Estimation of Bacterial Contamination in Ultrapure Water: Application

Nov 7, 1998 - We constructed and established a hybridoma cell line that produces immunoglobulin G-type anti-DNA antibody. By using this antibody, we c...
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Anal. Chem. 1998, 70, 5296-5301

Estimation of Bacterial Contamination in Ultrapure Water: Application of the Anti-DNA Antibody Takako Nogami,*,† Tokio Ohto,‡ Osamu Kawaguchi,† Yasushi Zaitsu,‡ and Shoichi Sasaki†

Central Research Laboratories, Organo Corporation, 4-9, Kawagishi 1-chome, Toda-City, Saitama 335-0015, Japan, and Fuji Electric Corporate Research and Development, Ltd., 2-2-1, Nagasaka, Yokosuka-City, 240-0194 Japan

We constructed and established a hybridoma cell line that produces immunoglobulin G-type anti-DNA antibody. By using this antibody, we could successfully detect a single bacterial cell in ultrapure water (UPW). The detection system is composed of a membrane-supported western blotting-type immunoassay and a two-dimensional photon analyzer with high resolution. It can detect and count every bacterial cell in a wide field of view on a trapping filter i.e., a circle with an 18-mm diameter. This means 10 fg (10-14 g) of bacterial DNA can be detected in the field. This system could be a useful tool for evaluating the number of bacteria contained by UPW and water used for medical purposes. Increasing demand for semiconductors has accelerated the development of the technologies involved in their manufacture. Ultrapure water (UPW), used for washing them, also has been forced to develop to satisfy the higher criteria including the levels of total organic carbon, particles, trace elements, and bacteria. Although almost all of the detection systems for UPW are now automated and are able to detect elements far beyond the ordinary sensing limits of 10 years ago, the detection of contaminating bacteria still depends on time-consuming techniques with ambiguous results. Conventional cultivation methods, the most commonly used for estimating bacterial contamination,1,2 are timeconsuming and imprecise. There are delays in obtaining results and there is a possibility of missing a population growing at a slower rate or of a population being viable but unable to grow in the cultivation conditions. Here we introduce an alternative antibody-horseradish peroxidase (HRP) method developed for detecting bacteria with high resolution, precision, and rapidity. This technique is based on the chemiluminescence enzyme immunoassay3,4 and the twodimensional photon-analyzing system. Marking the bacterial DNA with anti-DNA antibody conjugated with HRP, and with the photonproducing reaction catalyzed by the enzyme, we can precisely * Corresponding author: (phone) +81 48-446-1244; (fax) +81 48-441-2469. † Organo Corp. ‡ Fuji Electric Corporate Research and Development, Ltd. (1) Governal, R. A.; Yahya, M. T.; Gerba, C. P.; Shadman, F. J. Ind. Microbiol. 1991, 8, 223-227. (2) Yagi, Y.; Shinoda, T.; Saito, M. Proc. Semicond. Pure Water Chem. Conf. 1992, 1992, 148-163. (3) Hauber, R.; Miska, W.; Shleinkofer, L.; Geiger, R. J. Clin. Chem. Clin. Biochem. 1988, 26, 147-148. (4) Puget, K.; Michelson, A. M.; Avrameas, S. Anal. Biochem. 1977, 79, 447456.

