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Oligoclonal IgG bands in CSF: A diagnostic tool
16. Rolhberg, R. M., et al. 1973. Systemic immunity after local antigenic stimulation of the lymphoid tissue of the gastrointestinal tract. J. Immunol. 111:1906-1913. 17. Shen, L., and M. W. Fanger. 1981. Secretory IgA antibodies synergize with IgG in promoting ADCC by human polymorphonuclear cells, monocytes, and lymphocytes. Cell. Immunol. 59:75-81.
18. Tomasi, T. B., et al. 1965. Characteristics of an immune system common to certain external secretions. J. Exp. Med. 121:101-124. 19. Tseng, J. 1981. Transfer of lymphocytes of Peyer's patches between immunoglobulin allotype congenic mice: Repopulation of the lgA plasma cells in the gut lamina propria. J. Immunol. 127:2039-2043. 20. Yardley, J. H., el al. 1978. Local
(immunoglobulin A) immune response by the intestine to cholera toxin and its partial suppression with combined systemic and intraintestinal immunization. Infect. Immun. 19:589-597.
Oligoclonal IgG Bands in CSF: A Diagnostic Tool Pankaj D. Mehta, Ph.D. Bruce A. Patrick, M.S. New York State Institute f o r Basic Research in Developmental Disabilities 1050 Forest Hill Road Staten Island, New York 10314
The increase in 3,-globulin expressed as a percent of total protein in cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients was first demonstrated in 1942 by Kabat et al. (10), using free electrophoresis. Since then several additional reports (18, 28) have extended the original findings and demonstrated that a part of the immunoglobulin G (IgG) in MS-CSF is produced within the central nervous system (CNS). Lowenthal et al. (20), using agar gel electrophoresis, first observed that the increased IgG resolved in a number of subfractions with restricted heterogeneity. These findings suggested that electrophoretic analysis might be useful in the diagnosis of MS. When sera from healthy individuals are subjected to electrophoresis and scanned in a densitometer, a broad diffuse peak with no significant increase or decrease in -,/-globulin concentration (i.e., normal pattern) is seen. Three types of abnormal -,/-globulin profiles have been reported: a) monoclonal - - o n e narrow homogeneous peak, which is excessive output o f a single plasma cell clone and is commonly known as the M-component (e.g., in multiple myeloma and macroglobulinemia); b) polyclonal--a general increase in immunoglobulin
Copyright© 1982by G. K. Hall& Co.
level due to stimulation by many different clones (e.g., in rheumatoid arthritis and chronic active hepatitis); and c) oligoclonal--discrete populations of IgG bands with restricted heterogeneity (e.g., in CSF of MS and subacute sclerosing panencephalitis (SSPE). We herein report two assays, quantitation of IgG in CSF and demonstration o f oligoclonal bands in concentrated CSF, that are of considerable importance in the clinical diagnosis of MS and SSPE.
Quantitation of lgG in C S F CSF-IgG has been quantitated by electroimmunodiffusion, electroimmunoassay, and radial immunodiffusion. O f the three procedures, the most commonly used in the laboratory is radial immunodiffusion, since the method is simple and easy to perform. No special equipment is needed, and ready-made plates available from commercial sources (e.g., L-C Partigen Plates, Behring Diagnostics, Sommerville, N.J.) can measure low concentrations of IgG and albumin in CSF. The IgG in patients with nonneurologic disease is less than 10070 of total CSF protein, whereas in 85°70 of MS patients the range varies from 11 to 35070 of the total CSF protein. One of the simplest ways o f demonstrating intracerebral synthesis of IgG is to determine the IgG/albumin ratio in CSF. Tourtellotte (28), using the electrodiffusion method, showed that the IgG/ albumin ratio in MS-CSF was significantly increased compared to
that seen in the control group, suggesting that IgG is synthesized within the CNS in MS. Several investigators (5, 25) modified this technique and determined IgG/ albumin ratio in both CSF and serum. The IgG/albumin ratio in controls was 0.26 to 0.66, whereas the increased ratio o f 0.27 to 0.90 was found in 88°70 o f MS patients. A number o f investigators (6, 9, 15, 18) using a variety o f techniques confirmed the observation that most IgG is synthesized within the CNS in MS. Using this ratio, it is possible to distinguish patients with MS from those with other neurologic diseases; however, the test is not specific for MS, since increased IgG/albumin ratios have been observed in other chronic CNS infections such as SSPE, neurosyphilis, and meningitis. Electrophoresis of CSF Many methods are available for CSF electrophoresis. Cellulose acetate, agar, agarose, and polyacrylamide gel electrophoresis and isoelectric focusing procedures (7-9, 12, 16, 21, 22, 27, 33) have been used to demonstrate the presence of oligoclonal bands. Recently, Johnson et al. (8, 9) have described the agarose gel electrophoretic procedure, namely, P A N A G E L electrophoresis (Worthington Diagnostics, Freehold, N.J.) for detection o f oligoclonal IgG bands in CSF. The method is easy to perform and appears suitable for the clinical laboratory. In addition to agarose gel electro-
I01
phoresis, a number of investigators (7, 12, 16, 21, 22, 26) have recently used the isoelectric focusing (IEF) procedure to analyze the oligoclonal bands in CSF of MS patients. This technique represents a major advance in the high resolution of proteins, since it can detect microheterogeneity in proteins that appear to be homogeneous by other electrophoretic methods. Since the PANAGEL and IEF procedures are commonly used in a number of laboratories, important aspects of these methods and profiles of CSF and sera bands from MS and control patients are included here. P A N A G E L Electrophoresis CSF is usually concentrated 40-to60-fold by lyophilization, and then
I0/~1 of CSF (IgG content 15-20/~g) is subjected to the PANAGEL electrophoresis. The total time to complete the electrophoresis is 90 min. One of the major advantages of the procedure is that ready-made agarose gel plates and buffer are commercially available. The method is sensitive and shows 2 to 4 distinct oligoclonal bands in most patients with MS. PANAGEL electrophoresis is illustrated in Figure 1A. A sensitive procedure of immunofixation after PANAGEL (Figure 1B) has been employed by several investigators (8, 9, 17). The procedure is useful to detect and identify oligoclonal bands with their respective immunoglobulin classes by using specific heavy or light chain antisera.
