JP2007232501A - Adjustment method and detection method of detection object in environmental sample - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an adjustment method and a detection method of a detection object in an environmental sample capable of adjusting efficiently the detection object by automating a work and by removing impurities in the environmental sample. <P>SOLUTION: This adjustment method includes a step for recognizing the detection object 2, and bringing the environmental sample into contact with a carrier 4 to which a recognition material 3 bonded specifically to the detection object 2 is bonded; a step for separating the detection object 2 in the environmental sample from impurities 5; a step for eliminating and recovering the detection object 2 bonded to the carrier 4; a step for adjusting so that the recovered detection object 2 and a labeling material for labeling the detection object 2 are suitable to be bonded together; and a step for bonding the detection object 2 to the labeling material. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for adjusting a detection target in an environmental sample and a detection method. In particular, an adjustment method for automating operations, further purifying a detection target in a solution efficiently, and appropriately adjusting a binding force between the detection target in the solution and a label for labeling the detection target. And a detection apparatus.

  It is thought that environmental samples such as rivers and lakes that are raw water supply are contaminated with various chemical substances because various chemical substances exist in the environment. The environmental sample is also contaminated by protozoa such as Cryptosporidium, bacteria such as enterohemorrhagic Escherichia coli O157 and Legionella, and microorganisms such as viruses. Pollution and waterborne infections due to these contaminated environmental samples are a major problem.

Here, detection of harmful chemical substances and microorganisms contained in environmental samples is performed. Generally, a gas chromatograph-mass spectrometer (GC / MS) or a liquid chromatograph-mass spectrometer (LC / MS), which is a highly sensitive analyzer, is used. Used to detect chemical substances and microorganisms.
For example, in a gas chromatograph-mass spectrometer, first, to prepare a volatile derivative, trimethylsilylation or propylation is performed on an environmental sample, the mixture is separated by a gas chromatograph, and separated by mass by a mass spectrometer. Makes highly sensitive detection.

  Moreover, since the concentration of chemical substances and microorganisms contained in the environmental sample is very low (ng / L to μg / L (ppb to ppt)), when performing detection using the above apparatus, several environmental samples are used. It needs to be concentrated 1000 times. Furthermore, in order to detect with high reproducibility, it is necessary to remove impurities and interfering substances that affect the analysis result from the sample. Therefore, for example, solvent extraction or solid phase extraction is performed using an organic solvent such as hexane, dichloromethane, or acetone. Further, when purification or concentration of the sample is necessary, chromatography is continuously performed using a silica gel column or the like.

In particular, for protozoa such as Cryptosporidium and Giardia, which are the cause of emerging water-borne infections, testing methods (for example, “Cryptosporidium Provisional Countermeasure Guidelines”, Ministry of Health, Labor and Welfare, 1998) have been published.
This inspection method is roughly divided into a sample collection step, a filtration concentration step, an exfoliation suspension step, a separation and purification step, an immunofluorescence staining step, and a microscope observation step.
In the sample collecting step, 10 L is collected as a sample in a polyethylene container for river water or raw water for water, and 40 L is used for purified water or tap water.
In the filtration concentration step, the sample is filtered using a filtration device in which a predetermined disk filter is attached to a pressure or suction type filter holder. The disk filter is made of hydrophilic polytetrafluoroethylene (PTFE) having a diameter of 47 mm or 90 mm and a pore size of 5 μm.
In the exfoliation suspension step, the disc filter capturing the turbidity is put in a 50 mL centrifuge tube, and the turbidity containing Cryptosporidium is exfoliated from the disc filter by adding the elicitor and stirring vigorously. The elicitor contains 0.02% sodium pyrophosphate, 0.03% EDTA-3Na, 0.01% Tween80.
In the separation and purification step, Cryptosporidium is recovered from the suspension by immunomagnetic beads. An antibody that recognizes Cryptosporidium is bound to the immunomagnetic beads.
In the immunofluorescent staining step, Cryptosporidium is stained with a fluorescently labeled anti-Cryptosporidium antibody and observed using a fluorescence microscope.
Here, since the observation work with a fluorescence microscope requires a high degree of skill and the physical burden of having to observe for a long time, a flow cytometer is used for automatic measurement of fluorescently labeled cryptosporidium. It has been proposed. In the flow cytometer, first, fine particles are allowed to flow one by one together with the sheath liquid. Next, laser light or ultraviolet light is condensed on the fine particles, and optical information such as scattered light and fluorescence is detected. Thereby, the size, form, and properties of the particles can be examined. Furthermore, in the flow cytometer, the forward scattered light intensity corresponding to the particle size of the particles, the fluorescence intensity of the fluorescent substance that binds to the labeled antibody, and the fluorescence intensity of the algae that are the main contaminants contained in the environmental sample are measured. It can be measured simultaneously. Therefore, these optical information can be processed and fractionated into a region where Cryptosporidium is present and a region where impurities are present, and labeled Cryptosporidium can be detected.

