403
Clinica Chimica Acta, 92 (1979) 403-409 @ Elsevier/North-Holland Biomedical Press
CCA 10041
RADIOIMMUNOASSAY OF SERUM MYELIN BASIC PROTEIN AND ITS APPLICATION TO PATIENTS WITH CEREBROVASCULAR ACCIDENT
JOHN W. PALFREYMAN and C.D. FORBES a
a**, R.V. JOHNSTON a, J.G. RATCLIFFE
a, D.G.T. THOMAS b
a Departments of Biochemistry and Medicine, Glasgow Royal Infirmary, Castle Street, Glasgow G4 OSF and b Gough-Cooper Department of Neurosurgery, Queen’s Square, London WClN 3BG (U.K.) (Received September 22nd, 1978)
Summary Myelin basic protein-like immunoactivity was measured in the serum of patients after cerebrovascular accident (CVA) using a double antibody radioimmunoassay for myelin basic protein with a detection limit of 3 ng/ml serum. For up to 6 days after ictus, serum myelin basic protein levels in patients with severe CVA and patients who died as a result of CVA were significantly greater than those in control patients, patients with moderate CVA and patients surviving CVA. All patients with serum myelin basic protein levels greater than the range found in control subjects subsequently died. Serial dilutions of positive sera suggested that the immunoactivity differs from authentic myelin basic protein and may represent breakdown products of the protein. Serum from some patients with a previous history of moderate CVA had myelin basic protein binding activity consistent with the presence of antibodies to the protein.
Introduction The assay of biochemical markers in biological fluids following tissue damage has important applications in clinical chemistry. However, the biochemical assessment of brain damage has not received much attention as the only readily available markers, such as the enzyme lactate dehydrogenase or isoenzymes of creatine kinase, lack absolute tissue specificity. This limitation has been overcome in recent years by the development of sensitive radioimmunoassays for the brain specific protein, myelin basic protein (MBP) [l-4]. Thus, MPB or fragments of the protein have been detected in serum and CSF from patients with * Present address to Glasgow,
U.K.
which
correspondence
should
be sent:
MRC
Institute of
Virology,
Church
Street,
404
brain damage after head injury [4,5] and in CSF after subarachnoid haemorrhage [6]. In patients with head injury serum levels of MBP correlate well with the type and severity of brain damage and also with final outcome [ 51. In the present study we have investigated the possible clinical applications of a modified MBP assay to the assessment of patients following cerebrovascular accident. Materials and methods Radioimmunoassay for MBP The system used was similar to the serum MBP assay previously reported [ 41 with some modifications. 100 ~1 serum, 100 ~1 of buffer (0.05 M sodium phosphate (pH 7.4), 0.2 M NaCl, 50 g/l bovine serum albumin and 2 g/l calf thymus histone type II s) and 50 ~1 of rabbit anti-human MBP serum (KK 42. Pool II) diluted 1 : 40 000 in 0.05 M sodium phosphate buffer containing normal rabbit serum diluted 1 : 200 were incubated overnight at 4” C. After addition of iodinated MBP and a second overnight incubation, bound and free tracer were separated by double antibody precipitation [4]. A standard curve was constructed by serial dilutions of human MBP in pooled normal human serum. It was plotted as % specific 12’I-MBP bound (i.e. total tracer bound in the presence of antiserum - apparent tracer binding in the absence of specific antiserum) against mass of, or equivalent serum concentration of MBP (Fig. 1). The specific binding of ‘2SI-MBP (i.e. as above) was determined for each serum sample and MBP concentration calculated by interpolation from the standard curve. All MBP levels reported in this paper are those found in undiluted serum. The antiserum (KK 42, Pool II) used in the assay was from the same rabbit as that used in the previous study [ 41. It was, however, a more recent bleed taken after a further series of immunisations with human MBP and had a higher titre than the original antiserum. Assay of serum MBP binding activity The assay system contamed 100 ~1 of test serum, 100 ~1 of histone
buffer
40. 3 ;; 520 B 3 I
20
E- 10 yjb+ OO
0.2
0.5
1
2
5
10
Human MBP(W diluent; O----O, serum and ---o* delipidated Fig. 1. Standard curves for MBP assay. m------I, serum standard curves. Nonspecific binding of tracer (i.e. apparent binding in the absence of antibody) in each buffer has been deducted from each Point. Delipidated serum was prepared by high speed centrifugation of pooled normal human serum.
