JP4256802B2 - Sensitized latex and immunoassay - Google Patents

Description

  The present invention relates to an antibody-sensitized latex, an immunological technique, and an aerobic organism that rapidly and easily measures a bacterium belonging to the genus Nitrospira present in biological nitrogen treatment processes such as activated sludge, water, and soil. It relates to the operation management method of the treatment facility.

  In more detail, it is possible to accurately detect bacteria of the genus Nitrospira, which has required a long period of time and technical skill, with a simple operation. A method for accurately operating and managing biological nitrogen treatment processes such as activated antibody sludge, a method for measuring a novel antibody-sensitized latex, a nitrospira bacterium, and the like.

  Wastewater, garbage, etc. discharged from our living environment contain a lot of nitrogen compounds, which are discharged into the surrounding environment such as groundwater, rivers and lakes, and may cause serious environmental problems. As a countermeasure, biological nitrification and denitrification using activated sludge has been developed and used in various sewage treatment plants. Among the treatment methods, nitrifying bacteria present in activated sludge play an important role in the nitrification process.

  In the biological denitrification process, the nitrification reaction is at the rate-determining stage. Therefore, in order to stabilize the treatment process and further increase the efficiency, microorganism groups related to the nitrification reaction (nitrifying bacteria, ammonia oxidizing bacteria and Analysis of nitrite-oxidizing bacteria) is considered essential.

  Bacteria involved in the nitrification reaction are autotrophic bacteria, and have a low specific growth rate compared to heterotrophic bacteria that remove organic matter and are susceptible to environmental conditions such as pH and water temperature. It is very important to keep nitrifying bacteria at a high density.

  However, the evaluation and maintenance of these treatment tanks are determined by analyzing the input and output of the system, but at present the determination of the essential flora is not performed at all.

  Specifically, conventionally, as a method for quantifying nitrite-oxidizing bacteria in biological nitrogen treatment processes such as activated sludge treatment tanks in sewage treatment facilities and in soil, samples are generally collected and from 1 month to 2 months. After passing through a culture period of about 5 minutes, there has been known a method of indirectly measuring these samples from the presence or absence of consumption of nitrous acid (see Non-Patent Document 1).

  Conventionally, there has been an attempt to manage biological nitrogen treatment processes such as activated sludge and the trend of nitrifying bacteria in soil by using such a method. Since skill is required for the measurement of this, and a considerable number of days are required, it is currently impossible to take a quick response for daily management of the trend of nitrifying bacteria from the measurement results.

  Therefore, the inventors detected nitrosomonas bacteria (ammonia-oxidizing bacteria) and nitrobacter bacteria (nitrite-oxidizing bacteria), which are representative nitrifying bacteria, using antibodies that specifically recognize these bacteria. A method has been developed, and a method for quickly and simply detecting ammonia-oxidizing bacteria or nitrite-oxidizing bacteria by the method is provided (see Patent Document 1). Furthermore, a method of detecting ammonia-oxidizing bacteria and nitrite-oxidizing bacteria more easily by using antibody-sensitized latex in which the antibody is adsorbed on latex particles has been provided (see Patent Document 2).

  On the other hand, since 1990, with the introduction of analysis techniques using molecular biological techniques in the field of water environment, knowledge on the distribution and population of nitrifying bacteria is gradually being updated. Especially for nitrite-oxidizing bacteria, a new nitrospyra bacterium was found, and a nitrogen treatment process detected as a dominant species has been frequently reported. Therefore, in order to accurately grasp the nitrogen removal efficiency in the biological nitrogen treatment process, how to accurately measure the number of bacteria in the genus Nitrospira in addition to the bacteria in the genus Nitrosomonas and Nitrobacter This is an important issue.

However, in the measurement method described above, in the process of culturing using a sample such as activated sludge obtained from a biological nitrogen treatment process, cells other than nitrospira bacteria such as nitrosomonas bacteria and nitrobacter bacteria are dominant. As a result, there was a problem that an accurate result could not be obtained. In addition, since the nitrospira bacterium itself cannot be isolated by conventional methods, there is a problem in that an antibody that specifically recognizes the nitrospira bacterium cannot be obtained, and the bacterium cannot be detected or quantified.
Kimura Ryusuke et al., Soil Microbial Experimental Method, pp. 207-214 (1992) JP 05-322896 A Japanese Patent Application Laid-Open No. 08-029426

  In light of this situation, the present inventors will further develop a method for easily and rapidly measuring nitrospyra bacteria present in biological nitrogen treatment processes such as activated sludge, water, soil, etc. We conducted an intensive study for the purpose. As a result, the present inventors produced an antibody specific to a bacterium of the genus Nitrospira, and used the antibody-sensitized latex in which the antibody was adsorbed on latex particles, thereby allowing the target nitrospira genus to be obtained by a simple operation. The present inventors have found that bacteria can be measured quickly and have completed the present invention.