  The presence of Cryptosporidium can be confirmed by the Cryptosporidium test method described above. However, it cannot be determined whether Cryptosporidium is alive or has an ability to infect humans. Therefore, in order to obtain a more detailed test result, a growth activity test and an infectivity test may be performed in addition to the above-described cryptosporidium test. In the growth activity test of Cryptosporidium, a decapsulation test for judging the life and death based on the presence or absence of sporozoites (life forms) from oocysts (sac) of Cryptosporidium, DAPI (4,6-diamino-2-phenylindole) and PI There is a vital staining test for determining the viability from double staining of (propidium indole). On the other hand, Cryptosporidium infectivity tests include methods for evaluating infectivity by infection of laboratory animals such as mice or infection of cultured cells derived from human small intestine.

In addition, an enzyme immunoassay (ELISA method) has been developed as a highly sensitive analyzer. The ELISA method is a method for detecting a detection target with high sensitivity by an antigen-antibody reaction using an antibody that specifically binds to the detection target in a sample.
In the ELISA method, first, a detection target and an enzyme-labeled antigen are caused to react competitively with an immobilized antibody that recognizes and specifically binds to the detection target. Next, an enzyme substrate is added to the enzyme-labeled antigen captured by the immobilized antibody, and the enzyme-labeled antigen is colored. Here, the amount of the enzyme-labeled antigen that binds to the antigen decreases as the concentration of the detection target increases. Therefore, the concentration of the detection target in the sample can be calculated by comparing the absorbance of the enzyme-labeled antigen with the absorbance of the standard substance measured in advance. In addition, an immobilized antibody that recognizes and specifically binds to the detection target, an enzyme-labeled antibody that recognizes the complex of the detection target, or a detection target that is recognized by the immobilized antibody. There is also an ELISA method using an enzyme-labeled antibody that recognizes a site different from the site.

Even in the ELISA method used for simple measurement and screening of harmful chemical substances contained in environmental samples, contaminants contained in the environment are non-specific to immobilized antibodies or enzyme-labeled secondary antibodies. Adsorption may adversely affect the detection result. In addition, in order to measure harmful chemical substances at a lower concentration, pretreatment for removing and concentrating contaminants and interfering substances is necessary as in the case of instrumental analysis. For this reason, it is possible to easily measure harmful chemical substances, but there is a problem that a series of analytical operations require a high degree of skill and time, making it difficult to automate and save labor.
In addition, even in the detection of pathogenic microorganisms contained in environmental samples, algae that are abundant in environmental samples bind to labeled antibodies non-specifically to detect particles as false positives, or algae-derived fluorescence interferes with detection. Therefore, it is desired to detect with higher accuracy.

In order to prevent outbreaks due to water-borne infections, it is also necessary to monitor the causative microorganisms frequently in the water treatment process. However, since a technique for efficiently separating and concentrating microorganisms from a sample has not been established, separating and concentrating pathogenic microorganisms that are only slightly contained in environmental samples requires a high degree of skill and time.
Japanese Patent Laying-Open No. 2005-077301 JP 2005-195473 A

  In view of the above-described problems, the present invention automates operations, removes impurities in the environmental sample, efficiently adjusts the detection target, and detects the detection target with high accuracy. An object of the present invention is to provide a method for adjusting and detecting an object.

In order to achieve the above object, the present invention provides a method for adjusting a detection target in an environmental sample, which recognizes the detection target and binds a recognition target that specifically binds to the detection target. A step of contacting an environmental sample, a step of separating the detection object and impurities in the environmental sample, a step of desorbing and recovering the detection object bound to the carrier, and a recovered detection object An object and a label for labeling the detection target are adjusted so as to be suitable for binding to each other, and a step for binding the detection target and the label.
Examples of the detection target include cryptosporidium. As the recognition object, an anti-cryptospodium antibody is preferably used. In addition, a fluorescently labeled antibody is preferably used as the label. As the carrier, polystyrene having an amino group introduced is preferably used.

  Another aspect of the present invention is a method for detecting an object to be detected in an environmental sample. In the detection method, the environmental sample is adjusted by the adjustment method according to the present invention, and then detected from the fluorescence intensity of the fluorescently labeled antibody. A step of detecting an object is performed.