405
TABLE I RELATIONSHIP OF SEVERITY OF. OR DAY AFTER. ICTUS Diagnosis
n
OF CVA AND OUTCOME
Survivors
TO SERUM MBP LEVELS
Range
Patients with elevated MBP
4.4 11.5 *
U.D.-7.5 U.D.-40
0 11
4.4 11.2 *
U.D.--7.5 U.D.-40
0 11
Mean serum MBP wzll)
Moderate CVA Severe CVA
35 23
32 3
outcome
”
Moderate CVA
$urvival Death
35 23
32 3
ON THE DAY
* P < 0.05 compared with control patients. U.D. = undetectable (i.e. < 3 pg/l).
(see above) and 2 ng of 1251-MBP (10 000 cpm in 50 ~1 of histone buffer). After overnight incubation at 4°C free plus non-specific bound tracer were separated from serum bound tracer by addition of 200 ~1 of dextran coated charcoal. The charcoal suspension contained 1.5% charcoal, (Norit OL from Hopkin and Williams, Chadwell Heath, U.K.) 0.375% dextran (Pharmacia Fine Chemicals, Uppsala, Sweden) T70, and 30% horse serum (Wellcome Reagents Ltd., Beckenham, U.K.) in 0.25 M, pH 7.4 sodium phosphate buffer. Patients studied Blood samples were taken from 58 patients presenting at Glasgow Royal Infirmary with cerebrovascular accident (CVA). Patients were classified by diagnosis (severe or moderate CVA) and outcome (survival or death). Severity of CVA was determined by criteria which are based on those cited by Oxbury et al. [7]. Severe CVA was characterised by a markedly impaired level of consciousness and failure of conjugate ocular gaze towards the side of limb weakness. The distribution of patients within groups is shown in Table I. Where possible, blood samples were taken serially for at least six days after ictus. Serum was prepared from blood allowed to clot for 2-4 h at 4”C, frozen within 24 h and stored at -20°C until assay. Statistical significance of differences of grouped data was by non-parametric analysis using the Mann-Whitney U test. Results MBP assay The modifications to the assay outlined above resulted in a lower detection limit (mass of MBP giving 10% inhibition of specific tracer binding) of 300 pg, equivalent to a serum concentration of 3 ng/ml. The upper limit of the normal range for serum MBP estimated from 50 control samples from subjects without neurological, or other organ disease was 7 ng/ml. Despite modifications to the assay the diluent and serum standard curves were not coincident (Fig. 1). A standard curve in delipidated serum lies between these two curves (Fig. 1). Non-specific binding of tracer in patient and control sera in the absence of MBP
406
anti-serum varied between 6.9% and 14.7% (mean = 10.09%, s.d. = 1.81%). The mean recovery of 1.0 ng of unlabelled human MBP added to 100 ~1 of 12 different sera was 108%.
