ANALYTICALBIOCHEMISTRY

185,313-318

(1990)

A Method for the Quantitation of Hyaluronan Acid) in Biological Fluids Using a Labeled Avidin-Biotin Technique P. Kongtawelert

and P. Ghosh’

Raymond Purves Research Laboratories (University of Sydney, St. Leonards, NS W, 2065, Australia

Received

August

(Hyaluronic

of

Sydney) at the Royal North

Shore Hospital

14,1989

An improved method for the detection and quantitation of hyaluronan (hyaluronic acid) (HA) in biological fluids is described. The principle on which the method is based is that HA binds strongly to a biotinylated HAbinding protein (B-HABP) which was prepared from cartilage proteoglycans. HA was immobilized on polyvinyl chloride plates which had been precoated with poly-L-lysine. The unknown sample or HA standards together with excess B-HABP are then added. The BHABP that binds to the immobilized HA is then incubated with the enzyme-conjugated avidin (e.g., alkaline phosphatase), and the color which develops on addition of enzyme substrate (e.g., p-nitrophenyl phosphate) is determined by light absorption using a microtitration plate reader. The assay is not only convenient and reliable but is capable of measuring HA in solution at the picogram level. The assay was used to determine HA levels in human sera and synovial fluid taken from volunteers and patients with rheumatoid arthritis and osteoarthritis. 0 1990 Academic Press, Inc.

Hyaluronan or hyaluronic acid (HA)’ has been shown to be elevated in rheumatoid arthritis (RA), as well as a number of other diseases(l-4). The quantitation of HA in tissues and bodily fluids may therefore provide a useful marker for diagnostic purposes. The quantitation of microgram amounts of HA has been achieved by a vari-

ety of methods; the oldest and most widespread is colorimetry using modifications of the carbazole method for the determination of hexuronic acid (5). However, this method is not specific since other hexuronate-containing compounds and neutral sugars also generate color in this assay. Some selectivity may be achieved using specific hyaluronidases (e.g., from Streptomyces hyalurolyticus) to generate reducing end groups which can then can be quantitated using thiobarbiturate (6); however, the sensitivity of this procedure is limited to a microgram or more of HA. In recent years, methods employing proteins with a specific affinity for HA have been utilized for its estimation. Tengblad and Laurent (73) developed a radiometric assay for HA using HA-binding proteins (HABPs) isolated from bovine cartilage. This method has enabled submicrogram quantities of HA to be detected and quantitated in biological samples, but since the 1251-HABP has a relatively short half-life the assay must be performed with freshly prepared materials. An ELISA method has been devised by Delpech and coworkers (9), using the HA-binding protein hyaluronectin, and an enzyme-linked immunosorbent-inhibition assay (ELISIA) for quantitation of HA (10) has been developed in our laboratories by employing monoclonal antibody against HA-binding region of proteoglycans. We report here a simple and highly sensitive method for detection and quantitation of HA utilizing a labeled avidin-biotin technique. MATERIALS

i To whom correspondence should be addressed. ’ Abbreviations used: HA, hyaluronan or hyaluronic acid; ELISIA, enzyme-linked immunosorbent-inhibition assay; ELISA, enzymelinked immunosorbent assay; PBS, phosphate-buffered saline; RA, rheumatoid arthritis; OA, osteoarthritis; HABP, hyaluronic acid binding protein; PG, proteoglycan; KS, keratan sulfate; BSA, bovine serum albumin; GuHCl, guanidinine hydrochloride.

0003-2697/90 $3.00 Copyright 0 1990 by Academic Press, All rights of reproduction in any form

AND

METHODS

Isolation of HABP Proteoglycans (PGs) were extracted from human artitular cartilage with buffered 4 M guanidine hydrochloride (GuHCl), pH 7.4, and purified under associative and dissociative conditions by ultracentrifugation, as de313

Inc. reserved.

314

KONGTAWELERT

scribed previously by Heinegard (11). The purified PGs were digested with chondroitinase ABC to remove chondroitin sulfate chains and the products separated by Sepharose CL-2B column chromatography (12). The KS-core protein fraction was further partially digested with trypsin and degradation products purified by Sepharose CL-6B column chromatography (13). The protein (ODzso) peak which eluted in the void volume was pooled and dialyzed against distilled water at 4°C. The HABP obtained was purified further using HA-affinity chromatography (14), and the bound peak which eluted with 4 M GuHCl was then dialyzed against water, lyophilized, and stored frozen until required.

