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Distribution of cerebrospinal fluid oligoclonal IgM bands in neurological diseases: a comparison between agarose electrophoresis and isoelectric focusing

Distribution of cerebrospinal fluid oligoclonal IgM bands in neurological diseases: a comparison between agarose electrophoresis and isoelectric focusing

Journal of the NeurologicalSciences, 109 (1992) 83-87 83 © 1992 Elsevier Science Publishers B.V. All rights reserved 0022-510X/92/$05.00 JNS 03738 ...

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Journal of the NeurologicalSciences, 109 (1992) 83-87

83

© 1992 Elsevier Science Publishers B.V. All rights reserved 0022-510X/92/$05.00 JNS 03738

Distribution of cerebrospinal fluid oligoclonal IgM bands in neurological diseases: a comparison between agarose electrophoresis and isoelectric focusing M.K. S h a r i e f a n d E.J. T h o m p s o n Department of Clinical Neurochemistry, Instituw of Neurology and The National Hospitals for Neurology and Neurosurgety, Queen Square, London, UK (Received 26 August, 1991) (Revised, received 4 November, 1991) (Accepted 7 November, 1991)

Key words: lgM; Oligoclonal bands; Cerebrospinal fluid; Agarose gel electrophoresis; lsoelectric focusing; Multiple sclerosis

Summary lsoelectric focusing (IEF) and agarose gel e!ectrophoresis were used to detect oligoclonal lgM bands in cerebrospinal fluid and serum samples from 850 patients with diverse neurological diseases. Oligoclonal IgM bands in cerebrospinal fluid were mainly detected in patients with infectious and inflammatory disorders of the nervous system. Both IEF and agarose electrophoresis revealed similar frequencies of oligoclonal IgM bands. Bands detected by IEF were mainly seen in the anodal range. Despite higher resolving capacity, IEF was less specific than agarose gel electrophoresis. It is concluded that oligoclonal IgM bands have important diagnostic significance and that agarose gel electrophoresis is more suitable for their detection in routine clinical work and use in differential diagnosis.

Introduction

lmmunoglobulin (Ig) M is both phylogenetically and ontogenetically the most primitive immunoglobulin which appears at an earlier stage of the immune response. Persistent elevation of IgM levels usually indicate an ongoing immunologic stimulation, and these levels return to normal when the immunologic stimulation ceases (Hische et al. 1988). Therefore, intrathecal synthesis of IgM may be important in the diagnosis and the follow-up of infectious and inflammatory diseases of the central nervous system (CNS). In fact intrathecal production of lgM is a valuable indicator of recent antigenic stimulation within the CNS (Sharief and Thompson 1989; Lolli et al. 1991), and an important

Correspondence to: Dr. M.K. Sharief, The National Hospital for Neurology and Neurosurgery, Queen Square, London WC1N 3BG, UK. Tel.: (+44) 71 837 3611 ext. 3812; Fax: (+44) 71 833 1016.

parameter in monitoring disease activity of some inflammatory neurologie diseases (Sharief and Thompson 1991a,b). We have already shown that the oligoclonal IgM bands in cerebrospinal fluid (CSF) are more reliable than quantitative measurements in evaluating intrathecal lgM synthesis (Sharief et al. 1990). Thus, an accurate detection of oligoclonal IgM bands is important to determine intrathecal synthesis. Two methods for oligoclonai IgM bands detection in CSF were described. The first method involves agarose isoelectric focusing (IEF) and immunoblotting (Keir et al. 1982; Giles and Wroe 1990) while the other method utilizes agarose gel electrophoresis and a modified western blotting (Sharief et al. 1989). Hitherto, little is known about the distribution of CSF oligoclonal IgM bands in a large heterogenous neurologic population. The purpose of this study is to (1) analyze the pattern of oligoclonal lgM bands in CSF, (2) study the distribution of oligoclonal IgM bands in various neurologic disorders, (3) compare the sensitivity and specificity of agarose gel electrophoresis and

84 IEF in detecting CSF oligoclonal IgM bands, and (4) discuss the disadvantages of these methods in evaluating intrathecal synthesis of IgM.