  According to the adjustment method and the detection method of the detection object in the environmental sample according to the present invention, the operation can be automated, and the detection object can be efficiently purified by removing impurities in the environmental sample. In addition, since the binding force between the detection object and the label that labels the detection object can be adjusted appropriately, the detection object can be detected with high accuracy.

  Embodiments of a method for adjusting a detection target in an environmental sample and a detection method according to the present invention will be described below with reference to the accompanying drawings.

First, an embodiment of a method for adjusting an object to be detected in an environmental sample according to the present invention will be described generally with reference to FIG.
First, the separation column 1 is packed with a carrier 4 having a recognition object 3 that specifically binds to the detection object 2 [FIG. 1 (a)].
In preparing the environmental sample, the contaminant 5 may clog the separation column 1. Therefore, it is preferable to use the carrier 4 having a particle size of about 100 μm to 10 mm in consideration of the particle size of the impurities being several tens to several hundreds μm. As such a carrier 4, a ball made of resin is suitable. As the resin, a polystyrene having an amino group introduced can be mentioned as a suitable one.

  Examples of the recognition product 3 include immune antibodies, single chain antibodies prepared using gene recombination, peptide antibodies, receptors, template molecules prepared using molecular imprinting, and the like.

Next, an environmental sample including the detection object 2 and the contaminants 5 is injected into the separation column 1 [FIG. 1 (b)].
The separation column 1 is in a constant temperature layer, and the detection target 2 and the recognition target 3 are bonded at 37 ° C., for example, in a combination in which the detection target 2 is Cryptosporidium and the recognition target 3 is an antibody. During this time, the separation column 1 is shaken with a shaker. Here, the shaking means is not limited to the shaker, and a stirrer may be used.

  Next, the washing solution is passed through the separation column 1 to separate the detection target 2 bonded to the carrier 4 in the separation column 1 from the environmental sample [FIG. 1 (c)].

Further, a hydrochloric acid solution or the like is passed through the separation column 1, and the detection target 2 is separated (desorbed) from the carrier 4 to extract the detection target 2 into the reaction vessel 6 [FIG. 1 (d)].
For separation / extraction, in addition to using hydrochloric acid solution, it is also possible to separate the object to be detected and the recognition object using ionic strength change due to high concentration solution such as sodium chloride or polarity change due to ethylene glycol etc. A suitable object is selected according to the attributes of the detection object, the carrier used, and the recognition object.
The detection target is separated by the series of operations described above. That is, the detection target in the environmental sample is purified.

In the method for adjusting a detection target according to the present invention, a label is further bound to the detection target in the environmental sample purified by the series of operations of FIG.
In binding, first, the recovered detection object 2 and the label for labeling the detection object 2 are adjusted so as to be suitable for binding to each other.
That is, since the solution extracted into the reaction container 6 is acidic, the detection target object 2 and the labeling substance cannot be bonded, or the fluorescence intensity of the labeling object is decreased. Therefore, alkali is added to the reaction solution in the reaction solution 6 to bring the pH to near neutral. When the detection object 2 and the recognition object 3 are separated by ionic strength change or polarity change, the sample is adjusted using filtration with a filter, ultrafiltration, dialysis with a dialysis membrane, or the like.
In addition, as the labeling substance, a fluorescent substance that emits specific fluorescence when irradiated with light having a specific excitation wavelength, an enzyme that generates a color by adding a substrate and develops color, an enzyme that chemiluminescents by adding a substrate, or the like is used. For example, fluorescent substances such as fluorescein isothiocyanate (FITC) and phycoerythrin (PE), such as peroxidase, are used to generate color by adding a substrate to produce a dye, and luciferase is used to generate chemiluminescence by adding a substrate. is there.
In the method for detecting a detection object according to the present invention, after adjusting the environmental sample as described above, the detection object is detected from the fluorescence intensity of the fluorescently labeled antibody.

