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Demonstration of locally synthesized immunoglobulin M antibodies to Treponema pallidum in the central nervous system of patients with untreated neurosyphilis
Journal of Neuroimmunology, 7 (1984/85) 43-54
43
Elsevier JNI 00210
Demonstration of Locally Synthesized Immunoglobulin M Antibodies to Treponema pallidum in the Central Nervous System of Patients with Untreated Neurosyphilis F. Mi~ller 1, M. Moskophidis I and H.W. Prange 2 IDivtsion of Immunology of Infectious Diseases, Department of Medical Microbiology, lnsntute of l-lyglene, Hamburg and 2Department of Neurology, Umversity of G~ttmgen, GOttingen (F.R. G.)
(Received18 October, 1983) (Revised, received5 March, 1984) (Accepted 5 March, 1984)
Summaff Immunoglobulin M (IgM) antibodies to Treponema pallidum (TP) in serum and cerebrospinal fluid (CSF) can be evaluated quantitatively using a recently introduced enzyme-linked immunosorbent assay (ELISA). In 19 patients with untreated neurosyphilis, local synthesis of TP-specific IgM antibodies within the central nervous system (CNS) was demonstrated by estimation of the C S F / s e r u m ratio of the TP-IgM-ELISA titer/mg total IgM. The TP-specific IgM antibody levels/mg total IgM in the CSF were between 3- and 75-fold higher than those in the corresponding serum of the same patients. Patients with untreated syphilis but without CNS involvement showed equal amounts of TP-specific IgM antibody levels/mg total IgM in CSF and serum, and served as controls. Key words: Cerebrospinal f l u i d - Immunoassay - Immunoglobulin M - Neurosyphilis - Treponema pallidum
Correspondence: Prof. F. Mi)ller, Hygiene-lnstitut, Gorch-Fock-Wall 15/17, D-2000 Hamburg 36, F.R.G. 0165-5728/84/$03.00 © 1984 ElsevierSciencePublishers B.V.
44 Introduction
In the last few years oligoclonal immunoglobulin G (IgG) bands have been demonstrated in the cerebrospinal fluid (CSF) of patients with neurosyphilis (Oxelius et al. 1969; Vartdal et al. 1982; Prange et al. 1983). Analysis of these antibodies in electrofocused specimens by an immunofixation technique, have disclosed the production of oligoclonal IgG antibodies to Treponema pallidum (TP) in the central nervous system (CNS), reflecting a specific local immune response to the infectious agent (Vartdal et al. 1982; Pedersen et al. 1982). Recently it has been observed that a group of adequately treated syphilitic patients, without CNS involvement, had equal TP-specific IgG antibody levels/mg total IgG in CSF and serum, i.e., the TP-specific IgG portion of total IgG was the same in both body fluids. On the other hand, the CSF/serum ratio of TP-specific IgG antibody levels/mg total IgG was increased in 82% of the investigated patients with neurosyphilis, demonstrating local production of IgG antibodies to TP in the CNS. It could be shown "that TP-specific IgG antibody levels/mg total IgG were between 3- and 430-fold higher in CSF than those in serum of the same patients (Mi~ller and Moskophidis 1983; Prange et al. 1983). It has been technically complicated to estimate routinely the concentration of total IgM in the CSF. IgM immunoassays have made it possible to measure total IgM in CSF down to a level of 0.022 mg/1 (Kobatake et al. 1980; Sindic et al. 1982). With presently available serological techniques the demonstration of TP-specific IgM antibodies has been impossible because of the low antibody titers and the low sensitivity of the methods used. In previous experiments a TP-specific lgM enzymelinked immunosorbent assay (TP-IgM-ELISA) with high sensitivity and specifity has proved its value for the quantitative estimation of IgM antibodies to TP in serum and CSF (Mialler and Moskophidis 1984). Intrathecal synthesis of IgM antibodies to TP has been postulated to occur in untreated neurosyphilis (Oxelius et al. 1969), but the detection of these antibodies has been unsuccessful, even by the immunofixation technique, because IgM does not penetrate into polyacrylamide gel and hence can not be focused (Vartdal et al. 1982). In the present paper we report on investigations of local production of TP-specific IgM antibodies in the CNS of patients with untreated neurosyphilis, by estimation of the TP-IgM-ELISA titers/mg total IgM in CSF and serum, as well as their CSF/serum correlations. Materials and Methods
Serum and CSF samples Paired serum and CSF samples were obtained from neurological clinics: collected from the patients simultaneously. Specimens from 24 healthy adults and 12 adequately treated syphilitic patients without clinical evidence of CNS involvement, served as control groups for the estimation of the mean values and ranges (in mg/l) of normal albumin, total IgG and total IgM in serum and CSF, as well as their serum/CSF ratios.
