JPH06347463A - Optical inspection method - Google Patents

Abstract

PURPOSE:To accurately inspect antigen-antibody reaction by detecting optical echo out of an examined sample where antibodies are mixed with the mixture of a specimen and an optically sensitive material. CONSTITUTION:In general, since a specimen exhibiting antigen-antibody reaction, has no absorption of light in a visible region wave length, therefore, after optically sensitive material such as rhodamine 640 and the like has been mixed with the specimen, it is then mixed with antibodies to be formed into the specimen 8, so that optical echo inspection is made possible. Now, light from a light source 1 is divided in two by an optical divider 2 to be controlled by a controller 11. After light has passed through light delay means 3 delaying light by the specified quantity of delay, and also through a phase modulation means 4 modulating light by the specified quantity in phase, lights are then joined together by a light joining means 5. The joined light beam is made to pass through a lens 6, so that the light beam is focused on a sample 8 within a cooling means 7. Next, optical echo from the sample 8 is detected by a light detector 9, so that an output signal is forwarded to a data processing means 13 via an electrical signal processing means 12 and the like. Based on information on phase relaxation time, the intensity of optical echo and the like, the presence and/or the absence, the kind, and the degree of antigen and antibody reaction can be detected.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for optically examining the presence or type of an antigen-antibody reaction in a test sample by measuring optical echo from the test sample.

[0002]

2. Description of the Related Art The antigen-antibody reaction has been used as a method for inspecting substances (antigen, antibody) derived from the inside and outside of living tissue by utilizing its extremely high specificity, as an inspection method for various diseases. Various methods have been developed so far for the examination of antigen-antibody reaction. For example, there is a method using an antibody labeled with a fluorescent substance (a kind of probe) (fluorescent antibody). This is a direct method in which the antibody is labeled with a fluorescent substance in advance (labeled antibody) and then mixed (reacted) with the antigen,
There is an indirect method in which an antigen is mixed (reacted) with an antibody (primary antibody), and then a secondary antibody labeled with a fluorescent substance is further mixed (reacted).

The mixed (reacted) test sample is observed with a fluorescence microscope, and the presence or type of the antigen-antibody reaction is judged from the fluorescence amount or fluorescence pattern. When an antigen reacts with a corresponding antibody and aggregates, there is a method of determining the presence or absence of an antigen-antibody reaction by examining the pattern of the reaction, that is, the degree of aggregation.

[0004]

However, in the conventional method as described above, the observer observes the sample to be inspected with the naked eye or a microscope, so that it may be difficult to make a determination or a difference in determination may occur between individuals. Therefore, there was a problem that it was not possible to make an objective decision. The present invention has been made in view of the above problems, and provides a method for objectively and accurately testing for the presence or type of an antigen-antibody reaction.

[0005]

Therefore, as a result of diligent research, the present inventor measured the optical echo from the sample to be examined, and obtained information [phase relaxation time (T ₂ ), optical echo intensity ( It is possible to detect the presence or absence of an antigen-antibody reaction, and the type and degree, based on the ratio of the intensity of the optical echo derived from the zero phonon line to the intensity of the optical echo derived from the phonon sideband, etc.). Invented the invention.

That is, in the inspection method of the present invention, the first step of "mixing" the object to be inspected and the photosensitive substance, and the second step of mixing the mixture obtained in the first step with the antibody And the third step of detecting the optical echo from the test sample obtained in the second step and examining the presence or type of the antigen-antibody reaction.

[0007]

In the present invention, "mixing" means simply mixing, reacting, binding, staining, or labeling. The optical echo is extremely sensitive to the difference in the structure and constituent components of the substance, and these differences appear in the difference in phase relaxation time (T ₂ ).

The phase relaxation time (T ₂ ) will be described as an example with reference to FIG. Consider a case where a substance is excited by pulsed light (excitation light). First, at t _1, the pulsed light of E ₁ is irradiated,
Subsequently, at time t ₂ , pulsed light of E ₂ is irradiated. Next is irradiated with pulsed light of t ₃ at E _3, [t ₃ + t ₂ -t ₁ =
At time t ₃ + τ (τ = t ₂ −t ₁ )], light is emitted from the substance. This is a light echo. And
The optical echo intensity is attenuated in proportion to exp (-4τ / T ₂ ). When this τ is changed and the intensity of the optical echo is measured each time, T ₂ can be obtained. T ₂ is different for each substance or each state of the substance. Therefore, by measuring T ₂ , the substance or state can be detected. This measuring method is disclosed in JP-A-4-132020.

