JPS607230B2 - How to detect antigens or antibodies - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention detects immune reactions with high sensitivity, determines the concentration of immunoreactive biological particles in a specimen, and quantifies antigen-antibody reactions without the need for specific staining methods. The present invention relates to a method and apparatus for making a visible and durable record of measurements on a solid substrate surface.

The immune response involves a first immunoreactive biochemical particle (generally a protein) known as an antigen being directed against this antigen.
It is a very unique biochemical reaction in which it combines with a second protein known as an antibody to create an immunological anchor protein.

Immune responses that occur within biological systems, such as animals or humans, are of great importance in fighting disease. In biological systems, proteins in the outer shell,
That is, when an antigen enters the body, the biological system makes a specific antibody protein against this antigen, but the mechanism is not yet fully understood. Antibody protein molecules have chemical binding sides that are complementary to those of the antigen molecule such that the antigen and antibody bind to form an immunological complex. Most antigens are or have an essential part of a protein, whereas all antibodies are proteins.

Proteins are molecules of large molecular weight, ie, polymers consisting of chains of variable numbers of amino acids. Conventional immunology (e.g., pending U.S. Patent Application Serial Nos. 457,092, 266,278, 384113, and 445)
No. 204), it was said that any protein adheres to the surface of the substrate only as a monomolecular layer and that no other arbitrary proteins adhere to this initial protein layer. Proteins that are particularly reactive with a protein are immunoconjugated with it.According to the prior art techniques listed above, a substrate surface, such as a slide, to which a monolayer of one type of protein has been adsorbed, is used to immunobind with the protein that is specifically reactive with it. Based on the aforementioned discoveries, medical diagnostic devices have been developed to test suspect solutions for the presence of proteins. A two-molecule protein layer is formed on the body surface.If there is no particularly reactive protein in the solution,
After exposure to the solution, there is only the original monolayer on the substrate surface. The related techniques cited above describe optical, electrical, and chemical means for distinguishing between monolayers and bilayers of biological particles, but their sensitivities and economics vary. Since biological systems produce antibodies in response to the invasion of foreign proteins, it is important for medical diagnosis to detect antibodies in biological systems in order to determine what kind of antigen the biological system has been exposed to. It's worth it.

A typical example of diagnostic detection of antibodies is the detection of antibodies against syphilis or gonorrhea in human serum. Conversely, detection of certain antigens in biological systems is also valuable in medical diagnosis.
Examples of detecting antigens for medical purposes include the detection of HCG protein molecules in urine as a pregnancy test, and the detection of hepatitis-associated antigen (HAA) molecules in donor blood. To do this, a pair of suitable immunoreactive proteins must be found.

The only known sources of antibody proteins so far are living biological systems. More specifically, only the phylum vertebrates are currently known to have an immune response to the introduction of foreign proteins. For example, ``many antibodies are found in the serum of animals and humans exposed to the corresponding antigen; however, many antigens can be produced in a controlled manner in laboratory cultures. For example, antigens associated with hepatitis, like antibodies, can currently only be obtained from living higher biological systems. It is known that when introduced into a system, it acts as an antigen.

Therefore, in such vertebrate systems, antibodies that specifically react with a given antibody can be easily produced. In current practice, the collection and purification and diagnostic use of immunoreactive biological particles relies on the precipitation or aggregation properties of proteins that occur through immunological reactions. A classic example of such a diagnostic use is blood typing, in which a sample of blood is mixed with serum antibodies and blood type determined by observing what agglutination occurs in the sample. Another diagnostic use of immunoreactive biological particles is the HCG protein pregnancy test, which is currently implemented as a suppression test. This test is performed by mixing a certain amount of anti-Japanese CG serum into a urine specimen. A plurality of polystyrene beads coated with HCG protein are placed into a pre-prepared urine specimen. HCG in urine specimen
Polystyrene beads aggregate only if no protein is present. If HCG protein is not present in the urine specimen, the HCG protein rusts on the polystyrene beads with the anti-Japanese CG serum previously placed in the urine specimen, causing the beads to aggregate.
On the other hand, if HCG protein is present in the urine specimen, it will form an armor with the anti-Japanese CG serum that has been added in advance, and the added antiserum will rust with the HCG protein on the surface and cause it to aggregate. I can't. Furthermore, for other types of clinical tests for proteins, such as the detection of antigens associated with hepatitis, it is highly desirable to further improve the sensitivity of the test and be able to measure the concentration of the antibody or antigen.Agglutination tests The disadvantage is that the particles used tend to agglutinate for various reasons unrelated to immunological agglutination, making the test less reliable.Typically, agglutination tests are Although performed with great care by experienced personnel, diagnostic errors still occasionally occur.Immunization experiments have traditionally been performed in cellulose acetate membranes and gels.

