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WO2010038628A1 - Instrument produisant des particules de gel et dispositif de mesure de particule de gel l’utilisant - Google Patents

Instrument produisant des particules de gel et dispositif de mesure de particule de gel l’utilisant Download PDF

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Publication number
WO2010038628A1
WO2010038628A1 PCT/JP2009/066273 JP2009066273W WO2010038628A1 WO 2010038628 A1 WO2010038628 A1 WO 2010038628A1 JP 2009066273 W JP2009066273 W JP 2009066273W WO 2010038628 A1 WO2010038628 A1 WO 2010038628A1
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sample
gel particle
light
sample cell
reagent
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English (en)
Japanese (ja)
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徹 小幡
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Individual
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/59—Transmissivity
    • G01N21/5907—Densitometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/82—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a precipitate or turbidity

Definitions

  • the present invention is used in a gel particle measuring apparatus for measuring a target substance such as endotoxin or ⁇ -D-glucan in a sample to be measured by gelation reaction, and is effective in generating gel particles.
  • the present invention relates to a generator and a gel particle measuring apparatus using the same.
  • endotoxin is a fragment of bacterial cells that do not mainly stain Gram stain (Gram negative), and its component is a lipid polysaccharide called lipopolysaccharide, specifically lipid A (Lipid).
  • Lipid polysaccharide (LPS) in which a lipid called A) and a polysaccharide chain are linked via 2-keto-3-deoxyoctanoic acid (KDO), the lipid structure called Lipid A contained therein
  • LPS Lipid polysaccharide
  • KDO 2-keto-3-deoxyoctanoic acid
  • endotoxin is a substance that causes clinical symptoms with high fatality such as sepsis and bacteremia, it is clinically required to estimate endotoxin that has entered the body.
  • pharmaceuticals injections, etc.
  • medical devices vascular catheters, etc.
  • bacteria recombinant proteins, DNA used for gene therapy, etc.
  • the measurement method using the Limulus reagent is simply mixed with the patient's plasma as a sample, left standing, and then falls down after a certain period of time to confirm the presence or absence of gelation, causing gelation.
  • This is a so-called gelation method in which the endotoxin amount is estimated at the maximum dilution ratio.
  • a turbidimetric time analysis method that measures the endotoxin concentration by turbidity change associated with the gelation reaction using an optical measurement method is known. ing.
  • Patent Document 1 is not related to a gelation reaction measuring device, but measures the size and number of platelet aggregates in the process of platelet aggregation in blood and growing as a mass.
  • the sample is irradiated with irradiation light from a laser light source, and a part of the scattered light scattered 90 degrees laterally by platelets is detected by a photodetector, and the size of platelet aggregates is determined based on the detection result. The number is measured.
  • Patent Document 2 relates to a gelation reaction measuring apparatus using a turbidimetric time analysis method, and measures a temporal change in transmitted light intensity of a mixed liquid in which a specimen (sample) and a Limulus reagent are mixed, The endotoxin concentration of the specimen is measured from the amount of change in a predetermined time.
  • ⁇ -D-glucan is a polysaccharide (polysaccharide) that forms a cell membrane characteristic of fungi. By measuring this ⁇ -D-glucan, it is effective in screening for a wide range of fungal infections including not only common fungi such as Candida, Aspergillus and Cryptococcus but also rare fungi.
  • ⁇ -D-glucan In the measurement of ⁇ -D-glucan, it is also utilized that the blood cell extract component of horseshoe crab gels with ⁇ -D-glucan, and the gelation method, turbidimetric time analysis method, and chromogenic synthetic substrate method described above are used. Measured. Endotoxin and ⁇ -D-glucan measurement methods have similarities. For example, by using almost the same measurement hardware and removing the Factor G component from the blood cell extract components of horseshoe crab, A color reaction can be measured, and the endotoxin in the sample can be inactivated by pretreatment to measure a gelation reaction or a color reaction selective to ⁇ -D-glucan.
  • the conventional gelation method, turbidimetric time analysis method, and color synthesis substrate method have the following problems.
  • the gelation method and the turbidimetric time analysis method it takes a long time of about 90 minutes or more at a low concentration to form a gel. That is, the gelation time of the reaction solution is proportional to the concentration of the target substance in the sample to be measured, but neither the gelation method nor the turbidimetric time analysis method can detect the exact gelation start time from the point of sensitivity. Therefore, the amount of reaction is calculated from the time until the end of gelation and is used as a standard for the gelation time.
  • the turbidimetric time analysis method knows the first level at which the change begins and the level at which the change arrives, but it is difficult to understand the time at which each change begins and the end time. It was established as a quantitative method to replace the observation of the entire gelation by measuring a certain level of change (increase in turbidity) between the first and last levels. However, when the concentration of endotoxin is low, gelation of the entire system is prolonged, and the change in turbidity observed is slowed accordingly, making it difficult to measure, and the sensitivity is inevitably reduced accordingly.
