CN101273270A - Immune body sensitized emulsion - Google Patents
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- CN101273270A CN101273270A CNA2005800516743A CN200580051674A CN101273270A CN 101273270 A CN101273270 A CN 101273270A CN A2005800516743 A CNA2005800516743 A CN A2005800516743A CN 200580051674 A CN200580051674 A CN 200580051674A CN 101273270 A CN101273270 A CN 101273270A
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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 [mu]m 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
Technical field
The present invention relates to a kind of immune body sensitized emulsion (antibody-sensitized latex), it is used for detecting the described ammonia of the bacterium that belongs to Nitrosospira and is found at biological wastewater treatment systems, for example at current and activated sludge, and in the water at river, lake and beach and in mud environment (soil habitats).More specifically, the present invention relates to need skilled work and for a long time conventional method different, can be accurately and fast detecting belong to the immune body sensitized emulsion of the bacterium of Nitrosospira.The invention still further relates to by using described immune body sensitized emulsion to measure the method for the bacterium that belongs to Nitrosospira, and described mensuration is applied to method in the control of biological sewage processing procedure.
Background technology
Human habitat comprises waste water and the non-disposal garbage that contains organic substance or nitrogen compound.If they so just are discharged in river, lake and the underground water, can bring serious environmental problem.A kind of method that addresses this problem is to adopt activated sludge to carry out biological purification by nitrification and denitrogenation in its processing procedure.This process has been widely used in many sewage disposal devices.A step in this process is a nitrification, and the nitrifier that wherein moves in the activated sludge plays the part of important role.
Because nitrification has limited the speed of biological sludge treatment process, so must quantitatively improve the stability and the efficient of processing to the microorganism (for example nitrifier, ammonia oxidizing bacteria and NOB) that participates in nitrification.
The bacterium that participates in nitrification mainly is an autotrophy, so its specific growth rate is littler than the heterotroph bacterium of removing organic substance, and they are subjected to for example influence of pH and water temperature of environmental baseline.Therefore, the high density of keeping nitrobacteria in nitrated bio-reactor is extremely important.
At present, according to quantitative test, realized the operation of reactor and kept, but almost not have the information of use about micropopulation to the input and output of sewage disposal device.
Therefore, the mensuration of nitrite-oxidizing bacteria is necessary for the biological wastewater treatment systems in the reactor in activated sludge tank or the sewage disposal device and mud/mud environment (habitat).Conventional indirect determination method comprises sampling back cultivation 1 to 2 months and measures consumption (the Japanese article: people such as Ryusuke Kimura of this section nitrous acid in period, " Methods forExperiments on Soil Microorganisms ", Society for the Researchof Soil Microorganisms, Yokendo Co., Ltd., (1992) pp.207-214).
Attempted using said method in activated sludge or mud, to measure the contrast nitrifier, be used for biological sewage and handle.Unfortunately, said method needs skilled work to measure nitrifier and needs considerable time, therefore is not suitable for carrying out according to measurement result control every day of the instantaneity of nitrifier.
In order to tackle this situation, to develop a kind of method and detected the bacterium that belongs to Nitrosomonas (ammonium oxidation bacterium) and belong to Nitrobacter (nitrite-oxidizing bacteria), this method has been used a kind of antibody that can these bacteriums of specific recognition.Proposed to use this method also to measure ammonia oxidation bacteria and nitrite-oxidizing bacteria (JP-5322896-A) fast simply.Up to the present the another kind of method that detects of the employing short-cut method of Ti Chuing has adopted the sensitised antibodies latex, forms (JP-8029426-A) by latex particle and the described antibody that adsorbs above.
By the way, since nineteen ninety molecular biotechnology be introduced in during water environment analyzes, progressively upgrade about distribution of nitrifier and the knowledge of population.In new knowledge, there is the bacterium that belongs to Nitrosospira to belong to the discovery of (Nitrospira) about NOB.The nitrifying process of the processing water that occupies the majority for this bacterium has carried out a large amount of reports.Now, the bacterium of accurately measuring (counting) Nitromonas (Nitromonas) and nitrifier (Nitrobacter) and belonging to Nitrosospira learns accurately how the efficient of biological treatment process removal nitrogen is an important problem.
Unfortunately, above-mentioned conventional sense method does not produce suitable result, because in the cultivation of being carried out with the mud sample in the biologically pure device, other bacterium beyond the above-mentioned bacterium has accounted for majority.Another problem is, can't obtain any antibody that specific recognition belongs to the bacterium of Nitrosospira, and this is because in fact do not belong to the purification process of the bacterium of Nitrosospira.Therefore, with conventional method the bacterium that belongs to Nitrosospira is detected and mensuration has difficulties.
Summary of the invention
Consider foregoing, the inventor has carried out the method that the bacterium that belongs to Nitrosospira was developed fast and simply measured in a series of researchs, in biological wastewater treatment systems, found this bacterium, for example at current and activated sludge, and in the water at river, lake and beach and in the mud environment.The result, the inventor has found a fact, can be by using immune body sensitized emulsion simply this and method detection fast and mensuration target bacteria, this latex comprise a kind of liquid medium and suspend wherein, above be adsorbed with the latex particle of antibody, described antibody is specific to the bacterium that belongs to Nitrosospira.This discovery has caused the present invention.
