Int. Archs Allergy appl. Immun. 49: 453-463 (1975)
Natural Antibodies in Human Cerebrospinal Fluid to Rabbit Erythrocytes G. O. F ossan 1 and O. T onder 2 The Broegelmann Research Laboratory for Microbiology, The Department of Neurology, and The Gade Institute, Department of Microbiology, School of Medicine, The University of Bergen, Bergen
Abstract. With an indirect agglutination technique using rabbit antisera to hu man immunoglobulins, IgG antibodies to rabbit erythrocytes were detected in 211 of 221 unconcentrated cerebrospinal fluids from patients with diseases of the nerv ous system and controls. The titres varied from 0.25 to 128. The highest titre in the control group was 4, the geometric mean 1.5. The titres were highest in the groups with elevated total protein, for instance inflammatory diseases with mean titre of 9.25. The titre divided by total protein concentration did not vary much in the dif ferent groups, but tended to be higher than controls in multiple sclerosis, inflamma tory diseases and vascular brain lesions, and lower in the other groups. It did not in crease parallel to IgG in multiple sclerosis and inflammatory diseases.
Antibodies in cerebrospinal fluid (CSF) are normally derived from the blood [7], Owing to the small amounts concerned, however, antibodies present in serum are not regularly detected in normal unconcentrated CSF by the common techniques. Antibodies are detectable in the CSF mainly when the corresponding serum titres are high, or if the bloodbrain barrier is damaged [4, 20]. With more elaborate techniques, anti bodies to poliovirus [5] and to adenovirus [15], can be detected in most CSF. If antibodies of a certain specificity were detectable in the CSF as often as in serum, they would be useful in the study of the blood-brain barrier. Since antibodies to rabbit erythrocytes are found in almost all hu man sera [19] throughout life, we have studied a panel of CSF and sera. * Fellow of The Norwegian Research Council for Science and the Humanities. 2 The authors wish to thank Mrs. S. M orner and Mrs. T. T ynning for valuable technical assistance.
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Received: February 7, 1975.
454
F ossan/T onder
This paper deals with the demonstration and characterization of these an tibodies in CSF. Materials and Methods Patients 221 patients were studied. On the basis of clinical and laboratory diagnosis they were divided into nine groups. 1. Control group. This group comprised patients with minor psychoneurotic dis orders. The patients were originally suspected of having neurological illnesses, but subsequently diagnosed as nonneurological. The CSF total protein was below 45 mg/100 ml and the cell count was 0-3 cells/«l. For the present purpose these pa tients serve as ‘normals’. 2. Multiple sclerosis (MS). The patients were selected according to the criteria of A llison and M illar [2]; subgroup A as probable MS and subgroup B as possible MS. 3. Inflammatory diseases. This group comprised patients with serous meningitis, acute encephalitis, subacute encephalomyeloradiculitis and acute radiculoneuritis (Guillain-Barre). None had subacute sclerosing panencephalitis or bacterial meningi tis. 4. Diseases of the spine. This group comprised patients with degenerative disease of the spine, with or without signs of cervical or lumbar disc prolapse, and six pa tients with intraspinal tumour. This group was divided into two subgroups: A with CSF total protein less than 50 mg/100 ml and B with CSF total protein 50 mg/ 100 ml or above. 5. Sequelae of head injuries. These patients had signs of brain lesion. The lum bar puncture was performed at least 1 year after the head injury. 6. Brain lesions of miscellaneous aetiology. For the majority of patients in this group the aetiology was multiple or uncertain. Some had birth trauma. 7. Vascular brain diseases. Patients with cerebral embolism, haemorrhage, arterio sclerosis, hypertensive encephalopathy or sequelae following ruptured arterial aneurism were included in this group. 8. Chronic degenerative and heredodegenerative diseases. 9. Other neurologic diseases. This group comprised patients hospitalized in the neurological department who did not fit into any of the groups mentioned above. Cerebrospinal Fluids The CSF were collected by lumbar -25 °C. Fluids containing erythrocytes protein were routinely performed at Haukeland Hospital, using a modified and C loss [3].
