Hansotto Reiber, Ph.D.,
Neurochemisches Labor der Neurologischen Klinik,
Universit�t G�ttingen, Robert-Koch Strasse 40, 37075 G�ttingen,
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Acute, subacute and chronic inflammatory diseases of the central
nervous system (CNS) are often accompanied by humoral immune
response characterized by the following methods:
- The disease-related immunoglobulin class pattern (1)
- The specific antibodies against the causative antigen (IgG-,
IgA-, IgM-class) (2)
- Oligocloncal IgG (IgG-class) (3)
- Polyspecific immune response against a large variety of
non-causative antigens (4)
Intrathecally synthesized antibodies, detected in cerebrospinal
fluid (CSF), can have two different sources: a causative persisting
antigen or a polyspecific concomitant immune response. The
classical view of the immune response is the clonal selection of a
B lymphocyte clone producing specific antibodies against the
invading microorganism. As always several clones are found to fit
the specific antigen, we speak about an �oligoclonal� immune
reaction. In addition to the oligoclonal antibodies against the
specific causative antigen each immune reaction produces a wide
spectrum of different antibody-species not connected with the
causative antigen. This �polyspecific immune response� does not
depend on the presence of a corresponding persisting antigen (13)
and is of lower intensities than for the causative antigen (4,
The detection of oligoclonal immunoglobulin G (IgG) in CSF is a
basic part of a laboratory supported diagnosis of MS (3). An
unexplained high frequency of combined intrathecal measles, rubella
and varicella zoster virus (VZV) antibodies in CSF of patients with
(MS) (4) or autoimmune diseases with involvement of the CNS (6)
allows the diagnosis of a chronic inflammatory process (autoimmune
type) at the time of the first clinical manifestation (1, 4). This
is also important for diagnosis of cases with a monosymptomatic
start of the disease like an optical neuritis or an uveitis
intermedia and periphlebitis retinae (7).
It was a long way to understand that antibodies, which are
synthesized in brain and in blood, are not only directed against a
causative antigen (clonal selection) but also against other
antigens, which are not involved in the cause of the disease. The
first detection of intrathecal measles antibody synthesis (8) in
multiple sclerosis (MS) led to the hypothesis of a virus aetiology
of MS. Later observations showed a polyspecific antibody synthesis
against rubella, varicella zoster, herpes simplex, mumps viruses
(9) in the single MS-patient. Meanwhile there are also reports
about the intrathecal synthesis of autoantibodies against ds-DNA,
observed in a fraction of MS patients (4, 6).
It is the recognition of an immunological network (10), which
gives the clue to understand the polyspecific, oligoclonal immune
The detection of intrathecal antibody synthesis in CSF has a
long tradition. The linear Goldmann-Witmer-Index (11) frequently
used in ophthalmology (GW-I= Qspec / QIgG) is improved by the
corrected �Antibody-Index�, AI (2), established in CSF analysis to
avoid a false negative interpretation (7) in cases with a strong
intrathecal IgG synthesis.
The AI presents a relative value for the quantity of
intrathecally synthesized specific antibodies. With the invention
of the measurement of absolute antibody concentrations (5) the
evaluation of quantitative intrathecal antibody synthesis became
possible. With an improved calculation of the specific antibody
fraction in CSF, Fs, a virus-driven antibody synthesis can now be
discriminated from a polyspecific, network- related immune
These methodological improvements are based on the evaluation of
immunoglobulin quotients QIgG, QIgA, QIgM with a nonlinear,
hyperbolic discrimination function, QLim (2), which allows the
sensitive discrimination between blood- and brain- derived
immunoglobulin fractions (i.e., intrathecal synthesis of IgG, IgA
and IgM) in CSF. This replaces the earlier linear approaches, like
IgG-Index (Ref. in (1)), which lead to false interpretations in
cases of a blood/CSF barrier dysfunction as demonstrated in detail
The polyspecific, oligoclonal IgG response is seen in many
chronic, acute and subacute inflammatory diseases of the CNS, but
there are basic differences in quantity (Tab. 1) and the frequency
of an intrathecally synthesized antibody species (4, 7).