(A) -
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Fig. 1. A) P A N A G E L electrophoresis o f serum and CSF o f a patient with rheumatoM arthritis (1, 2); a patient with multiple myeloma (3, 4); patients with MS (5, 6 and 9, 10); and a normal individual (7, 8). Ten I~l o f undiluted sera and concentrated CSF (lgG concentration 10 to 15 ~g) were applied. B) lmmunofixation after P A N A GEL o f CSF f r o m the above patients. The lgG bands were identified using specific goat anti-human IgG serum. The same amount o f CSF was applied, except myeloma CSF (4) was diluted two-foM before the application. The solid arrows denote the oligoclonal bands, whereas the broken arrows represent the points o f application.
102
Although a combination of PANAGEL and immunofixation can detect oligoclonal bands in the CSF of 65-80°70 of patients with MS, the test is not specific for thedisease. In addition to MS, similar profiles of oligoclonal bands have also been found in other neurologic diseases, such as SSPE, progressive rubella panencephalitis (31), neurosyphilis, mumps, and tuberculosis meningitis (11, 30). However, these disorders are not likely to cause diagnostic confusion, since they can usually be distinguished from MS by other clinical and laboratory data (4, 9). Isoelectric Focusing Immunofixation
and
IEF is an electrophoretic technique where proteins are separated and focused according to their isoelectric points (pI) in a pH gradient established by carrier ampholytes. Polyacrylamide and agarose are found to be suitable media for the stabilization of the pH gradient. In our laboratory, the thin-layer IEF in polyacrylamide gels is prepared according to the modified method of Nilsson and Olsson (24), as previously described by Mehta et al. (23). The modifications include an appropriate combination of alkaline pH ampholytes to enhance separation in the alkaline pH region and the use of N,N'-diallytartardiamide (DATD) instead of N,N'-methylenebis (acrylamide) to increase the mechanical stability of the gel as well as its transparency. CSF samples are dialyzed against distilled water and concentrated 30-to-60-fold before IEF. Ten to twenty microliters of concentrated sample containing 5-10/zg of IgG are applied to IEF gels, whereas for immunofixation samples are further diluted 2-to-4-fold. All samples are applied in duplicate on the gel. After focusing is completed, the glass plate is cut, and one-half of the plate is fixed in a trichloroacetic acid solution and stained. These staining patterns are referred to as regular IEF. The other half of the plate, containing a duplicate set of
ClinicalImmunologyNewsletter
samples, is immunofixed with specifc anti-human heavy or light chain antisera, as described by Arnaud et al. (1). The plate is thoroughly washed and stained. This method is referred to as immunofixation. Typical profiles of IEF and immunofixation are shown in Figure 2. Immunofixation after IEF (Figure 2B) was also carried out in order to identify IgG bands.
Figure 3 shows IEF and immunofixation profiles of three MS patients. The number of oligoclonal bands and their intensities and isoelectric points differ from one sample to another. A complete correlation of oligoclonal IgG bands in IEF and immunofixation after IEF is seen. Although the protein concentrations of the samples used for immunofixation were two-fold less A
e
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2
,. t
7 9P
8 O,IIIP~
pH
I 9.3
I 8.5
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Fig. 2. A) IEF profiles o f serum and CSF from patient with multiple myeloma (1, 2); patients with MS (3, 4); normal individual's serum and isolated serum lgG (5, 6); and CSF from patient with non-neurologic disease (7); serum and CSF f r o m patient with rheumatoid arthritis (8, 9). Ten l~l o f 1:10 serum dilution and concentrated CSF (IgG concentration 6-10 t~g) were applied to the gel B) hnmunofixation after IEF o f CSF. Here, 10 I~l o f samples containing 3-5 Fg o f lgG were applied. The lgG bands were identified by using specific goat anti-huntan lgG serum.
Cop:Tight © 1982 by G. K. Hall & Co.
than those used for regular IEF, the intensity of a number of oligoclonal IgG bands is significantly increased after immunofixation. The presence of oligoclonal bands in CSF is not directly related to increased IgG concentration since bands have been detected in a number of MS patients with normal CSF-IgG levels. One of the largest series of CSF specimens analyzed (3) by IEF demonstrated that 91°70 of 262 MS patients had oligoclonal IgG bands whereas only 7°70 of 272 patients with other neurologic diseases showed oligoclonal band profiles. Recently, a number of investigators (7, 21, 23) analyzed CSF from patients with MS or other neurologic diseases by PANAGEL and IEF. The microheterogeneity of oligoclonal IgG bands observed in IEF was not seen in PANAGEL electrophoresis. Since our recent data (Mehta et al., unpublished data) on 70 coded CSFs showed that IEF detects oligoclonal bands in the CSF of 20°70 more MS patients than PANAGEL, this method is superior to PANAGEL for the diagnosis of MS. Although IEF is more sensitive than agar gel electrophoresis for the detection of oligoclonal bands, a number of investigators have recommended PANAGEL because of its simple operation, low cost, and because it is less time-consuming. Hbwever, PANAGEL profiles of CSF are often difficult to interpret because the bands are not as well resolved as in IEF. Since premade thin-layer IEF in polyacrylamide plates is available from a number of commercial sources, IEF may be useful in patients who are suspected of having MS or primary optic neuritis where PANAGEL is unable to show the presence of oligoclonal bands. Recently, a number of investigators (13, 14) have developed a sensitive agarose IEF method for the detection of oligoclonal bands in unconcentrated MS-CSF. This method does not appear to be suitable for clinical laboratories, however, since it involves preparation of IEF gel, radioactively labeled antibody, and
103
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/
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I 9.3
, I 8.5
I 7.8
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Fig. 3. A) IEF profiles o f CSF from three MS patients. Ten t~l o f concentrated CSF containing 6-10 t~g of lgG were applied. Note that each CSF oligoclonal profile differs in number of bands, intensity, and isoelectric point. B) hnmunofixation after IEF by using goat anti-human IgG serum. Ten t~l of CSF containing 3-4 t~g of lgG were applied. The intensity of a number of faint oligoclonal bands seen in regular IEF (A) is significantly increased after immunofixation.
