May 2, 1991 - the enzyme-linked immunosorbent assay (ELISA) (17), the ... at 4°C. Sera were diluted 1:100 with blocking buffer (see above). The samples ...
Vol. 30, No. 1
JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1992, p. 229-232
0095-1137/92/010229-04$02.00/0
Copyright © 1992, American Society for Microbiology
Ovalbumin Blocking Improves Sensitivity and Specificity of Immunoglobulin M Immunoblotting for Serodiagnosis of Patients with Erythema Migrans R. LANGE,'* H. BOCKLAGE,2 T. SCHNEIDER,' H. W. KOLMEL,l J. HEESEMANN,2 AND H. KARCH2 Abteilung fur Neurologie, Klinikum Rudolf Virchow, Freie Universitat Berlin, Spandauer Damm 130, 1000 Berlin 19,1 and Institut fur Hygiene und Mikrobiologie der
Universitat Wurzburg, 8700 Wurzburg,2 Germany Received 2 May 1991/Accepted 9 October 1991
To improve the serodiagnosis of erythema migrans, we evaluated how sensitivity and specificity of immunoblotting are influenced by antigen concentration and blocking conditions. We found that an antigen concentration of 0.5 ,ug per lane in concert with ovalbumin blocking of the nitrocellulose provided the best results. In this case, 81% of the erythema migrans patients had positive immunoglobulin M tests, whereas only 33% were positive in a flagellum enzyme-linked immunosorbent assay (ELISA) and 28% were positive in a sonicate ELISA.
applied to a 4% stacking gel and electrophoresed on a 10% separating gel at a constant current of 30 mA at 10°C (Minigel, 8 by 7 cm; Hoefer Scientific Instruments, San Francisco, Calif.). Subsequently, the proteins were transferred in a semidry condition to nitrocellulose sheets (0.45 ,Lm; Schleicher & Schull, Dassel, Germany) for 1.5 h with a current of 0.8 mA/cm2 at room temperature. To optimize the immunoblot assay, different blocking procedures and different B. burgdorferi antigen concentrations were evaluated. The blocking conditions were as follows: (A) 5 M glucose, (B) phosphate-buffered saline (PBS)-0.05% Tween 20, (C) 1% bovine serum albumin, or (D) 1% ovalbumin. An amount of 10, 5, 2, 1, or 0.5 ,ug of sonicated B. burgdorferi protein was applied to each slot. The sheets were blocked overnight at 4°C. Sera were diluted 1:100 with blocking buffer (see above). The samples were incubated overnight on a rocking platform at 4°C. The immunoblots were washed three times with PBS-0.05% Tween 20 and incubated for 1.5 h with anti-human immunoglobulins (Dianova, Hamburg, Germany) conjugated with alkaline phosphatase diluted 1:7,000 in blocking buffer (see above). The blots were washed three times, and the antigen-antibody reaction was visualized with naphthol-fast red substrate (Sigma). Sera were considered IgM positive when at least the flagellin band was detected. The IgG immunoblot was considered positive when sera reacted with the flagellin protein (41 kDa) and two bands with a corresponding molecular mass of 95, 38, 34, 31, 27, 24, 22, or 20 kDa. Since immunoblotting procedures vary from laboratory to laboratory, we assessed the significance of antigen concentration and blocking procedures. We used serum samples from patients with clinically confirmed neuroborreliosis (six serum samples) and Lyme arthritis (four serum samples). Ten serum samples from healthy blood donors were chosen from a collection of 100 blood donor specimens on the basis of recognition of three to five polypeptides (out of polypeptides of 80, 73, 68, 60, 58, 55, 41, and approximately 30 kDa) when IgG blots were blocked by procedures described by Grodzicki and Steere (7) and Zoller et al. (21). The outcome of these experiments revealed that the reaction patterns of sera obtained from patients with clinically defined Lyme borreliosis were not affected by antigen concentration or by
Lyme borreliosis can be successfully serodiagnosed in patients with lymphocytic meningoradiculitis (Bannwarth's syndrome) (stage II) and arthritis or acrodermatitis in stage III. Procedures such as the immunofluorescence assay (16), the enzyme-linked immunosorbent assay (ELISA) (17), the capture immunosorbent assay (3, 14), the immunoperoxidase test (19), the fluorescence immunoassay (12), the complement fixation test (1) and immunoblotting (7, 13, 15) were used to confirm clinical diagnosis. Because of the absence of immunoglobulin G (IgG) antibodies and the presence of antibodies directed to flagellin (2, 4-6) during early infection, the predictive value of these serological assays is rather dubious. Only the determination of IgM antibody titers by a flagellum IgM ELISA (8), an IgM antibody capture assay (3), or a modified ,u-capture ELISA (11) was useful in diagnosing the onset of Lyme borreliosis. Despite the suggestions of Grodzicki and Steere (7), Western immunoblot analysis has not gained general approval as the method of choice to detect the early stages of Lyme disease unambiguously. In this study, we report our attempts to optimize the sensitivity and specificity of the immunoblot technique for Borrelia burgdorferi proteins. Serum samples collected from 36 patients with solitary erythema migrans (EM) were examined. Sera were obtained from the preantibiotic period (3 to 14 days after the beginning of disease). Control sera were collected from 100 blood donors (from northern parts of Germany). All samples were analyzed independently in a parallel mode, by employing two commercially available ELISA systems (flagellum ELISA, Dako, Copenhagen, Denmark; sonicate ELISA, Virimmun, Frankfurt, Germany) and by immunoblotting with B. burgdorferi proteins separated on Tricine-sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (Tricine-PAGE) as described by Schaegger and von Jagow (18). Briefly, B. burgdorferi (tick isolate RT1 [Berlin]) cells were washed three times and solubilized in sample buffer containing 4% SDS, 2% 2-mercaptoethanol, 12% glycerol, and 0.01% Serva blue G adjusted to pH 6.8 with HCI. Twenty micrograms of protein *