  • the gelation method and the turbidimetric time analysis method are both suitable for urgent cases and for measuring a large number of specimens. Furthermore, in the turbidimetric time analysis method, nonspecific turbidity unrelated to endotoxin may occur, and there is a concern that measurement accuracy is lacking.
  • the measurement limit concentration of the gelation method is 3 pg / ml, and the measurement limit concentration of the turbidimetric time analysis method is about 1 pg / ml.
  • a turbidimetric time analysis method applied to the gelation reaction measuring apparatus even if the scattering photometry method shown in Patent Document 1 is applied, it is the above-described quantitative method instead of the change observation of the entire gelation.
  • the chromogenic synthetic substrate method has a measurement time of about 30 minutes as compared with the gelation method and turbidimetric time analysis method, but a false-positive reaction may occur and a measurement with high specificity should be performed.
  • the measurement preparation is complicated and the measurement limit concentration is 3 pg / ml, which is inferior to the turbidimetric time analysis method.
  • the present invention relates to a gel particle generating device capable of generating gel particles while uniformly and stably generating a gelation reaction when measuring a target substance in a sample by the gelation reaction, and a gel particle using the same A measuring device is provided.
  • a sealing member capable of injecting the sample into the reagent cell after sealing, and starting the stirring operation by the stirring member when the sample is injected into the sample cell, Whole if the solution is a gel particle production device, characterized in that so as to produce gel particles while suppressing the gelation of.
  • the invention according to claim 2 is the gel particle generating device according to claim 1, further comprising a holding cover that is attached to the opening edge of the reagent cell and holds the sealing member.
  • the invention according to claim 3 is a gel particle measuring apparatus for measuring a target substance in a sample by particleizing by a gelation reaction, the sample being arranged on a predetermined measurement stage and injecting the sample into a sample cell.
  • the gel particle generating device according to claim 1 and the measurement stage are provided outside the sample cell, and the entire mixed solution composed of the sample and the reagent is prevented from gelation by rotating the stirring member in the sample cell.
  • the light detection means for detecting the light passing through the mixed solution comprising the sample and the reagent in the sample cell out of the light from the coherent light source, and the detection output of the light detection means And a light fluctuation measuring means for measuring the light fluctuation component, and a gel particle generation determining means for determining the generation state of the gel particles in the mixed solution based on the measurement result of the light fluctuation measuring means.
  • the gel particle measuring device in the gel particle measuring apparatus according to the third aspect, the sample cell of the gel particle generating instrument has a transmission part through which light passes from one side to the other, and the light detection means includes the sample cell.
  • the gel particle measuring apparatus is provided on the opposite side of the coherent light source outside the transmission unit and detects light transmitted through the mixed solution composed of the sample and the reagent in the sample cell.
  • a component of the scattered light scattered by the gel particles and deviating in phase is directed to the light detection means.
  • grain measuring apparatus provided with the scattered light removal means from which water is removed.
  • the temperature can be adjusted so that the entire data cell disposed on the measurement stage is maintained at a predetermined constant temperature. It is a gel particle measuring apparatus provided with a suitable temperature control means.
  • gel particles when measuring the target substance in the sample by the gelation reaction, gel particles can be generated while the gelation reaction is uniformly and stably generated. According to the invention of claim 2, it is possible to effectively prevent the sealing member from being unnecessarily detached from the opening edge of the reagent cell. According to the third aspect of the invention, when measuring the target substance in the sample by the gelation reaction, it is possible to generate gel particles while causing the gelation reaction to occur uniformly and stably. The production state of gel particles can be accurately measured.
  • the light detection means detects the transmitted light of the mixed solution consisting of the sample and the reagent, and the gel is based on this detection output. Since the generation state of the particles is discriminated, the generation state of the gel particles can be measured with higher sensitivity than the method of detecting the scattered light by the light detection means. According to the invention which concerns on Claim 5, the detection accuracy in a photon detection means can be improved more, and the production
  • the invention according to claim 6 can effectively suppress the influence on the gelation reaction due to the temperature change when measuring the target substance in the sample by the gelation reaction.
  • (A) is explanatory drawing which shows the outline
  • (b) is embodiment of the gel particle measuring apparatus using the gel particle production
  • (A) is an explanatory view schematically showing the gelation reaction
  • (b) is an explanatory view showing the progress steps I to III of the gelation reaction
  • (c) is the reaction time and transmitted light intensity in the progress step of the gelation reaction, It is explanatory drawing which shows these relationships. It is explanatory drawing which shows typically the gelatinization reaction process of the endotoxin at the time of using a Limulus reagent.
  • (A) is a disassembled perspective view which shows the gel particle production
  • (b) is the cross-sectional explanatory drawing. It is explanatory drawing which shows the construction method of the gel particle production
  • (A) is front explanatory drawing of the gel particle measuring apparatus which concerns on Embodiment 1
  • (b) is plane explanatory drawing seen from B direction in (a).
  • 4 is a flowchart showing an example of data analysis processing of the gel particle measuring apparatus according to Embodiment 1.
  • (A) is explanatory drawing which shows an example of the gel particle measuring apparatus which concerns on a comparison form
  • (b) is the plane explanatory drawing seen from B direction in (a).