In this case, the specimen containing the antibody as well as its antigen is placed in different areas of the moistened membrane (or in well-shaped or concave holes in the gel).
In this conventional experiment, the sensitivity of the test is not as high as desired; The sedimentation line obtained by the immunoreaction is determined by measuring the cellulose acetate membrane or gel using C.I. A. Williams and M. W. Until it is suitably stained with a protein substance such as amide black, which is described in the book ``Methods in Immunology and Immunochemistry 1'' co-edited by Cheese, Volume M, pages 153 and 169, It is invisible to the naked eye. This staining method is
It adds one more step to the method of detecting such biological particles. Moreover, the formation of sedimentation lines in the gel is disadvantageous in that it is susceptible to unwanted bacterial growth in the gel during the time it is stored. Although the conventional substrates (slides) mentioned above have satisfactory performance in detecting bilayers of immunoreactive biological particles, the substrates described therein are not well suited for dual diffusion methods. Not suitable.

Furthermore, although the substrates (slides) described above have satisfactory performance in detecting bilayers of immunoreactive biological particles, the 2
It does not in itself indicate the concentration of biological particles in solution that form a monolayer.

It should be noted that the use of metallized slides alone is a poor means of determining the concentration of secondary particles in a specimen such as a blood sample.
L. in ``Federation Bulletin'' Magazine No. 3, No. 5 (September-October 1971). Broman et al., "Interactions among Human-Blood Proteins at Interactions and Journal of Immuno/Logical Methods 3" (King, 1973), pp. 227-232. A. L. Adams et al.'s paper “Three Sinful
A similar drawback occurs with the anodized tantalum slide described in ``Ways to Detect Anti-Body Antigen Complex on 1 Flat Surfaces''. It is less sensitive, particularly in detecting hepatitis, than the indium-gold alloy and indium oxide slides described in the aforementioned U.S. Patent Application Ser. Another article on conventional metallized slides is the article by Alexander Rosen, ``Immunology'', published in Physiological Chemistry and Physics 5 (King 1972), pages 243-258. Physical and mechanical reactions carried out at a solid liquid interface. Briefly, in accordance with its purpose, the present invention detects a second immune reaction in a test solution by directly visualizing the precipitation lines of key proteins formed on a metallized solid surface as a result of the immune reaction. A method and apparatus for detecting biological particles are provided.

To this end, a substrate with a metallized surface is first exposed to a certain amount of moisture localized by an overlying material such as a moist cellulose film or gel or glass slide. Cover with a fixed moisture layer. A specimen of the first solution containing the immunoreactive first biological particles is then fixed onto a moist layer, e.g. a first selected area of the moist membrane, or a gel or localized moisture layer. in which case a sample of the test solution suspected of containing second biological particles specific for the first particles is subsequently or simultaneously deposited into the first well;
Seed is deposited on the settled moisture layer at a second selected area or second well spaced apart from the first area. The two specimens are then diffused through the fixed moisture layer to the surface below the fixed moisture layer. During this diffusion, the biological particles pass through the settled moisture layer, and a precipitation line of the key protein is formed at the intersection of the diffused first and second biological particles. This sedimentation line can be seen with the naked eye, and when observed with the naked eye, even without the use of dyes.
Has good contrast against the background. Furthermore, a durable record of the detected immune response is created. Keep the device in the greenhouse during the diffusion process.

It is noted that the second biological particle is detected with much higher sensitivity than can be obtained using the secondary dual diffusion method.
Typical examples of solid surfaces mentioned above include microscope slides, glass plates or plates, plastic plates or plates, etc. Furthermore, using the method of the present invention, the concentration of immunoreactive second biological particles in a test solution can be determined by direct visual observation.

