Note: Descriptions are shown in the official language in which they were submitted.
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METHOD FOR MEASURING HYALURONIC ACID
Field of the Invention
,
This invention relates to a method for measuring hyaluronic
acid in biological fluids. More particularly, this invention
relates to a method for measuring hyaluronic acid in bioloyical
fluids using cartilage proteoglycan and an antibody reactive with
keratan sulfate.
Back round of the Invention
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Changes in serum hyaluronic acid (hereinafter also referred
to as hyaluronate) have been correlated with certain diseases.
For example, an increase in serum hyaluronic acid has been found
in patients having hepatic dysfunctions, such as cirrhosis.
Additionally, there is a correlation between the increase in
serum hyaluronic acid and cancer and rheumatoid arthritis.
Methods or the detection and quantitation of hyaluronic acid in
serum and other biological fluids are, therefore, desirable.
Cartilage proteoglycan core protein has a hyaluronic acid
binding region which specifically and reversibly binds to
hyaluronic acid. Keratan sulfate is a glycosaminoglycan
covalently linked to the cartilage proteoglycan core protein in
several areas and is concentra-ted in -the keratan sulfate-rich
region near the hyaluronic acid binding region.
Several radioassays have been disclosed for measuring the
amount of hyaluronate in biological samples. For example, the
Pharmacia HA Test 50 (Pharmacia Viagnostics AB, Uppsala, Sweden)
is a radiometric assay for the determination of hyaluronic acid
using 125I-labelled hyaluronic acid binding proteins from bovine
cartilage. See also, A. Tengblad, "Quantitative Analysis of
Hyaluronate in Nanogram Amounts", Biochem~ J. 185, 101-105
[1980).
In Thonar et al, "Quantification of keratan Sulfate in Blood
as a Marker o Cartilage Catabolism", Arthritis and Rh~umatism
28(12), 1367-137~ (1985~, there i5 described an enzyme-linked
immunosorbent-inhibition assay (ELISA) using a monoclonal
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antibody specific for keratan sulfa-te -to quantify keratan sulfate
present as single chains in adult human serum.
In Delpech et al, "Immunoenzymeassay of the
Hyaluronic-Hyaluronectin Interaction: Application to the
Detection of Hyaluronic Acid i~n Serum of Normal Subjects and
Cancer Patients", Anal. Biochem. 149, 555-565 (1985), -the authors
describe a method for investigating the binding of a hyaluronic
acid-binding glycoprotein, hyaluronectin, extracted from the
human brain, to hyaluronic acid using an enzyme-linked
immunosorbent assay technique. This reference states that
hyaluronectin is a protein component from the human brain which
combines in vitro with hyaluronic acid but not with other
glycosaminoglycans
Prior to the present invention, there was no non-isotopic
method for measuring the amount of hyalurona-te in biological
samples using cartilage proteoglycan.
Description of the Invention
This invention relates to a method for measuring hyaluronic
acid in a biological sample which comprises:
(a) linking hyaluronic acid to a solid support to produce a
coated solid support;
lb) incubating the sample with cartilage proteoglycan;
(c) exposing the incubated sample of s-tep (~) to the coated
solid support o~ step (a) to bind free proteoglycan to
the linked hyaluronic acid on the coa-ted solid support;
(d) exposing the product of step (c3 to a keratan sulfate-
reacting antibody to link the antibody to the keratan
sulfate of the proteoglycan bound -to the linked
hyaluronic acid on the coated solid support;
~e) determining the amount of antibody linked to the
keratan sulfate; and
(f3 correlating the amount of antibody linked to the
keratan sulfate to the amount of hyaluronic acid in the
sample.
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In a preferred enbodiment of the method of this invention,
microtiter plates are coated with hyaluronic acid. In the
inhibition step, in separate "dummy" (i.e., no linked
hyaluronate) plates, aliquots of a solution containing cartilage
proteoglycan are incubated with various known concentrations of
hyaluronic acid and with unknown samples of blood plasma or serum
for a period of time sufficient to permit substantially complete
reaction of the proteoglycan with the hyaluronic acid. Then,
equal amounts of the incubation mix-tures are applied to the
hyaluronate-coated plates and incubated for a period of time
sufficient for free proteoglycan which was not prereacted
~inhibited) with the hyaluronate in solution, to react with the
hyaluronate coated on the plates. The plates are then washed
thoroughly to remove all proteo~lycan, hyaluronate and
proteoglycan-hyaluronate complex that is not bound to the plates.
Next, aliquots of a solution containing a keratan
sulfate-reactive antibody ~preferably a monoclonal antibody) are
applied to the plates and incubated for a period of time
sufficient to permit substantially complete reac-tion of the
antibody with the keratan sulfate associated with the
proteoglycan bound to the hyaluronate on the plates. Monoclonal
antibodies specifically reactive with keratan sulfate are known
in the art. See, e.g., Bruce Ca-tesson, J. Biol. Chem. 258,
8848-8854 (1983).
As a means of visualizing the amount of an-tibody that is
bound to the plate, the plate is exposed to an excess of a second
antibody that reacts with the anti-keratan sulfate antibody and
which is appropriately labeled with an enzyme or other
non-isotopic marker. For example, if the first antibody is an
anti-keratan sulfate monoclonal mouse antibody, the enzyme label
may be linked to anti-mouse immunoglobulin. A preferred means of
labeling the second antibody is by linkage to an enzyme, such as
peroxidase, which catalyzes a colorimetric reaction by which its
presence is reported. An example of a suitable peroxidase
catalized reporter reaction is described in the Journal of
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siological Chemistry 257: 14173-141~0, 1982. Other enzymatic
reporter systems may be substituted for the peroxidase reporter
system as a means for visuali~ing keratan sulfate-bound
antibodies.
The absorbances of the plates can then be read with an MR600
Microplate Reader (Dynatech, Alexandria, Virginia), for example,
either directly or with the aid of a computer.
In this preferred assay system, the more hyalu onic acid
that is present in the sample, the less color that develops.
Although the degree of color development is a function of
hyaluronic acid concentration in the sample, it is a log linear
relationship. Accordingly, it is necessary to prepare a standard
curve from known hyaluronic acid standard concentrations against
which the color development in an unknown specimen can be
compared. The standard curve for this assay is generally log
linear over only a limited hyaluronic acid concentration range,
and in order that the level of hyaluronic acid in an unknown
sample can be read with accuracy from a standard curve, it is
preferred to perform the assay on serial dilutions of each sample
to assure that one or more of the serial dilutions of each sample
assay fall within the log linear range of the standard curve.
While this invention has been described in terms of certain
preferred enbodiments, modifications obvious to one skilled in
the art may be made without departing from the scope of the
invention. For example, an isotopic label on the second antibody
or on the keratan sulfate-reactive antibody can be used to
determine the amount of keratan sulfate-reactive antibody linked
to the keratan sulfate of the proteoglycan bound to the
hyaluronic acid linked to the coated solid support. The method
of this invention can be used to measure hyaluronic acid in the
biological fluids of humans and lower animals as well.
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