45 Diagnosis of syphilis was made when the Treponema pallidum haemagglutination assay (TPHA) and the fluorescent treponemal antibody absorption (FTA-ABS) test were positive in the patient's sera. Specimens from 4 untreated syphilitic patients without CNS involvement (normal cell count in the CSF, normal s e r u m / C S F ratios of albumin, total IgG, total IgM and TP-specific IgG antibody titer) were used as a control group, for the TP-specific IgM antibody levels/mg total IgM and the s e r u m / C S F ratios. Pairs of serum and CSF from another 19 patients with clinically and biochemically diagnosed untreated neurosyphilis (with pleocytosis in the CSF and abnormal s e r u m / C S F ratios for albumin, or total IgG or total IgM) were investigated for TP-specific IgM antibody titers, their levels/mg total IgM and their C S F / s e r u m ratios.
Quantitative determination of albumin, total IgG and total IgM Quantitation of albumin and total IgG in serum and CSF, as well as total IgM in serum, were carried out by single radial immunodiffusion using Partigen plates (Behringwerke, Marburg, F.R.G.).
ELISA for quantitative determination of total IgM in CSF Concentrations of total IgM in CSF were measured by a microenzyme-linked immunosorbent assay. Flexible polyvinylchloride microtiter plates (Becton-Dickinson, Oxnard, CA, U.S.A.) with U-shaped wells were coated at 4 ° C overnight with 100 ~1 of a rabbit anti-human IgM (/l-chain specific) serum dilution (Miles Laboratories, Elkhart, IN, U.S.A.). Coating dilution was prepared with phosphate-buffered saline (PBS; 137 mM NaC1, 2.7 mM KC1, 4.6 mM N a z H P O 4, 1.5 mM KH2PO4), pH 7.2, to contain 40/~g antibody/ml. Unoccupied sites on the wells were blocked with 200 p.1 PBS containing 1% (w/v) bovine serum albumin at 22°C for 1 h. Then the wells were washed 3 times with 200/11 PBS containing 0.05% (v/v) Tween 20. Hundred #1 of CSF were incubated undiluted, and in serial dilutions (1:2 to 1:128), from healthy adults or 1:2 to 1:2048 from syphilitic patients, in the coated wells at 22°C for 2 h. After this first immunoreaction the wells were washed 3 times with 200 ~1 0.05% Tween 20-PBS. Coupled IgM was identified by incubation at 22°C for 2 h with a horseradish peroxidase-conjugated rabbit anti-human IgM (/.t-chain specific; Daco Immunochemicals, Copenhagen, Denmark), diluted 1:1 000 with PBS. After this the wells were washed again 3 times with 200 ~1 0.05% Tween 20-PBS. The enzyme reaction was performed with 50 ~1 substrate solution (10 mg 1,2-phenylenediamine di§solved in 10 ml 0.1 M phosphate buffer, pH 6.0, containing 0.01% hydrogen peroxide) at 22°C for 10 min in the dark and stopped by addition of 100 ~1 2 M sulfuric acid. The wells were read photometrically at 492 nm in an automatic Titertek Multiscan MC against a blank of 50/xl substrate solution and 100 ~1 2 M sulfuric acid. Standard curves for each test performance were generated using purified human myeloma IgM (purity 98%) or a standardized mixture of human IgM (Behringwerke, Marburg, F.R.G.) and preparing serial dilutions (in PBS) from 0.5 to 128 ng
46 IgM/100 ~1. The assay was shown to be IgM-specific and the linear range was from 2 to 32 ng IgM. The optical density of the blank was below 0.200.
ELISA for the quantitative determination of TP-specific IgM antibodies in serum and CSF (TP-IgM-EL1SA) The assay has been described by Yokota et al, (1982) and was used quantitatively with some modifications (Mt~ller and Moskophidis 1984). Polystyrene beads were coated with an ultrasonicate of TP (Nichols strain) as antigen. The coated beads were incubated at 37 °C for 2 h in test tubes containing 300 /tl of serial serum dilutions (1:50 to 1:25600) or serial CSF dilutions (1:1 to 1:2048) in PBS. After washing of beads 4 times with a 0.14 M NaCI 400 #1 solution of a horseradish peroxidase-conjugated antihuman IgM (p-chain specific) were added for the identification of coupled TP-specific IgM antibodies and incubated at 37 °C for 1 h. After washing of the beads the enzyme reaction was performed with 400/~1 chromogen solution at 37°C for 1 h in the dark and the reaction was stopped by addition of 2 ml 0.05 M oxalic acid. The absorbance was read at 420 nm. Sensitivity and specificity of the TP-IgM-ELISA in serum and specificity in CSF were shown to be higher than 97%. Optical densities of specimens which exceeded the arithmetic mean plus its 5-fold standard deviation (~ + 5s) of at least 8 negative controls were considered to be positive. The IgM antibody titer was defined as the highest serum or CSF dilution showing a positive result. The presence of rheumatoid factor in the specimens was excluded by performing a quantitative rheumatoid antibody haemagglutination assay (RAHA; Fujirebio Inc., Tokyo, Japan). Estimation of TPHA-lgG titer / mg total lgG . Separation of IgG from IgM antibodies to TP in serum and CSF was carried out by Ultrogel AcA 34 filtration (Mialler 1982). Briefly, 0.7 of samples were filtrated through an 1.5 x 14 cm gel column using PBS, pH 7.3. Fractions of 1.3 ml were collected and the absorbance measured at 280 nm. Twenty-five ~1 of the IgG peak fraction with TP-specific IgG antibodies was investigated quantitatively in the T P H A (Fujirebio Inc., Tokyo, Japan). The TPHA-IgG titer/mg total IgG was calculated from the TPHA-IgG titer divided by total IgG (in mg) of 25 pA specimen. Estimation of TP-IgM-ELISA titer/mg total lgM The TP-IgM-ELISA titer/mg total IgM was calculated from serum or CSF titers divided by total IgM (in mg) of 100 ~1 specimen.