Next, an embodiment 100 of a system for carrying out a method for adjusting a detection target in an environmental sample and a detection method according to the present invention will be outlined with reference to FIG.
As main components of the system 100, in accordance with reference numerals, an environmental sample 101, a cleaning solution 102, a hydrochloric acid solution 103, a separation column 108, a sodium hydroxide solution 113, a recognition product solution 114, a cleaning solution 115, a reaction vessel 120, And the detection part 125 is shown in FIG.
Here, the mutual relations / functions of these components will be described later as the operation of the system according to the present embodiment, but the other components shown in FIG. 2 will be described below.
First, the thermostatic layer 122 is configured to include a separation column 108, a shaker 111, a reaction vessel valve 112, a reaction vessel 120, and a shaker 121. The carrier 109 is held on the separation column 108 by a net 110.
The environmental sample 101, the cleaning solution 102, and the hydrochloric acid solution 103 are connected to valves 104 to 106, respectively. Further, a pump 107 is connected to these valves 104 to 106. Thus, the environmental sample 101, the cleaning solution 102, or the hydrochloric acid solution 103 can be sent to the separation column 108.
The sodium hydroxide solution 113 and the recognized substance solution 114 are connected to valves 116 and 117, respectively. Further, a pump 118 is connected to these valves 116 and 117 so that the sodium hydroxide solution 113 or the recognized substance solution 114 can be sent to the reaction vessel 120.
Further, the cleaning solution 115 is configured to be sent to the reaction vessel 120 by a pump 119.
Furthermore, the detection unit 125 is connected to a pump 124 via a valve 123 and is configured to be able to send a solution from the reaction vessel 120 to the detection unit 125.

Next, an embodiment of the method for adjusting and detecting a detection target in an environmental sample according to the present invention by operating the system 100 of FIG. 2 including the above components will be described.
First, the pump 107 is operated, the valve 104 is opened, and the sample solution 101 is injected into the separation column 108.
Subsequently, the separation column 108 is shaken using the shaker 111, and the detection target object and the recognition object are combined at a predetermined temperature. Here, the detection object and the recognition object are as described in FIG. 1 described above.
Here, impurities other than the object to be detected that do not bind to the recognition object are disposed as waste liquid, and the valve 104 is closed and the valve 105 is opened, and the cleaning solution 102 is injected into the separation column 108 and the impurities from the separation column 108. Remove.

  Next, the valve 105 is closed, the valve 106 and the reaction vessel valve 112 are opened, and the hydrochloric acid solution 103 is injected into the separation column 108. Here, the hydrochloric acid solution 103 is used to adjust the solution in the separation column 108 to an appropriate pH to separate the detection target object and the recognition object. Subsequently, the solution containing the detection target is extracted into the reaction solution 120. Here, the thing other than the hydrochloric acid solution 103 can be used as described above with reference to FIG.

Further, the valve 106 and the reaction vessel valve 112 are closed and the valve 105 is opened again to wash the separation column 108. At this time, the hydrochloric acid solution is washed away, and the remaining contaminants can be washed away, so that the next sample can be prepared.
In order to promote the binding between the extracted detection object and the label, the pump 107 is stopped and the pump 118 is operated, the valve 105 is closed and the valve 116 is opened, and the sodium hydroxide solution 113 is put into the reaction vessel 120. inject. Subsequently, the valve 116 is closed, the valve 117 is opened, and the labeled solution 114 is injected into the reaction vessel 120.
Then, the reaction vessel 120 is shaken by the shaker 121, and the detection target and the label are combined at a predetermined temperature.
Although the shaker 121 is used here, the reaction container 120 may be stirred with a stirrer to combine the detection target and the label.

Next, the pump 118 is stopped and the pump 124 is operated, the valve 117 is closed and the valve 123 is opened, and the solution containing the detection target is injected into the detection unit 125. The detection unit 125 detects a detection target in the environmental sample by analyzing a signal emitted from a label bound to the detection target.
Further, the valve 123 is closed and the pump 119 is operated to wash the reaction vessel 120 a plurality of times. Thus, it is possible to prepare for the processing of the next environmental sample.

Next, examples in which the method for adjusting and detecting the detection target in the environmental sample according to the present invention are described below.
The purpose of this example was to detect Cryptosporidium in environmental samples. As the carrier packed in the separation column, polystyrene balls introduced with amino groups (diameter 6.3 mm, Sumitomo Bakelite) were used. Moreover, the mouse | mouth anti-Cryptosporidium monoclonal antibody (IgG-3 (kappa), biogenesis) was used for the immunity antibody (recognition material) couple | bonded with the support | carrier. The carrier and the immune antibody were covalently bonded using glutaraldehyde.

The sample solution (virtual environmental sample) has a contaminant number concentration on the order of 10 ⁴ (sample A) and a sample column having a contaminant number concentration on the order of 10 ³ (sample B). Prepared. A predetermined amount of Cryptosporidium was added to each sample.
Next, a label having a fluorescent substance FITC (EasyStain, Wako Pure Chemical Industries) was added to these samples, and an antigen-antibody reaction was performed at 37 ° C. for 30 minutes.

