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Previous Article | Next Article 
Journal of Clinical Microbiology, March 1999, p. 553-557, Vol. 37, No. 3
Planning and Development Division,
Received 20 July 1998/Returned for modification 16 September
1998/Accepted 19 November 1998
The usefulness of the measurement of urinary lactoferrin (LF)
released from polymorphonuclear leukocytes and of an
immunochromatography test strip devised for measuring urinary LF for
the simple and rapid diagnosis of urinary tract infections (UTI) was
evaluated. Urine specimens were collected from apparently healthy
persons and patients diagnosed as suffering from UTI. In the
preliminary study, the LF concentrations in 121 normal specimens and 88 specimens from patients (60 with UTI) were quantified by an
enzyme-linked immunosorbent assay. The LF concentration was
3,300.0 ± 646.3 ng/ml (average ± standard error of the
mean) in the specimens from UTI patients, whereas it was 30.4 ± 2.7 ng/ml and 60.3 ± 14.9 ng/ml in the specimens from healthy
persons and the patients without UTI, respectively. Based on these
results, a 200-ng/ml LF concentration was chosen as the cutoff value
for negativity. Each urine specimen was reexamined with the newly
devised immunochromatography (IC) test strip to calculate the indices
of efficacy. Based on the cutoff value, it was calculated that the
sensitivity, specificity, and positive and negative predictive values
of the IC test were 93.3, 89.3, 86.2, and 94.9%, respectively,
compared with the results of the microscopic examination of the urine
specimens for the presence of leukocytes. The respective indices for
UTI were calculated as 95.0, 92.9, 89.7, and 96.6%. The tests were
completed within 10 min. These results indicated that urine LF
measurement with the IC test strip provides a useful tool for the
simple and rapid diagnosis of UTI.
Urinary tract infections (UTI) are
among the most common diseases of humans, and large numbers of urine
specimens are processed daily in most clinical microbiology
laboratories. The screening of these specimens for microorganisms is a
repetitive procedure with a quantitative distinction between positive
and negative results (9, 23). The majority of the test
results are negative (11, 14), leaving positive specimens
for further processing. Most such tests are relatively simple and
reliable but require an overnight incubation of cultures before results
are available. Therefore, a rapid screening test would be advantageous
if it greatly reduced the time spent on specimens which prove to be negative (thereby allowing the majority of negative reports to be
issued the same day that the specimens are received) and required the
culture of only positive specimens. The detection of pyuria, in which
polymorphonuclear leukocytes (PMNs) are present due to inflammation of
the UT, may aid in the diagnosis of UTI (1, 16); pyuria
accompanied by a low or negligible bacterial count is a significant
finding (13, 15, 20, 25, 26). The presence of asymptomatic
and bacteriuritic pyuria may indicate infection rather than
colonization (10, 17, 27). However, the current methods for
detecting pyuria require some experience on the part of the
microscopist and can also be time-consuming. A rapid report of results
leading to a diagnosis of UTI would be useful to the physician and
patients because unnecessary therapy might be avoided.
A test using the esterase activity of PMNs combined with the
azo-coupling reaction (5, 19, 24) was reported to simplify the diagnosis of UTI. In addition to esterase, PMNs contain another component, the stable iron-binding protein lactoferrin (LF), in their
nuclei and secondary granules (6, 21). The present study was
carried out to test the usefulness of the measurement of urinary LF for
the diagnosis of UTI and to evaluate the immunochromatography (IC) test
strip devised for measuring urinary LF.
Urine specimens and UTI criteria.
The first-voided morning
urine specimens were obtained from apparently healthy persons (72 men
aged 24 to 59 and 49 women aged 20 to 55). Urine specimens of
outpatients were also collected from 45 men aged 40 to 83 (catheterized, 20%; midstream, 27%; and unspecified, 53%) and from
43 women aged 2 to 87 (catheterized, 42%; midstream, 2%; and
unspecified 56%). The specimens were stored immediately at 4°C and
submitted to the clinical laboratory within 2 h.
