Clinical Chemistry 43: 569-574, 1997;
(Clinical Chemistry. 1997;43:569-574.)
© 1997 American Association for Clinical Chemistry, Inc.
New sandwich ELISA for human urinary N-acetyl-ß-D-glucosaminidase isoenzyme B as a useful clinical test
Yoshito Numata1,a,
Atsushi Morita1,
Yoko Kosugi1,
Kazunori Shibata1,
Nozomu Takeuchi2 and
Kiyohisa Uchida1
1
Diagnostic Science Department, Shionogi & Co., Ltd., 2-5-1 Mishima, Settsu-shi, Osaka 566, Japan.
2
Department of Clinical Laboratory Medicine, Ehime
University School of Medicine, Shigenobu-cho, Ehime-Ken 791–02, Japan.
a Author for correspondence. Fax 06-319-4109.
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Abstract
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We have developed a new ELISA for quantifying
N-acetyl-ß-D-glucosaminidase (NAG)
isoenzyme B in human urine after raising monoclonal antibodies against
the isoenzyme from human placenta. Though the obtained antibodies
reacted not only to isoenzyme B but also to A, we could detect
isoenzyme B selectively by a two-step sandwich ELISA with a pair of
selected antibodies at low pH in the first reaction. The detected limit
was 0.5 µg/L for a sample volume of 25 µL. Within-run CVs ranged
from 2.5% to 5.4% and between-run CVs ranged from 6.2% to 9.1%.
Recoveries of NAG isoenzyme B added to each of three urine samples
ranged from 91% to 114%. The dilution curves of urine samples showed
good linearity. The cross-reactivity of NAG isoenzyme A was practically
negligible (2–3%). The mean value for NAG isoenzyme B in spot urines
from healthy adults was 2.9 µg/g creatinine. This ELISA method is
rapid and precise enough for routine determination of NAG isoenzyme B
in human urine.
Key Words: indexing terms: monoclonal antibody • renal damage • immunological activity
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Introduction
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N-Acetyl-ß-D-glucosaminidase (NAG;
EC 3.2.1.30) is a widely distributed lysosomal enzyme located
predominantly in the renal proximal tubules
(1).1
Increased NAG
enzymatic activity in urine has been found to be associated with
various kidney injuries (2)(3). It is
considered to be a sensitive marker of renal diseases, an early warning
of rejection after renal transplantation (4), and a sign
of drug nephrotoxicity (5). NAG enzymatic activity in
urines has been determined fluorometrically with
4-methylumbelliferyl-N-acetyl-ß-D-glucosaminide
(6), and colorimetrically with
p-nitrophenyl-N-acetyl-ß-D-glucosaminide
(7) or sodio
m-cresolsulfophthaleinyl-N-acetyl-ß-D-glucosaminide
(8).
NAG can be separated into two major isoenzymes, A and B, and several
other minor isoenzymes (I, P, S) (9). NAG isoenzyme A is
composed of two different subunits 
and ß, whereas NAG isoenzyme
B, and probably I, consist of two ß-subunits (10)(11)(12).
These isoenzymes differ in pI, substrate specificity, and thermal
stability. There have been many reports describing the possible
clinical significance of NAG isoenzyme analysis and especially of using
NAG isoenzymes B and I, which are present at 10–20% in normal human
urine, as more sensitive markers of various types of renal damage than
total NAG (13)(14)(15)(16)(17)(18).
The separation of NAG isoenzyme B from A has been performed by
ion-exchange chromatography, electrophoresis, and a method that takes
advantage of the thermoinstability of NAG isoenzyme A. However, these
methods are time consuming, not precise, and (or) not easily adapted to
routine clinical work. Therefore, rapid and highly sensitive methods
for more extensive evaluation are needed for NAG isoenzyme B [and (or)
I] determination. Here, we describe a new ELISA method for NAG
isoenzyme B (and I) that is based on a two-step sandwich assay
involving two anti-NAG monoclonal antibodies and microplates as the
solid phase.