  That is, the object of the present invention is to provide a novel nitrospira antibody-sensing antibody sensation for measuring bacteria of the genus Spirobacterium that enables rapid and simple detection of biological nitrogen treatment processes such as activated sludge, nitrospira spp. It is to provide a latex product. Another objective is to use the above-described antibody-sensitized latex for measurement to perform rapid and simple measurement of biological nitrogen treatment processes such as activated sludge and nitrospira bacteria in water and soil. It is to provide a static measurement method. Still another object is to provide an operation management method for an aerobic biological treatment tank using the number of nitrifying bacteria including Nitrospira as an index for daily management of an aerobic biological treatment facility.

  The antibody-sensitized latex for measuring a nitrospira bacterium according to the invention described in claim 1 is an antibody specific for a nitrospira bacterium having nitrite oxidation activity, having a specific gravity of about 1.5 g / ml and an average particle size of 1. It is characterized by being adsorbed on latex particles of 0.0 to 1.5 μm.

  An immunological measurement method using the antibody-sensitized latex for measuring nitrospira bacteria according to the invention described in claim 2 is a method in which the antibody-sensitized latex according to claim 1 is mixed with a test sample. After standing for a given time, the presence or absence of the occurrence of an aggregate image indicating that the individual latex particles have aggregated is confirmed.

  As described above, the present invention relates to an antibody-sensitized latex for measurement in which an antibody specific to a bacterium of the genus Nitrospira is adsorbed on latex particles. By using the antibody-sensitized latex, Therefore, it is possible to easily and conveniently perform detection and measurement of bacteria belonging to the genus Nitrospira, which has been technically required for skill. Another invention of the present invention is to produce a specific antibody against a nitrospira bacterium, to identify a biological nitrogen treatment process such as activated sludge, to identify a nitrospira bacterium in water or soil, and to rapidly increase the number of bacteria. And it makes it possible to measure appropriately. In yet another invention, the operation management of the aerobic biological treatment facility is facilitated by controlling the operating conditions of the aerobic biological treatment tank using the number of nitrifying bacteria including the genus Nitrospira as an index.

  The antibody-sensitized latex for measuring nitrospira bacteria of the present invention is an antibody specific for nitrospira bacteria having nitrite oxidation activity having a specific gravity of approximately 1.5 g / ml and an average particle diameter of 1.0 to 1.5 μm. It is adsorbed on latex particles, which makes it easy and simple to detect and measure nitrospyra bacteria, which conventionally required a long period of time and technical skill. it can.

  The method of the present invention may be an immunoassay method in which an antibody against a nitrospyra bacterium is reacted with the antibody. Examples of the specimen include biological nitrogen treatment processes such as activated sludge, water, and soil. Samples collected from can be used as they are or after dilution. More specifically, an immunoassay method for detecting and quantifying a nitrospyra bacterium present in a specimen with an antibody specific to these bacteria can be mentioned.

  Examples of the immunoassay include immunoagglutination such as latex agglutination and reverse passive latex (erythrocyte) agglutination, immunochromatography, fluorescent antibody method, enzyme antibody method, and radioimmunoassay. From the viewpoint of rapidity, immunoaggregation and immunochromatography are preferred. Of the immunoaggregation methods, the reverse passive latex (erythrocyte) aggregation method, which is an indirect agglutination reaction using erythrocytes or latex particles bound with an antigen or antibody, is particularly preferred.

  As the insoluble carrier used in the immunoaggregation method of the present invention, any of inorganic compound particles such as red blood cells and gold colloid particles and organic polymer particles such as latex particles may be used. Or it is preferable from the point of stability of the reagent after antibody sensitization.

  The bacterium of the genus Nitrospira referred to in the present invention is a genus of so-called nitrite-oxidizing bacteria, which is a microorganism having an action of producing nitric acid by nitrite oxidation activity, that is, nitrite oxidation. Currently, 43 DNA sequences of 16S-rRNA of nitrospira-like bacteria are registered in DDBJ (DNA Data Bank of Japan), and four strains (species) have been proposed from the results of phylogenetic analysis using the sequences. .