Serum levels after CVA Serum MBP levels in 58 patients on the day of ictus are shown in Table I. Levels of serum MBP above the normal range were found only in patients with severe CVA and all patients with elevated MBP died within 2 days of ictus. Levels of MBP in patients with severe CVA and patients who subsequently died were significantly greater (p < 0.05) than those in control patients, patients with moderate CVA and survivors after CVA. Thirty-four of the fiftyeight patients were followed serially. The absolute levels of serum MBP in these patients grouped according to outcome are illustrated in Fig. 2. No survivors had serum MBP levels higher than the upper limit of normal, and all patients with elevated levels subsequently died. The mean serum MBP levels from patients in the serial study are shown in Figs. 3a and 3b. Data is grouped according to diagnosis (Fig. 3a) or outcome (Fig. 3b). Patients with severe CVA and those who died have significantly elevated levels of serum MBP on days 2-6. However, not all patients in these two groups have elevated levels. Serum MBP levels from patients who died after severe CVA show a wide variation (Fig. 4). Levels, which are low on day 1 if the patient is to survive for more than two days, increase to a maximum within 2-5 days and remain elevated, in one case for up to 12 days. By contrast one patient shows no increase in serum MBP for up to 7 days after ictus. The nature of the immunoactivity in positive sera was investigated by comparison of inhibition curves for serially diluted sera with the serum standard curve (Fig. 5). The inhibition curves are not parallel with the standard curve indicating differences between the antigenic nature of MBP and the immunoactivity found in test serum. Overnight incubation of positive sera with dextran coated charcoal (0.5 ml sera and 50 ~1 of a suspension of 25 mg/ml charcoal containing 2.5 mg/ml dextran) removed all measurable immunoactivity. .
. .
.
o-1
2
5 3 4 Days After ktus
6
Fig. 2. Serial MBP levels in CVA patients. The broken line indicates the upper limit found in control patients. 0. survivors; 0, died.
407
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14
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moderate CVA; n----n, severe Fig. 3. (a) Mean serum MBP levels related to severity of CVA. m-------o, CVA. Between days 2 and 6 serum MBP levels in severe group are significantly greater @ < 0.05) than in controls or in moderate CVA group. Numbers of observations at each point are indicated. (b) Mean serum died. Between days 2 and 6. serum MBP MBP levels related to outcome. w-i?, survivors; o-----e, levels in patient who died are significantly greater @ < 0.05) than in controls or in the survivors. Numbers of observations at each point are indicated.
Serum MBP binding activity In control sera 11% of added lzSI-MBP remained in the supernatant after charcoal absorption. Increasing the charcoal concentration decreased this level to less than 5% but high charcoal concentrations are known to disturb antigenantibody equilibria of low avidity binders, so were not routinely used. Of 58 patients with CVA tested for serum *2SI-MBP binding activity five (8.6%)
Testserum
w-
6.25
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2$
401
. ,3, O-l
,
,
,
,
,
,
,
(
2
3
4
5
6
7
8
9
5
20
50
100
Serum MBP(pgII)
Days After lctus Fig. 4. Serial serum MBP levels in individual severe CVA who subsequently died.
IO
patients.
Serial MBP levels in 6 patients
(*+ 0, 0, 0, \-, l) with
Fig. 5. Parallelism of authentic human MBP and serum immunoactivity from ten patients with apparently high MBP levels. Inhibition of tracer ( 1 251 rabbit MBP) binding to MBP antisera by human MBP (i.e. standard curve). Other lines indicate inhibition of tracer binding by dilutions of sera from patients with high serum MBP levels.
