KR20080034970A - Antibody-sensitized latex - Google Patents

Abstract

A antibody-sensitized latex for use in the determination of a bacterium belonging to the genus Nitrospira, the latex comprising a latex particle having a specific gravity of about 1.5 g/ml and an average particle size of 1.0 to 1.5 mum and an antibody adsorbed onto the latex particle which is specific to a bacterium belonging to the genus Nitrospira and has a nitrite-oxidizing activity. The antibody-sensitized latex is mixed with a sample to be tested, allowed to stand for a predetermined period of time, and then confirmed on the presence or absence of the generation of an aggregation image that indicates the presence of aggregation of the latex particles. Thus, the antibody-sensitized latex can be used in the biological nitrogen removing process for active sludge or the like or used for detecting/determining the presence of a bacterium belonging to the genus Nitrospira in water, soil or the like readily in a simple manner.

Description

Antibody sensitizing latex {ANTIBODY-SENSITIZED LATEX}

The present invention relates to an antibody sensitized latex for quickly and easily measuring bacteria belonging to the genus Nitrospira present in biological nitrogen treatment processes such as activated sludge, water, soil and the like. Specifically, it is possible to accurately perform detection of bacteria of the genus Nitrospyra, which requires a long time and requires technical skill in the past, with a simple operation. In addition, a method for measuring nitrospira bacteria using the antibody-sensitized latex and a method for precisely operating and managing biological nitrogen treatment processes such as activated sludge are established.

Wastewater and wastes discharged from our living environment contain many nitrogen compounds, and these are discharged into the surrounding environment such as groundwater, rivers, lakes and swamps, which cause serious environmental problems. As a countermeasure, a biological nitriding and denitrification method using activated sludge has been developed and used in various sewage treatment plants. In the treatment method, nitriding bacteria present in the activated sludge play an important role in the nitriding process.

In the biological denitrification process, since the nitriding reaction is in the rate step, the microbial group (composed of nitrifying bacteria, ammonia oxidizing bacteria and nitrite oxidizing bacteria) affecting the nitriding reaction is required to stabilize the treatment process and to achieve higher efficiency. It is thought that the interpretation of is indispensable.

 In general, bacteria that affect the nitrification reaction are independent nutrient bacteria, and the nitrification reaction tank (reaction tank) is less proliferative compared to heterotrophic bacteria that remove organic matter, and is easily affected by environmental conditions such as pH and water temperature. It is very important to keep nitrided bacteria at high density.

However, although evaluation and maintenance of these treatment tanks are judged by the analysis of the input and output of the system, the determination of the important flora is not carried out at all.

Specifically, during the biological nitrogen treatment process such as an activated sludge treatment tank of a conventional sewage treatment plant or as a quantitative method of nitrite oxidizing bacteria in soil, a sample is generally taken and after a culturing period of one to two months, The method of measuring these indirectly from the presence or absence of the consumption of nitrous acid in the said sample was known (refer nonpatent literature 1).

In addition, there have been attempts to manage biological nitrogen treatment processes such as activated sludge and the trend of nitrifying bacteria in the soil by using such a method. However, the above method requires skill in measuring the number of nitrifying bacteria and a considerable number of days. In the present situation, it is necessary to quickly respond to routinely manage the trend of nitrided bacteria from the measurement results.

Here, the inventors detect detection using an antibody that specifically recognizes these bacteria against bacteria of the genus Nitrosomonas (ammonia oxidizing bacteria) and bacteria of the genus Nitrobacter (nitrite oxidizing bacteria). The method is developed and the method of detecting ammonia oxidizing bacteria or nitrite oxidizing bacteria quickly and easily by the said method is provided (refer patent document 1). Moreover, the method which detects ammonia oxidizing bacteria and nitrite oxidizing bacteria more easily by using the antibody sensitizing latex which made the said antibody adsorb | suck to latex particle has been provided (refer patent document 2).

On the other hand, since 1990, analytical techniques using molecular biological methods have been introduced into the aquatic environment, and knowledge about the distribution and population of nitrided bacteria has gradually been updated. Especially for nitrite oxidizing bacteria, bacteria of the genus Nitrospyra were newly discovered, and the nitrogen treatment process detected as an excellent point species was frequently reported. From this, in order to accurately grasp the removal efficiency of nitrogen in the biological nitrogen treatment process, an important problem is how to accurately measure the number of bacteria in the genus Nitrospyra as well as the bacteria in the genus Nitrosomonas and Nitrobacter. .

However, in the above-mentioned measurement method, cells other than Nitrosphyra bacteria, such as Nitrosomonas bacteria and Nitrobacterium bacteria, predominate in the process of culturing using samples such as activated sludge obtained from a biological nitrogen treatment process. There was a problem that accurate results were not obtained. In addition, since nitrospira bacteria themselves cannot be isolated by the conventional method, there is a problem that an antibody that specifically recognizes nitrospira bacteria cannot be obtained, so that the bacteria cannot be detected and quantified.