To make this determination, immunoreactive first biological particles are first adsorbed onto the surface of the metallized substrate to form a first monolayer thereof. Thereafter, a fixed moisture layer as described above is provided over the monolayer coated substrate. For moisture confinement, a drop of water moistens the area between the monolayer-forming metallized substrate and the confinement member by capillary action. The fixed moisture layer is then exposed to a sample of the test solution suspected of containing second biological particles specific to the first particles. 2nd in the test solution
particles, a second monolayer is formed on top of the first layer, against the background and where the area of the second layer is related to the concentration of the second biological particles. It has good contrast and is visible to the naked eye. The fixed moisture layer is exposed to the specimen by depositing a single drop of the sample on an area, by subsequent deposition in a well, or by depositing a drop of sample in a localized member, depending on the nature of the fixed moisture layer. You do this by placing your counterfeit in the hole. Furthermore, specimens can be deposited at the edge of the moisture layer.
Alternatively, if moisture is to be localized by an overlying member, a second
layers form much faster.

This solution exits the device along the edges of the slide or
Alternatively, it comes out from the end of a narrow groove formed on the lower surface of the upper slide. Alternatively, by connecting a pair of electrodes to a DC voltage source and placing them at opposite ends of a channel formed on the underside of the upper slide, the biological particles are moved along the electric field. The second layer can be quickly formed by electrophoresis. In each case, the concentration of the second particles is related to the size or area of the bilayer. While the novel features of the invention are particularly set forth in the claims, the structure and operation of the invention, together with other objects and advantages, will be best understood from the following description of the drawings.

Figures IA and IB depict an apparatus constructed in accordance with a first embodiment of the invention for detecting an immune response. In particular, the device of this first embodiment can be placed in a suitable container 1 such as a small glass, metal or plastic dish or tray.
0 or any other type of container made of a suitable solid material. The container has a generally vertically extending tongue portion for containing a liquid medium therein. Metal particles are deposited on the inside bottom surface of the container 10, forming a discontinuous layer 1 of metal particles to provide a metallized surface on the inside of the container 10. A typical example of the metallization of container 10 is a discontinuous layer of spherical particles of indium having an average thickness on the order of 2,000 to 4,000 angstroms. Instead, the surface of the container 10 may be metallized using one metal, an alloy of two metals, or one metal or an alloy of two metals with an oxide film of one of the metals. A continuous coating may also be utilized. As an example of metallizing with an alloy, a convenient alloy is an alloy of indium and gold. Typical examples of metallization using one metal and an oxide include a film of indium oxide several hundred angstroms thick, or nickel and nickel oxide.

A typical example of metallization using an alloy of two metals and an oxide film of one of the metals uses a gold-indium alloy and an indium oxide film.

In this case, the metallization is made as a continuous or discontinuous layer of indium particles, as described in the above-cited US patent application Ser. No. 445,204. Metallized container 1
0 is then placed on a suitable support and a small amount of gel sufficient to cover its metallized surface to a depth of less than 1 mm is poured into the container 10, thus forming a 1 mm thick gel. A fixed moisture layer 12 is formed.

The most common gels suitable for immunoreactivity testing are agar and agarose. The gel solution to be injected into the container 1 contains the biological components used in the test, as described on pages 14 and 148 of the aforementioned book "Methods in Immunology and Immunology." Depending on the solution of the particles, it may be a salt solution, a distilled water solution or a buffer solution.
After the gel solidifies, a plurality of closely spaced wells 13a-13e are formed in the thin layer of gel 12 by any conventional method described on page 149 of the above-mentioned book. The main differences between this invention and conventional diffusion in gel devices such as those described in the above-mentioned book are as follows.

m The device of the invention requires a metallized solid surface because visible sedimentation lines are formed on the metallized surface (although sedimentation lines are also formed within the gel) caused by the immunoreaction of biological particles. However, none of the related devices describes a metallized solid surface, but rather simply means for supporting the gel layer.

[2) In this invention, the gel layer is much thinner than the conventional gel layer.

