Acta neurol. scandinav. 58, 141-147, 1978

Department olf Neurology, University Hospital, Linkoping, Sweden

Localization of the immunoglobulins G, A and M, p -trace protein and Y -trace protein on isoelectric focusing

of serum and cerebrospinal fluid by immunofixation

MARIA ASSUNTA LAURENZI AND HANS LINK

The mobility of the immunoglobulins G, A and M, P-trace protein and y-trace protein on isoelectric focusing of serum and CSF was determined by immunofixation using specific antisera. Polyclonal IgG migrated as mul- tiple bands between pH 4.7-8.6, polyclmal IgA as multiple bands be- tween pH 4.9-6.1 in CSF and serum. IgM could not be identified in normal CSF or serum. p-trace protein gave three bands at pH 8.0, 8.4 and 7.4-7.5, respectively, while y-trace protein gave one single band at pH 9.5-> 9.5. Oligoclonal IgG in CSF in multiple sclerosis and neuro- syphilis migrated between pH 8.6-> 9.5 and was easily discriminated from other proteins.

Key words: Mobility of immunoglobulins - isoelectric focusing - immuno- fixation - cerebrospinal fluid - multiple sclerosis - oligoclonal immuno- globulins

Isoelectric focusing on thin-layer polyacrylamide gel has been proposed for analytical investigation of cerebrospinal fluid (CSF) proteins and for dern- onstration of oligoclonal immunoglobulin (Ig) bands in e.g. multiple scle- rosis (MS) (Delmotte 1971, 1975, Kjellin & Vesterberg 1972, 1974, Latner 1973, Delmotte & Gonsette 1977, Kjellin & Side'n 1977). The use of iso- electric focusing in this context necessitates the definition of the mobility of IgG in the normal individual and in the MS patient, as well as deter- mination of the mobility of those other proteins which migrate in the region of IgG.

In the present report the mobility on isoelectric focusing of the immuno- globulins G, A and M, p-trace protein and y-trace protein is characterized.

It is well established that the band pattern seen in the gammaglobulin region on agar gel electrophoresis of normal CSF consists of p-trace pro- tein which gives rise to at least three bands in the anodic part, y-trace protein which gives one single band in the cathodic part, and IgG which migrates as a diffuse zone in between (Link 1967). On agarose gel electro- phoresis of normal CSF, IgG appears as a zone at the cathodic side of the

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application spot, in between this and the single band of y-trace protein. In at least 90 % of MS patients, additional bands are demonstrable in the gammaglobulin region by electrophoresis of CSF on agar gel (Laterre et al. 1970, Link & Miiller 1971) as well as on agarose gel (Link 1973). In experi- ments consisting of isolation of IgG and subsequent electrophoretic analysis it has been shown that these additional bands consists of oligoclonal IgG (Link 1967). The mobility of IgG, IgA and IgM on agarose gel electro- phoresis of normal serum has been established (Laurel2 1972), while the mobility of these Ig on isoelectric focusing is unknown.

MATERIALS

IgG, IgA, IgM, P-trace protein and y-trace protein were identified in the patterns obtained at isoelectric focusing of pools of normal serum and pools of normal CSF, each pool containing identical volumes of serum or CSF from 10 different individuals. These constituted of neurological patients with a notma1 CSF cell count, a normal blood-brain barrier as determined by the CSFJserum albumin ratio (Link & Tibblirzg 1977), normal values of the CSF-IgG index (< 0.70), CSF-IgA index (< 0.62) and CSF-IgM index (< 1.0) (Fryde‘n ei al. 1978), normal serum values of IgG, IgA and IgM, and normal findings at agarose gel electrophoresis of CSF and serum. In addition, IgG, IgA, p-trace protein and y-trace protein were investigated in specimens of CSF and serum from normal individuals, MS patients and one patient with neurosyphilis. For IgM identification, CSF and serum from one patient with Mollaret’s chronic meningitis were also used.

Antisera against IgG, IgA and IgM were purchased from Dakopatts (Copenhagen, Denmark), while rabbit antisera against P-trace protein and 7-trace protein were a gift from Dr. J. E. Olsson, Department of Neurology, University of Lund, Sweden. Monospecificity of the antisera was tested by immunoelectrophoresis.

