JP4290019B2 - Fluorescence immunoassay method - Google Patents

Description

The present invention relates to a fluorescence immunoassay method for observing an antigen-antibody reaction on the surface of an optical waveguide.

  An analysis method using an immune reaction is excellent as a method for selectively and simply measuring a specific measurement object from a sample having a lot of impurities, and is becoming widespread in fields such as food, medicine, and the environment. Especially in the food field, due to the diversification of food, the spread of eating out and eating out, and the development of distribution, the causes of food poisoning tend to be different from the conventional ones, and mass food poisoning that is highly pathogenic over a large scale and wide area has occurred. Therefore, rapid inspection before shipment is required as a measure against food poisoning bacteria.

  As an analysis method using an immune reaction, a precipitation reaction, an agglutination reaction, or the like has been conventionally used as a direct or indirect measurement method of a conjugate by an antigen-antibody reaction. Furthermore, the measurement sensitivity is dramatically increased by a labeled immunoassay method in which the antigen-antibody reaction is quantified using a label such as a radioisotope, a fluorescent substance, a luminescent substance, or an enzyme. A radioimmunoassay method using a labeling method, an enzyme immunoassay method, a fluorescence immunoassay method, and the like are known, and several devices are commercially available.

  In fluorescence measurement, noise components are easily included due to high sensitivity, and various measures have been taken to ensure the reliability of the measurement value in the fluorescence immunoassay method. As one method, excitation by evanescent waves is used. Use. This method, in which light is totally reflected inside the optical waveguide and the fluorescent dye is excited by the evanescent wave that oozes out to the outer surface, can selectively observe the reaction that occurs only near the surface of the optical waveguide. Suitable for observation of reaction.

  In addition, by performing the total reflection in a multiplexed manner, the excitation light can be used more efficiently and the sensitivity can be improved. As an example of the fluorescence immunoassay method using multiple total reflection, a method using a fiber-type optical waveguide immersed in a sample solution is already known in Patent Document 1. Patent Document 2 discloses a fiber-type optical waveguide having a unique shape portion in order to increase the efficiency of total internal reflection.

US Pat. No. 4,582,809 US Pat. No. 6,136,611

  In the fluorescence immunoassay based on the antigen-antibody reaction, the specific binding substance is captured regardless of whether the object to be measured is live or dead. Therefore, there may be inconvenience if the dead food poisoning bacteria are detected without distinction from the live bacteria. For example, in the production process of food, when it is determined that a harmless bacteria killed by heat treatment is positive and the product is collected, the cost and damage of the collection become enormous.

  In the method of measuring microorganisms with high sensitivity, rapidity and simpleness by the fluorescence immunization method, in order to detect only viable bacteria, it is necessary to devise an operation procedure in the measurement.

An object of the present invention is to provide a fluorescence immunoassay method capable of solving the above-mentioned problems and measuring only viable bacteria with high measurement sensitivity and reliability by a simple operation.

The fluorescence immunoassay method according to the present invention for achieving the above object is the fluorescence immunoassay method for observing an immune reaction on the surface of the optical waveguide, wherein the optical waveguide and the specimen are brought into contact with each other and captured on the surface of the optical waveguide. A labeled antibody having a fluorescent chromophore is bound to the measured object, the fluorescent chromophore is excited by an evanescent wave generated by introducing excitation light into the optical waveguide, and propagates through the optical waveguide. A first step of observing the collected light, and a second step of culturing the specimen in the same container as the container for contact with the optical waveguide after the first step, The first step is repeated at least once after the second step, and the change in the amount of observation light accompanying the culture is measured.

According to the fluorescence immunoassay method of the present invention, viable bacteria can be detected with high sensitivity, speed, convenience, and reliability by measuring microorganisms grown by culture by fluorescence immunoassay using an optical waveguide.

The present invention will be described in detail based on the embodiments shown in the drawings.
FIG. 1 is a side view of the optical probe 1 and FIG. 2 is a plan view. The optical probe 1 is manufactured by, for example, polystyrene molding by injection molding, the upper part is a lens part 3 with the flange part 2 as a boundary, and the lower part is The optical waveguide 4 is used. The intermediate portion of the optical waveguide 4 is a rod portion 5 used for antibody fixation, and the tip is a light absorption portion 6.