408 TABLE SERUM
II ’ 2s I-MBP BINDING
Five of 58 patients showed serum ’ 2’ I-MBP binding of greater than 2 S.D. from the mean control valve (11.2 ? 4.9%). One patient (446) was studied seriallv all samples being positive for tracer binding. Patient number
Category
Previous stroke
% ’ * 5 I-MBP bound
406 408
Severe CVA/died Severe CVA/died
Yes Yes
29.1 22.6
425 440
Moderate Moderate
CVA/survived CVA/survived
Yes Yes
25.8 22.4
446
Moderate
CVA/survived
Yes
34.3 36.7 38.1
showed binding greater than two standard deviations of the mean level (Table II). All five patients had a previous clinical history of CVA. Scatchard analysis [8] on one patient indicated an equilibrium (dissociation) constant for antigen binding of 2 X lOWa l/mol. Discussion MBP assay
The MBP radioimmunoassay reported in this paper has some important differences from that described previously [ 41. Use of the more avid antiserum in the assay resulted in a more sensitive assay. The lower control levels of MBP reported here are due to (a) use of pooled normal human serum in the standard curve and (b) improved assay sensitivity which minimises the effect of nonspecific factors near the detection limit 191. The 28% of control sera which showed positive, though low, immunoactivity even in the modified assay system may contain serum components other than MBP interfering in the assay. Delipidation of pooled normal human serum produced a slight shift in the MBP standard curve, indicating that serum lipids may bind MBP, and increase the apparent levels of MBP immunoactivity in some sera. Significant levels of serum MBP binding activity were demonstrated in approximately 10% of sera from patients with a history of previous stroke. The dissociation constant for the binding agent is within the range for a low avidity antibody indicating the possible presence of MBP in serum at some time previously. This suggests that MBP may be released into the circulation after mild CVA. A more sensitive assay would be required to substantiate this. The nature of serum MBP binding factor requires further investigation to distinguish normal from possible pathological factors. However, there was no evidence that these binding factor(s) interfered with the assay for MBP, probably because their avidities are much lower than those of the rabbit anti MBP serum. Clinical results and their implications
This study provides good evidence that MBP occurs in the serum of some patients after CVA and that the apparent level is related to clinical severity and outcome. The nature of the measured serum immunoactivity needs further
409
definition since it differed from authentic MBP on serial dilution. Absorption of serum immunoactivity with charcoal is consistent with it being due to highly charged molecules such as MBP or its metabolites. Because of the non parallelism, the present assay can only be considered semiquantitative and it may be levels of serum MBP. However, the more accurate to describe “apparent” differences between patient groups in this study remain clinically significant whatever the precise nature of serum MBP immunoactivity. After severe CVA, the levels of serum MBP increase to reach a peak four to six days after ictus. The maintenance of elevated levels contrasts with the rapid clearance of systemically injected MBP in rabbits (less than 15 min) [lo]. This suggests either continuing infarction and breakdown of brain tissue after CVA or that acute brain damage is followed by increasing impairment of the blood brain barrier with leakage of MBP into the circulation. In either case, monitoring of MBP after CVA may prove useful in evaluating new methods of treatment designed to limit complications of CVA such as spread of infarction or cerebral oedema. It is also possible that the pattern of release of MBP into serum may reflect the nature of the underlying pathology (thrombosis, intracerebral or subarachnoid haemorrhage) which was not determined in the present study. We have previously reported that serum MBP levels after head injury may be a useful prognostic index [ 51, though the duration of the present assay restricts its routine use. This study indicates that serum MBP assay may also be useful prognostically after CVA. No patient with an elevated serum MBP level survived, though not all patients who died showed elevated levels on serial study. It may therefore be useful in supplementing clinical assessment in patients with severe CVA managed on life support systems. Acknowledgements We are grateful to Dr. J. Wallace, Blood Transfusion Service, Law Hospital for normal human serum and to the physicians who allowed access to their patients. J.W. Palfreyman was a recipient of a Higher Medicine Grant from the Greater Glasgow Health Board. References 1 2 3 4 5 6 7 8 9 10