[Non-Patent Document 1] Ryusuke Kimura et al., "Methods for Experiments on soil Microorganisms", Society for the Research of Soil Microorganisms, Yokendo Co., Ltd., (1992) pp. 207-214

Patent Document 1: Japanese Patent Application Laid-Open No. 05-322896

Patent Document 2: Japanese Patent Application Laid-Open No. 08-029426

In view of such a situation, the present inventors have made intensive studies for the purpose of developing a method for more easily and quickly measuring the bacteria of the genus Nitrospyra present in biological nitrogen treatment processes such as activated sludge, water, soil and the like. . As a result, the present inventors have found that by producing an antibody specific for a bacterium of the genus Nitrospyra and using an antibody sensitized latex in which the antibody is adsorbed to latex particles, it is possible to quickly measure the desired bacterium of the genus Nitrospyra with a simple operation. The present invention has been completed.

That is, an object of the present invention is to provide a novel antibody sensitized latex for nitrospyras bacteria measurement, which makes it possible to quickly and easily detect bacteria of the genus Nitrosphyra in biological nitrogen treatment processes such as activated sludge, water, soil and the like. It is in doing it. Another object of the present invention is to provide an immunological measurement method for quickly and easily measuring bacteria of the genus Nitrospyra such as biological sludge treatment processes such as activated sludge, water and soil by using the above-described antibody sensitized latex. Moreover, another object is to provide the operation management method of an aerobic biological treatment tank which made the number of nitride bacteria including Nitrosphyra genus the index with respect to daily management of an aerobic biological treatment facility.

The antibody-sensitized latex for measuring nitrospyras bacteria according to the present invention is an antibody-sensitized latex containing a liquid medium and latex particles suspended in the medium, wherein the latex particles have a specific gravity of about 1.5 g / ml and an average particle diameter of 1.0 to 1.5. It is characterized by adsorbing an antibody specific to nitrospira bacteria having a nitrite oxidizing activity. As a result, the detection and measurement of bacteria of the genus Nitrospyra, which requires a long time and requires technical skill, can be performed easily and simply.

In addition, the antibody-sensitized latex for nitrospyras bacterial measurement of the present invention can detect nitrospyras bacteria by immunological measurement. In other words, an immunological measurement method for reacting the antibody against the bacteria of the genus Nitrospyra may be used. As a sample, for example, a biological nitrogen treatment process such as activated sludge, or a sample taken from water or soil can be used as it is or diluted. have. More specifically, the immunoassay method etc. which detect and quantify the bacteria of the genus Nitrospyra which exist in a sample with the antibody specific to these are mentioned.

Examples of immunoassays include immunoaggregation methods such as latex agglutination and reverse-received latex (red blood cell) aggregation, immunochromatography, fluorescent antibodies, enzyme antibodies and radioimmunoassays. And immunochromatography. In particular, a reverse aggregation latex (erythrocyte) aggregation method, which is an indirect aggregation reaction using erythrocytes or latex particles to which antigens or antibodies are bound, is particularly preferable in the immunoaggregation method.

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 colloidal particles and organic polymer particles such as latex particles may be used, but latex particles may be used for the reagents after antigen or antibody sensitization. It is preferable at the point of stability.

The bacterium of the genus Nitrospyra as used in the present invention is a group of microorganisms having a nitrite oxidation activity, that is, a function of producing nitric acid by oxidation of nitrous acid, so-called nitrite oxidizing bacteria. Currently, 43 16S-rRNA nucleotide sequences of nitrospyran bacteria are registered in the DNA Data Bank of Japan (DDBJ), and four strains (species) have been proposed from phylogenetic analysis using the sequence.

However, the strains that succeeded in pure culture were only two weeks of freshwater Nitrospira moscoviensis and Nitrospira marina ATCC43039 isolated from the deep sea, and thus activated sludge, soil, and other sewage treatment reactors. It is not isolated from conventional biological nitrogen treatment processes. In the present invention, although nitrospyra moscobiensis was used as a bacterium of the genus Nitrospyra for producing an antibody, it should be noted that the antibody can be produced in the same manner even if the strain is used when a pure culture of the genus Nitrospyra is established. There is no.

Antibodies specific to these bacteria may be used as antibodies for detecting bacteria using antibodies obtained by immunizing animals such as rabbits, mice, rats, chickens, sheep and horses using the cells as antigens. Moreover, the antiserum manufactured by the well-known method (method of patent document 1, etc.) is further refine | purified by appropriate purification means, such as an affinity chromatography which combined the protein G, and is used.

Therefore, the method of the present invention is a method for detecting nitrospira bacteria using the antibody-sensitized latex, the operation of mixing the antibody-sensitized latex and the test sample, the operation of allowing the obtained mixed sample to stand still for a predetermined time, and the latex in the mixed sample that has been settled. It is characterized by including the operation of confirming the presence or absence of aggregation of particle | grains.