Biotinylation

of HABP Biotinylation of HABP was performed using the method described by Rappuoli et al. (15). HABP (13 mg), which was prepared as described above, was dissolved in 0.1 M sodium hydrogen carbonate buffer, pH 8.5 (1.3 ml), and mixed in a ratio of 1:3 (w/w) with N-hydroxysuccinimidobiotin (65 mg in 1.9 ml of dimethylsulfoxide) at room temperature for 18 h. The mixture was applied to a Sepharose G-25 column (PD-10 prepacked column, Pharmacia, Uppsala, Sweden) and eluted with phosphate-buffered saline (PBS, 0.15 M, pH 7.4). The excluded protein peak was collected, aliquots were introduced into Eppendorf tubes (100 ,ul each) and stored at -20°C or in the lyophilized form which can be reconstituted when needed. In this form the preparation is stable for at least 3 months.

Coating Microtitration

Plates with HA

Activated polyvinyl chloride immunoassay plates (Titertek, Zwanenburg, The Netherlands) were precoated with poly-L-lysine (Sigma, St. Louis, MO) by the addition of 100 Ill/well containing a solution of 50 pg/ml distilled water, pH 7.4. After incubation for 1 h at 37”C, the solution were flicked out and the plates air-dried. These plates could be kept for up to 2 weeks when stored at 4°C wrapped in Vitafilm plastic [Goodyear Tyre and Rubber Co. (Australia) Ltd., Thomastown, Victoria, Australia]. To the precoated plates was added 100 pl/ well of human umbilical cord HA (Sigma, St. Louis, MO; 50 pg/ml) dissolved in PBS, pH 7.4. Plates were then incubated at 37°C for 1 h and washed three times with PBS-Tween 20, and 1% BSA-PBS (100 pi/well) was added. A further incubation at 37°C for 1 h followed by five washes with PBS-Tween 20 and air-drying afforded the required HA plates. The coated plates should be wrapped in polythene film and stored at 4°C until needed.

Quuntitatiun of HA in Samples by the Labeled Avidin-Biotin Technique The principle of this method is illustrated in Fig. 1. Samples containing unknown amounts of HA or stan-

AND

GHOSH

dard known concentrations (between 6 and 200 pg/ml) of highly purified HA medical preparation (10 mg/ml; Healon, Pharmacia Fine Chemicals, Uppsala, Sweden) in PBS, pH 7.4, containing 6.0% BSA were pipetted into small polypropylene tubes with appropriate concentrations of biotinylated HABP [0.285 pg/ml of B-HABP (equal volume)] and incubated at 37°C for 3 h. Aliquots (100 ~1) of this reaction mixture were then applied to HA-coated and BSA-blocked plates and incubated at 37°C for 90 min. The plate wells were washed five times with PBS-Tween 20 (0.05% v/v) and the appropriate dilution (1:2000 of 0.5 mg protein/ml in PBS, pH 7.4) of alkaline phosphatase-conjugated avidin (100 pi/well; Sigma, St. Louis, MO) was added. The plates were then washed five times with PBS-Tween 20 and air-dried. Alkaline phosphatase substrate (1 mg/ml of p-nitrophenyl phosphate in 1 M diethanolamine and 0.001 M MgClz, pH 9.8,lOO pi/well) was added and plates were incubated at 37°C for 60 min. The reaction was stopped by the addition of 50 ~1 of 5 N NaOH. The absorbance at 405 nm was determined using a microtiter-plate reader (Titertek Twinreader, Flow Laboratories, Zwanenburg, The Netherlands). For this assay, triplicate results were averaged and the percentage inhibition was calculated by the formula % inhibition

= 100

_ [Abo5(B-HABP + sample) - Ako5(BSA)] X 100 [A405 (B-HABP + BSA) - A4,,5(BSA)] A standard inhibition curve for HA was constructed using log/linear coordinators and the HA levels in the test samples were determined by comparing their capacity to inhibit color development at AdO5nm relative to this standard curve.

