Journal of the Neurok)gical ,S'cIo~,e ~, l !~3 i i ~)9 i 1 2 i l~-,~Z
216
g 1991 Elsevier Science Publishers B.\ 1/022 510X,~1~$0~.>t~
JNS 03550
Affinity immunoblotting: rapid and sensitive detection of oligoclonal IgG, IgA and IgM in unconcentrated CSF by agarose isoelectric focusing R. Kaiser Department of Neurology, University of Freiburg, Freiburg (Germany)
(Received 2 October, 1990) (Revised, received 22 January, 1991) (Accepted 25 January, 1991)
Key words."IEF/affinity mediated immunoblotting; CSF; Oligoclonal IgG, IgM and IgA; Multiple sclerosis; Neuroborreliosis Summary
Two methods for the investigation of oligoclonal immunoglobulin bands in cerebrospinal fluid are compared. Isoelectric focusing (IEF) in agarose gels combined with a highly sensitive affinity immunoblotting proved to be superior to PAGE-IEF and silver staining regarding detection sensitivity and expenditure of labour. The procedure presented here allows examination of oligoclonal bands not only of IgG, but also of IgA and IgM antibodies in less than 4 h. Oligoclonal IgM bands could be detected in 6 patients with neuroborreliosis or neurosyphilis as well as in one patient with a peripheral neuropathy where other electrophoretic methods, including immunoelectrophoresis, were negative. Oligoclonal IgA bands were demonstrated only in 2 patients with a bacterial infection of the CNS. In patients with multiple sclerosis amounts of IgM and IgA in the CSF were too low to allow investigation of oligoclonal IgM or IgA bands. Introduction
The presence of oligoclonal IgG in cerebrospinal fluid (CSF), demonstrable by isoelectric focusing (IEF), is a well known phenomenon in various inflammatory diseases of the central nervous system (CNS) (Kostulas 1985). Over 90~o of patients with multiple sclerosis (MS) (Tourtellotte and Walsh 1984) and approximately one-third of those with acute meningitis show CSF oligoclonal bands (Fryd6n 1978). A variety of techniques including electrophoresis in agarose gels (Mattson 1981), in sodium dodecyl sulfatepolyacrylamide gels (SDS-PAGE)(Iivanainen 1981), isoelectric focusing (IEF) in PAGE (Laurenzi and Link 1978) and IEF in agarose gels (Kostulas and Link 1982) were used to analyze these bands. Some of these methods are hampered by the necessity to concentrate the CSF or by long incubation times for Coomassie blue or silver staining. These problems have been recently overcome by IEF in combination with an affinity mediated immunoblot (AMI), a technique primarily designed to investigate virus-specific oligoclonal IgG bands (Domes and ter Meulen 1984; DOrries et al. 1989; Kaiser et al. 1989). Correspondence to: Dr. Reinhard Kaiser, Department of Neurology, University of Freiburg, Hansastrasse 9, D-7800 Freiburg, F.R.G. Tel.: 0761-2705301; Fax: 270-7200.
A common feature in neuroborreliosis and some other chronic bacterial infections is intrathecal synthesis not only of IgG, but also of IgA and IgM antibodies (Felgenhauer and Sch~tdlich 1987; Pohl et al. 1986). Oligoclonal IgM might also be found in patients with a peripheral neuropathy in association with a "monoclonal gammopathy" (Kelly 1985). The widely used routine procedure for the investigation of oligoclonal IgG bands - focusing in polyacrylamide gels, immunofixation and silver staining - is, however, not suitable to demonstrate oligoclonal IgA and IgM bands. Due to the limited pore size of the gel these proteins of high molecular size mostly do not enter the gel. The aim of this study therefore was to investigate an affinity immunoblotting procedure combined with IEF as a suitable method for the rapid and sensitive demonstration of oligoclonal IgG, IgA and IgM bands in routine CSF sample examination.
Materials and methods
Samples Paired specimens of CS F and serum were obtained from fifty patients from whom samples were taken at the Department of Neurology, University of Freiburg, for analysis regarding the presence of oligoclonal IgG bands in CSF.
217 TABLE 1 MEAN AND RANGE OF" IgG, IgM and IgA INDICES
Normal values Study group: Multiple sclerosis 01 - 27)
IgG index
IgM index
IgA index
< 0.7
< 0.06
< 0.4
1.52 0.5-2.0
Neuroborreliosis, neurosyphilis (n = 10) Encephalitis, myelitis, GBS, meningitis, polyneuropathy (n = 13)
0.03 0.002-0.15
~).12 0.01-0.31
2.1
1.0
0.5-5.0
0.21-4,1
().44 O.02-1.0 I
0.6 0.5-1.3
0.04 0.01-0.15
0,13 0.007-0.32
TABLE 2 F I N D I N G S FROM CSF IgG STUDIES Patient
Diagnosis
IgG index
IgG-OCB
NO.
PAGE-IEF ].
2. 3. 4. 5. (L Y.
t}.
1!). II. 12. 13. 14. 15. 16. 17. I~. 19. 20. 21. 22. 23. 24. 25. 26. 27.
MS
29. 28. 3o. 31. 32. 33.
i
34. 35. 36. 37.
]
[
[
2.0 1.9 1.8 1.8 1.7 1.6 1.5 1.4 1.4 1.1 1.1 1.0 1.0 0.9 0.9 0.9 0.8 0.8 0,7 0.7 0.7 0.7 0.7 0.7 0.7 0.7 0.5
Neuroborreliosis
1.3 0.8 0.7 0.7 0,7 0.5
Neurosyphilis
2,6 3.3 5.0 2.2
Agarose-1 EF,'AMI
+ + + + t 4+
+ + 4 +
+
+
+ + +
+ + 4 +
218 TABLE 2 (continued) Patient No.
Diagnosis
lgG index
tgG-OCB PAGE-IEF
~\garo so- I E F:A M I
1.3 O.6 0.5
+ 0 0
o 0
0.4
0
0
38. 39. 40. 41.
Encephalitis
42.
Myelitis
0.5
0
*
Meningitis
0.7 0.5
+ 0
4 It
4
43. 44.
/ J"
45. 46. 47.
/ /
GBS
0.6 0.6 0.5
0 0 0
0 0 •
48. 49. 50.
] /
Polyneuropathy
0.5 0.5 0.6
0 0 0
• 0 0
< 0.7
0
0
Normal values
MS = multiple sclerosis; GBS = Guillain-Barr6 syndrome; 0 = no antibody bands detectable - polyclonal IgG staining; + = oligoclona[ lgG-bands (OCB) occurring predominantly in the CSF; * = OCB of similar staining intensity in CSF and serum.
Clinically defined MS (Poser et al. 1984) was diagnosed in 27 patients (study group 1), 10patients had neuroborreliosis or neurosyphilis (study group 2) and 13 subjects presented with other inflammatory diseases of the central or peripheral nervous system (study group 3) (Table 1 and 2). Samples were stored at - 2 0 °C and thawed prior to use. IgG, IgM, IgA and albumin levels were determined by laser nephelometry. In the case of IgM and IgA concentrations in the CSF lower than 10 mg/1 levels in paired sera and CSF were determined by ELISA. Intrathecal synthesis of antibodies was determined separately for each class of immunoglobulins according to the method of Delpech and Lichtblau: the following indices were taken as being indicative of local synthesis: IgG: >0.7, IgA: >0.4 and IgM: >0.06 (Forsberg et al. 1984; Sindic et al. 1984). The mean and range of these indices according to the three study groups are listed in Table 1.
The plates were then incubated for 1 hour at room temperature. Serum and CSF samples were diluted in PBS-Tw 1 : 10.000 and 1 : 100.000 (serum) and 1 : 10 and 1 : 100 (CSF). Reference sera (Behringwerke, Marburg, F.R.G) for each Ig class were adjusted to an IgA or IgM concentration of 200 #g/1 and diluted in 2-fold steps to a final IgA or IgM concentration of 6.25 #g/1. The plates were washed and samples and reference samples (100 #1 per well) were added. After 2 h plates were washed again and peroxidaselabelled rabbit anti human IgA or IgM antibodies (1:2000 in PBS-Tw, 100/21 per well, Dako, Hamburg, F.R.G.) were added for 1 h at room temperature. After repeated washing o-phenylenediamine (OPD) was added and the absorbance at 492 nm measured with a Titertek Multiscan reader (FlowLab). The amounts of IgM and igA in samples were determined from standard curves obtained from standard serum.