autoradiography. Studies using sensitive silver staining technique may enable us to detect oligoclonal bands in unconcentrated MS-CSF. Although the band patterns in the CSF of individual MS patients remain remarkably stable during the course of the disease, recent IEF data (21) have shown additional bands in CSF with increased duration of disease. A number of investigators (17, 18) have reported abnormal x- to~-type L-chain ratios in the CSF of more than 50% of MS patients. Several studies (16, 23) employing immunofixation after IEF have shown that the majority of individual oligoclonal bands in MSCSF possess one type of L-chain. The specificity of oligoclonal bands in MS-CSF is not known, although local production of antibodies to a number of viruses in CNS has been
104
demonstrated (34). The combined viral and brain-specific IgG appear to be less than 5% of total oligoclonal IgG. It is possible that the bands are not directed against any specific set of antigens but represent general activation of B-cell clones during attacks of MS. Subacute Sclerosing
Panencephalitis SSPE is a rare, slowly progressive disease of the CNS of children and young adults, caused by measles virus. The patients have unusually high titers of antibody to measles virus (2, 19, 32). Immunologic abnormalities include increased concentration of IgG in CSF and sera, and high IgG/albumin ratio in CSF, demonstrating the synthesis of IgG within the CNS (29). PANAGEL electrophoretic analysis has shown
the presence of 2-6 oligoclonal IgG bands in CSF and occasionally in serum (19, 22, 32). In our experience, CSF and serum from 20 SSPE patients studied by IEF showed 10-15 distinct oligoclonal band profiles (Figure 4) with respect to number, pI, and intensity. These results suggest that the same plasma cell clones are responsible for the synthesis of oligoclonal IgG in the CNS and serum of patients with SSPE. In contrast, MS patients showed oligoclonal IgG bands in serum that differed with respect to number, pI, intensity, and light chain type from those in corresponding CSF. The band patterns change during the clinical stage of the disease, and additional bands are seen during the course of the disease. The band profiles of MS and SSPE appear very similar in PANAGEL and IEF. However, recent studies (21) with the use of a scanner show that in IEF the IgG from CSF of SSPE patients have sharper peaks than those of MS patients. In SSPE, a number of oligoclonal bands are formed against measles virus, and most bands in CSF can be absorbed by measles virus (22, 32), These data strongly support the concept that measles virus is responsible for the formation of oligoclonal bands within the CNS in SSPE. Summary The increase in the IgG/albumin ratio and the presence of oligoclonal bands in CSF from MS patients now serve as important tools in the diagnosis of MS. Although a small percentage of patients with other neurologic disorders also demonstrate oligoclonal bands, electrophoresis of CSF is the single most useful test for making a clinical diagnosis of MS. The oligoclonal band profiles in CSF of MS and SSPE appear to be identical; however, SSPE can easily be differentiated from MS by very high measles antibody titers in CSF and serum. Although IEF shows better identification of a number of distinct oligoclonal bands than can
ClinicalImmunologyNewsletter
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Fig. 4. A) IEF profiles o f matched serum (S) and CSF (C) from two SSPE patients. In patient 1, ten 1*!o f serum and CSF containing 6-10 ~g of lgG, and in patient 2, serum containing 6tag of lgG and CSF with 3 pg o f lgG were applied to the gel B) lmmunofvcation after 1EF o f the same specimens using goat anti-human IgG serum, lgG concentration in serum was 4 tag whereas in CSF it was 2 ~g. Although the oligoclonal lgG bands in CSF from patient 2 in alkaline p H region o f 8.5-9.3 appeared to be absent in regular IEF (A), the bands are visible in immunoffL~ation profile.
be seen in a g a r o s e gel electrophoresis, commerical PANAGEL e l e c t r o p h o r e s i s a p p e a r s to be a convenient system for the a v e r a g e h o s p i t a l ' s clinical l a b o r a t o r y to use to d e m o n s t r a t e o l i g o c l o n a l b a n d s in CSF.
2.
3. ACKNOWLEDGMENT:This work was supported in part by grant #RG-1189 from the National Multiple Sclerosis Society.
References 1. Arnaud, P., et al. 1977. Immunofixation after electrofocusing: Improved method for specific detection of serum proteins with determination of isoelectric points. I. Im-
Copyright © 1982 by G. K. Hall & Co.
4.
5.
munofixation print technique for detection of alpha-l-protease inhibitor. J. Immunol. Methods 16:221-231. Connolly, J., et al. 1967. Measles antibody and antigen in subacute sclerosing panencephalitis. Lancet 1:542-544. Delmotte, P., and R. Gonsette. 1977. Biochemical findings in multiple sclerosis IV. Isoelectric focusing of the CSF gamma globulins in multiple sclerosis (262 cases) and other neurological diseases (272 cases). J. Neurol. 215:27-37. Ebers, G. C., and D. W. Paty. 1980. CSF electrophoresis in one thousand patients. Can. J. Neurol. Sci. 7:275-280. Ganrot, K., and C. B. Laurell. 1974. Measurement of IgG and
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15.
16.