Results
Healthy adults and treated syphilitic patients without CNS participation on the TP infection As shown in Table 1 the mean values and ranges of albumin, total IgG and total IgM, in serum and CSF, as well as the s e r u m / C S F ratios, of 12 patients with
47 r~
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adequately treated syphilis (without clinical or biochemical evidence of CNS involvement) did not differ from the findings in 24 healthy adults. In the group of healthy adults, the levels for total IgM in CSF ranged from 0.027 to 0.186 mg/1 with a mean value of 0.102 mg/I. The mean s e r u m / C S F ratio of total IgM was found to be 12 427. The relationship between total IgM concentrations of CSF and serum is shown in Fig. 1.
Untreated syphilitic patients without CNS participation on the TP infection In 4 syphilitic patients without clinical and biochemical evidence of CNS involvement a high TP-specific IgM antibody titer was found in serum, indicating an TP infection in need of treatment. But the albumin, total IgG and total IgM values in CSF of these patients do not differ from those of healthy adults (Table 2A, group 1). S e r u m / C S F ratios of TP-specific IgM and IgG were identical with those of total IgM and IgG, respectively. The mean s e r u m / C S F ratio of the TP-IgM-ELISA titer was calculated to be 10 628, i.e., in the normal range. Furthermore, the TPHA-IgG and the TP-IgM-ELISA titers/mg total IgG or IgM in serum and CSF were nearly equal, i.e., the C S F / s e r u m ratios were approximately 1 (Table 2B, group 1). Patients with untreated neurosyphilis On the basis of the b l o o d - C S F barrier function the 19 patients with untreated neurosyphilis were divided into two subgroups. 0.250 o
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All patients with untreated neurosyphilis and intact blood-CSF barrier (normal albumin serum/CSF ratio) showed an increased total IgM in CSF. In 4 of these patients (No. 1-4) total IgG in CSF was within, in another 7 (No. 5-11) this value was out of the normal range (Table 2A, group 2). The serum/CSF ratios of TP-IgM-ELISA titers were reduced resulting in increased CSF/serum ratios of TP-specific IgM/mg total IgM. It can be seen from Table 2B that the TP-IgM-ELISA titer/mg total IgM in CSF was 4- to 62-fold higher than in the corresponding serum. Only in the patient No. 1 (group 2) with syphilitic meningitis the CSF/serum ratio of TP-specific IgG/mg total IgG was normal (1.0). In the 10 other patients of this subgroup this ratio was increased. All patients with dysfunction of the blood-CSF barrier (abnormal albumin serum/CSF ratio) had an increased total IgM in CSF which could be demonstrated by the CSF-IgM index (Fryd6n et al. 1978; Link et al. 1979). The total IgG in CSF was increased as shown by an elevated CSF-IgG index (Tibbling et al. 1977) (Table 2A, group 3). The TP-IgM-ELISA titers/mg total IgM were 3- to 75-fold higher than those of the corresponding serum. The TP-specific IgG titers/mg total IgG in CSF were in most of the patients higher than in serum (except the patients 12 and 13 of Table 2B, group 3). The relationship between CSF TP-IgM-ELISA titers/mg total IgM and those of serum are shown in Fig. 2. Patients with untreated syphilis but without CNS / u') ~"
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52 involvement are located within the dash lines, indicating that the TP-specific IgM levels/mg total IgM in CSF and serum are equal. Patients with untreated neurosyphilis are located above the dash lines, indicating that the TP-specific IgM titers/mg total lgM are higher in CSF than those in serum.