Determination of IgA and IgM concentrations Microtiter plates (Nunc Immunoplates) were coated with rabbit antiserum against human IgM or IgA antibodies (Dako, Hamburg, F.R.G.) diluted 1:1000 in 0.05 M sodium carbonate buffer, pH 9.6. 100 #1 of this solution was added to each well. After incubation overnight at 4 ° C the antibody solution was removed from the wells and 0.1 ~o bovine serum albumin (BSA) in phosphate buffered saline (PBS: 1.38 g NazHPO4, 0.24 g NaH2PO4, 8.0 g NaC1 and 1000 ml distilled water, pH 7.6) containing 0.1 ~o Tween 20 (PBS-Tween) was added to block free protein binding sites.
Isoelectric focusing PA GE-IEF and silver staining A standardized procedure for PAGE-IEF and silver staining was used for the routine investigation of oligoclonal bands (Kruse et al. 1982). The polyacrylamide gel (Pharmacia-LKB PAG Plate, pH 3.5-9.5) was placed on a temperature controlled flatbed (Pharmacia-LKB Multiphor, Multitemp Circulator). Samples of CSF and serum (20/~1) adjusted to an IgG concentration of 20 mg/1 were applied on filter paper tabs 3 cm from the anode. The gel was
219 focused for a total of 2000 Vh at 4 °C. The power supply (Pharmacia) was set at 1500 V, 50 mA and 30 W. After focusing the gels were fixed and stained according to Kruse et al. (1982). This procedure takes about 30 h for the evaluation of results and does not allow detection of oligoclonal IgA or IgM bands.
for 30min at 37°C to reduce IgM and lgA to the monomeric form. C S F s a m p l e s containing less than 80 mg/1 of IgA or IgM were diluted to 10 mg/l (45 bd) and then mixed with 5#1 D T T (0.01, 0.002, 0.0004 and O.O0OO8 M).
Isoelectric focusing Agarose-lEF and ql]finity immunoblotting The method described by DOmes et al. (1984, 1989) was used with somc modifications as follows.
Antibody loading ((nitrocellulose fihers for immunoblotting Nitrocellulose (NC) filters (BA 83, Schleicher and Schall) were cut to 10 x 10 cm 2 sized sheets and loaded separately with goat antibodies to human IgG, IgM or IgA (Dianova, Hamburg, F.R.G., Nos. 109-002-011, 109-002003, 109-002-043) by overnight incubation at room temperature on a rocker platform. Antibodies were diluted in PBS to the following concentrations: 25, 50, 100, 200 #g/ml/10 cm 2 (corresponding to 20 #g/cm2). After overnight incubation, filters were rinsed briefly in PBS and unoccupied protein bindings sites on the filter were blocked with PBS-Tween for 1 h.
Preparation oJspecimens For screening of the optimal Ig concentrations samples were each adjusted to an IgG, IgA or IgM level of 40 mg./1 and then diluted in two fold steps to at least 5 rag/1. Samples to be assayed for oligoclonal IgM or IgA antibodies were studied both in the native state and after mild reduction with dithiothreitol (DTT). In this case specimens were diluted to 80 mg/1 mixed with an equal volume (50 #1) of D T T (0.1 , 0.02, 0.004 and 0.0008 M) and then incubated
Coa t i YN
uq/a
Thin agarose gels (0.5 mm) containing 0.9"{, IEFagarose (Pharmacia), 12~',, sorbitol, 1",, Nonidet P40 (NP 40, Sigma) and Pharmalyte (Pharmacia) were cast at 75 °C by capillary action between 2 glass plates onto a Gelbond sheet (FMC). For IEF o f l g G antibodies gels were supplemented with 6.2 ° 0 Pharmalyte pH 3-10, while focusing of IgM and IgA antibodies was carried out with gels containing 3.1 °~, Pharmalyte pH 3-10 plus 3.1", Pharmalyte pH 4-6.5. The plates were "aged" at 4 : C in a moist chamber for 1 h. Focusing was performed on a Multiphor apparatus (Pharmacia-LKB, Freiburg) at 17 :C. The electrode strips contained 0.5 M N a O H (catholyte) or 0.115 M H2SO 4 (anolyte). Samples (10 btl) were applied directly to the gel using a silicone application mask having 8 x 8 mm slots. After focusing the gel was dipped for a few seconds in distilled water to remove unfocused surface material. Subsequently, focused antibodies were blotted onto nitrocellulose strips with goat antibodies to either human lgG, lgM or IgA. The nitrocellulose sheet was laid on the agarose gel carefully to avoid air bubbles, covered with one sheet of cellulose acetate, 3 layers of Whatman filter C H R 1, a glass plate and a weight of approx. 200 g. After 60 min the nitrocellulose was removed and v~ashed for 1(I min in PBSTween. Bound antibodies were detected by incubating the sheets with the following dilutions of peroxidase-con-
IEF-PATTERN
2
k~
2,5 5 I
lit
20
J
-
I
IIII
=
Fig. 1. Effect of antibody loading of nitrocellulose sheets with regard to sensitivity' in detecting oligoclonal bands. NC sheets were coated with rising concentrations of goat anti human IgG, 20/*g/cm 2 corresponds to 200/~g/ml/10 cm a. A total amount of 100 ng IgG was focused. Peroxidasc-labelled goat anti-human-lgG was diluted 1 : 500. Black arrows indicate the pH gradient, from left to right: pH 6.5, pH 8.6.
220
Fig. 2. Sensitivity in detection of oligoclonal IgM bands: l0/~1 of CSF was diluted from 20 to 5 mg/l (50-200 ng of total lgM). NC sheets werc coated with goat anti-human IgM, 50 #g/ml/10 cm2. Peroxidase-labelled goat anti-human IgM was diluted 1:500. Black arrows indicate the pH gradient, from left to right: pH 4.0, pH 6,5. j u g a t e d goat anti-human IgG, IgA or IgM (Dianova, H a m b u r g , F R G , Nos. 109-035-003, 109-035-011,109-035043): 1 : 250, 1 : 500, 1 : 1000 and 1 : 2 0 0 0 in P B S / T w e e n . After 1 h at r o o m t e m p e r a t u r e on a rocking platform, blots were rinsed 3 times with P B S - T w e e n a n d then immersed in substrate solution (3 mg/ml 4-chloro- I- naphthol, 0.03'!~0 hydrogen peroxide in P B S ) for 15 min. The blots were w a s h e d with water and air-dried.
Results
Assay conditions In preliminary experiments 64 c o m b i n a t i o n s o f assay conditions were tested for each antibody class: antibody
loading o f N C - Ig concentrations o f samples - dilution o f peroxidase-labelled second antibody. Each p a r a m e t e r was examined in 4 dilutions.
Antibody loading of NC sheets The effects o f variations in the concentration of antibodies b o u n d to the N C were determined. Fig. 1 shows the results o f varying the antibody concentration on detection of h u m a n I g G antibodies. 10 #1 samples of C S F adjusted to an I g G concentration o f 10 mg/1 were separated by I E F and blotted to N C which h a d been c o a t e d with varying concentrations o f goat anti-human IgG. The peroxidaselabelled a n t i b o d y was diluted 1 : 500. The results showed that the optimal range o f antibody coating was between 25
Fig. 3. Sensitivity of IgG detection: 10 #1 of CSF was diluted from 40 to 5 rag/1 (50-400 ng of total IgG). NC sheets were coated with goat anti-human IgG, 50 #g/ml/10 cm2. Peroxidase-labelled goat anti-human IgG was diluted 1 : 500. Black arrows indicate the pH gradient, from left to right: pH 6.5, pH 8.6.