Moreover, as a more preferable aspect, the presence or absence of nitrospira bacteria in the process liquid in an aerobic biological process tank can be detected by using the sample which the said test sample collect | collected from the process liquid in an aerobic biological process tank.

Hereinafter, the case of the immunoaggregation method will be described in more detail. As the latex particles to be used, the specific gravity is 1.0 g or more / ml, preferably about 1.5 mg / ml, particularly 1.2 to 1.8 mg / ml of high specific gravity latex particles are used as suitable ones, and the average particle diameter is about 0.1 to 4.0. Latex particles of about mu m, preferably about 1.0 to 1.5 mu m, more preferably about 1.3 mu m on average are used as suitable ones, and those colored on latex particles are used as suitable ones. As such latex particle | grains, for example, "Bacto 0.81" (the Difco Laboratories product, the average particle diameter of 0.81 micrometers, specific gravity 1.0 g / ml), "IMMUTEX H series" (the JSR Corp. product, specific gravity 1.5g / ml, The average particle diameter can be exemplified by commercial products such as 0.94 µm to 4.9 µm, including colored latex) and "Polybeads" # 15706 and # 15709 (manufactured by Polysciences, Inc.), but are not limited thereto. It is possible to use whatever kind it has.

In order to produce antibodies specific for the bacteria of the genus Nitrospyra, the bacteria are first cultured using an inorganic salt liquid medium containing nitrous acid as a growth substrate, and sterilized and collected as necessary as antigens. Then, this antigen is administered by injection from the rabbit's ear vein, and after confirming the increase of the antibody value, the whole blood is collected, and after appropriate treatment such as centrifugation, inactivation is performed at 56 ° C to give antiserum. Moreover, it refine | purifies by suitable purification means, such as affinity chromatography which combined the protein G, and is used.

Then, the latex particles of the carrier for adsorbing the specific antibody or the method for producing the sensitized latex are not particularly limited, and are produced according to the method reported by the inventors (for example, the method described in Patent Document 2). can do.

The method of preparing these antibodies on the carrier particles to form the immunological aggregated reaction particles includes a physical adsorption method and a chemical bond method, but any of them can be suitably used for the immunological aggregated reaction of the present invention. In the case where latex particles are used as the aggregation reaction particles, the antigen or antibody can be sufficiently sensitized by physical adsorption.

When sensitizing an antibody to the said latex particle, it is preferable to carry out in a buffer. That is, a suspension of latex particles diluted to a suitable concentration and a solution of an antibody or antigen are mixed, left to stand for a while, and then washed to prepare sensitized latex particles. As a buffer used for dilution, generally, the thing which has an ionic strength and pH which does not inhibit the reaction with the antibody or antigen which are a supported antigen or an object to be measured of an antibody is selected. For example, TBS, PBS, GBS, phosphate buffer and boric acid buffer can be used. Among them, phosphate buffer, boric acid buffer and GBS are used as suitable because cohesion and self-aggregation of sensitized latex particles hardly occur. It is common to select in the range of pH 5-10, and especially the range of pH 6-9 is selected as a suitable thing.

As for the density | concentration of the latex particle at the time of sensitization, 0.01-0.5 (w / v)% is suitable, and the favorable result is obtained especially about 0.05-0.25 (w / v)%. In the case where the antibody is sensitized, the antibody protein concentration of the antibody solution is suitably 1.0 to 1000 µg. Otherwise, it is difficult to make a positive or negative judgment due to a decrease in sensitivity or self-aggregation of sensitized latex particles. In addition, about the temperature at the time of a sensitization reaction, although it carries out within the range of 0-60 degreeC, when it reacts at room temperature or temperature slightly higher than room temperature (-40 degreeC), a sensitive latex particle is obtained.

Using these sensitizing latexes, the reverse receiving latex agglomeration method is preferable for detecting bacteria and measuring the number of bacteria of the genus Nitrospyra. The reverse-received latex agglomeration method is a liquid reagent and a test substance containing immunological agglutination particles in which a substance (antibody, etc.) having a property of specifically binding to a test substance is bound to a high specific weight of a substance (latex particles, etc.). It is a method of mixing or containing the sample containing the target substance, and detecting or quantifying a target substance. In more detail, the diluted sample and the reagent containing the antibody sensitized latex are mixed at the appropriate stage to confirm the dilution degree in which the aggregate phase is observed, and on the other hand, the mixed test using the standard sample containing the bacteria solution of known bacteria is carried out. What is necessary is just to calculate a microbe number from a comparison with the result.

