An IgM-antibody capture enzyme immunoassay for serodiagnosis of Lyme borreliosis
Post on 28-Aug-2016
Qrodiagnosis and Immunotherapy in Infectious Disease ( 1989) 3,4 1342 I
An IgM-antibody capture enzyme immunoassay for serodiagnosis of Lyme borreliosis
Mats Karlsson and Marta Granstr6m2
Department ?f Infectious Diseases, Danderyd HospitaI, S-182 88 Danderyd, Sweden and Department of Bacteriology. Karoiinska Hospital, Stockholm. Sweden
An IgM capture enzyme immunoassay (ELISA) was compared to an indirect IgM ELISA for serodiagnosis of Lyme borreliosis using sonicated cells of Borrelia burgdorferi as antigen. Sera from eight of 30 (27%) patients with erythema migrans were positive by indirect IgM ELISA as compared to IO/30 (33%) positive by IgM capture. The corresponding numbers for sera from patients with neuroborreliosis were 14/37 (38%) positive by indirect IgM ELISA as compared to 20/37 (54%) positive by IgM capture. Three of 10 sera from patients with acrodermatitis chronica atrophicans were positive by indirect IgM ELISA but negative by capture IgM. The positive indirect IgM ELISA in these three sera was falsely positive due to rheumatoid factor reactivity. Sera from one of 43 controls with meningitis/encephalitis or multiple sclerosis, one of ten with syphilis, two of ten with rheumatoid arthritis and three of ten with Epstein-Barr virus infection were positive by indirect IgM ELISA. In IgM capture, sera from two controls with Epstein-Barr virus infection were positive. Altogether, seven of 73 (10%) control sera were positive by indirect IgM ELISA as compared to two of 73 (3%) by IgM capture. The results of the present study shows a tendency towards an increased sensitivity and specificity with a IgM capture assay.
Keywords: IgM-capture, serodiagnosis, Lyme borrehosis.
Borreliu burgdorferi, the etiological agent of Lyme borreliosis., is introduced by tick- bite into the skin of humans. At the site, an erythematous skin lesion, erythema migrans, may appear3. The spirochetes may spread from the skin and cause acute or chronic infection in other organs, most commonly in joints, the nervous system and the heart4. A chronic infection of the skin, acrodermatitis chronica atrophicans can develops.
The culture of spirochetes from patients is slow and insensitive6, so that serology is the usual way to confirm the clinical diagnosis8.9. The presence of specific IgG antibodies in serum is compatible with active disease but may also represent anamnestic antibodies after previous clinical or subclinical infection with B. hurgdorfedO~. Specific IgM antibodies might be better markers of current infection, but are often lacking in chronic disease. In addition, serology may be negative in early disease9.
413 0888~786/89/060413 + 09 $03.00/O CQ 1989 Academic Press Limited
414 M. Karlsson and M. Granstrbn
A purified flagellum antigen has been shown to improve the diagnostic sensitivity as compared to a sonicated whole ceil antigen in indirect ELISA2.3. We found this improvement to be most pronounced regarding detection of IgG antibodies in serum (personal observation). The flagellum ELISA was almost as sensitive as Western blot (immunoblotting) for detecting IgG antibodies, but less sensitive for IgM detection. The Western blot analysis showed the presence of specific antibodies in sera not demon- strated by indirect ELISA, irrespective of antigen type14. The aim of the present study was to evaluate if the use of an TgM capture enzyme immunoassay would result in an improved sensitivity and specificity. Such an antibody capture assay has previously been reported from patients with erythema migrans15.
Materials and methods
Patients and controls
Pretreatment sera from 77 patients with Lyme borreliosis were analysed. Thirty patients had erythema migrans (EM) without extracutaneous complications, 37 patients had neuroborreliosis, and ten patients had acrodermatitis chronica atrophicans (ACA). The diagnosis of EM and ACA was based on clinical evidence and the diagnosis of ACA was confirmed by histopathology. Neuroborreliosis (NB) was defined as (a) pleocytosis (> 5 leukocytes x lo6 I-) with lymphocytic predominance in cerebrospinal fluid (CSF) together with neurological signs and symptoms and/or general symptoms compatible with NB, or (b) neurological signs and symptoms within 3 months after onset of EM. Neurological signs and symptoms were defined as sensory or motor neuroradiculitis, myelopathy or cranial nerve neuritis. General symptoms compatible with NB were those of a subacute or chronic meningitis as described previously, which includes fatigue, loss of appetite, loss of weight, migrating musculoskeletal pains, headache and sometimes low grade fever6,7. In many cases fulfilling criteri(a), the diagnosis was strengthened by a history of a recent tick-bite and/or EM. Thirty five patients with NB had pleocytosis in their CSF (range 6821 leukocytes x 1061-, median 137), 15 patients with NB had a recent or present EM.