  However, there are only two strains that have succeeded in pure culture: fresh water nitrospira moscoviensis (Nitrospira moscoviensis) and nitrospira marina (Nitrospira marina ATCC43039) isolated from the deep sea. It has not been isolated from normal biological nitrogen treatment processes such as reactors. In the present invention, nitrospira moscobiensis was used as a nitrospira bacterium for preparing the antibody. However, when a pure culture of nitrospira is established, the antibody can be similarly used even if the strain is used. It goes without saying that can be prepared.

  Antibodies specific to these bacteria may be used to immunize animals such as rabbits, mice, rats, chickens, sheep, and horses using the above bacterial cells as antigens, and the obtained antibodies may be used as antibodies for detecting bacteria. Furthermore, the antiserum produced by a known method (such as the method described in Patent Document 1) is further purified by an appropriate purification means such as affinity chromatography bound with protein G and used.

  Hereinafter, the case of the immunoagglutination method will be described as an example in more detail. As latex particles to be used, specific gravity is 1.0 g / ml or more, preferably around 1.5 mg / ml, particularly high specific gravity latex particles of 1.2 to 1.8 mg / ml are preferably used. Latex particles having an average particle size of about 0.1 to 4.0 μm, preferably about 1.0 to 1.5 μm, more preferably about 1.3 μm on average are preferably used, and the latex particles are further colored. Is used as a preferred one. Examples of such latex particles include Bact latex 0.81 (manufactured by DIFCO, average particle size 0.81 μm, specific gravity 1.0 g / ml), and IMMUTEX H series (manufactured by JSR, specific gravity 1). 0.5 g / ml, the average particle size is 0.94 μm to 4.9 μm, including colored latex) and polybeads # 15706, # 15709 (manufactured by Polysciences), but not limited to this, Any of these types can be used as long as they have the same effects.

  To prepare an antibody specific to a bacterium belonging to the genus Nitrospira, first cultivate the bacterium using an inorganic salt liquid medium containing nitrite as a growth substrate, sterilize it if necessary, collect it, and use it as an antigen. . Next, this antigen is administered by injection from the ear vein of a rabbit, and after confirming an increase in antibody titer, whole blood is collected, subjected to appropriate treatment such as centrifugation, and then inactivated at 56 ° C. Antiserum. Further, it is used after being purified by an appropriate purification means such as affinity chromatography to which protein G is bound.

  Next, the latex particles of the carrier for adsorbing the specific antibody and the method for producing the sensitized latex are not particularly limited. For example, the method reported by the present inventors (described in Patent Document 2) Can be prepared according to the above method).

  There are a physical adsorption method and a chemical bonding method as a method for carrying these antibodies on carrier particles to form immunological agglutination reaction particles, both of which can be suitably used for the immunological agglutination reaction of the present invention. When latex particles are used as the agglutination reaction particles, the antigen or antibody can be sufficiently sensitized by a physical adsorption method.

  When the antibody is sensitized to the latex particles, it is preferably performed in a buffer solution. That is, a suspension of latex particles diluted to an appropriate concentration and an antibody or antigen solution are mixed and allowed to stand for a while before washing to produce sensitized latex particles. As the buffer used for dilution, a buffer having an ionic strength and pH that does not inhibit the reaction with the antibody or antigen, which is generally a supported antigen or antibody measurement target substance, is selected. For example, TBS, PBS, GBS, phosphate buffer and borate buffer can be used. Among them, phosphate buffer, borate buffer and GBS are preferably used because of the aggregation property of the sensitized latex particles and the difficulty of self-aggregation. In general, the pH is selected within the range of 5 to 10, and the pH range of 6 to 9 is particularly preferable.

  The concentration of latex particles at the time of sensitization is suitably from 0.01 to 0.5 (w / v)%, and good results are obtained particularly at about 0.05 to 0.25 (w / v)%. In addition, the antibody protein concentration of the antibody solution in the case of sensitizing the antibody is suitably 1.0 to 1000 μg. Otherwise, it is difficult to make a positive / negative judgment due to a decrease in sensitivity or self-aggregation of sensitized latex particles. Become. Furthermore, the temperature during the sensitization reaction is in the range of 0 to 60 ° C., but when the reaction is performed at room temperature or a temperature slightly higher than room temperature (˜40 ° C.), latex particles with good sensitivity can be obtained.