Therefore, another aspect of the present invention is a method for measuring the number of bacteria of the genus Nitrosphyra using the antibody-sensitized latex, which is to prepare a plurality of test samples diluted at different concentrations, and the antibody-sensitized latex to each of the test samples. Operation of mixing the mixed samples, pre-determining each of the obtained mixed samples, operation of confirming the presence or absence of agglomeration of latex particles in each of the mixed samples, and measuring and confirming in advance the agglomerations identified for each mixed sample. And a step of identifying the number of bacteria of the genus Nitrosphyra by comparing the measured images of the aggregates identified by the same operation using a standard sample containing the bacterial solution of known bacteria.

More specifically, an antigen or a standard sample diluted stepwise with a buffer solution is added to a U-type microtiter plate, an equal amount of reagent is dispensed into each hole, and then left at room temperature for 4 to 15 hours, followed by visual or 10 Observation and judgment are made using a loupe of about twice. Specifically, the aggregated phase of each hole is determined by making the aggregated phase in the case of only the buffer solution negative, and the number of bacteria is calculated from the dilution ratio of the test hole showing the positive aggregated phase and the result of the aggregation test using the standard antigen.

In the present invention, in addition to this, the latex particles and the test sample according to the present invention are mixed on a slide glass to determine whether agglomerates are formed under an optical microscope (microscopic latex method), or immunological aggregation such as immunochromatography. Needless to say, it can be used for various qualitative and quantitative methods using the reaction particles.

On the other hand, in the past, it has been known that latex particles exist as carriers to which antibodies have been generally adsorbed. However, examples of actually using them for the detection and identification of bacteria of the genus Nitrospyra have not been reported so far, and the effectiveness thereof has been reported by the present inventors. It was the first review. In addition, by using an antibody specific for bacteria of the genus Nitrospyra, it is possible to identify a target microorganism having a biological nitrogen treatment process of an aerobic biological treatment tank such as an activated sludge, an activity in water or soil (oxidation of nitrite), and to quickly determine the number of bacteria. The knowledge that it is possible to manage properly and appropriately is obtained for the first time by the present inventors.

In another embodiment of the present invention, after measuring the number of bacteria of the genus Nitrogen spp. In the treatment liquid in the aerobic biological treatment tank, the solids residence time and tank in the tank so that the number of bacteria of the genus Nitrogen sp. And managing at least one operating condition selected from the amount of dissolved oxygen in the chamber and the inflow load into the tank. On the other hand, the number of bacteria of the genus Nitrosphyra necessary for maintaining the target nitriding rate is preferably 10 ⁷ cells / mL or more.

More preferably, in addition to the nitrospira bacteria number, the nitrosomonas bacteria and nitrobacter bacteria number in the aerobic biological treatment tank are further measured to maintain the nitrisomonas genus necessary for maintaining the target nitriding rate. The method may further include an operation of managing at least one operating condition selected from the solids residence time in the tank, the amount of dissolved oxygen in the tank, and the inflow load into the tank so that the bacterial count, the nitrobacter bacterium, and the nitrospira bacteria count.

This makes it easy to manage the daily operation of the aerobic biological treatment facility as the index of the number of nitrifying bacteria including the genus Nitrospora.

In addition, aerobic biotreatment referred to herein 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, odors, iron and the like. The aerobic treatment is roughly classified into a method of oxidatively decomposing organic materials in a state in which a biofloc is suspended by aeration, and a method of oxidatively decomposing by attaching and growing microorganisms to a carrier to form a biofilm and contacting the wastewater. The former representative is the active sludge process and is widely used for sewage treatment. The latter is generally called the biofilm method.

The active sludge method described herein includes a standard activated sludge method, an oxygen activated sludge method, a long time aeration method, an oxidation ditch method, a batch activated sludge method, and the like, and a cyclic nitriding denitrification method and nitrification endogenous denitrification method. , Anaerobic-aerobic-aerobic and aerobic-aerobic sludge methods are included. In addition, since the biofilm method here is what immobilizes a microorganism to a carrier (also called a support body or a contact material), it is called the biofixing method in many cases in recent years. From the state of the carrier, the carrier is classified 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 (fluid bed).

In addition, a combination of aerobic treatment and anaerobic treatment, such as aerobic treatment of a high concentration wastewater using a biofilm for the purpose of nitrogen compound or phosphorus removal, has been utilized. Needless to say, it is widely applicable to.

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 Nitrosphyra is adsorbed to latex particles. The use of the antibody-sensitized latex requires a long time and requires technical skill. It is possible to easily and conveniently detect and measure bacteria of the genus Nitrosphyra. In addition, another invention can produce antibodies specific for the bacteria of the genus Nitrospyra, and can identify bacteria of the genus Nitrospyra such as biological nitrogen treatment processes such as activated sludge, in water and in soil. Moreover, in another invention, it is possible to measure these nitrospyras genus quickly and appropriately. Moreover, in another invention, the operation management of an aerobic biological treatment plant becomes easy by controlling the operating conditions of an aerobic biological treatment tank using the number of nitriding bacteria, including nitrospira, as an index.