Therefore, a target of the present invention provides a kind of new immune body sensitized latex and fast and simply detects the bacterium that belongs to Nitrosospira, described bacterium is found in biological wastewater treatment systems, for example at current and activated sludge, and in the water at river, lake and beach and in the mud environment, find.Another target of the present invention provides a kind of immunoassay and fast and simply measures the bacterium that belongs to Nitrosospira, described bacterium is found in biological wastewater treatment systems, for example at current and activated sludge, and in the water at river, lake and beach, in the mud environment, described immune body sensitized emulsion has been used in described immunoassays.Further object of the present invention provides a method, and according to operation every day of the quantity of nitrifier control aerobic bacteria biological sewage processing procedure, described nitrifier for example belongs to the bacterium of Nitrosospira.
According to the present invention, the immune body sensitized emulsion that is used to detect the bacterium that belongs to Nitrosospira comprises liquid medium and the latex particle that is suspended in wherein.The proportion of each latex particle is about 1.5g/mL, and the average particulate diameter scope is 1.0-1.5 μ m.Each latex particle also carries the antibody that adsorbs on it, and this antibody has specificity to the bacterium that belongs to Nitrosospira with nitrous acid oxidation activity.Immune body sensitized emulsion can with simply a kind of and fast mode detect the bacterium that belongs to Nitrosospira, do not need skilled work and long-time.
According to the present invention, the immune body sensitized emulsion that is used to detect the bacterium that belongs to Nitrosospira is applicable to by immunoassays measures described bacterium.Immunoassays relate to sample and belong to reaction between the bacterium specific antibody of Nitrosospira, therefore can measure and calculate described number of bacteria.The sample of immunoassays is taken from biological wastewater treatment systems, for example current and activated sludge, and the water that comes from river, lake and beach, and mud environment.Sample can be used as it is or dilutes use.Therefore, the present invention also provides a kind of immunoassay, under the help of the specific antibody of the bacterium that belongs to Nitrosospira, detect and working sample in described bacterium.
Immune body sensitized emulsion of the present invention is applied to immunoassay, and described immunoassay includes but not limited to immune agglutination, latex agglutination for example, reverse passive latex (red blood cell) aggegation, and immune chromatograph, immunofluorescence, EIA enzyme immunoassay and radiommunoassay.In these methods, immune agglutination and immune chromatograph meet simply, requirement fast.In the immune agglutination method, reverse passive latex (red blood cell) aggegation is particularly suitable, and this method has been used red blood cell or has been combined with antigen or the indirect agglutination of the latex particle of antibody.
According to the present invention, the soluble carrier that can be used to immune agglutination comprises red blood cell, inorganic compound particle, for example colloid gold particle, and organic polymer particles, for example latex particle.From antigen or immune body sensitized after the position of stability, latex particle is preferred.
According to the present invention, the bacterium that belongs to Nitrosospira is applied to the biological sewage processing procedure.Nitrous acid oxidation bacterium is the microorganism that nitrous acid is converted into nitric acid by oxidation.At present, in DDBJ (Japan DNA Data Bank), registered the base sequence of 16S-rRNA of 43 bacterial strains of the bacterium that belongs to Nitrosospira.The kind system that carries out according to these base sequences studies show that this Pseudomonas comprises 4 groups.
But known have only two bacterial strains to be identified by pure culture.They are the nitrated spirillums in Mo Kewen Nice (Nitrospira moscoviensis) and the nitrated spirillum in ocean (Nitrospira marina) ATCC43039 that separates in the deep-sea that separate in fresh water.Also do not isolate which kind of bacterial strain in them in the activated sludge from sewage-treating reactor or scale biological sewage treatment plant, earth and the mud.In the example of mentioning in the back, the nitrated spirillum in Mo Kewen Nice is used as the bacterium that belongs to Nitrosospira, is used to prepare antibody.Certainly, when in the future can pure culture during other any bacterial strain except that above-mentioned two kinds of bacterial strains, described bacterial strain can be used to prepare antibody.
The bacterium specific antibody that belongs to Nitrosospira obtains from animal, for example rabbit, mouse, rat, chicken, sheep or horse, with described bacterium as these animals of antigen immune.Can also from the antiserum that utilizes above-mentioned known method (JP-5322896-A) preparation, obtain it, utilize the protein g affinity chromatography purifying subsequently.
According to the preferred embodiment of the invention, finish the detection that utilizes the bacterium that belongs to Nitrosospira that above-mentioned immune body sensitized emulsion carries out by following step: sample and immune body sensitized emulsion are mixed, allow the gained potpourri place certain hour, observe the gathering that whether occurs latex particle in the potpourri.
Sample is preferably collected the treat liquid in comfortable aerobic bacteria wastewater treatment container or the pond, if exist the bacterium that belongs to Nitrosospira just can be detected like this in sample.
Be that the present invention preferably utilizes immune agglutination to belong to the detailed description of embodiment of the Bacteria Detection of Nitrosospira below.The latex particle that is used to detect should be hyperbaric, has the proportion that is not less than 1.0g/mL, about preferred 1.5g/mL, especially in the 1.2-1.8g/mL scope.Their average particulate diameter scope is approximately 0.1-4.0 μ m, and preferred 1.0-1.5 μ m is more preferably about 1.3 μ m.In addition, preferred latex particle is coloured.The latex particle that meets these requirements can buy on the market, and for example, " Bacto 0.81 " (trade mark) of DifcoLaboratories, average particulate diameter are 0.81 μ m, and proportion is 1.0g/mL; " IMMUTEX-H series " (trade mark) of JSR company, average particulate diameter is 0.94-4.9 μ m, proportion is 1.5g/mL, comprises coloured; And " Polybeads " (trade mark) #15706 and the #15709 of Polysciences company.The latex particle that the present invention uses is not limited to the above-mentioned commercial prod that exemplifies, and can use suitable arbitrarily article.
