U.S. patent number 4,778,768 [Application Number 06/732,003] was granted by the patent office on 1988-10-18 for method of monitoring the progressive destruction of articular cartilage in a joint.
This patent grant is currently assigned to Pharmacia AB. Invention is credited to Dick K. Heineg.ang.rd, Gert Lindblad.
United States Patent |
4,778,768 |
Heineg.ang.rd , et
al. |
October 18, 1988 |
Method of monitoring the progressive destruction of articular
cartilage in a joint
Abstract
Method of determining changes occurring in articular cartilage.
The method involves (a) quantifying proteoglycan monomer and/or
antigenic fragments thereof in a synovial fluid sample and (b)
correlating the values thus obtained with progressive destructions
in the articular cartilage appertaining to that sample fluid.
Inventors: |
Heineg.ang.rd; Dick K. (Lund,
SE), Lindblad; Gert (Upsala, SE) |
Assignee: |
Pharmacia AB (Upsala,
SE)
|
Family
ID: |
20352424 |
Appl.
No.: |
06/732,003 |
Filed: |
May 2, 1985 |
PCT
Filed: |
July 10, 1984 |
PCT No.: |
PCT/SE84/00257 |
371
Date: |
May 02, 1985 |
102(e)
Date: |
May 02, 1985 |
PCT
Pub. No.: |
WO85/01353 |
PCT
Pub. Date: |
March 28, 1985 |
Foreign Application Priority Data
Current U.S.
Class: |
436/501; 435/4;
435/7.92; 435/7.93; 435/7.95; 435/961; 436/506; 436/507; 436/509;
436/518; 436/538 |
Current CPC
Class: |
G01N
33/6887 (20130101); G01N 2800/105 (20130101); Y10S
435/961 (20130101) |
Current International
Class: |
G01N
33/68 (20060101); G01N 033/566 () |
Field of
Search: |
;435/4,7
;436/506,507,509,518,512,538 |
References Cited
[Referenced By]
U.S. Patent Documents
|
|
|
4153417 |
May 1979 |
Hallgren et al. |
4499186 |
February 1985 |
Tedorescu et al. |
|
Other References
Baker, et al., "Methods in Enzymology, vol. 83, (1983), Academic
Press, pp. 216-235. .
Greiling et al., X1 International Congress of Clinical Chemistry,
(1982), Walter de Gruyter, pp. 635-650. .
Adam, et al., Articular Synovium Int. Symp., Bruges, (1981), pp.
129-141..
|
Primary Examiner: Warden; Robert J.
Assistant Examiner: Wieder; Stephen C.
Attorney, Agent or Firm: Philpitt; Fred
Claims
We claim:
1. An immunological method for monitoring the progressive early
phase destruction of articular cartilage in a joint which comprises
the steps of
(a) obtaining a first synovial fluid sample from a joint,
(b) on at least one later occasion obtaining a least one other
synovial fluid sample from said joint,
(c) reacting said fluid samples with an antibody specific to
proteoglycan monomers recognized to contain three peptide regions
or specific to antigenic fragments thereof existing in the synovial
fluid samples withdrawn in accordance with steps (a) and (b),
(d) quantifying said monomers or fragments thereof, and
(e) comparing the quantities found in accordance with step (d),
whereby the degree of progressive early phase destruction of the
articular cartilage in said joint at said later occasion may be
ascertained by observing the magnitude of the increased
proteoglycan values relative to the proteolgycan value found for
said first fluid sample.
2. A method according to claim 1 wherein the immunological assay is
performed with an antibody preparation directed specifically
against at least one of the three peptide regions of the
proteoglycan monomer.
3. A method according to claim 2 wherein antibody-binding sites in
said proteoglycan monomer or in said antigenic fragments thereof
have been unmasked by depolymerizing the chondroitin sulfate side
chains of the proteoglycan in order to facilitate a
proteoglycan-antiproteoglycan reaction.
4. A method according to claim 2 wherein the immunological assay
method is a heterogenuous method.
5. A method according to claim 4 wherein proteoglycan monomer or
antigenic fragments thereof are bound to an insoluble support and
are allowed to compete with the sample proteoglycan for
antiproteoglycan, whereupon the antiproteoglycan thus insolubilized
is quantified with the aid of labeled antibodies directed against
the antiproteoglycan.