1 is a diagram showing the relationship between the particle size of a latex particle and a measurement lower limit. The vertical axis represents the lower limit value (cells / ml) measured by the bar graph, and the symbol ◆ represents the particle size of the latex (µm), respectively. The abscissa is the product number of the latex particles.

Fig. 2 is a graph showing measured values of bacteria of the genus Nitrospyra by the RPLA method, FISH method and MPN method. In the figure, the abscissa of each drawing shows facilities A to D for performing standard activated sludge method, b for facilities E to K for oxidizing dichroic method, and C for facilities A and C for performing A ₂ O method. The active sludge sample number collected from the reaction tanks of 12 facilities (each figure, A-L of a horizontal axis) of the facilities L which perform the separation method is shown. The vertical axis of each figure shows the number of bacteria of the genus Nitrospyra (cells / ml in the RPLA method and the FISH method, and MPN / 100 ml in the MPN method). In each figure, graph a shows the RPLA method, graph b shows the FISH method, and graph c shows the MPN method.

3 is a graph showing the relationship between the number of bacteria of the genus Nitrosphyra and nitriding rate. The vertical axis represents the number of bacteria (cells / ml), the horizontal axis represents the treated water Kjeldahl nitrogen removal rate (nitration rate) (%), and the dots in the figure represent nitrite oxidizing bacteria.

Hereinafter, although an Example demonstrates this invention still in detail, this invention is not limited at all by the said Example.

Example 1

Preparation of cells used for antigen

Nitrosspira mosbybiensis was incubated in the media of Table 1. This week Dr. Universitat Hamburg Obtained from Eva Spieck (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. Reference). The culture was divided into three stages (100 mL scale seed culture, 1 L scale intermediate culture, 4 L scale final culture), and the culturing temperature of all the stages was 37 ° C and the incubation period was 7 to 10 days.

The final culture was stopped when the growth of the bacteria was considered to be the maximum, and the culture was centrifuged at 24,000 × g for 10 minutes at 4.0 ° C. to recover the bacteria. After 30 mL of PBS was added and the bacteria were dispersed by pipetting, an equivalent amount of 0.6% formalin was added and the mixture was sufficiently stirred. After leaving this solution at 37 ° C for 1 hour, the solution was further fixed at 4.0 ° C until the bacteria died. The fixed cells were recovered by centrifugation at 24,000 × g for 10 minutes, washed with PBS, and then the cells were centrifuged at 24,000 × g for 10 minutes as an antigen. Furthermore, it adjusted to PBS so that the light absorbency of 660 nm might be set to 0.5, and it was set as the test antigen.

The purity of the antigen was confirmed using DNA probes NSR1156 and Ntspa0662 specific to nitrospira. That is, the hybridization test was performed using the said probe, and it was confirmed whether the bacteria other than nitrospyra moscobiences (DSM10035) were mixed in the bacterial suspension.

Example 2

Preparation method of polyclonal antibody

KBL: Two Jw rabbits were used. The dose of the antigen was 0.5 mL per dose and injected from the vein every 7 to 10 days. Serum was collected experimentally from rabbits under immunization, and the increase in antibody value against nitrospira moscobiensis DSM10035 strain was examined by enzyme linked immunosorbent assay (ELISA).

Specifically, an antigen adjusted to absorbance (A _{660 nm} ) = 0.05 in a carbonic acid / bicarbonate buffer was dispensed in 100 μl per well into a 96-hole plate and adsorbed at 4.0 ° C. for 16 hours. After washing each well with a washing solution (PBS (-) containing 0.05% Triton-X100), 120 μl / well of a blocking solution (carbonated / bicarbonate buffer containing 1.0% BSA) was added, followed by 1 hour at 37 ° C. Reacted.

After washing the wells, 90 μl of serum diluted with PBS (−) containing 0.05% Triton-X100 and 1.0% BSA was added per well, and reacted at 37 ° C. for 1.5 hours. After the wells were washed, 100 µl / well of peroxidase-labeled secondary antibody (anti-rabbit immunoglobulin G (IgG) -amount IgG) was added, shaded, and further reacted at 37 ° C for 1.5 hours.

After the completion of the reaction, 100 µl / well of a citrate buffer of O-phenylenediamine containing hydrogen peroxide was added to the washed wells, shaded, and left to stand at 37 ° C for 10 minutes for color development. 50 µl / well of 2.5 M sulfuric acid was added to stop the reaction, and then the absorbance (A _{660 nm} ) of 492 _nm was measured. In addition, the antibody value was shown by the inverse of the dilution magnification of the serum whose color intensity of A _{492 nm} shows 0.5. When no increase in the antibody value was confirmed, all blood was collected from the heart. Inactivation was performed at 56 ° C to prepare antiserum corresponding to bacteria of the genus Nitrospyra.