In the conventional case, the thickness is at least 1 mm, and the above-mentioned book states that it is in the range of 1 to 3 mm,
The fixed moisture layer of this invention is less than 1 mm. This noticeable difference in thickness is due to the fact that in the device of the present invention, the gel as a fixing moisture layer is utilized solely for diffusion, i.e. the specimen of immunoreactive biological particles is spread along the metallized solid surface. is used to keep the moisture immobile so that reproducible sedimentation lines can be formed, whereas in conventional devices, the sedimentation lines are formed within the gel and are therefore not visible. This is due to the fact that a thicker gel layer is required to form a sufficiently thick sedimentation line. As a result of the things listed in {31 above, '2}, the sedimentation lines formed in this invention become a durable and permanent record of the immune response;
Does not require staining to be visible.

In conventional devices, the gel often needs to be stained in order to make the sedimentation lines visible to the naked eye, and even when stained, the contrast between the lines and the background is lower than in the present invention. Far inferior. ■ In this invention, the wells are formed completely through the gel layer.

In the secondary device, the bottom of the well must be sealed from the surface of the plate supporting the gel above it, as described on page 151 of the above-mentioned book. Although the apparatus of the present invention works satisfactorily with the bottom of the well sealed from the metallized surface of the vessel 10, sealing the bottom of the well in this manner is not necessary and as shown in all drawings of this application. It is preferable to form the wells so that they completely pass through the gel layer. This significant difference regarding the wells is due to the fact that in the device of the present invention, visible sedimentation lines are formed on the solid surface of the metallization, whereas in the conventional method they are formed within the gel itself. be. The wells 13a to 13e formed in the gel layer 12 have a generally circular cross section and approximately the same diameter, and may be as small as one millimeter or as large as several millimeters.

The apparatus immediately prior to depositing a specimen of immunoreactive biological particles into a well is shown in FIGS. IA and IB. 3A and 3B show a second embodiment of the apparatus of the invention in which the material constituting the metallized solid surface is a metallized substrate or slide as described in U.S. Patent Application Serial No. 445,204. An example is shown. In particular, the base body 30 has a generally flat top surface and is made of metal, glass, plastic, or any other suitable material. Base body 30'
Preferably, it is a glass slide, such as a common microscope cover glass. The flat top surface of substrate 30 is metallized as described in the above-referenced US patent application. This one
As an example, metallization involves the use of a discontinuous layer of metal particles or beads, typically indium, or a first layer of indium beads overlaid with a thin gold coating. A layer of indium beads (or a continuous layer of indium of a certain thickness) is covered with a thin film of gold, alloyed with indium, and a thin film of indium oxide is applied. (4) It can be composed of a metal such as nickel and a film of its oxide. Metallization with indium particles is often preferred when using particles of roughly uniform size, whereas indium-gold alloys and indium oxide coated substrates are used, such as when testing for hepatitis. In particular, it is often preferred for particles of very different dimensions. When metallizing with a discontinuous layer of indium particles, a translucent substrate material such as glass or plastic must be used, and the indium particles deposited on the surface of the substrate should be on the order of 1000 angstroms in diameter. There is no requirement that the particle size be exact, as long as the diameter of the particle is more than half the wavelength of visible light. When metallized with indium particles, the color is light brown. When metallizing with an indium-gold alloy and indium oxide, the thickness of the indium is approximately twice the thickness of the gold when initially deposited (the thickness of the indium is approximately 2000△
, gold is about 1000 A) and the indium oxide coating is several hundred angstroms thick to obtain the bronze color of these coatings. As described in U.S. Patent Application Ser. If the values are different, slides of different colors with different sensitivities will be obtained. Base body 30
The metallized coating 31 on the upper surface of the
The upper surface of the metallization coating is somewhat uneven when formed of only metal balls, or of this metal ball and a coating of a second metal, such as gold, and an oxide coating of indium. It becomes a rule.

Instead, when forming the metallized coating with a continuous layer of indium having a constant thickness, the gold and indium oxide coating has a generally flat top surface. Any type of metallized substrate 30 can be used with the present invention.
The substrate 30 may be as small as 1' square.
Details of the metallization of the substrate and its preparation are described in the pending US patent applications listed at the beginning of this specification. A device using the metallized substrate 30 is made in the same manner as in the first embodiment. That is, a thin layer 12 (less than 1 mm) of gel is formed over the metallized surface of the substrate, and two or more wells 13a, 13b are formed, preferably completely passing through the gel layer. Figures 5A and 58
The figure shows a device according to a third embodiment of the invention for detecting an immune response.