  The reverse passive latex agglutination method is preferred for detecting nitrospyra bacteria and measuring the number of bacteria using these sensitized latexes. The reverse passive latex agglutination method is a liquid containing immunological agglutination reaction particles in which a substance (such as an antibody) that specifically binds to a test substance is bound to a high-specific gravity labeling substance (such as a latex particle). In this method, a reagent and a sample containing a test substance are mixed to detect or quantify the target substance. More specifically, a sample diluted at an appropriate stage and a reagent containing antibody-sensitized latex are mixed to confirm the dilution at which an aggregated image is observed, while a standard containing a bacterial solution of a known number of bacteria. A mixing test using a sample is performed, and the number of bacteria may be calculated from comparison with the result.

  More specifically, an antigen or standard sample diluted stepwise with a buffer solution is added to a U-shaped microtiter plate, an equal amount of reagent is dispensed into each well, and then allowed to stand at room temperature for 4 to 15 hours. After that, observation and determination are performed using the naked eye or a magnifier of about 10 times. Specifically, the agglutination image in the case of only the buffer solution is regarded as negative, the agglutination image of each hole is determined, and from the result of the agglutination test using the dilution factor of the test hole that showed a positive agglutination image and the standard antigen Calculate the number of bacteria.

  In the present invention, in addition to this, the latex particles according to the present invention and a test sample are mixed on a slide glass, and a method of determining the success or failure of an aggregated image by an optical microscope (microscopic latex method), or immunochromatography Needless to say, the present invention can be applied to various qualitative and quantitative methods using immunological agglutination reaction particles.

  Conventionally, it has been known that latex particles generally exist as a carrier on which antibodies are adsorbed. However, there have been reports of examples in which this is actually used for detection and identification of bacteria of the genus Nitrospira. The effectiveness has not been studied for the first time by the present inventors. In addition, by using antibodies specific to the bacteria of the genus Nitrospira, we identify target microorganisms that have biological nitrogen treatment processes such as activated sludge, activity in water and soil (oxidation of nitrite), and their number The inventors have for the first time obtained the knowledge that it is possible to establish a method for quickly and appropriately managing the risk.

  Furthermore, in the present invention, the number of nitrospira bacteria in the aerobic biological treatment tank is measured, and the solid residence time in the tank, the tank so that the nitrospira bacteria number necessary for maintaining the target nitrification rate is obtained. At least one operating condition selected from the amount of dissolved oxygen in the tank and the inflow load into the tank. More preferably, in addition to nitrospyra, the number of nitrosomonas and nitrobacter is measured, and the number of nitrosomonas, nitrobacter and nitrospira necessary to maintain the target nitrification rate. At least one operating condition selected from the solid residence time in the tank, the dissolved oxygen amount in the tank, and the inflow load into the tank is controlled so that the number of bacteria belongs. This facilitates the daily operation management of the aerobic biological treatment facility using the number of nitrifying bacteria including Nitrospira as an index.

  The aerobic biological treatment mentioned here is a treatment method in which various aerobic microorganisms are involved in the presence of dissolved oxygen to oxidatively decompose and remove organic substances, ammonia nitrogen, odor, iron, etc. is there. The aerobic treatment can be broadly divided into a method of oxidizing and decomposing organic substances in a state where biological flocs are suspended by aeration, and a biofilm is formed by attaching microorganisms to a carrier to form a biofilm, which is then contacted with wastewater to oxidatively decompose It is divided into methods. The former representative is the activated sludge method, which is widely used in sewage treatment. The latter is generally called biofilm method.

  The activated sludge method mentioned here includes standard activated sludge method, oxygen activated sludge method, long-time aeration method, oxidation ditch method, batch activated sludge method, etc., as well as circulating nitrification denitrification method, nitrification endogenous Denitrification methods, anaerobic-anoxic-aerobic methods, anaerobic-aerobic activated sludge methods are included. The biofilm method here is often called a biofixation method recently because microorganisms are immobilized on a carrier (also called a support or contact material). When classified according to the state of the carrier, it can be divided into a case where the carrier is immersed in the treatment tank (fixed bed) and a case where the carrier is moved in the treatment tank (fluidized bed).

  In addition, a combination of aerobic treatment and anaerobic treatment, such as a method of anaerobic treatment after anaerobic treatment of high-concentration wastewater using a biofilm mainly for the purpose of removing nitrogen compounds and phosphorus, is utilized. It goes without saying that it can be widely applied to biological treatment methods including such aerobic biological treatment.