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detect and estimate all kinds of bacteria trapped on a testing filter without cultivation. EXPERIMENTAL SECTION Establishment of Immunoglobin G (IgG)-Type Anti-DNA Antibody Production Cell Line. Mice. The MRL/l mouse is a model for an autoimmune disease (systemic lupus erythematosus) and makes antibodies for its own DNA.5 The mice were obtained from Charles River Japan Inc. After the symbolic disease became apparent (age of 4-5 months), the spleen cells were prepared and used for cell fusion. Cell Fusion. PAI(JCRB0113), a myeloma cell line of BALB/c origin was used as a partner cell. The MRL/l spleen cells and PAI were fused electrically. Briefly, 3 × 108 unfractionated spleen cells and 5 × 107 myeloma cells in serum-free minimum essential medium (MEM; Gibco) were combined and then co-pelleted. After being washed twice with serum-free MEM and once with 0.3 M sucrose, the cells were suspended in 3 mL of 0.3 M sucrose. The suspension was transferred to an electric fusion chamber equipped with a Krabo electropolator BM-850. The conditions were as follows: 40 VAC with 1-MHz frequency for 15 s to arrange the pearl beads’ cell alignment and then 700 VDC for 30 µs. After cell fusion, the cell suspension was transferred carefully to 100 mL of MEM solution supplemented with 20% fetal calf serum (FCS; Gibco) and hypoxanthine, aminopterin, and thymidine (HAT). After standing for 1 h at room temperature, the suspension was mixed with 108 feeder cells prepared from the spleens of Balb/c mice, then poured into 96-well microplates, and cultured. Three days later, hybridoma colonies appeared in every well and they were screened for anti-DNA antibodies. Screening for Antibodies for DNA. The aim of the screening was to select cell lines that produced the antibody with affinity for both double- and single-stranded DNA (see below). Sonicated herring sperm DNA, treated with S1 nuclease was used as doublestranded DNA. Samples of the same DNA were denatured by heat and used as single-stranded DNA. The DNA-coated microtiter plates were prepared as follows: 50 µL of phosphate-buffered saline (PBS) containing both 50 µg of double- and single-stranded DNA was added to the wells of polystyrene microtiter plates; after standing at 4 °C overnight, the solution was discarded. The plates were then blocked with PBS containing 10% FCS for 1 h at room temperature, and the solution was discarded again. (5) Andrezejewski, C., Jr.; Stollar, B. D.; Lalor, T. M.; Schwartz, R. S. J. Immunol. 1980, 124, 1499-1502. 10.1021/ac9805854 CCC: $15.00

© 1998 American Chemical Society Published on Web 11/07/1998

Culture fluids (50-µL aliquots) were transferred from the culture wells to the DNA-coated microtiter plates, and the plates were then incubated for 1 h at 37 °C. The wells were washed 3 times with PBS before the second antibody, a mixture of antimouse IgG, IgM, and IgA conjugated with HRP, was added, and the wells were incubated for 1 h at 37 °C and again washed with PBS 3 times. A 0.4 mg/mL aliquot of o-phenylenediamine and 0.01% of hydrogen peroxide were dissolved into a buffer solution composed of 0.1 M citrate and 0.2 M disodium hydrogen phosphate (pH 4.8) and used as the substrate for HRP. The wells that turned yellow were scored as positive and screened further. Cloning and Establishment of MN-1, a Hybridoma Cell Line Producing IgG-Type Anti-DNA Antibody. During the first screening, 20 wells were determined as positive. To increase the hybridoma populations contained in the positive wells, the culture volumes were increased gradually to 1 mL and were used for further screening. After the third screening, the positive populations were cloned 3 times and established as the anti-DNA antibody-producing cells. Finally, three cell lines that produced IgG-type antibody were obtained. One of the cell lines, MN-1, was used for further investigation. The antibody produced by MN-1 was the only one confirmed to have affinity for both doubleand single-stranded DNA. Preparation of the HRP-Conjugated Anti-DNA Antibody. Ascites fluid (20 mL) obtained from Balb/c mice inoculated with MN-1 was partially purified with DEAE-Sephacel (Pharmacia) ionexchange chromatography. After being dialyzed against 20 mM Tris hydrochloride buffer (pH7.5), the ascites fluid was charged onto the DEAE-Sephacel column (200 mL) which was equilibrated with the same buffer. The protein fraction adsorbed to the gel was eluted with linear sodium chloride gradients (0-0.5 M). The DNA-binding activity of each fraction (2 mL) was tested as described above. The fractions possessing DNA-binding activity were collected, concentrated, and further purified with a protein A affinity column (Pierce) according to the instruction manual. The purified antibody was conjugated with HRP. The antibody concentration was adjusted to 10 mg/mL in 0.01 M sodium bicarbonate solution (pH 9.5). A 30-mg or greater sample of HRP (Pierce) was treated with 0.01 M sodium periodate to oxidize the sugar side chains and dialyzed against 1 mM sodium acetate (pH4.4). Then, 0.01 M of disodium carbonate was added to adjust the pH to 9.0. The antibody solution (3 mL of a 10 mg/mL solution) and 3 mL of the HRP solution (10 mg/mL) were mixed completely for 2 h at room temperature. After being reduced with sodium borohydrate (final concentration of 0.2 mg/mL) at 4 °C for 2 h, the anti-DNA antibody conjugated with HRP was purified with Ultrogel AcA44 (IBF) gel filtration chromatography. Solid-Phase ELISA for Testing the Avidity of the Anti-DNA Antibody. A microtiter plate was coated with stepwise DNA gradient. Aliquots (50 µL) of PBS solutions containing 0.01, 0.1, 0.5, 1.0, 5.0, 10.0, 50, and 100 µg of double-stranded DNA (herring sperm; see above), respectively, were poured into respective wells and then air-dried. The wells were blocked as described above. Antibody solutions containing 0.1, 0.5, or 1.0 µg/mL antibody conjugated with HRP were added to the wells. After standing at 37 °C for 1 h, the wells were washed with PBS 3 times, and then 100 µL of the substrate for HRP was added. The reaction was stopped after 5 min by adding 50 µL of 6 N sulfuric acid. The