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Materials and Methods
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antibodies
Monoclonal antibodies against NAG isoenzyme B were raised by using
partially purified NAG isoenzyme B from human placentas (Sigma Chemical
Co., St. Louis, MO). Hybridomas were produced in accordance with
conventional procedure (19). In the screening method,
wells of microplates (module plate F8; Nunc, Roskilde, Denmark) were
coated with goat anti-mouse immunoglobulins. Hybridoma supernatants
were added to the wells and the wells were incubated at room
temperature for 2 h. The wells were washed with PBS (10 mmol/L, pH
7.4) containing 0.5 mL/L Tween 20. Partially purified NAG isoenzyme was
then added, followed by further incubation. After washing, the specific
antibodies were detected by assaying NAG enzymatic activity in the
wells.
Isotype determination was carried out by the enzyme immunoassay method
with an isotyping kit (PharMingen, San Diego, CA).
purified nag isoenzymes a and b
NAG isoenzymes A and B were purified from human placentas by
procedures including chromatographies on concanavalin A–Sepharose
(Pharmacia, Uppsala, Sweden), an immunoadsorbent column, Mono Q
Sepharose (Pharmacia), and Superose 12 column (Pharmacia) by the method
of Kinoshita et al. (12). For preparation of the
immunoadsorbent column, anti-NAG monoclonal antibody, Hex 33, was
immobilized to a protein G-Sepharose 4 Fast Flow column (Pharmacia)
according to Schneider et al. (20). The purities were
checked by sodium dodecyl sulfate–polyacrylamide gel electrophoresis
(SDS-PAGE). The protein concentrations were determined by the method of
Lowry et al. (21).
fab'–peroxidase conjugate
A monoclonal antibody (Hex 32, IgG1, 
, 18 mg) was conjugated
with horseradish peroxidase (HRP; EC 1.11.1.7) as described by Kato et
al. (22).
reagents
Buffers and solutions used in the ELISA were: assay buffer I:
citrate buffer (0.4 mol/L, pH 5.2) containing bovine serum albumin
(BSA) (1 g/L) and Kathon CG (1 g/L; Rohm and Haas, Philadelphia, PA);
assay buffer II: PBS containing BSA (1 g/L) and Kathon CG (1 g/L);
washing solution: PBS containing Tween 20 (0.5 g/L); substrate
solution: citrate buffer (0.01 mol/L, pH 5.0) containing
2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), diammonium salt
(ABTS) (0.004 mol/L; Boehringer Mannheim, Mannheim, Germany) and
H2O2 (0.002 mol/L); and stop solution: citrate
buffer (0.01 mol/L, pH 5.0) containing NaN3 (0.05 g/L).
When binding activities of NAG isoenzyme at various pHs were examined,
the assay buffers used were citrate, 0.4 mol/L containing BSA (1 g/L),
pH range 4.8–5.6; and phosphate, 0.01 mol/L containing NaCl (9 g/L)
and BSA (1 g/L), pH range 6.8–8.0.
microplates coated with anti-nag monoclonal antibody
Each well of a microplate was filled with a solution (200 µL) of
anti-NAG antibody Hex 31 (10 mg/L in PBS) and incubated overnight at
4 °C. After removal of the antibody solution, the wells were washed
three times by filling them with washing solution and aspirating it
out. PBS (300 µL) containing saccharose (100 g/L) was added to each
well of the microplates, which were then incubated at room temperature
for 1 h. After aspiration, the microplates were dried in a
desiccator under vacuum overnight and stored at 4 °C.
