fold in each seropositive patient within 6 months after treatment, and the findings were accurately predicted by the IgM ELISA. The results confirmed that the ELISA was an effective alternative for detection of OspC borreliacidal antibodies produced during early Lyme disease in humans and also provided strong evidence that a significant decline in the response coincides with successful treatment of the illness.     

Establishment of enzyme-linked immunosorbent assay using purified recombinant 83-kilodalton antigen of Borrelia burgdorferi sensu stricto and Borrelia afzelii for serodiagnosis of Lyme disease.

The 83-kDa antigen of Borrelia burgdorferi was expressed as a recombinant protein in Escherichia coli and purified for use in an enzyme-linked immunosorbent assay (p83-ELISA). Antibodies to the 83-kDa antigen of both the immunoglobulin G (IgG) and IgM isotypes could be detected in all stages of Lyme disease. Sensitivity varied, depending on the clinical stage of illness. In early stages, as defined for 118 patients with erythema migrans, it was found to be 20% (24 of 118 patients: 7 with IgM, 16 with IgG, and 1 with IgM and IgG). Of the patients with late-stage Lyme arthritis and acrodermatitis chronica atrophicans, 94% (16 of 17:2 with IgM and IgG and 14 with IgG) and 86% (36 of 42:2 with IgG and IgM and 34 with IgG) revealed positive results in the p83-ELISA, respectively. p83 displays sequence heterogeneity according to the genomospecies, but when the reactions of serum specimens from acrodermatitis chronica atrophicans patients and arthritis patients with p83 derived from representative strains of B. burgdorferi sensu stricto and Borrelia afzelii in ELISAs were compared, no differences in specificity and sensitivity were seen. When 82 serum specimens from healthy controls were tested, none had IgG and only 3 (4%) had IgM antibodies, indicating a high specificity. Positive reactions with antibodies against Treponema pallidum (1 of 37 patients; IgG) and Epstein-Barr virus (1 of 44 patients; IgM) and with autoantibodies of various specificities (1 of 53 patients; IgG) were seen with < 3% of the serum samples te11111111111111111111 high speficicity for B. burgdorferi.2+ 13% for IgM antibodies, the IgM p83-ELISA provided little diagnostic information for Lyme disease, whereas the IgG p83-ELISA appears to be a suita ;e test for serodiagnosis of advanced-stage Lyme disease.     

Improved immunoglobulin M serodiagnosis in Lyme borreliosis by using a mu-capture enzyme-linked immunosorbent assay with biotinylated Borrelia burgdorferi flagella.

A mu-capture enzyme-linked immunosorbent assay (ELISA) for detection of serum immunoglobulin M (IgM) antibodies to Borrelia burgdorferi by using biotinylated purified B. burgdorferi flagella was developed. The diagnostic performance of the mu-capture ELISA was compared with that of a conventional indirect ELISA. Sera from untreated patients with erythema migrans (n = 50), neuroborreliosis (n = 100), and acrodermatitis chronica atrophicans (ACA; n = 48) were investigated. The cutoff of the ELISAs was adjusted to a diagnostic specificity of 98% on the basis of examination of 200 serum specimens from healthy controls. The mu-capture ELISA increased the diagnostic sensitivity in patients with erythema migrans from 32 to 48% (P less than 0.01) and in patients with neuroborreliosis from 37 to 57% (P less than 0.001). Because of an increased signal/noise ratio, the mu-capture ELISA yielded a significantly better quantitative discrimination of individual positive measurements from the cutoff (P less than 0.001). The increased signal/noise ratio was most likely a consequence of the elimination of IgG competition for the test antigen. This may also explain why 12% of patients with ACA showed significantly increased specific IgM levels only by the mu-capture ELISA. Of patients with ACA, 27% had IgM rheumatoid factor. The mu-capture principle with a directly labeled antigen showed no interference with IgM rheumatoid factor, in contrast to the indirect ELISA. The high diagnostic performance and ease of this three-step mu-capture ELISA make it suitable for routine anti-B. burgdorferi IgM serodiagnosis.     

Detection of Borrelia burgdorferi in urine of Peromyscus leucopus by inhibition enzyme-linked immunosorbent assay.