  FIG. 3 is a configuration diagram of a basic measurement optical system. The optical probe 1 is fixed in the container 11 containing the specimen using the flange portion 2. A semiconductor laser light source 13 that emits a 635 nm laser beam via a beam splitter 12 is disposed above the lens portion 3, and a detector 14 made of, for example, a photodiode is disposed on the side of the beam splitter 12. The optical probe 1 performs excitation light introduction and fluorescence collection in the container 11 in the optical waveguide 4 which is a sensor part, and the entire optical system becomes compact, stable and easy to maintain. Become.

  Further, since the specimen is located outside the rod portion 5 in the container 11, it has an advantage that various operations such as immersion and washing can be easily performed. On the other hand, however, there is a disadvantage in that reflected light and scattered light of the excitation light are easily mixed with fluorescence. This disadvantage is considerably solved by disposing a black body in the light absorption part 6 on the end face of the optical waveguide 4 and introducing an optical filter in the measurement optical system. However, it is substantially effective when dealing with a small amount of fluorescence. It often becomes a problem.

In addition, even when the dead food poisoning bacteria are used for an application that does not cause a problem, the specific binding substance fixed to the optical waveguide 4 is used to improve that the measurement object is detected without distinction between life and death. In addition, if viable bacteria are present in the specimen, they are proliferated by culture. That is, of the amount of light observed by the detector 14, the fact that the amount of fluorescence from the fluorescence-labeled antibody observed by the antigen-antibody reaction with the living bacteria increases according to the growth of the living bacteria. The basic measurement procedure of this embodiment is as shown in the flowchart of FIG.
(1) The optical waveguide 4 is brought into contact with the specimen in the container 11, a labeled antibody having a fluorescent chromophore is bound to the measurement object captured on the surface of the optical waveguide 4, and excitation light is introduced into the optical waveguide 4. The fluorescent chromophore is excited by the evanescent wave generated in this way, and the light collected by propagating through the optical waveguide 4 is observed by the detector 14.
(2) If the amount of light is the same as the previous one by this observation, the measurement is terminated because there is no viable bacteria. If the amount of light has changed, step (3) is performed.
(3) The specimen is cultured in the container 11 under suitable conditions.
(4) The above steps (1) and (2) are performed again.
A series of steps is repeated at least once, and the change in the amount of observation light accompanying the culture is measured by the detector 14, and if there is an increase in the amount of observation light, it is attributed to the growth of viable bacteria.

  In this measurement procedure, when non-specific adsorption that is not attributable to the antigen-antibody reaction of the labeled antibody is strong, an increase in fluorescence due to this may be observed. When there is such a fear, it is desirable to contact with the labeled antibody only prior to the main measurement and saturate the nonspecific adsorption in advance.

  If there is a possibility that fluorescent impurities may be present in the sample, measure the blank sample that does not include the measurement target by exercising the same procedure as the main measurement prior to the main measurement. Also good. This operation is also effective when the non-specifically adsorbed component once bound is eluted for some reason. The net amount of fluorescence can also be derived by subtracting the amount of fluorescence obtained in the blank measurement from the amount of fluorescence obtained in the subsequent measurement.

  The method for capturing the measurement object on the surface of the optical waveguide 4 can use direct physical adsorption or capture using an adsorbent prepared on the surface in advance, but is specific to the measurement object fixed on the surface in advance. Capturing with a substance that binds to the antibody, more preferably capturing with an antibody, is desirable as a highly selective method. After the contact between the optical waveguide 4 and the specimen and after the contact between the optical waveguide 4 and the labeled antibody solution, it is effective to perform cleaning with an appropriate liquid before proceeding to the next operation. Absent.