  • FIG. It is a graph which shows the result of having measured the transmitted luminous intensity for every reaction time about various endotoxin density
  • FIG. It is explanatory drawing which shows the example of a calibration curve preparation using the graph figure shown in FIG. It is a graph which shows the result of having measured the transmitted luminous intensity for every reaction time about various endotoxin density
  • FIG. It is explanatory drawing which shows the example of a calibration curve preparation using the graph figure shown in FIG.
  • FIG. It is explanatory drawing which shows an example of the production
  • FIG. It is explanatory drawing which shows the example of application of this invention in a blood coagulation reaction.
  • (A) (b) is explanatory drawing which shows the example of application of this invention in an antigen antibody reaction.
  • FIG. 1A is an explanatory diagram showing an outline of a gel particle generating device according to an embodiment to which the present invention is applied.
  • a gel particle generating instrument 11 is used in a gel particle measuring apparatus that measures and measures a target substance in a sample by gelation reaction.
  • the gel particle generating device 11 generates gel particles, and a sample S is injected and accommodated therein.
  • a reagent 2 that is stored in advance in the sample cell 1 and gels by reacting with a target substance in the sample S,
  • a stirring member that stirs the mixed solution W so as to suppress gelation of the entire mixed solution W (see FIG.
  • the whole mixed solution W is that so as to produce gel particles while suppressing the gelation of.
  • the target substance of the present case includes a wide range of substances as long as they undergo a gelation reaction with the predetermined reagent 2 to generate gel particles. Examples include endotoxin and ⁇ -D-glucan. In addition to this, blood coagulation reactions and antigen-antibody reactions also correspond to gelation reactions, so the components in sample S that lead to these gelation reactions are also included. It can be the target substance in this case. Further, the sample cell 1 may be entirely composed of a transmissive member, but is not limited to this, and it is sufficient that the sample cell 1 has at least a transmissive portion in a portion through which light is transmitted. The constituent material may be glass or resin.
  • the shape may be appropriately selected such as a circular cross-section, a polygon such as a rectangle, and the cross-sectional shape does not necessarily have to be uniform, and a constricted portion or the like may be provided in part.
  • the reagent 2 may be appropriately selected as long as it causes a gelation reaction (aggregation reaction) with the target substance in the sample S, and may be a solid such as a lyophilized powder or a liquid.
  • the stirring member 3 includes a wide range of materials that are incorporated in the sample cell 1 and that give a stirring action to the mixed solution W composed of the sample S and the reagent 2.
  • a stirring bar having magnetism may be used, and stirring driving means 12 (see FIG. 1B) for driving it may be provided outside.
  • the sealing member 4 should just be sealed in the state which accommodated the stirring member 3 and the reagent 2 in the sample cell 1, for example, plug materials, such as rubber
  • the holding cover 5 may be detachable from the sample cell 1 and once removed from the sample cell 1 when the sample S is injected from the sealing member 4, but the injection operability of the sample S is improved. From the viewpoint of simplicity, it is preferable that the hole 5a partially faces the sealing member 4 and the sample S is injected using the hole 5a without removing the holding cover 5. .
  • FIG.1 (b) is explanatory drawing which shows the outline
  • a gel particle measuring apparatus measures a target substance in a sample S by particleizing by a gelation reaction.
  • the gel particle measuring apparatus is arranged on a predetermined measurement stage and the sample S is placed in a sample cell 1.
  • the entire mixed solution W composed of the sample S and the reagent 2 is gelated by rotating the stirring member 3 provided in the sample cell 1 of the injected gel particle generating instrument 11 and the measurement stage and rotating in the sample cell 1.
  • the stirring drive means 12 that stirs the mixed solution W so as to suppress the mixing, and the mixed solution W that is provided outside the permeation portion of the sample cell 1 and includes the sample S and the reagent 2 in the sample cell 1.
  • a coherent light source 13 that emits coherent light to the light, and light Bm that has passed through the mixed solution W composed of the sample S and the reagent 2 in the sample cell 1 out of the light Bm from the coherent light source 13.
  • a light detection means 14 that emits light, a light fluctuation measurement means 15 that measures a light fluctuation component based on a detection output of the light detection means 14, and a measurement result of the light fluctuation measurement means 15, based on the measurement result of the light fluctuation measurement means 15.
  • Gel particle generation determining means 16 for determining the generation state of the gel particles is provided.
  • the above-described embodiment is used as the gel particle generating device 11.
  • the stirring drive means 12 what drives the stirring member 3 of the gel particle production
  • One method is to apply a rotational force to the rod.
  • the degree of agitation of the agitating member 3 by the agitation driving means 12 needs to suppress the entire mixed solution W composed of the sample S and the reagent 2 in the sample cell 1 from gelation.
  • the coherent light source 13 is not limited to the laser light from the laser light source as long as it emits coherent light, and can be created by passing monochromatic light such as light from a sodium lamp through a pinhole.
