Journal of Immunological Methods, 144 (1991) 63-67 © 1991 Elsevier Science Publishers B.V. All rights reserved 0022-1759/91/$03.50

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JIM 07002

A new qualitative method for detecting IgD in unconcentrated cerebrospinal fluid Milica Mavra, Richard Luxton, Geoffrey Keir, Vojislav Miletic * and Edward J. T h o m p s o n Department of Special Chemical Pathology, The National Hospital for Neurology and Neurosurgery, London, U.K. (Received 21 March 1991, revised received 11 June 1991, accepted 1 July 1991)

We report a method which is capable of demonstrating the isoelectric focusing (IEF) pattern of immunoglobulin D in unconcentrated cerebrospinal fluid (CSF) samples containing as little as 0.1-0.5 ng of total IgD. The method used was an immuno-sandwich technique, with alkaline phosphatase enzyme amplification. Oligoclonal and polyclonal IgD patterns were seen in CSF samples. No cross-reactivity with other immunoglobulins (IgG, IgA and IgM) was detected. Key words: IgD; Isoelectric focusing; Cerebrospinal fluid

Introduction

Investigations of the quantity and quality of immunoglobulins in cerebrospinal fluid (CSF) have been of value in the diagnosis of neurological diseases, especially in various inflammatory and demyelinating nervous system diseases (Thompson and Johnson, 1982; Link, 1987). Little is known about the clinical significance of immunoglobulin D (IgD) in CSF (Out,1987; Lolli et al., 1989). This is mainly due to its extremely low concentration in CSF (Nerenberg and Prasad, 1975; Nerenberg et al., 1978; Lolli et al., 1989). The only reports using isoelectric focusing (IEF) to study IgD in CSF are restricted to cases with paraproteinemia, where increased levels of IgD, both in serum and CSF were present (Schipper

and Prange, 1984; Merelli et al., 1986). Even in these cases, the CSF was concentrated in order to detect IgD. This report describes a technique which is capable of demonstrating the IEF pattern of IgD in unconcentrated CSF samples containing as little as 0.1-0.5 ng of total IgD. IgD is first separated using IEF in an agarose gel and then transferred to an Immobilone-P membrane, precoated with rabbit anti-human IgD. After washing, human IgD is detected using an alkaline phosphatase labelled anti-human IgD antibody. This method permits IEF studies of human IgD in CSF without the disadvantages of preconcentration.

Materials and methods Correspondence to: M. Mavra, Department of Special Chemical Pathology, The National Hospital for Neurology and Neurosurgery, Queen Square, London WC1N 3BG, U.K. * Present address: Institute for Blood Transfusion, Svetog Save 2, 11 000 Beograd, Yugoslavia.

CSF samples were randomly chosen for investigation. Specimens were aliquoted into four tubes, two tubes were treated with e-amino caproic acid (EACA, 30 mmol/l) diluted 1/10 in

64 the sample in order to prevent the degradation of IgD (Spiegelberg, 1977). One each of the treated and untreated aliquots were stored at 4°C and analysed within 7 days for IgD. One of the other two aliquots were frozen for two to three months. Both the frozen and unfrozen aliquots were analysed after that period for a second time. A number of serum samples were prepared and run in parallel with the corresponding CSF specimens.

Coating of membrane A sheet (9 x 20 cm) of Immobilone-P membrane (Millipore, Watford) was cut from a roll and immersed in methanol for 1 rain. The methanol was washed from the membrane with three or four washes in physiological saline. To 50 ml physiological saline 0.2 ml rabbit anti-human IgD antibody (Dako code no. AO93, High Wycombe) was added, into which the wet Immobilone-P membrane was submerged. The membrane was incubated overnight on a rocker at 4°C. The following morning the membrane was removed from the solution of coating antibody and immersed in blocker (2% skimmed milk solution in physiological saline) and rocked at room temperature for one hour while the IEF of sampies was performed.