  For the contact between the specimen and the optical waveguide 4, an arrangement in which the container 11 containing the specimen is rotated and the rod-shaped optical waveguide 4 is placed in a portion parallel to the rotational axis except on the rotational axis in the container 11 is suitable. . The specimen container 11 used for the measurement of the fluorescence signal can be removed and moved to an incubator, and culture can be performed under appropriate conditions. Alternatively, culture can be performed in a fluorescence immunoassay apparatus having a container 11 with a temperature control mechanism inside, and at this time, there is no need to take out the container 11 once. Therefore, fluorescence immunoassay including culture is automated. It becomes possible to do.

  As the container 11 into which the specimen is placed, a container having an open top can be used. However, in order to prevent contamination when culturing is performed, a structure that can be opened and closed without changing the container 11 is more preferable.

  In addition, the sample is a liquid medium containing foods, environmental materials such as appliances and production lines, water such as drinking water, soil, and feces. Liquid materials can be used as they are, and fluids, powders, and solid specimens having high viscosity can be diluted with a diluent and homogenized by a stomacher. Tools and production lines such as cloths and cutting boards that can be collected by the stamp method can be used, and these are added to a liquid general bacterial culture medium or a selective culture medium as a specimen.

  FIG. 5 is a configuration diagram of the measuring apparatus. The output from the detector 21 including the configuration of FIG. 3, that is, the output of the detector 14 is connected to the personal computer 23 via the signal processor 22. The output of the personal computer 23 is connected to a motor 24 that rotates the sample container 11. Further, a temperature control unit 25 and a liquid feed pump 26 are connected to the sample container 11 of the detection unit 21, and a buffer solution is supplied from a reservoir tank 27 to the liquid feed pump 26.

  The optical waveguide 4 is immersed in the sample container 11 filled with the sample, and captures the measurement object. At this time, the specimen container 11 is rotated by the motor 24 to facilitate the capture. Furthermore, the conjugate | bonded_body of an antigen antibody reaction is formed by contacting with a labeled antibody, and the light quantity of a fluorescence signal is measured in the detector 14 of the detection part 21. FIG. Here, cleaning of the optical waveguide 4, introduction of 635 nm laser light from the semiconductor laser light source 13, and concentration of fluorescence are performed. The fluorescence is detected by the detector 14 from the lens portion 3 of the optical waveguide 4 through the lens and the filter. In addition, the culture is performed using the temperature control unit 25 under appropriate conditions.

[Experiment 1]
An Escherichia coli O157: H7 antibody (manufactured by Kirkegaard & Perry Lab. Inc.) is immobilized on the surface of the optical waveguide 4, and the unbound portion of the antibody on the surface of the optical waveguide 4 is 0.5% Tween 20 containing 0.4% casein. The following operation was performed with a measuring apparatus using what was blocked with 0.01 M phosphate buffer containing (trade name).

(A) First, in order to obtain a signal due to nonspecific adsorption, the optical waveguide 4 contains 2 μg / ml fluorescently labeled antibody (Amersham Biosciences, Inc .: labeled with Cy5 bisfunctional reactive dye) in the container 11. It was immersed in a buffer solution and allowed to stand at 25 ° C. for 5 minutes.
(B) The container 11 was washed with 2 ml of buffer solution.
(C) Again, the sample container 11 was filled with the buffer solution, and the fluorescence signal was measured three times. This confirmed in advance a slight signal increase and saturation due to nonspecific adsorption.
(D) Subsequently, in order to obtain a signal from the antigen-antibody reaction, a 10 ml sample to which Escherichia coli O157: H7 and viable cells inactivated in TSB medium (manufactured by Nissui Pharmaceutical Co., Ltd .: Tryptosoya bouillon) were added was used as a sample. Injected into container 11. The optical waveguide 4 was immersed in the specimen, and the specimen container 11 was rotated by the motor 24 at 100 rpm for 5 minutes.
(E) The container 11 was washed with 2 ml of buffer solution.
(F) A buffer containing 2 μg / ml of fluorescently labeled antibody was placed in the container 11, the optical waveguide 4 was immersed therein, and allowed to stand at 25 ° C. for 5 minutes.
(G) The container 11 was washed with 2 ml of buffer solution.
(H) Again, the buffer solution was filled in the sample container 11 and the fluorescence signal was measured.