  Hereinafter, although an example explains the present invention still in detail, the present invention is not limited at all by the example.

Example 1. Preparation of Bacteria Used for Antigen Nitrospira moscoviensis was cultured in the medium shown in Table 1. This strain was distributed from Dr. Eva Spieck, University of Hamburg, Germany (see Non-Patent Document 2). The culture was divided into three stages (100 mL scale seed culture, 1 L scale intermediate culture, 4 L scale final culture), and in each stage, the culture temperature was 37 ° C. and the culture period was 7 to 10 days.
Ehrich S., D. Behrens, E. Lebedeva, W. Ludwig, and E. Bock. (1995) A new obligately chemolitoautotrophic, nitrite-oxidizing bacterium, Nitrospira moscoviensis sp. Nov. And its phylogenetic relationship. Arch. Microbiol. 164 : pp 16-23.

  The final culture was stopped when the growth of the bacteria was considered to be maximum, and the culture was centrifuged at 24,000 × g for 10 minutes at 4.0 ° C. to collect the bacteria. After adding 30 mL of PBS and dispersing the bacteria by pipetting, an equal amount of 0.6% formalin was added and sufficiently stirred. This solution was allowed to stand at 37 ° C. for 1 hour, and then fixed at 4.0 ° C. until the bacteria were killed. The fixed cells were collected by centrifugation at 24,000 × g for 10 minutes, washed with PBS, and then centrifuged again at 24,000 × g for 10 minutes for use as an antigen. Furthermore, it adjusted with PBS so that the light absorbency of 660 nm might be set to 0.5 as a test antigen.

  Antigen purity was confirmed using DNA probes NSR1156 and Ntspa0662 specific for nitrospira. That is, a hybridization test was performed using the probe, and it was confirmed whether bacteria other than DSM10035 were mixed in the bacterial suspension.

Example 2 Preparation method of polyclonal antibody Two KBL: Jw (Japanese white breed) rabbits were used. The dose of the antigen was 0.5 mL per time, and injection was performed from the ear vein every 7 to 10 days. Serum was experimentally collected from immunized rabbits, and the degree of increase in antibody titer against nitrospira moscoviensis strain DSM10035 was examined by enzyme linked immunosorbent assay (ELISA) ELISA. Specifically, an antigen adjusted to an absorbance (A _{660 nm} ) = 0.05 with a carbonate / bicarbonate buffer solution was dispensed at 100 μL per well in a 96-well plate and adsorbed at 4.0 ° C. for 16 hours. After washing each well with a washing solution (PBS (-) containing 0.05% Triton-X100), a blocking solution (a carbonate / bicarbonate buffer containing 1.0% BSA) was added at 120 μL / well, and the mixture was incubated at 37 ° C. The reaction was carried out for 1 hour. After washing the wells, 90 μL of serum serially diluted with PBS (−) containing 0.05% Triton-X100 and 1.0% BSA was added and reacted at 37 ° C. for 1.5 hours. After washing the wells, a peroxidase-labeled secondary antibody (anti-rabbit immunoglobulin G (IgG) -sheep IgG) was added at 100 μL / well, followed by further reaction at 37 ° C. for 1.5 hours in the dark. After completion of the reaction, 100 μL / well of a citrate buffer solution of O-phenylenediamine containing hydrogen peroxide was added to the washed wells, and the mixture was allowed to stand at 37 ° C. for 10 minutes in the dark to develop color. After stopping the reaction by adding 50 μL / well of 2.5 M sulfuric acid, the absorbance at 492 nm (A _{660 nm} ) was measured. The antibody titer was shown as the reciprocal of the dilution ratio of serum having a color development intensity of A _{492 nm} of 0.5. When no increase in antibody titer was observed, whole blood was collected from the heart. It was inactivated at 56 ° C. to obtain an antiserum corresponding to a bacterium of the genus Nitrospira.

Example 3 FIG. Purification of Immunoglobulin G (IgG) IgG was fractionated from the antiserum by affinity chromatography coupled with protein G (Mab TrapG kit, Amersham Pharmacia). This purified IgG was used as an anti-nitrospira antibody and used in the following experiments.