  • the light detection means 14 transmitted light or gel particles that have passed through the mixed solution W composed of the sample S and the reagent 2 out of the light from the coherent light source and transmitted through the gel particles generated in the mixed solution W are used. What is necessary is just to detect the scattered light scattered.
  • the light fluctuation measuring means 15 measures a light fluctuation component based on the detection output of the light detection means 14, and includes a technique of averaging or smoothing the detection output and filtering.
  • the gel particle generation discriminating means 16 broadly includes one that discriminates the generation state of the gel particles.
  • the generation state of the gel particles widely includes the generation start (appearance) time of the gel particles, the change in the generation process, the generation end time, the generation amount, and the like.
  • “determining the generation state of the gel particles” means not only directly determining the information on the generation state of the gel particles, but also information that can be determined based on the generation state of the gel particles (for example, quantitative information of the target substance) It also includes discriminating.
  • the light attenuation change point may be determined as the appearance timing of the gel particles based on the measurement result of the light fluctuation measuring means 15.
  • the gel particle generating instrument 11 is disposed, from the viewpoint of keeping the measurement conditions constant, temperature adjustment that allows temperature adjustment so that the entire sample cell 1 is maintained at a predetermined constant temperature.
  • An embodiment comprising means 18 is preferred.
  • the temperature control means 18 a thermostat is mentioned, for example.
  • the light detection means 14 is preferably one that mainly detects transmitted light that passes through the mixed solution W composed of the sample S and the reagent 2 out of the light from the coherent light source 13. This transmitted light detection method has the following advantages over the scattered light detection method. (1) Originally, the transmitted light component is larger than the scattered light component.
  • the light detection means 14 employs the transmitted light detection method
  • a part of the scattered light scattered by the gel particles may be detected by the light detection means 14 as stray light, but most of the detection output is Since it is a transmitted light component, even if a stray light component is partially included, there is little influence.
  • the scattered light detection method needs to adopt a special container structure with a small container thickness with little attenuation, but the transmitted light detection method has no such limitation.
  • the transmitted light detection method may be rougher.
  • a component of the scattered light scattered by the gel particles and shifted from the phase between the light detection unit 14 and the sample cell 1 is directed to the light detection unit 14.
  • the aspect provided with the scattered light removal means 17 from which is removed is mentioned.
  • the scattered light removing means 17 there is a polarizing filter that cuts the scattered light component and allows only the transmitted light component to pass.
  • adopted the transmitted light detection system although the one part of the scattered light scattered by the gel particle may be detected by the light detection means 14 as stray light, the influence by a stray light component is avoided.
  • correction by the light fluctuation measuring means 15 can be considered, but this embodiment is preferable in that the stray light component is surely removed with a simple configuration. Further, from the viewpoint of viewing the measurement result obtained by the light fluctuation measuring means 15, it is preferable to include a display means 19 for displaying the measurement result obtained by the light fluctuation measuring means 15.
  • the gelation reaction is schematically shown in FIG.
  • the reagent 2 that specifically reacts with the target substance St of the sample S
  • the target substance St is specifically different from the reagent 2 at a ratio depending on the concentration of the target substance St in the sample S.
  • a reaction occurs.
  • the reagent 2 receives a stimulus from the target substance St, activates a predetermined factor, and at the timing when a predetermined enzyme is activated due to this, for example, a water-soluble protein is decomposed by the enzyme. It is converted into an insoluble protein, leading to the appearance of gel particles G.
  • the endotoxin gelation reaction process is schematically shown in FIG.
  • the endotoxin stimulation shown in (1) is transmitted to the Limulus reagent, first, as shown in (2), factor C (Factor C) is activated to become activated factor C (Activated Factor C), Next, by the action of the activation factor C, as shown in (3), factor B (Factor B) is activated to become activated factor B. Thereafter, due to the action of activating factor B, Pro-Clotting enzyme becomes Clotting enzyme as shown in (4), and this Clotting enzyme degrades Coagulogen (water-soluble protein) as shown in (5). To produce Coagulin (insoluble protein).
  • This Coagulin appears as gel particles G when the entire gelation is inhibited by stirring, and polymerizes and gels as shown in (6) when allowed to stand. That is, when the target substance St of the sample S is endotoxin, by giving a constant stirring state to the mixed solution W, the gelation of the entire mixed solution W is inhibited, and in this state, in the Limulus reagent 2 When the stimulation of endotoxin is transmitted, it is possible to produce Coagulin (insoluble protein) gel particles G around the Clotting enzyme. After Coagulin (insoluble protein) is generated as gel particles G, the gel particles G are sequentially formed. It is understood that it undergoes a reaction process that is generated.
  • the rate at which endotoxin stimulation is transmitted to Limulus Reagent 2 depends on the endotoxin concentration. The higher the endotoxin concentration, the faster the Limulus reaction rate, and the appearance timing of gel particles G composed of Coagulin (insoluble protein). Was found to be fast. Therefore, if a light change (for example, a change in transmitted light) is accurately detected, it is possible to grasp the appearance timing of the gel particle G composed of the Coagulin (insoluble protein) as the generation start point of the gel particle G. This is the basic measurement principle of the gel particle measuring apparatus according to the present embodiment.