17.
albumin content o f cerebrospinal fluid, and its interpretation. Clin. Chem. 20:571-573. Hershey, L. A., and J. L. Trotter. 1980. The use and abuse of the cerebrospinal fluid IgG profile in the adult: A practical evaluation. Ann. Neurol. 8:426-434. Hosien, Z. Z., and K. P. Johnson. 1981. Isoelectric focusing o f cerebrospinal fluid proteins in the diagnosis of multiple sclerosis. Neurology 31:70-76. Johnson, K. P., et al. 1977. Agarose electrophoresis of cerebrospinal fluid in multiple sclerosis: A simplified method for demonstrating cerebrospinal fluid oligoclonal immunoglobulin bands. Neurology 27:273-277. Johnson, K. P., and B. J. Nelson. 1977. Multiple Sclerosis: Diagnostic usefulness of cerebrospinal fluid. Ann. Neurol. 2:425-431. Kabat, E. A., D. H. MoorE, and H. Landow. 1942. An electrophoresis study of the protein components in cerebrospinal fluid and their relationships to the serum proteins. J. Clin. Invest. 21:571-577. Kinnman, J., H. Link, and A. Fu,'den. 1981. Characterization of antibody activity in oligoclonal immunoglobulin G synthesized within the central nervous system in a patient with tuberculous meningitis. J. Clin. Microbiol. 13:30-35. Kjellin, K. G., and A. Siden. 1977. Aberrant CSF protein fractions found by electrofocusing in multiple sclerosis. Eur. Neurol. 15:40-50. Kostulas, V. K., and H. Link. 1982. Agarose isoelectric focusing of unconcentrated CSF and radioimmunofixation for detection of oligoclonal bands in patients with multiple sclerosis and other neurological diseases. J. Neurol. Sci. 54:117-127. Lasne, Y., et al. 1981. A sensitive method for characterization of oligoclonal immunoglobulins in unconcentrated cerebrospinal fluid. J. Neurochem. 36:1872-1874. Laterre, E. C., et al. 1970. Electrophoretic morphology of gamma globulins in cerebrospinal fluid of multiple sclerosis and diseases of the nervous system. Neurology 20:982-990. Laurenzi, M. A., et al. 1980. Oligoclonal IgG and free light chains in multiple sclerosis demonstrated by thin-layer polyacrylamide gel isoelectric focusing and immunofixation. Ann. Neurol. 8:241-247. Link, H., and M. A. Laurenzi. 1979. Immunoglobulin class and light chain type of oligoclonal bands
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in CSF in multiple sclerosis determined by agarose gel electrophoresis and immunofixation. Ann. Neurol.
6:107-110. 18. Link, H., and R. Muller. 1971. Immunoglobulins in multiple sclerosis • and infections o f the nervous system. Arch. Neurol. 25:326-344.
24.
19. Link, H., M. Panelius, and A. A. Saimi. 1973. Immunoglobulins and measles antibodies in subacute sclerosing panencephalitis. Arch. Neurol. 28:23-30. 20. Lowenthal, A., M. Van Sande, and D. Karcher. 1960. The differential diagnosis o f neurological diseases by fractionating electrophoretically the CSF globulins. J. Neurochem. 6:51-56. 21. Mattson, D. H., et al. 1981. Comparison of agar gel electrophoresis and isoelectric focusing in multiple sclerosis and subacute sclerosing panencephalitis. Ann. Neurol. 9:34-41.
22. Mehta, P. D., A. Kane, and
25.
26.
27.
28.
H. Thormar. 1977. Quantitation of measles virus specific immunoglobulins in serum, CSF and brain extract from patients with subacute sclerosing panencephalitis. J. Immunol. 118:2254-2261.
23. Mehta, P. D., B. A. Patrick, and H. M. Wisniewski. 198I. Isoelectric
29.
focusing and immunofixation of cerebrospinal fluid and serum in multiple sclerosis. J. Clin. Lab. Immunol. 6:17-22. Nilsson, K., and J. E. Olsson. 1978. Analysis for cerebrospinal fluid proteins by isoelectric focusing in polyacrylamide gel: Methodological aspects and normal values with special reference to the alkaline region. Clin. Chem. 24:1134-1139. Olsson, J. E., and B. Pettersson. 1976. A comparison between agar gel electrophoresis and CSF serum quotients of IgG and albumin in neurological diseases. Acta. Neurol. Scand. 53:308-322. Siden, A., and K. G. Kjellin. 1978. CSF protein examinations with thin layer isoelectric focusing in multiple sclerosis. J. Neurol. Sci. 39:131-146. Thompson, E. J. 1977. Laboratory diagnosis of multiple sclerosis: Immunological and biochemical aspects. Br. Med. Bull. 33:28-33. Tourlellotte, W. W. 1975. What is multiple sclerosis? Laboratory criteria for diagnosis, pp. 9-26. In A. N. Davison, et al. (eds.), Multiple Sclerosis Research. Elsevier Scientific Publishing Co., Amsterdam. Tourtellolte, W. W., et al. 1968. Subacute sclerosing panencephalitis:
30.
31.
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33.
34.
Brain immunoglobulin-G, measles antibody and albumin. Neurology 18:117-121. Vandvik, B., el al. 1978. Mumps meningitis: Prolonged pleocytosis and occurrence of mumps virusspecific oligoclonal IgG in the cerebrospinal fluid. Eur. Neurol. 17:13-22. Vandvik, B., el al. 1978. Progressive rubella virus panencephalitis: Synthesis of oligoclonal virus-specific IgG antibodies and homogeneous free light chains in the central nervous system. Acta Neurol. Scand. 57:53-64. Vandvik, B., and E. Norrby. 1973. Otigoclonal lgG antibody response in the central nervous system to different measles virus antigens in subacute sclerosing panencephalitis. Proc. Natl. Acad. Sci. USA 70:1060-1063. Vandvik, B., and S. Skrede. 1973. EIectrophoretic examination of the cerebrospinal fluid proteins in multiple sclerosis and other neurological diseases. Eur. Neurol. 9:224-241. Varldai, F., B. Vandvik, and E. Norrby. 1980. Viral and bacterial antibody response in multiple sclerosis. Ann. Neurol. 8:248-255.
