Detection of Measles Antibodies in Cerebrospinal Fluid and Serum by a Radioimmunoassay C. CUNNINGHAM-RUNDLES, C. JERSILD, B. DUPONT, J. B. POSNER & R. A. GOOD Sloan-Kettering Institute for Cancer Research, New York, New York, USA

Cunningham-Rundles, C., Jersild, C., Dupont, B., Posner, J. B. & Good, R. A. Detection of Measles Antibodies in Cerebrospinal Fluid and Serum by a Radioimmunoassay. Scand. J. Immunol. 4, 785-790, 1975. Evidence that different structural components of the measles virus may act as antigens has been provided by the serologic methods of hemagglutination inhibition hemolysin inhibition, and nucleocapsid complement fixation. Using radioiodinated measles viral antigens, an immune precipitation assay has been designed that is capable of discriminating among various reactivities to measles viral structural components in serum or cerebrospinal fluid (CSF) and of distinguishing whether IgG and IgM antibody is involved. This technique has been applied to the study of measles antibodies in CSF and sera of patients with multiple sclerosis (MS) and other neurologic diseases. From data presented here, it was found that both groups of patients have individual reactivity to measles proteins, present in CSF and serum, whereas threh normal CSF samples were found not to have such antibodies. It appears that oligoclonal immunoglobulins in CSF of MS patients may be detected by this method, a n d one patient with MS was found to have CSF IgM anti-measles antibodies. C . Cunningham-Rundles, M.D., Sloan-Kettering Institute for Cancer Research, 1275 York Aue., N e w York, N Y 10021, USA

Many recent reports have indicated that sera of patients with multiple sclerosis (MS) and their siblings have higher measles antibody titers than their matched controls (1, 2, 13, 14). Serologic measurements of measles viral titers in MS sera by hemagglutination inhibition, hemolysin inhibition, and nucleocapsid complement fixation has shown that the individual antibody response to these structural components of the measles virus is independently variable and that one or more of these antibody fractions may be absent (16). This individual pattern of antibody response to the measles virus would appear to be a normal phenomenon, since MS patients, their siblings, and control groups cannot be distinguished by their reactivity in such tests (16). Specific measles virus antibodies can also be detected in cerebrospinal fluid (CSF) obtained from MS patients, and a similar variation in antibody response to individual mea-

2

sles components is found in this fluid (17). From several kinds of evidence, it appears that these antibodies result, at least in part, from local synthesis within the central nervous system (CNS) (4, 11). This finding has been amplified by the observation that the CSF of MS patients contains electrophoretically homogeneous immunoglobulin components on agarose gel electrophoresis ( 9 , 20). At least two such immunoglobulin bands are found in concentrated CSF in about 90% of patients with MS ( 9 ) . It has been tempting to relate such immunoglobulins to the several antibodies directed against the components of the measles virus. However, Norrby & Vandvik (1 3) have recently shown that such oligoclonal bands are not readily removed from CSF by absorption to measles virus, although this technique was found successful in deleting the oligoclonal bands seen in CSF from patients with subacute sclerosing panencephalitis. This discrepancy

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C. Cunningbarn-Rundles, C. Jersild, B. Dupont,

may be due to technical difficulties, or it may indicate different antigenic specificities for the immunoglobulins produced in MS. Whereas sera of MS patients may have both IgG and IgM antibodies against measles, normal control sera have been found to have only IgG antimeasles antibodies (5, 12). This has been taken as evidence that there may be a persistence of measles viral antigen in patients with MS (12). Since CSF anti-measles IgM has not been demonstrated in MS, it has been proposed that viral antigens may persist outside the CNS (5, 12). This study was undertaken to reevaluate the presence of measles antibodies in the CSF and sera of MS patients by a radioimmune technique that is capable of discriminating among various reactivities to measles viral components and distinguishing whether IgG and IgM antibody is involved.