6. An immunological method for estimating the progressive early
phase destruction of the articular cartilage in a joint which
comprises the steps of
(a) obtaining a synovial fluid sample from a first healthy
joint,
(b) obtaining at least one synovial fluid sample from a second
joint,
(c) reacting said fluid samples with an antibody specific to
proteoglycan monomers recognized to contain three peptide regions
or specific to antigenic fragments thereof existing in the synovial
fluid samples withdrawn in accordance with steps (a) and (b),
(d) quantifying said monomers or fragments thereof, and
(e) comparing the quantities found in accordance with step (d),
whereby progressive early phase destruction of the articular
cartilage in said second joint is indicated by a proteoglycan value
in the sample of step (b) that is higher than the proteoglycan
value obtained from the sample from said healthy joint.
7. A method according to claim 6 wherein the immunological assay is
performed with an antibody preparation directed specifically
against at least one of the three peptide regions of the
proteoglycan monomer.
8. A method according to claim 6 wherein antibody-binding sites in
said proteoglycan monomer or in said antigenic fragments thereof
have been unmasked by depolymerizing the chondroitin sulfate side
chains of the proteoglycan in order to facilitate a
proteoglycan-antiproteoglycan reaction.
9. A method according to claim 6 wherein the immunological assay
method is a heterogenous method.
10. A method according to claim 9 wherein proteoglycan monomer or
antigenic fragments thereof are bound to an insoluble support and
are allowed to compete with the sample proteoglycan for
antiproteoglycan, whereupon the antiproteoglycan thus insolubilized
is quantified with the aid of labled antibodies directed against
the antiproteoglycan.
Description
The present invention is concerned with a method of clinically
detecting changes occurring in articular cartilage (=the cartilage
of skeletal joints). In particular, the invention is concerned with
the detection of changes involving progressive destruction of the
cartilage, that is, changes indicating a condition of increasing
degradation of articular cartilage.
Articular cartilage comprises as its major component an
extracellular matrix which assumes an important functional role and
the composition of which is ccntrolled by a relatively small number
of cells. This matrix is composed of (i) collagen forming a fibrous
network which is of importance for the volume stability of the
tissue, and (ii), as a further major component, proteoglycan having
a large amount of mutually repellant electric charges due to which
the tissue acquires its elasticity and its ability to resist
compression. Articular cartilage, moreover, contains several other
proteins, generally without known functions. An exception to this
are the link proteins which participate in the formation of
proteoglycan aggregates and contribute to the stability of these
aggregates. Such aggregate formation appears to be a prerequisite
in order for the proteoglycan and its negatively charged groups to
be fixed in the tissue.
Other proteins present in articular cartilage are unknown in
respect of their structure and function. Examples of these are two
proteins having an apparent molecular weight of about 60 kDa.
Moreover, proteins have been detected which are present in most
types of cartilage and one of which has a molecular weight of about
36 kDa. Fibronectin, too, can be detected; but its occurrence is
actually more conspicuous in other types of connective tissue and
in blood plasma.
The structure of cartilage proteoglycans is known nowadays in its
major aspects. The molecular unit is the so-called proteoglycan
monomer which consists of a central protein core and a large number
of carbohydrate side chains carrying a large number of negative
charges and attached to the core at one of their ends. The central
protein core can be conceived as being subdivided into three
regions according to its carbohydrate side chains and its amino
acid sequence. The side chains comprise two main types, viz.,
chondroitin sulfate and keratan sulfate side chains. These two
types of side chains are concentrated each to one region of the
protein core, to thus form a chondroitin sulfate rich region at one
end of the core and a smaller keratan sulfate rich region located
between said first-named region and the third core region, this
latter being the hyaluronic acid-binding region which does not
possess any carbohydrate side chains of the aforesaid types. The
predominant proteoglycans contain about 100 chondroitin sulfate
chains each with 100 negative charges, and about 50 keratan sulfate
chains each with about 5 negative charges, the total of charged
groups being about 10,000. Via the hyaluronic acid-binding region a
great number of proteoglycan monomers are bound to hyaluronic acid
which consists of a long polysaccharide chain. The aggregates thus
formed have molecular weights exceeding 100.times.10.sup.6 Da. The
overall compositional pattern is further complicated by the fact
that articular cartilage contains two antigenically different
populations of aggregating proteoglycans differing slightly inter
se in respect of the nature of their side chains. By means of an
immunochemical method small amounts of proteoglycans have been
characterized in biopsies from cartilage (Biochem. J. (1979), p.
35-45, and Biochem. J. 187 (180), p. 687-694).
Degradation of cartilage structures is believed to involve the
whole extracellular matrix,, although different parts are degraded
in different stages of a cartilage disease. In an early phase,
mainly proteolycans are degraded, while the collagen remains in the
matrix. Unpublished results indicate that in the very beginning
only the two carbohydrate rich regions are degraded and excreted to
the synovial fluid, while the hyaluronic acid binding region
remains bound to the extracellular matrix for a longer time.