Example 3

Purification of Immunoglobulin G (Immunoglobulin G: IgG)

IgG was fractionated from antiserum by affinity chromatography (Mab Trap G Kit, Amersham Pharmacia) bound to protein G. This purified IgG was used as an antinitrospira antibody for the following experiments.

Example 4

Cross reactivity test

The cross reactivity of the antinitrospira antibody and each bacterium shown in Table 2 was examined by ELISA method. The cross-reactivity of the anti-nitrospira antibody was 100% as the antibody value (inverse of the dilution ratio indicating the absorbance (OD ₄₉₂ = 0.5) by color reaction using ELISA method) against nitrospira moscobiensis (DSM10035 strain). At this time, the cross-reactivity of the antibody and each cell was expressed in%. In general, there have been many reports of gram-negative bacillus such as Pseudomonas, Alcaligenes, Flavobacterium, etc., from the water purification process such as activated sludge.

However, as shown in Table 3, the anti-nitrospira antibody produced here was found to have no specific cross-reactivity to these bacteria and had high specificity. The antiserum having high specificity is based on the reaction characteristics such as the aggregation reaction for each bacterium of the antibody as a direct index or the color reactions correlated with the reaction characteristics as an index. It has been found to be useful for the simple detection of bacteria of the genus Nitrospyra in biological nitrogen treatment processes such as activated sludge, in water and in soil.

Example 5

Preparation of Latex Reagent for Nitrospira

In order to increase the detection sensitivity of the latex reagent for nitrospira, the amount of protein sensitized to the latex particles and the particle size of the latex particles were optimized. Preparation of the antibody sensitizing latex reagent was performed according to the method of well-known patent document 2.

Specifically, the anti-nitrospira antibody was diluted with GBS to 50 to 500 µg / mL. One volume of the polystyrene high specific gravity latex solution (JSR Corp., specific gravity 1.5 g / ml) diluted to 0.25% w / v with GBS was mixed with one volume of the antibody diluent, and reacted at 37 ° C for 1.0 hour. After completion of the reaction, a blocking solution (0.1 M GBS containing 1.0% BSA) was added to block the nonadsorbed portion of the antibody on the latex particles with BSA.

After blocking for 30 minutes, unbound antibody was removed by centrifugation at room temperature for 2,700 × g for 10 minutes. The antibody-bound latex particles were repeatedly centrifuged twice with a preservative solution (0.1 M GBS pH 8.2 containing 1.0% BSA, 0.08% NaN ₃ ), and finally, the particle concentration was 0.05% (w / v). It was suspended in the stock solution and used as the latex reagent for nitrospira. This operation was performed with respect to latex particle | grains of each particle diameter shown in Table 4.

Example 6

Detection of Nitrospyra by Reverse Receive Latex Agglomeration

Formalin-fixed nitrospira moscobiensis (DSM11035 strain) of known bacterial counts was serially diluted 2 using a preservative solution. 50 µl of each dilution was dispensed into a 96-well UV plate (SJ103-20 Sanko Junyaku Co., Ltd.), and then an equal amount of a high specific gravity latex reagent sensitized with various concentrations of antibodies was added. The mixture was stirred sufficiently to the extent that the mixed liquid did not scatter, and then reacted at room temperature for 4.0 hours to obtain the minimum detectable bacterium. This test was carried out for each particle diameter shown in Table 4, and the particle size with the highest detection sensitivity and the antibody protein sensitization concentration were examined.

As a result, as shown in Fig. 1, it became clear that the measurement sensitivity of the reagent was high in proportion to the particle diameter between the diameters of 0.9 to 1.4 mu m. On the contrary, when the diameter exceeded 1.4 micrometers, the fall of the sensitivity was confirmed. In addition, since the detection sensitivity is changed depending on the amount of antibody sensitizing even the same particles, it is preferable to prepare a latex reagent for nitrospira having an arbitrary detection sensitivity by appropriately adjusting the particle size and the amount of the sensitizing antibody protein of the latex particle sensitizing the antibody. It was possible.

In addition, product No. By using H1009 high specific gravity latex particles, it has been found that the detection sensitivity of latex reagents can be increased up to 7.0 x 10 ⁵ cells / mL. In the past, we have collected activated sludge from wastewater treatment facilities in various parts of Japan, and analyzed the nitrifying bacteria group inhabiting the sludge by fluorescence in-situ hybridization method (hereinafter abbreviated as FISH method). As a result, it was found that the number of bacteria of the genus Nitrosphyra was changing between 10 ^{6 and} 10 ⁸ cells / mL. Therefore, the detection sensitivity of the latex reagent for nitrospir obtained at this time is considered to be a level practically useful as a method for detecting and quantitating nitrospirs which are reproduced in biological nitrogen treatment processes such as activated sludge and in water and soil.