  • the measurement principle of such a gel particle measuring apparatus is, for example, the measurement principle of the conventional gelation method or turbidimetric time analysis method (in the reaction process with Limulus reagent 2, the effect of the activated enzyme under the stationary condition). Finally, it is gelled, which is completely different from the embodiment in which this gelled state is measured by turbidity.
  • the measurement principle of the gel particle measuring apparatus using the transmitted light detection method is schematically shown in FIG.
  • the gel particle measuring apparatus according to the present embodiment as shown in Step I of FIG. 2B, when there is no gel particle in the mixed solution W composed of the sample S and the reagent (not shown), it is not shown. because never transmitted light Bm 1 from the coherent light source is blocked by the gel particles, the transmitted light intensity of the transmitted light Bm 1 is kept substantially constant (see FIG. 2 (c) I step P 1). Then, as shown in Step II of FIG.
  • the gel particle measuring apparatus includes a gel particle generating instrument 100 (see FIG. 4), and measures, for example, the concentration of endotoxin as a target substance of a sample by a gelation reaction using a Limulus reagent.
  • the gel particle generating device 100 has a sample cell 101 into which a sample containing endotoxin is injected, for example, as shown in FIGS.
  • the sample cell 101 is a bottomed cylindrical body that is integrally formed of, for example, a glass material and has an open top, and has a bottomed cylindrical body.
  • a flange portion 102 is formed on the top, and a lower portion of the flange 102 is formed.
  • a constricted portion 103 is formed, a small-diameter hole portion 104 is formed in the flange portion 102 and the constricted portion 103, and a large-diameter space portion 105 having a larger diameter than the small-diameter hole portion 104 is formed inside.
  • a sample 106 containing an endotoxin and a reagent 106 that causes a gelation reaction are preliminarily aseptically stored, for example, in the form of lyophilized powder, and a stirring bar 107 using a magnetic material is preliminarily stored. Is done.
  • a sealing plug 108 made of an elastic material such as rubber is fitted in the small diameter hole 104 of the sample cell 101.
  • the sealing plug 108 has a substantially T-shaped cross section, and its head portion 108a is placed on the flange portion 102 of the sample cell 101, and its leg portion 108b is inserted in close contact with the small diameter hole portion 104. Yes.
  • a notch 108c is provided in a part of the leg 108b of the sealing plug 108.
  • the flange portion 102 of the sample cell 101 and the head portion 108a of the sealing plug 108 are covered with a cap-like holding cover 109 made of, for example, aluminum, and this holding cover 109 is fitted into the peripheral wall of the flange portion 102 of the sample cell 101.
  • the sealing plug 108 is held and held from the outside.
  • a hole 109 a is formed at the center of the holding cover 109 so as to face the head 108 a of the sealing plug 108.
  • this type of gel particle generating device 100 accommodates the reagent 106 and the stirring rod 107 with the small-diameter hole 104 of the sample cell 101 opened, and in this state, the sample cell 101.
  • the small-diameter hole 104 is sealed with a sealing plug 108, and the sealing plug 108 is covered with a holding cover 109.
  • Such a gel particle generating instrument 100 is supplied to the user as an accessory or a measurement kit of the gel particle measuring apparatus.
  • the hole 109a of the holding cover 109 is used to drill the sealing plug 108 with a punch such as an injection needle.
  • the sample S is injected by the injector 110 through the perforations.
  • the sealing specification of the sealing plug 108 may be set so that the sample cell 101 maintains a predetermined negative pressure level with respect to the atmospheric pressure.
  • the gel particle measuring apparatus is configured as shown in FIGS.
  • the gel particle generating instrument 100 is installed on a predetermined measurement stage.
  • the gel particle generating instrument 100 is arranged in a thermostat 115 and is a mixed solution composed of a sample S and a reagent (not shown).
  • W is placed in a constant constant temperature environment (for example, 37 ° C.) to make the measurement conditions constant.
  • Reference numeral 120 denotes an agitation driving device for driving the magnetic stirring rod 107 in the sample cell 101 to agitate the mixed solution W in the sample cell 101.
  • the stirring drive device 120 is a stirring drive source (magnetic) that applies a stirring force by magnetic force to a stirring bar (stirrer bar) 107 made of a magnetic material built in the bottom wall in the sample cell 100. It is configured as a stirrer.
  • reference numeral 130 denotes a laser light source that is provided on one side of the side wall of the sample cell 101 and emits coherent light
  • 140 is provided on the opposite side of the laser light source 130 with the sample cell 101 interposed therebetween.
  • This is a transmitted light detector that detects transmitted light B from.
  • the transmitted light detector 140 for example, optical components such as photodiodes can be widely used.
  • the coherent light Bm from the laser light source 130 is irradiated along a path that crosses the substantially diameter portion of the sample cell 101, and the light diameter is generated.
  • a sufficiently large value for example, about 1 mm
  • the gel particle diameter for example, about 0.5 to 20 ⁇ m).