Sera from patients with EM were drawn between a few days and 2 months (median 3 weeks) after onset of EM, sera from patients with NB were drawn between a few days and 11 months (median 3 weeks) after onset of neurological symptoms, and sera from patients with ACA were drawn between 6 months and 20 years (median 2 years) after onset of clinical symptoms.
Sera from 35 patients with meningitis/encephalitis of non-borrelia etiology, eight patients with multiple sclerosis (MS), ten patients with syphilis (SY), ten patients with primary Epstein-Barr virus infection (EBV infection), and ten patients with rheumatoid factor positive arthritis (RA) served as controls. Seven controls with meningitis/ encephalitis had herpes simplex type one encephalitis, six varicella-zoster encephalitis, one herpes simplex type 2 meningitis, four enterovirus meningitis, four morbilli encephalitis, three parotitis meningitis, three viral tick-borne encephalitis, one influenza A encephalitis, one mycoplasma encephalitis, two tuberculous meningitis, one men- ingococcal meningitis, one cryptococcal meningitis and one post rabies vaccination encephalitis.
All sera were analysed for the presence of IgM and IgG antibodies by indirect ELISA, but only for IgM antibodies in the capture assay. Sera from patients with ACA were
IgM assay for Lyme borrebsis 415
analysed for IgM rheumatoid factor reactivity (IgM RF). All patients and controls were from the Stockholm area in Sweden.
Indirect enzyme-linked immunosorhent assay
The indirect ELISA used has been described in detail previously. Briefly, microtiter plates (Dynatech, USA), were coated with 100 ul of a sonicate antigen (approximately 10 ug protein ml- of spirochetes diluted in phosphate-buffered saline (PBS) (pH 7.2). Borrelia burgdorferi strain STGl52, isolated from Ixodes ricinus in Sweden, was used for antigen preparation. Serum samples were diluted l/l000 in PBS with 0.5% Tween 20. Antibody-antigen reactions were made visible with alkaline phosphatase conjugated goat antihuman IgM or IgG (Sigma, USA) diluted l/l000 and I/500, respectively, p- Nitrophenyl phosphate (Sigma) was used as substrate. The absorbance at 405 nm (OD value) was measured in a Titertec Multiscan (Flow laboratories, UK). Positive and negative control sera were included in each test, and the time for substrate incubation was adjusted to that of these controls. The upper limit of normal values (cut-off) was defined as the 98th percentile of OD values in sera from 50 individuals admitted to hospital due to disorders not related to Lyme borreliosis. The cut-off level corresponded to an OD value of 0.47.
ZgM antibody capture enzyme immunoassay
Microtiter plates (Dynatech) were coated overnight at room temperature (22C) with 100 ul of affinity isolated goat IgG, directed against human IgM (u-chain specific, Sigma), diluted l/l 50 in carbonate buffer (pH 9.5). The wells were washed three times in 0.9% NaCl with 0.05% Tween 20, and similarly washed after the sequentially addition and incubation (h) at 37C of 100 ul of the following reagents diluted in PBS with 0.05% Tween 20: (a) patient sera diluted l/l00 (2 h) (b) 10 ug ml- of sonicate antigen prepared from B. burgdorferi strain STG152 (1 h), (c) polyclonal anti-borrelia rabbit serum diluted l/SO0 (1 h), (d) alkaline phosphatase conjugated, human serum adsorbed, goat anti-rabbit serum (Sigma) diluted l/l000 (1 h) and (e) p-nitrophenyl phosphate as substrate (1 h). The anti-borrelia rabbit serum was prepared by injecting a New Zealand white rabbit five times (0, 2w, 6w, 9w, 40~) with ca. 0.5 mg of borrelia sonicate, (strain STG 152), emulsified in Freunds complete adjuvant. Two percent goat serum (State Veterinary Laboratory, Uppsala, Sweden) and 2% human serum, both negative for antibodies to B. burgdorferi by ELISA, were added to the rabbit serum to reduce unspecific binding. The optimal concentrations of reagents were determined on the basis of the best resolution obtained between positive and negative control sera. OD values were measured, and upper limit of normal values established as described above. The cut-off level corresponded to an OD value of 0.35.
Fischers two tailed exact test was used for comparison of the number of patients positive by the two assays. OD values obtained in the two assays were compared by Sign testix. Differences P> 0.05 were considered not significant.