Discussion
Sensitive, specific immunoassays for the quantitation of total IgM (IgM ELISA) and TP-specific IgM (TP-IgM-ELISA) in CSF have made it possible to correlate the results of these assays and to evaluate local synthesis of TP-specific IgM antibodies in the CNS. Both assays have been shown to be suitable, not only for the estimation of increased, but also of normal levels with reproducible results. IgM ELISA values from 0.027 to 0.186 m g / l with a mean of 0.102 m g / l were observed in the group of 24 healthy adults. Appropriate mean values have been reported by Nerenberg and Prasad (1978) (0.170 mg/i), Mingioli et al. (1978) (0.0513 mg/1), Kobatake et al. (1980) (0.240 mg/l) and Sindic et al. (1982) (0.0975 mg/l). Our results correspond to these findings. The mean s e r u m / C S F ratio for total lgM in the patients of our group was 12427, and hence was in accord with the value of 11 443 reported by Sindic et al. (1982). Compared with the results in the group of healthy adults, the mean values for albumin, total IgG and total IgM in serum and CSF, as well as their s e r u m / C S F ratios, from 12 adequately treated and 4 untreated syphilitic patients, were in the normal range. Because of the occasional low titers of TP-specific IgM in serum and CSF, an assay with high sensitivity and specificity is necessary for the determination of these antibodies. The used TP-IgM-ELISA fulfills these conditions. The sensitivity of the assay in serum is |0-fold higher than that of the indirect immunofluorescence techniques (Mialler and Moskophidis 1984). The passive transfer of TP-specific IgM antibodies from serum to CSF, through the intact b l o o d - C S F barrier, depends on their concentration in serum. Four untreated syphilitic patients without clinical or biochemical symptoms of neurosyphilis and normal b l o o d - C S F barrier function had high titers ( ~ 1:6400) of TP-specific IgM antibodies in serum (group 1, Tables 2A and B). From this group, the normal passive transfer of TP-specific IgM antibodies could be estimated as 10628 to 1. This transfer rate cannot be distinguished from that for total IgM, i.e., the portion of TP-specific IgM a n t i b o d y / m g total IgM is the same in serum and CSF. The C S F / s e r u m ratio is therefore approximately 1. The same principle of normal s e r u m / C S F transfer was formerly shown for TP-specific IgG antibodies in treated syphilitic patients without CNS involvement (Mialler and Moskophidis 1983). On the other hand, the TP-specific IgM titers/mg total IgM was, in all untreated neurosyphilitic patients, between 3- and 75-fold higher in CSF than in serum. This can only be explained by the assumption that local production of TP-specific IgM antibodies takes place in CNS.
53 It should be emphasized that the correlation of the TP-specific IgM a n t i b o d y titer with the total IgM does not depend on the function of the b l o o d - C S F barrier, as can be seen from Fig. 2. Therefore, by estimating the C S F / s e r u m ratio of TP-specific lgM a n t i b o d y / m g total IgM, local p r o d u c t i o n of TP-specific IgM antibodies in C N S can be assumed, not only in neurosyphilitic patients with n o r m a l b l o o d - C S F barrier f u n c t i o n (Tables 2A a n d B, group 2) b u t . a l s o in those patients with d y s f u n c t i o n of the b l o o d - C S F barrier (Tables 2A and B, group 3). The patients No. 1, 12 a n d 14 (Table 2B) are of particular interest. They show only local synthesis of TP-specific IgM but not of IgG antibodies. This might be explained by the very early stage of the C N S involvement. Finally, it should be stated that local p r o d u c t i o n of TP-specific IgM antibodies may be d e m o n s t r a t e d by the C S F - I g M index ( F r y d 6 n et al. 1978; Link et al. 1979), if instead of the total IgM (in m g / l ) , the TP-specific IgM titers are inserted in the formula but it should be kept in m i n d that, using this C S F - I g M index, no correlation of TP-specific IgM with the total IgM is possible.
Acknowledgements We greatly acknowledge the excellent technical assistance of Miss D. O r t m a n n a n d Mrs. J. Dolberg.
References Fryd6n, A., H. Link and E. Norrby, Cerebrospinal flmd and serum lmmunoglobulins and antibody titers in mumps meningitis and aseptic meningitis of other etiology, Infect. Immunol., 21 (1978) 852-861. Kobatake, K., Y. Shinohara and S. Yoshimura, lmmunoglobulins in cerebrospinal fluid -- Enzyme-lmmunoassay, J. Neurol. Sci., 47 (1980) 273-283. Link, H., B. Wahren and E. Norrby, Pleocytosis and immunoglobulin changes in cerebrospinal fluid and herpes virus serology m patients with Guillain-Barr6 syndrome, J. Clin. Microbiol., 9 (1979) 305-316. Mingioli, E.S., W. Strober, W.W. Tourtellotte, J.N. Whltaker and D.E. McFarhn, Quantitation of IgG, IgA and igM in the CSF by radioimmunoassay, Neurology, 28 (!978) 991-995. Mtiller, F., Der 19S(IgM)-FTA-ABS-Testm der Serodiagnostik der Syphilis -- Technik, Fehlermoglichkeiten und diagnostische Aussage, Immun, lnfekt., 10 (1982) 23-34. Mialler. F. and M. Moskophidis, Estimation of the local production of antibodies to Treponemapalhdum in the central nervous system of patients with neurosyphilis, Brit. J. Vener. Dis., 59 (1983) 80-84. Mialler, F. and M. Moskophidis, Evaluation of an enzyme-linked immunosorbent assay for immunoglobulin M antibodies to Treponema palhdum in human syphdis, Brit. J. Vener. Dis., 60 (1984) m press. Nerenberg, S.T. and R. Prasad, Radiolmmunoassay for lg classes G, A, M, D, and E m spinal fluids -Normal values of different age groups, J. Lab. Clin. Med., 86 (1978) 887-898. Oxelius, V.A., M. Rorsman and A.B. Laurell, Immunoglobulins of cerebrospinal fluid in syphilis, Brit. J. Vener. Dis., 45 (1969) 121-125. Pedersen, N.S., S, Kam-Hansen, H. Link and M. Mavra, Specificity of immunoglobulins synthestzed within the central nervous system in neurosyphihs, Acta Path. Microblol. Immunol. Scand., Sect. C, 90 (1982) 97-104. Prange, H.W.. M. Moskophidis, H.I. Schlpper and F. Muller, Relationship between neurological features and intrathecal synthesis of IgG antibodies to Treponema palhdum in untreated and treated human neurosyphihs, J. Neurol., 230 (1983) 241-252.