221 and 100/~g/ml/10 c m 2 with 50/,g/ml/10 c m 2 probably being the best loading concentration. In the case of lgA and IgM antibodies similar results were obtained; between 25 and 50/~g/ml of goat antibodies to human lgA or lgM were necessary to coat 10 cm 2 of NC area to demonstrate Ig class specific oligoclonal antibodies. Increasing the amounts of loading antibodies ( > 100/~g/ml/10 cm e) resulted in a loss of sensitivity in detecting focused antibodies but slightly improved the demonstration of their oligoclonal nature. Coating of NC with less than 10 #g/ml/10 c m 2 of Ig class specific antibodies gave an unacceptably high costaining of polyclonal antibodies making it difficult to detect single antibody bands. Loading conditions of antibodies on NC sheets are given in/xg/ml/cm= to indicate that both factors, amounts of antibodies (Hg) and volume of buffer (ml) will influence zmtibody binding to the NC sheet (data not shown).
1~ concentrations.fi~r lran,sJ'er procedure.s Employing an optimal antibody loading of NC of 50 Hg/ml/10 cm e the optimal range of lg antibodies to be [\)cused was determined. 50-100 ng (5-10 mg/1) of IgG, lgA or lgM were found suitable for application on IEF to reveal clearly xisible oligoclonal bands. Lowering the amounts ol" focused antibodies to 25 ng resulted in an visible loss of bands (Fig. 2). If more than 200 ng of IgG, IgA or IgM antibodies were focused the amounts of polyclonal antibodies increased considerably so that single bands were difficult to differentiate (Fig. 3). For comparative studies of oligoclonal IgG bands in PAGE-IEF and agarose-IEF (50 patients) the IgG concentration was therefore adjusted to 10 mg/l for focusing in agarose gels.
ant i
-
IqC
Sign(lk;ance 0/" second antibody dihttion The minimal concentration of second antibody for den> onstrating oligoclonal bands is largely dependent on the amounts of Ig bound to NC. Using the above mentioned optimal conditions for coating of NC sheets and focusing of samples peroxidase labelled antibodies had to be diluted 1:500 to 1:1000 (Fig. 4). Higher dilutions reduced the staining intensity and number of detectable bands while lower antibody dilutions increased the amounts of costained polyclonal immunoglobulins. This was shown for IgG, lgA and IgM antibodies.
El]~,cts q/ sample treatment with dithiothreitol IgA and lgM antibodies not treated with DTT prior to IEF did not enter the gel and mostl? left at the point of application. After treatment with rising conccntrations of DTT samples focused mainly in the pH-region 4-6.5. Best results were obtained after diluting CS F and serum samples to 10 mg/I of IgA or IgM (45 IH) and reducing the pcntameric IgM and dimeric IgA to monomeric forms with 0.0004 M DTT (5 ~H) for 30 rain at 37 C . Identical specimens were applied at the anode and cathode of the same gel and focused for 1 h. Affinity blotting revealed bands of identical pI in both samples indicating optimal sample treatment. Increasing the concentration of DTT produced a high "background staining" and a loss of oligoclonal lgM or IgA bands. Discrimination of oligoclonal IgA and lgM bands was improved by spreading the pH gradient of the gel between pH 4 and 6.5.
IEF-PATTERN
1:258 1:508
1:188
,~ 4>'
~4~
Fig. 4. Significance of second antibody' dilution: Peroxidase-labelled goat anti-human lgG was diluted 1:250 to 1 "2000. Antibody loading of NC shccts: 50/tgml I() cm e, a total amount of 100 ng lgG was focused. Black arrows indicate the pH gradient, from left to right: pH 6.5, pH 8.6.
222
Fig. 5. Oligoclonal IgG bands of a patient with multiple sclerosis. Comparison of results from PAGE-IEF and silver staining with agarose-lEF and affinity mediated immunoblotting. Isoelectric focusing was performed with 400 ng of IgG in case of PAGE-IEF and with 200 ng of IgG in case of agarose IEF.
Fig. 6. Oligoclonal bands of IgG, IgM and IgA in a patient with neuroborreliosis. Investigation by IEF and affinity mediated immunoblotting.
223
Comparison of results ,from PA GE-IEF and agarose-IEF Oligoclonal bands of high intensity in the CSF generally were visible both by PAGE-lEE and silver staining as well as by agarose lEE and affinity-mediated blotting (AMI). However, oligoclonal IgG bands of low intensity were better detected by agarose lEE and AMI. Silver staining of CSF and serum proteins focused in polyacrylamide gels frequently creates bands which are not related to the IgG fraction. Fig. 5 demonstrates a comparison of results from IEF in polyacrylamide gels and lEE in agarose gels of a patient with nmltiple sclerosis (No. 20). Using PAGE-IEF and silver staining 37 of 50 patients (74°~,) tested revealed oligoclonal IgG bands predominantly in the CSF. In all of these cases the IgG index was
> 0.7. Using agarose-IEF and AMI oligoclonal IgG bands were demonstrated in 42 patients (8400). In 39 subjects bands were visible predominantly in the CSF. The lgG index was elevated in 38 patients (Table 2). Of the two patients with oligoclonal IgG bands detected by agaroseIEF and affinity immunoblotting but not by PAGE-lEE, one (No. 16) had multiple sclerosis (IgG index 0.9) and the other (No. 33) presented with an acute neuroborreliosis (IgG index 0.5). In 3 patients IgG bands occurred with a similar staining intensity in CSF and sermn indicating derivation of these antibodies from the serum. One of these patients (No. 42) presented with a myelitis probable due to viral infection, one (No. 47) suffered from Guillain-Barr6 syndrome and one patient (No. 48) had a polyneuropathy in association with an lgG,"IgM gammopathy. In this
"1ABLE 3 F I N D I N G S FROM CSF IgM AND lgA STUDIES
Patient No.
Diagnosis
lgM index
IgM-OCB
lgA index
IgA-OCB
0.(11 (].02 0.11 0.003 0.01 0.08 0.03 0.01 0.01 0.006 0.03 O. 15 0.05 0.04 0.01 0.02 0.002 0.03 0.03
n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
0.21 0.08 0.22 0.15 0.04 0.20 0.23 (I.25 0.12 0.12 II.O 1 0.06 0.05 0.02 0.06 0.06 0,24 0,16 O. 12
11.d. n.d. n.d n.d n.d. n.d n.d. n.d. n.d. n.d n.d. n.d. n.d. n,d. n.d. n,d. n d. n.d. n.d.
20. 21. 22. 23.
0.006 0.06 0.006 0.01
n.d. n.d. n.d. n.d.
0.03 O. 14 0.05 I). 13
n.d. n.d. n.d. n.d.
24. 25. 26. 27.
0.04 0.03 0,007 0.05
n.d. n.d. n.d. n.d.
i). 16 0.31 0.03 0.08
n.d. n.d. n,d. n.d.
28. 29.
4.11
0.55 1.01
0 +
0.72 0.33 0.31 0.02
0 0 0 0
0.25 0.11 1.00 0.12
0 0 + 0
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. I1. 12. 13. 14. 15. 16. 17. 18. 19.
3(1. 31. 32. 33. 34. 35. 36. 37.
MS
Neuroborreliosis
Neurosyphilis
2.20 1.00 0.52 0.71 0.54 0.25 (I.25 0.52 0.21
224 TABLE 3 (continued) Patient No. 38. 39. 40.
Diagnosis
IgM index
IgM-OCB
IgA index
IgA-O('B
Encephalitis
0.15 0.01 (I.06 0.09
n.d. n.d. n.d. n.d.
(I.14 0.05 0.(18 0.03
n.d. n.d n.d. n.d.
42.
Myelitis
0.02
n.d.
0.15
n.d.
43. ] 44. f
Meningitis
0.01 0.05
n.d. n.d.
0.21 0.06
n.d. n.d.
41.
45. ] 46. / 47.
GBS
0.01 0.02 0.02
n.d. 0 0
0.07 0.16 0.28
n.d. 0 0
48. | 49. / 50.