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Measurement of Urinary Lactoferrin as a Marker
of Urinary Tract Infection
ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
10/mm3, and a bacterial count of 103
CFU/ml of at least one clearly predominating organism before antibiotic
therapy; (ii) for acute pyelonephritis, which is also one of the
uncomplicated UTIs, female population aged 16 to 69, fever of 37°C
with flank pain, PMNs at 10/mm3, and a bacterial count
of 104 CFU/ml; (iii) for complicated UTI; both male and
female populations having at least one underlying disease of the UT,
PMNs at 10/mm3, and a bacterial count of
104 CFU/ml.
Preparation of PMN lysate. PMNs were collected from normal heparinized venous blood according to the method of Ferrante and Thong (4). The PMNs were suspended in 100 mM borate-buffered saline (BBS), pH 8.2, containing 0.07% Zwittergent 3-14 (Calbiochem-Novabiochem, La Jolla, Calif.), 0.07% CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate} (Dojindo, Kumamoto, Japan), 0.07% BIGCHAP (Dojindo), 0.2% Triton X-100 (Bio-Rad, Hercules, Calif.), and 0.5% bovine serum albumin (BSA; Sigma, St. Louis, Mo.) (this is hereafter referred to as the "sample dilution buffer") followed by sonication for 1 min to lyse PMNs and release LF.
Preparation of polyclonal antibody to LF. Female Japanese white rabbits (5 weeks old; 2.5-kg body weight) were immunized subcutaneously with 2 mg of LF (Sigma) in Freund's complete adjuvant (Difco, Detroit, Mich.). Two and 4 weeks later, the rabbits were boosted subcutaneously with 0.5 mg of LF in complete adjuvant. One week after the final immunization, whole blood was collected for sera. The antibody was purified from the sera by precipitation at between 20 and 33% saturation with (NH4)2SO4. The antibody was then diluted with 50 mM carbonate-bicarbonate buffer, pH 9.6, for use as the immobilized antibody and was also biotinylated with a protein biotinylation module (Amersham, Little Chalfont, Buckinghamshire, United Kingdom) for use as the second antibody of the sandwich enzyme-linked immunosorbent assay (ELISA).
Preparation of monoclonal antibodies to LF. Female BALB/cA mice (4 weeks old) were immunized subcutaneously with 100 µg of LF in complete adjuvant. Four and 8 weeks later, the mice were immunized again in the same way. Three days after the final immunization, splenocytes of the mice were mixed with P3-X63-Ag8-6.5.3 myeloma cells at a ratio of 5:1 and fused in polyethylene glycol 1500 (Boehringer, Mannheim, Germany) by a modification of the method of Koeler and Milstein (12). Positive hybridomas were selected by limiting dilution and further incubated in Celgrosser-H serum-free medium (Sumitomo Pharmaceutical, Osaka, Japan) at 37°C in a humidified 5% CO2 atmosphere. Antibodies were purified from the supernatants of cell cultures by an affinity chromatographic technique with an AvidChrom IgPure Isolation kit (Unisyn Technologies, San Diego, Calif.), which has low-molecular-weight ligands on the surface of Sepharose C1-4B to capture immunoglobulin G (IgG), according to the method of Ngo and Khatter (18).
Among the 14 monoclonal antibodies prepared, 17B04-08 [IgG1(
)] was
used as the antibody immobilized on an Immunodyne ABC membrane (3-µm
pore size) (Pall Gelman Sciences, New York, N.Y.), which is made from
nylon66 and has a high density of chemically activated covalent binding
sites against amino groups, and 32D01-10 [IgG2a()] was used as the
antibody conjugated with AuroBeads G10 colloidal gold particles
(Amersham) for the sandwich-type IC system.
Sandwich ELISA of LF. The polyclonal antibody to LF (10 µg/ml) was immobilized in each well of 96-well microtiter plates (catalog no. 3590; Costar, Cambridge, Mass.) overnight at 4°C followed by blocking with 1% gelatin (enzyme immunoassay grade; Bio-Rad) for 1 h at 37°C. After the washing of each well, urine specimens diluted with the sample dilution buffer were allowed to react for 1 h at 37°C. After the wells were washed, biotinylated polyclonal antibody (2 µg/ml) was added and allowed to react for 1 h at 37°C. After another washing, alkaline phosphatase-avidin complex (1:1,000) (Dako, Glostrup, Denmark) was added and allowed to react for 30 min at 37°C. After a final washing, p-nitrophenylphosphate solution (1 mg/ml in 9.7% diethanolamine and 0.01% MgCl2 solution, pH 9.8) was added and allowed to react for 10 min. After the reaction was stopped by the addition of 4 M NaOH solution, the absorbance of each well at 405 nm was measured with a microplate reader (EL 312e; Bio-Tek Instruments, Winooski, Vt.).