Patients

Paired CSF and serum samples were obtained from 850 patients between the years 1986 and 1990. Their diagnoses are listed in Table 1. Multiple sclerosis was diagnosed according to the criteria of McDonald and Halliday (1979). Degenerative CNS diseases included Alzheimer's, Parkinson's, Huntington's, and motor neurone diseases. CNS tumours included primary glial neoplasms, meningiomas, neurinoma, and lymphoblastic malignancies. The category of "other neurological diseases" included epilepsy, migraine, benign intracranial hypertension, myasthenia gravis, metabolic encephalopathies, and various movement disorders.

Methods

All samples were examined by both IEF and agarose gel electrophoresis in a blinded fashion. IEF was carried out in agarose gel (Giles and Wroe 1990). In brief, 15/~! CSF concentrated to a total protein content of 10 g/l and serum diluted to the same protein content were electrofocused in agarose gel containing ampholytes with a pH range of 3-10 (Pharmacia). Separated proteins were blotted with polyvinyl difluoride membrane (Miilipore, UK) which was blocked with 2% gelatin solution before oligoclonal IgM bands were stained using a double antibody technique (Olsson et al. 1984). Agarose gel electrophoresis for the detection of oligoclonal IgM bands was carried out on unconcentrated CSF as already described (Sharief et al. 1989). Briefly, adequate volumes of CSF and diluted serum containing 10 ng total IgM were electrophoresed using agarose gel of high electroendosmosis. A volume of 25 /tl CSF and 10 /~1 1:300 diluted serum are usually

TABLE 1 COMPARISON BETWEEN THE PREVALENCE OF OLIGOCLONAL IgM BANDS ON AGAROSE GEL ELECTROPHORESIS (AGE) AND ISOELECTRIC FOCUSING (IEF) RELATED TO CSF TOTAL PROTEIN (TP) AND TOTAL lgM CONCENTRATION (mg/I FOR CSF AND g/I FOR SERUM LEVELS) Diagnostic category

Total No. of

Mean CSF TP

patients

(g/I)

CSF

Serum

180

0,46

0.54

1,74

58

62

2

3

CNS infections Active neurosyphilis Treated neurosyphilis Encephalitis Meningitis Neuro-borreliosis HTLV-1 myelitis

23 17 48 52 9 8

0.51 0.44 0.63 0.71 0.53 0.47

0.73 0.42 0.54 0.59 0.65 0.51

1.78 1.26 2.04 1.92 2.11 1.62

100 0 75 79 78 75

91 12 65 71 67 63

0 6 6 12 22 13

9 18 10 4 0 13

Inflammatory CNS diseases Neuro-sareoidosis Cerebral lupus Cerebral Beh~et's Others

19 12 9 24

0.51 0.53 0.58 0.48

0.47 0.52 0.55 0.42

1.24 1.37 1.41 1.46

53 58 44 63

58 42 56 54

11 17 11 17

16 33 0 8

Guillain-Barr6 syndrome CNS tumours Peripheral neuropathies Vertebral compression Degenerative CNS diseases Cerebrovascular diseases Myopathies Other neurological diseases Normal controls

38 26 49 52 64 35 30 135 20

0.76 0.46 0.34 0.37 0.28 0.36 0.31 0.32 0.26

0.58 0.49 0.30 0.34 0.31 0.46 0.34 0.29 0.24

1.69 1.42 1.50 1.32 1.41 1.24 1.37 1.26 1.06

26 12 0 0 0 0 0 2 0

37 19 8 10 9 9 7 7 0

31 27 6 8 11 14 3 11 0

39 35 4 12 14 14 13 9 0

Total

850

0.46

0.45

1.51

32

34.8

9

11

Multiple sclerosis

Mean Total lgM

Group I e (% positive) AGE

Group II a (% positive) IEF

AGE

a Group I represents intrathecal synthesis of oligoclonal IgM bands and Group !1 represents leakage of bands from serum.

