JPS6132621B2 - - Google Patents

Description

[Detailed description of the invention]

The present application concerns the detection of substances derived from microbial fluids or tissues and loaded with fluorescent chromium dyes. According to the invention of this application, antigens, antibodies, hormones,
Detection of enzymes, drugs and other substances is performed. Many infectious diseases of bacterial or viral origin are
It causes antibodies to be produced in the patient's serum. This creates immunity if challenged again in the future by the same infectious agent or infectious antigen. One method of detecting the presence of a specific antigen is to add a specific antibody that binds to the antigen. If this antibody has a radioactive element that does not interfere with immunity (RIA technique)
Alternatively, if a fluorescent dye is added, the bound complex can be detected with a suitable detector. When using fluorescent adducts, the best semi-quantitative measurements in the prior art are mostly determined by visual inspection on a microscope slide. Known fluorescent test methods include quantitative or, in the best case, semi-quantitative analysis. For example, in most fluorescence techniques performed on microscope slides, the detector is a laboratory technician who records the degree of fluorescence as 0, +1, +2, +3, or +4. If blood titers or concentrations of antibodies are to be determined, a number of slides are prepared containing various concentrations of the test substance. Thus, the technician can prepare the serum or bacterial broth medium with distilled water for 11 minutes.
4, 1:16 or 1:28, etc., to estimate a +4 reaction in the microscope. The ability to quickly, accurately, automatically and quantitatively read titers using a fluorometer without diluting serum would be of great benefit to medical scientists and clinical specialists. Known fluorometers were designed to measure liquid systems and were not able to detect fluorophores derived from microbial fluids or tissues on solid surfaces. One reason for this defect is that excessive (e.g., substantially 99% or more) emitted fluorescent light is lost between the fluorescent material and the detector portion that converts the light into an electrical signal. It's something you get used to. There are many reasons why RIA techniques are not completely satisfactory. One is that the presence of small amounts of antigen yields only a small number of counts per second. Since the "noise" of the system is the square root of the number of signal counts, accuracy errors become large at these low signal levels. Furthermore,
Due to half-life issues, radioisotopes have a short shelf life and require special authorization, handling and disposal. The present invention relates to a method for testing sample material derived from fluorescent microbial fluids or tissues. In particular, the invention relates to a method and a fluorometry system for detecting substances of this type. The fluorescence measurement method of the present invention measures a sample coated on a solid substrate. This method includes a light source that excites fluorescent light in the substrate and a photoconductor that guides the light from the light source of the sample. Detector means measure the intensity of the fluorescence emitted by the substance and display the measurement. The detector means comprises known means for converting the light intensity into an electrical signal. Fluorescent light is transmitted from the sample to the conversion means by suitable light-conducting means whose rear end is adjacent to the sample. This terminal end is identified herein by the phrase "incoming end" of the detector. The distance between the sample and the electrical conversion means defines the optical path, but this optical path does not include a gap distance that would allow excessive loss of useful fluorescence passing through the optical path (ie, more than 95% cumulative loss). Therefore, if the fluorescent light is transmitted from the sample to the conversion means by means of a light-conducting means, such as a fiber optic cable, the gap distance at either end of the cable will not exceed the levels mentioned above. The coated substrate examined with a fluorometer consists of a single sample coated on a body member (carrier, support). Alternatively, in order to provide a main body member capable of detecting and measuring one or more types of sample substances, the main body member may have a plurality of, for example, band-shaped application areas in which different substances are applied at different distances. A single application area may have different randomly dispersed materials loaded with different fluorescent chromium dyes. A particularly effective fluorescence measurement system includes bifurcated fiber optic cables for conducting light from the light source to the sample and emitted fluorescence from the sample to the detector. One of the branches transmits light from the light source to the sample, and the other transmits fluorescent light to the detector. These branches are
It is combined with a normal fiber bundle at the rear end at the light input end. In this way, the area of coincidence of excitation and emission is maximized and the gap distances are significantly closer. Another preferred fiber optic system has at least two fiber optic cables for transmitting the emitted light to the detector and means disposed between the cables for alternating the input to the detector. This method allows at least two coated areas on a single substrate to be read without moving the substrate, such as when comparing a standard amount of sample with one or more unknown samples. Similarly, the means for transmitting light that excites fluorescence and the means for transmitting light that receives fluorescence are both branched optical cables, and the configuration is such that light of multiple wavelengths is sent to the sample and different fluorescence signals are received. You can also. The invention and some aspects of practicing the invention will now be described with reference to the accompanying drawings. The present invention relates to a fluorescence measurement system and method for quantitatively detecting and measuring a fluorescent sample material coated in a layer on a substrate. The phrase "fluorescent sample material" as specified herein is derived from microbial secretions or tissues, alone or in combination with other materials;
Refers to a substance that emits fluorescence when excited by light of a selected wavelength in a solid layered form. Common fluorescent sample substances include self-fluorescent substances derived from microbial secretions such as tetracycline, substances obtained by adding fluorescent chromium dye to such secretions before or after isolation, and Mention may be made of substances originating from the secreted fluid and bound in the layer to homogeneous fluorescent chromium dye adducts, such as antigens or antibodies already attached to the fluorescent chromium dye. The present description will proceed in particular with respect to the last-mentioned substances. One embodiment of the invention, shown by way of example in FIGS. 1, 2 and 3, has a body member or ball 10 made of plastic, e.g. ) etc., supporting a dry film or coating 11 of antibodies against the antigen to be measured. All balls are applied at approximately the same temperature (37°C)
Since the incubation times are approximately the same, for example 30 minutes, each ball will have essentially the same amount of antibody. This is important for obtaining quantitative results. For example, in the ball 10 shown in Fig. 4, if the diameter is 10 mm, the total surface area is