was
Corresponding author. 229
J. CLIN. MICROBIOL.
NOTES
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FIG. 1. Reactivity of a positive control serum with B. burgdorferi under various blocking conditions and antigen concentrations. Amounts of 10, 5, 2, 1, and 0.5 ,ug of total protein of B. burgdorferi sonic extracts were loaded onto each slot (lanes 1 to 5). Nitrocellulose sheets were blocked overnight at 4°C. Blocking conditions were as follows: (A) 5 M glucose; (B) PBS-0.05% Tween 20; (C) 1% bovine serum albumin; (D) 1% ovalbumin. Blocking reagents were diluted in PBS-0.05% Tween 20. Sera were diluted 1:100 with blocking buffer.
When blocking was with 1% ovalbumin (procedure D), however, only a unique band of 80 kDa remained visible (Fig. 2D). No binding to B. burgdorferi proteins at about 30 kDa and between 55 and 95 kDa could be observed. Therefore, ovalbumin blocking provides a reliable means to discriminate between true- and false-positive immunoblot results. Compared with immunoblotting, only 12 (33%) of the 36 patients with EM were IgM positive by the flagellum ELISA (Table 1). According to the cutoff levels defined by the manufacturer's recommendations (cutoff level A), the ELISA with whole cells was even less sensitive (Table 1). In an effort to increase the sensitivity, we used the cutoff value determined by the mean extinction of the negative controls plus 3 standard deviations (cutoff level B). Unlike results with the manufacturer's cutoff, significantly more patients had positive IgM tests (56% versus 33%, Table 1). Under these conditions, 13 of 100 serum samples from blood donors
various blocking conditions. Representative reaction patterns are shown in Fig. 1. Lyme borreliosis sera reacted specifically with the 95-, 70-, 41-, 38-, 34-, 31-, 24-, 22-, and 20-kDa protein bands. In contrast, the results of immunoblots of the control group depended heavily on the blocking procedure. Figure 2 represents one serum sample which demonstrates the effect of the blocking procedure on the number of bands occurring in the immunoblot. If the nitrocellulose was blocked with 5 M glucose (procedure A), three polypeptides (68, 41, and approximately 31 kDa) were detected. Application of PBS containing 0.05% Tween 20 (procedure B) gave rise to four proteins (73, 68, 41, and approximately 31 kDa), whereas by using 1% BSA (procedure C), an additional band of 80 kDa could be recognized. Grodzicki and Steere proposed that an immunoblot scores positive for Lyme borreliosis only when more than three specific bands can be detected (7). By blocking procedures A to C, the blood donor serum (Fig. 2) seemed to be positive.
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FIG. 2. Reactivity of a negative control serum with B. burgdorferi under various blocking conditions and antigen concentrations, as given in the legend to Fig. 1.
VOL. 30, 1992
NOTES
TABLE 1. Sensitivity and specificity of detection of anti-B. burgdorferi antibodies in EM patients by using different test systemsa Serum samples from:
Negative controls (n= 100)b
EM (n = 36)
Ig class
Cutoff level
% Sensitivity and specificity
Flagellum Sonicate Immunoblot ELISA
ELISA
IgM
A B
6 6
4 13
0
IgG
A B
1 2
0 0
2
IgM
A B
33 56
29 50
81
a Flagellum cutoff level A relied on measurements of 200 Danish blood donors, whereas cutoff A of the sonicate ELISA was determined by the following formula: optical density of negative control serum + optical density of positive control seruml2. Cutoff level B was determined by standard methods (mean optical density of control sera [n = 100] + 3 standard deviations). b Individuals without clinical evidence of Lyme borreliosis.