In particular, the apparatus includes a metallized substrate 10 and a fixed moisture layer 11 covering at least a portion of the metallized surface of the substrate member.
It consists of a moistened cellulose membrane. Base 1
It has a generally flat top surface and is made of metal, glass, plastic or other similar material. Substrate 10 is preferably in the form of a glass slide, such as a common cover glass for commercially available microscopes. The flat top surface of the substrate 10 is metallized as described for the first and second embodiments of the invention. For example, this metallization is {
1) A discontinuous layer, i.e. a metal particle or bead, typically using indium, or ■ A thin film of gold overlaid on a first layer of indium beads, or [3] Indium beads. (or a continuous layer of indium with a certain thickness) is alloyed with this indium to provide a thin film of gold,
Furthermore, it can be constructed with a thin film of indium oxide, or with a metal such as nickel and its oxide film. Metallization with indium particles is often preferred when the biological particles are of approximately the same size, whereas indium-gold alloys and indium oxide coated substrates are used, such as when testing for hepatitis. This is often preferred when the particle sizes are very different, such as. As mentioned earlier, when metallizing with a discontinuous layer of indium particles, it is necessary to use a transparent substrate material such as glass or plastic, and the indium particles deposited on the substrate have a diameter of about 1000△. However, there is no requirement that the particle size be as long as the particle has a diameter equal to a large fraction of the wavelength of visible light. When metallized with indium particles, the color is light brown. When metallizing with indium-gold alloy and indium oxide, the indium thickness is approximately twice the gold thickness when first deposited (the indium thickness is about 2000△, the gold is about 1
000A) and the indium oxide coating is several hundred angstroms thick to achieve the bronze color of these coatings. The degree of oxidation of the first metal determines the color of the oxide film, as described in U.S. Patent Application Ser. For each layer, slides of different colors with different sensitivities are obtained. If the metallized coating 10b on the top surface of the substrate 10 is formed of indium beads or a coating of indium-gold alloy and indium oxide, the top surface of the metallized coating will be somewhat irregular.

Alternatively, when such a metallized coating is formed with a continuous layer of indium, gold and indium oxide of constant thickness, the coating is generally flat on the top surface. Any type of metallized substrate 10 may be utilized with the present invention. The same can be said for other embodiments of the invention. The cellulose membrane 11 is a very thin suitable membrane of cellulose or a cellulose derivative such as cellulose'acetate, and may be any common porous paper such as oven paper or simply tissue paper. Membrane 1
The dimensions of 1 may be the same as or different from the base 10. Membrane 1
1 is made as thin as possible to the extent that it can be conveniently used, and its thickness is less than 1 mm and thinner than the thickness of the paper used in the conventional double diffusion method. In typical applications, membrane 11 is shown as being smaller in length and width than substrate 10 for convenience in describing the apparatus of the present invention. The base body 10 is 0. It can be as small as the size of a square mark. Details of the method of metallizing the substrate, as well as its preparation, are described in the previously cited pending US patent applications, and reference is made to those applications. The cellulose waist 11 is moistened before or after being placed on the substrate 10.

The membrane 11 should be placed smoothly on the metallized surface with great care so that it is in contact with the metallized surface along its entire lower surface. After assembly, the substrate with the fixed moisture layer is placed in a greenhouse and a sample of the first solution containing the immunoreactive first biological particles is placed in the first well, e.g., the fixed moisture layer is a gel. It is deposited either in the central well 13a, in some cases, or over the first selected area if the fixed moisture layer is a moistened cellulose film.

Each specimen described in this section can consist of one or more drops of the corresponding solution. Immediately after or simultaneously depositing the first sample into the fixed moisture layer (e.g., well 13a in FIG. IA or membrane 11 in FIG. 5A), a sample of the test solution, i.e. A second specimen suspected of containing immunoreactive second biological particles is placed on top of the fixed moisture layer (e.g., in wells 13c, 13b in FIG. IA or on top of membrane 11 in FIG. 5A). (separate from the specimen) and allow the two specimens to diffuse through the layer. Generally, the first solution and the test solution also contain other (non-specific) biological particles.