Polyneuropathy
0.01 0.02 0.03
* 0 0
0.22 0.12 0.007
0 (i 0
< 0.06
0
Normal values
< 0.4
0
IgM/lgA-OCB, oligoclonal bands (OCB) of IgM/IgA antibodies; 0, polyclonal staining oflgM or Igm antibodies; +, OCB occurring predominantly in the CSF; *, OCB detectable predominantly in serum; n.d., lgM or IgA antibody concentration in the CSF too low ( < 5 mg/l) to be detectable by IEF,
patient affinity immunoblotting not only demonstrated oligoclonal IgG bands (pH region 8-10) but also oligoclonal IgM bands (pH region 4-6) (Table 3). In both cases antibody bands were derived from the serum. Immunoelectrophoresis of the serum was negative in respect to a monoclonal IgM gammopathy.
Oligoclonal IgM and IgA bands Due to the low concentrations of IgM and IgA in the CSF ( < 5 mg/1) oligoclonal bands of these immunoglobulin classes could be investigated only in a limited number of patients (15 of 50). Oligoclonal IgM bands in the CSF (pH4-6.5) were detected in 6 of 10patients with neuroborreliosis or neurosyphilis (Nos. 28, 29, 30, 32, 34, 35) all of whom had elevated IgM indices (mean: 1, 42; range: 0.52-4.11). As mentioned above, one patient with a peripheral neuropathy displayed oligoclonal IgM bands predominantly in serum. The other patients tested revealed a polyclonal staining of IgM antibodies in CSF and serum. Oligoclonal IgA bands in the CSF were detectable only in one patient (No. 29) with neuroborreliosis (IgA index: 0.7) and one (No. 36) with neurosyphilis (IgA index: 1.0). IgA bands were only of weak intensity. Fig. 6 illustrates the finding of oligoclonal IgG, IgM and IgA bands in a patient with neuroborreliosis (No. 29). In none of these cases could oligoclonal IgM or IgA antibody bands be detected by PAGE-IEF.
Discussion
The aim of this study was to compare a recently developed immunoblot technique with a routine IEF method for demonstrating oligoclonal immunoglobulin bands. The most commonly used procedure to investigate oligoclonal bands in daily routine examination is IEF in polyacrylamide gels with silver staining. Although this technique is reliable and sensitive, it takes about 30 h to provide results. As shown by the finding of an additional 5 patients with oligoclonal bands demonstrable only by IEF and immunoblotting the assay described here might be even more sensitive and requires less than 4 h for performance. Another aim of this study was to modify the immunoblot technique in order to investigate otigoclonal lgA and IgM antibody bands. Large molecules (> 200 kDa) are difficult to focus in polyacrylamide IEF gels due to sieving effect of the cross-linked gels. This problem can be partly overcome by the use of low percentage agarose gels (0.8-0.9°~o). However in the case of IgM (900 kDa) this procedure was found to be inadequate for separating these immunoglobulins (data not shown). Sample treatment with 0.0004 M DTT (see above) reduce the disulfide bonds of the 5 subunits of the IgM pentamer, facilitating the focusing of the monomers in the IEF gel. The use of a higher concentration of DTT resulted in a loss of single IgM bands and an increase of diffuse antibody staining. In this case further disulfide bonds (i.e., inter- and intrachain disulfide bonds)
225
might be broken, producing inmmnoglobulin breakdown products. The inability to demonstrate oligoclonal IgM or IgA bands by PAGE-IEF and silver staining is connected with the fact that this staining procedure does not discriminate between certain proteins, i.e. lgG, IgA or IgM. Another aspect is that CS F samples with detectable amounts of lgM or IgA antibodies would require a 10-fold lower dilution to examine oligoclonal IgM or IgA bands than to investigate lgG bands (different concentration of these immunoglobulin classes in the CSF). As all proteins will be stained (silver staining) high amounts of other proteins (i.e., IgG) would prevent detection of single IgM or lgA bands. The finding of an oligoclonal IgM immune response in the CS F of 6 and a polyclonal IgM imnmne response in the CSF or a further 4patients with neuroborreliosis or neurosyphilis might indicate different stages of disease or different immune mechanisms involved. Further studies, using NC sheets coated with Borrelia burgdor/'eri antigen, have shown that these bands indeed are at least partially reactive with this pathogenic agent (manuscript in preparation). Follow up studies will show whether this observalion is of diagnostic or prognostic value in patients with CNS infection. Patients with oligoclonal IgM bands in the CSF always had elevated IgM indices (Table 3). In onc patient who suffered from a polyneuropathy and a monoclonal IgG gannnopathy demonstrated by immunoelectrophoresis, I EF and immunoblotting disclosed not only oligoclonal lgG but also clearly visible IgM bands, predominantly in the serum. Amounts of IgM were too low to be detectable by immunoelectrophoresis. Further studies are in progress to investigate the reactivity of these bands to peripheral nerve proteins. Oligochmal lgA bands were hardly discovered in the CSF of any patient tested. Preliminary studies done with sera of a patients with a monoclonal lgA gammopathy had confirmed the reproducibility of this technique. However, in this study only 2 patients with a bacterial infection of the CNS (Borrelia burgdor/eri and Treponema pallidum) had weakly visible IgA bands in the CSF. The finding of a loss of detectable oligoclonal bands by coating of NC with increasing concentrations of goat antibodies to human inmmnoglobulins demands further explanation. One possibility is that higher concentrations of "'capture antibodies" might bind to a higher number of free epitopes on human immunoglobulins preventing the binding of"detection antibodies". Another explanation might be that the fraction of antibodies used for the coating of NC are not completely specific for human immunoglobulins (G, A or M) but also contain antibodies to irrelevant proteins. In conclusion, IEF in combination with affinity immunoblotting is a simple, sensitive and rapid technique for the analysis of oligoclonal lgG, IgA and IgM antibody bands in inflammatory diseases of the CNS. The presence of 100 ng of lgG, lgM or lgA (10 rag/l) allows detection of oligoclonal bands in less than 4 h.
Acknowledgements This study was supported by grant no 01KI9001 from the German Federal Ministry for Research and Technology (Bundesministerium ftir Forschung mad Technology).