Example 7

Measurement of the number of bacteria of the genus Nitrosphyra

In order to confirm the effectiveness of the immunoassay according to the present invention, bacteria of the genus Nitrospyra in active sludge were detected and quantified by reverse receiving latex agglutination (RPLA method). The test sample was the sum of four standard activated sludge methods (a degree), six oxidization dichroic methods (b), one A ₂ O method (c) and one membrane separation (d) Activated sludge collected from reaction tanks of 12 facilities (angles A to L on each horizontal axis) was used.

For the activated sludge from each facility, in addition to the quantification of bacteria by reverse-received latex agglutination, according to the method of Amann et al. Using the DNA probe Ntspa0662 specific for bacteria of the genus Nitrospyra as a comparative control test (Amann R. (1995) In situ identification of micro-organisms by whole cell hybridization with rRNA-targeted nucleic acid probes; see Molecular Microbial Ecology Manual 3.3.6: pp. 1-15. In addition, the number of nitrite oxidizing bacteria was determined by the maximization method (MPN method), which is a culture method.

As a result of measuring the nitrite oxidizing bacteria in the active sludge by the RPLA method (FIG. 2), the bacteria of the genus Nitrospyra were detected in all the samples, and the number of bacteria was 10 ⁶ to 10 ⁸ orders. In the FISH method, bacteria were detected from 14 samples, and the measured values were almost identical to those of the RPLA method in 6 samples. Moreover, about the other sample, the tendency for the measured value of the FISH method to become about 1 order lower than the RPLA method was confirmed. Moreover, the influence by the difference of the processing method was also confirmed.

On the other hand, the MPN method showed a value 1-5 orders or more lower than the reverse receiving latex agglomeration method. Regarding the measured value by the MPN method, when the number of nitrided bacteria in the mixed culture sample is measured by the MPN method, it is known that the measurement efficiency is 2 to 5 orders smaller than the antigen antibody method (Araki N., Ohashi A., Harada H., and Izarul M., "Cell count of nitrifying bacteria in the biofilm by the FISH method and observation of their special distribution", Journal of Water and Environmental Technology, vol. 22, No. 2, Japan Society of Water Enviornment (1999) pp. 152-159). The same results were obtained in the experiments conducted this time, and it was found that the reverse receiving latex aggregation method is more suitable than the MPN method as a method for measuring nitrided bacteria.

On the other hand, in general, the FISH method is a detection method using 16S-ribosomal DNA and RNA sequencing information between bacteria, and is considered to be a very useful method as an analysis method for environmental microorganisms in molecular biology. However, in this method, since it is essential that a large number of targets (ribosomal RNA) exist in the cell, it is not possible to detect it in cells (cells in starvation state, etc.) that do not sufficiently contain the ribosomal RNA molecules. It is mentioned as. On the other hand, microorganisms inhabiting biological nitrogen treatment processes, such as activated sludge, inhabits under some environmental stress due to the diversity of inflow water quality, etc., and therefore, sufficient ribosome RNA is not synthesized in the cells. Therefore, it is estimated that there exists a bacterium of the genus Nitrospyra which is not detected enough by the FISH method even if it also has a nitrite oxidation activity.

In addition, since the antibody was used in the reverse-receiving latex aggregation method, it was thought that there was a possibility of measuring the dead microorganisms, although the activity was already lost. For this reason, it was thought that the measured value of nitrospira bacteria by the reverse receiving latex flocculation method and the measured value by the FISH method did not coincide, and the reverse receiving latex flocculation method was higher than the measured value by the FISH method.

However, this development aims at identifying the bacteria of the genus Nitrosphyra during the water purification process such as activated sludge, underwater, and soil, and establishing a method for quickly and appropriately managing the bacteria. It was confirmed that the reverse-received latex aggregation method using the antibody-sensitized latex particles for bacteria measurement of the genus Nitrosphyra of the present invention was excellent in terms of speed and simplicity.

Example 8

Application to operation management of sewage treatment facilities

The relationship between the nitrospyras bacterial water and the amount of Kjeldahl nitrogen in the treated water was determined for the nitriding reaction tanks of the sewage treatment facility (ten facilities). The number of samples was taken from each facility at spring, summer, autumn and winter and a total of 40 samples were analyzed. As a result, as shown in Fig. 3, it became clear that 90% or more of Kjeldahl nitrogen was removed from the reaction tank in which the number of bacteria of the genus Nitrospyra was maintained at 10 ⁷ cells / mL or more. In addition, 80% or more of Kjeldahl nitrogen was removed from a tank of about 90% in a tank maintained at 10 ^{6-10 7} cells / ml.

The nitriding rate is represented by the following formula.

Nitriding rate (%) = (Kjeldahl nitrogen concentration of influent-Kjeldahl nitrogen concentration of treated water) / (Kjeldahl nitrogen concentration of influent) x 100

Therefore, it is thought that the nitriding rate of the nitrification reaction tank can be maintained at 80% or more by adjusting the operating conditions of the nitrification reaction tank so that the number of bacteria of the genus Nitrospyra is 10 ⁶ cells / mL or more, more preferably 10 ⁷ cells / mL or more. do.