Figure 1. Schematic of the Biocell counter. The Biocell counter is a hypersensitive two-dimensional photon analyzer. This system is composed of a two-dimensional photon counter tube, some optical elements, a CCD camera, and an image processor (a personal computer).

color strength of each well was determined by measuring the 490nm absorbance. Detection of Bacteria with the Antibody-HRP Method. Nitrocellulose (45-mm-diameter) filters with funnels (Milifllex; Millipore) were used to filter UPW and trap bacteria. After filtration, the filters were cut off from the funnels and placed on Petri dishes. Bacteria trapped on the filter surface were lysed with the lytic solution composed of 0.2 N sodium hydroxide and 1% sodium dodecyl sulfate, by soaking the filters in 3 mL of the solution for 5 min at room temperature. This treatment was for the purpose of fixing the bacterial DNA onto the filter surface. The lytic solution was aspirated, and the filters were washed twice with PBS by soaking them in 5 mL of PBS briefly. Then the filters were blocked with 8 mL of PBS containing 10% bovine serum albumin (BSA) and 3% Tween 20 for 30 min at room temperature with gentle agitation. The blocking solution was replaced by 8 mL of PBS-based antibody solution containing 0.5% BSA and 0.04 µg/mL anti-DNA antibody conjugated with HRP. The filters were incubated for 2 h at room temperature with gentle agitation to mark bacterial DNA with the antibody. The excess antibody was washed out with 8 mL of PBS containing 3% Tween 20 4 times (15 min each). Finally, the filters were placed on a paper blotter to absorb the remainder of the washing solution and then dipped in the chemiluminescence substrate reagent composed of hydrogen peroxide and luminol (ECL detection reagent; Amersham). After quenching for 1 min, the filter was placed on the stage of a hypersensitive two-dimensional photon image analyzer (the Biocell counter; described below), and the photons were accumulated for 20 s. The obtained two-dimensional photon image was automatically analyzed to give the number of bacteria by the image processor with which the system was equipped. Detection of Bacteria with the Conventional Cultivation Method. Samples were filtered as described above. The filters were cut off from the funnels, settled on the nutrient agar (Difco) plates, and incubated for 3 days at 30 °C. The colonies that appeared on the filter surface were counted and described as the number of contaminated bacteria. Hypersensitive Two-Dimensional Photon Image Analyzer. The Biocell counter, a hypersensitive two-dimensional photon (6) Niedhardt, F. C. Escherichia coli and Salmonella typhimurium, Cellular and Microbiology; American Society for Microbiology: Washington, DC, 1987; Part I.