J. B. Posner 6 R. .4.

Good

For viral antigen preparations, several pilot methods were used in order to choose a method in which all viral components were exposed and radioiodinated to high specific activity. For this, various concentrations of Nonidet P-40 (NP40, Shell Chemicals), from 0.0576 to 2.5% in phosphate-buffered saline (PBS), and 196 sulfododecy1 sulfate (SDS) were used to disrupt the virus before iodination. In the method selected for radioimmune precipitation assay, the purified virus was suspended in PBS made up to 0.2576 in NP40, heated to 37°C for 60 min, and centrifuged at 20,000 g 2 hr in a Sorvall RC 2-B centrifuge to remove nondisrupted particles. The supernatant of this preparation, which was subsequently used for all experiments, was divided into aliquots and stored at -20°C until use. 0.2 to 0.5 ml of this was iodinated as needed by the lactoperoxidase method, in which 1 mCi 1251 (New England Nuclear), 0.1 mg lactoperoxidase (CalbioMATERIALS AND METHODS chem), and two aliquots of HzOz were used Cerebrospinal fluid and blood samples were (15 ) . In one experiment the chloraniine-T obtained from 10 MS patients and 10 patients iodination method (8) was used to compare with other neurologic diseases, most of which iodination efficiency and completeness of iodiwere primary or secondary malignancies of the nation of the component measles proteins. All CNS. Samples considered normal were from viral preparations were analyzed by polyone individual who had a lumbar puncture in acrylamide gel electrophoresis before use in the evaluation of headaches (final diagnosis, the radioimmunoassay to ensure that the six tension headaches), one individual who was predominant proteins known to be present in evaluated for an enlarged sella turcica, and one measles virus were present in each preparation individual who had a myelogram procedure (7, 19). Molecular weight markers run in for a possible prolapsed intervertebral disc but companion gels were transferrin, serum albuwas found normal. All CSF was concentrated min, p chain, 7 chain or ovalbumin, light 20-fold in an Amicon B15 concentrator. IgG chains, and cytochrome c. concentrations in serum and in concentrated Rabbit anti-human IgG and IgM, heavyCSF were determined by radial immunodiffu- chain-specific, were purchased from Pentex and sion (10). were checked for specificity by immunoelecMeasles virus, Edmonston strain 84F, was trophoresis, using standard normal serum as purchased from Electronucleonics, Rockville, the antigen. Md., and was a sucrose density gradient-banded The concentrated CSF or serum, diluted 1:3 preparation grown in monkey kidney cells. The in PBS and heat-inactivated at 56°C for 45 final suspension contained 107 viral particles/ min, was incubated with 105 cpm of dialyzed ml, based on electron microscopy. A control viral antigen at a volume ratio, of 1:50 for virus, mouse mammary tumor virus, was ob- 30 min at 37°C. Rabbit anti-human IgG or tained from Dr. William Feller of Georgetown IgM was then added and incubation continued University, Washington, D.C. This preparation for 30 min at 37"C, and then at 4°C overwas also sucrose density gradient-banded and night. The immune precipitates were washed contained 10*-109 particleslml. three times in 1 ml PBS, dissolved in 0.1%

Radioimmunoassay for Measles Antibodies

SDS and 0.1M urea, and electrophoresed in 12 cm 0.176 SDS, O.5M urea, and 55% acrylamide gels (18) at 0.83 mA/cm; bromphenol blue (BTB) was used as a tracking dye in some experiments. The gels were then sliced into 2-mm slices on a Gilson (model 3loO/GMAGCB) gel cutter; and the slices were individually counted in a Packard 5285 autogamma spectrometer. Iodinated standard proteins run in companion gels were used to monitor each experiment. Controls were viral antigen preparations incubated with rabbit antihuman IgG and precipitated with goat antirabbit sera, and duplicate experiments using CSF samples from MS and other patients, which were incubated with a viral preparation prepared from mouse mammary tumor virus and precipitated as described above.