IC of LF. All solutions and buffers used to prepare the IC test strip were filtered with 0.22-µm-pore-size membrane filters before use.
17B04-08 antibody (3 mg/ml in BBS) was deposited on an Immunodyne ABC membrane (5 by 35 mm) as a 1-mm-wide line at about the midpoint of the length of the membrane as the detection zone of LF. As the control zone, rabbit anti-mouse Ig (3 mg/ml in BBS) (Dako) was deposited on the membrane as a 1-mm-wide line 5 mm downstream from the detection zone. After drying for 1 h at room temperature and for 10 min at 37°C, the membrane was blocked with 0.5% monoethanolamine in BBS, pH 8.5, for 30 min at room temperature and further with 0.5% Hammarsten's casein (Merck, Darmstadt, Germany) in 100 mM maleic acid and 150 mM NaCl solution, pH 7.5, for 30 min at room temperature with continuous agitation. After being rinsed once with BBS and three times with distilled water, the membrane was dried at 37°C. The 32D01-10 antibody was dialyzed against 2 mM Na2B4O7 buffer, pH 9.0 (borax buffer) at 4°C, followed by ultracentrifugation at 100,000 × g for 1 h at 5°C in an ultracentrifuge equipped with a 50Ti rotor (L8-50M/E; Beckman Instruments, Palo Alto, Calif.). The supernatant of the solution was collected and further diluted with ultracentrifuged borax buffer. A 1-ml aliquot of 32D01-10 antibody solution (240 µg/ml) was added to a 10-ml aliquot of AuroBeads G10 colloidal gold suspension (pH 9.0) in a glass tube. The mixture was then stirred continuously for 2 min and stored for 10 min at room temperature. After the addition of a 1.5-ml aliquot of 10% BSA solution, pH 9.0, the antibody-gold conjugate was washed with 20 mM Tris-HCl-150 mM NaCl buffer containing 1% BSA, pH 8.0, in a series of three centrifugation steps at 45,000 × g at 5°C for 30 min. The final pellet was resuspended in the same buffer, and the absorbance of the suspension at 520 nm was adjusted to 10. A 2-µl aliquot of the antibody-gold conjugate suspension was deposited on the glass fiber absorbent pad (5 by 34 mm) at the downstream end and allowed to dry at room temperature. A polystyrene support (5 by 80 mm) was laminated on the back of the antibody-immobilized membrane, and the antibody-gold conjugate pad was attached to the support at the upstream end with 2 mm overlapping with the membrane. Then another absorbent pad made from cellulose (5 by 20 mm) was incorporated with the downstream end of the support, with 7 mm overlapping with the membrane, to serve as a sink for any excess volume of liquid sample. A 100-µl aliquot of each urine specimen diluted 1:4 with the sample dilution buffer and the PMN lysate was deposited on the upstream end of the antibody-gold conjugate pad. After a 10-min migration of the antibody-gold conjugate with LF through the membrane at room temperature, the lines at both the detection zone and the control zone caused by the capture of the antibody-gold conjugate were evaluated by the naked eye.Data analysis. The data from the microplate reader were processed on a Macintosh computer, and a reduction was performed with DELTA SOFT II (BioMetallics, Princeton, N.J.). The reduction was a four-parameter algorithm with the absorbance at 405 nm on the y-axis and the LF concentration (in namograms per milliliter) on the x-axis.
Statistical analysis. The quantitative detection range of LF by a sandwich ELISA and the significance of the difference between each pair of mean values of urinary LF were analyzed by using Student's t test, where the standard error of the mean (SEM) was used as a measure of variance among the means.
Indices of efficacy. The indices of efficacy of the IC test of LF in comparison with the results of urinary PMNs and those for predicting UTI were calculated based on the methods described by Ranshoff and Feinstein (22).
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RESULTS |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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Polyclonal and monoclonal antibodies to LF. The polyclonal antibody and 14 monoclonal antibodies to LF did not cross-react with transferrin or bovine LF when examined by an ELISA method with these antigens immobilized in the wells of microtiter plates. Moreover, the two monoclonal antibodies, 17B04-08 and 32D01-10, used for the IC test strip were shown to have specificities for different epitopes on the LF molecule when examined by a sandwich ELISA.
