Journal of Clinical Laboratory Analysis 9:193-195 (1995)

Novel Homogeneous Enzyme lmmunoassay: Chitinase Activity Enhancement lmmunoassay (CAEIA)

Sakari Sekine,' Takeshi Watanabe,' and Hirosato Tanaka2 'Department of Biosystem Science, Graduate School of Science and Technology; and 'Department of

Agricultural Chemistry, Faculty of Agriculture, Niigata University; Niigata, Japan

A novel homogeneous enzyme immu- noassay has been developed that takes ad- vantage of the observation that the activity of a bacterial chitinase could be enhanced by the binding of a ligand conjugate of a monoclonal antibody to the enzyme (L- MAbchi). The enhancement was sup- pressed by an antiligand antibody (AbL). Free ligand molecules competed for AbL combining with L-MAbchi, and the sup- pression of the enhancement was re-

duced. Thus the concentration of free ligand could be estimated. Biotin was used as a ligand in this model experiment. When this monoclonal antibody was conjugated with a ligand (e.g., biotin), we furthermore found that the enhancement could be sup- pressed by an antiligand antibody (AbL). When free ligand molecules are added to the system, competition with L-MAbchi results to reduce the suppression of en- hancement. o 1995 Wiley-Liss, Inc.

Kev words: monoclonal antibodv. chitinaseA1. biotin. Baci//us circulans WL-12

INTRODUCTION

Enzyme immunoassay techniques have been widely used as alternatives to radioimmunoassay techniques for assay- ing various substances of clinical interest. They are gener- ally classified into two types: heterogeneous techniques where physical separation of free and bound materials is required and homogeneous techniques, where physical separation is not required. Thus homogeneous enzyme immunoassay tech- niques can offer significant advantages to clinical laborato- ries owing to the rapidity and simplicity of their protocols. To date, various enzyme, including lysozyme (l), hexoki- nase, glucose-6-phosphate dehydrogenase (2), P-amylase (3), P-D-galactosidase ( 4 3 , and peroxidase (6) have been used as labels or indicators in such immunoassays.

Here, we report a novel homogeneous enzyme immunoas- say technique (chitinase activity enhancement immunoassay, CAEIA), which utilizes a monoclonal antibody to enhance the activity of chitinase. To our knowledge, this is the first example of enzyme-enhancing activity by a monoclonal an- tibody. Briefly, the system includes chitinase A1 of Bacillus circulans WL-12 (cloned in Escherichia coli HB 101) (7), a monoclonal antibody against it which enhances its activity conjugated to a ligand of interest (biotin), G-2-5M (8), and an antibody against biotin,AbL. As described previously (8), two out of 11 monoclonal antibodies against chitinase A1 of B. circzilans enhanced the enzyme activity significantly. This observation, coupled with the fact that chitinase was practi- cally nonexistent in human body fluid, lead us to develop immunoassay. Using biotin as a model analytefligand, we

covalently linked it to the chitinase-enhancing monoclonal antibody G-2-5M to make L-MAbchi, which still remains in enhancing activity. An antibody directed against biotin, the analyte and ligand, was found to suppress the enhance- ment of chitinase activity by L-MAbchi presumably through steric hindrance. In the presence of free biotin, the degree of the suppression of the enhancement was re- duced. A definite correlation was obtained between the concentration of biotin and the chitinase activity in the reaction mixture with fixed concentrations of chitinase, L-MAbchi and AbL. Thus biotin concentration in the as- say mixture could be estimated.

MATERIALS AND METHODS

Chitinase and Monoclonal Antibody Against Chitinase

prepared and purified as previously described (8). Chitinase A1 and monoclonal antibody (G-2-5M) were

Biotin Conjugation to G-2-5M (MAbchi)

MAbchi solution was dialyzed against 0.1 M carbonate buffer, pH 8.5, at 4°C overnight. After dialysis, 0.2 ml (600 pg) of

Received September 15, 1999; accepted September 28, 1994.

Address reprint requests to Hirosato Tanka, Department of Agricultural Chemistry, Faculty of Agriculture, Niigata University, 8050 Ikarashi-2, Niigata 950-21, Japan.