416 M. Karlsson and M. Granstrijm
The number of sera from patients and controls positive by indirect IgM ELISA, and by IgM capture ELISA are shown in Tables 1 and 2. Sera from 8/30 (27%) patients with erythema migrans were positive by indirect IgM ELISA as compared to IO/30 (33%) by IgM capture (P= 0.77). The corresponding number for sera from patients with neuroborrelios were 14/37 (38%) positive by indirect IgM ELISA as compared to 20/37 (54%) by IgM capture (P=O*24). None of ten sera from patients with ACA were positive by IgM capture as compared to 3/10 by indirect IgM ELISA (P=O.20). These three sera and another serum from a patient with ACA, negative in both IgM assays, were IgM RF-positive.
Sera from 5/24 patients with EM or NB which were positive by indirect IgM ELISA were negative by IgM capture ELISA. Three of these five patients had EM with a duration of a few days, 2 and 6 weeks, respectively. The other two patients had NB with neurological symptoms for 1 and 10 weeks, respectively. The former of these had a significantly elevated serum IgG titer by indirect ELTSA.
Table 1. Comparison of indirect ELISA and IgM capture ELISA in sera from patients with Lyme borreliosis and controls
No. of positive patients and controls by
Disease/manifestation No. Indirect IgM IgM capture
Erythema migrans (EM) 30 8 10 Neuroborreliosis (NB) 37 14 20 Acrodermatitis chronica
atrophicans (ACA) 10 3 0 Meningitis/encephalitis, multiple sclerosis 43 1 0 Syphilis (SY) 10 1 0 Epstein-Barr virus
infection (EBV) 10 3 2 Rheumatoid arthritis (RA) 10 2 0
Table 2. Comparison of indirect IgM ELISA and IgM capture ELISA in sera from patients with erythema migrans (EM) and neuroborreliosis (NB) in relation to the duration of EM and
No. of positive patients by
Duration of disease in weeks
Indirect IgM ELISA
IgM capture ELISA
62 3112 6112 2112 8112 >24,6 l/2 2110 212 3/10
IgM assay for Lyme borreliosis 417
Sera from 13/30 patients with EM or NB which were positive by IgM capture were negative by indirect IgM ELISA. Five of these 13 patients had EM with a duration of 2, 3, 4, 5 and 8 weeks, respectively. The latter three patients had positive serum IgG titers by indirect ELISA. The other eight patients with positive serum titers by IgM capture and negative titers by indirect IgM ELISA had NB with neurological symptoms for 3,8, 8, 21, 29. 48 and 322 days respectively. The latter two patients had positive serum IgG titers by indirect ELISA.
If only sera from patients with EM or NB negative by IgG ELISA are considered, 171 47 (36%) were positive by indirect IgM ELISA, as compared to 21/47 (45%) by IgM capture. The corresponding numbers in sera from patients with EM or NB positive by indirect IgG ELISA were 5/20 (25%) positive by indirect IgM ELISA as compared to 9/ 20 (45%) by IgM capture.
The OD values of sera obtained in the two IgM assays (Figure 1) were compared by Sign test18. The indirect IgM ELISA showed significantly higher OD values in sera from patients with ACA (P=O.Ol) [Figure l(b)] but no significant differences were found regarding the OD values in sera from other patients or from controls.
Sera from l/43 controls with meningitis/encephalitis or multiple sclerosis, l/l0 with syphilis, 3110 with EBV-infection and 2110 with RA were positive by indirect IgM ELISA. Sera from two controls with EBV-infection were positive by IgM capture. Altogether, 7/73 (10%) control sera were positive by indirect IgM ELISA as compared to 2/73 (3%) by IgM capture (P=O.l6). Sera from 2/43 controls with meningitis/ encephalitis, 3/10 with syphilis and l/l0 with RA were positive by indirect IgG ELISA. Sera from one control with syphilis and one with RA were positive for both IgM and IgG by indirect ELISA but negative by IgM capture. None of the controls had either a history of, or current symptoms compatible with Lyme borreliosis.
The present study shows a slightly improved sensitivity with an antibody capture assay as compared to indirect ELISA for detecting IgM antibodies to B. burgdorferi in serum. The improvement was not statistically significant, and less pronounced than previously reported by Berardi et al., using a similar assay with a sonicate antigen15. The improved sensitivity in that study was mainly found in sera from patients with erythema migrans and clinical evidence of disseminated disease. However, a comparison with the present study is difficult, since OD values are not given in the referred study, and the results were calculated in a different manner.