Workshops and Meetings Research and Therapeutic Issues in Diabetes: An International Conference. C h a r l o t t e s v i l l e , Virginia. October 12-15, 1982. Contact: M r s . Shirley N e w m a n , International Diabetes Conference,
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18. Tomasi, T. B., et al. 1965. Characteristics of an immune system common to certain external secretions. J. Exp. Med. 121:101-124. 19. Tseng, J. 1981. Transfer of lymphocytes of Peyer's patches between immunoglobulin allotype congenic mice: Repopulation of the lgA plasma cells in the gut lamina propria. J. Immunol. 127:2039-2043. 20. Yardley, J. H., el al. 1978. Local
(immunoglobulin A) immune response by the intestine to cholera toxin and its partial suppression with combined systemic and intraintestinal immunization. Infect. Immun. 19:589-597.
Oligoclonal IgG Bands in CSF: A Diagnostic Tool Pankaj D. Mehta, Ph.D. Bruce A. Patrick, M.S. New York State Institute f o r Basic Research in Developmental Disabilities 1050 Forest Hill Road Staten Island, New York 10314
The increase in 3,-globulin expressed as a percent of total protein in cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients was first demonstrated in 1942 by Kabat et al. (10), using free electrophoresis. Since then several additional reports (18, 28) have extended the original findings and demonstrated that a part of the immunoglobulin G (IgG) in MS-CSF is produced within the central nervous system (CNS). Lowenthal et al. (20), using agar gel electrophoresis, first observed that the increased IgG resolved in a number of subfractions with restricted heterogeneity. These findings suggested that electrophoretic analysis might be useful in the diagnosis of MS. When sera from healthy individuals are subjected to electrophoresis and scanned in a densitometer, a broad diffuse peak with no significant increase or decrease in -,/-globulin concentration (i.e., normal pattern) is seen. Three types of abnormal -,/-globulin profiles have been reported: a) monoclonal - - o n e narrow homogeneous peak, which is excessive output o f a single plasma cell clone and is commonly known as the M-component (e.g., in multiple myeloma and macroglobulinemia); b) polyclonal--a general increase in immunoglobulin
Copyright© 1982by G. K. Hall& Co.
level due to stimulation by many different clones (e.g., in rheumatoid arthritis and chronic active hepatitis); and c) oligoclonal--discrete populations of IgG bands with restricted heterogeneity (e.g., in CSF of MS and subacute sclerosing panencephalitis (SSPE). We herein report two assays, quantitation of IgG in CSF and demonstration o f oligoclonal bands in concentrated CSF, that are of considerable importance in the clinical diagnosis of MS and SSPE.
Quantitation of lgG in C S F CSF-IgG has been quantitated by electroimmunodiffusion, electroimmunoassay, and radial immunodiffusion. O f the three procedures, the most commonly used in the laboratory is radial immunodiffusion, since the method is simple and easy to perform. No special equipment is needed, and ready-made plates available from commercial sources (e.g., L-C Partigen Plates, Behring Diagnostics, Sommerville, N.J.) can measure low concentrations of IgG and albumin in CSF. The IgG in patients with nonneurologic disease is less than 10070 of total CSF protein, whereas in 85°70 of MS patients the range varies from 11 to 35070 of the total CSF protein. One of the simplest ways o f demonstrating intracerebral synthesis of IgG is to determine the IgG/albumin ratio in CSF. Tourtellotte (28), using the electrodiffusion method, showed that the IgG/ albumin ratio in MS-CSF was significantly increased compared to
that seen in the control group, suggesting that IgG is synthesized within the CNS in MS. Several investigators (5, 25) modified this technique and determined IgG/ albumin ratio in both CSF and serum. The IgG/albumin ratio in controls was 0.26 to 0.66, whereas the increased ratio o f 0.27 to 0.90 was found in 88°70 o f MS patients. A number o f investigators (6, 9, 15, 18) using a variety o f techniques confirmed the observation that most IgG is synthesized within the CNS in MS. Using this ratio, it is possible to distinguish patients with MS from those with other neurologic diseases; however, the test is not specific for MS, since increased IgG/albumin ratios have been observed in other chronic CNS infections such as SSPE, neurosyphilis, and meningitis. Electrophoresis of CSF Many methods are available for CSF electrophoresis. Cellulose acetate, agar, agarose, and polyacrylamide gel electrophoresis and isoelectric focusing procedures (7-9, 12, 16, 21, 22, 27, 33) have been used to demonstrate the presence of oligoclonal bands. Recently, Johnson et al. (8, 9) have described the agarose gel electrophoretic procedure, namely, P A N A G E L electrophoresis (Worthington Diagnostics, Freehold, N.J.) for detection o f oligoclonal IgG bands in CSF. The method is easy to perform and appears suitable for the clinical laboratory. In addition to agarose gel electro-
I01
phoresis, a number of investigators (7, 12, 16, 21, 22, 26) have recently used the isoelectric focusing (IEF) procedure to analyze the oligoclonal bands in CSF of MS patients. This technique represents a major advance in the high resolution of proteins, since it can detect microheterogeneity in proteins that appear to be homogeneous by other electrophoretic methods. Since the PANAGEL and IEF procedures are commonly used in a number of laboratories, important aspects of these methods and profiles of CSF and sera bands from MS and control patients are included here. P A N A G E L Electrophoresis CSF is usually concentrated 40-to60-fold by lyophilization, and then
I0/~1 of CSF (IgG content 15-20/~g) is subjected to the PANAGEL electrophoresis. The total time to complete the electrophoresis is 90 min. One of the major advantages of the procedure is that ready-made agarose gel plates and buffer are commercially available. The method is sensitive and shows 2 to 4 distinct oligoclonal bands in most patients with MS. PANAGEL electrophoresis is illustrated in Figure 1A. A sensitive procedure of immunofixation after PANAGEL (Figure 1B) has been employed by several investigators (8, 9, 17). The procedure is useful to detect and identify oligoclonal bands with their respective immunoglobulin classes by using specific heavy or light chain antisera.