  • the transmitted light detector 140 has a detection surface capable of detecting the light flux region of the transmitted light Bm from the laser light source 130, and the detection accuracy of the transmitted light detector 140 is within the passage area of the transmitted light Bm. Or it is set to such an extent that a change in the amount of transmitted light due to the presence or absence of several gel particles can be detected.
  • a polarizing filter 150 is disposed between the sample cell 101 and the transmitted light detector 140. This polarizing filter 150 removes the stray light of the component toward the transmitted light detector 140 by the scattered light scattered by the gel particles G generated in the mixed solution W from the light Bm from the laser light source 130.
  • the principle of stray light removal by the polarizing filter 150 is based on the fact that when the coherent light Bm from the laser light source 130 is scattered by the gel particles G, the phase of the scattered light is shifted, and stray light having a phase component other than the phase of the transmitted light Bm.
  • the ingredients are cut.
  • optical components such as a condensing lens and a mirror may be arranged between the laser light source 130, the transmitted light detector 140, and the sample cell 101 as necessary in determining the optical path and the irradiation light diameter.
  • optical components such as a condensing lens and a mirror may be arranged between the laser light source 130, the transmitted light detector 140, and the sample cell 101 as necessary in determining the optical path and the irradiation light diameter.
  • Reference numeral 160 denotes a data analysis device that takes in the detection output from the transmitted light detector 140 and executes the data analysis processing shown in FIG. 7, for example, and 170 is a display that displays the analysis results analyzed by the data analysis device 160.
  • This data analysis device 160 is constituted by a computer system including a CPU, ROM, RAM, I / O interface, etc.
  • the data analysis processing program shown in FIG. The data analysis processing program is executed by the CPU based on the detection output from the device 140.
  • the detection output from the transmitted light detector 140 is, for example, converted from current to voltage by an amplifier (not shown), then AD converted by an AD converter, and taken into the data analyzer 160.
  • the gel particle measuring apparatus After injecting the sample S containing endotoxin into the sample cell 101 of the gel particle generating device 100 shown in FIGS. 6A and 6B, when a start switch (not shown) is turned on, the gel particle measuring device The measurement sequence is started. In this measurement sequence, the stirring rod 107 is rotated by the stirring drive device 120 to stir the mixed solution W composed of the sample S and the Limulus reagent in the sample cell 101. For this reason, while the whole mixed solution W is stirred uniformly, gelatinization as the whole mixed solution W is suppressed.
  • the sample cell 101 stimulation of endotoxin is transmitted to the Limulus reagent, the Limulus reaction as shown in FIG. 3 occurs, and gel particles G are sequentially generated in a state where gelation of the entire mixed solution W is suppressed. Go.
  • the timing at which, for example, one gel particle G is generated within the passage area of the light Bm from the laser light source 130 is grasped as the generation start point of the gel particle G, and the attenuation change point of the transmitted light Bm This is the timing.
  • the data analysis device 160 reads the detection output from the transmitted light detector 140 as transmitted light amount data (digital data) as shown in FIG.
  • the calibration curve indicates the relationship between the endotoxin concentration (ETX concentration) and the time threshold until reaching the attenuation change point of the transmitted light Bm. Based on these correlations, the endotoxin concentration (ETX concentration) is determined.
  • the display 170 switches and displays data such as time-series data of transmitted light amount data and time-series measurement data of fluctuation components of transmitted light amount data.
  • EX concentration endotoxin concentration
  • the gel particle measuring apparatus stirs the mixed solution W composed of the sample S and the Limulus reagent under a predetermined constant temperature environment, and gel particles composed of Coagulin particles produced in the mixed solution W. It detects that the transmitted light Bm is partially blocked by the appearance of G and diminishes, and tries to catch the start time of gelation.
  • a coherent and strong light such as a laser beam is used, and in order to detect a minute change, particularly in a change at a low concentration.
  • the stray light component is removed by the polarizing filter 150 using the fact that the scattered stray light strikes the gel particle G made of Coagulin particles and the phase is shifted, only the transmitted light component from the laser light source 130 is included in the transmitted light detector 140. Is incident, and the change in transmitted light is accurately detected accordingly.
  • a gel particle measuring apparatus injects a sample S containing endotoxin into a sample cell 201 of a gel particle generating instrument 200 (a state in which a reagent and a stirring rod 207 are previously stored in a sample cell 201). Then, the mixed solution W is stirred by the stirring rod 207 driven by the stirring drive device 220 and light Bm ′ from the laser light source 230 is irradiated into the mixed solution W to be generated by the Limulus reaction.
  • a part of the scattered light scattered laterally by the gel particles G is detected by the photodetector 240, and the detection output of the photodetector 240 is taken into the data analysis device 260 to calculate the production time of the gel particles G To do.
  • the gel particle measuring apparatus since the scattered light originally has a smaller ratio than the transmitted light and only a part of the scattered light can be measured, it is necessary to suppress the attenuation of the scattered light as much as possible. Therefore, in this comparative form, a strict optical circuit is necessary to prevent the scattered light from being attenuated, and as shown in FIG. 8B, a position perpendicular to the surface of the mixed solution W of the sample cell 201 is obtained.