Isoelectric focusing IEF was carried out on a 1% agarose gel pH gradient 3.0-10.5 (Keir et al., 1990). For 30 ml gels (22.5 x 11.4 cm) 3.6 g Sorbitol (Sigma, Poole) and 0.3 g IEF agarose (Pharmacia, Milton Keynes) were dissolved in 30 ml 10% glycerol (BDH, Dagenham) using a boiling water bath to melt the agarose. The mixture was placed in a 65°C water bath to cool before adding 2.0 ml ampholyte pH 3.0-10.0 and 0.5 ml ampholyte pH 8.0-10.5 (Pharmacia). The IgD level in most of the samples was assayed by a sandwich ELISA method (Sharief et ai., 1991). A volume of unconcentrated CSF containing between 0.1-0.5 ng IgD, with an upper limit of 20 /zl, was applied to the gel. The volume of fluid applied was limited to avoid distortion of the patterns. In cases where a serum sample was run with the corresponding CSF, serum was diluted to give an equivalent amount of total IgD per lane (approximately 1/1000). An IEF pH marker

(BDH, Dagenham) was run in parallel for the measurement of pH range. The samples were applied 2 cm from the anode by means of sample application mask (Pharmacia). The application slots were elongated by cutting out the film between the large and small slots, so that samples up to 20/~1 could be applied. Before putting the application mask in position, the gel surface was carefully blotted with a piece of nitrocellulose membrane cut to the dimensions of the gel. Focusing was carried out for one hour at 35 W / g e l . T h e voltage was set to a maximum of 1250 V to minimize heating. The application mask was removed after 20 min of run.

Transfer of focused IgD to lmmobilone-P membrane When focusing was complete, the surface of the gel was preblotted briefly with nitrocellulose membrane to remove surface material. The sheet of Immobilone-P membrane was then removed from the blocker, blotted to remove excess milk, and laid on the gel, excluding air bubbles. The membrane was covered with a damp sheet of fine filter paper (Whatman 50, Maidstone, Kent) and then ten layers of dry thick filter paper (Postlip, Cheltenham) were applied, followed by a glass plate and weight of 1500 g. The gel was compressed for 20 min on the flat bed electrophoresis cooling plate.

Detection of lgD The Immobilone-P membrane was washed in several changes of tap water, followed by two changes of physiological saline and two changes of diluted blocker (0.2% skimmed milk) for total time of 10 min. Alkaline phosphatase conjugated antibody to IgD (Sigma code no. A-6406) was used at a dilution of 1/1000 (50 /~1 in 50 ml physiological saline containing 0.2% dried milk). Incubation was allowed to proceed for 1 h at room temperature. The Immobilone-P membrane was again washed in water, physiological saline and distilled water for 15 rain.

Alkaline phosphatase staining One each of commercially available tablets of nitroblue tetrazolium (NBT) and 5-bromo-4chloro-3-indolyl phosphate (BCIP) (Sigma) were

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dissolved in 50 ml 0.05 M carbonate buffer p H 9.2. The IgD patterns started to appear after approximately 15 min and the staining procedure was carried out until the patterns became clear, at least for 1 h. Samples with a very low concentration of IgD were left in the staining solution for several hours, and if nothing appeared, the incubation was continued overnight, after which the majority of samples were clearly visible. After staining, the I m m o b i l o n e - P m e m b r a n e was washed in water, laid on a glass plate, blotted with filter p a p e r and dried with hot air.

Investigation of cross-reactivity with other immunoglobulins To investigate whether other major immunoglobulins (IgG, IgA and IgM) might contribute to the I E F patterns when unconcentrated CSF was analysed for IgD, sera from patients with paraproteinemias G, A and M were run on the same gel as sera from three cases of paraproteinemia D, which were used as 'positive' controls. The amounts of the controls for IgD, IgA, IgG and IgM were adjusted to be approximately 50 ng per sample. To test the specificity of the capture antibody we run a number of samples twice on the same gel. Oligoclonal I g G was used instead of monoclonal I g G which can give an atypical reaction with the detecting antiserum. After blotting, one m e m b r a n e was stained for IgD and the other m e m b r a n e was stained with anti-IgG or IgA or IgM or anti-K and anti-A light chains of immunoglobulins.