Example 4 Cross-reactivity test The cross-reactivity between the anti-nitrospira antibody and each bacterium shown in Table 2 was examined by ELISA. The cross-reactivity is defined as the antibody titer against the anti-nitrospira antibody nitrospira moscoviensis (DSM10035 strain) (absorbance (OD ₄₉₂ = 0.5) representing the absorbance by color reaction applying ELISA method) is 100%. The cross-reactivity between the antibody and each cell was shown in%.

  In general, there are many cases where Gram-negative bacilli such as Pseudomonas, Alkaligenes, and Flavobacterium are reported as dominant species from water purification processes such as activated sludge. However, as shown in Table 3, it was revealed that the anti-nitrospira antibody produced this time has high specificity without any cross-reactivity with these bacteria. These antisera having high specificity can be obtained by directly using reaction characteristics such as agglutination reaction of the antibody against each bacterium as an index, or using a color reaction correlated with the reaction characteristics as an index, and sensitizing an antibody for detection. It has been found that latex is useful for conveniently detecting biological nitrogen treatment processes such as activated sludge, nitrospira bacteria in water and soil.

Embodiment 5 FIG. Preparation of Latex Reagent for Nitrospira To increase the detection sensitivity of the latex reagent for nitrospira, the amount of protein sensitized to latex particles and the particle size of latex particles were optimized. The antibody-sensitized latex reagent was prepared in accordance with the method described in Patent Document 2.

  Specifically, the anti-nitrospira antibody was diluted with GBS so as to be 50 to 500 μg / mL. 1 volume of the antibody diluted solution is mixed with 1 volume of polystyrene high specific gravity latex solution (JSR, specific gravity 1.5 g / ml) diluted to 0.25% w / v with GBS and reacted at 37 ° C. for 1.0 hour. I let you. After completion of the reaction, a blocking solution (0.1 MGBS containing 1.0% BSA) was added to block the antibody non-adsorbed portion on the latex particles with BSA. After a blocking reaction for 30 minutes, the unbound antibody was removed by centrifugation at 2,700 × g for 10 minutes at room temperature. The antibody-bound latex particles were repeatedly washed twice with a stock solution (0.1 M GBS pH 8.2 containing 1.0% BSA and 0.08% NaN3), and finally the particle concentration was 0.05%. (W / v) was suspended in a stock solution to obtain a latex reagent for nitrospira. This operation was performed on latex particles having various particle sizes described in Table 4.

Example 6 Detection of Nitrospira by Reverse Passive Latex Aggregation Method A known number of formalin-fixed nitrospira moscoviensis (DSM10035 strain) was diluted to a factorial of 2 using a stock solution. After 50 μL of each diluted solution was dispensed into a 96-well UV plate (SJ103-20 Sanko Junyaku Co., Ltd.), equal amounts of high specific gravity latex reagents sensitized with various concentrations of antibodies were added. The mixture was sufficiently stirred to such an extent that the mixture did not scatter, and then allowed to react at room temperature for 4.0 hours to determine the minimum detectable bacterial amount. This test was carried out for each particle size shown in Table 4 to examine the particle size with the highest detection sensitivity and the antibody protein sensitization concentration.

As a result, as shown in FIG. 1, it was revealed that the measurement sensitivity of the reagent is increased in proportion to the particle diameter when the diameter is from 0.9 to 1.4 μm. On the other hand, when the diameter exceeded 1.4 μm, a decrease in sensitivity was observed. In addition, since the detection sensitivity varies depending on the amount of antibody to be sensitized even with the same particle, the nitrospira having an arbitrary detection sensitivity can be obtained by appropriately adjusting the particle size of the latex particle to sensitize the antibody and the amount of sensitized antibody protein. It was possible to prepare a latex reagent for use. Product No. It was clarified that the detection sensitivity of latex reagent can be increased up to 7.0 × 10 ⁵ cells / mL by using high specific gravity latex particles of H1009. In the past, we have collected activated sludge from wastewater treatment facilities in various parts of Japan, and analyzed the nitrifying bacteria inhabited in the sludge by the fluorescent in-situ hybridization method (hereinafter abbreviated as FISH method). As a result, it has been clarified that the number of bacteria of the genus Nitrospira changes between 10 ⁶ to 10 ⁸ cells / mL. Therefore, the detection sensitivity of the latex reagent for nitrospira obtained this time is considered to be sufficiently practical as a biological nitrogen treatment process such as activated sludge, and a method for detecting and quantifying nitrospira inhabiting in water and soil. It was.

Example 7 Measurement of the number of Nitrospira bacteria in the actual sample In order to confirm the effectiveness of the immunoassay method according to the present invention, the bacteria of the genus Nitrospira in the activated sludge were detected and quantified by the reverse passive latex agglutination method.