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Figure 2. Quantitative analysis of DNA with the anti-DNA antibody. Solid-phase ELISA was carried out as described in the text. The antibody concentration was (A) 0.1, (B) 0.5, and (C) 1 µg/mL.

analyzer, was developed to combine with the antibody-HRP method for detecting bacteria without cultivation. The DNA content of one bacterium is 10-14 g or lower.6 To detect this small an amount of DNA, a highly specialized optical system had to be developed. Hardware. The schematic figure of the Biocell counter is shown in Figure 1. This system is composed of a two-dimensional photon counter tube, some optical elements, a CCD camera, and an image processor. A faint light, chemically produced by HRP conjugated with the antibody, is multiplied by 103-105 through an image intensifier in the tube and consequently illuminates a luminescent glass plate at the end of the tube. This luminescent image is caught by the CCD camera, digitally accumulated in a memory, and processed by the image processor to extract the light spots caused by the antibody-HRP method. The photon counter tube has a resolution of 15 line pairs/mm while the CCD camera has 370 000 pixels that give a sufficiently high resolution compared with the photon counter image. Image Processing. A cumulated image is treated as follows to extract authentic light spots caused by the antibody-HRP method. 5298 Analytical Chemistry, Vol. 70, No. 24, December 15, 1998

Figure 3. Basic theory of the antibody-HRP method. Bacteria are filtered and trapped on a filter (A) and lysed with the lytic solution (B). Bacterial DNA is denatured and fixed on the filter surface (C). The anti-DNA antibody conjugated with HRP attaches to, and marks, the DNA (D). In this figure, the antibody molecules are represented by Y letters and HRP molecules are represented by dark circles. HRP catalyzes the photon-producing reaction repeatedly, and photons are accumulated at the original positions of the bacteria (E). The photon image is processed, and the number of the light spots is expressed as the number of bacteria (F).

At first, the noise reduction image is made from the cumulated native image by processing standard arithmetic filtering operators. In this process, matrices (3 × 3), the sizes of which are much smaller than the expected light spots, are smoothed to nullify shot noise and to define the light spots. The background image is also made from the native image by a method similar to that used to make the noise reduction image. The difference is that the matrices (15 × 15, the sizes of which are much larger than the expected light spots) are smoothed to nullify large gloomy light spots coming from unknown materials other than bacterial cells. Subtracting the background image from the noise reduction image finally gives an image including objective light spots without the shot noise and the background undulation. This final image is suitable for the binary conversion process. The binary conversion and counting processes are performed according to ordinary methods of image-processing technique.

Figure 4. Photographic images of the cultured E. coli on the filters. The cultured E. coli cells were tested by the antibody-HRP method. The calculated numbers in the view field are (A) 2, (B) 15, (C) 38, (D) 135, (E) 250, and (F) 280.

RESULTS Characteristics of the Anti-DNA Antibody. The solid-phase ELISA was carried out to test the avidity of the obtained antibody to DNA. The results are shown in Figure 2. Although the sensitivity varied with the dosage of anti-DNA antibody, it was found that the anti-DNA antibody detected DNA quantitatively in the range of 0.01-100 µg of DNA. These experimental results demonstrated that the antibody obtained in this study had a high avidity for DNA and therefore could be a useful tool to detect and quantitate DNA. The antibody shows affinity for both double- and singlestranded DNA. Anti-DNA antibodies having this type of affinity for DNA are supposed to bind to the phosphate-sugar backbone of DNA, a common antigenic determinant of a wide variety of

nucleotides, and are useful tools for measuring DNA.7,8 The wide spectrum for DNA species, together with the high avidity, makes the antibody suitable for detection of a variety of bacteria inhabiting UPW.1,2 Antibody-HRP Method. The basic theory is depicted in Figure 3. Bacteria trapped on a filter surface are lysed with a solution composed of a strong base and a detergent. The highly packed bacterial DNA is burst, expanded, and exposed on the filter surface.9 The base at the same time denatures the bacterial (7) Koike, T.; Nagasawa, R.; Nagata, N.; Shirai, T. Immunol. Lett. 1982, 4, 9397. (8) Du Clos, T. W.; Rubin, R. L.; Tan, E. M. J. Immunol. Methods 1986, 88, 185-192. (9) Delius, H.; Worcel, A. Cold Spring Harbor Symp. Quantum Biol. 1973, 38, 53-58.