0 1995 Wiley-Liss, Inc.

194 Sekine et al.

MAbchi solution and 2.9 mg of sulfosuccinimidyl-6- (biotinamido) hexanoate (Pierce Chemical Company Co., Rock- ford, IL) in 0.12 ml of the s’me buffer were mixed and stirred for 2 hr at room temperature to form the conjugate. To stop the reaction, 10 mg of glycine powder was added. The mixture was dialyzed against 0.02 MTris buffer, pH 7.5, containing 0.15 M NaCl and 0.1% NaN3 at 4°C overnight to remove any free sulfosuccinimidyl-6-(biotinamido) hexanoate.

Assays

Protein concentration were determined by the method of Bradford (9) or by the absorance at 280 nm using extinction coefficients of = 14 for IgG.

Chitinase assay was performed as previously described (8) with p-nitrophenyl-N,N”N’’-triacetyl chititriose [p-NP- (GlcNAc)3] (Yaiszu Suisan Industry Co., Yaizu, Japan) as sub- strate. To assay the chitinase activity in human serum, 25 p1 of human serum was added to 1.1 ml of 0.1 mM [p-NP-(GlcNAc),] in 0.1 M phosphate buffer, pH 6.0, and the mixture was incu- bated at 37°C. The absorbance at 337 nm (A317 “,J was moni- tored and chitinase activity was expressed as the initial rate of increase in A337nm (AA337nm/min). N-actyl-P-D-gluco- saminidase (NAG) was measured using “NAG TEST SH1ONOGI”reagent kit (Shionogi Seiyalcu Co., Osaka, Japan) in which sodium-m-cresolsulfonphthaleinyl N-acetyl-P-D- glucosamide was used as a substrate. Serum samples were ob- tained from apparently normal human adults ages 20-50.

Reagents

Antibiotin antibody (goat) was obtained from Organon Teknika N. V. Cappel Products (Durham, NC), normal goat IgG from Rockland Co. (Gilbertsville, PA), and (+)-biotin from Wako Pure Chemical Industries Co. (Osaka, Japan). Streptavidin was purchased from Sigma Chemical Co. (St. Louis, MO).

RESULTS

Enhancement of Chitinase Activity by Biotinylated G-2-5M (L-MAbchi)

Figure 1 shows the increase chitinase activity with increas- ing concentrations of L-MAbchi and the leveling off of enzyme activity at a level -3.5-fold that of normal activity.

Suppression of Enhancement of Chitinase Acitivity by L-MAbchi by Antibiotin Antibody (AbL)

As described above, chitinase activity was enhanced -3.5- fold by the addition of L-MAbchi. When increasing amounts of AbL were added, the chitinase activity decreased and lev- eled off roughly at the level without L-MAbchi (see Fig. 2A), indicating that the enhancement of the chitinase activity by

0 200 400 h(N1

L-MAbchi conc. (pg/rnl)

Fig. 1. The enhancement of chitinase activity by L-MAbchi. Chitinase (5 pg) in 25 p1 of 0.1 M sodium phosphate buffer, pH 6.0, was incubated at 3 7 T in 25 pl of 0.1 M sodium phosphate buffer, pH 6.0, containing varying concentrations of L-MAbchi. After 30 min, 1 ml of substrate solution [p- NP-(GlcNAc)l] was added to this solution and the initial rate was deter- mined at 337 nm.

L-MAbchi was suppressed by AbL. As a control experiment, normal goat IgG was added in place of AbL and no suppres- sion of the enhanced chitinase activity was observed (data not shown). But when increasing amounts of streptavidin, which is known to bind specifically to biotin, were added, chitinase activity decreased (see Fig. 2B).

Assay of Biotin by CAEIA

When the concentrations of L-MAbchi,AbL and the chitinase were fixed in the buffer solution and the concentration of biotin was increased, chitinase activity increased as shown in Figure 3. The result can be explained by the fact that biotin combines

h

‘2 .