The bacterium specific antibody that can adopt certain methods to prepare to belong to Nitrosospira.Wherein a kind of as follows.At first, with nitrite as the breeding substrate the inorganic salt liquid nutrient culture media in culture of bacteria.The bacterium (selectable) that sterilization is cultivated is also collected, as antigen.This antigen is expelled in the vein in the rabbit ears.After definite antibody titer increases, collect whole blood.The blood of centrifugal collection also produces antiserum 56 ℃ of following deactivations.Finally, by in conjunction with protein g affinity chromatography purifying antiserum.Obtain required antibody by this way.
The latex particle specific antibody that absorption obtains like this as carrier.Can be used for this purpose latex particle or body sensitized emulsion by any means preparation, the publicity of for example above-mentioned patent document (JP-8029426-A) institute.
The particle that carries specific antibody that can be used for immune agglutination by the method or the preparation of chemical bond method of physisorption, two kinds of methods all satisfy requirement of the present invention.When using latex particle to carry out aggegation, physisorption can make the latex particle can be by antigen or the fully sensitization of antibody institute.
Preferably, in buffer solution, carry out the activation process of latex particle and antigen or antibody.That is, be cushioned latex particle suspension and antigen or the antibody-solutions mixing of solution dilution to suitable concn.Mix after a period of time, clean latex particle and obtain required body sensitized emulsion particle.The buffer solution that is used to dilute be have appropriate ions intensity and pH value, antigen or antibody moved on to reaction on the particle and antigen or antibody test reaction not have the solution that damages.The example of sort buffer liquid comprises TBS, PBS, GBS, phosphate buffered solution and borate buffer solution.Preferably GBS, phosphate buffered solution and borate buffer solution in them, they are not easy to cause the aggegation of body sensitized emulsion particle or from aggegation.The pH value of buffer solution should be between 5-10, preferred pH 6-9.
The latex particle of being ready for sensitization should have the concentration of 0.01-0.5% (w/v), preferred 0.05-0.25% (w/v).The antibody-solutions of being ready for sensitization should have the concentration that antibody protein is 1.0-1000 μ g/mL.Concentration outside the above-mentioned scope can cause the self aggregation of sensitivity decline and body sensitized emulsion particle, can blur like this positive and negative judgement.In addition, sensitized reaction should carry out under 0-60 ℃, and especially room temperature reaches about 40 ℃.Suitable reaction temperature is essential for high responsive latex particle.
Preferably, adopt reverse passive latex agglutination to use the body sensitized emulsion of gained to belong to detection and the mensuration of the bacterium of Nitrosospira.This method is by mixing sample mutually with the solution (reagent) that contains particle that can immune agglutination, attempt to detect or working sample in target substance, described particle is the high density marker thing (for example latex particle) that is combined with material (for example antibody) that can specificity combining target material on it.Particularly, this method comprises the multiple test sample of preparation, described test sample is being different aspect the dilutability with testing sample, with test sample and the reagent mix that contains immune body sensitized emulsion, obtain a plurality of potpourris, seek the dilution of sample degree of in potpourri, observing latex agglutination subsequently.On the other hand, the reference sample is carried out and above-mentioned identical operations, each has known bacterial cell quantity with reference to sample.Therefore, calculate bacterial cell quantity in the sample according to dilutability and known bacterial cell quantity.
According to the preferred embodiment of the invention, adopt the method that comprises the steps to use immune body sensitized emulsion to measure the bacterium that belongs to Nitrosospira: prepare multiple test sample, described test sample is being different aspect the testing sample dilutability; With every kind of test sample and the reagent mix that contains immune body sensitized emulsion, obtain a plurality of potpourris; Allow potpourri place predetermined a period of time; Whether observation aggegation occurs in each potpourri; According to above-mentioned steps multiple control sample is operated subsequently, every kind of control sample has the known bacterial cell quantity that belongs to Nitrosospira, and the potpourri aggegation result that observes and the aggegation result of control test are compared; According to the observed result of potpourri with corresponding to the comparison between the result of the control test of the aggegation that in potpourri, observes, measure the cell quantity of the bacterium that belongs to Nitrosospira in the test sample then.
Can finish observation by the microtiter plate that use has a hole of many circular concave bottoms.First group of hole adds a kind of in the potpourri that divides loading amount, and every kind all contains immune body sensitized latex and a kind of test sample, and described test sample is cushioned solution dilution respectively to mutually different dilutability.Add a kind of in the potpourri that divides loading amount in second group of hole, every kind all contains immune body sensitized latex and a kind of control sample, and described control sample has known bacterial cell quantity.After at room temperature leaving standstill 4-15 hour, with the naked eye or magnifier (* 10) detect the aggegation whether potpourri in the hole exists latex particle.Observed result has shown bacterial cell quantity.The observation performance that only adds the hole of buffer solution is regarded as negative, and the aggegation in this observation performance and other hole is compared.Quantity according to the bacterial cell that contains in the dilutability of test sample and the known bacterial cell quantity calculation sample in the control sample.
Above-mentioned detection and mensuration process can be replaced by any other method, for example microscope latex method, immunochromatography and immune agglutination.First method has adopted glass sheet to replace microtiter plate, and sample and immune body sensitized particle mix thereon, therefore observes aggegation under optical microscope.