In MS (4) as well as in autoimmune diseases with involvement of
the CNS (6), i.e., in chronic inflammatory diseases, we observe a
high frequency of measles-, rubella- and VZV intrathecal antibody
synthesis among antibodies against other virotropic viruses or
other microorganisms, like toxoplasma gondii (4), or chlamydia
pneumonia (14). This frequency is not seen in any other acute or
subacute chronic disease.
The frequency of measles antibodies in MS is 78%, for rubella
60%, for VZV 55% and for HSV 28%. In other acute or subacute
inflammatory diseases (HSV-E, SSPE, neurosyphilis,
neurotuberculosis) we find the antibody response for these single
species with the frequency below 5% and for a combination of two
antibody species (e.g., measles and rubella) below 0.1%.
In MS patients we observed with an increasing intrathecal IgG
fraction an increasing frequency of the combination of all three
antibody species (measles, rubella, zoster = M, R, Z) compared with
those patients who had a low intrathecal IgG response with only one
or a combination of two of these three antibodies (4). With
increasing intrathecal IgG synthesis there is also an increasing
amount of antibodies produced for these MRZ species, indicated by
an increasing AI (4).
With the invention of the quantitation, i.e., characterization
of the intensity of the intrathecal antibody response (5) and the
improvement of the calculation (7) for FS (Methods), we get new
perspectives for detection of intrathecally synthesized antibodies,
regarding its diagnostic as well as its pathophysiological
Table 1. Mean intensity of the intrathecal virus-specific
antibody synthesis in acute, subacute and chronic inflammatory
diseases nof the CNS. Comparison of the Antibody-index (AI) and the
specific intrathecal antibody fraction (FS) against the causative
antigen in subacute sclerosing panencephalitis (SSPE), herpes
simplex encepahalitis (HSV-E) and the Fuchs heterochromic cyclitis
of the eye (FHC) besides multiple sclerosis (MS) with a
polyspecific immune reaction against non-causal antigens.
1 Data in the aqueous humor of the eye in the Fuchs
heterochromic cyclitis (7)
2 Correspondingly, in MS patients with uveitis or
periphlebitis the following data are found in aqueous humor:
Rubella-AI = 3.0 (0.7-35); Rubella-FS = 0.06 (0.01-0.25)%
In Table 1 we compare directly the Antibody-Index (AI) with the
specific antibody fraction (FS) for chronic, acute and subacute
diseases. From these data we learn that also in acute or subacute
diseases with a persistent causative antigen, only less than 30% of
the intrathecally synthesized total IgG represents the specific
antibodies (20% measles antibodies in SSPE, 9% herpes simplex
antibodies in HSV-E, or 2.6% rubella antibodies in aqueous humor of
the Fuchs heterochromic cyclitis (FHC) of the eye). But it is clear
by these data that the intensity of antibody synthesis against the
causative antigen is up to 60-fold higher than the intensity of the
polyspecific antibody synthesis in a chronic inflammatory process,
like MS (FS < 0.5%). The specific fraction, FS, allows a better
discrimination between these both causes of the antibody response
than the AI. AI is a relative value depending on the ratio of the
amounts of the antibodies in CSF, which derive from blood and brain
FS contributes an important diagnostic aspect to discriminate the
polyspecific antibody synthesis from the antibody synthesis against
a causative antigen. Nevertheless, for the general diagnostic
approach, AI remains the most sensitive parameter to detect an
intrathecal antibody synthesis.
As a second important information from these data in Tab. 1, we
learn that also in case of a specific antibody response against the
causative antigen the larger fraction of the intrathecally
synthesized IgG represents a polyspecific antibody response against
the non-causative antigens, which do not need the persistence of an
antigen in the immune system (13).
As a particular clue of these investigations, e.g., about the
rubella antibody synthesis in the eye of the FHC patients, we get a
biological example for theoretical approaches (15), which try to
explain the dynamics, which in the immune response can lead to a
chronic inflammatory process.
With these approaches we are on the way to a new understanding
of an immunological network-based immune response in chronic
11.2.1 Oligoclonal IgG
According to the international consensus, oligoclonal IgG is
detected with isoelectric focussing and immune detection (Andersson
et al. 1994). As interpretation criteria the 5 types shown in
figure 1 are accepted as state of the art.
Figure 1. Isoelectric focussing on agarose gels with immunoblot:
The figure represents the classical types 1 � 5 (Andersson et al.
- Type 1: No bands in CSF and serum.