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DNA, and the denatured DNA adheres to the filter. The expanded DNA is easily accessible to the anti-DNA antibody. The bacterial DNA is so large and spreads so widely that an amount of antiDNA antibody is able to attach to the molecule. All HRP gather on the DNA with the carrier antibody and catalyze the photonproducing reaction repeatedly in the presence of hydrogen peroxide and luminol, and photons accumulate on the positions where bacteria originally existed. The Biocell counter surveys the whole area of an 18-mm diameter, processes automatically the signals of the accumulated photons, and expresses the number of light spots as the number of bacteria. In this system, we can detect a single molecule of bacterial DNA that exists in the area. All of the detection operations can be completed within 4 h. In this system, the naked DNA is stable against hydrolytic enzymes and chemicals as it tightly sticks to the membrane. Treatments with deoxyribonuclease I, proteinase K, pepsin, and formic acid did not exclude the DNA molecules from the membrane surface (data not shown). This stability contributes to the accuracy of the system and to its ability to detect one bacterial cell on the filter. Detection of Cultured Esherichia coli with the AntibodyHRP Method. Figure 4 shows photographs of the light spots made by the cultured E. coli trapped on membrane filters. The images were photographed by a videoprinter attached to the Biocell counter. Approximately 100, 500, 1000, 3000, and 5000 bacterial cells (estimated from a colony-forming unit) were trapped on the filters (B-F) with (A) as a blank membrane. A few faint spots were found to exist in (A), probably due to the original bacterial contamination of the filter. As the area of the field of view is one-tenth that of the filter, the real numbers are obtained by multiplying 10 times the number counted in the images. The calculated total number of B membrane was 150 cells, which was near the number estimated from the colony-forming unit. The calculated numbers of the cells trapped on filters C-F were 380, 1350, 2500, and 2800, respectively. There existed a linear relationship between the results from the antibody-HRP method and those from the cultivation method (colony-forming unit) with cell numbers less than 3000 trapped on one filter. When the bacterial cell numbers trapped on the filter exceeded 3000, the overlapping spots could not be discriminated by this imageanalyzing program. The same experiment carried out with Pseudomonas putida and Bacillus subtilis gave similar results. In this experiment, E. coli cells from early log-phase culture were used for calculating the cell number both by the antibodyDNA method and by the cultivation method. As actively growing cells dominated the culture and most of them were able to form colonies, the numbers of the detected cells with the antibodyHRP method well reflected the numbers of the colony-forming unit. Conversely, when a water sample from a natural source is measured, the number of bacteria estimated with the antibodyHRP method is usually larger than with the colony-forming unit. A large number of unculturable bacteria are supposed to inhabit an environment and this might cause the underestimation of cultivation methods10,11 (discussed below). Estimation of the Contaminated Bacteria in UPW. The numbers of bacteria contaminating UPW from three semiconduc(10) Pepper, I. L.; Josephson, K. L.; Bailey, R. L.; Burr, M. D.; Pillai, S. D.; Tolliver, D.; Pulido, S. Proc. Semicond. Pure Water Chem. Conf. 1993, 1993, 50-62. (11) Roszak, D. B.; Colwell, R. R. Microbiol. Rev. 1987, 51, 365-379.

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Table 1. Estimation of the Bacterial Contamination in UPW Plantsa detected count/L sampling point

colony-forming unit

antibody-HRP method

1 2 3 4 5 6 7 8 9

Plant A 20 20 40 6 250 4 150 0 4

1100 4500 200 400 9200 880 1720 960 540

1 2 3 4 5 6 7 8 9 10 11

Plant B 25 5000 0 1000 1000 3 500 0 0 10000 0

250 20000 0 1000 600 100 2000 200 100 1000000 0

1 2 3 4 5 6

Plant C 0 2 2 2 1 4

30 20 20 30 20 110

a UPW from three plants were tested for bacterial contamination with the antibody-HRP method as well as the conventional cultivation method. While a 20-L sample was taken from plant B for the cultivation method, the result is expressed as the number of detected bacteria per liter, for convenience.