By the way, known latex particle is used as carrier, and antibody adsorbs on it.But, do not report also that up to the present latex particle is used to detect and measure the bacterium that belongs to Nitrosospira.The inventor has confirmed the effect of latex particle first.In other words, the bacterium specific antibody that the inventor finds to belong to Nitrosospira can be used for that selectivity detects and fast measuring can be carried out the peculiar microorganism of nitrite-oxidizing, described microorganism is found in the biological wastewater treatment systems, for example current and activated sludge, and in the water at river, lake and beach, and in the mud environment, and this measurement result can be used to control the biological sewage processing procedure.
According to another embodiment preferred of the present invention, the method of using immune body sensitized latex to detect the bacterium that belongs to Nitrosospira further comprises the step of the cell quantity of measuring the bacterium that belongs to Nitrosospira in the pending liquid that is found in the aerobic bacteria treatment reactor, come the operation of controlling reactor, and according at least one operating conditions in below the control of the cell quantity of bacterial detection: the solid retention time in the reactor, the dissolved oxygen content in the reactor and the inflow of reactor, make the cell quantity of bacterium maintain, to keep nitrated rate predetermined in the processing on the necessary cell quantity of the liquid in the reactor.Preferably, in order to keep nitrated rate predetermined in the processing, the necessary cell quantity that belongs to the bacterium of Nitrosospira of the liquid in the reactor maintains 10
7Cell/mL or this are more than quantity.
Can revise above-mentioned steps, so not only can measure the cell quantity of the bacterium that belongs to Nitrosospira, can also be nitrosation sporangium and nitrifier, they all are found in the pending liquid in the aerobe treatment reactor, and mensuration according to bacterial cell quantity, come the operant response device by at least one operating conditions in the inflow of the solid retention time in the controlling reactor, dissolved oxygen content in the reactor and reactor, make the liquid in the reactor contain essential amount of bacteria, keep nitrated rate predetermined in the processing.
Said process makes the following possibility that becomes: come suitable control is carried out in operation every day of aerobic bacteria treatment facility as index by using main nitrobacteria cell quantity, described main nitrobacteria comprises bacterium and other bacterium that belongs to Nitrosospira.
By the way, term " handle " with aerobic bacteria refer to a kind of by means of multiple aerobic bacteria, the sewage disposal process that decomposes and remove by oxidation of organic compounds matter, ammonium nitrogen, smell and iron.Be classified as two big classes with the aerobic bacteria processing.To be bacterium swim in oxidation and the decomposition of carrying out organic substance in the oxygenation liquid with the form of flocculate to the first kind.Second class is to carry out organic substance oxidation and decomposition by allowing waste water contact with microbial film, and described microbial film is made up of stilt and aerobe, the wherein thereon attached and breeding in the above of aerobe.The former example is an activated-sludge method, and the latter is commonly called biomembrance process.
The activated sludge process of herein mentioning comprises activated-sludge method, oxygen activation mud method, extended aeration process, oxidation ditch process and batch activated-sludge method of standard.It also comprises the nitrated-denitrogenation of circulation, nitrated denitrogenation of inducing, anaerobic-anoxic-aerobic method and anaerobic-aerobic activated-sludge method.Biomembrance process is commonly called the immobilization method now, because it has used the microorganism that is fixed on stilt or carrier or the contact material.Stilt can be dipped in (or fixed bed) reactor or move (thermopnore) in reactor.
When using microbial film to remove nitrogen compound and phosphorus from concentrate waste water, anaerobion is handled and handles in conjunction with aerobic bacteria usually.Certainly, the method according to mensuration bacterium of the present invention can be used to comprise in the biological sewage processing procedure of anaerobion processing.
As mentioned above, the invention provides the novel antibodies body sensitized emulsion that is used to detect, it contains latex particle and adsorbs antibody on it, and described antibody specificity is at the bacterium that belongs to Nitrosospira.This latex is different from skilled operation of needs and routine techniques for a long time, can detect and measure the bacterium that belongs to Nitrosospira in mode simply and fast.According to the present invention, because with the reaction that belongs to the bacterium specific antibody of Nitrosospira, might detect and measure the bacterium that belongs to Nitrosospira, the described bacterium that belongs to Nitrosospira is found in the biological wastewater treatment systems, for example current and activated sludge, and in the water at river, lake and beach and in the mud environment.In addition, according to the present invention, might measure the cell quantity of the bacterium that belongs to Nitrosospira with method simply and fast.In addition, according to the present invention, might be by using the operating conditions of this mensuration bacterial cell quantity as the control indexes treatment pond.The control of this device operation and aerobic bacteria sewage-treatment plant.
Realize the optimal mode of invention
To use more following embodiment to describe characteristics of the present invention and advantage as a reference in more detail.Certainly, technical scope of the present invention is not limited among these embodiment.
Embodiment 1: the preparation bacterium is as antigen
Below containing, cultivate the nitrated spirillum in Mo Kewen Nice in the nutrient culture media of composition shown in the table 1.This bacterial strain comes from Eva doctor Spieck of Hamburg university.(referring to Ehrich S., D.Behrens, E.Lebedeva, W.Ludwig, and E.Bock " A newobligately chemolitoautotrophic, nitrite-oxidizing bacterium,
Nitrospira moscoviensisSp.nov.and its phylogeneticrelationship " Arch.microbiol.164 (1995), pp.16-23) cultivation is divided into three phases (seed culture of 100-mL scale, the final cultivation of cultivation and 4-L scale in the middle of the 1-L scale).Cultivation temperature is 37 ℃, and the culture period that comprises all stages is 7 to 10 days.