1EF

85 TABLE 2 PREDOMINANTpH DISTRIBUTIONOF OLIGOCLONALIgM BANDSDETECTED BY ISOELECTRICFOCUSING

adequate for routine work. Separated proteins were cross-linked to a nitrocellulose membrane by glutaraidehyde, to prevent protein loss, then oligoclonal IgM bands were immunostained by a peroxidase-conjugated F(ab') 2 fragment of anti-human IgM antibody (Sigma, UK).

Patient group a (total No.) Group ! (296) Group II (9O) Group I!I

Results Banding patterns for IgM obtained by the two methods are shown in Figs. I and 2. Oligoclonal bands were defined as 2 or more bands as detected by IEF. According to the finding of agarose gel electrophoresis, patterns could be divided into four main groups. Group I includes CSF oligoclonal bands that are either absent from or substantially more in number than corresponding serum indicating local synthesis (Laurenzi and Link 1978; Sharief et al. 1989) while group II represents the presence of identical oligoclonal bands in both CSF and serum samples suggesting leakage of bands from serum. Group III involves the presence of a single monoclonal band in both CSF and corresponding serum and group IV represents normal electrophoresis pattern. Patterns detected by IEF were also divided into 4 main categories similar to agarose gel electrophoresis except that the monoclonal band in group III is distributed as a fractionated pattern (Fig. 3). Table 1 summarises the results of agarose electrophoresis and IEF for the detection of oligoclonal lgM bands in the study population. In patients with inflammatory or infectious CNS disorders, agarose gel electrophoresis and IEF revealed IgM bands at similar frequencies. However, IEF revealed lgM bands in some patients who had no evidence of intrathecai immune response such as patients with peripheral neuropathy, vertebral compression, or myopathy. These patients had completely normal routine CSF tests including CSF cell count and CSF total protein and y-globulin concentrations. A monoclonal pattern (Group l i d suggestive of an lgM paraprotein was detected by IEF and

(14)

Percentageof patients with lgM bands in CSF pH 3-6.4 pH 6.5-7.9 pH 8.0-9.5 pH > 9.5 63 29 7 1 47

39

12

2

71

29

0

0

Group I: intrathecal synthesis of oligoclonal bands; Group !I: leakage of bands from serum; Group I!I: monoc!onal pattern. agarose electrophoresis in 8 patients with peripheral neuropathy and 6 patients with CNS tumours. Oligoclonal IgM bands detected by IEF were mainly seen in the anodal range and most pl values were between 3 and 6.5 pH units (Table 2). Table 3 shows the sensitivity and specificity of IEF and agarose electrophoresis in detecting oligoclonal IgM bands in patients with inflammatory and infectious CNS diseases.

Discussion Agarose gel electrophoresis and IEF as carried out in this study gave similar frequencies of 01igoclonal lgM bands in patients with inflammatory and infectious CNS diseases. However, agarose electrophoresis was more specific than IEF in detecting oligoclonal lgM bands in patients with putative intrathecal immune response. In IEF, proteins migrate under an applied voltage to their isoelectric point in a pre-established pH gradient. This focusing effect of homogeneous molecules gives rise to discrete protein bands with much greater resolution than with electrophoretic methods. Therefore, IEF successfully separated more discrete bands of lgM in positive CSF and serum samples. The possibility of artefact formation or pro-

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mW .....

C

S

I

C

S

2

C

S

3

C S

C $

4

5

~i~i~ ~

C

_-~---o

S

6

C

S

7

Fig. 1. A selection of oligoclonal lgM bands in paired CSF (C) and serum (S) samples detected by agarose gel electrophoresis. Samples from patient no. 1 represent negativecontro!s. Sites of sample application are represented by short dashes and individualbands are indicatedby arrow heads. Migrationof proteins is toward the cathode(anode shownas +).