It is ^314mm2 . Regardless of the plastic material used, the ball or body member 10 is not a microscopic object, typically about 5 to 20 mm in diameter, so that an area of at least 1 square millimeter can be observed with a fluorometer. . In this embodiment, this ball is dropped into a pot 12 having a diameter of 12 mm. When detecting fluorescent light using a ball in a pot, the pot must be made of a material, such as glass, that does not emit fluorescent light at the measurement wavelength and prevents transmission of 30% or more of the fluorescent light. 1 ml of patient or subject serum 13 is added to coat the ball 10 and incubated for 5 minutes at room temperature with gentle rocking of the acupoint. If Australian antigen 14 is present, the antigen is ball 10
It binds to antibody 11 above. A lid 16 with a 5 mm hole 17 is placed on the open end 18 of the pot 12 and the pot 12 is inverted to allow the serum to flow out. A small second hole 20 is provided in the lid 16 to allow air to circulate. It is preferable that the inner surface 21 on the base 22 of the acupoint 12 is round or approximately semicircular, so that when the ball is used to measure fluorescence within the acupoint 12, the position of the ball 10 is determined and the fluorescence is emitted. The ball does not roll during testing. After incubation with the serum of the subject, the pot is washed with, for example, a phosphate buffered aqueous solution or distilled water, and then the washing water is allowed to flow out through the hole 17 with a diameter of 5 mm, leaving the ball 10 with a diameter of 10 mm inside the pot 12. Next, 1 ml of an antibody solution containing a substance that emits fluorescence under ultraviolet light is added to the pot. As this type of fluorescing substance or fluorescent labeling substance, for example, sodium fluorescein isocyanate and other substances can be used. However, sodium fluorescein isocyanate is preferred as it is excited at 460 nanometers and emits fluorescence at 520 nanometers. The material in the pot 12 is incubated again as above, the liquid is allowed to flow out through the hole 17 and the bulb is washed as above. If the Australian antigen is present, ball 10 will thus support antigen 14 and fluorescent antibody 23.
The acupoint 12 and the body member 10 are now ready to be investigated using a fluorometric method. In the fluorescence measurement method, the pot 12 is placed at a position where the fiber optic cable 24 transmits ultraviolet light from a light source 27, which is the desired light source, and gelatin or gel is placed to ensure that only the excitation wavelength light reaches the bulb or main body member. When a beam of light is transmitted through the filter 28 and then through the wall of the vase 12 and impinges on the coated surface of the body member 10, the beam excites the bonded complex 23 to emit fluorescence. Second fiber optic cable 25
is preferably arranged at a small angle, that is, at an angle of 30° or less, from the cable 24, and only the emitted fluorescent light is transmitted to the photomultiplier tube 29 via the fiber optic cable 25.
Gelatin filter 30 that reliably reaches
Sends the emitted fluorescent light through. The photomultiplier tube 29 converts the intensity of the emitted fluorescent light into an electrical signal.
This signal is sent to the filter 31, processor 31
a [This is, for example, peak-to-peak
A device that converts an alternating current signal into a direct current signal through a -to-peak detector and makes the relationship between the fluorescent light intensity and the direct current voltage a linear relationship, for example by a fourstep diode linearizer. It is. ], is sent to an amplifier 32 and an analog-to-digital converter, and is displayed directly on a titer scale on a digital panel meter 34 (see FIG. 7). In the embodiment shown in FIG. 3, a nylon cylinder 3 is used instead of the ball 10 as the main body member.
5 is used. In this embodiment, the cylinder 3
The upper surface of the cylinder 35 is coated with the same fluorescent substance used to apply the antibody coating 38 on the lower part of the cylinder 35, and is measured by a detection device used for testing or analysis, which in this example is a fluorometer. A standard fluorescent coating 36 having a known potency is applied when applied. It is preferable to leave a blank space 37 on the surface of the cylinder 35 that separates the coating film 36 and the lower coating film 38 . The lower coating 38 contains a streptococcal fluorescent antibody, which allows only the lower area to be immersed in an antibody solution to determine the presence of the suspect antigen or antibodies in the serum being tested. The body member 10 is prepared in the same manner as described above with respect to body member 10, except that it is immersed. In this embodiment, an ultraviolet light source 27, a fiber optic cable 24, and a filter 28 are provided. Two cables 39 and 47 are provided for filters 39a and 47a. One cable 39 transmits the fluorescent light from the standard fluorescent coating to the photomultiplier tube 29 and the other cable 47
The fluorescent light emitted from the lower coating film 38 is transferred to the photomultiplier tube 2.
9. A cutting wheel 46 operated by a motor 45 rotates to alternately direct streams of light from each coating 36 and 38 into tube 29. In this way, a direct comparison can be made between the standard part and the test part. In the embodiment shown in FIG.
and a paddle-shaped main body member 40 having a leg portion 42 and a wide flat head portion 43 provided at the other end,
The head 43 supports a coating 44 of the sample. In this embodiment, the two cables, the fiber optic cable 24 for the excitation beam and the fiber optic cable 25 for the emitted fluorescence beam, are parallel to each other, ie they make an angle of 0 DEG to each other. Cables 24 and 25
It is also possible to arrange them coaxially, and this arrangement is convenient. Other elements of the apparatus and method are as previously described and illustrated. Another example of a multiple test stylistic element is shown in FIG. In this embodiment, the stylistic element is a cube 50 located at the other end of the handle 51. cube 50
has four faces, of which faces 52 and 53 are observable. Each side has a different sample. 4
Different types of fluorophore adducts can be provided and the fluorometer is equipped with a filter wheel having four selected wavelength ranges to isolate the energy going to the photomultiplier tube 29. Each test can be read sequentially by the operator rotating the cube 50 or other shaped polyhedral body member. Similarly, the handle may be attached to a cylinder, sphere or other substrate. Means for moving the substrate coated with layers of materials derived from different microorganisms may be employed for use in changing the surface exposed to the fiber optic cable. Broadly speaking, the strip cylinder shown in FIG. 3 and the cube shown in FIG. It can be said that it shows two shapes. The shape of the substrate can be any shape as long as it has a first area of fluorescent chromium dye-loaded sample material and at least one area of fluorescent chromium dye-loaded material. In different areas of this kind,
A standard area containing a predetermined amount of material of the same type as the fluorescent chromium dye-added material may also be provided. In this way, for example, bands 36 and 3 in FIG.