When the propagation of bacterium is estimated to reach maximal value, suspend finally and cultivate.4.0 reclaimed bacterium in 10 minutes with the centrifugal nutrient culture media of 24,000 * g under ℃.Bacterium joins in the 30mL buffer solution (PBS) and inhales and break up.In gained solution, add 0.6% formalin of equivalent and stir evenly.Gained solution left standstill under 37 ℃ 1 hour, fixed down at 4.0 ℃ subsequently, up to bacterium death.Centrifugal 10 minutes of 24,000 * g reclaims fixing bacterium, cleans under 24,000 * g centrifugal once more 10 minutes subsequently with PBS.The bacterium of being reclaimed is used as antigen.
Dilute this antigen with PBS, making the absorbance value of gained solution at 660nm wavelength place is 0.5.The antigen that obtains like this is used for detecting.
Table 1
Medium component (1000mL) is used for
The nitrated spirillum in Mo Kewen Nice, bacterial strain DMS 10035
| NaNO 2 0.2g CaCO 3 0.007g NaCl 0.5g MgCl 2·7H 2O 0.05g KH 2PO 4 0.15g | MnSo 4·H 2O 33.8μg H 3BO 3 49.4μg ZnSO 4·7H 2O 43.1μg (NH 4) 6Mo 7O 24·4H 2O 37.1μg CuSO 4·5H 2O 25.0μg FeSO 4·7H 2O 973.0μg |
pH＝8.6
Use belongs to the specificity DNA probing needle NSR1156 of bacterium of Nitrosospira and the purity that Ntspa0662 confirms antigen.In other words, use these probes to hybridize and observe any bacterial strain that except Mo Kewen Nice nitrated spirillum bacterial strain DMS 10035, does not contain other in the bacterial suspension.
Embodiment 2: the preparation polyclonal antibody
Use two kinds of rabbit KBL:Jw (Japanese candida species) preparation polyclonal antibody.Each antigen of injecting 0.5mL dosage in ear vein is spaced apart 7 to 10 days.The increase situation that periodic sampling rabbit immune serum is observed the antibody titer of Mo Kewen Nice nitrated spirillum bacterial strain DMS 10035 by ELISA (the crosslinked immunosorbent assay of enzyme).
Be performed as follows ELISA.Dilute antigen with carbonate-bicarbonate buffer, the absorption value that makes gained solution is A
660nm=0.05.Antigenic solution joins in 96 holes of microtiter plate, every hole 100 μ L.The solution that leaves standstill adding under 4.0 ℃ adsorbed in 16 hours.Clean each hole with the PBS (-) that contains 0.05%Triton-X100.Every hole adds 120 μ L confining liquids (carbonate-bicarbonate buffer that contains 1.0%BSA), reacts 1 hour down at 37 ℃ subsequently.
After the cleaning, every hole adds the serum of 90 μ L with PBS (-) serial dilution that contains 0.05%Triton-X100 and 1.0%BSA, reacts 1.5 hours down at 37 ℃ subsequently.After cleaning once more, two anti-(the anti-rabbit immunoglobulin G (IgG)-sheep IgG) that every hole adds 100 μ L peroxidase marks reacted 1.5 hours down at 37 ℃ subsequently in the dark.
After reaction is finished, in the hole that each has cleaned, add citrate buffer solution and the O-phenylenediamine that 100 μ L contain hydrogen peroxide, under 37 ℃, leave standstill in the dark subsequently and induced the generation color in 10 minutes.The 2.5M sulfuric acid that every hole adds 50 μ L comes stopped reaction, measures the absorbance value (A at 492nm wavelength place subsequently
492nm).By the way, antibody titer is with the absorbance value (A of serum
492nm) be that the inverse of 0.5 o'clock dilution ratio is represented.When antibody titer did not increase, the blood of collecting whole blood and collecting 56 ℃ of following deactivations from heart prepared the antiserum of the bacterium that belongs to Nitrosospira.
Embodiment 3: preparation immunoglobulin G (IgG)
The affinity chromatography (Mab Trap G kit is available from Amersham Pharmacia) that the antiserum that obtains in embodiment 2 carries out Protein G obtains the IgG part.The IgG of the purifying that is obtained in the experiment below is used as the antibody of the family dependents of military personel in the liberated areas in the bacterium of Nitrosospira.
Embodiment 4: the cross reaction experiment
The cross reactivity of every kind of bacterium that the family dependents of military personel in the liberated areas who utilizes ELISA to detect to obtain in embodiment 3 shows in the bacterial antibodies of Nitrosospira and table 2.Cross reactivity represents for the number percent of the relative antibody titer of every kind of bacterium with antibody, and the family dependents of military personel in the liberated areas is 100% in the antibody of the bacterium of Nitrosospira for the antibody titer of Mo Kewen Nice nitrated spirillum bacterial strain DMS 10035.Antibody titer is that the ELISA color reaction provides absorbance value (OD
492) be that the inverse of 0.5 o'clock dilution is represented.By the way, often report in the activated sludge of from water purifying device, collecting it mainly is gram-Negative bacillus (for example pseudomonad, Bacillus alcaligenes and Flavobacterium).
Table 2 is used to detect the bacterial strain of the family dependents of military personel in the liberated areas in the cross reaction of the bacterial antibodies of Nitrosospira
Testing result is shown in following table 3.The family dependents of military personel in the liberated areas that table 3 it should be noted that in embodiment 3 preparation does not almost demonstrate the cross reactivity of those listed bacterial strains of his-and-hers watches 2 in the bacterial antibodies of Nitrosospira.This shows that it is a high degree of specificity to the nitrated spirillum in Mo Kewen Nice.Now, have been found that, the high specific antiserum can be used to belong to the simple detection of the bacterium of Nitrosospira, the described bacterium that belongs to Nitrosospira is found in biological wastewater treatment systems, for example at current and activated sludge, and in the river, in the water at lake and beach, with in the mud environment, because it can cause the aggegation with relevant bacterium, this can be as the direct indicator that detects, can also cause the chromogenic reaction relevant with the response feature of aggegation in addition, this can be as the indirect index that detects, and it can be prepared to immune body sensitized latex and is used for detecting.