  • the sample cell 201 must also adopt a special container structure with a small container thickness k with little attenuation, and the installation accuracy of the laser light source 230 and the photodetector 240 must be extremely increased.
  • the container structure of the sample cell 101 should not be provided with a special structure and should be a strong container having a thick container thickness.
  • the laser light source 130 and the transmitted light detector 140 may be installed on the sample cell 101 in a straight line toward the substantially diameter direction of the sample cell 101, and the installation accuracy associated with the replacement of the sample cell 101 can be improved. It can be rough to some extent.
  • the gel particle measuring apparatus for the gel particle generating instrument 100 for one specimen is shown.
  • a plurality of specimens for example, a plurality of specimens (samples)
  • a multi-sample cell in which the sample cells 101 of the gel particle generating device 100 are integrated is prepared, and a laser light source 130 and a transmitted light detector 140 are arranged corresponding to each sample cell, and a plurality of specimens (samples) are simultaneously obtained.
  • the substance to be measured is disclosed as endotoxin, but the present invention is not limited to this.
  • the same measurement hardware and the same or similar limulus reagent is used.
  • the substance to be measured can be ⁇ -D-glucan.
  • Example 1 This example is a realization of the gel particle measuring apparatus according to the first embodiment.
  • the conditions of the examples are as follows.
  • Laser light source 130 Red light or green light Transmitted light detector 140: Photodiode Speed of stirring bar (stirrer bar) 107: 1000 rpm ⁇ Constant temperature condition: 37 °C
  • a change in transmitted light intensity was examined with a gel particle measuring device with respect to the Limulus reagent when various endotoxin concentrations (10 ⁇ 1 ⁇ 0.1 pg / ml) were added.
  • FIG. 9 is a plot of the transmitted light intensity over time, 10 pg / ml twice, 1 pg / ml, and 0.1 pg / ml.
  • the change in the transmitted light intensity under each condition shows a tendency to attenuate and decrease at a certain time when there is a portion that maintains a substantially constant level.
  • the attenuation change point of the transmitted light intensity corresponds to the gel particle generation start point (gelation start time), and is assumed to mean light attenuation at the start of gelation.
  • gelation start time the gel particle generation start point
  • the gelation start times (reaction times) t 1 (10), t 2 (10), t (1), and t (0.1) were determined, respectively.
  • a calibration curve is created using the values of the gelation start times t 1 (10), t 2 (10), t (1), and t (0.1) obtained from the graph of FIG. (See FIG. 10).
  • the X-axis is the ETX concentration (log conversion), which is the endotoxin concentration
  • the Y-axis is the gelation start time (log conversion)
  • a linear relationship is obtained, and the correlation coefficient is ⁇ 0.9804.
  • a high correlation is shown, demonstrating the usefulness of this calibration curve.
  • Example 2 This example is a realization of the gel particle measuring apparatus according to the first embodiment.
  • the conditions of the example are as follows as in the first example.
  • Laser light source 130 Red light or green light Transmitted light detector 140: Photodiode Speed of stirring bar (stirrer bar) 107: 1000 rpm ⁇ Constant temperature condition: 37 °C
  • the change in transmitted light intensity was examined with a gel particle measuring apparatus.
  • FIG. 11 is a plot of transmitted light intensity over time for each endotoxin concentration.
  • the data of endotoxin concentration 10 ⁇ 1 ( ⁇ ) overlaps with other data, and is not visible.
  • the value of the gelation start time obtained from the graph of FIG. 11 (in this example, data of endotoxin concentrations of 10 ⁇ 1 , 10 ⁇ 2 , 10 ⁇ 3 , 10 ⁇ 4 unit) is used.
  • the results shown in FIG. 12 were obtained.
  • Example 3 An example of the generation state of the gel particles is shown in FIG. 13 using the gel particle generation device 100 used in the first embodiment.
  • No. 1 shows the state which stirred the mixed solution which consists of a reagent and water with the stirring rod for the predetermined time (about 20 minutes) to the gel particle production
  • No. 2 shows a state in which a mixed solution consisting of a reagent and an endotoxin concentration of 10 pg / ml was stirred with a stirring rod for a predetermined time in the gel particle generating apparatus according to this example.
  • No. 3 (comparative example) is No. The state which measured the mixed solution similar to 2 using the turbidimetric time method is shown.
  • gel particles having a higher turbidity appear in the generation state of the gel particles by the gel particle generation device of the present example as compared to the gel of the whole solution of the comparative example.
  • the gel particles are biased to the lower side of the mixed solution. This is considered to be because the generated gel particles were precipitated because the rotation of the stirring rod was stopped when photographing.
  • the present invention is widely applicable to measuring devices for measuring target substances capable of generating gel particles by gelation reaction, including gel particle measuring devices for measuring endotoxin, ⁇ -D-glucan and the like using Limulus reagent.
  • it can be applied in blood coagulation reactions and antigen-antibody reactions.