Two different areas such as 8 can be observed to directly compare the strength of the standard and test sections. One technique for performing the multiple measurements described above is to use the same photomultiplier tube in combination with a cutting wheel and multiple fiber optic cables. Alternatively, multiple photomultiplier tubes and fiber optic cables can be used without the use of a cutting wheel. As mentioned above, instead of a substrate for detecting a specimen with fluorescent chromium dye added to a standard sample,
Different test specimens of the same type or different types can also be deposited in separate areas of the same sample substrate support or body member. As discussed below, the wavelength of the fluorescent excitation light can also be varied for different areas. A modified fluorometer 60 is shown in FIG. 8 that uses one branched fiber optic cable 61 instead of two separate cables 24 and 25. cable 61
The unitary portion 62 is guided to a solid substrate 63 having a fluorescent surface 64 and directed outward from there. One branch 65 of the cable 61 is connected to a lamp 66
or any other light source to suit (blue)
It is sent to a fluorescent surface 64 via a filter 67. A second branch 70 of the same cable 61 transmits the fluorescent light emitted from surface 64 via a suitable (green) filter 71 and lens 72 to a solid state or photomultiplier detector 73. Although there are readily apparent differences, the operation is essentially the same as in FIG. FIG. 9 shows another modification of the fluorescence measuring device 80. In this embodiment, fiber optic cable 61 replaces branch fiber optic cables 81, 82, 83 and 84. The bundled base 86 of the cables is led to a base 87 with a fluorescent surface 88 and from there directed outwards. Branches 81 and 82 direct light from lamps 89 and 90, respectively, or other light sources to a fluorescent surface 88. Branches 83 and 84 of the same cable 86 transmit the fluorescent light emanating from surface 88 through suitable lenses 91 and 92, respectively, to solid state or photomultiplier detectors 93 and 94. One method of using the very convenient apparatus shown in FIG. 9 is to observe a surface 88 in which a plurality of substances of microbial origin are randomly distributed. Each substance is attached to a fluorescent chromium dye that fluoresces in response to different wavelengths of light. Thus, lamps 89 and 90 emit fluorescent excitation of different wavelengths, and the plurality of fluorescent lights are simultaneously received by detectors 93 and 94 via light transmission branches 83 and 84, respectively. A plurality of randomly distributed fluorescent chromium dye adducts can also be read using a single branch fiber optic cable as shown in FIG. In this case, a single lamp or light source is used in place of lamps 89 and 90, and multiple filters are used to obtain light of the appropriate wavelength to excite each fluorescent chromium dye in the sample. Similarly, if the wavelength to which the detector responds is synchronized with the selected fluorescent chromium dye being excited, optical transmission cables 83 and 84 can be
It is also possible to replace two cables and replace the detectors 93 and 94 with one detector. An important feature of the fluorescence measurement system according to the invention is to maximize the fluorescence light received from the sample. This feature is particularly important when the fluorophore is present at very low concentrations. It has been found that this objective can be achieved by avoiding gap distances in the optical path between the fluorescent material and the means for converting the light intensity into an electrical signal for quantitative measurements. If a fiber optic cable is used to transmit light from an input end adjacent to the sample to the conversion means, this gap includes the distance between the input end and the sample material and the distance between the optical cable and the conversion means. Become. It has been found that the cumulative fluorescence loss through the gap distance in the optical path at the average fluorescence intensity should not exceed a minimum of 95% of the effective fluorescence flowing along the optical path. This loss does not include the loss due to observing only the fluorescent sample surface portion. Taking this loss into account, it relates only to the fluorescent light rays leaving the sample within the area defined by the entrance edge periphery that projects above the sample surface. Gap loss of fluorescence through the entire optical path
Although suppression to 95% is a significant improvement over conventional fluorometers, it is desirable to suppress this loss to 50-90% or less, most preferably 10% or less. If the loss level is set to this level, even a very small amount of sample can be detected and measured quantitatively. The above discussion relates primarily to fiber optic cables and light pipes and refers to the importance of proximity, measured in gap distance from the receiving surface to the receiving surface. The light transmission system may include, for example, a lens that focuses the collected light, a bell that reflects the light and changes its direction, and a hole that allows the light to pass through after being dispersed. When using hemispherical elements that receive light from light-diffusing surfaces, the amount of light these elements capture is determined by the hemispherical surface that produces a radius equal to the gap distance between the emitting surface and the receiving element. is approximately proportional to the portion of π steradians defined by the circumference projected on . Based on the above relationship, a circumference that results in a loss of no more than 95% of the emitted fluorescence corresponds to a circumference that forms a solid angle of about 0.3 steradians. When the cross section is not circumferential, the solid angle is specified by the equivalent circumferential area. Another technique for avoiding fluorescence loss is to avoid placing a solid medium in the gap between the sample coating and the entrance end of the detector that would prevent the transmission of excess amounts of fluorescence light.