The present IgM capture assay also shows a tendency towards an improved specificity in comparison to the indirect ELISA, as evaluated in controls with meningitis encephalitis, MS, syphilis, EBV-infection or rheumatoid arthritis. Furthermore, sera from 3/10 patients with ACA which were positive by indirect IgM ELISA were negative by IgM capture. These three sera were IgM RF positive, and the results reflect an increased specificity of the capture assay due to a reduced interference by IgM RF. It has previously been shown that sera from patients with ACA may include IgM RF that causes false positive IgM titers by indirect ELISA13. RF reactivity may also be present in serum during several acute and chronic infections. including EBV-infection and syphilis.
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0.5 I.0 I.5 2.0
OD-value capture IgM ELISA
I I I 0.5 I.0 I.5
OD-value capture IgM ELISA
IgM assay for Lyme borreliosis
o-5 1.0 I.5
OD-value capture IgM ELISA
Figure I, OD-values in sera from patients and controls analysed by indirect IgM ELISA and Capture IgM ELISA. The upper limit of normal values in indirect IgM ELISA is indicated by a horizontal line. The upper limit of normal values in Capture IgM ELISA is indicated by a vertical line. (a) sera from 30 patients with erythema migrans and 37 patients with neuroborreliosis. (b) sera from 10 patients with acrodermatitis chronica atrophicans. (c) sera from 43 controls with meningitis/encephalitis of non-borrelia etiology or multiple sclerosis, ten controls with syphilis, ten controls with EBV-infection and ten controls with rheumatoid arthritis.
Only a limited number (2/10) of sera from patients with RF-positive rheumatoid arthritis were positive by indirect IgM ELISA. This was most likely due to the high serum dilution (l/1000) used in our assay, and the number of false-positive results in indirect IgM ELISA due to IgM RF may be higher in assays with lower serum dilutions. The problem with false-positive IgM titers in indirect ELISA due to IgM RF will only be seen in the presence of specific IgG antibodies or if unpurified antigens are used in the presence of crossreactive and non-specific IgG antibodies.
Another theoretical advantage of the IgM capture assay is the exclusion of competi- tion by specific IgG antibodies for binding sites. In fact, the present capture assay did identify IgM antibodies in five additional sera from patients with EM or NB. negative by indirect IgM ELISA and positive by indirect IgG ELISA.
In the present study, we did not apply the capture assay for detecting IgG antibodies in serum since preliminary results showed a decreased sensitivity for IgG capture as compared to indirect IgG ELISA (data not shown). This finding is in accordance with the results of Berardi et ~1.~. An analysis of their data showed that the indirect ELISA was in fact slightly superior to the capture assay for IgG antibodies5*9. Also. theoretically, a capture assay for IgG should not improve sensitivity like a capture assay for IgM, since the ratio of specific IgG/total IgG is expected to be much lower than the ratio of specific IgM/total IgM.
420 M. Karlsson and M. Granstriim
Some sera, positive by both IgM assays, showed lower OD values by the capture assay than by indirect ELISA. This finding could be due to the limited amount of specific IgM captured by the immobilized anti-IgM in the well of the microtiter plate. In the case of high levels of specific IgM, the indirect ELISA system with binding of IgM antibodies direct to the antigen might result in binding of greater amounts of IgM and consequently higher OD-values.
We conclude that the present IgM antibody capture assay shows a tendency to both an improved sensitivity and improved specificity as compared to indirect IgM ELISA. However, the slight improvement as compared to the standard assay has to be weighted against the more complicated procedure for the capture assay. The capture assay could probably be further improved by use of purified antigens and enzyme conjugated monoclonal antibodies to B. burgdorferi.
We thank Eva Asbrink, Dept of Dermatology, South Hospital, Stockholm, who kindly provided clinical data and sera from 30 patients with EM and 10 patients with ACA. We also thank Erika Szanto, Section of Rheumatology, Department of Internal Medicine, Danderyd Hospital, Stockholm, who kindly provided clinical data and sera from 10 patients with rheumatoid arthritis and Jan Andersson, Dept of Infectious Diseases, Danderyd Hospital, Stockholm, who kindly provided clinical data and sera from 10 patients with primary EBV infection. We also thank Ingrid MGllegBrd, Dept of Bacteriology, Danderyd Hospital, for excellent assistance in performing the ELISA test.
This study was funded by grants from Fiirenade Liv Mutual Group Life Insurance Company, Stockholm, Sweden, and from Leila and Bertil Ehrengrens Memory founda- tion, Swedish Society for Medical Research, Karolinska Institute, Stockholm, Sweden.
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(Manuscript accepted 7th December 1989)