puncture, divided into portions and kept at were excluded. The determinations of total the Department of Clinical Biochemistry, sulphosalicyl method described by Barren
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 10:02:41 AM
Antisera Rabbit antiserum to human serum was produced by immunization with whole serum in Freund’s complete adjuvant. Polyvalent antiserum to human immunoglob-
Natural Antibodies in CSF
455
ulins (Ig) was prepared by immunization of rabbits with rabbit erythrocytes agglu tinated by human serum as previously described [10]. Antiserum to IgG was pro duced using umbilical cord sera for sensitization of the rabbit erythrocytes. These antisera were absorbed with rabbit erythrocytes agglutinated by secretory IgA from human milk [9]. Antisera to human IgA and IgM were produced by immunization of rabbits with their own cells agglutinated by antibodies within the IgA or IgM class, respectively, according to the technique described earlier [18]. The specificity of the sera were checked by ring test precipitation, double diffusion in agar against serum and serum fractions and by immunoelectrophcresis using several dilutions of antigen and antisera. All sera were stored at -25 °C. Mercaptoelhanol Treatment A mixture of equal volumes of 0.2 m 2-mercaptoethanol (ME) and CSF was in cubated for 30 min at 37 °C. For some experiments CSF was dialyzed against 0.1 m ME followed by dialysis against 0.1 M iodoacctamide [21]. The CSF was then di alyzed against phosphate-buffered saline (PBS) pH 7.2. Indirect Agglutination of Rabbit Erythrocytes Using the Antiglobulin Test To avoid concentrating CSF the twofold ‘dilution’ row started with four parts of CSF by placing 0.4, 0.2 and 0.1 ml of undiluted CSF in the first, second and third tube, respectively, and preparing twofold dilutions in 0.1 ml of PBS from the fourth tube. To all tubes 0.1 ml of an 0.5%> suspension of rabbit erythrocytes prepared as earlier [19] was then added. After incubation at room temperature for 15 min the erythrocytes in each tube were washed twice in 10 vol of PBS. Finally, 0.1 ml of a 1/200 dilution of antiserum to human IgG was added to each tube. This antiserum had a titre of 8,000 in Coombs’ test with rabbit erythrocytes sensitized by 1/8 agglu tinating unit of human IgG antibodies (fraction II, gamma-Kabi, 16.5°/o solution, AB Kabi, Stockholm). 2°/o inactivated normal rabbit serum in PBS was used as di luent for the antiserum. The racks were left for 1 h at room temperature and then at 4 °C overnight. The agglutination was recorded using the patterns of the sedi mented erythrocytes on the bottom of the tubes after the tubes had been tilted in an almost horizontal position for 10-15 min. Unagglutinated erythrocytes drained down the tube bottoms in teardrop-like patterns, leaving the agglutinated erythro cytes adherent to the glass. The titre was defined as the reciprocal of the highest CSF dilution which gave agglutination (AREC titre).
Quantitation of Immunoglobulins This was performed essentially as described by F ahey and M c K elvey [6], except that Petri dishes were used instead of glass plates. The final concentration of agar (Difco Special Noble) in the agar-antiserum mixture was l°/o in PBS, 2 ml being
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 10:02:41 AM
Inhibition Test To the twofold dilutions of CSF prepared as described above 0.1 ml of a 1/5 di lution of antiserum specific for IgA or IgM was added. The mixture were incubated at 37 °C for 15 min and rabbit erythrocytes were added. Thereafter the procedure for indirect agglutination was followed.
456
F ossan/T ônder
used for each dish (diameter 45 mm). The wells (diameter 3 mm) were punched out at the same distance from the centre of the dish, 4/tl being placed in each well. Pooled sera from blood donors were used as standard. This serum pool was as signed the following Ig concentrations: IgG 1,000 mg/100 ml, IgA 200 mg/100 ml and IgM 100 mg/100 ml. The standard was regularly compared with stabilized standard human scrum purchased from Behringwerke, Marburg, FRG. The lower limit for detection of IgA and IgM was 0.25mg/100ml and for IgG 0.5 mg/100 ml. At the lower limit of detection the variation (1 SD) in 10 quantitations of the same sample was approximately 20°/o. The variation between duplicate quantitations made in ten different CSF samples with a mean IgG concentration of 1.8 mg/100 ml and a mean IgA and IgM concentration of 0.5 mg/100 ml was approximately 10%. Gel Filtration Prior to gel filtration CSF was concentrated by negative pressure dialysis through collodium membranes [13]. The proteins in CSF were separated on an 80X1.7 cm Sephadex G-200 column using 0.05 m Tris HC1 buffer pH 8.0 contain ing 0.14 m NaCl and 0.02% Na-azide. Fractions of 3 ml were collected at a flow rate of 4 ml/cm2/h.
Experiments and Results Presence of Antibodies CSF did not agglutinate rabbit erythrocytes directly, except when there was a marked increase in total protein. The indirect agglutination de scribed above, using antiserum to human serum, was therefore developed. AREC titres could be determined in 211 of 221 CSF samples, the distri bution of number of individuals according to titres being shown in table I. The highest titre recorded in the control group was 4, while the disease group showed some higher titres and a wider distribution.