It has been found that glasses of moderate thickness, 1.27 mm (0.05 inch) or less, or certain plastics, such as polystyrene, cause less than 30% fluorescence loss. Although it is preferable to avoid the presence of solid media of this type, this loss can be accepted if necessary or expedient for the system. For example, in the embodiment shown schematically in FIG. 1, it is convenient to use a thin-walled pot to hold the circular coating substrate. In that case, it must be made of a material that does not cause a loss of more than 30% of the fluorescence. FIG. 10 schematically depicts a typical fiber bundle 86 with a branch cable 80, in which a number of fibers have been enlarged for clarity. Fibers are represented by closed circles, open circles, circles with an "x" and circles with an "r". It is clear that in this arrangement the four different types of fibers are randomly arranged. Although not shown, special optical effects can also be achieved using fibers arranged concentrically or with different diameters. Studies of fluorescence measurement methods have shown that an important factor contributing to this percentage loss is the ratio of the effective diameter of the input end of the emitted light receiver to the gap distance between the samples. The phrase "effective diameter" means the diameter of an entrance end with a circular cross-section or the equivalent diameter in the case of a non-circular cross-section. This phrase is approximated by the following equation. Area: πd ² /4 The effective diameter d′ of a non-circular cross section is

It is specified by [Formula]. The relationship between the gap distance and the effective diameter ratio is based on the approximate relationship that the intensity of fluorescent light is inversely proportional to the square of the distance from the fluorescent material. This approximation can be achieved by minimizing the angle of reflection, ie the angle between the ray of light transmitted to the fluorophore to excite the fluorophore and the ray of light received by the entrance end of the detector means. Does not take into account light increase. This number was found to be less important than the gap distance. The effective diameter of the light entrance end is
This is clearly important because as the area increases, the amount of light captured increases. Using the above calculation, the loss of emitted fluorescence can be calculated as
The gap distance adjacent to the sample should be 95% or less.
The ratio of the gap distance to the effective diameter of the light entrance end is approximately 5:1.
It corresponds to: Similar calculations are performed to determine theoretical ratios for other fluorescence loss percentages. It should be understood that this ratio is only an approximation. Gap distances in the optical path, for example between the fiber optic cable and the detector section that converts light into electrical signals, and between lenses and mirrors used in the optical path, can also be calculated using the same formula. The branched fiber optic system shown in FIGS. 8-10 is particularly effective for minimizing gap distance and minimizing loss of emitted fluorescent light. This is based on the principle that only the area that can be received by the detector, which is sent to the fluorescent substance to excite the fluorescent light, corresponds to the observation area of the light entrance end of the detector. Relatively small values of gap distance can be achieved using a separate fiber optic cable as shown in FIG. As the gap distance decreases, the overall coincident area of the separate excitation and emission beam cables decreases continuously. It is clear that this fact is a limiting factor for the gap distance and thus the possibility of causing excessive fluorescence loss of very small amounts of sample material. On the other hand, by using a branched cable consisting of a plurality of optical transmission fibers terminating in a normal fiber bundle at the light input end, the fluorometers can be placed very close to each other without causing a lack of consistency. be able to. The only constraint on this is that as the gap distance approaches zero, the thin fibers of the fiber bundle act as independent cables. For example, in the case of the multi-branched embodiment shown in FIG. 9, ordinary fiber bundles are particularly effective. A cable having separate input and output ends for each branch of cable 80 requires sufficient substantial gap distance to ensure sufficient matching area. The above description has cited fiber optic cables as the preferred light transmission means. For distances that do not require conventional fiber optic bundles, other optical transmission means such as light pipes and lenses may also be used. The foregoing description also illustrates the fluorescence measurement method of the present invention by means of microbial secretions or tissue loaded with certain fluorescent chromium dyes. This substance is used in tests to determine the presence of pathogenic bacteria or their toxins in body fluids, such as serum, urine, or other fluids, or to determine the concentration of other substances in secretions. In general, this type of fluorescent test substance belongs to the class of substances that are selectively or spatially compatible and paired with the mating substance. Pairs of this type are antigen-antibody, enzyme-substrate, binding protein-hormone, binding protein-vitamin, enzyme-inhibitor, etc. However, other fluorescent sample materials may also bind to the substrate in pairs, such as by physical capture or sorption. The systems and methods of the present invention are applicable to the detection of a wide variety of fluorescent sample materials. Areas of application include drugs for acute diseases such as morphine, methazone, cocaine and barbiturates;
Drugs used to control certain chronic diseases or conditions, such as digoxin (heart disorders), insulin (digitalis) and diphenylhydantoin (epilepsy); hormones such as thyroxine and triiodothyroxine; aldosterone, Steroids and hormones such as cortisol, testosterone, estriol and progesterone; peptides, hormones and proteins such as adrenocorticotropin, angiotensin, gastrin, choroidal gonadotropins, follicle-stimulating hormone, neurophycin, placental lactodiene, growth hormone and thyroid storage hormone. Hormones; vitamins such as cyanocobalamin and folic acid; enzymes such as chymotrypsin, creatine phosphokinase, alkaline phosphatase and conjugate dehydrogenase; antigens such as carcinoembryonic antigen, hepatitis-related antigens and alpha fetoprotein. Antibodies such as anti-toxoplasmosis antibodies, anti-thyroid antibodies, anti-toxoplasmosis antibodies; Bacteria, fungi, protozoa, cell forming bodies such as red blood cells and alpha leukocytes; fibrinogen and anti-hemophilic factors, fatty acids proteins, serum proteins such as immunoglobulins and thyroxine-binding globulin; cell degradation products such as myoglobin, bacterial toxins and lytic enzymatic digests. Becoming fluorescent through proteins, substances, inhibitors, enzymes, antigens, or antibodies that are fluorescent or bind specific fluorescent labels, or through direct labeling. physical adsorption, special protein binding, immunoadsorption, substrate or inhibitor binding, physical entrapment within the pores of the matrix, ion conversion, and other methods before and after direct or indirect labeling. Other materials can also be used as long as they can be deposited onto the surface by. Among the various types mentioned above, an important system is the antigen-
It is an antibody pair. Generally, an antigen is defined as a substance that can evoke an antibody response. The term antigen reaction is intended to include not only the narrow meaning of antibody production and binding with antibodies in vivo, but also in vitro conditions that can be used to bind substances that are not normally antigens to other substances. For example, thyroxine-binding globulin (TBG), a normal component of human blood, can be employed as the first layer of a sandwich on a solid surface to bind thyroxine ( _T4 ), another normal component of human blood. I can do it. Thus, _T4 -TBG binding is an analog of antigen-antibody binding, and _T4 can be analyzed fluorometrically in the fluorometers described herein. As such, a protein binding reaction can generally be treated as a relationship between a substance and a protein that tends to bind the substance. The antigen-antibody reaction also serves as an important example of general cases involving the binding of certain drugs, hormones, and enzymes to their respective substrates and immunological substances, and is a general concept. should be understood as encompassing or indicating a generic concept. In one embodiment described above, a fluorescent chromium dye doped material (e.g. antigen) is applied in a film onto the external surface of a substrate or solid body member, such as a ball, cylinder or flat plate, which acts as a mobile substrate. . The substance is a first type of protein (e.g. an antigen) suspected of being present in the serum of the subject or patient.