54 Smdic, C,J.M., C.L. Camblaso, A. Depr~, E.C. Laterre and P.L. Masson, The concentration of IgM in the cerebrospinal fluid of neurological patients, J. Neurol. Scl., 55 (1982) 339-350. Tibbling, G., H. Link and S. Ohman, Principles of albumin and IgG analysis in neurological disorders, Part 1 (Establishment of reference values), Scand. J. Clin. Lab. Invest., 37 (1977) 385-390. Vartdal, F., B. Vandvik, T.E. Mlchaelsen, K. Loe and E. Norrby, Neurosyphdis - - Intrathecal synthesis of oligoclonal antibodies to Treponema palhdum, Ann. Neurol., 11 (1982) 35-40. Yokota, M., Y. Kasahara, Y. Tsuji, M. Kobayashl and T. Tomizawa, The measurement of IgG and IgM anttbodles to Treponema palhdum by enzyme-hnked immunosorbent assay (ELISA) simultaneously. In: E. Levy (Ed.), Advances in Pathology, Vol. 1, Pergamon Press, Oxford, New York, 1982, pp. 125-128.
43
Elsevier JNI 00210
Demonstration of Locally Synthesized Immunoglobulin M Antibodies to Treponema pallidum in the Central Nervous System of Patients with Untreated Neurosyphilis F. Mi~ller 1, M. Moskophidis I and H.W. Prange 2 IDivtsion of Immunology of Infectious Diseases, Department of Medical Microbiology, lnsntute of l-lyglene, Hamburg and 2Department of Neurology, Umversity of G~ttmgen, GOttingen (F.R. G.)
(Received18 October, 1983) (Revised, received5 March, 1984) (Accepted 5 March, 1984)
Summaff Immunoglobulin M (IgM) antibodies to Treponema pallidum (TP) in serum and cerebrospinal fluid (CSF) can be evaluated quantitatively using a recently introduced enzyme-linked immunosorbent assay (ELISA). In 19 patients with untreated neurosyphilis, local synthesis of TP-specific IgM antibodies within the central nervous system (CNS) was demonstrated by estimation of the C S F / s e r u m ratio of the TP-IgM-ELISA titer/mg total IgM. The TP-specific IgM antibody levels/mg total IgM in the CSF were between 3- and 75-fold higher than those in the corresponding serum of the same patients. Patients with untreated syphilis but without CNS involvement showed equal amounts of TP-specific IgM antibody levels/mg total IgM in CSF and serum, and served as controls. Key words: Cerebrospinal f l u i d - Immunoassay - Immunoglobulin M - Neurosyphilis - Treponema pallidum
Correspondence: Prof. F. Mi)ller, Hygiene-lnstitut, Gorch-Fock-Wall 15/17, D-2000 Hamburg 36, F.R.G. 0165-5728/84/$03.00 © 1984 ElsevierSciencePublishers B.V.
44 Introduction
In the last few years oligoclonal immunoglobulin G (IgG) bands have been demonstrated in the cerebrospinal fluid (CSF) of patients with neurosyphilis (Oxelius et al. 1969; Vartdal et al. 1982; Prange et al. 1983). Analysis of these antibodies in electrofocused specimens by an immunofixation technique, have disclosed the production of oligoclonal IgG antibodies to Treponema pallidum (TP) in the central nervous system (CNS), reflecting a specific local immune response to the infectious agent (Vartdal et al. 1982; Pedersen et al. 1982). Recently it has been observed that a group of adequately treated syphilitic patients, without CNS involvement, had equal TP-specific IgG antibody levels/mg total IgG in CSF and serum, i.e., the TP-specific IgG portion of total IgG was the same in both body fluids. On the other hand, the CSF/serum ratio of TP-specific IgG antibody levels/mg total IgG was increased in 82% of the investigated patients with neurosyphilis, demonstrating local production of IgG antibodies to TP in the CNS. It could be shown "that TP-specific IgG antibody levels/mg total IgG were between 3- and 430-fold higher in CSF than those in serum of the same patients (Mi~ller and Moskophidis 1983; Prange et al. 1983). It has been technically complicated to estimate routinely the concentration of total IgM in the CSF. IgM immunoassays have made it possible to measure total IgM in CSF down to a level of 0.022 mg/1 (Kobatake et al. 1980; Sindic et al. 1982). With presently available serological techniques the demonstration of TP-specific IgM antibodies has been impossible because of the low antibody titers and the low sensitivity of the methods used. In previous experiments a TP-specific lgM enzymelinked immunosorbent assay (TP-IgM-ELISA) with high sensitivity and specifity has proved its value for the quantitative estimation of IgM antibodies to TP in serum and CSF (Mialler and Moskophidis 1984). Intrathecal synthesis of IgM antibodies to TP has been postulated to occur in untreated neurosyphilis (Oxelius et al. 1969), but the detection of these antibodies has been unsuccessful, even by the immunofixation technique, because IgM does not penetrate into polyacrylamide gel and hence can not be focused (Vartdal et al. 1982). In the present paper we report on investigations of local production of TP-specific IgM antibodies in the CNS of patients with untreated neurosyphilis, by estimation of the TP-IgM-ELISA titers/mg total IgM in CSF and serum, as well as their CSF/serum correlations. Materials and Methods
Serum and CSF samples Paired serum and CSF samples were obtained from neurological clinics: collected from the patients simultaneously. Specimens from 24 healthy adults and 12 adequately treated syphilitic patients without clinical evidence of CNS involvement, served as control groups for the estimation of the mean values and ranges (in mg/l) of normal albumin, total IgG and total IgM in serum and CSF, as well as their serum/CSF ratios.