- Type 2: Oligoclonal IgG-bands in CSF, not in
- Type 3: Oligoclonal bands in CSF (like type 2) and additional
identical oligoclonal bands in CSF and serum (like type
- Type 4: Identical oligoclonal bands in CSF and
serum.Interpretation:No intrathecal IgG-synthesis but systemic
- Type 5: Monoclonal bands in CSF and
Figure 2. Isoelectric focussing on polyacrylamidgel with
silverstain: In contrast to the immune detection (Fig. 1 and Fig.
2) we find in the protein stain the albumin range (also place for
application of the samples) at about pH < 5.0. In the alcaline
range (at pH 9.3) we find cystatin C (gamma-trace-protein) as a
single band in CSF (CSF marker).
The pH-range of the gradient starts on the right side with pH 3.5
(anode (+)) and reaches pH 10.5 (catode (-)).
In case of immunoblot the pattern is inversed. The
nitro-cellulose-acetate foil is attached at pH 6.5.
Figure 2 shows an isoelectric focussing with subsequent protein
detection by silver stain. Both methods have the same sensitivity,
but the silver stain is more capricious as an immune detection.
Other electrophoretic techniques are not sufficient to identify
olicoclonal IgG. It depends on the method how many bands (at least
2 bands) are necessary to detect reliably oligoclonal IgG. A single
isolated band in CSF is per definition not oligoclonal IgG.
Oligoclonal IgG is more sensitive than the Antibody-Index (AI)
against the specific antigen in case of chronic diseases (4), but
in cases of acute inflammatory processes the Antibody-Index of the
antibody species against the causative antigen is more sensitive
than the oligoclonal IgG (16) e.g., in varicella zoster caused
facial nerve palsy, with 100% increased Antibody-Index, only 50% of
the patients had oligoclonal IgG detectable.
Definition of the (corrected) Antibody-Index (AI)
AI = Qspec /QIgG (QIgG <
AI = Qspec /QLim (QIgG >
||AB(CSF) / AB(ser), specific antibody-CSF/serum quotient
||IgG(CSF) / IgG(ser), empirical immunoglobulin CSF/serum
quotient for IgG, IgA or IgM
||upper hyperbolic discrimination line of the reference range for
blood-derived immunoglobulins (IgG, IgA or IgM)
Reference range and interpretation
Method-related range of precision( x�
2s): AI = 1.0 � 0,3
Clinically defined reference range
Normal AI =
0.7 � 1.3
synthesis AI = 1.5
Values of AI < 0.5 are an indication of non-matched CSF/serum
samples or of analytical faults. Values reach a higher sensitivity
by combined evaluation of several Antibody-Index values as shown in
the three cases in the table, where in case I a rubella-AI = 1.4 is
the clear indication of an intrathecal antibody synthesis with
reference to the other normal Antibody-Index values. Less
reliability is found in the case II with a rubella-AI = 1.5
compared to the three other high Antibody-Index values. Case III
represents a typical combination of non-matched CSF/serum samples
(in spite of repetition the measles-AI remains < 0.5).
Table 2. Evaluation examples of the Antibody-Index (AI)
11.2.3 Fraction of specific intrathecal antibodies in CSF.
The specific fraction, F, in % is the ratio of the intrathecally
synthesized concentration of specific antibodies
(ABLoc), and the intrathecally synthesized concentration
of total IgG (IgGLoc). This calculation of F, for
comparison of means in different groups, refers to
Qmean, the mean function (12) of the reference range
instead of the upper limit QLim used for AI: F=
ABLoc(mean) / IgGLoc(mean) � 100 in %. With
Qmean (IgG) = (0.65 (QAlb2 +8) 0.5
� 1.4) � 10-3, we calculate IgGLoc (mean) =
(QIgG � Qmean ) � IgG(ser) in mg/L or
ABLoc (mean) = (Qspec � Qmean) �
AB(ser) in mg/L.
The antibody concentrations are determined as quantitative
values in mg/l.
Quantitation of IgG class antibodies in CSF and serum.The
absolute amount of measles-, rubella-, VZV- and HSV antibodies was
measured with a modified ELISA, established by Conrad et al (1994):
The microtiter plate was divided into five stripes: coated with
anti-Human IgG, measles antigen, rubella antigen, varicella zoster
antigen and herpes simplex antigen, respectively (Jacobi et
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