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Fig. 1. A) P A N A G E L electrophoresis o f serum and CSF o f a patient with rheumatoM arthritis (1, 2); a patient with multiple myeloma (3, 4); patients with MS (5, 6 and 9, 10); and a normal individual (7, 8). Ten I~l o f undiluted sera and concentrated CSF (lgG concentration 10 to 15 ~g) were applied. B) lmmunofixation after P A N A GEL o f CSF f r o m the above patients. The lgG bands were identified using specific goat anti-human IgG serum. The same amount o f CSF was applied, except myeloma CSF (4) was diluted two-foM before the application. The solid arrows denote the oligoclonal bands, whereas the broken arrows represent the points o f application.
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Although a combination of PANAGEL and immunofixation can detect oligoclonal bands in the CSF of 65-80°70 of patients with MS, the test is not specific for thedisease. In addition to MS, similar profiles of oligoclonal bands have also been found in other neurologic diseases, such as SSPE, progressive rubella panencephalitis (31), neurosyphilis, mumps, and tuberculosis meningitis (11, 30). However, these disorders are not likely to cause diagnostic confusion, since they can usually be distinguished from MS by other clinical and laboratory data (4, 9). Isoelectric Focusing Immunofixation
and
IEF is an electrophoretic technique where proteins are separated and focused according to their isoelectric points (pI) in a pH gradient established by carrier ampholytes. Polyacrylamide and agarose are found to be suitable media for the stabilization of the pH gradient. In our laboratory, the thin-layer IEF in polyacrylamide gels is prepared according to the modified method of Nilsson and Olsson (24), as previously described by Mehta et al. (23). The modifications include an appropriate combination of alkaline pH ampholytes to enhance separation in the alkaline pH region and the use of N,N'-diallytartardiamide (DATD) instead of N,N'-methylenebis (acrylamide) to increase the mechanical stability of the gel as well as its transparency. CSF samples are dialyzed against distilled water and concentrated 30-to-60-fold before IEF. Ten to twenty microliters of concentrated sample containing 5-10/zg of IgG are applied to IEF gels, whereas for immunofixation samples are further diluted 2-to-4-fold. All samples are applied in duplicate on the gel. After focusing is completed, the glass plate is cut, and one-half of the plate is fixed in a trichloroacetic acid solution and stained. These staining patterns are referred to as regular IEF. The other half of the plate, containing a duplicate set of
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samples, is immunofixed with specifc anti-human heavy or light chain antisera, as described by Arnaud et al. (1). The plate is thoroughly washed and stained. This method is referred to as immunofixation. Typical profiles of IEF and immunofixation are shown in Figure 2. Immunofixation after IEF (Figure 2B) was also carried out in order to identify IgG bands.
Figure 3 shows IEF and immunofixation profiles of three MS patients. The number of oligoclonal bands and their intensities and isoelectric points differ from one sample to another. A complete correlation of oligoclonal IgG bands in IEF and immunofixation after IEF is seen. Although the protein concentrations of the samples used for immunofixation were two-fold less A
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Fig. 2. A) IEF profiles o f serum and CSF from patient with multiple myeloma (1, 2); patients with MS (3, 4); normal individual's serum and isolated serum lgG (5, 6); and CSF from patient with non-neurologic disease (7); serum and CSF f r o m patient with rheumatoid arthritis (8, 9). Ten l~l o f 1:10 serum dilution and concentrated CSF (IgG concentration 6-10 t~g) were applied to the gel B) hnmunofixation after IEF o f CSF. Here, 10 I~l o f samples containing 3-5 Fg o f lgG were applied. The lgG bands were identified by using specific goat anti-huntan lgG serum.
Cop:Tight © 1982 by G. K. Hall & Co.
than those used for regular IEF, the intensity of a number of oligoclonal IgG bands is significantly increased after immunofixation. The presence of oligoclonal bands in CSF is not directly related to increased IgG concentration since bands have been detected in a number of MS patients with normal CSF-IgG levels. One of the largest series of CSF specimens analyzed (3) by IEF demonstrated that 91°70 of 262 MS patients had oligoclonal IgG bands whereas only 7°70 of 272 patients with other neurologic diseases showed oligoclonal band profiles. Recently, a number of investigators (7, 21, 23) analyzed CSF from patients with MS or other neurologic diseases by PANAGEL and IEF. The microheterogeneity of oligoclonal IgG bands observed in IEF was not seen in PANAGEL electrophoresis. Since our recent data (Mehta et al., unpublished data) on 70 coded CSFs showed that IEF detects oligoclonal bands in the CSF of 20°70 more MS patients than PANAGEL, this method is superior to PANAGEL for the diagnosis of MS. Although IEF is more sensitive than agar gel electrophoresis for the detection of oligoclonal bands, a number of investigators have recommended PANAGEL because of its simple operation, low cost, and because it is less time-consuming. Hbwever, PANAGEL profiles of CSF are often difficult to interpret because the bands are not as well resolved as in IEF. Since premade thin-layer IEF in polyacrylamide plates is available from a number of commercial sources, IEF may be useful in patients who are suspected of having MS or primary optic neuritis where PANAGEL is unable to show the presence of oligoclonal bands. Recently, a number of investigators (13, 14) have developed a sensitive agarose IEF method for the detection of oligoclonal bands in unconcentrated MS-CSF. This method does not appear to be suitable for clinical laboratories, however, since it involves preparation of IEF gel, radioactively labeled antibody, and
103
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Fig. 3. A) IEF profiles o f CSF from three MS patients. Ten t~l o f concentrated CSF containing 6-10 t~g of lgG were applied. Note that each CSF oligoclonal profile differs in number of bands, intensity, and isoelectric point. B) hnmunofixation after IEF by using goat anti-human IgG serum. Ten t~l of CSF containing 3-4 t~g of lgG were applied. The intensity of a number of faint oligoclonal bands seen in regular IEF (A) is significantly increased after immunofixation.