were 1gM positive in the sonicate ELISA but only 6 were positive in the flagellum ELISA. There was no difference in the specificity of the flagellum ELISA (six of six were false-positive IgM reactions). As shown in Fig. 3B, none of the blood donors had IgM antibodies against flagellin. Eighty-one percent of the EM patients reacted clearly with the flagellin (Fig. 3A) and some reacted with proteins of 70 or 68 kDa. Seven patients were seronegative by IgM immunoblotting; i.e., no antibodies could be detected by standard ELISA procedures or by ,u-capture ELISA (data not shown). These data demonstrate that not all patients with EM can be reliably diagnosed by ELISA. In contrast, serological analysis by IgM immunoblotting significantly increases the sensitivity of the diagnosis. Although none of the blood donors showed positive IgM tests by immunoblot-
A
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22 20 FIG. 3. IgM immunoblot of B. burgdorferi with representative from EM patients (A) and negative controls (B). Proteins were separated by Tricine-PAGE. A Western immunoblot with serum from a patient with definite Lyme arthritis is shown on the left. The numbers indicate the molecular mass of the immunoreactive polypeptides. Negative controls consisted of 100 blood donors without any clinical symptoms of Lyme borreliosis. sera
There are some possible explanations for the increased sensitivity and specificity of the IgM immunoblot we developed. The high sensitivity may be caused by a lack of competition with IgG specific to the 41-kDa antigen which may give false low or false negative results in the ELISA. Furthermore, the absolute concentration of specific IgM needed to give a positive signal was lower than that needed in the ELISA. We have verified that the increased specificity depends on the blocking conditions. Incubation with ovalbumin may eliminate the possibility of false-positive reactions due to antibodies with low affinity to cross-reactive flagellin epitopes. Furthermore, a surplus of antigen is responsible for false-positive reactions when blocking procedures A to C are used (Fig. 2). Thus, it is useful to lower the antigen concentration if one wants to increase specificity without a concomitant loss of sensitivity. Especially, a surplus of OspA protein could provide a false-positive signal (Fig. 2A to C). The IgM response of most of our EM patients is highly specific. Before patients with EM are considered anti-B. burgdorferi negative by ELISA procedures, immunoblotting for IgM antibodies should be performed as an affirmative test (20).
i
I
41
ting, some of them reacted as false positives in ELISAs if the cutoff levels were decreased. This emphasizes the high degree of specificity of the immunoblot technique. Hansen and Asbrink detected IgG and IgM antibodies in sera from 107 EM patients by using purified native flagella from B. burgdorferi and compared the data with those from a sonic extract ELISA (9). They found that the sensitivity for IgM detection increased from 16.6 to 44.8%. In our study, 33% of the patients yielded IgM antibodies by the flagellum ELISA, and if we decreased the cutoff levels, a higher sensitivity rate (56%) resulted. The control group contained 6% false-positive reactions. In another attempt to improve serodiagnosis of early Lyme disease, Berardi et al. (3) described an IgM capture assay to improve serology. Twenty (67%) of their patients with EM had elevated IgM antibody levels (3). Recently, Hansen et al. reported that the ,u-capture ELISA increases the sensitivity in patients with EM from 32 to 48% when compared with the indirect IgM flagellum ELISA (11).
This work was supported by a Berlin-Forschung (12/89) and FNK grant from the Free University of Berlin.
Kd 95
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231
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J. CLIN. MICROBIOL. sis. Serodiagn. Immunother. Infect. Dis. 3:413-421. 15. Karlson, M., I. Mollegard, G. Stiernstedt, and B. Wretlind. 1989. Comparison of Western blot and enzyme linked immunosorbent assay for diagnosis of Lyme borreliosis. Eur. J. Clin. Microbiol. 8:871-877. 16. Lane, R. S., E. Lennette, and J. E. Madigan. 1990. Interlaboratory and intralaboratory comparison of indirect immunofluorescence assays for serodiagnosis of Lyme disease. J. Clin. Microbiol. 28:1774-1779. 17. Magnarelli, L. A., and J. F. Anderson. 1988. Enzyme-linked immunosorbent assays for the detection of class-specific immunoglobulins for Borrelia burgdorferi. Am. J. Epidemiol. 127: 818-825. 18. Schaegger, H., and G. von Jagow. 1987. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166:368-379. 19. Schmidli, J., T. Hunziker, and H. Gerber. 1987. Immunoperoxidase slide test for detecting antibodies to Borrelia burgdorferi. J. Infect. Dis. 1:218-219. 20. Schutzer, S. E., P. K. Coyle, A. L. Belman, M. G. Golightly, and J. Drulle. 1990. Sequestration of antibody to Borrelia burgdorferi in immune complexes in seronegative Lyme disease. Lancet 335:312-315. 21. Zoller, L., S. Burkard, and H. Schafer. 1991. Validity of Western immunoblot band patterns in serodiagnosis of Lyme borreliosis. J. Clin. Microbiol. 29:174-182.