tor manufacturers were measured with the antibody-HRP method. Table 1 describes the results obtained from the three semiconductor manufacturers. In parallel, the colony-forming unit was determined with the cultivation method for a comparative reference. In plant A, replicate 1-L UPW samples were drawn from the nozzles of the midway sampling points and supplied for detection. In plant B, samples were taken as in plant A; the sample for the antibody-HRP method was 1 L and the sample for the cultivation method was 20 L. In plant C, replicate 1-L samples from the use points were taken for the test. The relationship between the results obtained with the antibody-HRP method and those obtained with the cultivation method was in good accordance. The samples marked by a large colony-forming unit inevitably represented a larger number of bacteria in the antibody-HRP method. Conversely, the samples determined to be contaminated with less bacteria in the antibodyHRP method showed less or none in the colony-forming unit. In most cases, however, the numbers of the bacteria obtained with the antibody-HRP method were larger than those obtained with the cultivation method. As with the antibody-HRP method, bacteria need not be cultured before detection; even the cells that could not grow in the cultural conditions were efficiently counted. It has been noted that many of the bacterial populations in the

environment cannot grow under artificial conditions.11,12 Especially for UPW, in which anaerobic and oligotrophic bacteria are the main inhabitants,1,2 the colony-forming unit may not have the sensitivity to detect the contaminating bacteria. DISCUSSION The number of bacteria that exist in 1 L of UPW might be one or lower.2 Usually, these low-level contaminants are estimated after being concentrated on filters. Even though 100 L of UPW is filtered, bacterial cells trapped on the filter surface might total no more than 100. It is impossible to use a microscope or a scanning electron microscope to detect bacteria existing with such sparsity, and these techniques are not able to discriminate bacteria from other small particles. Thus, colony-forming ability, a detectable proof of existing bacteria, is the only means to estimate bacterial contamination in UPW. Although alternatives to the cultivation methods have been suggested, none of them could be applied to estimate bacterial contamination in UPW. The recently introduced ATP bioluminescence technique12 cannot be applicable for UPW because of the low sensitivity; the detection limit of the ATP method is 103 living cells,1 whereas 1 L of UPW might contain as little as one bacterial cell.2 The polymerase chain reaction (PCR) technique, effective to assess a special bacterium in the environment, cannot elucidate the precise number of bacteria contaminating the sample.10,13 Having the capability to increase its number in a short time, and having the possibility of causing (12) Stanley, P. E. J. Biolumin. Chemilumin. 1989, 4, 375-380. (13) Saiki, R. K.; Gefland, D. H.; Stoffel, S.; Scharf, S. J.; Higuchi, R.; Horn, G. T.; Mullis, K. B.; Erlich, H. A. Science 1988, 239, 487-494.

a UPW defect, bacterial contaminants have to be monitored more strictly and rapidly. Focusing on apparently dead bacteria, a detection system completely independent of the colony-forming unit should be developed. The antibody-HRP method, combined with the hypersensitive optical system, made it possible to detect and count these sparsely distributed bacteria with high resolution. Since this system captures the bacterial DNA instead of bacterial cells themselves, it has a wide detection spectrum. It can detect all kinds of bacteria, including growing cells, dead cells, and resting cells. The results in Table 1 indicate that bacterial contamination in UPW is more severe than normally considered. Since the demands on UPW quality are becoming more stringent, it is time to reconsider the conventional idea that a colony-forming unit is an adequate criterion for estimating bacterial contamination in UPW. The possibility that the bacteria, which are missed by the cultivation method, might happen to burst the size of the population and make trouble during chip-manufacturing processes, cannot be ignored. In terms of sensitivity, detection spectrum, and rapidity, the antibody-HRP method, together with the Biocell counter, is considered to be the most suitable tool to control and monitor bacterial contamination in UPW.

Received for review May 27, 1998. Accepted September 17, 1998. AC9805854

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