autoradiography. Studies using sensitive silver staining technique may enable us to detect oligoclonal bands in unconcentrated MS-CSF. Although the band patterns in the CSF of individual MS patients remain remarkably stable during the course of the disease, recent IEF data (21) have shown additional bands in CSF with increased duration of disease. A number of investigators (17, 18) have reported abnormal x- to~-type L-chain ratios in the CSF of more than 50% of MS patients. Several studies (16, 23) employing immunofixation after IEF have shown that the majority of individual oligoclonal bands in MSCSF possess one type of L-chain. The specificity of oligoclonal bands in MS-CSF is not known, although local production of antibodies to a number of viruses in CNS has been
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demonstrated (34). The combined viral and brain-specific IgG appear to be less than 5% of total oligoclonal IgG. It is possible that the bands are not directed against any specific set of antigens but represent general activation of B-cell clones during attacks of MS. Subacute Sclerosing
Panencephalitis SSPE is a rare, slowly progressive disease of the CNS of children and young adults, caused by measles virus. The patients have unusually high titers of antibody to measles virus (2, 19, 32). Immunologic abnormalities include increased concentration of IgG in CSF and sera, and high IgG/albumin ratio in CSF, demonstrating the synthesis of IgG within the CNS (29). PANAGEL electrophoretic analysis has shown
the presence of 2-6 oligoclonal IgG bands in CSF and occasionally in serum (19, 22, 32). In our experience, CSF and serum from 20 SSPE patients studied by IEF showed 10-15 distinct oligoclonal band profiles (Figure 4) with respect to number, pI, and intensity. These results suggest that the same plasma cell clones are responsible for the synthesis of oligoclonal IgG in the CNS and serum of patients with SSPE. In contrast, MS patients showed oligoclonal IgG bands in serum that differed with respect to number, pI, intensity, and light chain type from those in corresponding CSF. The band patterns change during the clinical stage of the disease, and additional bands are seen during the course of the disease. The band profiles of MS and SSPE appear very similar in PANAGEL and IEF. However, recent studies (21) with the use of a scanner show that in IEF the IgG from CSF of SSPE patients have sharper peaks than those of MS patients. In SSPE, a number of oligoclonal bands are formed against measles virus, and most bands in CSF can be absorbed by measles virus (22, 32), These data strongly support the concept that measles virus is responsible for the formation of oligoclonal bands within the CNS in SSPE. Summary The increase in the IgG/albumin ratio and the presence of oligoclonal bands in CSF from MS patients now serve as important tools in the diagnosis of MS. Although a small percentage of patients with other neurologic disorders also demonstrate oligoclonal bands, electrophoresis of CSF is the single most useful test for making a clinical diagnosis of MS. The oligoclonal band profiles in CSF of MS and SSPE appear to be identical; however, SSPE can easily be differentiated from MS by very high measles antibody titers in CSF and serum. Although IEF shows better identification of a number of distinct oligoclonal bands than can
ClinicalImmunologyNewsletter
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Fig. 4. A) IEF profiles o f matched serum (S) and CSF (C) from two SSPE patients. In patient 1, ten 1*!o f serum and CSF containing 6-10 ~g of lgG, and in patient 2, serum containing 6tag of lgG and CSF with 3 pg o f lgG were applied to the gel B) lmmunofvcation after 1EF o f the same specimens using goat anti-human IgG serum, lgG concentration in serum was 4 tag whereas in CSF it was 2 ~g. Although the oligoclonal lgG bands in CSF from patient 2 in alkaline p H region o f 8.5-9.3 appeared to be absent in regular IEF (A), the bands are visible in immunoffL~ation profile.
be seen in a g a r o s e gel electrophoresis, commerical PANAGEL e l e c t r o p h o r e s i s a p p e a r s to be a convenient system for the a v e r a g e h o s p i t a l ' s clinical l a b o r a t o r y to use to d e m o n s t r a t e o l i g o c l o n a l b a n d s in CSF.
2.
3. ACKNOWLEDGMENT:This work was supported in part by grant #RG-1189 from the National Multiple Sclerosis Society.
References 1. Arnaud, P., et al. 1977. Immunofixation after electrofocusing: Improved method for specific detection of serum proteins with determination of isoelectric points. I. Im-
Copyright © 1982 by G. K. Hall & Co.