or it may be a second type of substance (eg, an antibody against that type of antigen).
The substance may also be a hormone, enzyme, or other protein of interest. The second standard body (scale body), which has already been exposed during manufacture to an antigen solution of known titer, already has all three layers and, with proper manipulation, can be used for example +4 at a titer of 128. , can read known values on the instrument. Adjust the instrument if necessary to correct the scale. The immunized body exposed to the patient's serum is then inserted into a fluorometer and the resulting titer is read on a quantitatively calibrated digital meter. For example, in the case of cylinders, plates, cubes, or other suitable non-circular geometries, the method described above using multiple areas coated with fluorescent chromium dye-added material may be employed.
For example, as described above, a portion of the body member, such as a cylinder or plate, may be coated with a standard fluorescent chromium dye having a known scale value. Antigens, fluorescent antibodies, etc. may be applied to other parts of this type of body in the longitudinal direction for testing in the same manner as the spherical immune body. A fiber optic cable is directed to each of the two parts of the cylinder to first read the internal scale and then read the test results.
The signal to the photomultiplier is alternately switched using a "cut" wheel or rotating sector disc or by electronic switch switching. By using this internal standard, the step of removing the necessary test pot and replacing it with a standard pot when not in use can be omitted. When using antibodies or antigen solutions,
The concentration is a concentration widely known in the art. For example, the titer is 1:128 for the Staphylococcus test and 1:8 for Neisseria gonorrhoeae. The standard technique for coupling sample material to a substrate is referred to by immunologists as the "Sandermansch technique." In this case, antibodies against the specific pathogen whose presence is to be determined are coated onto the substrate. Many plastics have properties that allow them to directly bind complex proteins, of which antibodies are one type. A typical example of this is the test for the ``Australian antigen'' in the blood, which is considered prima facie evidence of hepatitis infection. The anti-Australia antigen-antibody is attached in a film on a plastic substrate. Serum from a subject suspected of having the antigen is applied onto the antibody film. If the antigen is present, it will immunologically bind to the antibody film. After washing with water to remove any unbound material, yet another fluorescently tagged antibody is added. This antibody binds to the antigen if it is present. A final wash is performed to remove any unbound fluorescence-imparting antibody. If fluorescence is emitted from the coating film, it is detected by the fluorescence measuring method according to the present invention. In addition to the above-mentioned "Sandermansch Technique",
Other standard immunofluorescence techniques can also be used, such as indirect techniques. When it is desired to measure antibodies against syphilis in a subject, for example, Treponema pallidum is prepared and its antigen is applied to the main body member. The body member is used to culture the subject's serum and then washed. Next, a fluorescent non-human antibody (from goat or rabbit) is added, incubated and washed. If human antibodies against syphilis are present in the serum, the antibodies will attach to the main body parts due to the immune reaction and be captured by fluorescent antibodies obtained from goats, for example, so that the titer of syphilis can be reduced. Can be read quantitatively. This is a modification of the standard FTA-ABS test (fluorescent syphilis antibody-absorption), in which case the system of the body part and the immunofluorometer according to the invention allows for accurate quantification. Any infectious disease that produces antibodies can be easily analyzed using this technique. Diseases of this type that are relevant to public health include syphilis, gonorrhea, strep throat infections, dysentery, salmonella infections, typhoid fever, rabies, serum hepatitis, influenza, etc. The invention is also useful for quality control in the food and pharmaceutical industries. Furthermore, as another application example using the above-mentioned technology,
Valuable not only for diagnosing the presence of pathogenic microorganisms (antigens), but also for determining the protective immunity (antibody titer) of the body against pathogenic microorganisms resulting from dilberate innoculation or smallpox. That could be a method. For example, a doctor can inject a child with D.P.T. serum to provide immunity to diphtheria toxin, pertussis or spasmodic cough, and tetanus. Therefore, the doctor estimates that all three of the above types have been acquired. Testing sera for each can, in fact, determine if all three antigens are effective enough to generate immune antibodies. Existing technology does not allow for this simple method of differentiation. Although not included in antigens or antibodies in the strict sense, the following combinations can be cited as examples of the system according to the present invention that employs protein binding methods. (1) Thyroxine ( _T4 ) and thyroxine-binding globulin (TBG) (2) Thyroxine ( _T4 ) and thyroxine-binding prealbumin (TBPA) (3) Intrinsic factor and vitamin B-12 (4) Insulin and alpha-2- Macroglobulin (5) Cortisol and trans-cortin (6) Haptoglobin and hemoglobin In each case, one of the pair (the first type of protein) is bound to a solid substrate, and the other of the pair (the first type of protein) is bound to a solid substrate. (a second type of protein capable of binding to a second type of protein) is removed from the serum and allowed to bind, which is then exposed to a fluorescing molecule of the first type of protein. These examples and other common uses are quite similar to the antibody-serum antigen-labeled antibody system described for some of the immunological reactions. Other variations use other fluorescent labels, such as those exemplified below. As a fluorescent label,
Lissamine Rhodamine B, D.A.N.S. (1
Mention may be made of fluorescent amine dyes which are often used in fluorescence microscopy. The first two have more orange or red emission spectra than yellow, green or green fluorescence, and the latter two have blue or green emission spectra. The modifications required to the fluorometer described herein are to change the excitation and emission filters used and to change the fluorescent label attached to the antibody in the reaction vessel. Another important modification is microanalysis using fluorometers. In this case, instead of a large fiber optic bundle, for example 10 mils (0.010
Bundles or sometimes single fibers of very small diameter (up to 1 inch) are used. A small diameter microball (eg 50-100 microns in diameter) is coated with the antibody for the test in question. The reason why the acupoint and the reaction agent ball are made so small is to enable immunofluorescence analysis of a minute sample of serum. This is a very attractive method because it is much easier to obtain blood from a finger prick than from an arm venipuncture for population differential planning. This method allows population identification of infants for pediatric testing of diseases in newborns or very young children. It may be desirable to determine the presence of one or more microorganisms. For example, symptoms that send a patient to a urologist suggest a kidney infection, urinary tract infection, or bladder infection. To determine whether any microorganisms respond, place the urine specimen on a number of different growth media using conventional microbial plates.