Table /. Number of patients according to titres of antibodies in CSF to rabbit erythrocytes, detected by Coombs’ test (AREC titres) AREC titres
Natural Antibodies in Human Cerebrospinal Fluid to Rabbit Erythrocytes G. O. F ossan 1 and O. T onder 2 The Broegelmann Research Laboratory for Microbiology, The Department of Neurology, and The Gade Institute, Department of Microbiology, School of Medicine, The University of Bergen, Bergen
Abstract. With an indirect agglutination technique using rabbit antisera to hu man immunoglobulins, IgG antibodies to rabbit erythrocytes were detected in 211 of 221 unconcentrated cerebrospinal fluids from patients with diseases of the nerv ous system and controls. The titres varied from 0.25 to 128. The highest titre in the control group was 4, the geometric mean 1.5. The titres were highest in the groups with elevated total protein, for instance inflammatory diseases with mean titre of 9.25. The titre divided by total protein concentration did not vary much in the dif ferent groups, but tended to be higher than controls in multiple sclerosis, inflamma tory diseases and vascular brain lesions, and lower in the other groups. It did not in crease parallel to IgG in multiple sclerosis and inflammatory diseases.
Antibodies in cerebrospinal fluid (CSF) are normally derived from the blood [7], Owing to the small amounts concerned, however, antibodies present in serum are not regularly detected in normal unconcentrated CSF by the common techniques. Antibodies are detectable in the CSF mainly when the corresponding serum titres are high, or if the bloodbrain barrier is damaged [4, 20]. With more elaborate techniques, anti bodies to poliovirus [5] and to adenovirus [15], can be detected in most CSF. If antibodies of a certain specificity were detectable in the CSF as often as in serum, they would be useful in the study of the blood-brain barrier. Since antibodies to rabbit erythrocytes are found in almost all hu man sera [19] throughout life, we have studied a panel of CSF and sera. * Fellow of The Norwegian Research Council for Science and the Humanities. 2 The authors wish to thank Mrs. S. M orner and Mrs. T. T ynning for valuable technical assistance.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 10:02:41 AM
Received: February 7, 1975.
454
F ossan/T onder
This paper deals with the demonstration and characterization of these an tibodies in CSF. Materials and Methods Patients 221 patients were studied. On the basis of clinical and laboratory diagnosis they were divided into nine groups. 1. Control group. This group comprised patients with minor psychoneurotic dis orders. The patients were originally suspected of having neurological illnesses, but subsequently diagnosed as nonneurological. The CSF total protein was below 45 mg/100 ml and the cell count was 0-3 cells/«l. For the present purpose these pa tients serve as ‘normals’. 2. Multiple sclerosis (MS). The patients were selected according to the criteria of A llison and M illar [2]; subgroup A as probable MS and subgroup B as possible MS. 3. Inflammatory diseases. This group comprised patients with serous meningitis, acute encephalitis, subacute encephalomyeloradiculitis and acute radiculoneuritis (Guillain-Barre). None had subacute sclerosing panencephalitis or bacterial meningi tis. 4. Diseases of the spine. This group comprised patients with degenerative disease of the spine, with or without signs of cervical or lumbar disc prolapse, and six pa tients with intraspinal tumour. This group was divided into two subgroups: A with CSF total protein less than 50 mg/100 ml and B with CSF total protein 50 mg/ 100 ml or above. 5. Sequelae of head injuries. These patients had signs of brain lesion. The lum bar puncture was performed at least 1 year after the head injury. 6. Brain lesions of miscellaneous aetiology. For the majority of patients in this group the aetiology was multiple or uncertain. Some had birth trauma. 7. Vascular brain diseases. Patients with cerebral embolism, haemorrhage, arterio sclerosis, hypertensive encephalopathy or sequelae following ruptured arterial aneurism were included in this group. 8. Chronic degenerative and heredodegenerative diseases. 9. Other neurologic diseases. This group comprised patients hospitalized in the neurological department who did not fit into any of the groups mentioned above. Cerebrospinal Fluids The CSF were collected by lumbar -25 °C. Fluids containing erythrocytes protein were routinely performed at Haukeland Hospital, using a modified and C loss [3].