787

100 80

60 40

20 12 8

4

.0

c\I

I

X

0

8MC

64-

2

2

RESULTS

Fig. 1A (left and right) shows the six major proteins of the measles virus as demonstrated by lactoperoxidase iodination of NP40-disrupted virus. Although the degree of iodination of each peak varied in different experiments, in all viral preparations six peaks were found, which were of molecular weights 75,00080,000; 70,000; 60,000; 53,000; 46,00050,000; and 35,000-40,000. These may correspond with measles proteins P76, GP60, P60, GP53, P51, and P45 (7, 19). (The molecular weight times 10-3 is preceded by P (for proteins) or GP (glycoproteins) .) Fig. 1B-E shows the results of CSF IgG precipitation of radioiodinated measles virus in four selected MS patients. Fig. IF-I illustrates the corresponding result obtained by IgG precipitation of serum from the same patient. The remaining six patients, for whom data are not presented, had precipitation patterns similar to those shown. From the results obtained from CSF precipitation, it appears that IgG antibody recognition may be directed against various combinations of measles components in different MS patients. Serum and CSF precipitations from the same individual are similar, although for Patient K.B. (Fig. 1E and I), the 75,000-

10

50 10 30 Fraction number

30

50

Fig. 1. Part A (left and right) represents the six radioiodinated polypeptides of the measles virus; the molecular weights of these are given in the text. B-E shows the measles components precipitated by the cerebrospinal fluid (CSF) or four multiple sclerosis (MS) patients; F-I shows the components precipitated by serum of the same patients. Patient initials are in the center of each figure. Molecular weight markers used here were transferrin ( 8 5 , 0 0 0 ) , serum albumin (60,000), p chain (53,000), ovalbumin (45,000), and light chains (23,000), with mobilities at fraction 10, 27, 38, 45, and 57, respectively.

80,000-dalton measles component is clearly precipitated, whereas in the matched serum the precipitation is less apparent. This may be caused by an antigen precipitated by an oli-

788

C. Cunningham-Rundles, C . Jersild, B. Dupont, J . B . Posner C R. A . Good

Fraction number Fig. 2. A-E shows the measles components precipitated by the cerebrospinal fluid (CSF) of patients with other neurologic diseases. The patient initials are to the right in each figure. Molecular weight markers were included as described in Fig. 1.

goclonal IgG present in the CSF sample. (Agarose gel electrophoresis for oligoclonal proteins was not carried out for this study.) Fig. 2A-D shows the measles components precipitated by CSF obtained from five patients with other neurologic disorders. (Diagnoses are listed in Table I.) Comparing Fig. 1B-E with Fig. 2A-D, it is clear that MS patients cannot be distinguished from patients with other CNS disorders by this method since a variety of precipitation patterns is evident in both groups. Nor is the result of the radioassay dependent on CSF IgG concentration, as can be Seen in Table 1. The three normal CSF samples had no recognizable measles precipitation in this assay, and control samples using radiolabeled measles virus, rabbit anti-human IgG, and ‘sandwich‘ antibody goat anti-rabbit sera were negative. A final control, a similar radioimmunoassay using iodinated mouse mammary tumor virus, was also negative. Fig. 3 shows the result of IgG and IgM precipitation using CSF from one MS patient (Patient R.C.). Also included for comparison is the precipitation result obtained for serum IgM. Although both serum and CSF IgM is found in diminished concentrations in comparison with IgG in these fluids, CSF IgM has a somewhat different antigenic reactivity than

Table I. Cerebrospinal (CSF) protein and IgG concentrations Patient

Diagnosis

Radial CSF IgG, mgfloo ml

Total protein mg/100 ml ~

R.C. M.C. L.F.

K.B. A.K R.G. J.B. M.C. M.D. N.O.* M.H.* M.P.*

Multiple sclerosis Multiple sclerosis Multiple sclerosis Multiple sclerosis Epidural effusion Meningeal carcinomatosis Polyneuropathy associated with myeloma Spinal-cord metastasis Polyneuropathy, etiology unknown Enlarged sella, negative evaluation Tension headaches Prolapsed intervertebral disc

* These patients are considered to have ‘normal’ CSF.