Preparation

of Biological

Samples for Analysis

The samples received from the clinics were centrifuged, divided into aliquots, and stored at -20°C until analysis. They were then diluted (tenfold) with PBS, pH 7.4, in the first or second order and diluted with 6% BSA in the final order. RESULTS

An important reagent for this assay was the HA-binding protein which was prepared as described herein or according to methods by others (13,14). Figure 2 shows the elution profile obtained by HA-affinity column chromatography of trypsin-digested HABP. As is evident, HABP was eluted from the column by 4 M GuHCI. To determine the capacity of the poly-L-lysine-precoated plates for HA, they were incubated with various concentrations of human umbilical cord HA. The amounts of

QUANTITATION

1 I

MICROTITER

OF

315

HYALURONAN

PLATE PROTEINS

+

/f STANDARD HA ORUNKNOWN

PRECOATEDWITH POLY-L-LYSINE

EXCESS BIOTINYLATEDHABP

q IN E!l BIOTIN

COATED

WITH

AND BLOCKED

HYAL~ONAN

WITH

I % BSA

ALKALINE PHOSPHATASE CONJUGATED AVIDIN

P-NITROPAENYL

PHOSPHATE

P-NITROPHENOL (OD 405nm)

FIG.

1.

Diagrammatic

representation

of the principle

used for the quantitation

HA adhering to the poly+lysine-precoated plates was determined by adding B-HABP, enzyme-conjugated avidin and enzyme substrate, as described under Materials and Methods. The saturation curve obtained for HA using the poly-L-lysine-precoated plates is shown in Fig. 3. It was found that the plates were saturated at HA concentrations greater than 50 pg/ml. This concentration of HA was therefore used in the standard method which had a low coefficient of variation from well to well (data not shown).

of HA by the labeled

avidin-biotin

technique.

To determine the optimal dilution of B-HABP and enzyme-conjugated avidin, various dilutions of HABP were applied to HA-coated plates and incubated with different dilutions of enzyme-conjugated avidin. Figure 4 shows the titration curve obtained. A B-HABP dilution of 1:40 was selected together with an enzyme-conjugated avidin dilution of 1:2000 as observed by the absorption at 405 nm using a development time of 60 min at 37°C. Under these conditions optimum quantitation of HA in standards and unknown samples was obtained.

KONGTAWELERT

AND

GHOSH 3.0,

” FRACTION

FIG. 2. Elution profile cording to the conditions

5

NUMBER

of HA-binding protein on HA-Sepharose described under Materials and Methods.

10

20

40

DILUTION

ac-

Figure 5 shows a typical standard inhibition curve for HA using the standard conditions described. It was found that through use of this method it was possible to reproducibly quantitate between 10 and 200 pg/ml of HA in biological samples. In sera taken from healthy volunteers the intra- and interassay coefficients of variation obtained using this assay were 14 + 6 and 19 f 9%, respectively. HA recovery was determined by adding known amounts of purified HA solutions (Healon) to samples. The recovery obtained varied between 75 and 98% of the theoretical value. The mean value of HA in diluted normal sera was found to be 24 + 12 rig/ml by our labeled avidin-biotin technique and 22 + 16 rig/ml using a commercial radioassay kit (HA test, Pharmacia, Sweden; see Table 1). Using the same serum samples a correlated coefficient of 0.785 was obtained using values generated with our assay and the commercial kit. Values for HA obtained by two independent methods were found not to be significantly different from each other using the Student paired t test (Table 1). The values determined for human sera and synovial fluids taken from RA and OA patients are shown in Table 2. In the small number of samples

80 FACTOR

160

3’20

640

1280 2560

OF BIOTINYLATED

FIG. 4. Relationship between dilution phosphatase-conjugated avidin at dilutions and 1:4000 (A) on an HA plate.

512010240

HABP

of B-HABP of 1:lOOO

and alkaline (m), 1:2000 (O),

examined, a higher concentration of HA was noted in sera obtained from RA patients than in sera from OA patients (Table 2). However, the HA concentrations in sera from both RA and OA patients were statistically (P < 0.0001) higher than that in sera of health volunteers (Table 1). There did not appear to be any difference between the levels of HA in synovial fluids derived from RA and OA joints. DISCUSSION

An improved method to quantitate HA is described which was developed by modification of existing techniques (7-10) and by utilization of the strong binding of avidin to biotin (16,17). HA is an anionic macromolecule which normally will not bind directly to the polyvinyl chloride or polystyrene plates. Immobilization of this macromolecule to wells of microtitration plates has previously been described using cationic proteins (18); how-

100, 90

0.65 k s 8

0.60 -

CONCENTRATION

OF HA (uplm0 CONCENTRATION

FIG. 3. Saturation curve obtained for the optimal concentration of human umbilical cord HA in PBS, pH 7.4, for adsorption on poly-Llysine (50 fig/ml; 100 pg/well)-precoated polyvinyl chloride immunoassay plates.