Subsequent culturing is required for 24 to 48 hours. Furthermore,
Different microorganisms require different antibiotic treatments. The fluorometer may be equipped with a filter wheel to allow selection of several different wavelengths for several specific fluorochromes, such as those exemplified below, which may be of interest in causing urinary tract infections. Three microorganisms are present, namely E. coli, pseudomonas and staphylococcus.
By attaching each label to a different antibody against the genus Staphylococcus and Staphylococcus, one, two, or all three types can be measured and identified simultaneously. Examples of fluorescent labels used in this case are: (1) Fluorescein isothiocyanate (yellow to green) (2) Lissamine rhodamine B-200 (deep orange) (3) D・A・N・S (1 -dimethylamino-naphthalene-5-sulfonic acid), (red) (4) Ortho-phthalaldehyde (blue to green) (5) Fluoresamine (red to green) It is convenient to prepare a test body with several bands of individual samples, as described elsewhere in the book for the case of a single measurement. Standard bands may be added or removed as desired. Alternatively, the body can be provided with a coating consisting of a mixture of several samples. It is also possible to culture a single body part in a mixture of antibodies against several microorganisms, and if all antibodies are present in equal concentrations, there is enough on the polymeric chains of the plastic to pick up an equal amount of each antibody. There must be a bond position. The coated substrate according to the present invention should be distinguished from microbeads and the like.
The area is at least 1 mm. In the case of a ball or cylinder, the diameter is preferably 5 to 20 mm or less, and in the case of a flat plate or cube, the width is preferably 20 mm or less. A minimum area of 1 mm provides a microscopic surface of the detector, allowing collection of the fluorescent beam from large conglomerates of attached molecules, thus reducing sample-to-sample sampling errors. The use of polymers (plastics) to bond sample materials to substrates has been described herein. Polymeric materials of this type may include polymethyl methacrylate, polystyrene, polyamides (nylon) or other known polymers capable of binding substances by physical adsorption of proteins. As other substrates which can bind the test substance directly or with the aid of mediated protein binding, mention may be made of older types of adsorbents such as activated carbon, silica, alumina, ion exchange resins or dextran. . Proteins can also be immobilized on materials by physical adsorption, as reported in Science News, May 18, 1974, page 324.
Also, a number of techniques have been developed to immobilize or fix proteins that can serve as coating layers on substrates.
For example, the technology for immobilizing enzymes is developed by The Chemical Co., Ltd.
The Chemical Rubber
"Immobilized Enzymes" by Zaborsky, published in 1973 by
(Immobilized Euzymes). For example, a report on proteins such as antibodies and antigens suitable for this purpose was published by Campbell and Ueliki, 1942, Academic Press, New York City, New York.
"Immunology and Immunochemistry" by Weliky)
(Immunology and Immunochemistry), Volume 1. As these authors point out, the attachment of proteins to other substances, such as enzyme substrates and enzyme inhibitors, may be due to their entrapment within the pores of the stretched substrate matrix and to chemical locations on the substrate. or by chemical immobilization of a physically adsorbed substance coating the substrate with a drug such as glutaraldehyde. Covalent bonds can also be created using ceramic materials, such as glass, as described by Weetal in US Pat. No. 3,652,761. Other substrates prepared by the above-described or other known techniques capable of achieving surface adhesion of test substances are also suitable. The first three examples are illustrative of the actual process of carrying out the invention with different techniques and reading the test results with the apparatus described herein.