Prior to the invention no methods were available to detect changes
at this early stage and only later in the destructive process
arthroscopy could be used as a diagnostic tool. The macroscopic,
probably irreversible changes appear as a consequence of further
degradation involving both proteoglycan and collagen. At the later
stages nearly all the proteoglycan will be lost meaning that the
cartilage will become rather static with respect to its
proteoglycan content.
Degradation of structures in articular cartilage is seen typically
in all diseases resulting chronically in the destruction of the
joint structures. As examples of such disorders may be mentioned
rheumatoid arthritis, psoriasis arthritis, and osteoarthrosis.
Acute inflammation of a joint, too, is often accompanied by a
destruction of cartilage, although in most cases this will not
develop into the chronically destructive disease. It is not known
which factors are crucial for the acutely inflamed joint to either
proceed to healing or develop into the chronic process. Examples of
diseases involving acute joint inflammation are yersinia arthritis,
pyrophosphate arthritis, gout (Arthritis urica), septic arthritis
and various arthrites of traumatic etiology. Among other factors
potentially conducive to the destruction of articular cartilage may
be mentioned, for instance, treatment with cortisone; this has been
known for a long time to accelerate the degenerative process in
osteoarthrosis. It should be noted, however, that the actual
conditions prevailing in cases of arthritis with severe
inflammation of the joint are of a rather more complex character,
since in those cases injection of cortisone appears to have an
overall positive effect. An object of the present invention is to
improve the diagnostic possibilities of cartilage degenerative
processes, particularly in early stages, and to provide a means of
checking up on or monitoring the effects of therapeutical measures
taken.
Heretofore diseases of the joints have mostly been diagnosed by
indirect methods. For example, inflammatory processes have been
detected by demonstration of increased amounts of leucocytes in
samples of synovial fluid. In addition, the function of the joint
has been determined, and also the sensation of pain from the joint
has been studied. As regards cartilage biopsies, these have been
available only in a very late stage, usually in connection with
actual surgery; only in that late stage has it been pcssible, thus,
to study changes in the composition of the cartilage. The problem
inherent in this type of biopsy is (i) that it has to be taken from
the very portion of the cartilage in which the changes have
occurred, and (ii) that such biopsies are often taken in so late a
stage that most of the primary changes have already subsided.
A rudiment of an alternative method was published in 1967 by
Sandson (Science 155 (1967), p. 839-841). Sandson described a "new
component". This was detected by cross reaction with an antibody
preparation prduced by immunization of rabbits with a partially
purified cartilage extract. Preparations of the type employed have
later been shown to contain a large number of different proteins.
Results obtained with these preparations would generally tend to
vary, and for this reason it has now for a long time been customary
to adopt a different technique in the cartilage field; in view of
this circumstance it is not possible now to give any definition of
the contents of these preparations. Sandson found that the "new
component" was likely to be present in increased amounts in certain
disease conditions. At the same time he showed, together with Janis
R. et.al. (Science 158 (1967), p. 1464-67), that the lining cells
of the synovial membrane contained a component antigenically
related to the aforesaid "new component". Sandson and Janis
suggested that this component might pass into the synovial cavity
from the synovial membrane. These lining cells are not known to
contain proteoglycan of the kind that is typical of articular
cartilage, and as a consequence the Sandscn method has not provided
a means of measuring specifically proteoglycan monomers deriving
from articular cartilage.
A similar methodology has recently been applied by Gysen P. et al.
(Articular Synovium Int. Symp. (1982) p. 129-41) who used a
competitive solid phase immunoassay (labelled proteoglycan and a
solid phase antiproteoglycan antiserum) in order to determine the
proteoglycan content of synovial fluids from patients suffering
from rheumatoid arthritis and osteoarthrosis. In their study they
only found very low values which were related to an enhanced
phagocytosis by certain cells not being part of the articular
cartilage. On the basis of research results obtained in connection
with the present invention, these low values must be interpreted as
if the studied cartilage was in a very late phase of
destruction.
Both Gysen, P. et al. and Sandson thus only examined very late
stages of cartilage destruction.
It will thus be appreciated that numerous disadvantages have been
involved with earlier methods for specifically measuring changes
occurring in an articular cartilage. In many respects these
disadvantages are eliminated by the present invention. The method
of this invention for determining changes occurring in articular
cartilage is characterized in that the concentration or quantity of
cartilage proteoglycan monomer or antigenic fragments thereof is
determined specifically in a synovial fluid sample from the
synovial cavity bordering on the articular cartilage in question,
and that the value obtained is compared with a reference value from
an earlier analysis. The term "proteoglycan" will hereinafter be
used synonymously for the aforesaid fragments and for the monomer.