elisa for determination of nag isoenzyme b
In the typical assay procedure, all incubations were performed at
30 °C. Each well of the antibody-coated microplates was washed once
with washing solution and assay buffer I (150 µL) and aliquots of
purified NAG isoenzyme B or urine (25 µL each) were added to the
wells. Incubation was for 2 h (first reaction). After this the
wells were washed four times with washing solution, and Hex 32
Fab'-HRP conjugate (25 ng) in assay buffer II (100 µL)
was added. The samples were left standing for 1 h (second
reaction). The wells were again aspirated and washed four times, then
substrate solution (100 µL) added to each well. After 15 min of
incubation (enzyme reaction), stop solution (100 µL) was added and
the absorbance at 415 nm was measured with immunoreader MTP-32 (Corona
Electric Co., Ibaraki, Japan). Experiments were performed in duplicate
except where noted otherwise.
nag isoenzyme analysis
NAG isoenzyme separation was carried out by ion-exchange
chromatography with a fast protein liquid chromatography (FPLC) system
(Pharmacia). The concentrated urine samples were dialyzed against 10
mmol/L phosphate buffer, pH 6.0 (buffer A), at 4 °C overnight. A
Mono Q column (0.5 x 5.0 cm) was equilibrated with buffer A and 2
mL of dialyzed sample was applied to the column. NAG isoenzymes were
eluted with 5 mL of buffer A and then with a linear NaCl gradient in
buffer A at a flow rate of 1 mL/min. Fractions (1-mL) were collected
and NAG activities were determined by both the enzyme assay and the
ELISA.
nag enzyme assay
Enzymatic activity of NAG (total NAG) was measured by the
sodio
m-cresosulfophthaleinyl-N-acetyl-ß-D-glucosaminide
method (8) with a commercially available kit (Shionogi,
Osaka, Japan).
calculation of urinary nag isoenzyme b enzymatic
activities
Urinary NAG isoenzyme B enzymatic activities were calculated by
using the following equations:
Chromatographic method.
NAG isoenzyme B (U/L)
= total NAG (U/L) x percentage of isoenzyme B (%) x
10-2.
ELISA method.
NAG isoenzyme B (U/L) = NAG isoenzyme B
(g/L) x 164 (specific activity, U/mg) x 10-3.
Percentage of NAG isoenzyme B (%) = NAG isoenzyme B (U/L) x
102/total NAG (U/L)
samples
The urine samples were obtained from Ehime University School of
Medicine. The samples were kept frozen at -80 °C until analysis.
Under this condition, NAG isoenzyme B was stable for at least 1 month.
Human sample acquisition was conducted in accordance with the policies
and procedures of the Institutional Review Board for the use of Human
Subjects in Research at Diagnostic Science Department, Shionogi & Co.,
Ltd.
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Results
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characterization of anti-nag monoclonal antibodies
Cell fusions repeated twice gave six hybridoma clones that
produced antibodies (Hex 31–36) reacting to the antigen (NAG isoenzyme
B). Though a partially purified antigen was used as an immunogen, we
could obtain anti-NAG monoclonal antibodies by assaying captured NAG
enzymatic activities. All clones were IgG1 type with a light chain
and reacted not only to NAG isoenzyme B but also to A. Among the
preliminary sandwich assays involving combinations of the obtained
antibodies, the assay involving Hex 31 and Hex 32 as an immobilized
antibody and a labeled one, respectively, showed a dose-dependent
response to NAG isoenzyme B with great sensitivity. Therefore, we tried
to develop an ELISA for NAG isoenzyme B with the pair of Hex 31 and Hex
32.
purities of nag isoenzymes a and b prepared from human placentas
Purified antigen as a calibrator was necessary for the ELISA. We
purified NAG from human placentas by using an immunoadsorbent column.
Finally, the NAG isoenzymes were purified 5900-fold for isoenzyme A and
6500-fold for isoenzyme B, respectively. The specific activities of
isoenzymes A and B in our system were 142 and 164 U/mg protein.
SDS-PAGE of NAG isoenzyme A showed two bands with molecular masses of
52 kDa and 60 kDa, which corresponded to the and ß chains (Fig. 1
, lane 1). Only the ß chain was seen for NAG isoenzyme B (lane
2). After reduction of NAG isoenzymes A and B, the ß chain
dissociated into two bands, ßa and ßb
chains with molecular masses of 30 kDa and 28 kDa, respectively (lanes
3, 4). We used purified NAG isoenzyme B as the calibrator material of
our ELISA.