An inhibition enzyme-linked immunosorbent assay was developed to detect Borrelia burgdorferi, the etiologic agent of Lyme borreliosis, in urine from white-footed mice (Peromyscus leucopus). Of the 87 urine specimens tested from 87 mice collected in widely separated tick-infested sites in Connecticut, 57 (65.5%) contained detectable concentrations of spirochetal antigens. Forty-seven (62.7%) of 75 serum samples analyzed contained antibodies to B. burgdorferi. In culture work with tissues from bladders, kidneys, spleens, or ears, 50 of 87 mice (57.5%) were infected with B. burgdorferi. Thirty-eight (76%) of 50 infected mice had antigens of this spirochete in urine, while 36 (72%) individuals had infected bladders. Of those with infected bladders, 24 (66.7%) mice excreted subunits or whole cells of B. burgdorferi into urine. Successful culturing of B. burgdorferi from mouse tissues, the presence of serum antibodies to this bacterium, and detection of antigens to this spirochete in urine provide further evidence that multiple assays can be performed to verify the presence of B. burgdorferi in P. leucopus.     

Serodiagnosis of erythema migrans and acrodermatitis chronica atrophicans by the Borrelia burgdorferi flagellum enzyme-linked immunosorbent assay.

The diagnostic performance of an enzyme-linked immunosorbent assay (ELISA) using purified Borrelia burgdorferi flagella as test antigen was compared with that of a B. burgdorferi sonic extract ELISA. We tested sera from 200 healthy controls, 107 patients with erythema migrans (EM), 50 patients with acrodermatitis chronica atrophicans (ACA), and 98 patients with various dermatological disorders without clinical evidence of active Lyme borreliosis. The flagellum ELISA was significantly more sensitive than the sonic extract ELISA. With sera from patients with EM, the diagnostic sensitivity for immunoglobulin G (IgG) antibody detection increased from 11.2 to 35.5% (P less than 0.001) and for IgM antibody detection it increased from 16.6 to 44.8% (P less than 0.001). In the flagellum ELISA, the number of positive tests increased significantly (P less than 0.005) when the duration of EM exceeded 1 month, but still only about 50% of patients with longstanding (1 to 12 months) untreated EM were IgG seropositive. Concomitant general symptoms did not affect the antibody level, whereas patients with multiple erythema were more frequently seropositive. All sera from patients with EM which were positive in the sonic extract ELISA were also positive in the flagellum ELISA. Not only did the overall number of positive tests increase, but the flagellum ELISA yielded a significantly better quantitative discrimination between seropositive patients and controls (P less than 0.002). IgG antibodies to the B. burgdorferi flagellum were found in all sera from patients with ACA, indicating persistence of an antiflagellum immune response in late stages of Lyme borreliosis. IgM reactivity in sera from patients with ACA was shown to be unspecific and the result IgM rheumatoid factor. A rheumatoid factor was detected in sera from 32% of patients with ACA, compared with 7.5% of patients with EM. The improved diagnostic performance, the ease of standardization of the flagellum antigen, and the lack of strain variation make the B. burgdorferi flagellum a needed reference antigen for growing routine serology in Lyme borreliosis.     

Borrelia burgdorferi enzyme-linked immunosorbent assay for discrimination of OspA vaccination from spirochete infection.

Recombinant Lyme disease vaccines based on purified preparations of outer surface protein A (OspA) have been shown to be effective in preventing transmission of Borrelia burgdorferi in experimental animal models and are now being tested in humans. Since the most widely used screening tests for Lyme disease are based on a whole-cell sonicate of B. burgdorferi, serologic false positivity in vaccinated persons could result from reactivity to OspA within the antigen preparation. In order to avoid serologic false positivity in vaccinated subjects, we developed an immunoassay based on a low-passage-number, naturally occurring variant of B. burgdorferi which lacks the plasmid encoding OspA and OspB. The use of an antigen preparation derived from this organism provided sensitive and specific detection of B. burgdorferi seropositivity in experimental animals and in human Lyme disease cases. The OspA-B-negative enzyme-linked immunosorbent assay (ELISA) also appeared to be capable of discriminating the vaccinated state from vaccine failure and natural infection in experimental animals. Sera from human subjects participating in a vaccine trial gave false-positive results with an ELISA based on an OspA-containing strain, but no such reactivity was observed when the OspA-negative ELISA was used. We conclude that low-passage-number OspA-B-negative isolates in immunoassays may become useful for the immunologic discrimination of the vaccinated state, natural infection, and vaccine failure.     