In the light of what is known to date, an increased concentration
or quantity (level) means that the cartilage is subject to
progressive destruction. The said increase is measured in relation
to the reference value obtained for the/a joint having healthy
cartilage. A decreased concentration relatively to that same
reference value may indicate that proteoglycans have an impaired
ability to migrate into the synovial fluid. During the priority
year research work has shown that a decreased amount (relatively
that same value for a healthy joint) may point to the fact that the
total amount of proteoglycans in the cartilage is substantially
decreased, indicating tissue loss. As a consequence only a lower
amount of proteoglycans remains to be subjected to the destructive
process meaning that, with regard to proteoglycan, the destruction
is at or close to its endpoint.
Thus in acccrdance with the present invention in its general aspect
specific proteoglycans from articular cartilage are quantified in a
sample of synovial fluid by means of known per se methods,
whereupon the value thus obtained is compared with a reference
value from an earlier analysis, either on the same point or as a
mean value obtained for healthy joints, a difference between these
values reflecting a change that has taken place in the articular
cartilage corresponding to said sample. Various embodiments of the
invention are set forth in the appended claims.
In its preferred embodiments the invention is characterized in that
the determining of the content of proteoglycan monomer or its
antigenic fragment is carried out with the aid of an immunochemical
assay method. According to these embodiments of the invention, at
least one antibody preparation (henceforth to be called
antiproteoglycan) having specificity for antigenic determinants on
the proteoglycan is added to the sample in order to thus react with
proteoglycan present in the sample, the amount of proteoglycan in
the sample being then determined in a known per se manner. The
antiproteoglycan employed may be directed specifically against
structures in one, two or three regions of the three-pestide
regions of the proteoglycan monomer. One of the preferred
embodiments of the invention comprises determining the
concentration or amount of the region(s) corresponding to the
specificity of the antiproteoglycan employed. As already indicated,
when very early stages of destructions are to be determined, it is
most preferable to use an antiproteoglycan specific for one or more
determinants present in any of the two carbohydrate rich regions of
the proteoglycan core.
It will be seen from the above explanations that for determining
the proteoglycan monomer or fragment content the invention in its
preferred embodiments utilizes an antigenantibody reaction, thus
these modes encompass a method selected from the large group of
immunological assays. A multitude of different methods belonging to
this group are known to persons skilled in the art. All of these
methods are potentially applicable to the invention, although some
of them are more eminently suitable than others, for example
because of the specificity inherent in a special technique, or
because of a high degree of sensitivity or selectivity. Methods to
be mentioned in this context are e.g. those employing a labeled
reactant, for instance an antigen or antibody labeled with an
analytically traceable label group; examples of such labels are a
radioactive isotope, an enzyme, an enzyme substrate, an enzyme
inhibitor, a fluorescent or chemoluminescent group, a particle, a
bacteriophage or other known labels. In the context of the present
invention either the proteoglycan or the antiproteoglycan or an
antibody preparation reacting with an antiproteoglycan may
preferably be labeled and used according to the invention When
measuring fragmented proteoglycans by the use of labeled
proteoglycan, it is imperative to use either labeled forms of the
fragments to be, determined or to use antibody only reacting with
antigenic sites, common for such fragments and labeled
molecules.
In immunological assay methods of the type as mentioned above, the
labeled reactant such as for instance a labeled antigen is caused
to react with its immunochemical counterpart, for instance its
antibody. If labeled antigen and antibody are used, the reaction
between them will give a labeled antigen-antibcdy complex, with or
without residual free labeled antigen and free antibody remaining
in the reaction mixture. In this context "free" means that the
antigen or antibody, resp., does not form part of the complex.
After the reaction, the analytically traceable group is determined
in free labeled antigen or in the complex. The labeled reactant and
its amount have been chosen so that the value measured is a
function of that to be determined.
For the discrimination between free labeled reactant and
complex-bound labeled reactant two main types of practical methods
are available, viz., homogeneous and heterogeneous methods.
Homogeneous methods utilize the phenomenon that the activity of a
label group will vary depending on whether or not said group is
bound in a complex. It is thus possible to assay for complex-bound
and/or free labeled reactants without proceeding to their physical
separation from each other.