Table 3 family dependents of military personel in the liberated areas is in the cross reactivity of the bacterial antibodies of Nitrosospira
Embodiment 5: preparation belongs to the latex reagent of the bacterium of Nitrosospira
Prepared several latex reagents of the bacterium that is used to belong to Nitrosospira, the amount of the albumen of their latex sensitivity is different, and the diameter of latex particle also is different, so they have high detection sensitivity.According to the publicity of above-mentioned patent documentation JP-A-8029426 institute by being prepared as follows immune body sensitized reagent.
With GBS the family dependents of military personel in the liberated areas that embodiment 3 prepares is diluted to 50-500 μ g/mL in the bacterial antibodies of Nitrosospira.The antibody-solutions of dilution and 0.25% (w/v) latex solution equal-volume mixing that contains high-density polystyrene latex particle (proportion is 1.5g/mL, available from JSR company) of diluting with GBS.Potpourri reacted 1.0 hours down at 37 ℃.Add lock solution (the 0.1M GBS that contains 1.0%BSA) subsequently, BSA has just sealed the part that the latex clone is not adsorbed antibody like this.
Seal after 30 minutes, at room temperature, remove unconjugated antibody with the centrifugal latex of 2,700 * g 10 minutes.(contain 1.0%BSA and 0.08%NaN with preserving solution
30.1M GBS, pH 8.2) twice of the isolated latex particle that is adsorbed with antibody on it of eccentric cleaning.Suspend them in the preservation solution identical with mentioned component, and final solution contains 0.05% (w/v) particle like this.The solution of Huo Deing is used as the latex reagent of the bacterium that is used to belong to Nitrosospira like this.Repeat said process and be prepared as follows the different latex particle of the grain size shown in the face table 4.
Table 4 latex particle diameter
Embodiment 6: utilize reverse passive latex agglutination to detect the bacterium that belongs to Nitrosospira
The nitrated spirillum strain DSM 10035 in Mo Kewen Nice with the formalin fixed of preserving 2 times of known cell quantities of serial dilution of solution.The gained dilute solution is divided in the adding 96 hole micropore UV plates (JS103-20 is available from Sanko Junyaku company limited) (every hole 50 μ L).Every hole adds (dilutability difference) hyperbaric immune body sensitized latex reagent of the variable concentrations of equivalent.Slight vibration does not splash and after thoroughly stirring evenly, allows potpourri at room temperature react 4.0 hours.Reaction result has shown the smallest cell number that can be detected.Every kind of latex sample shown in the his-and-hers watches 4 carries out above-mentioned detection and studies relation between the concentration of the diameter of the sensitiveest latex particle of detection and responsive antibody protein.
As shown in Figure 1, testing result shows that the susceptibility and the diameter of reagent are proportional in 0.9 to, 1.4 μ m diameter ranges.On the contrary, susceptibility descends when diameter surpasses 1.4 μ m.In addition, the latex particle that diameter is identical, detection sensitivity relies on the quantity of responsive antibody and changes.Therefore, if to being carried out suitable control, just might prepare the latex reagent of bacterium that is used to belong to Nitrosospira with conceivable detection sensitivity arbitrarily by the quantity of the diameter of immune body sensitized latex particle and responsive antibody protein.
It should be noted that especially among Fig. 1 that the latex reagent with production code member H1009 (high density latex particle) preparation has maximum detection sensitivity 7.0 * 10
5Cell/mL.This shows, the latex reagent that is used to belong to the bacterium of Nitrosospira according to the present invention has the sensitivity of the bacterium that practical high-caliber detection and mensuration belongs to Nitrosospira, the described bacterium that belongs to Nitrosospira is found in biological wastewater treatment systems, for example at current and activated sludge, and in the water at river, lake and beach, with in the mud environment, the cell quantity scope that in fact belongs to the bacterium of Nitrosospira in the many sewage disposal devices in all Japan in the activated sludge of collecting is 10
6To 10
8Cell/mL.This cell quantity is based on inventor's research of utilizing fluorescence in situ hybridization (FISH) method to carry out.
Embodiment 7: the cell quantity that belongs to the bacterium of Nitrosospira in the working sample
Adopt reverse passive latex agglutination (RPLA) method to detect and measured the bacterium of in activated sludge, finding that belongs to Nitrosospira and proved effect according to immunoassays of the present invention.(Fig. 2 a) collects the activated sludge sample to use standard activated sludge process from 4 equipment, 6 equipment use oxidation ditch process (Fig. 2 b), and 1 equipment uses A
2O method (Fig. 2 c), an equipment uses membrane separation process (Fig. 2 d).Be marked as symbol A to L at Fig. 2 a these 12 equipment in the 2d.
Not only adopt the RPLA method also to adopt the FISH method to detect the bacterial cell quantity that belongs to Nitrosospira of the activated sludge sample of from some equipment, collecting, be used for comparison.The people proposed (referring to Amann R. " In situ identification ofmicro-organisms by whole cell hybridization with rRNA-targetednucleic acid probes " as Amann etc., Molecular Microbial Ecology Manual 3.3.6 (1995) pp.1-15), the FISH method has been used dna probe Ntspa 0662, and this probe specificity is at the bacterium that belongs to Nitrosospira.Also adopt the cell quantity that has detected the NOB of sample based on most probable number (MPN) method of cultivating.