In either case, the goal is to determine the presence and amount of gonococcal infection. Example 1 Sandersch Technique Process for Neisseria gonorrhoeae: 1 A 10 mm diameter nylon ball coated with Neisseria gonorrhoeae antibody is inserted into a pot. 2 Add enough patient serum to coat the ball. Incubate for 5 minutes at room temperature and allow the liquid to drain. 3. Add enough fluorescent gonococcal antibody solution to coat the ball. Incubate for 5 minutes at room temperature and drain off the liquid. 4. Wash thoroughly with buffer solution, then pour out the solution. 5 Place the pot into a fluorescence meter and read the titer. If antibodies are present in the subject's serum, indicating the presence of gonococcal infection, the meter will indicate a specific titer concentration with a "+". Example 2 Thyroxine Sanderutsch Technique This example is the same as Example 1 except for the following differences. In step 1, the solid body is coated with thyroxine-binding globulin (TBG) instead of gonococcal antibodies. In step 3, a fluorescent antibody against fluorescent TBG or _T4 is used (instead of a fluorescent antibody solution). Step 5 therefore displays the titer of thyroxine. Example 3 Indirect method steps for Neisseria gonorrhoeae: 1. Drop a 10 mm nylon ball into the pot. This ball is pre-coated with gonococcal antigen (dry film of bacteria). 2 Add enough serum to coat the ball. After 5 minutes of incubation at room temperature, the serum is poured off. If antibodies against gonococcal infection are present in the patient's serum, the antibodies will adhere to the applicator ball. 3. Add fluorescently labeled non-human immunoglobulin (obtained from rabbit or goat and commercially available from many sources). Incubate for 5 minutes and pour out the liquid. 4. Wash thoroughly with buffer solution, then pour out the solution. 5 Place the pot on the fluorescence meter and read the result. Example 4 Indirect methods against other bacteria Using appropriate antigens and antibodies in each case, the technique described in Example 3 can be used to treat virulence praxis, Streptococcus, staphylococcal infections and syphilis (Syphilis) infections. This method is well suited for Example 5 Competitive inhibition step: 1 A 10 mm diameter nylon ball coated with gonococcal antibody is dropped into the first pot. 2 Add 1 ml of serum from the control person and 1 ml of the prepared fluorescently labeled antibody solution to the second pot. Mix briefly and add the contents of the first jar. 3. Incubate at room temperature for 5 minutes and pour off the liquid. 4. Wash thoroughly with buffer solution and pour out the solution. 5. Place the pot into a fluorescence meter and read the results. Note: In this example, if the subject is antigen-free (uninfected), all antibody binding sites on the nylon ball will bind to the labeled antigen and therefore produce the maximum fluorescent signal. emanate. The more antigen (infection) there is in the subject's serum, the more completely the binding site will bind to non-fluorescent antigen molecules. Therefore, the greater the titer, the greater the reduction in the fluorescent signal. In this embodiment, it is necessary to use a known electronic circuit to imprint a scale curve indicating that the lower the signal value, the greater the degree of infection. Example 6 This series of measurements shows that the sensitivity of the method according to the invention to fluorophores on solid substrates is different from that of two known fluorometers used for the same purpose using a thin layer chromatography plate scanning accessory. For, the order is 1
It shows that the improvement has been improved to a different degree. The dye used was fluorescein isothiocyanate (FITC) diluted in barbital buffer at pH 8.6.
It is. Aliquots of the various dilutions were spotted onto the surface of polymethyl methacrylate and allowed to dry.
This spot was excited at a wavelength of 480 nm, and the fluorescence emission was read at a wavelength of 520 nm. I got the following readings.

【table】

[Table] Note) *Percentage of signal was calculated from 10.0 nanograms after subtracting the blank continuation value.
The low level detection ability achieved by the present invention is that the light incident end of the optical fiber used on the sample surface is 0.8 mm.
This is largely due to the close proximity and subsequent conservation of captured light energy. The Turner fluorometer is designed to capture only a portion of the emitted fluorescence that passes through a 0.2 x 0.2 cm slit at a 45 degree angle 7 cm from the sample surface. The Aminco-Bowman fluorometer uses an approximately diameter
Although it is designed to capture most of the emitted fluorescence at the input end of a 3.175 mm (1/8 inch) fiber optic cable, it is located where an acupoint would normally be placed to observe a liquid sample. A fiber optic cable is used as an accessory to transmit the captured luminescence to the mirror. The light beam reflected from this mirror radiates in three dimensions, and only a small part of it is about 1.9 cm from the mirror.
(3/4 inch) away into the first member of another light transmission means, a 0.2 cm x 1 cm entrance hole. Because of its construction, the Turner and Amin-Kobaumann fluorometer described above has been calculated to fail to transmit more than 1% of the picked up emission light to the detector. Example 7 These measurements demonstrate the importance of unprotected proximity to obtain maximum fluorescent signal from fluorescent materials on solid substrates. A 0.635 cm (1/4 inch) diameter fiber optic bundle was used to measure a 6 mm diameter spot. The observed substance is
Goat anti-rabbit globulin to which 1.0 micrograms of fluorescein isothiocyanate had been added was diluted with a phosphate buffer solution of pH 7.6, spotted on a polymethyl methacrylate surface etched with acid, and dried. Excitation and observation were performed through randomly placed fibers in a bifurcated fiber optic bundle. The excitation wavelength was 480 nm and the measured emission wavelength was 520 nm. The readings shown below were obtained.

[Table] Example 8 This example shows the fluorescence measurement of an antibody-antigen reaction when an antigen in a solution is labeled with a fluorescent label and reacted with an antibody immobilized on the surface. . In this so-called "direct" method, the surface fluorescence is proportional to the antigen concentration in solution. Polyamide strips were coated with anti-streptolysin O by soaking for 30 minutes while stirring and gradually adding 1% glutaraldehyde diluted 1:8 in a saline solution. . After washing with water and drying the strip, the strip is exposed to solutions of streptolysin O toxin at various concentrations diluted in distilled water. To attack the antigen label, 2 ml of each concentration was first mixed with 0.2 ml of a fluorescent amine dye solution (40 mg dissolved in 100 ml of acetone).
I reacted. The strips are then added, stirred for 30 minutes, removed, washed with water and dried. These strips are then placed in an instrument and excited with a wavelength of 375 nm,
Fluorescence was measured at a wavelength of 475 nm. The results shown below were obtained.