Heterogeneous methods involve separation of complex-bound labeled
reactant from free labeled reactant. This separation may be
accomplished in exxentially two different ways: (1) One separation
method utilizes a soluble or insoluble reagent which selectively
precipitates or adsorbs (a) the complex (containing labeled
reactant) but not free labeled reactant, or (b) free labeled
reactant but not complex-bound labeled reactant. The terms
"selective precipitation" and "selective adsorption" mean that one
species is precipitated or adsorbed to a greater extent than the
other. Examples of precipitating reagents are polyethylene glycol
and precipitating antiserum directed against some complex component
that is not labeled. (2) The other method utilizes an insoluble or
insolubilizable support to which is bound a non-labeled antigen,
non-labeled antibody or non-labeled anti-antibody. Just as in
method (1) two phases are formed, one of these phases being
enriched in free labeled reactant and the other one being enriched
in complex-bound labeled reactant. The two phases are then
separated from each other, whereupon the analytically traceable
group is determined in at least one of the phases.
The supports employed and their use belong to prior art technique.
Suffice it to mention here that hydrophobic plastics surfaces may
be employed such as for instance surfaces of polyvinyl chloride or
polystyrene to which an immunochemical reactant can be adsorbed or
bound. Other types of supports are insoluble supports containing
hydroxyl, amino, carboxyl or amide groups; to these the
immunochemical reactants may be bound covalently. In some cases it
is advantageous to adsorb the immunochemical reactant to the
support.
According to still another way of subdividing or grouping
immunological assay methods employing labeled reactants, one such
group comprises competitive methods and the other group comprises
non-competitive methods. The term "competitive" refers to the
phenomenon that a pair of different immunochemical reactants are
able to compete for a site on an immunochemical counterpart that is
common to both. Examples of such reactants are labeled and
non-labeled antigen. A competitive method with them will thus
involve inhibition of a reaction between labeled antigen and the
corresponding antibody by non-labeled antigen. Still another
example of this type of reactants are soluble and insolubilized
antigen. Among immunological methods that do not utilize labeled
reactants may be mentioned various electrochemical, immunodiffusion
and electrophoresis methods. Particularly worth mentioning among
these latter methods are Laurell's rocket method (Laurell C-B,
Anal. Biochem. 15 (1966), p. 45- ) and Mancini's diffusion method
(Mancini G. et.al., Immunochemistry 2 (1965), p. 235- ). Other
examples are the so-called agglutination methods.
In these assay methods it is in some cases possible to equivalently
make use of biospecific affinities other than the antigen-antibody
affinity. Examples of pairs of ccmpounds having such other-type
affinity to each other are protein A -IgG, carbohydrate - lectin,
Clq - immunocomplex, RF factor -immunocomplex, biotin - avidin,
etc.
In the hitherto best known and most advantageous embodiment of the
invention, a labeled reactant and a competitive heterogeneous
system are employed. In this embodiment of the invention a
proteoglycan bound to a support is allowed to compete with the
proteoglycan of the sample in respect of the added amount of
antiproteoglycan. The antiproteoglycan will bind to the support in
an amount inversely proportional to the proteoglycan content in the
sample. The thus resultant support-bound antiproteoglycan is
detected and quantified with an anti-antibody directed against the
antiproteoglycan and labeled with an analytically traceable group.
For best results it is suitable to preincubate the sample with
antiproteoglycan before the support-bound antigen (support-bound
proteoglycan) is added.
Proteoglycan corresponding to separated monomeric subpopulations
and their different peptide regions is produced in a manner known
per se, utilizing for example density gradient centrifugation in
CsCl, zonal rate centrifugation, fragmentation with proteolytic
enzymes followed by centrifugation and/or gel chromatography. In
the embodiment of the invention employing the immunochemical assay
route it is recommendable before carrying out the assay to
depolymerize the chondroitin sulfate side chains of the
proteoglycan, e.g. by means of chondroitinase digestion to thus
unmask antibody-binding sites. This applies both to the sample and
to the proteoglycan participating in the antigen-antibody reaction
of the assay.
As regards the production of antiserum and antibodies specifically
directed against one of the peptide regions of the monomer, this
too is a known per se method. However, it has been found that for
certain modes of the invention it may be advantageous to immunize
with intact proteoglycan monomers. An antiserum thus produced may
contain antibodies specific for the different determinants of the
proteoglycan; it may thus be employed as a universal reagent in the
method according to the present invention. Very probably the use of
preparations of so-called monoclonal antibodies or polyclonal
antibodies raised against substructures of the proteoglycan and
directed against different determinants on the proteoglycan will be
conducive to advantageous embodiments. Among the different regions
of the proteoglycan core, the hyaluronic acid binding region has
the highest immunogenicity. This means that in order to obtain a
good antibody preparation for the measurement of proteoglycan
fragments deriving from the other two regions, an antigenic
preparation only containing determinants from these two regions
should be used for immunizing purposes.