The results are shown in Fig. 2 a to 2d.The detection that utilizes the RPLA method to carry out has detected the bacterium that belongs to Nitrosospira in all samples, and detected cell quantity belongs to 10
6To 10
8Cell/mL the order of magnitude.Also adopt in 12 samples and utilize the detection of FISH method to come bacterial detection, detected cell quantity conforms to the bacterial cell quantity that obtains with the RPLA method in 6 samples.In other sample, the testing result that adopts the FISH method is than the low order of magnitude of RPLA method.By the way, this result does not change because of sewage water treatment method.
On the contrary, the testing result of MPN method is than low 1-5 the order of magnitude of RPLA method gained result.During known nitrifier in being applied to the Mixed culture sample, the cell quantity that the MPN method obtains hangs down 2-5 the order of magnitude (referring to Araki N. than the result of Ag-Ab method, Ohashi A., Harada H., and Izarul M., " Cell count of nitrifying bacteriain the biofilm by the FISH method and observation of theirspecial distribution ", Journal of Water and EnvironmentalTechnology, vol.22, No.2, Japan Society of Water Environment (1999) pp.152-159).The experiment of carrying out has obtained and top identical result in this embodiment.Therefore, obviously, more suitable than MPN method for the detection RPLA method of nitrifier.
The FISH detection method depends on 16S rDNA between the different bacterium and the base sequence of RNA is mutually different this fact, so the molecular biological analysis of environmental microorganism is considered to very useful.But this is based on following hypothesis: have many targets (rRNA) in the cell, so it is not suitable for the cell (for example hungry cell) that does not have enough rRNAs.On the other hand, those microorganisms that live in the activated sludge that is used for the biological nitrogen processing procedure do not contain enough rRNAs usually, because they are subjected to the environmental pressure that causes because of flowing water difference.Therefore, infer to have the bacterium that belongs to Nitrosospira in this experiment, but do not detected fully by the FISH method with enough nitrous acid oxidation activities.
Use the RPLA method of antibody to count to lose activity but still have residual antigenic dead cell.This may be the reason why detected cell quantity that belongs to the bacterium of Nitrosospira of RPLA method is sent out high than FISH.
Yet, the RPLA method of verified immune body sensitized emulsion particle used according to the invention is good at aspect quick and simple, as long as want Rapid identification and be determined at the bacterium of finding in the biological wastewater treatment systems that belongs to Nitrosospira, for example at current and activated sludge, and in the water at river, lake and beach and in the mud environment, find.
Embodiment 8: the operational administrative that is applied to sewage disposal device
Relation between the cell quantity of the bacterium that belongs to Nitrosospira that the research that experimentizes is found in nitrification tank and the amount of the kjeldahl nitrogen (Kjeldahl nitrogen) in inflow and the processing water.From 10 equipment, collect sample in each season (spring, summer, autumn, winter), therefore analyze 40 kinds of samples altogether.The results are shown in Fig. 3.It should be noted that among Fig. 3 that the cell quantity on the bacterium that belongs to Nitrosospira surpasses 10
7In the reactor of cell/mL, handle the kjeldahl nitrogen of having removed above 90%.And significantly, the cell quantity about 90% is 10
6To 10
7Kjeldahl nitrogen above 80% in the reactor between cell/mL is removed.
With the nitrated rate of following equation expression.
Wherein,
A: flow into the kjeldahl nitrogen concentration in the water
B: handle the kjeldahl nitrogen concentration in the water
Above-mentioned showing, if by this method operation nitrator, the bacterial cell quantity that belongs to Nitrosospira surpasses 10
6Cell/mL is preferably above 10
7Cell/mL can keep and is higher than 80% nitrated rate.
Because the growth rate of nitrifier is more much lower than the bacterium of decomposing organic substance, if should be met so use identical reactor to resemble biological nitration-C-BOD process oxygenolysis C-BOD and carry out the represented condition of nitrated, following formula (1) (nitrifier can be survived) simultaneously in system.
SRTN≥1/μN …(1)
Wherein,
SRTN: have in the reactor solid of nitrifier hold-up time (my god)
μ N: the specific growth rate of nitrifier
Carrying out in the process of wastewater treatment SRT and nitrifier identical with activated sludge.As a result, for quickening nitrated and keeping the processing procedure of nitrifier, must set up (1) SRT that satisfies formula.
Because the growth rate of nitrifier is influenced by water temperature, so must prolong the necessary solid retention time (SRT) in reactor of nitrifier in the low water temperature stage.Usually, be the sewage disposal device of standard activated-sludge method design, operate that described SRT utilizes experience to obtain according to the relation between SRT and the nitrated temperature according to the SRT that is enough to finish nitrifying process for every kind of water temperature.This method of controlling operation thereof differs based on empirical formula and from the SRT that it draws fully and produce maximum nitrated rate surely all reactor.
On the contrary, immune body sensitized emulsion of the present invention can be fast and the nitrifier in the assaying reaction device simply, and resulting data can allow SRT regulate, and reactor has kept for the suitable necessary amount of bacteria of nitrated rate like this.Therefore, controlling reactor separately.By the way, though can use MPN method, FISH method and quantitative PCR method to measure nitrifier, they are not suitable for the use simply and easily in common purifier apparatus, because that their need is long-time, skilled work and expensive equipment and reagent.