Results

In the majority of CSF samples, IgD was clearly seen. The most satisfactory results were obtained with volumes of unconcentrated CSF between 10 and 20 ~1 containing approximately 0.1-0.5 ng IgD per sample. In the case of CSF samples with higher concentration than this it was necessary to adjust the volume applied or even to dilute the sample in order to avoid overloading of Immobilone-P m e m b r a m e , resulting in a loss of resolution. The low level IgD samples, with no detectable IgD patterns, were rerun loaded with a maximum of 2 0 / z l of sample and then incubated

1 2 3 + Fig. 1. IEF IgD patterns in unconcentrated CSF specimens, 10/zl. CSF specimens with three different amounts of IgD per lane: (1) CSF sample containing 0.05 ng IgD; (2) CSF sample was overloaded and contained 1.2 ng IgD; (3) CSF sample containing 0.4 ng IgD.

in the staining solution overnight. Fig. 1 shows an example of the different staining intensities. Most of the IgD appeared in the p H region between 5.0 and 7.0. Occasionally the IgD was found over a wider p H range, reaching p H 8.3. All three IgD myeloma patterns were found in the p H range 5.0 and 6.0. A parallel run which was stained for I g G showed a different pattern in the p H range of 6.0-10.0. Oligoclonal IgD bands could be easily discerned by looking at the dried sheet of Immobilone-P membrane. The presence of two or more separate IgD bands in the CSF with no counterpart in the homologous serum was regarded as suggestive evidence of intrathecal synthesis of IgD (see Fig. 2.) IgD bands were observed in the

66 Discussion

c

s

cs

1 2 Fig. 2. Sample 1 shows oligoclonal lgD band patterns after IEF in agarose of unconcentrated CSF (C). Paired serum from the same individual (S) shows no bands, i.e. a polyclonal pattern. Sample 2 shows a polyclonal IgD pattern in both CSF and serum.

same region as the polyclonal IgD and also in different regions. Investigations of CSF samples analysed within seven days of a lumbar puncture and repeated after 2 or 3 months, revealed a clear difference in the intensity and the resolution of the patterns. The most distinctive patterns were present in fresh samples. Freezing and thawing of the sample made the patterns less clear and decreased their intensity. When oligoclonal IgD bands were detected in CSF, they became less distinct after 2 or 3 months storage and in some cases disappeared. The presence of inhibitor in most of the samples was not apparently correlated with preservation of the IgD patterns.

IgD occurs at low concentrations in serum (0.25% of the total serum proteins) and yet it is found on the surface of most B lymphocytes as a m e m b r a n e immunoglobulin, usually in association with another immunoglobulin class (Roth et al., 1982; Blattner and Tucker, 1984). It has been proposed that surface IgD has a role in the regulation of B cell populations, and that the secreted form of IgD is a humoral antibody found in the serum of patients with acute and chronic viral infections (Patrick et al., 1990). To understand its role in CSF, it is necessary to overcome the difficulty of detecting IgD in CSF, and only techniques of high sensitivity are able to determine its concentration with acceptable accuracy. The present method, enables IgD I E F patterns in unconcentrated CSF to be detected for the first time. We have adapted an enzyme-amplified immuno-sandwich technique to achieve sufficient sensitivity and thereby avoided the need to concentrate CSF or apply large volumes. Since IgD is usually found within the p H range 5.0-8.5. (Merelli et al., 1986) our choice of wider p H range gels requires explanation. Our initial assays using a p H range from 4.0 to 8.5 gave extended lgD patterns, but the bands detected were of low resolution due to spreading. When the p H range of the gel was extended to 10.5 by incorporating extra basic ampholytes, the p H range of interest was compressed to a smaller width on the gel, and this gave some additional 'concentrating' effect to the lgD patterns, allowing bands to be clearly seen if present. To obtain optimal I E F separation conditions we applied a higher power than that used for our routine IgG I E F runs (35 W versus 19 W per gel). Using optimal cooling and limiting the voltage to 1250 V, this high power presented no problems. The choice of Immobilone-P m e m b r a n e was made after testing a number of different membranes for the transfer of focused proteins. A nitrocellulose m e m b r a n e was effective only in cases of 10-100-fold concentrated CSF samples even when glutaraldehyde was used to enhance the binding of the protein to the m e m b r a n e (Sharief et al., 1989). Other membranes showed