As the test sample, activated sludge collected from reaction tanks of a total of 12 facilities including 4 standard activated sludge method facilities, 6 oxidation ditch method facilities, 1 A ₂ O method facility and 1 membrane separation method facility was used. In addition, as a comparative control test, the number of bacteria of the genus Nitrospira was determined by the FISH method according to the method of Amann et al. Using a DNA probe Ntspa0662 specific to the genus Nitrospira (see Non-Patent Document 3). Furthermore, the number of nitrite-oxidizing bacteria was determined by the most probable number method (MPN method) which is a culture method.
Amann R. (1995) In situ identification of micro-organisms by whole cell hybridization with rRNA-targeted nucleic acid probes; Molecular Microbial Ecology Manual 3.3.6: pp 1-15.

As a result of measuring nitrite-oxidizing bacteria in the activated sludge of the actual treatment facility by the RPLA method (FIG. 2), bacteria of the genus Nitrospira were detected from all samples, and the number of bacteria was on the order of 10 ⁶ to 10 ⁸ . In the FISH method, bacteria were detected from 14 samples, and the measured values of the 6 samples almost coincided with the values of the RPLA method. In addition, about the other sample, the tendency for the measured value of FISH method to become about 1 order lower than RPLA method was recognized. Moreover, the influence by the difference in a processing method was not recognized.

On the other hand, the MPN method showed a value 1 to 5 orders lower than the reverse passive latex agglomeration method. Regarding the measurement value by the MPN method, it is known that the measurement efficiency is 2 to 5 orders of magnitude smaller than the antigen-antibody method when the number of nitrifying bacteria in the mixed culture sample is measured by the MPN method (non-patent document). 4). In the experiment conducted this time, similar results were obtained, and it was shown that the reverse passive latex agglutination method was more suitable than the MPN method for measuring nitrifying bacteria.
Nobuo Araki, Akiyoshi Ohashi, Hideki Harada, Izarul Machdar (1999) Counting the number of nitrifying bacteria in biofilm and observing the spatial distribution using FISH method; Journal of Japan Society on Water Environment 22: (2) pp152-159.

  On the other hand, in general, the FISH method is a detection method utilizing the fact that the base sequence information of 16S-ribosomal DNA and RNA differs between bacteria, and is a very useful method as a molecular biological environmental microorganism analysis method. It is considered. However, in this method, since it is essential that a large number of targets (ribosomal RNA) are present in the cell, detection is required in cells that do not sufficiently contain the ribosomal RNA molecule (such as a starved cell). The problem is that it is not possible. On the other hand, microorganisms that inhabit biological nitrogen treatment processes such as activated sludge always inhabit some kind of environmental stress due to the diversity of influent water quality, etc., so sufficient ribosomal RNA is synthesized in the cells. Often not. Therefore, it has been speculated that there are bacteria of the genus Nitrospira that are not sufficiently detected by the FISH method even though they have nitrite oxidation activity this time.

  In addition, since the passive passive latex agglutination method uses an antibody, it may be possible to measure dead cells that have already lost activity but remain antigenic. For these reasons, the measured values of the nitrospyra bacteria by the reverse passive latex agglutination method and the measured values by the FISH method do not match, and the reverse passive latex agglutination method has a higher value than the measured value of the FISH method. it was thought.

  However, regarding the current development goal of identifying the bacteria of the genus Nitrospira in water, soil, etc. during water purification processes such as activated sludge and establishing a method for quickly and appropriately managing the number of bacteria, It was confirmed that the reverse passive latex agglutination method using the antibody-sensitized latex particles for the measurement of bacteria of the genus Nitrospira of the present invention is an excellent method in terms of rapidity and simplicity.

Example 8 FIG. Application to operation management of sewage treatment facilities The relationship between the number of Nitrospira bacteria and the amount of Kjeldahl nitrogen in the effluent was determined for the nitrification reaction tanks of sewage treatment facilities (10 facilities). As for the number of samples, one sample was collected from each facility for spring, summer, autumn and winter, and a total of 40 samples were analyzed. As a result, as shown in FIG. 3, it was revealed that 90% or more of Kjeldahl nitrogen was removed from the reaction tank in which the number of bacteria of the genus Spirobacterium was maintained at 10 ⁷ cells / mL or more. Moreover, in the tank in which the number of bacteria of 10 ⁶ to 10 ⁷ cells / ml is maintained, 80% or more of Kjeldahl nitrogen is removed in about 90% of the tanks.