References DOmes, R. and V. ter Meulen (1984) Detection and identification o1" virus-specific oligoclonal lgG in unconeentrated cerebrospinal fluid by immunoblot technique. J. Neuroimmunol., 7: 77-84. DOrries, R., R. Kaiser and V. ter Meulen (19891 Human immunodeficiency virus infection: affinity-mediated imumnoblot detects intrathecal synthesis of oligoclonal lgG specific for individual viral proteins. AIDS Res. hum. Retrovir., 5 (3): 303-310. Felgenhauer, K. and H.-J. Schfidlich ( 1987)The compartimental IgM and IgA response within the central nervous system. J. Neurol. Sci., 77: 125-135. Forsberg, P.. A. Henriksson, H. t.ink and S. ()lman (19841 Reference values for CSF-IgM, CSF-lgM/serum-lgM ratio and its application to patients with multiple sclerosis and aseptic menmgoencephalitis. Scan. J. Clin. Lab. Invest., 44: 7-12. Fryddn, A.. H. Link and E. Norrby (1978) Cerebrospinal fluid and serum immunoglobulins and antibody titers in mmnps meningitis and aseptic meningitis of other etioh)gy. Infect. lmmun., 21: 852-861. livanainen, M.V., W. Wallen, M.E. Leon, J. Keski-Oja, V. Calabrese, M. Krasny, E. Waybright, J. Selhorst, J.W. Harbison, D.L Madden and J.L. Sever (19811 Micromethod for detection of oligoclonal IgG in unconcentrated CSF by polyacrylamide gel electrophoresis Arch. Neurol., 38: 427-430. Kaiser, R.. R. D6rries, W. Ltier, S. Poser, K. Felgenhauer and V. ter Meulen (1989) Analysis of oligoclonal antibody bands against individual HIV structural proteins in the CSF of patients infected with HIV. J. Neurol., 236: 157-160. Kelly J.J. (19851 Peripheral neuropathies associated with monoclonal proteins: a clinical review. Muscle Nerve, 8: 138-150. Kostulas, V. (1985) Oligoclonal lgG bands in eerebrospinal fluid: methodological and clinical aspects. Acta Neurol. Stand., 72 (suppl. 1031: 1-112. Kostulas, V.K. and H. Link ( 19821 Agarose isoelectric focusing of unconccntrated CS F and radioimmunofixation tbr detection of oligoclonal bands in patients with multiple sclerosis and other neurological diseases. J. Neurol. Sci., 54:117-127. Kruse. H., H. Reiber and H.I. Schipper (1982) Bestimmung oligoclonaler IgG-Subfraktionen in Liquor und Serum, LKB Application Note, LKB, Sweden. Laurenzi, M,A. and H. Link (19781 Localization of the immunogloculins G, A and M, fl-trace protein and gamma-trace protein on isnelectric focusing of serum and cerebrospinal fluid by immunofixation. Acta Neurol. Scand., 58: 141-147. Mattson, D.H., R.P. Roos and B.G.W. Arnason (1981) Comparison of agar gel electrophoresis and isoelectric focusing in multiple sclerosis and subacute sclerosing panencephalitis. Ann. Neurol., 9: 34-41. Pohl, P., E. Schmutzhard and G. Stanek (19861 Ccrebrospinal fluid findings in neurological manifestations of lyme disease. Zbl. Bakteriol. Hyg., A263: 314-320. Poser, C.M., D.W. Pat}', L.C. Scheinberg, W.t. Mcl)onald and G.C. Fbers (Eds.) (1984)The Diagnosis of Multiple Sclerosis, Thiemc Stranon Inc., New York, NY. Sindic, CJ.M., D.L. Delacroix, J.P. Vaerman. E.C. l,aterre and P.L. Masson ( 19841 Study oflgA in the cerebrospinal fluid of neurological patients with special reference to size, subclass and local production. J. Neuroimmunol., 7: 65-74. Tourtellotte, W.W. and M.J. Walsh (19841 Cerebrospinal fluid profile in nmltiple sclerosis. In: Poser, C.M. et al. (Eds.) The Diagnosis of nmltiple Sclerosis, Thieme Stranon Inc.. Ne~ York. NY, pp. 165-178.
216
g 1991 Elsevier Science Publishers B.\ 1/022 510X,~1~$0~.>t~
JNS 03550
Affinity immunoblotting: rapid and sensitive detection of oligoclonal IgG, IgA and IgM in unconcentrated CSF by agarose isoelectric focusing R. Kaiser Department of Neurology, University of Freiburg, Freiburg (Germany)
(Received 2 October, 1990) (Revised, received 22 January, 1991) (Accepted 25 January, 1991)
Key words."IEF/affinity mediated immunoblotting; CSF; Oligoclonal IgG, IgM and IgA; Multiple sclerosis; Neuroborreliosis Summary
Two methods for the investigation of oligoclonal immunoglobulin bands in cerebrospinal fluid are compared. Isoelectric focusing (IEF) in agarose gels combined with a highly sensitive affinity immunoblotting proved to be superior to PAGE-IEF and silver staining regarding detection sensitivity and expenditure of labour. The procedure presented here allows examination of oligoclonal bands not only of IgG, but also of IgA and IgM antibodies in less than 4 h. Oligoclonal IgM bands could be detected in 6 patients with neuroborreliosis or neurosyphilis as well as in one patient with a peripheral neuropathy where other electrophoretic methods, including immunoelectrophoresis, were negative. Oligoclonal IgA bands were demonstrated only in 2 patients with a bacterial infection of the CNS. In patients with multiple sclerosis amounts of IgM and IgA in the CSF were too low to allow investigation of oligoclonal IgM or IgA bands. Introduction
The presence of oligoclonal IgG in cerebrospinal fluid (CSF), demonstrable by isoelectric focusing (IEF), is a well known phenomenon in various inflammatory diseases of the central nervous system (CNS) (Kostulas 1985). Over 90~o of patients with multiple sclerosis (MS) (Tourtellotte and Walsh 1984) and approximately one-third of those with acute meningitis show CSF oligoclonal bands (Fryd6n 1978). A variety of techniques including electrophoresis in agarose gels (Mattson 1981), in sodium dodecyl sulfatepolyacrylamide gels (SDS-PAGE)(Iivanainen 1981), isoelectric focusing (IEF) in PAGE (Laurenzi and Link 1978) and IEF in agarose gels (Kostulas and Link 1982) were used to analyze these bands. Some of these methods are hampered by the necessity to concentrate the CSF or by long incubation times for Coomassie blue or silver staining. These problems have been recently overcome by IEF in combination with an affinity mediated immunoblot (AMI), a technique primarily designed to investigate virus-specific oligoclonal IgG bands (Domes and ter Meulen 1984; DOrries et al. 1989; Kaiser et al. 1989). Correspondence to: Dr. Reinhard Kaiser, Department of Neurology, University of Freiburg, Hansastrasse 9, D-7800 Freiburg, F.R.G. Tel.: 0761-2705301; Fax: 270-7200.
A common feature in neuroborreliosis and some other chronic bacterial infections is intrathecal synthesis not only of IgG, but also of IgA and IgM antibodies (Felgenhauer and Sch~tdlich 1987; Pohl et al. 1986). Oligoclonal IgM might also be found in patients with a peripheral neuropathy in association with a "monoclonal gammopathy" (Kelly 1985). The widely used routine procedure for the investigation of oligoclonal IgG bands - focusing in polyacrylamide gels, immunofixation and silver staining - is, however, not suitable to demonstrate oligoclonal IgA and IgM bands. Due to the limited pore size of the gel these proteins of high molecular size mostly do not enter the gel. The aim of this study therefore was to investigate an affinity immunoblotting procedure combined with IEF as a suitable method for the rapid and sensitive demonstration of oligoclonal IgG, IgA and IgM bands in routine CSF sample examination.
Materials and methods
Samples Paired specimens of CS F and serum were obtained from fifty patients from whom samples were taken at the Department of Neurology, University of Freiburg, for analysis regarding the presence of oligoclonal IgG bands in CSF.
217 TABLE 1 MEAN AND RANGE OF" IgG, IgM and IgA INDICES
Normal values Study group: Multiple sclerosis 01 - 27)
IgG index
IgM index
IgA index
< 0.7
< 0.06
< 0.4
1.52 0.5-2.0
Neuroborreliosis, neurosyphilis (n = 10) Encephalitis, myelitis, GBS, meningitis, polyneuropathy (n = 13)
0.03 0.002-0.15
~).12 0.01-0.31
2.1
1.0
0.5-5.0
0.21-4,1
().44 O.02-1.0 I
0.6 0.5-1.3
0.04 0.01-0.15
0,13 0.007-0.32
TABLE 2 F I N D I N G S FROM CSF IgG STUDIES Patient
Diagnosis
IgG index
IgG-OCB
NO.
PAGE-IEF ].
2. 3. 4. 5. (L Y.
t}.
1!). II. 12. 13. 14. 15. 16. 17. I~. 19. 20. 21. 22. 23. 24. 25. 26. 27.
MS
29. 28. 3o. 31. 32. 33.
i
34. 35. 36. 37.
]
[
[
2.0 1.9 1.8 1.8 1.7 1.6 1.5 1.4 1.4 1.1 1.1 1.0 1.0 0.9 0.9 0.9 0.8 0.8 0,7 0.7 0.7 0.7 0.7 0.7 0.7 0.7 0.5
Neuroborreliosis
1.3 0.8 0.7 0.7 0,7 0.5
Neurosyphilis
2,6 3.3 5.0 2.2
Agarose-1 EF,'AMI
+ + + + t 4+
+ + 4 +
+
+
+ + +
+ + 4 +
218 TABLE 2 (continued) Patient No.
Diagnosis
lgG index
tgG-OCB PAGE-IEF
~\garo so- I E F:A M I
1.3 O.6 0.5
+ 0 0
o 0
0.4
0
0
38. 39. 40. 41.
Encephalitis
42.
Myelitis
0.5
0
*
Meningitis
0.7 0.5
+ 0
4 It
4
43. 44.