Since the growth rate of nitrifying bacteria is considerably smaller than the rate of growth of bacteria decomposing organic matter, nitrifying bacteria are counted when oxidative decomposition and nitrification of C-BOD is performed in the same reaction tank as in biological nitrification and denitrification processes. The condition (expression 1) which can be maintained inside is required.

SRTN ≥ 1 / μN-(Equation 1)

SRTN: Solids retention time (SRT) (d) required to keep nitrifying bacteria in the reaction tank system

μN: non-proliferative 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 nitriding in the process, it is necessary to set an SRT that satisfies Equation 1.

Since the growth rate of the nitrided bacteria is affected by the water temperature, the solids residence time (SRT) required to keep the nitrided bacteria in the reaction tank becomes longer in the low temperature period. At present, from the relationship between the water temperature and the solids residence time for the nitrification reaction in a sewage treatment facility designed as a standard activated sludge method, the value of SRT for each water temperature that can cause nitriding can be empirically quantified. Management of the driving conditions is performed. However, since this method is based on empirical formulas only, it is not possible to obtain the best nitrification rate in all reaction tanks by the value of SRT derived from this equation.

In the present invention, however, the nitrifying bacteria in the reaction tank can be measured quickly and simply, and the SRT can be adjusted to maintain the nitrifying bacteria in an amount capable of maintaining a good nitriding rate based on the measurement data. It is possible to perform operation management suitable for the operation.

In addition, the measurement of the number of nitrided bacteria can be used, such as the most conventional method of measuring the nitrided bacteria, fluorescence in-situ hybridization, quantitative PCR. However, these methods could not be easily measured in a facility because the measuring period was long, or required by skilled or expensive equipment and reagents.

However, the operation management method of the active sludge process which uses the number of bacteria as an index is realized for the first time by this invention, and it can be said that it is a more efficient method compared with the existing method.

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 Nitrosphyra is adsorbed to latex particles. It is possible to easily and conveniently detect and measure bacteria of the genus Nitrosphyra.

In addition, the present invention is to prepare a specific antibody to the bacteria of the genus Nitrospyra, to identify the bacteria of the genus Nitrospyra in the biological nitrogen treatment process such as activated sludge, water and soil, and to measure the number of bacteria quickly and appropriately To make it possible.

In addition, the present invention facilitates the operation and management of the active sludge process by quickly and simply measuring the number of nitrided bacteria in the reaction tank of the sewage treatment plant and controlling the operating conditions such as the solid residence time of the activated sludge process as an index. Done.

Claims (7)

In an antibody sensitized latex containing a liquid medium and latex particles suspended in the medium: The latex particles have a specific gravity of about 1.5 g / ml and an average particle diameter of 1.0 to 1.5 µm, and the antibody sensitization for measuring bacteria belonging to the genus Nitrospyra, characterized by adsorbing a specific antibody to the genus Nitrospyra bacteria having nitrite oxidation activity. Latex. As a method for detecting nitrospira bacteria using the antibody sensitizing latex according to claim 1: Mixing the antibody sensitized latex and a test sample; An operation of allowing the obtained mixed sample to stand still for a predetermined time; And A detection method of the genus Nitrospyra, comprising the step of confirming the presence or absence of aggregation of latex particles in the stationary mixed sample. The detection method according to claim 2, wherein the test sample is a sample collected from a treatment liquid in an aerobic biological treatment tank. As a method for measuring the number of bacteria of the genus Nitrospyra using the antibody sensitizing latex according to claim 1: Preparing a plurality of test samples diluted to different concentrations; Mixing the antibody sensitizing latex to each of the test samples; An operation of allowing each obtained mixed sample to stand still for a predetermined time; An operation of confirming the presence or absence of aggregation of latex particles in each of the mixed samples that have been left to stand; And An operation for identifying the number of bacteria of the genus Nitrospyra by comparing the measured phase of the aggregates identified for each mixed sample with the measured phase of the aggregates identified by the same operation using a standard sample containing a known bacterial count of the known bacterial count. Nitrospyras bacteria measurement method comprising a. The method according to claim 4, wherein the test sample is a sample collected from a treatment liquid in an aerobic biological treatment tank. 6. The solids residence time in the tank and dissolved in the tank according to claim 5, wherein the number of nitrospira bacteria in the treatment liquid in the aerobic biological treatment tank is measured, and then the number of solids bacteria in the tank is maintained so that the number of nitrospira bacteria required to maintain the target nitriding rate. A method for measuring Nitrogenspira bacteria, further comprising the step of managing one or more operating conditions selected from the amount of oxygen and the inflow load into the tank. The method of measuring nitrospyra genus bacteria according to claim 6, wherein the number of nitros pyra genus bacteria necessary for maintaining the target nitriding rate is 10 ⁷ cells / mL or more.

Images