[Table] Example 9 This example shows the fluorescence measurement of bacteria in suspension, which were bound onto a surface and reacted with a fluorescently labeled antibody using the so-called "Sand-Germany" technique. be. The fluorescence on the surface is proportional to the bacterial concentration of the sample. Samples of Streptococcus betahaemolyticus type A were cultured in tryptic broth, autoclaved, centrifuged, and washed with phosphate buffered saline (PBS). Cells in PBS
Reconstituted to concentrations of 10 ⁷ , 10 ⁵ and 10 ⁵ cells/ml. The DEAE-cellulose pieces were added to a 1:16 dilution of antiserum in phosphate buffer with 0.5 ml of 50% diluted with stirring.
The strips were coated with Streptococcus A antiserum by immersion for 15 minutes while glutaraldehyde was slowly added. After washing, each piece was incubated for 5 minutes in 5 ml of each of the above concentrations, and then washed with PBS. Each piece was then incubated for 5 minutes in a 1:4 dilution of fluorescein isocyanate-labeled Streptococcus A antibody in phosphate buffer, washed with buffer, and air-dried. Each piece was then placed into the instrument and measured, giving the following readings:

Table: Example 10 This example describes the fluorescence measurements of multiple surfaces exposed to a common solution containing several substances desired to be measured. Four cellulose squares, each containing a different covalently linked immunoglobulin antibody (non-human sheep I _g A, I _g E, I _g G and I _g M) and placed on a piece of plastic, were diluted in the appropriate buffer. Place in the serum sample. After 2 hours of incubation, the pieces are removed and the four squares are washed with buffer. The sheep anti-human _Ig A, Ig was then loaded with an equal active concentration of fluorescein isocyanate diluted in a suitable buffer _.
React with E, I _g G and I _g M solutions. Wash each piece once more, air dry, and read each area individually on the instrument. Thus, the relative concentrations of the various immunoglobulins are determined. Example 11 This example describes the fluorescence measurement of a common surface exposed to a solution containing several substances for which individual measurements are desired. A circular plate of polymethyl methacrylate containing a random mixture of three different covalently bound anti-drug antibodies (rabbit anti-morphine, anti-cocaine and anti-barbiturate) placed on a piece of polymethyl methacrylate was Expose to urine sample diluted in buffer. After 1 hour of incubation, the pieces are removed and the discs are washed with buffer. This is then incubated in a solution containing a mixture of rabbit antibodies against the drugs. Each antibody is labeled with a different fluorescent chromium dye. That is, fluoresamine-adducted rabbit antimorphine,
Using a solution diluted with a suitable buffer solution, fluorescein/isocyanate-added rabbit anti-cocaine and lissamine-rhodamine B200-added rabbit anti-barbiturate were used.
Perform time culture. Each piece is removed, washed with buffer and dried. These are then placed in the instrument one after another, first excited at 390 nm and reading the emission at 485 nm, then excited at 480 nm and read at 520 nm, and finally
Excite at 520nm and read at 595nm. The presence of morphine, cocaine or barbiturates is thus determined. The above description and drawings are provided for illustrative purposes, and it is understood that changes and modifications may be made within the technical scope of the present invention without departing from the spirit of the claims. . Those skilled in the technical field to which the present invention pertains will be able to make changes to the structure of the present invention and widely change the embodiments and applications of the present invention without departing from the spirit and technical scope of the present invention. It is easy to do so. The disclosure and description herein are presented purely for illustrative purposes and are not intended to limit the invention in any way.

[Brief explanation of the drawing]

FIG. 1 is a schematic top view of an embodiment of the invention, with the pot lid removed. Second
The figure is a partial elevational sectional view taken along line 2-2 in FIG. FIG. 3 is a schematic illustration of another embodiment of the invention in which the solid body member is a cylinder. FIG. 4 is a schematic cross-sectional view of a solid body member coated with antibodies, with the size and thickness of the antigen and fluorescent antibodies attached in accordance with one aspect of the present invention exaggerated for illustration purposes; Shown. FIG. 5 is a schematic perspective view of another embodiment of the object and device according to the invention, consisting of a flat plate with a handle. FIG. 6 is a schematic perspective view of yet another embodiment employing a cube with a handle for use in multi-sample testing. FIG. 7 is a diagram showing an embodiment of the detection device of FIGS. 1 and 3. FIG.
8 and 9 are schematic diagrams similar to FIG. 1 showing a modification of the fluorometer. Figure 10 shows 10-
10 is a cross-sectional view of the conventional fiber optic cable of FIG. 9 taken along line 10; 10... Ball (body member), 11... Dry coating film, 12... Pot, 13... Serum, 14... Antibody, 16... Lid, 17... Hole, 18... Open end, 20 ...pore, 23...binding complex, 24,
25...Fiber optical cable, 27...Light source, 28
...Filter, 29...Photomultiplier tube, 30...
...Filter, 31...Filter, 31a...
Processor, 32...Amplifier, 33...Analog-to-digital converter, 34...Digital panel meter, 35...Nylon cylinder (main body member), 36...Standard fluorescent coating, 37...Blank area , 38... Antibody coating film, 39, 47... Cable, 39a, 47a... Filter, 40... Paddle-shaped main body member, 41... Handle, 42... Leg, 43
... Head, 44 ... Paint film, 45 ... Motor, 46
... Cutting wheel, 50 ... Cube, 51 ...
Pattern, 52, 53... face.

Claims (1)

[Scope of Claims] 1. A method for fluorometric measurement of an unknown quantity of a sample, comprising: (a) dispersing the antibody or antigen into at least one non-granular, non-intumescent, impermeable, continuous, flat surface of the body; (b) to the antibody or antigen-binding surface of step (a) an antigen or antibody in solution that is reactive with said antibody or antigen and a fluorescent chromated antibody or antigen in solution; (c) after completion of step (b), a solution containing the unbound fluorescent chromium-conjugated antibody or antigen to the reacted surface; (d) washing the reacted surface of step (c); and (e) transporting the body part to a detection station of a fluorometer for detection from the washed surface of step (d). A method characterized by quantitatively measuring the amount of fluorescent light emitted.