  Next, using a flow cytometer (FACS Calbur, Becton Dickinson), the forward scattered light intensity of the fluorescently labeled Cryptosporidium, the FITC fluorescence intensity, and the fluorescence intensity (FL3) at 650 nm were measured. When FACS Calibur is used, an argon laser (488 nm) is used as the light source of the light irradiation unit, and signals to be detected are FSC (forward scattered light), SSC (side scattered light), and FL1 (530 ± 15 nm). ), FL2 (585 ± 21 nm) and FL3 (650 nm or more), and each signal was detected by a corresponding photodetector.

Since FL1 represents fluorescence derived from FITC, the FL1 intensity of Cryptosporidium bound to the FITC-labeled antibody is increased. On the other hand, FL3 is a photosynthetic organ-constituting substance derived from dinoflagellate and represents fluorescence of PerCP (peridinin chlorophyll protein), which is a fluorescent substance, and can therefore be used for detecting chlorophyll in algae.
Since FSC corresponds to the particle size of the particle, the FSC intensity corresponding to the particle size of Cryptosporidium was grasped by measuring Cryptosporidium in advance. The intensity of fluorescently labeled Cryptosporidium with FSC, FL1, and FL3 was measured using a flow cytometer and compared. Regions 32 and 34 where Cryptosporidium is present were set in each two-dimensional dot plot.

In this measurement, measurement results of FSC, FL1, and FL3 were used.
3 shows a two-dimensional dot plot of FSC and FL1 of sample A, and FIG. 5 shows a two-dimensional dot plot of FSC and FL1 of sample B. 4 shows a two-dimensional dot plot of FL3 and FL1 of sample A, and FIG. 6 shows a two-dimensional dot plot of FL3 and FL1 of sample B.
According to this, when the number concentration of contaminants is lowered using a separation column, it can be seen that false positive particles are reduced in the regions 32 and 34 where Cryptosporidium is present and the detection accuracy is improved. Therefore, it can be seen that the detection accuracy of the detection object can be improved according to the adjustment method and the detection method of the detection object in the environmental sample of the present invention.

It is the schematic explaining the outline | summary of the purification method of the detection target object in the environmental sample which concerns on this invention. BRIEF DESCRIPTION OF THE DRAWINGS It is the schematic explaining one Embodiment about the system for implementing the adjustment method and detection method of the detection target object in the environmental sample which concern on this invention. It is a graph which shows the two-dimensional dot plot of FSC of sample A, and FL1 about the Example of this invention. It is a graph which shows the two-dimensional dot plot of FSC of sample B, and FL1 about the Example of this invention. It is a graph which shows the 2-dimensional dot plot of FL3 of sample A, and FL1 about the Example of this invention. It is a graph which shows the two-dimensional dot plot of FL3 of sample B, and FL1 about the Example of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Separation column 2 Detection object 3 Recognized object 4 Carrier 5 Contaminant 6 Reaction solution 100 System 101 Environmental sample 102 Hydrochloric acid solution 103 Cleaning solution 104 Valve 105 Valve 106 Valve 107 Pump 108 Separation column 109 Carrier 110 Net 111 Shaker 112 Valve 113 Sodium hydroxide solution 114 Labeled substance 115 Cleaning solution 116 Valve 117 Valve 118 Pump 119 Pump 120 Reaction vessel 121 Shaker 122 Constant temperature bath 123 Valve 124 Pump 125 Detector 32 Region where Cryptosporidium is present 34 Region where Cryptosporidium is present

Claims (6)

Recognizing a detection target and contacting an environmental sample with a carrier bound with a recognition target that specifically binds to the detection target;
Separating the detection object and contaminants in the environmental sample;
Desorbing and collecting the detection object bound to the carrier;
Adjusting the recovered detection target and the label for labeling the detection target to be suitable for binding to each other;
A method for adjusting a detection target in an environmental sample, comprising the step of binding the detection target and the label.   The adjustment method according to claim 1, wherein the detection object is Cryptosporidium.   The method according to claim 1 or 2, wherein the recognition product is an anti-cryptospodium antibody.   The adjustment method according to claim 1, wherein the label is a fluorescently labeled antibody.   The adjusting method according to claim 1, wherein the carrier is polystyrene having an amino group introduced therein.   A step of adjusting an environmental sample by the method for adjusting a detection target in an environmental sample according to any one of claims 1 to 5, and then detecting the detection target from the fluorescence intensity of the fluorescently labeled antibody is performed. A method for detecting an object to be detected in an environmental sample.