METHODS

Zsoelectric focusing was carried out on LKB Ampholine ready-prepared thin-layer poly- acrylamide gels (PAC plates, LKB, Stockholm, Sweden) according to the manufac- turer’s instructions. The pH range was 3.5-9.5. The anode-electrode solution consisted of 1M H,PO,, the cathode-electrode solution of 1M NaOH. For routine separation, CSF was concentrated by ultrafiltration in collodium bags (Sartorius Membran Filter, Gottingen, West Germany) to an IgG level of 3-4 @I. Serum was diluted with phy- siological saline to the corresponding IgG concentration. CSF and serum samples from the same patient were always run side by side on the same plate; 15 p1 of con- centrated CSF and of diluted serum, corresponding to 60-80 pg of IgG, were ab- sorbed on filter paper pieces (10 X 5 mm) which were applied on the gel at a pH interval between 6.2-6.4.. Pooled blood donor serum, serum IgG (Kabi, Stockholm, Sweden) with a concentration of 4 g/l, and carboxyhemoglobin were run as references on every plate. The separation was started at about 200 V and 50 mA, and the volt- age was increased every 10 min until, after 1 h, about 1200 V and 18 mA were reached. Separation was continued at this voltage for an additional 30 min until the carboxyhemoglobin had formed several sharp bands. The electrofocusing apparatus was connected with a cooling system to allow separation at about 8 O C . After 90 min

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the pH gradient between the anode and the cathode was measured by a surface electrode (type LOT 403-30 M8, Ingold, Zurich, Switzerland) at the temperature of the experiment, i.e. 8OC. Thereafter, isoelectric focusing was carried out for an ad- ditional 10 min. The proteins were then fixed, stained and dried according to the manufacturer's instructions.

Zmmunofixation for identification of the migration of individual proteins on iso- electric focusing was carried out according to Arnaud et al. (1977) with slight modi- fication. For the initial separation on isoelectric focusing, CSF was concentrated and serum diluted to obtain a concentration of the individual protein to be identified of 1-2 g/l. If possible, 20-40 pug of the protein to be identified was thus applied. Due to the low concentrations of some proteins in CSF, up to 30 p l of concentrated CSF was sometimes applied. A too high protein content had to be avoided because excess of protein in relation to antiserum in the subsequent immunofixation procedure causes dissolution of immunoprecipitate. After isoelectric focusing, the temperature of the polyacrylamide gel was immediately kept constant at 8OC in order to prevent protein diffusion. Cellulose acetate strips (Sepharose 111, Gelman Instrument Company, Ann Arbor, Michigan, USA) were dipped in the corresponding antiserum and applied to the gel surface. The gel plate was incubated for 1 h in a moist chamber at room temperature, and then two glass plates were put on the gel for 30 min to apply uniform pressure. Thereafter, the strips were carefully removed, and the strips and the gel plate were washed separately in 2 1 of physiological saline over night. The strips and the gel were stained with Coomassie Brilliant Blue (R 250) in ethanol- water-acetic acid (3:91), destained in ethanol-water-acetic acid (3:9:1), and stored in preserving solution consisting of glycerol in the destaining solution (1:lO). The pat- terns obtained from development of immune complexes in the cellulose acetate strips were compared with those in the gel.

RESULTS Mobility of ZgG

The results obtained on immunofixation after isoelectric focusing for iden- tification of the mobility of IgG, IgA, IgM, p-trace protein and y-trace protein in normal CSF and in serum are given in Table 1.

IgG migrated in the form of more than 15 bands between pH 4.7-8.6. The bands differed regarding number and mobility in different normal CSF and serum samples and pools, and the lowest pH at which IgG bands were demonstrable varied between 4.7-5.5. A constant feature was, however, that no IgG bands were demonstrable at a pH higher than 8.6, either in CSF or in serum.

In the MS CSF specimens investigated, IgG yielded oligoclonal bands between pH 8.6 and > 9.5.

As is shown in the foIIowing paper (Laurenzi & Link 1978), the number of bands varied between different MS patients in this pH region. The IgG pattern of the corresponding serum was found to be similar to that of nor- mal serum in the two specimens investigated. The CSF specimen from the neurosyphilis patient displayed an IgG pattern similar to that of MS CSF specimens.

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Table 1. Locaiization of IgG, I g A , &trace protein and y-trace protein on isoelectric focusing of normal CSF and serum

Protein Band pattern found on isoelectric focusing

Multiple bands between pH 4.7-8.6 in CSF and serum

Multiple bands between pH 4.9-6.1 in CSF and serum

No band in CSF or serum

1 band pH 8.0, 1 band pH 8.4, and 1 band pH 7.4-7.5 in CSF No band in serum

1 band pH 9.5-> 9.5 in CSF. No band in serum

IgG

IgA

IgM

P-trace protein

y-trace protein

Mobility of ZgA

IgA migrated as 6-13 bands between pH 4.9-6.1 on investigation of CSF as well as serum from normals, from MS patients and from the patient with neurosyphilis. Thus the number of bands differed between different CSF and serum samples and pools. No differences were observed regarding the mobility of IgA in CSF compared with serum.