OF HA (pg/ml)

FIG. 6. A typical standard inhibition curve obtained by the standard labeled avidin-biotin technique using Healon in 6% BSA as standard reagent.

QUANTITATION

OF

ever, these proteins can introduce nonspecific interactions which can interfere with the determination of HA. This problem was overcome in the present method by using poly-L-lysine-precoated plates to immobilize the HA. A similar technique was reported by Epstein and Lunney (19) for the development of an ELISA for cell surface antigens. The conditions established for this assay employed only very small amounts of B-HABP which is a distinct advantage as native HABP is relatively difficult to prepare in large quantities. In contrast to other assays (7,8), we used HABP derived from human articular cartilage; however, we have found that bovine HABP is just as effective. Under the standard conditions of the assay it was found that serum samples required 1000X dilution to enable HA levels to be accurately determined. The high dilution of serum samples required in our assay eliminated

the need to precipitate

with trichloroacetic

proteins

from

samples

317

HYALURONAN

TABLE2 Hyaluronan Levels in Pathological Human Sera and Synovial Fluids as Determined by the Labeled Avidin-Biotin Tech-

nique Hyaluronan Diagnosis”

Serum (rig/ml)

Synovial fluid (rdml)

J.N.

RA

150

N.D.6

M.W. H.L.

OA OA

G.P.

RA

Subject

G.L.

RA

G.H.

RA

E.W. J.M.

OA RA

J.S.

RA

39

acid prior to analysis as is required

560 275 610 255 210

N.D. 72

Mean f SD

in other assays (10). Our method also avoids the problems associated with the presence of naturally occurring antibodies against HA in biological fluids of some spe-

450 250 300

56 124 132 72 88

91-c39

363+154

DRA, rheumatoid arthritis; OA, osteoarthritis. * Not determined.

cies (20). The antibodies can interfere with the immunologically of HA.

based

assays

(9),

giving

an underestimation

The major advantage of the present method against other published be conveniently

HA

assays

stick) provides analysis of HA,

a sufficient

is its ability

to allow

HA to

determined in extremely small volumes of biological fluids. For example, we have shown that 10 ~1 of blood (equivalent to that obtained from a finger amount

for

reproducible

The level of HA is reported to be elevated in rheumaneoplastic diseases, and liver diseases (l4). Using the present assay we were able to confirm previous reports (4) that sera of patients with RA contained higher levels of HA than sera from healthy patients or toid arthritis,

TABLE

1

OA patients (Tables 1 and 2). While the reasons have yet to be clarified, the increased mobilization of HA into the blood from the synovial space may be related to inflammation which enhances the permeability of the synovial membrane (21), as well as increases interstitial lymphatic drainage of HA (22) in arthritis. Certainly there appears to be some correlation between joint inflammation and circulating HA levels as determined by experimental studies in animals (23,24). Irrespective of the explanation, it is clear that measurement of the level of HA in biological fluids may provide a useful marker for monitoring the onset and progression of a number of important diseases and disorders. This being the case, the availability of a simple, sensitive, and accurate assay for this glycosaminoglycan can only improve our understanding and treatment

Levels of Hyaluronan in Sera of Normal Healthy Volunteers as Determined by a Commerical Radioassay and the Labeled Avidin-Biotin Technique (LABT) Hyaluronan

of these

1. Engstrom-Laurent,

(rig/ml)

D.B. G.K. J.M. M.S. N.H. P.G. P.K.

Mean f SD

Sex

Age

Radioassay test

M F M F F M M

37 30 33 32 32 47

18 13 14 8 16 31 55

29

22f16

LABT

2.

34 14

3.

11 11

4.

present

method

would

Ann.

A., Feltelius, N., Hallgren, R., and Wasteson,

Rheum.

Dis. 44,614.

Frebourg, T., Delpech, B., Bercoff, E., Senant, J., Bertrand, P., Deugnier, Y., and Bourreille, J. (1986) Hepatology 6,392. Engstrom-Laurent, A., Loof, L., Nyberg, A., and Schroder, T. (1985) Hepatology 54,638. Engstrom-Laurent, A., and Hallgren, R. (1985) Ann. Rheum. Die. 44,83.

25 31 39 23+

The

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