  • -Blood coagulation reaction (Fig. 14)- Prothrombin in plasma becomes thrombin through activation of various blood coagulation factors, and fibrin aggregates. Supplementing this point, the plasma coagulation system proceeds through the following initial phase, amplification phase, and propagation phase. ⁇ Starting period> (Exogenous pathway) When cells are damaged in the blood coagulation cascade, tissue factor binds to factor VIIa (factor VII activated).
  • factor VIIa activates factor IX to form factor IXa.
  • Factor IXa activates factor X to form factor Xa.
  • a negatively charged solid eg, rock or sand
  • prekallikrein and high molecular weight kininogen activate Factor XII to Factor XIIa.
  • Factor XIIa activates factor XI to form factor XIa.
  • Factor XIa activates factor IX to form factor IXa.
  • ⁇ Amplification period> Thrombin activates factor XI to form factor XIa.
  • Factor XIa activates factor IX to form factor IXa.
  • this particle measurement method can measure the degree of aggregation by mixing it with a suitable diluted plasma and a certain amount of agglutination-promoting reagent (for example, ADP, collagen, epinephrine, etc.). Is done. Therefore, in this example, the gel particle generating instrument 100 in which a certain amount of ADP or the like is aseptically placed together with the magnetic stirring rod 107 in the sample cell 101 and lyophilized or the like is processed is created. It is possible to measure the degree of aggregation ability by introducing plasma diluted appropriately at the site through the upper sealing plug 108 and measuring the generation time of aggregates with the same gel particle measuring apparatus as in the first embodiment. Become.
  • agglutination-promoting reagent for example, ADP, collagen, epinephrine, etc.
  • FIG. 15 -Antigen-antibody reaction
  • specific antibodies 310 against various antigens 300 are associated to promote inactivation of the antigen 300 as an insoluble precipitate, and have a protective action on the living body.
  • the specific antibody 310 is prepared in advance using this phenomenon, the generated precipitate is proportional to the amount of the antigen 300 present, and therefore, various methods for quantifying the antigen 300 have been devised.
  • various detection methods and sensitive detection devices have been developed.
  • the precipitation formation of the antigen-antibody reaction is regarded as gelation particle formation, it is considered that a gel particle measuring apparatus and a gel particle generating instrument that stably form particles and measure them are applicable.
  • a detection reaction of a type in which an antibody 330 is bound to a microbead 320 such as a resin and an antigen-antibody reaction is caused with the antigen 300 on the bead surface. It is easy to grasp by changing the pattern of particle formation, and can be applied to the method. Therefore, in the sample cell 101 of the gel particle generating device 100, together with the magnetic stirring rod 107, a certain amount of antibody solution bound to the antibody 330 or the microbead 320 is aseptically placed.
  • a test solution such as plasma diluted to a certain degree is introduced through the upper sealing plug 108, and the rate of aggregate formation due to the antigen-antibody reaction is measured, for example, with the gel particle measuring apparatus of the first embodiment.
  • the rate of decrease in transmitted light is measured in order to capture the rate of gel particle generation.

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Abstract

L'invention concerne un instrument produisant des particules de gel possédant une cellule cylindrique pour échantillon (1) dans laquelle un échantillon (S) est injecté et contenu et qui comprend une partie de transmission dont au moins une partie est capable de transmettre une lumière, un réactif (2) préalablement introduit dans la cellule pour échantillon (1) qui réagit avec une substance souhaitée se trouvant dans l'échantillon (S) et qui se gélifie, un organe d'agitation (3) préalablement introduit dans la cellule pour échantillon (1) qui agite une solution mélangée (W) pour empêcher la gélification de la totalité de la solution mélangée (W) composée de l'échantillon (S) injecté et du réactif (2), et un organe d'étanchéité (4) qui ferme hermétiquement une ouverture de la cellule pour échantillon (1) dans laquelle se trouve le réactif (2) et l'organe d'agitation (3) et à travers lequel l'échantillon (S) peut être injecté dans la cellule pour échantillon (1) après la fermeture hermétique de l'ouverture. L'opération d'agitation par l'organe d'agitation (3) commence quand l'échantillon (S) est injecté dans la cellule pour échantillon (1), et des particules de gel sont produites sans que la totalité de la solution mélangée (W) se gélifie.
PCT/JP2009/066273 2008-09-30 2009-09-17 Instrument produisant des particules de gel et dispositif de mesure de particule de gel l’utilisant Ceased WO2010038628A1 (fr)

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WO2012053515A1 (fr) * 2010-10-18 2012-04-26 Obata Toru Réactif de mesure de particules de gel et procédé de mesure l'utilisant
CN119246330A (zh) * 2024-10-09 2025-01-03 集美大学 一种基于光散射效应的胶凝时间测定仪和方法

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CN106680246A (zh) * 2016-12-31 2017-05-17 北京科兴中维生物技术有限公司 一种检测类毒素絮状单位的装置及其应用

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CN119246330B (zh) * 2024-10-09 2025-10-28 集美大学 一种基于光散射效应的胶凝时间测定仪和方法

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