Sandwich ELISA of LF. To quantitatively assess the detectable range of LF, standard solutions of LF at concentrations from 411.5 pg/ml to 300 ng/ml were examined. As shown in Fig. 1, a well-fitted standard curve was obtained (n = 6; r = 0.998; P < 0.001). The quantitative detection range of LF was from 4.8 to 117 ng/ml (n = 6; P < 0.001).
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IC of LF.
To determine the minimum detectable concentration of
LF, various amounts of standard LF (0, 20, 50, 100, and 200 ng and 100 µg of LF/ml) were examined. The line produced by capturing the antibody-gold conjugate was observed at the control zone of all test
strips, suggesting that the antibody-gold conjugate migrated by
capillary action through the membrane beyond the detection zone without
aggregation. Another line was revealed on the detection zone by the
production of the sandwich immunocomplex of LF and two monoclonal
antibodies when the concentration of LF was 50 ng/ml. In addition,
the detectable concentration range extended to >100 µg/ml without
false-negative results due to the prozone phenomenon. These results
indicated that this IC system for the detection of LF has a minimum
detectable concentration of 50 ng of LF/ml and a wide dynamic range.
104 PMNs/ml could be detected.
Each normal urine specimen containing <20 ng of LF/ml of urine
fortified with various amounts of LF (0, 100, 200, 400, and 800 ng of
LF/ml of urine) was diluted 1:4 and examined. The results showed that
the detectable concentration of LF in the urine was 200 ng of LF/ml of urine.
The LF of each urine specimen quantified by ELISA was reexamined with
the IC test strip. The results are summarized in Table 3 as a comparison of the presence of
urinary LF with the results of microscopic examination of urinary PMNs
and with the UTI diagnosis status. All 121 normal specimens were
negative for LF, and 25 of the 28 PMN-negative specimens and 26 of the
28 UTI-negative specimens were assessed as negative based on the IC
test strip results. Fifty-six of the 60 PMN-positive specimens and 57 of the 60 UTI-positive specimens were assessed as positive. The
sensitivity, specificity, and predictive values of negative and
positive findings of urinary LF in comparison with the results of
urinary PMNs were 93.3, 89.3, 86.2, and 94.9%, and the values for
predicting UTI were 95.0, 92.9, 89.7, and 96.6%, respectively. Thus,
all indices of efficacy were very high, and the urinary LF examination
results reflect the presence of urinary PMNs and predict UTI.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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In this study, we determined the feasibility of using urinary LF as a marker for UTI diagnosis and we evaluated the utility of a simple and rapid test for LF by IC. Because urine specimens contain many substances which interfere with the determination of LF, the analysis of LF in urine specimens has to be carried out in a detergent-rich sample dilution buffer, even though 0.1% Triton X-100 detergent solution alone can release LF from PMNs within 1 min (7).
In the preliminary study, the LF concentration in urine specimens was quantified. For this purpose, we developed a sandwich ELISA with a rabbit polyclonal antibody to LF. The quantitative detection range of LF was from 4.8 to 117 ng/ml. Hetherington et al. reported that 106 PMNs contain about 4.9 µg of LF (8). Therefore, this range requires about 5 × 103 to 1.2 × 105 PMNs/ml of urine and is sufficient to quantify LF in normal urine specimens. In UTI conditions, in which an excessive amount of LF is excreted from PMNs, the volume of the urine specimen to be analyzed can be reduced. The results of the recovery test for LF in normal urine specimens (recovery rates, 88.4 to 124.8%) indicate that the sandwich ELISA method is precise and accurate.
Furthermore, LF in the PMN lysate obtained from 104
PMNs/ml could be detected by the IC test strip. This PMN density
corresponds to about 50 ng of LF/ml, and the results correlated well
with the minimum detectable concentration of LF evaluated with the LF
standard solutions. These findings indicate that the IC test strip can
detect LF released from PMNs precisely.