Comparison of Western blot and enzyme-linked immunosorbent assay for diagnosis of Lyme borreliosis

The usefulness of Western blot in the serological diagnosis of Lyme borreliosis was evaluated compared with an ELISA using a whole cell sonicate antigen. Fifty-three of 68 (78 %) patients with neuroborreliosis had positive IgM and/or IgG immunoblots and 40 of 68 (59 %) had positive IgM and/or IgG ELISA titers in serum. Eight of 44 (18 %) controls with meningitis/encephalitis of non-borrelia etiology had positive IgM and/or IgG immunoblots and 4 of 44 (9 %) had positive IgM and/or IgG ELISA titers in serum. Western blot was more sensitive than ELISA, the difference being most pronounced in sera from patients with neurological disease for four weeks or less. Both patients and controls lived in an area endemic for Lyme borreliosis and some ELISA negative but Western blot positive controls were thought to have been previously exposed toBorrelia burgdorferi. However, the specificity for current disease was not improved by Western blot. In conclusion, Western blot does not seem to be the method of choice for screening purposes in a routine laboratory but can be used as a complement to ELISA for serodiagnosis in patients with disease of short duration.     

Serologic analysis of white-tailed deer sera for antibodies to Borrelia burgdorferi by enzyme-linked immunosorbent assay and western immunoblotting.

White-tailed deer serum samples were collected in the Minneapolis-St. Paul, Minn., metropolitan area during the fall and winter months from 1989 to 1992 and analyzed for antibodies to Borrelia burgdorferi, the etiologic agent of Lyme borreliosis. Ninety-eight percent of the serum samples were collected from regions where currently the vector tick, Ixodes dammini, is nonexistent. Antibodies to B. burgdorferi were detected in 2.2% of 508 samples by enzyme-linked immunosorbent assay, and their presence was confirmed by Western immunoblot analysis. Western immunoblotting yielded mean numbers of reactive bands of 0.1 and 6.0 for samples that were negative and positive for antibodies by enzyme-linked immunosorbent assay, respectively. The molecular weights of the antigens in many of the reactive bands from positive samples were similar to the molecular weights of antigens reactive with samples from humans with Lyme borreliosis. An antibody response to the major outer surface proteins A and B was not detected. Serologic analysis of deer sera may provide a valuable method for surveillance programs designed to monitor the spread of B. burgdorferi in nature.     

Serodiagnosis of Lyme disease by ELISA using Borrelia burgdorferi flagellum antigen

Antibodies to a 41,000 (41 kD) polypeptide in flagella of Borrelia burgdorferi were measured in patients with Lyme disease in Japan by flagellum ELISA. The IgG and IgM Classes of antibodies to a flagellum antigens were detected in the sera as early as 0.5 months after infection. The IgG antibodies continued to exist in their sera for more than one year, while the IgM antibodies quickly faded out from their sera. With respect to a diagnostic specificity of the flagellum ELISA, false positive reactions showing more than 10% were observed in sera with high levels of IgG or IgM, and with anti-syphilis antibody. This method, however, was unaffected by sera with high levels of IgA, rheumatoid factor or anti-nuclear antibody. In three cases of patients with erythema migrans preceded by tick-bite, and treated with antibiotics, seronegative results were observed by a immunoperoxidase (IP) test. Since two of them showed the positive level of IgM antibody by the flagellum ELISA, this method seems to be more sensitive and useful than the IP test for serodiagnosis of the Lyme disease.     

Comparison of indirect immunofluorescent-antibody assay, enzyme-linked immunosorbent assay, and Western immunoblot for the diagnosis of Lyme disease in dogs.

Enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescent-antibody assay (IFA), and Western immunoblot were used to test serum samples from 128 dogs for the presence of antibody to Borrelia burgdorferi. Sera included 72 samples from dogs suspected of having Lyme disease, 32 samples from dogs residing in areas in which Lyme disease was not considered endemic, and 24 samples from dogs with clinical and serologic evidence of immune-mediated disease (n = 10), Rocky Mountain spotted fever (n = 5), or leptospirosis (n = 9). Results of Western immunoblotting were used as the standard against which performances of ELISA and IFA were measured. ELISA was significantly more sensitive than IFA (84.8 versus 66.7%), although both tests were equally specific (93.5%). Eight samples that were positive by Western immunoblot were simultaneously negative by ELISA and IFA. Of these eight, four were from dogs suspected of having immune-mediated disease, two were from dogs suspected of having leptospirosis, and two were from dogs suspected of having Lyme disease. These results may indicate that sera from dogs with immune-mediated disease, and to a lesser extent sera from those with leptospirosis, cross-react with B. burgdorferi antigens. Alternatively, Western immunoblot results may not truly reflect Lyme disease status, particularly in the case of dogs with immune-mediated diseases. At present, however, the use of Western immunoblotting as a diagnostic standard for dogs offers the best alternative to a clinical definition of disease.     

Use of the enzyme-linked immunosorbent assay for the early diagnosis ofMycoplasma pneumoniae infection

IgM and IgG antibodies toMycoplasma pneumoniae were measured in 147 sera from four groups of patients by means of an indirect enzyme-linked immunosorbent assay (ELISA) and the results compared with those obtained by other methods. A good correlation was demonstrated between the complement fixation test and ELISA-IgM and to a lesser extent ELISA-IgG; for the metabolic inhibition test the reverse was the case. The indirect haemagglutination test appeared to detect mainly IgM antibodies. Low levels of IgM antibodies were dectected by ELISA in 60 sera of children not suffering from pneumonia. However, if only high titres (> 800) were regarded as indicative ofMycoplasma pneumoniae infection, a presumptive diagnosis could have been made in 42 of 73 single acute phase sera from patients. Comparable results were obtained with IHA. The diagnostic level of IgM antibodies was reached during the second week of the disease. It is concluded that examination of a single serum sample by either ELISA-IgM or IHA may assist in early diagnosis ofMycoplasma pneumoniae infection.     

Borrelia burgdorferi VlsE antigen for the serological diagnosis of Lyme borreliosis

In this study, raising and development of antibody response to Borrelia burgdorferi infection in 66 Italian patients suffering from culture-confirmed Lyme borreliosis erythema migrans (EM) was investigated. Sixty-two of 66 cultures obtained from biopsies were identified as B. afzelii by PCR. A total of 175 serially collected serum samples were tested by using two different sets of commercial assays: Enzygnost Lyme link VlsE/IgG and Enzygnost Borreliosis IgM (DADE Behring, Marburg, Germany) and LIAISON Borrelia IgG and IgM (Diasorin, Saluggia, Italy). Considering only samples obtained at first presentation when EM was clinically evident, 49/66 patients (72.4%) were IgG or IgM positive by Enzygnost, whereas 33/66 (50.0%) patients were IgG or IgM positive by LIAISON. Taking into account the follow-up period, eight patients sero-converted for IgG or IgM by Enzygnost and four by LIAISON. Similar and very good specificity values were obtained by all methods. Testing sera obtained from blood donors (n = 300) and from patients suffering from some of the most common biological conditions possibly resulting in false-positive reactivity in Lyme disease serology (n = 100) showed that Enzygnost Lyme link VlsE/IgG was the more specific (98.3%), followed by LIAISON Borrelia IgG (96.5%), and considering IgM tests, Enzygnost Borreliosis IgM showed to be 95.3%% specific, whereas the LIAISON Borrelia IgM was 92.8% specific. Recombinant VlsE antigens obtained from all three B.burgdorferi genospecies pathogenic to humans (included in Enzygnost Lyme link VlsE/IgG) greatly improved serodiagnosis of Lyme disease.     