Mobility of ZgM

IgM could not be identified in CSF or in serum from normals, from MS patients and from the patient with Mollaret’s chronic meningitis. In CSF from the neurosyphilis patient, IgM migrated as two faint bands, one at pH 5.2 and one at pH 5.4. This patient displayed oligoclonal IgM demon- strable by immunofixation after agarose gel electrophoresis in CSF but not in serum. Isoelectric focusing of serum from this patient did not yield any demonstrable IgM bands.

Mobility of p-trace protein

p-trace protein migrated regularly as one band at pH 8.0 and one band at pH 8.4. In addition, one normal CSF pool and one MS CSF specimen revealed one additional band between pH 7.4-7.5. No p-trace protein bands were demonstrable in serum specimens.

Mobility of y-trace protein

7-trace protein migrated as one broad band at pH 9.5-> 9.5 in all CSF specimens investigated. No additional y-trace bands were found. The serum specimens did not reveal any y-trace protein.

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DISCUSSION

It is obvious from the present investigation that immunofixation according to Arnaud el al. (1977) is a valuable method for identification of the mo- bility of individual proteins on isoelectric focusing. Such characterization is obligatory if isoelectric focusing of CSF and serum is to be used for diag- nostic purposes in, e.g., MS or other inflammatory CNS disorders.

It is apparent that IgG and IgA yielded much more complex band pat- terns on isoelectric focusing compared with those obtained on electro- phoresis using agar gel or agarose gel as supporting medium. The two MS patients included gave additional bands between pH 8.6 and 9.5, where IgG could not be demonstrated on investigation of normal CSF or serum. Therefore, isoelectric focusing may be supposed to be a useful method fol identification of oligoclonal IgG, provided that this pH region is examined. Oligoclonal IgG bands appearing in a pH region lower than 6.4 can not be distinguished from bands corresponding to other proteins.

The neurosyphilis patient included yielded oligoclonal IgG bands in the same pH region as the MS patients, when CSF was investigated. Thus, isoelectric focusing seems not to give any specific pattern regarding mobility of oligoclonal IgG bands in various types of inflammatory CNS disorders. This is also true for agar gel and agarose gel electrophoresis.

IgA migrates on isolelectric focusing in an area where IgG and probably also other proteins are migrating. IgA can therefore not be recognized by isoelectric focusing except when additional immunofixation is carried out. The mobility of oligoclonal CSF IgA has not yet been investigated.

In contrast to agarose gel electrophoresis, where IgM present in serum and also in CSF can be recognized at least after immunofixation, isoelectric focusing is less suitable for identification of IgM. The patient with neuro- syphilis displayed two distinct IgM bands on agarose gel electrophoresis of CSF but not in serum, while isoelectric focusing of CSF yielded only two faint, unsure bands. It may be assumed that the demonstration of oligo- clonal IgA or IgM on isoelectric focusing of CSF involves greater difficulties compared with agar gel or agarose gel electrophoresis.

The present investigation has shown that 8-trace protein, which con- stitutes 5-10 % of the total protein in normal CSF (Link 1967), and y-trace protein can easily be distinguished from IgG in normal CSF and from oligo- clonal IgG in CSF from patients with inflammatory CNS disorders by means of isoelectric focusing.

ACKNOWLEDGMENT

This study was supported from the Swedish Medical Research Council (Project No. 3381) and from the Swedish Institute by a scholarship to M.A.L.

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REFERENCES

Arnaud, P., G. B. Wilson, J. Koistinen & H. Fudenberg (1977): Immunofixation after electrofocusing: improved method for specific detection of serum proteins with determination of isoelectric points. I. Immunofixation print technique for detection of alphal-protease inhibition. J. Immunol. Methods 16, 221-231.

Delmotte, P. (1971): Gel isoelectric focusing of cerebrospinal fluid proteins. Z. Klin. Chem. Klin. Biochem. 9, 334-336.

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Delmotte, P. & R. Gonsette (1977): Biochemical findings in multiple sclerosis. IV. (Iso- electric focusing of the cerebrospinal fluid gammaglobulins in MS (262 cases) and other neurological diseases (272 cases)). J . Neurol. 215, 27-37.

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Received June 17, accepted September 5, 1978 Hans Link, M.D.

Department of Neurology University Hospital S-58185 Linkoping Sweden