The present invention utilizes this optical echo. In order to measure the optical echo, the test sample must have absorption at the wavelength of the excitation light (generally laser light). That is, the object to be measured (test sample) must be a light absorber for the excitation light. Generally, a test sample to be tested for an antigen-antibody reaction does not absorb light having a wavelength in the visible region. Currently, there is no laser for measuring an optical echo that has a wavelength in the ultraviolet region, and therefore the above-described test sample does not absorb excitation light. Therefore, in the present invention, the light-sensitive substance having absorption at the wavelength of the excitation light and the test object are “mixed” in advance. Since this mixture becomes a light absorber for the wavelength of the excitation light, it becomes possible to measure the light echo immediately after the antigen-antibody reaction.

Hereinafter, the present invention will be specifically described by way of examples, but the present invention is not limited thereto.

[0011]

EXAMPLE 1 FIG. 1 shows that the applicant of the present invention is Japanese Patent Application No. 4-28470.
1 is a configuration diagram of an optical echo measurement device proposed in Japanese Patent Publication No. 1 for measuring an optical echo from a test sample. Light source 1
Has a spatial coherence and a predetermined temporal coherence,
Light having a coherence shorter than the optical phase relaxation time of the light absorber of interest is supplied to the test sample.
The light source 1 is composed of, for example, a dye laser excited by a mode-locked argon ion laser. The optical delay means 3 is
Corner cube 3 fixed to a movable stage (not shown)
a and a displacement device 3b that displaces the movable stage, and delays the light due to the change in the position of the corner cube 3a.

The phase modulating means 4 is a corner cube 4
a, a piezoelectric element 4b, and an AC drive source 4c. One end of the piezoelectric element 4b is fixed and the corner cube 4a is fixed to the other end. In the phase modulation means 4, the AC drive source 4c applies an AC voltage of a predetermined frequency f to the piezoelectric element 4b to vibrate the piezoelectric element 4b at the frequency f. Further, the corner cube 4a is vibrated at the frequency f by the vibration of the piezoelectric element 4b. The vibration of the corner cube 4a phase-modulates the light at the frequency f. In this embodiment, the phase modulation frequency is 21 KHz.

The photodetector 9 is a photomultiplier tube (photomultiplier). The sharp cut filter 14 transmits only the light on the longer wavelength side of the irradiation laser light.
The lock-in amplifier 10 is a signal processing unit that extracts a modulation component that is twice the modulation frequency of the phase modulation unit 4. In this embodiment, the lock-in amplifier 10 is used for detecting the optical echo.
The lock-in frequency of was set to 42 KHz.

The light emitted from the light source 1 is split into two light beams by the light splitter 2. An optical delay unit 3 is arranged on the transmission optical path of the optical splitter 2 so as to generate a predetermined amount of optical delay. Further, on the reflected light path of the light splitter 2, the phase modulation means 4 is arranged so as to generate a predetermined amount of phase modulation. The optical delay means 3 and the phase modulation means 4 are controlled by the control means 11. The two light beams that have passed through the optical delay unit 3 and the phase modulation unit 4 are combined by the optical combiner 5.

The light flux multiplexed by the optical multiplexer 5 is reflected by the lens 6
It is focused on the sample 8 to be inspected in the cooling device 7.
Instead of the lens 6, an optical system of a microscope may be used and light may be condensed while observing the test sample. The optical echo from the test sample passes through the sharp cut filter 14 and is detected by the photodetector 9. The output signal of the photodetector 9 is
It is processed by the lock-in amplifier 10. The output signal of the lock-in amplifier 10 is sent to the data processing device 13 via the electric signal processing device 12.

The phase relaxation time (T ₂ ) of the test sample is obtained by recording the change in the intensity of the optical echo caused by sequentially changing the optical path length difference between the two light beams by the optical delay device. Cultured human laryngeal cancer-derived cells (HEp-2) were used as test objects. As the antibody, an anti-nucleolar antibody and an anti-DNA antibody contained in serum were used. The cultured human laryngeal cancer-derived cells (H
Ep-2) was "mixed" with XRITC, a dye for cell staining (photosensitive substance). This "mixed" and the above two antibodies are mixed respectively. The test sample thus obtained was used. When XRITC is represented by a chemical formula,

[0017]

[Chemical 1]

[0018] When these two types of test samples were inspected by the above-described optical echo measurement device, the two could be clearly distinguished from each other due to the difference in the phase relaxation time (T ₂ ). That is, it was possible to distinguish between different types of antigen-antibody reaction. In addition, as a dye (photosensitive substance) for cell staining, rhodamine 640, Texas red and its derivatives, 5 (6) carboxyrhodamine succinimidyl ester [5 (6) CARBOXY-X-RHO
DAMINE SUCCINIMIDYL ESTE
Similar results were obtained using R]. Rhodamine 6
40 is represented by a chemical formula,