Schmid. G., Thomas, G.. Hempel, K. and Gruninger, W. (1974) Eur. Neural. 12.173-185 Cohen, S.R., McKhann. G.M. and Guamieri, N. (1975) J. Neurochem. 25, 371-376 Whitaker, J. (1977) Neurology 27, 911-920 Palfreyman. J.W., Thomas, D.G.T. and Ratcliffe, J.G. (1978) Clin. Chim. Acta 82, 259-270 Thomas, D.G.T., Palfreyman, J.W. and Ratcliffe, J.G. (1978) Lancst i, 113-115 dohen, S.R., Herndon, R.M. and McKhann, G.M. (1976) New Eng. J. Med. 295.1455-1457 Oxbury, J.M., Greenhall. R.C.D. and Grainger, K.M.R. (1975) Br. Med. J. 3,125-127 Scatchard. G. (1949) Ann. N.Y. Acad. Sci. 51. 660-672 Hunter. W.M. and Bennie. J.G. (1978) J. Endocrinol., in press Eng, L.F.. Lee, Y.L., Williams, K.. Fukayama. G., Gerstl. B. and Kies, M. (1978) in Advances in Experimental Medicine and Biology. My&nation, and Demyelination. Plenum Publishing Corporation. London. in press
Clinica Chimica Acta, 92 (1979) 403-409 @ Elsevier/North-Holland Biomedical Press
CCA 10041
RADIOIMMUNOASSAY OF SERUM MYELIN BASIC PROTEIN AND ITS APPLICATION TO PATIENTS WITH CEREBROVASCULAR ACCIDENT
JOHN W. PALFREYMAN and C.D. FORBES a
a**, R.V. JOHNSTON a, J.G. RATCLIFFE
a, D.G.T. THOMAS b
a Departments of Biochemistry and Medicine, Glasgow Royal Infirmary, Castle Street, Glasgow G4 OSF and b Gough-Cooper Department of Neurosurgery, Queen’s Square, London WClN 3BG (U.K.) (Received September 22nd, 1978)
Summary Myelin basic protein-like immunoactivity was measured in the serum of patients after cerebrovascular accident (CVA) using a double antibody radioimmunoassay for myelin basic protein with a detection limit of 3 ng/ml serum. For up to 6 days after ictus, serum myelin basic protein levels in patients with severe CVA and patients who died as a result of CVA were significantly greater than those in control patients, patients with moderate CVA and patients surviving CVA. All patients with serum myelin basic protein levels greater than the range found in control subjects subsequently died. Serial dilutions of positive sera suggested that the immunoactivity differs from authentic myelin basic protein and may represent breakdown products of the protein. Serum from some patients with a previous history of moderate CVA had myelin basic protein binding activity consistent with the presence of antibodies to the protein.
Introduction The assay of biochemical markers in biological fluids following tissue damage has important applications in clinical chemistry. However, the biochemical assessment of brain damage has not received much attention as the only readily available markers, such as the enzyme lactate dehydrogenase or isoenzymes of creatine kinase, lack absolute tissue specificity. This limitation has been overcome in recent years by the development of sensitive radioimmunoassays for the brain specific protein, myelin basic protein (MBP) [l-4]. Thus, MPB or fragments of the protein have been detected in serum and CSF from patients with * Present address to Glasgow,
U.K.
which
correspondence
should
be sent:
MRC
Institute of
Virology,
Church
Street,
404
brain damage after head injury [4,5] and in CSF after subarachnoid haemorrhage [6]. In patients with head injury serum levels of MBP correlate well with the type and severity of brain damage and also with final outcome [ 51. In the present study we have investigated the possible clinical applications of a modified MBP assay to the assessment of patients following cerebrovascular accident. Materials and methods Radioimmunoassay for MBP The system used was similar to the serum MBP assay previously reported [ 41 with some modifications. 100 ~1 serum, 100 ~1 of buffer (0.05 M sodium phosphate (pH 7.4), 0.2 M NaCl, 50 g/l bovine serum albumin and 2 g/l calf thymus histone type II s) and 50 ~1 of rabbit anti-human MBP serum (KK 42. Pool II) diluted 1 : 40 000 in 0.05 M sodium phosphate buffer containing normal rabbit serum diluted 1 : 200 were incubated overnight at 4” C. After addition of iodinated MBP and a second overnight incubation, bound and free tracer were separated by double antibody precipitation [4]. A standard curve was constructed by serial dilutions of human MBP in pooled normal human serum. It was plotted as % specific 12’I-MBP bound (i.e. total tracer bound in the presence of antiserum - apparent tracer binding in the absence of specific antiserum) against mass of, or equivalent serum concentration of MBP (Fig. 1). The specific binding of ‘2SI-MBP (i.e. as above) was determined for each serum sample and MBP concentration calculated by interpolation from the standard curve. All MBP levels reported in this paper are those found in undiluted serum. The antiserum (KK 42, Pool II) used in the assay was from the same rabbit as that used in the previous study [ 41. It was, however, a more recent bleed taken after a further series of immunisations with human MBP and had a higher titre than the original antiserum. Assay of serum MBP binding activity The assay system contamed 100 ~1 of test serum, 100 ~1 of histone
buffer
40. 3 ;; 520 B 3 I
20
E- 10 yjb+ OO
0.2
0.5
1
2
5
10
Human MBP(W diluent; O----O, serum and ---o* delipidated Fig. 1. Standard curves for MBP assay. m------I, serum standard curves. Nonspecific binding of tracer (i.e. apparent binding in the absence of antibody) in each buffer has been deducted from each Point. Delipidated serum was prepared by high speed centrifugation of pooled normal human serum.