New method for detection of Borrelia burgdorferi antigen complexed to antibody in seronegative Lyme disease

Serologic tests for Lyme disease are problematic. Because of cross-reactive antigens Borrelia burgdorferi (Bb) shares with other organisms, Lyme disease can be overdiagnosed. However, in addition to specificity problems, serologic tests for early Lyme disease can be falsely negative due to lack of sensitivity of ELISAs and Western blots. Most routine antibody tests are designed to detect free antibodies, and in early, active disease, circulating antibodies may not be free in serum but sequestered in complexes with the antigens which originally triggered their production. This difficulty may be overcome by first isolating immune complexes (IC) from the serum and using this fraction for testing. Free Borrelia-specific antibodies can then be liberated from the immune complexes which may enhance test sensitivity in patients with active disease. We developed a technique that captures the antibody component of IC on immunobeads, and subsequently releases the antigen component of IC. Immunoblotting with monoclonal antibody detected at least one antigen to be OspA, thus definitively demonstrating a Borrelia-specific antigen in circulating IC in early Lyme disease. This test is also useful in demonstrating Bb antigen in otherwise seronegative Lyme disease patients.     

Borrelia burgdorferi RevA Significantly Affects Pathogenicity and Host Response in the Mouse Model of Lyme Disease

The Lyme disease spirochete, Borrelia burgdorferi, expresses RevA and numerous outer surface lipoproteins during mammalian infection. As an adhesin that promotes bacterial interaction with fibronectin, RevA is poised to interact with the extracellular matrix of the host. To further define the role(s) of RevA during mammalian infection, we created a mutant that is unable to produce RevA. The mutant was still infectious to mice, although it was significantly less well able to infect cardiac tissues. Complementation of the mutant with a wild-type revA gene restored heart infectivity to wild-type levels. Additionally, revA mutants led to increased evidence of arthritis, with increased fibrotic collagen deposition in tibiotarsal joints. The mutants also induced increased levels of the chemokine CCL2, a monocyte chemoattractant, in serum, and this increase was abolished in the complemented strain. Therefore, while revA is not absolutely essential for infection, deletion of revA had distinct effects on dissemination, arthritis severity, and host response.     

Measurement of rotavirus antibody by an enzyme-linked immunosorbent assay blocking assay.

A new method for the measurement of rotavirus antibody is described, utilizing the system of enzyme-linked immunosorbent assay (ELISA). In this method, serum is incubated with a fixed amount of rotavirus antigen, and the amount of antibody is determined by measuring the amount of unneutralized antigen. Such an assay system proved to be as efficient as the other available rotaviral antibody systems. The ELISA blocking assay also has the advantages of not requiring purified or gnotobiotic antigen and of being able to measure rotaviral antibody in all animal species.     

Hemagglutination and proteoglycan binding by the Lyme disease spirochete, Borrelia burgdorferi.

The ability of the Lyme disease spirochete to attach to host components may contribute to its ability to infect diverse tissues. We present evidence that the Lyme disease spirochete expresses a lectin activity that promotes agglutination of erythrocytes and bacterial attachment to glycosaminoglycans. Among a diverse collection of 21 strains of Lyme disease spirochete, hemagglutinating activity was easily detected in all but 3 strains, and these three strains were noninfectious. The ability to agglutinate erythrocytes was associated with the ability of the spirochete to bind to the sulfated polysaccharide dextran sulfate and to mammalian cells. Soluble dextran sulfate was a potent inhibitor of both hemagglutination and attachment to mammalian cells, while dextran had no effect on either activity, suggesting that dextran sulfate may inhibit attachment by mimicking host cell glycosaminoglycans. Consistent with this, the spirochete bound to immobilized heparin, and soluble heparin inhibited bacterial adhesion to mammalian cells. The bacterium did not bind efficiently to Vero cells treated with heparinase or heparitinase or to mutant CHO cell lines that are deficient in proteoglycan synthesis. Sulfation of glycosaminoglycans was critical for efficient bacterial recognition, as Vero cells treated with an inhibitor of sulfation, or a mutant CHO cell line that produces undersulfated heparan sulfate, did not mediate maximal spirochetal binding. Binding of the spirochete to extracellular matrix also appeared to be dependent upon this attachment pathway. These findings suggest that a glycosaminoglycan-binding activity which can be detected by hemagglutination contributes to the attachment of the Lyme disease spirochete to host cells and matrix.