45 Diagnosis of syphilis was made when the Treponema pallidum haemagglutination assay (TPHA) and the fluorescent treponemal antibody absorption (FTA-ABS) test were positive in the patient's sera. Specimens from 4 untreated syphilitic patients without CNS involvement (normal cell count in the CSF, normal s e r u m / C S F ratios of albumin, total IgG, total IgM and TP-specific IgG antibody titer) were used as a control group, for the TP-specific IgM antibody levels/mg total IgM and the s e r u m / C S F ratios. Pairs of serum and CSF from another 19 patients with clinically and biochemically diagnosed untreated neurosyphilis (with pleocytosis in the CSF and abnormal s e r u m / C S F ratios for albumin, or total IgG or total IgM) were investigated for TP-specific IgM antibody titers, their levels/mg total IgM and their C S F / s e r u m ratios.
Quantitative determination of albumin, total IgG and total IgM Quantitation of albumin and total IgG in serum and CSF, as well as total IgM in serum, were carried out by single radial immunodiffusion using Partigen plates (Behringwerke, Marburg, F.R.G.).
ELISA for quantitative determination of total IgM in CSF Concentrations of total IgM in CSF were measured by a microenzyme-linked immunosorbent assay. Flexible polyvinylchloride microtiter plates (Becton-Dickinson, Oxnard, CA, U.S.A.) with U-shaped wells were coated at 4 ° C overnight with 100 ~1 of a rabbit anti-human IgM (/l-chain specific) serum dilution (Miles Laboratories, Elkhart, IN, U.S.A.). Coating dilution was prepared with phosphate-buffered saline (PBS; 137 mM NaC1, 2.7 mM KC1, 4.6 mM N a z H P O 4, 1.5 mM KH2PO4), pH 7.2, to contain 40/~g antibody/ml. Unoccupied sites on the wells were blocked with 200 p.1 PBS containing 1% (w/v) bovine serum albumin at 22°C for 1 h. Then the wells were washed 3 times with 200/11 PBS containing 0.05% (v/v) Tween 20. Hundred #1 of CSF were incubated undiluted, and in serial dilutions (1:2 to 1:128), from healthy adults or 1:2 to 1:2048 from syphilitic patients, in the coated wells at 22°C for 2 h. After this first immunoreaction the wells were washed 3 times with 200 ~1 0.05% Tween 20-PBS. Coupled IgM was identified by incubation at 22°C for 2 h with a horseradish peroxidase-conjugated rabbit anti-human IgM (/.t-chain specific; Daco Immunochemicals, Copenhagen, Denmark), diluted 1:1 000 with PBS. After this the wells were washed again 3 times with 200 ~1 0.05% Tween 20-PBS. The enzyme reaction was performed with 50 ~1 substrate solution (10 mg 1,2-phenylenediamine di§solved in 10 ml 0.1 M phosphate buffer, pH 6.0, containing 0.01% hydrogen peroxide) at 22°C for 10 min in the dark and stopped by addition of 100 ~1 2 M sulfuric acid. The wells were read photometrically at 492 nm in an automatic Titertek Multiscan MC against a blank of 50/xl substrate solution and 100 ~1 2 M sulfuric acid. Standard curves for each test performance were generated using purified human myeloma IgM (purity 98%) or a standardized mixture of human IgM (Behringwerke, Marburg, F.R.G.) and preparing serial dilutions (in PBS) from 0.5 to 128 ng
46 IgM/100 ~1. The assay was shown to be IgM-specific and the linear range was from 2 to 32 ng IgM. The optical density of the blank was below 0.200.