405
TABLE I RELATIONSHIP OF SEVERITY OF. OR DAY AFTER. ICTUS Diagnosis
n
OF CVA AND OUTCOME
Survivors
TO SERUM MBP LEVELS
Range
Patients with elevated MBP
4.4 11.5 *
U.D.-7.5 U.D.-40
0 11
4.4 11.2 *
U.D.--7.5 U.D.-40
0 11
Mean serum MBP wzll)
Moderate CVA Severe CVA
35 23
32 3
outcome
”
Moderate CVA
$urvival Death
35 23
32 3
ON THE DAY
* P < 0.05 compared with control patients. U.D. = undetectable (i.e. < 3 pg/l).
(see above) and 2 ng of 1251-MBP (10 000 cpm in 50 ~1 of histone buffer). After overnight incubation at 4°C free plus non-specific bound tracer were separated from serum bound tracer by addition of 200 ~1 of dextran coated charcoal. The charcoal suspension contained 1.5% charcoal, (Norit OL from Hopkin and Williams, Chadwell Heath, U.K.) 0.375% dextran (Pharmacia Fine Chemicals, Uppsala, Sweden) T70, and 30% horse serum (Wellcome Reagents Ltd., Beckenham, U.K.) in 0.25 M, pH 7.4 sodium phosphate buffer. Patients studied Blood samples were taken from 58 patients presenting at Glasgow Royal Infirmary with cerebrovascular accident (CVA). Patients were classified by diagnosis (severe or moderate CVA) and outcome (survival or death). Severity of CVA was determined by criteria which are based on those cited by Oxbury et al. [7]. Severe CVA was characterised by a markedly impaired level of consciousness and failure of conjugate ocular gaze towards the side of limb weakness. The distribution of patients within groups is shown in Table I. Where possible, blood samples were taken serially for at least six days after ictus. Serum was prepared from blood allowed to clot for 2-4 h at 4”C, frozen within 24 h and stored at -20°C until assay. Statistical significance of differences of grouped data was by non-parametric analysis using the Mann-Whitney U test. Results MBP assay The modifications to the assay outlined above resulted in a lower detection limit (mass of MBP giving 10% inhibition of specific tracer binding) of 300 pg, equivalent to a serum concentration of 3 ng/ml. The upper limit of the normal range for serum MBP estimated from 50 control samples from subjects without neurological, or other organ disease was 7 ng/ml. Despite modifications to the assay the diluent and serum standard curves were not coincident (Fig. 1). A standard curve in delipidated serum lies between these two curves (Fig. 1). Non-specific binding of tracer in patient and control sera in the absence of MBP
406
anti-serum varied between 6.9% and 14.7% (mean = 10.09%, s.d. = 1.81%). The mean recovery of 1.0 ng of unlabelled human MBP added to 100 ~1 of 12 different sera was 108%.