/ J"
45. 46. 47.
/ /
GBS
0.6 0.6 0.5
0 0 0
0 0 •
48. 49. 50.
] /
Polyneuropathy
0.5 0.5 0.6
0 0 0
• 0 0
< 0.7
0
0
Normal values
MS = multiple sclerosis; GBS = Guillain-Barr6 syndrome; 0 = no antibody bands detectable - polyclonal IgG staining; + = oligoclona[ lgG-bands (OCB) occurring predominantly in the CSF; * = OCB of similar staining intensity in CSF and serum.
Clinically defined MS (Poser et al. 1984) was diagnosed in 27 patients (study group 1), 10patients had neuroborreliosis or neurosyphilis (study group 2) and 13 subjects presented with other inflammatory diseases of the central or peripheral nervous system (study group 3) (Table 1 and 2). Samples were stored at - 2 0 °C and thawed prior to use. IgG, IgM, IgA and albumin levels were determined by laser nephelometry. In the case of IgM and IgA concentrations in the CSF lower than 10 mg/1 levels in paired sera and CSF were determined by ELISA. Intrathecal synthesis of antibodies was determined separately for each class of immunoglobulins according to the method of Delpech and Lichtblau: the following indices were taken as being indicative of local synthesis: IgG: >0.7, IgA: >0.4 and IgM: >0.06 (Forsberg et al. 1984; Sindic et al. 1984). The mean and range of these indices according to the three study groups are listed in Table 1.
The plates were then incubated for 1 hour at room temperature. Serum and CSF samples were diluted in PBS-Tw 1 : 10.000 and 1 : 100.000 (serum) and 1 : 10 and 1 : 100 (CSF). Reference sera (Behringwerke, Marburg, F.R.G) for each Ig class were adjusted to an IgA or IgM concentration of 200 #g/1 and diluted in 2-fold steps to a final IgA or IgM concentration of 6.25 #g/1. The plates were washed and samples and reference samples (100 #1 per well) were added. After 2 h plates were washed again and peroxidaselabelled rabbit anti human IgA or IgM antibodies (1:2000 in PBS-Tw, 100/21 per well, Dako, Hamburg, F.R.G.) were added for 1 h at room temperature. After repeated washing o-phenylenediamine (OPD) was added and the absorbance at 492 nm measured with a Titertek Multiscan reader (FlowLab). The amounts of IgM and igA in samples were determined from standard curves obtained from standard serum.
Determination of IgA and IgM concentrations Microtiter plates (Nunc Immunoplates) were coated with rabbit antiserum against human IgM or IgA antibodies (Dako, Hamburg, F.R.G.) diluted 1:1000 in 0.05 M sodium carbonate buffer, pH 9.6. 100 #1 of this solution was added to each well. After incubation overnight at 4 ° C the antibody solution was removed from the wells and 0.1 ~o bovine serum albumin (BSA) in phosphate buffered saline (PBS: 1.38 g NazHPO4, 0.24 g NaH2PO4, 8.0 g NaC1 and 1000 ml distilled water, pH 7.6) containing 0.1 ~o Tween 20 (PBS-Tween) was added to block free protein binding sites.
Isoelectric focusing PA GE-IEF and silver staining A standardized procedure for PAGE-IEF and silver staining was used for the routine investigation of oligoclonal bands (Kruse et al. 1982). The polyacrylamide gel (Pharmacia-LKB PAG Plate, pH 3.5-9.5) was placed on a temperature controlled flatbed (Pharmacia-LKB Multiphor, Multitemp Circulator). Samples of CSF and serum (20/~1) adjusted to an IgG concentration of 20 mg/1 were applied on filter paper tabs 3 cm from the anode. The gel was
219 focused for a total of 2000 Vh at 4 °C. The power supply (Pharmacia) was set at 1500 V, 50 mA and 30 W. After focusing the gels were fixed and stained according to Kruse et al. (1982). This procedure takes about 30 h for the evaluation of results and does not allow detection of oligoclonal IgA or IgM bands.
for 30min at 37°C to reduce IgM and lgA to the monomeric form. C S F s a m p l e s containing less than 80 mg/1 of IgA or IgM were diluted to 10 mg/l (45 bd) and then mixed with 5#1 D T T (0.01, 0.002, 0.0004 and O.O0OO8 M).
Isoelectric focusing Agarose-lEF and ql]finity immunoblotting The method described by DOmes et al. (1984, 1989) was used with somc modifications as follows.
Antibody loading ((nitrocellulose fihers for immunoblotting Nitrocellulose (NC) filters (BA 83, Schleicher and Schall) were cut to 10 x 10 cm 2 sized sheets and loaded separately with goat antibodies to human IgG, IgM or IgA (Dianova, Hamburg, F.R.G., Nos. 109-002-011, 109-002003, 109-002-043) by overnight incubation at room temperature on a rocker platform. Antibodies were diluted in PBS to the following concentrations: 25, 50, 100, 200 #g/ml/10 cm 2 (corresponding to 20 #g/cm2). After overnight incubation, filters were rinsed briefly in PBS and unoccupied protein bindings sites on the filter were blocked with PBS-Tween for 1 h.
Preparation oJspecimens For screening of the optimal Ig concentrations samples were each adjusted to an IgG, IgA or IgM level of 40 mg./1 and then diluted in two fold steps to at least 5 rag/1. Samples to be assayed for oligoclonal IgM or IgA antibodies were studied both in the native state and after mild reduction with dithiothreitol (DTT). In this case specimens were diluted to 80 mg/1 mixed with an equal volume (50 #1) of D T T (0.1 , 0.02, 0.004 and 0.0008 M) and then incubated
Coa t i YN
uq/a
Thin agarose gels (0.5 mm) containing 0.9"{, IEFagarose (Pharmacia), 12~',, sorbitol, 1",, Nonidet P40 (NP 40, Sigma) and Pharmalyte (Pharmacia) were cast at 75 °C by capillary action between 2 glass plates onto a Gelbond sheet (FMC). For IEF o f l g G antibodies gels were supplemented with 6.2 ° 0 Pharmalyte pH 3-10, while focusing of IgM and IgA antibodies was carried out with gels containing 3.1 °~, Pharmalyte pH 3-10 plus 3.1", Pharmalyte pH 4-6.5. The plates were "aged" at 4 : C in a moist chamber for 1 h. Focusing was performed on a Multiphor apparatus (Pharmacia-LKB, Freiburg) at 17 :C. The electrode strips contained 0.5 M N a O H (catholyte) or 0.115 M H2SO 4 (anolyte). Samples (10 btl) were applied directly to the gel using a silicone application mask having 8 x 8 mm slots. After focusing the gel was dipped for a few seconds in distilled water to remove unfocused surface material. Subsequently, focused antibodies were blotted onto nitrocellulose strips with goat antibodies to either human lgG, lgM or IgA. The nitrocellulose sheet was laid on the agarose gel carefully to avoid air bubbles, covered with one sheet of cellulose acetate, 3 layers of Whatman filter C H R 1, a glass plate and a weight of approx. 200 g. After 60 min the nitrocellulose was removed and v~ashed for 1(I min in PBSTween. Bound antibodies were detected by incubating the sheets with the following dilutions of peroxidase-con-
IEF-PATTERN
2
k~
2,5 5 I
lit
20
J
-
I
IIII
=
Fig. 1. Effect of antibody loading of nitrocellulose sheets with regard to sensitivity' in detecting oligoclonal bands. NC sheets were coated with rising concentrations of goat anti human IgG, 20/*g/cm 2 corresponds to 200/~g/ml/10 cm a. A total amount of 100 ng IgG was focused. Peroxidasc-labelled goat anti-human-lgG was diluted 1 : 500. Black arrows indicate the pH gradient, from left to right: pH 6.5, pH 8.6.