15.0 1.G 4.7 2.4 12.3 11.0 61.0 1.0

< < 1.0 < 1.0 < 1.0 < 1.0

77 23 25 36

90 1,010 180

50 14 24 46

53

Radioinmunoassay for Measles Antibodies

789

either CSF IgG or serum IgM. It would appear that CSF IgM has biologic activity against the measles virus in the CSF of some MS patients. DISCUSSION Using the radioimmune precipitation assay de- NI 0 scribed here, it is possible to demonstrate measles antibodies in CSF and serum. Evidence x that different structural components of the E measles virus may act as antigens is in accord a 0 with the reports of Salmi et al. (16, 1 3 , who found variable responses to different antigens of the measles virus by serologic methods (hemagglutination inhibition, hemolysin inhibition, and nucleocapsid complemedt fixation). I The technique described here may offer a 1 0 30 50 means of relating the serologic tests to specific measles proteins. Although the six peptides Fraction number found in viral preparations had molecular Fig. 3. The measles components precipitated by the weights similar to those reported for measles cerebrospinal fluid (CSF) IgG of Patient R.C. are virus, the proteins used here have not been represented by the solid line, the CSF IgM precipifurther identified. Since all preparations had tation by a dotted and dashed line, and the serum IgM precipitation by a dottet line. Molecular weight the six components and no extraneous peaks markers were included as described in Fig. 1. were found, impurities, if any, were thought to be minimal. Haire et al. (6) have reported that, using fivefold concentrated CSF, 80.6% of MS patients, 34.5% of patients with other neurologic proteins; the correlated radioimmunoassay is diagnosis, and 0% of normal individuals were shown in Fig. 2. No other patients included found to have anti-nieasIes antibodies by in- in this study were thought to have a comprodirect immunofluorescence. By the present mised blood brain barrier). It is also possible technique, using 20-fold concentrated CSF, 10 that as the number of patients examined is MS patients and 10 patients with other neu- increased, negative samples with measles antirologic disorders were found to have such bodies in CSF will be found among the paantibodies, whereas the three normal samples tients with CNS diseases. In addition, normal were found to be negative. individuals with high serum titers to measIes This positive result for the first two groups may also be found to have detectable CSF is undoubtedly dut to the use of more con- antibodies by this technique. (Serum antibody centrated CSF in this assay, but worth con- titers have not been determined in this prelimisidering is the possible contamination of CSF nary study.) From results described here, it appears that with blood products (due either to a damaged blood brain barrier or to an unrecognized trau- IgG antibody directed toward certain measles matic lumbar puncture. Patient A.K. in Fig. components play be present in CSF but not 2 illustrates this possibility, since her first in serum of some MS patients (Fig. ID). lumbar puncture was negative in this system. Although a restricted antibody of this type However, after a somewhat traumatic pneu- could be a clonal immunoglobulin, experimenmoencephalogram, her spinal fluid protein in- tal proof is needed to show that banded imcreased, probably due to transudation of serum munoglobulin seen on agarose gel electropho7

790

C. Cunninghjm-Rundles, C. Jersild, B. Dupont, J . B. Posner & R. '4. Good

resis has specificity for such a measles component. This technique appears to be applicable to this investigation.' Another observation that has resulted from these exp,erimental findings is that anti-measles IgM is present in the CSF of one of the MS patients studied (Fig. 3 ) . Interestingly, CSF IgG and IgM do not have the same specificity, nor do serum IgM and CSF IgM. The importance of this observation is uncertain. It should be noted that this patient (Patient R.C.) had a markedly elevated spinal fluid IgG and was clinically in an exacerbation at the time of this analysis. Nine other MS patients examined in this way have had no demonstrable CSF IgM, but this must still be considered inconclusive. ACKNOWLEDGEMENTS This research was supported by National Cancer Institute grants NCI-CA-17404-01 and NCI-CA-08748-10, the American Cancer Society, and a National Foundation-March of Dimes grant from the Foundation for The Advanced Study of Cancer. REFERENCES