[0019]

[Chemical 2]

[0020] The chemical formula of Texas Red is

[0021]

[Chemical 3]

It becomes 5 (6) carboxyrhodamine succinimidyl ester is represented by a chemical formula:

[0023]

[Chemical 4]

It becomes Further, the photosensitive substance may be any substance as long as it can measure a light echo. For example, it may be an enzyme. In the case of an enzyme, after "mixing" the enzyme, a drug may be mixed to form a light absorber for excitation light. Further, in the present embodiment, when measuring the optical echo, using the device as described above, but is not limited to this,
Anything that can measure an optical echo may be used.

[0025]

[Second Embodiment] FIG. 2 shows that the present applicant filed Japanese Patent Application No. 4-98643.
It is the optical echo measurement device proposed in the publication. Unlike the device of the first embodiment, in this device, the light transmitted through the test sample is detected by the photodetector 9.

When a test sample similar to that of Example 1 was measured with this apparatus, it was possible to distinguish between them as in Example 1. That is, it was possible to distinguish between different types of antigen-antibody reaction. Further, when the test sample was a very strong scatterer, the scattered light was condensed by the lens and the light was detected by the photodetector 9, and the two could be similarly distinguished.

The other structure is the same as that of the first embodiment.

[0028]

[Third Embodiment] FIG.
This is the optical echo measurement device proposed in Japanese Patent No. 4 publication. This device has the same arrangement as the confocal laser scanning microscope, and detects only the light that has passed through the pinholes at the confocal position on the sample to be detected by the photodetector.

When the same test sample as in Example 1 was measured with this apparatus, it was possible to distinguish between the two as in Example 1. That is, it was possible to distinguish between different types of antigen-antibody reaction. Other configurations are the same as those in the first embodiment.

[0030]

Example 4 Cultured human laryngeal cancer-derived cells (HEp-2) were used as test objects. As the antibody, those containing antinuclear antibody in serum and those not containing antinuclear antibody in serum were used. The object to be inspected and XRITC, which is a dye (photosensitive substance) for cell staining, were “mixed” with each other. Each "mix" is mixed with serum containing anti-nucleolar antibody. The test sample thus obtained was used.

These test samples were tested in Examples 1 and 2 and 3.
When a test was conducted in the same manner as above, since the photosensitizer was not present in the test sample in which the antinuclear antibody was not present, the light echo was not measured, and the light echo was detected in the test sample in which the antinuclear antibody was present. Was measured. Therefore, the two could be clearly distinguished by the presence or absence of optical echo. That is, it was possible to distinguish the presence or absence of an antigen-antibody reaction.

[0032]

INDUSTRIAL APPLICABILITY As described above, the present invention provides a test method capable of detecting the presence or type of antigen-antibody reaction. By using this method, the examination of the antigen-antibody reaction, which has hitherto relied on the observation with the naked eye or the microscope, can be expressed in an objective numerical value, and an accurate examination can be performed.

[Brief description of drawings]

FIG. 1 is a conceptual diagram of an optical echo measurement device by the applicant.

FIG. 2 is a conceptual diagram of an optical echo measurement device by the applicant.

FIG. 3 is a conceptual diagram of an optical echo measurement device by the applicant.

FIG. 4 is a principle diagram of a conventional optical echo measurement.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Light source 10 ... Lock-in amplifier 2 ... Optical splitter 11 ... Control means 3 ... Optical delay means 12 ... Electric signal processing device 3a ... Corner cube 13 ... Data processing device 3b ... Displacer 14 ... Filter 4 ... Phase modulator 15 ... Mirror 4a ... Corner cube 16, 17 ...
Objective lens 4b ... Piezoelectric element 18 ... Lens 4c ... AC drive source 19 ... Semitransparent mirror 5 ... Optical multiplexer 20 ... Optical trap 6 ... Lens 21 ... Scanning Means 7 ... Cooling device 22 ... Scan drive means 8 ... Test sample 23 ... Pinhole 9 ... Photodetector

Claims (3)

[Claims] 1. A first step of "mixing" a test object and a photosensitive substance; a second step of mixing the mixture obtained in the first step with an antibody; and the second step. And a third step for detecting the presence or type of an antigen-antibody reaction by detecting a light echo from the test sample obtained in the above step. 2. The optical inspection apparatus according to claim 1, wherein the object to be inspected is a cultured cell. 3. The optical inspection method according to claim 1, wherein the antibody is contained in serum.