Serum levels after CVA Serum MBP levels in 58 patients on the day of ictus are shown in Table I. Levels of serum MBP above the normal range were found only in patients with severe CVA and all patients with elevated MBP died within 2 days of ictus. Levels of MBP in patients with severe CVA and patients who subsequently died were significantly greater (p < 0.05) than those in control patients, patients with moderate CVA and survivors after CVA. Thirty-four of the fiftyeight patients were followed serially. The absolute levels of serum MBP in these patients grouped according to outcome are illustrated in Fig. 2. No survivors had serum MBP levels higher than the upper limit of normal, and all patients with elevated levels subsequently died. The mean serum MBP levels from patients in the serial study are shown in Figs. 3a and 3b. Data is grouped according to diagnosis (Fig. 3a) or outcome (Fig. 3b). Patients with severe CVA and those who died have significantly elevated levels of serum MBP on days 2-6. However, not all patients in these two groups have elevated levels. Serum MBP levels from patients who died after severe CVA show a wide variation (Fig. 4). Levels, which are low on day 1 if the patient is to survive for more than two days, increase to a maximum within 2-5 days and remain elevated, in one case for up to 12 days. By contrast one patient shows no increase in serum MBP for up to 7 days after ictus. The nature of the immunoactivity in positive sera was investigated by comparison of inhibition curves for serially diluted sera with the serum standard curve (Fig. 5). The inhibition curves are not parallel with the standard curve indicating differences between the antigenic nature of MBP and the immunoactivity found in test serum. Overnight incubation of positive sera with dextran coated charcoal (0.5 ml sera and 50 ~1 of a suspension of 25 mg/ml charcoal containing 2.5 mg/ml dextran) removed all measurable immunoactivity. .
. .
.
o-1
2
5 3 4 Days After ktus
6
Fig. 2. Serial MBP levels in CVA patients. The broken line indicates the upper limit found in control patients. 0. survivors; 0, died.
407
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14
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*
.
’
5
6
7
%
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_
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7
0
Days After lctus
lctus
moderate CVA; n----n, severe Fig. 3. (a) Mean serum MBP levels related to severity of CVA. m-------o, CVA. Between days 2 and 6 serum MBP levels in severe group are significantly greater @ < 0.05) than in controls or in moderate CVA group. Numbers of observations at each point are indicated. (b) Mean serum died. Between days 2 and 6. serum MBP MBP levels related to outcome. w-i?, survivors; o-----e, levels in patient who died are significantly greater @ < 0.05) than in controls or in the survivors. Numbers of observations at each point are indicated.
Serum MBP binding activity In control sera 11% of added lzSI-MBP remained in the supernatant after charcoal absorption. Increasing the charcoal concentration decreased this level to less than 5% but high charcoal concentrations are known to disturb antigenantibody equilibria of low avidity binders, so were not routinely used. Of 58 patients with CVA tested for serum *2SI-MBP binding activity five (8.6%)
Testserum
w-
6.25
1qo
2$
401
. ,3, O-l
,
,
,
,
,
,
,
(
2
3
4
5
6
7
8
9
5
20
50
100
Serum MBP(pgII)
Days After lctus Fig. 4. Serial serum MBP levels in individual severe CVA who subsequently died.
IO
patients.
Serial MBP levels in 6 patients
(*+ 0, 0, 0, \-, l) with
Fig. 5. Parallelism of authentic human MBP and serum immunoactivity from ten patients with apparently high MBP levels. Inhibition of tracer ( 1 251 rabbit MBP) binding to MBP antisera by human MBP (i.e. standard curve). Other lines indicate inhibition of tracer binding by dilutions of sera from patients with high serum MBP levels.