220
Fig. 2. Sensitivity in detection of oligoclonal IgM bands: l0/~1 of CSF was diluted from 20 to 5 mg/l (50-200 ng of total lgM). NC sheets werc coated with goat anti-human IgM, 50 #g/ml/10 cm2. Peroxidase-labelled goat anti-human IgM was diluted 1:500. Black arrows indicate the pH gradient, from left to right: pH 4.0, pH 6,5. j u g a t e d goat anti-human IgG, IgA or IgM (Dianova, H a m b u r g , F R G , Nos. 109-035-003, 109-035-011,109-035043): 1 : 250, 1 : 500, 1 : 1000 and 1 : 2 0 0 0 in P B S / T w e e n . After 1 h at r o o m t e m p e r a t u r e on a rocking platform, blots were rinsed 3 times with P B S - T w e e n a n d then immersed in substrate solution (3 mg/ml 4-chloro- I- naphthol, 0.03'!~0 hydrogen peroxide in P B S ) for 15 min. The blots were w a s h e d with water and air-dried.
Results
Assay conditions In preliminary experiments 64 c o m b i n a t i o n s o f assay conditions were tested for each antibody class: antibody
loading o f N C - Ig concentrations o f samples - dilution o f peroxidase-labelled second antibody. Each p a r a m e t e r was examined in 4 dilutions.
Antibody loading of NC sheets The effects o f variations in the concentration of antibodies b o u n d to the N C were determined. Fig. 1 shows the results o f varying the antibody concentration on detection of h u m a n I g G antibodies. 10 #1 samples of C S F adjusted to an I g G concentration o f 10 mg/1 were separated by I E F and blotted to N C which h a d been c o a t e d with varying concentrations o f goat anti-human IgG. The peroxidaselabelled a n t i b o d y was diluted 1 : 500. The results showed that the optimal range o f antibody coating was between 25
Fig. 3. Sensitivity of IgG detection: 10 #1 of CSF was diluted from 40 to 5 rag/1 (50-400 ng of total IgG). NC sheets were coated with goat anti-human IgG, 50 #g/ml/10 cm2. Peroxidase-labelled goat anti-human IgG was diluted 1 : 500. Black arrows indicate the pH gradient, from left to right: pH 6.5, pH 8.6.
221 and 100/~g/ml/10 c m 2 with 50/,g/ml/10 c m 2 probably being the best loading concentration. In the case of lgA and IgM antibodies similar results were obtained; between 25 and 50/~g/ml of goat antibodies to human lgA or lgM were necessary to coat 10 cm 2 of NC area to demonstrate Ig class specific oligoclonal antibodies. Increasing the amounts of loading antibodies ( > 100/~g/ml/10 cm e) resulted in a loss of sensitivity in detecting focused antibodies but slightly improved the demonstration of their oligoclonal nature. Coating of NC with less than 10 #g/ml/10 c m 2 of Ig class specific antibodies gave an unacceptably high costaining of polyclonal antibodies making it difficult to detect single antibody bands. Loading conditions of antibodies on NC sheets are given in/xg/ml/cm= to indicate that both factors, amounts of antibodies (Hg) and volume of buffer (ml) will influence zmtibody binding to the NC sheet (data not shown).
1~ concentrations.fi~r lran,sJ'er procedure.s Employing an optimal antibody loading of NC of 50 Hg/ml/10 cm e the optimal range of lg antibodies to be [\)cused was determined. 50-100 ng (5-10 mg/1) of IgG, lgA or lgM were found suitable for application on IEF to reveal clearly xisible oligoclonal bands. Lowering the amounts ol" focused antibodies to 25 ng resulted in an visible loss of bands (Fig. 2). If more than 200 ng of IgG, IgA or IgM antibodies were focused the amounts of polyclonal antibodies increased considerably so that single bands were difficult to differentiate (Fig. 3). For comparative studies of oligoclonal IgG bands in PAGE-IEF and agarose-IEF (50 patients) the IgG concentration was therefore adjusted to 10 mg/l for focusing in agarose gels.
ant i
-
IqC
Sign(lk;ance 0/" second antibody dihttion The minimal concentration of second antibody for den> onstrating oligoclonal bands is largely dependent on the amounts of Ig bound to NC. Using the above mentioned optimal conditions for coating of NC sheets and focusing of samples peroxidase labelled antibodies had to be diluted 1:500 to 1:1000 (Fig. 4). Higher dilutions reduced the staining intensity and number of detectable bands while lower antibody dilutions increased the amounts of costained polyclonal immunoglobulins. This was shown for IgG, lgA and IgM antibodies.
El]~,cts q/ sample treatment with dithiothreitol IgA and lgM antibodies not treated with DTT prior to IEF did not enter the gel and mostl? left at the point of application. After treatment with rising conccntrations of DTT samples focused mainly in the pH-region 4-6.5. Best results were obtained after diluting CS F and serum samples to 10 mg/I of IgA or IgM (45 IH) and reducing the pcntameric IgM and dimeric IgA to monomeric forms with 0.0004 M DTT (5 ~H) for 30 rain at 37 C . Identical specimens were applied at the anode and cathode of the same gel and focused for 1 h. Affinity blotting revealed bands of identical pI in both samples indicating optimal sample treatment. Increasing the concentration of DTT produced a high "background staining" and a loss of oligoclonal lgM or IgA bands. Discrimination of oligoclonal IgA and lgM bands was improved by spreading the pH gradient of the gel between pH 4 and 6.5.
IEF-PATTERN
1:258 1:508
1:188
,~ 4>'
~4~
Fig. 4. Significance of second antibody' dilution: Peroxidase-labelled goat anti-human lgG was diluted 1:250 to 1 "2000. Antibody loading of NC shccts: 50/tgml I() cm e, a total amount of 100 ng lgG was focused. Black arrows indicate the pH gradient, from left to right: pH 6.5, pH 8.6.
222
Fig. 5. Oligoclonal IgG bands of a patient with multiple sclerosis. Comparison of results from PAGE-IEF and silver staining with agarose-lEF and affinity mediated immunoblotting. Isoelectric focusing was performed with 400 ng of IgG in case of PAGE-IEF and with 200 ng of IgG in case of agarose IEF.
Fig. 6. Oligoclonal bands of IgG, IgM and IgA in a patient with neuroborreliosis. Investigation by IEF and affinity mediated immunoblotting.
223
Comparison of results ,from PA GE-IEF and agarose-IEF Oligoclonal bands of high intensity in the CSF generally were visible both by PAGE-lEE and silver staining as well as by agarose lEE and affinity-mediated blotting (AMI). However, oligoclonal IgG bands of low intensity were better detected by agarose lEE and AMI. Silver staining of CSF and serum proteins focused in polyacrylamide gels frequently creates bands which are not related to the IgG fraction. Fig. 5 demonstrates a comparison of results from IEF in polyacrylamide gels and lEE in agarose gels of a patient with nmltiple sclerosis (No. 20). Using PAGE-IEF and silver staining 37 of 50 patients (74°~,) tested revealed oligoclonal IgG bands predominantly in the CSF. In all of these cases the IgG index was
> 0.7. Using agarose-IEF and AMI oligoclonal IgG bands were demonstrated in 42 patients (8400). In 39 subjects bands were visible predominantly in the CSF. The lgG index was elevated in 38 patients (Table 2). Of the two patients with oligoclonal IgG bands detected by agaroseIEF and affinity immunoblotting but not by PAGE-lEE, one (No. 16) had multiple sclerosis (IgG index 0.9) and the other (No. 33) presented with an acute neuroborreliosis (IgG index 0.5). In 3 patients IgG bands occurred with a similar staining intensity in CSF and sermn indicating derivation of these antibodies from the serum. One of these patients (No. 42) presented with a myelitis probable due to viral infection, one (No. 47) suffered from Guillain-Barr6 syndrome and one patient (No. 48) had a polyneuropathy in association with an lgG,"IgM gammopathy. In this
"1ABLE 3 F I N D I N G S FROM CSF IgM AND lgA STUDIES
Patient No.