T. Measles antibodies in multiple sclerosis. Proc. Soc. exp. Biol. (N.Y.)111, 562, 1962. 2. Ammitzboll, T. & Clausen, J. Measles antibody in serum of multiple sclerosis patients, their children, siblings and parents. Actu neurol. srand. 48, 47, 1972. 3. Brody, J. A., Sever, J. L. & Hensen, T. E. Virus antibody titers in multiple sclerosis patients, siblings, and controls. J . Amer. Med. Ass. 216, 1441, 1971. 4. Cohen, S. & Bannister, R. Immunoglobulin synthesis within the central nervous system in disseminated sclerosis. h n c e t I, 366, 1967. 5. Haire, M., Fraser, K. B. & Millar, J. H. D. Measles and other virus-specific immunoglobulins in multiple sclerosis. Brit. med. J . 3 , 612, 1973. 6. Haire, M., Millar, J. H . D. & Merrett, J. D. Measles virus-specific IgG in cerebrospinal fluid in multiple sclerosis. Brit. J . Med. 4, 192, 1974. 1. Adams, J. M. & Imagawa, D.

Received 14 June 1975 Received in revised form 8 September 1975

7. Hall, W. W. & Martin, S. J. Purification and characterization of measles virus. J . gen. Virol. 19, 175, 1973. 8. Hunter, W. M. & Greenwood, F. C. Preparation of iodine-131 labelled human growth hormone of high specific activity. Nuture (Lond.) 194, 495, 1962. 9. Link, H . Oligoclonal immunoglobulin G in multiple sclerosis brains. J . neurol. Scj. 16, 103, 1972. 10. Mancini, G., Carbonara, A. D. Heremans, J. F. Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochenzistry 2, 235, 1965. ll.McAlpine, D., Lumsden, C. E. & Acheson, E. D. Multiple Sclerosis. London, 1965. 12. Millar, J. M. D., Fraser, K. B., Haire, M., Connolly, J. H. Shirodaria, P. V. & Hadden, D. S. M. Immunoglobulin M specific for measles and mumps in multiple sclerosis. Brit. med. J . ZI 378, 1971. 13. Norrby, E. & Vandvik, B. in Perspectilies in Multiple Sclerosis. National Institutes of Health Conference, Bethesda, Maryland, February 1975. 14. Panelius, M., Salmi, A,, Halonen, P., Kivalo, F., Rinne, U. K. & Penttinen, K. Virus antibodies in serum specimens from patients with multiple sclerosis, from siblings and matched controls. A final report. Act3 neurol. smnd. 49, 85, 1973. 15. Phillips, D. R. & Morrison, M. The arrangement of proteins in the human erythrocyte membrane. Biochem. biophys. Res. Commun. 40, 284, 1970. 16. Salmi, A. A,, Gollmar, Y . , Norrby, E. & Panelius, M. Antibodies against three different structural components of measles virus in patients with multiple sclerosis, their siblings, and matched controls. Arta path. microbiol. srand. 81, 627, 1973. 17. Salmi, A. A,, Norrby, E. & Panelius, M. Identification of different measles virus-specific antibodies in the serum and cerebrospinal fluid from patients with subacute sclerosing panencephalitis and multiple sclerosis. Infect. Zmman. 6, 248, 1972. 18. Shapiro, A. L., Vinuela, E. & Maizel, J. V. Molecular weight estimation of polypeptide chains by electrophoresis in SDS polyacrylamide gels. Biochem. biophys. Res. Commun. 28, 815, 1967. 19. Waters, D. J. & Bussell, R. H. Polypeptide composition of measles and canine distemper virus. L'irology 5 5 , 554, 1973. 20. Zettervall, 0. & Link, H. Electrophoretic distribution of kappa and lambda immunoglobulin determinants in serum and cerekospinal fluid in multiple sclerosis. Clin. exp. Immunol. 7 , 365, 1970.