408 TABLE SERUM
II ’ 2s I-MBP BINDING
Five of 58 patients showed serum ’ 2’ I-MBP binding of greater than 2 S.D. from the mean control valve (11.2 ? 4.9%). One patient (446) was studied seriallv all samples being positive for tracer binding. Patient number
Category
Previous stroke
% ’ * 5 I-MBP bound
406 408
Severe CVA/died Severe CVA/died
Yes Yes
29.1 22.6
425 440
Moderate Moderate
CVA/survived CVA/survived
Yes Yes
25.8 22.4
446
Moderate
CVA/survived
Yes
34.3 36.7 38.1
showed binding greater than two standard deviations of the mean level (Table II). All five patients had a previous clinical history of CVA. Scatchard analysis [8] on one patient indicated an equilibrium (dissociation) constant for antigen binding of 2 X lOWa l/mol. Discussion MBP assay
The MBP radioimmunoassay reported in this paper has some important differences from that described previously [ 41. Use of the more avid antiserum in the assay resulted in a more sensitive assay. The lower control levels of MBP reported here are due to (a) use of pooled normal human serum in the standard curve and (b) improved assay sensitivity which minimises the effect of nonspecific factors near the detection limit 191. The 28% of control sera which showed positive, though low, immunoactivity even in the modified assay system may contain serum components other than MBP interfering in the assay. Delipidation of pooled normal human serum produced a slight shift in the MBP standard curve, indicating that serum lipids may bind MBP, and increase the apparent levels of MBP immunoactivity in some sera. Significant levels of serum MBP binding activity were demonstrated in approximately 10% of sera from patients with a history of previous stroke. The dissociation constant for the binding agent is within the range for a low avidity antibody indicating the possible presence of MBP in serum at some time previously. This suggests that MBP may be released into the circulation after mild CVA. A more sensitive assay would be required to substantiate this. The nature of serum MBP binding factor requires further investigation to distinguish normal from possible pathological factors. However, there was no evidence that these binding factor(s) interfered with the assay for MBP, probably because their avidities are much lower than those of the rabbit anti MBP serum. Clinical results and their implications
This study provides good evidence that MBP occurs in the serum of some patients after CVA and that the apparent level is related to clinical severity and outcome. The nature of the measured serum immunoactivity needs further
409
definition since it differed from authentic MBP on serial dilution. Absorption of serum immunoactivity with charcoal is consistent with it being due to highly charged molecules such as MBP or its metabolites. Because of the non parallelism, the present assay can only be considered semiquantitative and it may be levels of serum MBP. However, the more accurate to describe “apparent” differences between patient groups in this study remain clinically significant whatever the precise nature of serum MBP immunoactivity. After severe CVA, the levels of serum MBP increase to reach a peak four to six days after ictus. The maintenance of elevated levels contrasts with the rapid clearance of systemically injected MBP in rabbits (less than 15 min) [lo]. This suggests either continuing infarction and breakdown of brain tissue after CVA or that acute brain damage is followed by increasing impairment of the blood brain barrier with leakage of MBP into the circulation. In either case, monitoring of MBP after CVA may prove useful in evaluating new methods of treatment designed to limit complications of CVA such as spread of infarction or cerebral oedema. It is also possible that the pattern of release of MBP into serum may reflect the nature of the underlying pathology (thrombosis, intracerebral or subarachnoid haemorrhage) which was not determined in the present study. We have previously reported that serum MBP levels after head injury may be a useful prognostic index [ 51, though the duration of the present assay restricts its routine use. This study indicates that serum MBP assay may also be useful prognostically after CVA. No patient with an elevated serum MBP level survived, though not all patients who died showed elevated levels on serial study. It may therefore be useful in supplementing clinical assessment in patients with severe CVA managed on life support systems. Acknowledgements We are grateful to Dr. J. Wallace, Blood Transfusion Service, Law Hospital for normal human serum and to the physicians who allowed access to their patients. J.W. Palfreyman was a recipient of a Higher Medicine Grant from the Greater Glasgow Health Board. References 1 2 3 4 5 6 7 8 9 10
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