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no advantages over Immobilone-P membrane which was the most sensitive, easier to handle and the least expensive. Prior to IEF, the membrane was precoated with antibody and this proved to be effective in capturing free IgD from the sample. Various IgD detection techniques were investigated. The best results were achieved with a silver-enhanced immunogold assay and the alkaline phosphatase assay. The former method was very sensitive but restricted by the instability of the staining solution when it was necessary to use long incubation times. The silver enhancing reagents proved to be capricious and caused artefacts. The approach finally selected was found to be very sensitive, selective and reproducible. Using different immunoglobulins including oligoclonal IgG, free K and 3. light chains of immunoglobulins and paraprotein immunoglobulins (IgG, IgA and IgM), no cross-reactivity was found with IgD. The main advantage of the alkaline phosphatase method was the ability to adjust the staining procedure to the amount of IgD in the CSF samples, so that IgD could be detected in all CSF samples, even in those with amounts of IgD less than 10 n g / m l by ELISA. Under the conditions described the antibody dilutions used were optimal. Use of higher dilutions reduced the sensitivity or had no effect on the patterns, while lower dilutions led to an increased background. This sensitive and specific qualitative method for the study of IgD may be useful in elucidating the role of IgD in CSF and its involvement in different neurological conditions.

Acknowledgements We acknowledge Dr. M. Sharief for the quantitative measurements of IgD in CSF.

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Keir, G., Luxton, R. and Thompson, E.J. (1990) Isoelectric focusing of cerebrospinal fluid immunoglobulin G: an annotated update. Ann. Clin. Biochem. 27, 436. Link, H. (1987) Cerebrospinal fluid in immunological CNS diseases. In: J.A. Aarly, W.M.H. Behan, P.O. Behan (Eds.), Clinical Neuroimmunology. Blackwell, Oxford, p. 444. Lolli, F., Halawa, I. and Link, H. (1989) Intrathecal synthesis of IgG, IgA, IgM and IgD in untreated multiple sclerosis and controls. Acta Neurol. Scand. 80, 238. Merelli, E., Sola, P., Montagnani, G. and Torelli, G. (1986) Peripheral neuropathy in IgD myeloma. Cerebrospinal fluid paraprotein analysis in three cases. Acta Neurol. Scand. 74, 25. Nerenberg, S.T. and Prasad, R. (1975) Radioimmunoassay for Ig classes G, A, M, D and E in spinal fluid. Normal values of different age groups. J. Lab. Clin. Med. 86, 887. Nerenberg, S.T., Prasad, R. and Rothman, M.E. (1978) Cerebrospinal fluid IgG, IgA, IgM, IgD and IgE levels in central nervous system disorders. Neurology, 28, 988. Out, T.A., Hische, E.A., van Walbeek, H.K. and van der Helm, H.J. (1987) Immunoglobulin D in cerebrospinal fluid. Clin. Chim. Acta. 165, 289. Patrick, B.A., Mehta, P.D., Sobczyk, W., Kulczycki, J., Woyciechowska-Camenga, J., Camenga, D. and Thormar, H. (1990) Measles-virus specific immunoglobulin D antibody in cerebrospinal fluid and serum from patients with subacute sclerosing panencephalitis and multiple sclerosis. J. Neuroimmunol. 26, 69. Roth, P., Tonda, P. and Pernis, B. (1982) Membrane IgD expression and dynamics in clones of human B-lymphoblastoid cells. In: G.J. Thorbecke and G.A. Leslie (Eds.), Immunoglobulin D: Structure and function. NY Acad. Sci. 399, 175. Schipper, H.I. and Prange, H.W. (1984) Cerebrospinal fluid paraprotein in neurological disorders. J. Neurol. Sci. 64, 305. Sharief, M.K., Keir, G. and Thompson, E.J. (1989) Glutaraldehyde-enhanced immunofixation: a sensitive new method for detecting oligoclonal IgM. J. Neuroimmunol. 23, 149. Sharief, M.K., Hentges, R. and Thompson, E.J. (1991) The relationship of interleukin-2 and soluble interleukin-2 receptors to intrathecal immunoglobulin synthesis in patients with multiple sclerosis. J. Neuroimmunol. 32, 43. Spiegelberg, H.L. (1977) The structure and biology of human IgD. Immunol. Rev. 37, 3. Thompson, E.J. and Johnson, M.H. (1982) Electrophoresis of CSF proteins. Br. J. Hosp. Med. 28, 600.