puncture, divided into portions and kept at were excluded. The determinations of total the Department of Clinical Biochemistry, sulphosalicyl method described by Barren
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 10:02:41 AM
Antisera Rabbit antiserum to human serum was produced by immunization with whole serum in Freund’s complete adjuvant. Polyvalent antiserum to human immunoglob-
Natural Antibodies in CSF
455
ulins (Ig) was prepared by immunization of rabbits with rabbit erythrocytes agglu tinated by human serum as previously described [10]. Antiserum to IgG was pro duced using umbilical cord sera for sensitization of the rabbit erythrocytes. These antisera were absorbed with rabbit erythrocytes agglutinated by secretory IgA from human milk [9]. Antisera to human IgA and IgM were produced by immunization of rabbits with their own cells agglutinated by antibodies within the IgA or IgM class, respectively, according to the technique described earlier [18]. The specificity of the sera were checked by ring test precipitation, double diffusion in agar against serum and serum fractions and by immunoelectrophcresis using several dilutions of antigen and antisera. All sera were stored at -25 °C. Mercaptoelhanol Treatment A mixture of equal volumes of 0.2 m 2-mercaptoethanol (ME) and CSF was in cubated for 30 min at 37 °C. For some experiments CSF was dialyzed against 0.1 m ME followed by dialysis against 0.1 M iodoacctamide [21]. The CSF was then di alyzed against phosphate-buffered saline (PBS) pH 7.2. Indirect Agglutination of Rabbit Erythrocytes Using the Antiglobulin Test To avoid concentrating CSF the twofold ‘dilution’ row started with four parts of CSF by placing 0.4, 0.2 and 0.1 ml of undiluted CSF in the first, second and third tube, respectively, and preparing twofold dilutions in 0.1 ml of PBS from the fourth tube. To all tubes 0.1 ml of an 0.5%> suspension of rabbit erythrocytes prepared as earlier [19] was then added. After incubation at room temperature for 15 min the erythrocytes in each tube were washed twice in 10 vol of PBS. Finally, 0.1 ml of a 1/200 dilution of antiserum to human IgG was added to each tube. This antiserum had a titre of 8,000 in Coombs’ test with rabbit erythrocytes sensitized by 1/8 agglu tinating unit of human IgG antibodies (fraction II, gamma-Kabi, 16.5°/o solution, AB Kabi, Stockholm). 2°/o inactivated normal rabbit serum in PBS was used as di luent for the antiserum. The racks were left for 1 h at room temperature and then at 4 °C overnight. The agglutination was recorded using the patterns of the sedi mented erythrocytes on the bottom of the tubes after the tubes had been tilted in an almost horizontal position for 10-15 min. Unagglutinated erythrocytes drained down the tube bottoms in teardrop-like patterns, leaving the agglutinated erythro cytes adherent to the glass. The titre was defined as the reciprocal of the highest CSF dilution which gave agglutination (AREC titre).
Quantitation of Immunoglobulins This was performed essentially as described by F ahey and M c K elvey [6], except that Petri dishes were used instead of glass plates. The final concentration of agar (Difco Special Noble) in the agar-antiserum mixture was l°/o in PBS, 2 ml being
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 10:02:41 AM
Inhibition Test To the twofold dilutions of CSF prepared as described above 0.1 ml of a 1/5 di lution of antiserum specific for IgA or IgM was added. The mixture were incubated at 37 °C for 15 min and rabbit erythrocytes were added. Thereafter the procedure for indirect agglutination was followed.
456
F ossan/T ônder
used for each dish (diameter 45 mm). The wells (diameter 3 mm) were punched out at the same distance from the centre of the dish, 4/tl being placed in each well. Pooled sera from blood donors were used as standard. This serum pool was as signed the following Ig concentrations: IgG 1,000 mg/100 ml, IgA 200 mg/100 ml and IgM 100 mg/100 ml. The standard was regularly compared with stabilized standard human scrum purchased from Behringwerke, Marburg, FRG. The lower limit for detection of IgA and IgM was 0.25mg/100ml and for IgG 0.5 mg/100 ml. At the lower limit of detection the variation (1 SD) in 10 quantitations of the same sample was approximately 20°/o. The variation between duplicate quantitations made in ten different CSF samples with a mean IgG concentration of 1.8 mg/100 ml and a mean IgA and IgM concentration of 0.5 mg/100 ml was approximately 10%. Gel Filtration Prior to gel filtration CSF was concentrated by negative pressure dialysis through collodium membranes [13]. The proteins in CSF were separated on an 80X1.7 cm Sephadex G-200 column using 0.05 m Tris HC1 buffer pH 8.0 contain ing 0.14 m NaCl and 0.02% Na-azide. Fractions of 3 ml were collected at a flow rate of 4 ml/cm2/h.
Experiments and Results Presence of Antibodies CSF did not agglutinate rabbit erythrocytes directly, except when there was a marked increase in total protein. The indirect agglutination de scribed above, using antiserum to human serum, was therefore developed. AREC titres could be determined in 211 of 221 CSF samples, the distri bution of number of individuals according to titres being shown in table I. The highest titre recorded in the control group was 4, while the disease group showed some higher titres and a wider distribution.
Table /. Number of patients according to titres of antibodies in CSF to rabbit erythrocytes, detected by Coombs’ test (AREC titres) AREC titres