Diagnosis
lgM index
IgM-OCB
lgA index
IgA-OCB
0.(11 (].02 0.11 0.003 0.01 0.08 0.03 0.01 0.01 0.006 0.03 O. 15 0.05 0.04 0.01 0.02 0.002 0.03 0.03
n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
0.21 0.08 0.22 0.15 0.04 0.20 0.23 (I.25 0.12 0.12 II.O 1 0.06 0.05 0.02 0.06 0.06 0,24 0,16 O. 12
11.d. n.d. n.d n.d n.d. n.d n.d. n.d. n.d. n.d n.d. n.d. n.d. n,d. n.d. n,d. n d. n.d. n.d.
20. 21. 22. 23.
0.006 0.06 0.006 0.01
n.d. n.d. n.d. n.d.
0.03 O. 14 0.05 I). 13
n.d. n.d. n.d. n.d.
24. 25. 26. 27.
0.04 0.03 0,007 0.05
n.d. n.d. n.d. n.d.
i). 16 0.31 0.03 0.08
n.d. n.d. n,d. n.d.
28. 29.
4.11
0.55 1.01
0 +
0.72 0.33 0.31 0.02
0 0 0 0
0.25 0.11 1.00 0.12
0 0 + 0
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. I1. 12. 13. 14. 15. 16. 17. 18. 19.
3(1. 31. 32. 33. 34. 35. 36. 37.
MS
Neuroborreliosis
Neurosyphilis
2.20 1.00 0.52 0.71 0.54 0.25 (I.25 0.52 0.21
224 TABLE 3 (continued) Patient No. 38. 39. 40.
Diagnosis
IgM index
IgM-OCB
IgA index
IgA-O('B
Encephalitis
0.15 0.01 (I.06 0.09
n.d. n.d. n.d. n.d.
(I.14 0.05 0.(18 0.03
n.d. n.d n.d. n.d.
42.
Myelitis
0.02
n.d.
0.15
n.d.
43. ] 44. f
Meningitis
0.01 0.05
n.d. n.d.
0.21 0.06
n.d. n.d.
41.
45. ] 46. / 47.
GBS
0.01 0.02 0.02
n.d. 0 0
0.07 0.16 0.28
n.d. 0 0
48. | 49. / 50.
Polyneuropathy
0.01 0.02 0.03
* 0 0
0.22 0.12 0.007
0 (i 0
< 0.06
0
Normal values
< 0.4
0
IgM/lgA-OCB, oligoclonal bands (OCB) of IgM/IgA antibodies; 0, polyclonal staining oflgM or Igm antibodies; +, OCB occurring predominantly in the CSF; *, OCB detectable predominantly in serum; n.d., lgM or IgA antibody concentration in the CSF too low ( < 5 mg/l) to be detectable by IEF,
patient affinity immunoblotting not only demonstrated oligoclonal IgG bands (pH region 8-10) but also oligoclonal IgM bands (pH region 4-6) (Table 3). In both cases antibody bands were derived from the serum. Immunoelectrophoresis of the serum was negative in respect to a monoclonal IgM gammopathy.
Oligoclonal IgM and IgA bands Due to the low concentrations of IgM and IgA in the CSF ( < 5 mg/1) oligoclonal bands of these immunoglobulin classes could be investigated only in a limited number of patients (15 of 50). Oligoclonal IgM bands in the CSF (pH4-6.5) were detected in 6 of 10patients with neuroborreliosis or neurosyphilis (Nos. 28, 29, 30, 32, 34, 35) all of whom had elevated IgM indices (mean: 1, 42; range: 0.52-4.11). As mentioned above, one patient with a peripheral neuropathy displayed oligoclonal IgM bands predominantly in serum. The other patients tested revealed a polyclonal staining of IgM antibodies in CSF and serum. Oligoclonal IgA bands in the CSF were detectable only in one patient (No. 29) with neuroborreliosis (IgA index: 0.7) and one (No. 36) with neurosyphilis (IgA index: 1.0). IgA bands were only of weak intensity. Fig. 6 illustrates the finding of oligoclonal IgG, IgM and IgA bands in a patient with neuroborreliosis (No. 29). In none of these cases could oligoclonal IgM or IgA antibody bands be detected by PAGE-IEF.
Discussion
The aim of this study was to compare a recently developed immunoblot technique with a routine IEF method for demonstrating oligoclonal immunoglobulin bands. The most commonly used procedure to investigate oligoclonal bands in daily routine examination is IEF in polyacrylamide gels with silver staining. Although this technique is reliable and sensitive, it takes about 30 h to provide results. As shown by the finding of an additional 5 patients with oligoclonal bands demonstrable only by IEF and immunoblotting the assay described here might be even more sensitive and requires less than 4 h for performance. Another aim of this study was to modify the immunoblot technique in order to investigate otigoclonal lgA and IgM antibody bands. Large molecules (> 200 kDa) are difficult to focus in polyacrylamide IEF gels due to sieving effect of the cross-linked gels. This problem can be partly overcome by the use of low percentage agarose gels (0.8-0.9°~o). However in the case of IgM (900 kDa) this procedure was found to be inadequate for separating these immunoglobulins (data not shown). Sample treatment with 0.0004 M DTT (see above) reduce the disulfide bonds of the 5 subunits of the IgM pentamer, facilitating the focusing of the monomers in the IEF gel. The use of a higher concentration of DTT resulted in a loss of single IgM bands and an increase of diffuse antibody staining. In this case further disulfide bonds (i.e., inter- and intrachain disulfide bonds)
225
might be broken, producing inmmnoglobulin breakdown products. The inability to demonstrate oligoclonal IgM or IgA bands by PAGE-IEF and silver staining is connected with the fact that this staining procedure does not discriminate between certain proteins, i.e. lgG, IgA or IgM. Another aspect is that CS F samples with detectable amounts of lgM or IgA antibodies would require a 10-fold lower dilution to examine oligoclonal IgM or IgA bands than to investigate lgG bands (different concentration of these immunoglobulin classes in the CSF). As all proteins will be stained (silver staining) high amounts of other proteins (i.e., IgG) would prevent detection of single IgM or lgA bands. The finding of an oligoclonal IgM immune response in the CS F of 6 and a polyclonal IgM imnmne response in the CSF or a further 4patients with neuroborreliosis or neurosyphilis might indicate different stages of disease or different immune mechanisms involved. Further studies, using NC sheets coated with Borrelia burgdor/'eri antigen, have shown that these bands indeed are at least partially reactive with this pathogenic agent (manuscript in preparation). Follow up studies will show whether this observalion is of diagnostic or prognostic value in patients with CNS infection. Patients with oligoclonal IgM bands in the CSF always had elevated IgM indices (Table 3). In onc patient who suffered from a polyneuropathy and a monoclonal IgG gannnopathy demonstrated by immunoelectrophoresis, I EF and immunoblotting disclosed not only oligoclonal lgG but also clearly visible IgM bands, predominantly in the serum. Amounts of IgM were too low to be detectable by immunoelectrophoresis. Further studies are in progress to investigate the reactivity of these bands to peripheral nerve proteins. Oligochmal lgA bands were hardly discovered in the CSF of any patient tested. Preliminary studies done with sera of a patients with a monoclonal lgA gammopathy had confirmed the reproducibility of this technique. However, in this study only 2 patients with a bacterial infection of the CNS (Borrelia burgdor/eri and Treponema pallidum) had weakly visible IgA bands in the CSF. The finding of a loss of detectable oligoclonal bands by coating of NC with increasing concentrations of goat antibodies to human inmmnoglobulins demands further explanation. One possibility is that higher concentrations of "'capture antibodies" might bind to a higher number of free epitopes on human immunoglobulins preventing the binding of"detection antibodies". Another explanation might be that the fraction of antibodies used for the coating of NC are not completely specific for human immunoglobulins (G, A or M) but also contain antibodies to irrelevant proteins. In conclusion, IEF in combination with affinity immunoblotting is a simple, sensitive and rapid technique for the analysis of oligoclonal lgG, IgA and IgM antibody bands in inflammatory diseases of the CNS. The presence of 100 ng of lgG, lgM or lgA (10 rag/l) allows detection of oligoclonal bands in less than 4 h.
Acknowledgements This study was supported by grant no 01KI9001 from the German Federal Ministry for Research and Technology (Bundesministerium ftir Forschung mad Technology).
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