ELISA for the quantitative determination of TP-specific IgM antibodies in serum and CSF (TP-IgM-EL1SA) The assay has been described by Yokota et al, (1982) and was used quantitatively with some modifications (Mt~ller and Moskophidis 1984). Polystyrene beads were coated with an ultrasonicate of TP (Nichols strain) as antigen. The coated beads were incubated at 37 °C for 2 h in test tubes containing 300 /tl of serial serum dilutions (1:50 to 1:25600) or serial CSF dilutions (1:1 to 1:2048) in PBS. After washing of beads 4 times with a 0.14 M NaCI 400 #1 solution of a horseradish peroxidase-conjugated antihuman IgM (p-chain specific) were added for the identification of coupled TP-specific IgM antibodies and incubated at 37 °C for 1 h. After washing of the beads the enzyme reaction was performed with 400/~1 chromogen solution at 37°C for 1 h in the dark and the reaction was stopped by addition of 2 ml 0.05 M oxalic acid. The absorbance was read at 420 nm. Sensitivity and specificity of the TP-IgM-ELISA in serum and specificity in CSF were shown to be higher than 97%. Optical densities of specimens which exceeded the arithmetic mean plus its 5-fold standard deviation (~ + 5s) of at least 8 negative controls were considered to be positive. The IgM antibody titer was defined as the highest serum or CSF dilution showing a positive result. The presence of rheumatoid factor in the specimens was excluded by performing a quantitative rheumatoid antibody haemagglutination assay (RAHA; Fujirebio Inc., Tokyo, Japan). Estimation of TPHA-lgG titer / mg total lgG . Separation of IgG from IgM antibodies to TP in serum and CSF was carried out by Ultrogel AcA 34 filtration (Mialler 1982). Briefly, 0.7 of samples were filtrated through an 1.5 x 14 cm gel column using PBS, pH 7.3. Fractions of 1.3 ml were collected and the absorbance measured at 280 nm. Twenty-five ~1 of the IgG peak fraction with TP-specific IgG antibodies was investigated quantitatively in the T P H A (Fujirebio Inc., Tokyo, Japan). The TPHA-IgG titer/mg total IgG was calculated from the TPHA-IgG titer divided by total IgG (in mg) of 25 pA specimen. Estimation of TP-IgM-ELISA titer/mg total lgM The TP-IgM-ELISA titer/mg total IgM was calculated from serum or CSF titers divided by total IgM (in mg) of 100 ~1 specimen.
Results
Healthy adults and treated syphilitic patients without CNS participation on the TP infection As shown in Table 1 the mean values and ranges of albumin, total IgG and total IgM, in serum and CSF, as well as the s e r u m / C S F ratios, of 12 patients with
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adequately treated syphilis (without clinical or biochemical evidence of CNS involvement) did not differ from the findings in 24 healthy adults. In the group of healthy adults, the levels for total IgM in CSF ranged from 0.027 to 0.186 mg/1 with a mean value of 0.102 mg/I. The mean s e r u m / C S F ratio of total IgM was found to be 12 427. The relationship between total IgM concentrations of CSF and serum is shown in Fig. 1.
Untreated syphilitic patients without CNS participation on the TP infection In 4 syphilitic patients without clinical and biochemical evidence of CNS involvement a high TP-specific IgM antibody titer was found in serum, indicating an TP infection in need of treatment. But the albumin, total IgG and total IgM values in CSF of these patients do not differ from those of healthy adults (Table 2A, group 1). S e r u m / C S F ratios of TP-specific IgM and IgG were identical with those of total IgM and IgG, respectively. The mean s e r u m / C S F ratio of the TP-IgM-ELISA titer was calculated to be 10 628, i.e., in the normal range. Furthermore, the TPHA-IgG and the TP-IgM-ELISA titers/mg total IgG or IgM in serum and CSF were nearly equal, i.e., the C S F / s e r u m ratios were approximately 1 (Table 2B, group 1). Patients with untreated neurosyphilis On the basis of the b l o o d - C S F barrier function the 19 patients with untreated neurosyphilis were divided into two subgroups. 0.250 o
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All patients with untreated neurosyphilis and intact blood-CSF barrier (normal albumin serum/CSF ratio) showed an increased total IgM in CSF. In 4 of these patients (No. 1-4) total IgG in CSF was within, in another 7 (No. 5-11) this value was out of the normal range (Table 2A, group 2). The serum/CSF ratios of TP-IgM-ELISA titers were reduced resulting in increased CSF/serum ratios of TP-specific IgM/mg total IgM. It can be seen from Table 2B that the TP-IgM-ELISA titer/mg total IgM in CSF was 4- to 62-fold higher than in the corresponding serum. Only in the patient No. 1 (group 2) with syphilitic meningitis the CSF/serum ratio of TP-specific IgG/mg total IgG was normal (1.0). In the 10 other patients of this subgroup this ratio was increased. All patients with dysfunction of the blood-CSF barrier (abnormal albumin serum/CSF ratio) had an increased total IgM in CSF which could be demonstrated by the CSF-IgM index (Fryd6n et al. 1978; Link et al. 1979). The total IgG in CSF was increased as shown by an elevated CSF-IgG index (Tibbling et al. 1977) (Table 2A, group 3). The TP-IgM-ELISA titers/mg total IgM were 3- to 75-fold higher than those of the corresponding serum. The TP-specific IgG titers/mg total IgG in CSF were in most of the patients higher than in serum (except the patients 12 and 13 of Table 2B, group 3). The relationship between CSF TP-IgM-ELISA titers/mg total IgM and those of serum are shown in Fig. 2. Patients with untreated syphilis but without CNS / u') ~"
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52 involvement are located within the dash lines, indicating that the TP-specific IgM levels/mg total IgM in CSF and serum are equal. Patients with untreated neurosyphilis are located above the dash lines, indicating that the TP-specific IgM titers/mg total lgM are higher in CSF than those in serum.