86 pH 3.0 -

5.0

,.*

7.0

II, |

9.5

C

S 1

C

S 2

C

S 3

C

S

C

4

S

C

5

S

C

6

S 7

Fig. 2. Oligoclonal IgM bands detected by isoelectric focusing of CSF (C) and serum (S) samples from the same patients shown in Fig. |. Bands that are common to all samples (arrows) are regarded as artifacts. Sample application is indicated by *.

tein-ampholyte interaction has been suggested (Hare et al. 1978; Righetti and Gianazza 1978). Carrier ampholytes, like all charged molecules, may form avid complexes with protein molecules in a reaction that is strongly pH dependent (Hare et al. 1978). This fact may account for the presence of multiple bands in patients with non-inflammatory CNS diseases when IEF was used.

pH

+

3.0 4¢1-

5.0

--

i:~:~ •

7.0

There are other inherent difficulties when IEF is used to detect lgM bands. Since IgM is poorly soluble under conditions of low ionic strength required for IEF, it frequently precipitates near its isoelectric point resulting in some poorly resolved regions of separation. Moreover, IEF has low resolution for acidic proteins such as IgM, probably due to the presence of negative!y charged chemical groups that are part of the agarose molecule, such as sulphate, pyruvate and uronic acid residues. Other disadvantages of IEF include cofocusing of diverse Ig molecules that have similar pl point, and microheterogeniety with fractionation of monoclonal Ig molecules. Certain points should be observed whCn'~fet¢-eting oligoclonal lgM bands in the CSF. A control Oligoclonal band-positive and control negative specir,lens should he included in each electrophoretic or focusing set to serve as quality control. Furthermore, methods used to visualize IgM bands should be standardized according to the lgM content of the test samples. Using fixed dilution or volumes related to the total TABLE 3 SPECIFICITY AND DIAGNOSTIC SENSITIVITY OF AGAROSE GEL ELEC-'TROPHORESIS AND ISOELECTRIC FOCUSING IN DETECTING OLIGOCLONAL IgM BANDS IN PATIENTS WITH INFLAMMATORY/INFECTIOUS CNS DISEASES Method

Specificity Sensitivity (%) (%)

9.5 A

B

Fig. 3. Isoelectric focusing (A) and electrophoretic (B) patterns of a CSF sample from a patient with fgM paraproteinaemia. Bands that are present in all focused samples (arrows) are regarded as artifacts.

Agarose gel electrophoresis 96 lsoelectric focusing 84

Multiple Infections Inflammatory sclerosis of CNS a diseases 58

81

56

62

71

53

a Treated neurosyphilis not included.

87

protein content could result in false negative results as locally produced lgM is affected by varying metabolic turnover in different stages of disease activity. Increased IgM catabolism, binding to target structures, or increased concentration of proteolytic enzymes results in reduced CSF content of IgM. This fact may explain the relatively reduced sensitivity of IEF in patients with CNS infections since the IEF method was standardized according to CSF total protein, rather than IgM concentration. The use of double-antibody detection system in the IEF method has been shown to cross-react with IgG paraprotein bands despite the use of a/~-chain-specific first antibody (Giles and Wroe 1990). Such cross-reactivity, which is partially mediated by Fc antibody fragment, may be the cause of reduced specificity of IEF when compared to agarose gel electrophoresis. In contrast, the use of the F(ab') 2 fragment of anti-human lgM antibody to immunostain IgM bands in the agarose electrophoresis method was not associated with any cross-reactivity (Sharief et al. 1989). Another potentially serious interference in the IEF method is the need to concentrate CSF before focusing. Concentration of the CSF may induce selective loss of ~/-globulin and lead to cross-reaction with lgG bands (Giles and Wroe 1990). Concentration procedures also require a relatively large volume of CSF (up to 2.5 ml) and could be time-consuming. The use of unconcentrated CSF is, therefore, another advantage of the method of agarose gel electrophoresis. In conclusion, the detection of CSF oligoclonal lgM bands may provide important information in inflammatory neurologic diseases. IEF offers no major advantages to warrant replacement of agarose electrophoresis in detecting lgM bands, and may even provide misleading results. We therefore propose the use of agarose gel electrophoresis for demonstration of CSF oligoclonal IgM bands in routine clinical work. IEF clearly allows identification of many more bands than does agarose electrophoresis and its usefulness will probably be in etiologic and pathogenic studies of C'NS diseases rather than as a diagnostic tool. The main drawback of IEF is the occurrence of bands in diseases not typically associated with immune abnormalities.

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