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munofixation print technique for detection of alpha-l-protease inhibitor. J. Immunol. Methods 16:221-231. Connolly, J., et al. 1967. Measles antibody and antigen in subacute sclerosing panencephalitis. Lancet 1:542-544. Delmotte, P., and R. Gonsette. 1977. Biochemical findings in multiple sclerosis IV. Isoelectric focusing of the CSF gamma globulins in multiple sclerosis (262 cases) and other neurological diseases (272 cases). J. Neurol. 215:27-37. Ebers, G. C., and D. W. Paty. 1980. CSF electrophoresis in one thousand patients. Can. J. Neurol. Sci. 7:275-280. Ganrot, K., and C. B. Laurell. 1974. Measurement of IgG and
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albumin content o f cerebrospinal fluid, and its interpretation. Clin. Chem. 20:571-573. Hershey, L. A., and J. L. Trotter. 1980. The use and abuse of the cerebrospinal fluid IgG profile in the adult: A practical evaluation. Ann. Neurol. 8:426-434. Hosien, Z. Z., and K. P. Johnson. 1981. Isoelectric focusing o f cerebrospinal fluid proteins in the diagnosis of multiple sclerosis. Neurology 31:70-76. Johnson, K. P., et al. 1977. Agarose electrophoresis of cerebrospinal fluid in multiple sclerosis: A simplified method for demonstrating cerebrospinal fluid oligoclonal immunoglobulin bands. Neurology 27:273-277. Johnson, K. P., and B. J. Nelson. 1977. Multiple Sclerosis: Diagnostic usefulness of cerebrospinal fluid. Ann. Neurol. 2:425-431. Kabat, E. A., D. H. MoorE, and H. Landow. 1942. An electrophoresis study of the protein components in cerebrospinal fluid and their relationships to the serum proteins. J. Clin. Invest. 21:571-577. Kinnman, J., H. Link, and A. Fu,'den. 1981. Characterization of antibody activity in oligoclonal immunoglobulin G synthesized within the central nervous system in a patient with tuberculous meningitis. J. Clin. Microbiol. 13:30-35. Kjellin, K. G., and A. Siden. 1977. Aberrant CSF protein fractions found by electrofocusing in multiple sclerosis. Eur. Neurol. 15:40-50. Kostulas, V. K., and H. Link. 1982. Agarose isoelectric focusing of unconcentrated CSF and radioimmunofixation for detection of oligoclonal bands in patients with multiple sclerosis and other neurological diseases. J. Neurol. Sci. 54:117-127. Lasne, Y., et al. 1981. A sensitive method for characterization of oligoclonal immunoglobulins in unconcentrated cerebrospinal fluid. J. Neurochem. 36:1872-1874. Laterre, E. C., et al. 1970. Electrophoretic morphology of gamma globulins in cerebrospinal fluid of multiple sclerosis and diseases of the nervous system. Neurology 20:982-990. Laurenzi, M. A., et al. 1980. Oligoclonal IgG and free light chains in multiple sclerosis demonstrated by thin-layer polyacrylamide gel isoelectric focusing and immunofixation. Ann. Neurol. 8:241-247. Link, H., and M. A. Laurenzi. 1979. Immunoglobulin class and light chain type of oligoclonal bands
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in CSF in multiple sclerosis determined by agarose gel electrophoresis and immunofixation. Ann. Neurol.
6:107-110. 18. Link, H., and R. Muller. 1971. Immunoglobulins in multiple sclerosis • and infections o f the nervous system. Arch. Neurol. 25:326-344.
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19. Link, H., M. Panelius, and A. A. Saimi. 1973. Immunoglobulins and measles antibodies in subacute sclerosing panencephalitis. Arch. Neurol. 28:23-30. 20. Lowenthal, A., M. Van Sande, and D. Karcher. 1960. The differential diagnosis o f neurological diseases by fractionating electrophoretically the CSF globulins. J. Neurochem. 6:51-56. 21. Mattson, D. H., et al. 1981. Comparison of agar gel electrophoresis and isoelectric focusing in multiple sclerosis and subacute sclerosing panencephalitis. Ann. Neurol. 9:34-41.
22. Mehta, P. D., A. Kane, and
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H. Thormar. 1977. Quantitation of measles virus specific immunoglobulins in serum, CSF and brain extract from patients with subacute sclerosing panencephalitis. J. Immunol. 118:2254-2261.
23. Mehta, P. D., B. A. Patrick, and H. M. Wisniewski. 198I. Isoelectric
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focusing and immunofixation of cerebrospinal fluid and serum in multiple sclerosis. J. Clin. Lab. Immunol. 6:17-22. Nilsson, K., and J. E. Olsson. 1978. Analysis for cerebrospinal fluid proteins by isoelectric focusing in polyacrylamide gel: Methodological aspects and normal values with special reference to the alkaline region. Clin. Chem. 24:1134-1139. Olsson, J. E., and B. Pettersson. 1976. A comparison between agar gel electrophoresis and CSF serum quotients of IgG and albumin in neurological diseases. Acta. Neurol. Scand. 53:308-322. Siden, A., and K. G. Kjellin. 1978. CSF protein examinations with thin layer isoelectric focusing in multiple sclerosis. J. Neurol. Sci. 39:131-146. Thompson, E. J. 1977. Laboratory diagnosis of multiple sclerosis: Immunological and biochemical aspects. Br. Med. Bull. 33:28-33. Tourlellotte, W. W. 1975. What is multiple sclerosis? Laboratory criteria for diagnosis, pp. 9-26. In A. N. Davison, et al. (eds.), Multiple Sclerosis Research. Elsevier Scientific Publishing Co., Amsterdam. Tourtellolte, W. W., et al. 1968. Subacute sclerosing panencephalitis:
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Brain immunoglobulin-G, measles antibody and albumin. Neurology 18:117-121. Vandvik, B., el al. 1978. Mumps meningitis: Prolonged pleocytosis and occurrence of mumps virusspecific oligoclonal IgG in the cerebrospinal fluid. Eur. Neurol. 17:13-22. Vandvik, B., el al. 1978. Progressive rubella virus panencephalitis: Synthesis of oligoclonal virus-specific IgG antibodies and homogeneous free light chains in the central nervous system. Acta Neurol. Scand. 57:53-64. Vandvik, B., and E. Norrby. 1973. Otigoclonal lgG antibody response in the central nervous system to different measles virus antigens in subacute sclerosing panencephalitis. Proc. Natl. Acad. Sci. USA 70:1060-1063. Vandvik, B., and S. Skrede. 1973. EIectrophoretic examination of the cerebrospinal fluid proteins in multiple sclerosis and other neurological diseases. Eur. Neurol. 9:224-241. Varldai, F., B. Vandvik, and E. Norrby. 1980. Viral and bacterial antibody response in multiple sclerosis. Ann. Neurol. 8:248-255.
Workshops and Meetings Research and Therapeutic Issues in Diabetes: An International Conference. C h a r l o t t e s v i l l e , Virginia. October 12-15, 1982. Contact: M r s . Shirley N e w m a n , International Diabetes Conference,
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