.- 1; $ 1 0 1 , , . y o 1 , , , 6 0 u o

0 50. 100 150 Streptavidin conc. (pgirnl)

0 5 10 Anti-biotin antibody conc. ( m g / m l )

Fig. 2. Suppression of L-MAbchi enhdncement of chitinase activity by anti-biotin antibody (AbL) or streptavidin.Twenty fivepl of L-MAbchi (154 pgiml) in 0.02 M Tris-HC1 buffer, pH 7.5, containing 0.15 M NaCl and varying amounts of AbL (A) or streptavidin (R) in 50 pl of 0.1 M sodium phosphate buffer, pH 7.4, containing 0.14 M NaCl were mixed and incu- bated at 37°C for 30 min. Then 25 p1 of chitinase (5 pg) in 0.1 M sodium phosphate buffer, pH 6.0, was added to this solution and incubated at 37°C. After 30 min, 1 ml of substrate solution [pNP-(GlcNAc)3] was added to this solution to determine the initial rate at 337 m.

Homogeneous Enzyme lmmunoassay 195

DISCUSSION A novel homogeneous enzyme immunoassay technique,

which we designated as “chitinase activity enhancement im- munoassay” (CAEIA), was developed. In this assay sys- tem, biotin was chosen as an analyte and chitinase A1 of B . circulans WL-12 as the enzyme since chitinase activity was practically nonexistent in human serum, its nucleotide se- quence has been determined (7), its enzyme structure has been studied in some detail (lo), and monoclonal antibod- ies against it were available to the authors (8). The mono- clonal antibody G-2-5M, also referred to as MAbchi in this report, was found to enhance the activity of chitinase A1 (8) as was its biotinylated derivative L-MAbchi. An antibody directed against biotin, AbL, was found to suppress the en- hancement activity and streptavidin was also found to sup- press it. The enhancement activity was found to be suppressed by the materials known to bind specifically to biotin. Chiti- nase activity increased when the concentration of biotin was increased in the reaction mixture with fixed concentrations of chitinase, L-MAbchi andAbL. Thus a standard assay curve for biotin in relation to chitinase activity was obtained. We found that chitinase activity was practically nonexistent in human serum and that NAG did not interfere with this assay. Thus by replacing biotin with other clinically important sub- stances in human serum, this technique may be successfully applied as a novel homogeneous assay technique.

50

I C 30 a x f 20 .- - ”

0 0 10 20 30 40 50

Biotin conc. (pg/rnl) ‘

Fig. 3. Biotin assay by CAEIA. Various amounts of biotin in 25 p1 of 0.9% NaCl solution and AbL (10 mg/ml) in 50 PI of 0.1 M sodium phos- phate buffer, pH 7.4, containing 0.14 M NaCl were incubated at 37OC for 1 hr. Then L-MAbchi (154 pg/ml) in 25 pl of 0.02 M Tris-HC1 buffer, pH 7.5, containing 0.15 M NaCl was added and incubated at 37°C for 30 min. After incubation, chitinase (5 pg) in 25 pl of 0.1 M sodium phosphate buffer, pH 6.0, was added and incubated at 37°C for 30 min. Finally 1 ml of substrate solution [p-NP-(GlcNAc)3] was added to this solution and the initial rate was determined at 337 nm.

withAbL, and thus the amount of free L-MAbchi that enhances the chitinase activity is increased. The concentration of biotin could be estimated using this relationship.

Chitinase Activity in Human Sera

Chitinase and NAG activity were measured in 10 samples of fresh human sera using [p-NP-(GlcNAc)3] as a substrate for chitinase assay. The various units of NAG activity were detected in human sera, but no chitinase activity was detected (Table 1).

TABLE 1. Chitinase and NAG Activities in Human Sera

NAG activity Chitinase activity Human sera (un) ( A A ~ ~ ~ ~ ~ x

1 29.1 0.2 2 7.7 -0.8 3 14.1 -0.4 4 27.4 -0.6 5 39.8 0.6 6 22.0 -0.2 7 5.9 -0.2 8 40.3 0.2 9 24.0 -0.4

10 27.7 -0.2 chitinase A1 (5 pg) NT 15.0 blank” NT 0.4

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’0.1 M phosphate buffer, pH 6.0. NT = not tested.