  After the measurement was completed, the specimen was cultured at 42 ° C. for 90 minutes in an incubator, and then a measurement for obtaining a fluorescent signal by the antigen-antibody reaction was performed again. In addition, after each measurement was completed, the colony-forming units of the specimen were counted using an agar medium to obtain the number of bacteria.

  FIG. 6 shows the fluorescence signal intensity before and after the culture as an increase from the measured value of the nonspecific adsorption. Moreover, the number of bacteria of the specimen used for each measurement is shown, and both the signal and the number of bacteria are increased by the culture of the specimen. At this time, no increase in signal due to non-specific adsorption was observed.

[Experimental example 2]
In the same manner as in Experimental Example 1, the Escherichia coli O157: H7 antibody was immobilized, and the signal increase due to the nonspecific adsorption was confirmed using the optical waveguide 4 subjected to the block treatment. Subsequently, in order to obtain a signal from the antigen-antibody reaction, 10 ml of a sample prepared by adding inactivated Escherichia coli O157: H7 and viable cells to a raw scallop scallop that was added 9 times TSB medium and homogenized by a stomacher Was injected into the specimen container 11 and the fluorescence signal was measured in the same manner as in Experimental Example 1.

  After completion of the measurement, the specimen was moved to a temperature-controlled part in the measurement apparatus, cultured at 42 ° C. for 90 minutes, and then subjected to measurement for obtaining a fluorescent signal by antigen-antibody reaction again. Moreover, after completion | finish of each measurement, the colony formation unit of the test substance was counted using the agar medium, and the number of bacteria was calculated | required.

  FIG. 7 shows the fluorescence signal intensity before and after the culture as an increase from the measured value of the nonspecific adsorption. Moreover, the number of bacteria of the specimen used for each measurement is shown. As the sample was cultured, both the signal and the number of bacteria increased, and no increase in signal due to nonspecific adsorption was observed.

It is a side view of an optical probe. It is a top view. It is a block diagram of a measurement optical system. It is a flowchart figure of a measurement operation. It is a block diagram of a measuring device. It is a graph figure of the measurement data in example 1 of an experiment. It is a graph figure of the measurement data in example 2 of an experiment.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Optical probe 2 Flange part 3 Lens part 4 Optical waveguide 5 Rod part 6 Light absorption part 11 Container 12 Beam splitter 13 Semiconductor laser light source 14 Detector 21 Detection part 22 Signal processing part 23 Personal computer 24 Motor 25 Temperature control part 26 Liquid sending pump

Claims (5)

In a fluorescence immunoassay method for observing an immune reaction on the surface of an optical waveguide, the labeled optical antibody and a specimen are brought into contact with each other, and a labeled antibody having a fluorescent chromophore is bound to a measurement target captured on the surface of the optical waveguide. A first step of exciting the fluorescent chromophore with an evanescent wave generated by introducing excitation light into the optical waveguide, and observing the light collected by propagating through the optical waveguide; A second step of culturing the specimen in the same container as the container for contact with the optical waveguide after the step, and repeating the first step at least once after the second step, A method for measuring fluorescence immunity, characterized by measuring a change in the amount of observed light accompanying the measurement.   Prior to the first step, the evanescent wave generated by bringing the labeled antibody into contact with the surface of the optical waveguide and introducing excitation light into the optical waveguide propagates through the optical waveguide and is collected. The fluorescence immunoassay method according to claim 1, further comprising a step of performing a series of observation steps at least once and measuring in advance the amount of observation light caused by the non-specific adsorption component.   3. The fluorescence immunoassay method according to claim 2, wherein the measurement is automatically interrupted when the amount of light observed due to the non-specific adsorption component is larger than a preset value.   The fluorescence immunoassay method according to claim 1, wherein a substance that specifically binds to the measurement target is fixed in advance in the optical waveguide.   The fluorescence immunoassay method according to claim 4, wherein the substance that specifically binds to the measurement target is an antibody.

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