The synovial fluid sample is obtained in a manner known per se, for
instance by suction from the synovial cavity. Synovial fluid is a
highly viscous liquid which is very difficult to handle. It is
therefore recommendable to decrease the viscosity of the sample,
for instance by treatment with chondroitinase; but an important
point to be observed is that the decrease in viscosity must not
have any adverse effect on the quantification procedure. Thus if an
immunochemical method is employed for the quantification it is
imperative that the antibody-binding sites on the proteoglycan are
not destroyed.
The invention is further illustrated in the below working
examples.
EXAMPLE 1
Determining changes in animal articular cartilage
A. Preparing antigens
Proteoglycans were extracted from bovine nasal cartilage or canine
hip articular cartilage. The extracts were purified by means of
CsCl density gradient centrifugation under dissociative conditions
in guanidinium chloride (4M aqueous solution). Subpopulations of
proteoglycans rich in chondroitin sulfate (referred to as D1-S1)
and proteoglycans rich in keratan sulfate (D1-S2) were prepared
separately. The aforesaid preparations were made in conformity with
the teachings of Heineg.ang.rd, D. et.al. (Seminars Arthr. Rheum.
11:1 (1981) (Suppl. 1) p. 31-33). Proteoglycan preparations
employed for immunization were made in the same manner as above and
subjected to final purification by means of gel chromatography on
Sephadex.RTM. G-200 (dextran cross-linked with epichlorohydrin,
Pharmacia Fine Chemicals AB, Uppsala, Sweden) which was eluted with
4M guanidinium chloride (Wieslander J. et.al., Biochemical Journal
179 (1979) p. 35-45). Before a preparation was employed as an
antigen in an assay in accordance with the present invention the
chondroitin sulfate chains in the proteoglycans were removed by
digestion with chondroitinase ABC (Miles Chemicals, USA). For this
purpose, the preparations were dissolved in 0.05M Tris-HCl, pH 7.5,
to a concentration of 1 mg/ml, and were digested for 6 hrs at 37
.degree. C. Dilutions of the digested samples were employed without
further treatment.
B. Synovial Fluid
Samples of synovial fluid were taken from the left knee joint of
each of nine dogs which had been subjected to surgery in the left
knee joint so as to develop osteoarthrosis therein. Similar samples
were taken from the right knee joint as controls. The samples from
both knee joints were taken on the day of surgery, whereupon the
posterior cruciate ligament of the left joint was cut off in
accordance with Nuki (as a reference see McDevitt et.al., J. Bone
Joint Surg. Br. Vol. 58-B (1976) p. 94-101). After 3, 6, 10, 15,
19, 26 and 29 weeks samples were taken by aspiration of available
synovial fluid from both the left joint which had been operated on
and the right joint which was to serve as control. In several
instances sampling attempts were unsuccessful which is indicated by
gaps in Tables 1 and 2. The reason why these attempts failed is
that the volumes of synovial fluid available were very small,
especially in the control joints. Prior to being analyzed the
samples were digested with chondroitinase for the purposes of
depolymerizing hyaluronate and removing the chondroitin sulfate
side chains, all this in accordance with one of the preferred
embodiments of the invention. The digestion procedure comprised, as
a first step, a partial digestion for reducing the viscosity of the
synovial fluid to thus facilitate handling of the sample. This was
accomplished by adding 2 .mu.l (1% of the sample volume) of an
aqueous solution containing 0.01 (nominal) units of chondroitinase
ABC in 1.25M Tris-HCl, pH 8.0. After 4 hrs at 37.degree. C., 50
.mu.l samples were taken from the digest and were mixed with 400
.mu.l of an aqueous solution containing 0.1M Tris-HCl, pH 8.0, and
0.01 units of chondroitinase ABC. After incubation at 37.degree. C.
for another 4 hrs digestion of the chondroitin sulfate chains was
complete, and the samples were then frozen at -30.degree. C. to be
kept in that state until they were to be used.
C. Preparing Antisera
Antibodies were raised in rabbits immunized with protecglycan from
canine hip articular cartilage (The proteoglycan was obtained
analogously to the procedure in A omitting the chondroitinase
digestion). An initial injection of 1.5 mg proteoglycan monomer in
Freund's complete adjuvant was followed by two monthly injections
of 1.0 mg proteoglycan monomer in Freund's incomplete adjuvant.
Thereafter the antibody titer was sufficient for being used in an
immunoassay. The serum was not purified before being employed in
accordance with the invention.