Along with appearance of the present invention, become a reality according to accurate cell quantity control sewage disposal process.Obviously, this new control method is more effective than conventional method.In a word, the invention provides the immune body sensitized emulsion that is used to measure, comprise latex particle and the antibody that adsorbs on it, this antibody is specific to the bacterium that belongs to Nitrosospira.Different with special skilled work of needs and the routine techniques measured for a long time, immune body sensitized emulsion can be fast and is simply detected and measure the bacterium that belongs to Nitrosospira.
By with the reaction of the bacterium specific antibody that belongs to Nitrosospira, can be fast and be determined at the bacterium of finding in the biological wastewater treatment systems that belongs to Nitrosospira simply according to immune body sensitized emulsion of the present invention, for example at current and activated sludge, and in the water at river, lake and beach and in the mud environment, find.
Another benefit of the present invention is, can be used as a kind of index by the cell quantity of said determination, controls sewage disposal device in a simple manner.
The accompanying drawing summary
Fig. 1 is a bar chart, has shown the diameter of latex particle and the relation between the detection lower limit, and the left side ordinate represents to detect lower limit, and (cell/mL), the right side ordinate is represented latex diameter (μ m), uses ◆ mark, abscissa are represented the production number of latex particle.
Fig. 2 a is a bar chart to 2d, each has all shown the cell quantity of the bacterium of measuring with RPLA method, FISH method and MPN method that belongs to Nitrosospira, wherein Fig. 2 a purifier apparatus A of representing the standard activated-sludge method to the abscissa of 2d respectively to the purifier apparatus E of D, oxidation canal (oxidationditch) method to J, A
2The purifier apparatus K of O method and the purifier apparatus L of membrane separation process, from them, collect activated sludge, ordinate is represented the cell quantity (cell quantity (MPN/mL) of cell/mL) and MPN method, and bar a, b and c represent the testing result of RPLA method, FISH method and MPN method respectively of RPLA method and FISH method.
Fig. 3 has shown cell quantity of the bacterium that belongs to Nitrosospira and the relation between the nitrated rate, wherein ordinate is represented cell quantity (cell/mL), rate (%) that horizontal ordinate is represented nitrated (removing kjeldahl nitrogen from handle water), and the curve among the figure has been represented 40 samples that are used to detect.
Claims (7)
1. one kind is used for the immune body sensitized latex that detection belongs to the bacterium of Nitrosospira (Nitrospira), comprise liquid medium and suspension latex particle wherein, wherein the proportion of each described latex particle is about 1.5g/mL, the average particulate diameter scope is 1.0-1.5 μ m, wherein each described latex particle carries absorption antibody thereon, and described antibody is specific to the bacterium that belongs to Nitrosospira with nitrous acid oxidation activity.
2. one kind is detected the method for the bacterium that belongs to Nitrosospira by the defined immune body sensitized emulsion of claim 1, and described method comprises the steps:
Described immune body sensitized emulsion and test sample mixed obtain potpourri;
Allow potpourri leave standstill predetermined a period of time; And
Observe the aggegation that whether occurs latex particle in the potpourri.
3. according to the method for claim 2, wherein test sample is collected processed liquid in the comfortable aerobic bacteria wastewater treatment container.
4. one kind is detected the method for the cell quantity of the bacterium that belongs to Nitrosospira by means of the defined immune body sensitized emulsion of claim 1, and described method comprises the steps:
Prepare multiple test sample, described test sample is being different aspect the dilutability of testing sample;
Every kind of test sample and described immune body sensitized emulsion mixed obtain multiple potpourri;
Allow potpourri leave standstill predetermined a period of time;
Whether observation the aggegation of latex particle occurs in every kind of potpourri;
Relatively have the observed result of potpourri of aggegation and the result of control test, wherein multiple control sample has been carried out operation as above-mentioned step, every kind of control sample is specified the known bacterial cell quantity that belongs to Nitrosospira; And
According to the observed result of potpourri with corresponding to the comparison between the result of the control test of the aggegation that in potpourri, observes, measure the cell quantity of the bacterium that belongs to Nitrosospira in the test sample.
5. according to the method for claim 4, processed liquid in the comfortable aerobic bacteria wastewater treatment of the wherein said sample collection container.
6. according to the method for claim 5, belong to the cell quantity of bacterium of Nitrosospira in detection after, further comprise the steps:
Detection according to bacterial cell quantity, control the operating conditions that at least one is selected from the inflow of solid retention time, the dissolved oxygen content in the reactor and reactor in the reactor, make the cell number value of bacterium maintain on the necessary cell quantity of nitrated rate predetermined in the processing of the liquid-retentive in the reactor.
7. according to the method for claim 6, the cell quantity that wherein belongs to the bacterium of Nitrosospira is maintained at 10
7Cell/mL or more, this is essential for the predetermined nitrated rate of the liquid-retentive in the reactor.
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| CN103460046A (en) * | 2011-03-31 | 2013-12-18 | 积水医疗株式会社 | Latex particle for measurement reagent, sensitized latex particle, and measurement reagent for immunonephelometry |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103460046A (en) * | 2011-03-31 | 2013-12-18 | 积水医疗株式会社 | Latex particle for measurement reagent, sensitized latex particle, and measurement reagent for immunonephelometry |
| CN103460046B (en) * | 2011-03-31 | 2016-08-03 | 积水医疗株式会社 | Measure reagent latex particle, sensitizing latex particle and immunoturbidimetry with measuring reagent |
| US10048268B2 (en) | 2011-03-31 | 2018-08-14 | Sekisui Medical Co., Ltd. | Latex particle for measurement reagent, coated latex particle, and measurement reagent for immunoturbidimetric method |
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