For the diagnosis of UTI with LF as a marker, it is important to define
the cutoff value of the urinary LF concentration for the separation of
negative and positive results. To define the cutoff value, we
calculated the mean + 5SD (SEM × 
n) value using the results obtained from the healthy
population measured by the sandwich ELISA. Although this value was
calculated as 180.4 ng of LF/ml of urine, it is difficult to detect a
difference of 20 ng of LF/ml of urine with the IC test strip after
urine specimens are diluted before examination. Therefore, the cutoff
value was defined as 200 ng of LF/ml of urine instead of 180 ng of
LF/ml to minimize false-positive results. Based on this cutoff value and the minimum detectable LF concentration of the IC test strip, urine
specimens should be diluted 1:4 with the sample dilution buffer before
analysis. The LF concentration in the urine detectable by the IC test
strip is well correlated with the minimum detectable concentration
determined with standard LF and the cutoff value determined according
to the results of the ELISA. This indicates that urinary LF can be
detected precisely by the IC test strip without serious interference by
other substances present in urine.
The LF concentrations in urine specimens from UTI patients were much higher than those in specimens from both patients without UTI and healthy subjects. When the cutoff value for detecting the presence of urinary LF was set at 200 ng of LF/ml of urine, the LF positivity and negativity correlated well not only with the microscopic examination results of PMNs but also with UTI diagnosis. Furthermore, LF is stable in urine specimens even after the structures of PMNs are destroyed. For example, our preliminary experiments showed that the residual rates of LF from a normal urine specimen which was fortified with 200 ng of LF/ml of urine were 113.8% after storage at 45°C for 3 days and 94.4% after three cycles of freezing and thawing. Therefore, it is not always necessary to specify the time of specimen collection and the storage temperature. In addition, interfering reactions caused by reducing agents or antibiotics, as observed in the leukocyte esterase activity test (19), are not a problem, because this detection system for LF is based on a specific antibody-antigen reaction. Indeed, all nine of the urine specimens which were collected after antibiotic medication and which were positive in the microscopic examination of PMNs were positive for LF. Therefore, it is possible to diagnose an inflammation on mucosal surfaces of the UT even after antibiotic medication. Cohen et al. (3) noted that the amount of LF in vaginal mucus ranged from 3.8 to 218 µg/mg of protein. This suggests that it is necessary to use care in the collection of specimens from female patients to avoid false-positive results caused by contamination of LF by vaginal secretions. In the present study, however, no false-positive result was observed in any of the urine specimens from females examined. However, the LF results of all six urine specimens from outpatients for whom the method of specimen collection was not specified were negative, although their bacterial culture results were all positive. These results should be considered as contamination by bacteria in urine specimens rather than false-negative results of LF, because all six specimens were also negative in the microscopic examination of PMNs.
For the detection of LF, several methods, such as an ELISA (8) and a latex agglutination assay (7), have already been developed. Even though the ELISA can detect 3 ng of LF/ml quantitatively, this method is not recommended for the rapid diagnosis of UTI due to its complicated and time-consuming procedure. In the latex agglutination assay, the minimum detectable concentration of LF is 310 ng/ml. Therefore, it is impossible to detect the cutoff line of 200 ng of LF/ml of urine even if the urine specimen is examined directly without further dilution. The IC test strip has a minimum detectable concentration of 50 ng of LF/ml and is able to detect the cutoff line even after fourfold dilution of a urine specimen. In addition, all examination processes can be completed within 10 min after specimen collection.
In conclusion, urinary LF is a sensitive marker for the diagnosis of UTI caused by inflammatory pathogens. The IC test strip provides a useful tool for the simple and rapid diagnosis of UTI. Large numbers of urine specimens are processed daily for the overnight bacterial culture test to determine the inflammatory pathogens, even though the majority of test results are negative. And the microscopic examination of PMNs requires skill and experience on the part of the microscopist. Therefore, the use of the IC test strip to detect LF could greatly reduce the time, cost, and effort of routine urinalysis and could avoid unnecessary antibiotic medication of UTI-negative patients.
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ACKNOWLEDGMENTS |
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We are greatly indebted to R. Sakazaki of the Nippon Institute of Biological Sciences and K. Tamura of the National Institute of Infectious Diseases for their valuable suggestions. We also thank Y. Ohkubo and H. Matsuoka of Wakunaga Pharmaceutical Co. for their technical support in the use of the immunochromatography system.
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FOOTNOTES |
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* Corresponding author. Mailing address: Research and Development Department, Iatron Laboratories, Inc., 1460-6, Mitodai Mito, Tako, Katori, Chiba 289-2247, Japan. Phone: (81) 479-76-3666. Fax: (81) 479-76-3468.
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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