CA1303984C - Method for measuring hyaluronic acid - Google Patents
Method for measuring hyaluronic acidInfo
- Publication number
- CA1303984C CA1303984C CA000569875A CA569875A CA1303984C CA 1303984 C CA1303984 C CA 1303984C CA 000569875 A CA000569875 A CA 000569875A CA 569875 A CA569875 A CA 569875A CA 1303984 C CA1303984 C CA 1303984C
- Authority
- CA
- Canada
- Prior art keywords
- antibody
- hyaluronic acid
- keratan sulfate
- linked
- sample
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
Landscapes
- Peptides Or Proteins (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
ABSTRACT
A method for measuring hyaluronic acid in a biological sample which comprises (a) coating a solid support with hyaluronic acid; (b) incubating the sample with cartilage proteoglycan; (c) exposing the incubated sample to the coated solid support; (d) then exposing the coated solid support to a keratan sulfate-reactive antibody; (e) determining the amount of antibody linked to keratan sulfate; and (f) correlating the amount of antibody linked to the keratan sulfate to the amount of hyaluronic acid in the sample.
A method for measuring hyaluronic acid in a biological sample which comprises (a) coating a solid support with hyaluronic acid; (b) incubating the sample with cartilage proteoglycan; (c) exposing the incubated sample to the coated solid support; (d) then exposing the coated solid support to a keratan sulfate-reactive antibody; (e) determining the amount of antibody linked to keratan sulfate; and (f) correlating the amount of antibody linked to the keratan sulfate to the amount of hyaluronic acid in the sample.
Description
3 ~ 3 q3 ,~
.
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 g 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 *Trade Mark ,~
~L3~33~
, ...
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.
1~3~
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 ~L3~3~
~ ~ J
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.
*Trade Mark
.
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 g 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 *Trade Mark ,~
~L3~33~
, ...
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.
1~3~
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 ~L3~3~
~ ~ J
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.
*Trade Mark
Claims (5)
1. 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;
b) incubating the sample with cartilage proteoglycan;
c) exposing the incubated sample of step (b) to the coated solid support of step (a) to bind free proteoglycan to the linked hyaluronic acid on the coated solid support;
d) exposing the product of step (c) 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 f) correlating the amount of antibody linked to the keratan sulfate to the amount of hyaluronic acid in the sample.
a) linking hyaluronic acid to a solid support to produce a coated solid support;
b) incubating the sample with cartilage proteoglycan;
c) exposing the incubated sample of step (b) to the coated solid support of step (a) to bind free proteoglycan to the linked hyaluronic acid on the coated solid support;
d) exposing the product of step (c) 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 f) correlating the amount of antibody linked to the keratan sulfate to the amount of hyaluronic acid in the sample.
2. A method as recited in claim 1 wherein the antibody is a monoclonal antibody.
3. A method as recited in claim 1 wherein known concentrations of hyaluronic acid are measured in like manner, a standard curve is determined, and hyaluronic acid concentration in the sample is determined in reference to the standard curve.
4. A method as recited in claim 1 wherein the amount of the antibody linked to the keratan sulfate is determined by exposing a second antibody reactive with the antibody linked to the keratan sulfate to bind the second antibody to the antibody linked to the keratan sulfate and visualizing the bound second antibody.
5. A method as recited in claim 4 wherein the second antibody carries an attached enzyme and visualization of the bound second antibody is effected by exposing the support to a substrate system that expresses a color in the presence of the enzyme and determining the level of color expressed within a fixed time period.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US7047687A | 1987-07-07 | 1987-07-07 | |
| US070,476 | 1987-07-07 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1303984C true CA1303984C (en) | 1992-06-23 |
Family
ID=22095524
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000569875A Expired - Lifetime CA1303984C (en) | 1987-07-07 | 1988-06-20 | Method for measuring hyaluronic acid |
Country Status (2)
| Country | Link |
|---|---|
| JP (1) | JPH061273B2 (en) |
| CA (1) | CA1303984C (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2005795A1 (en) * | 1988-12-19 | 1990-06-19 | Peter Ghosh | Sandwich-elisa method for the detection and/or quantification of keratan sulphate peptides in biological fluids |
-
1988
- 1988-06-20 CA CA000569875A patent/CA1303984C/en not_active Expired - Lifetime
- 1988-07-05 JP JP63167631A patent/JPH061273B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPH061273B2 (en) | 1994-01-05 |
| JPS6429767A (en) | 1989-01-31 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKLA | Lapsed |