D. Enzyme Immunosorbent Method (ELISA)
Microtiter plates employed were polyvinyl chloride plates (Dynatech
(M 29), Alexandria, Va, USA). The wells were coated (24 hrs, room
temperature) with 200 .mu.l of a proteoglycan preparation
corresponding to chondroitinsulfate-rich proteoglycans (2 .mu.g/ml;
D1-S1) or to keratansulfate-rich proteoglycans (0.5 .mu.g/ml;
D1-S2), both produced in accordance with paragraph A above,
optionally with omission of the chondroitinase treatment mentioned
in said paragraph.
Stock solutions (0.5 mg/ml in 0.05 Tris-HCl, pH 8.0) of these
proteoglycan preparations were digested with chondroitinase ABC,
(0.01 units/mg) for 4 hrs at 37.degree. C. before being further
diluted (0.05M Tris-HCl, pH 8.0) and employed for coating the
wells. The coated microtiter plates were carefully rinsed with an
aqueous solution containing 0.15M sodium chloride, 0.05 %
Tween.RTM. 20 (polyoxyethylene sorbitan monolaurate) in order to
remove non-specific non-bound proteoglycan. Synovial fluid samples
(50 .mu.l) digested with chondroitinase in accordance with B above
were diluted with 750 .mu.l of an aqueous solution containing 0.10M
sodium chloride, 0.05M sodium phosphate, 0.05 % (w/v) Tween.RTM.
20, pH 7.5. Samples (110 .mu.l) of the dilutions were mixed with an
equal volume of a dilution in the same buffer of
rabbitanti(proteoglycan) specific for the proteoglycan monomer and
obtained in accordance with paragraph C above. After preincubation
for 24 hrs at room temperature 200 .mu.l of the mixture (in
triplicate) were added to the wells of the microtiter plate. After
a 60 min. incubation period at room temperature the plates were
rinsed as stated above. Next followed an addition of 200 .mu.l of a
1/150 dilution of swine-antirabbit IgG conjugated with alkaline
phosphatase (Orion Diagnostica, Helsinki, Finland) in an aqueous
solution containing 0.10M sodium chloride, 0.05M sodium phosphate,
0.05% Tween.RTM. 20, 2 mg/ml bovine serum albumin, pH 7.5. After a
further 60 minutes at room temperature the wells were rinsed again,
and this was then followed by addition of 200 .mu.l of an enzyme
substrate solution containing 1 mg/ml p-nitrophenyl phosphate, 1M
diethanolamine, 0.5M MgCl.sub.2 and having a pH of 9.8. Absorbance
at 405 nm was measured at the start and after a 60 min. period of
incubation at room temperature. The increase in absorbance served
as a basis for the calculations. A standard curve was obtained by
means of performing the procedure also with samples containing
known amounts of canine proteoglycan monomer (digested with
chondroitinase ABC). These standard samples were included in each
microtiter plate. All samples were analyzed in triplicate, the mean
value then being employed for calculations carried out with the aid
of a spline function. The results are set forth in Tables 1 and
2.
EXAMPLE 2
Tests were made analogous to Example 1, with the only difference
that polystyrene cuvettes were coated instead of microtiter plates.
The cuvettes were employed as the solid phase in accordance with
the invention. The results were showed a somewhat large coefficient
of variation than those obtained in Example 1.
EXAMPLE 3
Tests were run in the same manner as in Example 1 except that
combinations of homologous antigens were employed, that is, canine
proteoglycan for coating, samples from dogs, and canine
proteoglycan for immunization. The results obtained were similar to
those of Example 1.
EXAMPLE 4
Determining Changss in Human Articular Cartilage
Synovial fluids from a number of patients were analyzed in a manner
analogous to that described in Example 1. The diagnoses recorded
were confirmed by arthroscopy and/or clinically. The results are
set forth in Table 3. They demonstrate highly increased values for
disease conditions involving destruction of joints.
EXAMPLE 5
Detection of Changes During Treatment with Cortisone
Cortisone (Celestona.RTM. biphase, Schering AG, Berlin, Federal
Republic of Germany; and Depomedron.RTM., The Upjohn Company,
Kalamazoo, Mich., U.S.A.) were injected in normal vertebral joints
of horses. In the case of Celestona.RTM. biphase injections the
dose was 4 ml/injection (6 mg/ml), whereas in the case of
Depomedron.RTM. injections the dose has 2 ml/injection (4 mg/ml).