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Figure 1. SDS-PAGE of isolated NAG isoenzymes.
Electrophoresis was carried out under nonreducing conditions
(lanes 1 and 2) or reducing conditions
(lanes 3 and 4). Proteins were separated
by SDS-PAGE in a 10–20% gradient polyacrylamide gel and visualized
with Coomassie brilliant blue. Lanes 1 and
3, NAG isoenzyme A; lanes 2 and
4, NAG isoenzyme B.
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optimization of assay phs
We tested the ELISA for cross-reactivity of NAG isoenzyme A at
different pH values. When the pH of the second reaction was fixed at pH
7.4, the cross-reactivity of NAG isoenzyme A in the first reaction
amounted to 14.0%, 6.4%, and 2.4% at pH 7.0, 6.0, and 5.2,
respectively (Fig. 2
A). Lower reactivity to NAG isoenzyme B was observed at pH 4.8.
When the first reaction was carried out at a fixed pH of 5.2 and the
cross-reactivity with NAG isoenzyme A was examined from pH 5.2 to 8.0
in the second reaction (Fig. 2B
), there was little change in the
cross-reactivity. We therefore selected pH 5.2 for the first reaction
to decrease the cross-reactivity with NAG isoenzyme A and pH 7.4 for
the second reaction to increase the sensitivity.
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Figure 2. Binding activities of NAG isoenzymes at various pHs in the
first (A) and second (B) reactions.
ELISA was carried out as described in the text except for variation in
pH. Solutions of NAG isoenzyme A (40 g/L) and isoenzyme B (40 g/L) were
used. Arrows indicate the pHs used under the calibration
conditions.
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assay characteristics
As shown in Fig. 3
, the absorbance at 415 nm against the amount of calibrator
exhibited a linear relation in the range of 0 to 80 µg/L. The lower
detection limit of this ELISA, defined as the concentration at the mean
+ 3SD of 20 determinations of the zero calibrator, was 0.5 µg/L. The
cross-reactivity with human NAG isoenzyme A was ~2–3% in the
present ELISA, and no cross-reactivity with NAG from bovine kidney was
observed (data not shown). The precision of the present ELISA was
estimated with three different urine samples containing NAG isoenzyme
B. The CVs for within- and between-assay series were 2.5–5.4% (n
= 8) and 6.2–9.1% (n = 14), respectively, as shown in Table 1
. Recoveries of exogenously added NAG isoenzyme B from urine
samples containing three different concentrations of endogenous NAG
isoenzyme B were estimated (Table 2
). The recoveries ranged from 91% to 114%. Dilution curves of
three urine samples showed good linearity, as shown in Fig. 4
. Possible interfering substances were investigated by the usual
addition technique. Uric acid (1 g/L), ascorbic acid (10 g/L), glucose
(100 g/L), albumin (10 g/L), creatinine (10 g/L), and hemoglobin (50
g/L) had no effect on the present ELISA. The components of our assay
were stable for at least a year at 4 °C.
ion-exchange chromatography of human urinary nag isoenzymes
To examine the isoenzyme selectivity of the present ELISA, the
elution patterns of urinary NAG isoenzymes analyzed by ELISA were
compared with those analyzed by the NAG enzyme assay. Fig. 5
shows ion-exchange chromatograms of a urine sample from a
patient with renal damage. The ELISA responded to NAG isoenzymes B and
I, but not to isoenzyme A. This result indicates that the amount of NAG
isoenzyme B (and I) can be determined selectively in urinary samples.
The linear regression between the values measured with the
chromatographic method (x) and those determined by ELISA
(y) yielded y = 1.12x + 0.59 U/L
for 20 urine samples.