Discussion
Sensitive, specific immunoassays for the quantitation of total IgM (IgM ELISA) and TP-specific IgM (TP-IgM-ELISA) in CSF have made it possible to correlate the results of these assays and to evaluate local synthesis of TP-specific IgM antibodies in the CNS. Both assays have been shown to be suitable, not only for the estimation of increased, but also of normal levels with reproducible results. IgM ELISA values from 0.027 to 0.186 m g / l with a mean of 0.102 m g / l were observed in the group of 24 healthy adults. Appropriate mean values have been reported by Nerenberg and Prasad (1978) (0.170 mg/i), Mingioli et al. (1978) (0.0513 mg/1), Kobatake et al. (1980) (0.240 mg/l) and Sindic et al. (1982) (0.0975 mg/l). Our results correspond to these findings. The mean s e r u m / C S F ratio for total lgM in the patients of our group was 12427, and hence was in accord with the value of 11 443 reported by Sindic et al. (1982). Compared with the results in the group of healthy adults, the mean values for albumin, total IgG and total IgM in serum and CSF, as well as their s e r u m / C S F ratios, from 12 adequately treated and 4 untreated syphilitic patients, were in the normal range. Because of the occasional low titers of TP-specific IgM in serum and CSF, an assay with high sensitivity and specificity is necessary for the determination of these antibodies. The used TP-IgM-ELISA fulfills these conditions. The sensitivity of the assay in serum is |0-fold higher than that of the indirect immunofluorescence techniques (Mialler and Moskophidis 1984). The passive transfer of TP-specific IgM antibodies from serum to CSF, through the intact b l o o d - C S F barrier, depends on their concentration in serum. Four untreated syphilitic patients without clinical or biochemical symptoms of neurosyphilis and normal b l o o d - C S F barrier function had high titers ( ~ 1:6400) of TP-specific IgM antibodies in serum (group 1, Tables 2A and B). From this group, the normal passive transfer of TP-specific IgM antibodies could be estimated as 10628 to 1. This transfer rate cannot be distinguished from that for total IgM, i.e., the portion of TP-specific IgM a n t i b o d y / m g total IgM is the same in serum and CSF. The C S F / s e r u m ratio is therefore approximately 1. The same principle of normal s e r u m / C S F transfer was formerly shown for TP-specific IgG antibodies in treated syphilitic patients without CNS involvement (Mialler and Moskophidis 1983). On the other hand, the TP-specific IgM titers/mg total IgM was, in all untreated neurosyphilitic patients, between 3- and 75-fold higher in CSF than in serum. This can only be explained by the assumption that local production of TP-specific IgM antibodies takes place in CNS.
53 It should be emphasized that the correlation of the TP-specific IgM a n t i b o d y titer with the total IgM does not depend on the function of the b l o o d - C S F barrier, as can be seen from Fig. 2. Therefore, by estimating the C S F / s e r u m ratio of TP-specific lgM a n t i b o d y / m g total IgM, local p r o d u c t i o n of TP-specific IgM antibodies in C N S can be assumed, not only in neurosyphilitic patients with n o r m a l b l o o d - C S F barrier f u n c t i o n (Tables 2A a n d B, group 2) b u t . a l s o in those patients with d y s f u n c t i o n of the b l o o d - C S F barrier (Tables 2A and B, group 3). The patients No. 1, 12 a n d 14 (Table 2B) are of particular interest. They show only local synthesis of TP-specific IgM but not of IgG antibodies. This might be explained by the very early stage of the C N S involvement. Finally, it should be stated that local p r o d u c t i o n of TP-specific IgM antibodies may be d e m o n s t r a t e d by the C S F - I g M index ( F r y d 6 n et al. 1978; Link et al. 1979), if instead of the total IgM (in m g / l ) , the TP-specific IgM titers are inserted in the formula but it should be kept in m i n d that, using this C S F - I g M index, no correlation of TP-specific IgM with the total IgM is possible.
Acknowledgements We greatly acknowledge the excellent technical assistance of Miss D. O r t m a n n a n d Mrs. J. Dolberg.
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54 Smdic, C,J.M., C.L. Camblaso, A. Depr~, E.C. Laterre and P.L. Masson, The concentration of IgM in the cerebrospinal fluid of neurological patients, J. Neurol. Scl., 55 (1982) 339-350. Tibbling, G., H. Link and S. Ohman, Principles of albumin and IgG analysis in neurological disorders, Part 1 (Establishment of reference values), Scand. J. Clin. Lab. Invest., 37 (1977) 385-390. Vartdal, F., B. Vandvik, T.E. Mlchaelsen, K. Loe and E. Norrby, Neurosyphdis - - Intrathecal synthesis of oligoclonal antibodies to Treponema palhdum, Ann. Neurol., 11 (1982) 35-40. Yokota, M., Y. Kasahara, Y. Tsuji, M. Kobayashl and T. Tomizawa, The measurement of IgG and IgM anttbodles to Treponema palhdum by enzyme-hnked immunosorbent assay (ELISA) simultaneously. In: E. Levy (Ed.), Advances in Pathology, Vol. 1, Pergamon Press, Oxford, New York, 1982, pp. 125-128.