Every other joint was injected with cortisone and every other with
only physiological saline. Injections were administered once a week
for 3 weeks, and at the same time synovial fluid samples were
taken. The content of proteoglycan antigen was determined in the
manner as indicated in Example 1D, but the antigen employed was
proteoglycan monomer isolated from equine articular cartilage. The
antiproteoglycans employed were directed against a mixture of
aggregating proteoglycans derived from bovine nasal cartilage and
prepared in a manner similar to that of Example 1A. The values
obtained are set forth in Table 4. They show that cortisone
injections in healthy joints result in a substantially increased
release of proteoglycan into the synovial fluid, thus revealing
that the articular cartilage is being progressively destroyed. In a
similar manner it is possible to monitor treatments given for the
prevention of proteoglycan degradation in articular cartilage.
TABLE 1
__________________________________________________________________________
Concentration of proteoglycan fragments in synovial fluid samples.
Wells coated with proteoglycan rich in chondritin sulfate (D1-S1)
Values expressed as .mu.g/ml of proteoglycan equivalents. SYNOVIAL
Difference FLUID between Number of after- this sample surgey weeks
DOG NO. and that when samples I II III IV V VI VII VIII IX taken
before were taken l r l r l r l r l r l r l r l r l r surgery
__________________________________________________________________________
Before surgery 77 -- 37 71 -- 72 -- -- -- -- -- -- -- -- -- -- -- p
= 0.1 3 252 107 83 81 -- -- 177 -- 105 26 73 -- -- -- 90 23 -- p
< 0.025 6 197 -- 95 73 134 123 123 -- 113 82 105 -- 71 37 84 50
24 p < 0.15 10 152 73 87 89 104 67 -- -- -- 76 32 65 19 -- -- 48
35 p < 0.05 15 179 30 74 88 230 -- 128 -- 205 -- 83 -- 52 32 --
-- 31 29 p < 0.025 19 135 90 94 111 149 -- 198 -- 70 -- 94 -- 69
61 118 73 39 -- p < 0.15 26 86 44 73 -- -- 88 -- 115 -- 48 29 60
-- 53 -- 26 33 p > 0.2 29 -- 291 145 63 41 121 -- 75 23 28 19 79
-- -- -- 35
__________________________________________________________________________
p = levels of significance l = left r = right
TABLE 2
__________________________________________________________________________
Concentration of proteoglycan fragments in synovial fluid samples.
Wells coated with proteoglycan rich in keratan sulfate (D1-S1).
Values expressed as .mu.g/ml of proteoglycan equivalents. SYNOVIAL
Difference FLUID between Number of after- this sample surgey weeks
DOG NO. and that when samples I II III IV V VI VII VIII IX taken
before were taken l r l r l r l r l r l r l r l r l r surgery
__________________________________________________________________________
Before surgery 49 -- 53 -- 55 -- 56 -- -- -- -- -- -- -- -- -- --
-- 3 137 41 74 84 -- -- 168 -- 104 40 29 -- -- -- 60 -- 43 -- p
< 0.15 6 54 -- 134 49 86 70 148 -- 88 103 58 -- 79 43 119 -- 68
23 p < 0.1 10 66 58 81 -- 68 61 53 -- -- -- 45 31 58 32 -- -- 37
42 p < 0.15 15 61 27 69 101 127 -- 89 -- 166 -- 53 -- 58 43 --
-- 53 31 p < 0.1 19 53 -- 94 81 75 -- 94 -- 95 -- 52 -- 57 92 94
184 38 -- p < 0.1 26 42 34 45 -- -- -- 80 -- 93 -- 25 24 50 --
42 -- 30 30 p > 0.2 29 -- 95 117 -- 40 38 92 -- 103 38 27 27 114
-- -- -- 37 -- p
__________________________________________________________________________
< 0.2 p = levels of significance l = left r = right
TABLE 3 ______________________________________ Proteoglycan
Diagnosis of patients .mu.l/ml
______________________________________ Swollen joint, normal
cartilage 41 Swollen joint, no changes in cartilage 35 Normal 33
Rupture of meniscus, throughout with lesion 81 of substance
Yersinia arthritis 353 Pyrophosphate arthritis, synovitis 172
Rheumatoid arthritis 100 Visible cartilage destruction 81 ("torn
cartilage, with lesions of femoral condyles); gout
______________________________________
TABLE 4 ______________________________________ Proteoglycan
concentration .mu.g/ml Week Cortisone Cortisone No. inj Placebo inj
Placebo ______________________________________ Inj 1 0 103 120 99
105 Inj 2 1 2 795 104 1 515 76 Inj 3 2 2 660 116 3 137 58 3 2 829
218 3 626 72 4 465 58 189 57 5 130 70 768 38 6 41 54 72 46 Inj 1 0
49 37 57 69 Inj 2 1 1 161 47 581 65 Inj 3 2 1 466 44 1 640 69 3 1
259 43 941 57 4 54 32 83 47 5 38 25 48 43 6 29 23 38 37
______________________________________
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