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Figure 5. Ion-exchange chromatography patterns of urinary NAG
isoenzymes in a patient with renal damage.
Fractions were subjected to enzyme assay and ELISA. Each point
indicates enzymatic activity. A, B, and
I indicate NAG isoenzymes A, B, and I, respectively.
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concentrations of nag isoenzyme b in human urine samples
NAG isoenzyme B excretion in the normal control group was 2.9
µg/g creatinine (or 3.1 µg/L) for spot collection (n = 40,
Table 3
). The values showed no sex-related differences. The mean value
of the percentage of isoenzyme B in total NAG was 15.8%, which agrees
with earlier results obtained by the ion-exchange chromatography method
(23). Values of NAG isoenzyme B from pathological urine
samples were also measured (Table 3
). Most of the urine from patients
with renal diseases, especially pyelonephritis, showed significant
increases in both values and percentages of NAG isoenzyme B.
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Discussion
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Ellis et al. (13) reported that in pathological
urine, the excretion of NAG isoenzyme B was increased roughly in
parallel with the increase in total NAG. Gibey et al. (16)
demonstrated a significant increase in isoenzyme B activity after
treatment with aminoglycoside antibiotics. Vigano et al.
(15) showed a significant increase of NAG isoenzyme B in
upper urinary tract infections. Others have also reported increase of
urinary NAG isoenzyme B and (or) I during rejection of transplanted
kidneys (17) or in glomerular diseases (23).
Although the analytical method for NAG isoenzyme separation, for
example, electrophoresis or ion-exchange chromatography, can provide
interesting information on the underlying diseases, they are too time
consuming and uneconomical for clinical laboratories. Therefore,
quantitative methods that satisfy the fundamental analytical criteria
of within- and between-run precisions, sensitivity, specificity, and
accuracy need to be developed for the determination of NAG isoenzymes,
especially isoenzyme B.
We raised monoclonal antibodies against human placental NAG to develop
a new sandwich immunoassay for NAG isoenzyme B. All of the antibodies
reacted with both NAG isoenzymes A and B, probably due to the presence
of a common ß-subunit. Isoenzyme B-specific antibodies could not be
established, but we were able to develop a sandwich ELISA for selective
determination of the NAG isoenzyme B by using a selected antibody pair
at pH 5.2 in the first reaction. Although it was not clear why the
cross-reactivity could be reduced at low pH, the decrease in
immunoreactivity seems to be due to a conformational change of NAG
isoenzyme A.
In human urine, the amount of NAG detected by the present ELISA also
reflected those of isoenzymes B and I (Fig. 5
). NAG isoenzyme I, an
acidic variant of NAG isoenzyme B, may result from sialylation or
phosphorylation at the carbohydrate moiety (12). Our ELISA
method is rapid, sensitive, and precise without the need for
concentration and dialysis of urinary samples. Previously, Isaksson et
al. (24)(25) raised monoclonal antibodies
against human placental NAG and developed enzyme immunoassays for NAG
isoenzymes A and B. However, these immunoassays were not sandwich
assays but antigen-capture assays, in which the analytical accuracy was
unclear and the detection was based on both the enzymatic and
immunological activities of NAG. In general, immunological
activity is thought to be stable relative to enzymatic activity as
described for prostatic acid phosphatase (26).
Consequently, our immunoassay is expected to have two advantages over
the measurement of total NAG enzymatic activity: high selectivity for
isoenzyme B and detection on the basis of the immunological activity
alone.
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Acknowledgments
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We are very grateful to Akira Noto for his useful discussions, and
we thank Yasushi Iwao and Naoko Takemoto for their technical support.
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Footnotes
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1 Nonstandard abbreviations: NAG,
N-acetyl-ß-D-glucosaminidase; SDS-PAGE,
sodium dodecyl sulfate–polyacrylamide gel electrophoresis; HRP,
horseradish peroxidase; and BSA, bovine serum albumin. 
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Abstract
Introduction