The nitrification rate is expressed by the following formula.
Nitrification rate (%) = (Kjeldahl nitrogen concentration in influent water-Kjeldahl nitrogen concentration in treated water) / (Kjeldahl nitrogen concentration in influent water) x 100
Therefore, from the above results, if the operating conditions of the nitrification reaction tank are adjusted so that the number of bacteria of the genus Nitrospira is 10 ⁶ cells / mL or more, more preferably 10 ⁷ cells / ml or more, the nitrification rate is 80% or more. It was thought that it could be maintained.

Since the growth rate of nitrifying bacteria is considerably smaller than the growth rate of bacteria that decompose organic matter, oxidative degradation of C-BOD and nitrification are performed in the same reaction tank as in the biological nitrification / denitrification process. When performing, the condition (Formula 1) which can hold | maintain nitrifying bacteria in a system is needed.
SRTN ≧ 1 / μN − (Formula 1)
SRTN: Solid residence time (SRT) required to maintain nitrifying bacteria in the reaction tank system (d)
μN: Specific growth rate of nitrifying bacteria

  In the activated sludge process, the SRT of the nitrifying bacteria and the SRT of the activated sludge process are the same. Therefore, in order to maintain the nitrifying bacteria and promote nitrification in the process, it was necessary to set the SRT satisfying Equation 1. .

  Since the growth rate of nitrifying bacteria is affected by the water temperature, the solid residence time (SRT) required for holding the nitrifying bacteria in the reaction tank becomes longer in the low water temperature period. At present, the SRT value for each water temperature that can cause complete nitrification is empirically quantified from the relationship between the water temperature and solids retention time for the nitrification reaction in a sewage treatment facility designed as a standard activated sludge process. Management of operating conditions based on these values has been carried out. However, since this method is based solely on empirical formulas, the best nitrification rate was not obtained in all reaction tanks by the SRT value derived from this formula.

  However, in the present invention, the nitrifying bacteria in the reaction tank can be measured quickly and easily, and based on the measurement data, the SRT can be adjusted so that an amount of nitrifying bacteria that can maintain a good nitrification rate can be retained. It becomes possible to perform operation management suitable for each individual reaction tank.

  For the measurement of the number of nitrifying bacteria, the most probable number method, the fluorescent in-situ hybridization method, the quantitative PCR method, etc., which are existing nitrifying bacteria measurement methods, can also be used. However, these methods cannot be easily measured in a facility because of a long measurement period, skill, expensive equipment, and reagents.

  Therefore, the operation management method of the activated sludge process using the number of bacteria as an index is realized for the first time by the present invention, and can be said to be a more efficient method than the existing methods.

  As described in detail above, the present invention relates to an antibody-sensitized latex for measurement in which an antibody specific to a bacterium of the genus Nitrospira is adsorbed on latex particles, and by using the antibody-sensitized latex, Therefore, it is possible to easily and conveniently perform detection and measurement of nitrospira bacteria, which require a long period of time and technical skill.

  In addition, the present invention produces a specific antibody against a nitrospira bacterium, identifies a biological nitrogen treatment process such as activated sludge, identifies nitrospira bacteria in water and soil, and quickly and appropriately counts the number of bacteria. It makes it possible to measure.

  In addition, the present invention quickly and easily measures the number of nitrifying bacteria in a reaction tank of a sewage treatment facility, and uses this as an index to control the operating conditions such as the solids residence time of the activated sludge process, thereby enabling the activated sludge process. Operation management becomes easier.

It is a diagram which shows the relationship between the particle size of a latex particle, and a measurement lower limit. It is a diagram which shows the measured value of the bacterium of the genus Nitrospira by RPLA method, FISH method, and MPN method. It is a diagram which shows the relationship between the number of nitrospyra bacteria and a nitrification rate.

Claims (2)

  An antibody specific to a nitrospyra bacterium having nitrite oxidation activity is adsorbed on latex particles having a specific gravity of about 1.5 g / ml and an average particle size of 1.0 to 1.5 μm. Antibody-sensitized latex for measurement of bacteria belonging to it.   The antibody-sensitized latex according to claim 1 is mixed with a test sample and allowed to stand for a predetermined time, and then the presence or absence of an aggregated image indicating that individual latex particles are aggregated is confirmed. An immunological assay characterized by

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