Typically, the first solution is rabbit antiserum;
The test solution is human serum. While the specimen diffuses through the settled moisture layer, the first biological particles in the first specimen, as well as other (non-specific) biological particles, diffuse through the layer and reach the metallized solid surface ( (e.g. 11 in Fig. IA, 31 in Fig. 4B or 10b in Fig. 5B), forming a monolayer of the particles thereon (e.g. 13a' in Fig. 2A or 12a' in Fig. 6A). do. Similarly, any second particles as well as other (non-specific) particles present in the test solution or in the second specimen will diffuse through the membrane and become adsorbed onto the surface of the metallized slide, causing the A monolayer of the particles (eg, 13b' in FIG. 2A or 13a in FIG. 6A) is formed thereon. In the area where the diffused samples intersect, the sedimentation line of Tsuba protein (for example, 15 in Figure 2A or 4)
14 in Figure A or 14) in Figure 6A is formed. This is several layers thick and is caused by the immunoreaction of the first and second particles. Fixed moisture layer (e.g. gel 12 or membrane 1
1) The specimen diffuses radially outward, forming a circular pattern, and the sedimentation line is straight or curved, depending on the type of particles and their concentration. The time it takes for diffusion to complete and a sedimentation line to form depends on the types of first and second biological particles involved, the concentration of each particle in each solution, and the amount of specimen above the settled moisture layer. It is a function of temperature as well as spacing.
That is, if the specimens are closely spaced, the intersection of the two diffusions will occur sooner, and therefore the sedimentation line will form sooner than if the specimens were further apart. The specimens may be introduced at a very small distance (a few millimeters apart). The time it takes for a sample to diffuse and form a sedimentation line is typically several hours, but this process can be sped up to several minutes using lightning electrophoresis or pressurization of the solution. The wetting agent in the fixed moisture layer may be distilled water, a salt solution, or a buffered solution that does not react with the specimen, so that controlled diffusion of the specimen within the layer occurs, thus providing reproducible results. This moisture remains constant. After the sedimentation lines are formed on the metallized surface of the substrate, the fixed moisture layer, gel 12 or film 11 is peeled off from the surface of the substrate and the side of the substrate to which the sedimentation lines are attached is washed, typically with distilled water. and drying by blowing air onto the substrate, preferably at room temperature.

The solid metallized surface of the substrate is then visually inspected. This inspection can be carried out by direct observation with the naked eye, and the light reflected from the metallized surface Z or transmitted through the metallized surface is observed. For example, when metallized with indium particles, it is observed using transmitted light, but when metalized with indium-gold alloy and indium oxide, it is observed using reflected light. The color of the sediment is mainly related to the color of the metallization surface. The sedimentation lines of the Tsuba proteins are visible to the naked eye, have good contrast against the background, and biological particles are detected with much higher sensitivity than can be obtained with traditional dual diffusion methods.

Furthermore, unlike the conventional dual diffusion method, there is no need for staining of the sedimentation lines since the contrast between the sedimentation lines and the background is much better than in the secondary case. As previously mentioned, the sensitivity of the device of the present invention is superior to that of other conventional double diffusion methods, especially those published in the above-mentioned book, "Men's 2's In."

Immunology. And Imiyuno Chemistry” Part 3
The sensitivity is much higher than that of the method described in Vol. Therefore, only a small amount of biological particles need to be present to be detectable.
3 Another advantage of this invention is that the sedimentation lines on the metallized surface provide a durable record of the detected immunoreaction. In the detection method described above,
It was assumed that the first solution was a known solution containing the first biological particles.

In this case, both the first and second solutions may be test solutions suspected of containing the first and second particles. In this case, if a sedimentation line is formed, it can be seen that these particles were actually contained in each solution; if a sedimentation line is not formed, one or both solutions may contain the particles. All I know is that it wasn't included. The known solution is the first
~ This first particle may be made in a laboratory culture medium or, as previously mentioned, may be removed from a living higher biological system and may be commercially available in highly purified form. It can be used commercially, or if it is not commercially available, it can be made chemically pure. Typical solutions of the first biological particles include salt solutions of water or other liquids that are suitable for and do not react with the first biological particles;
Or it may be a sample of human serum. 1st and 2nd before
Biological particles, referred to as biological particles, can be easily grown or otherwise isolated and collected;
or antigens, antibodies, viruses, bacteria, hormones, enzymes or other biological particles present in human serum or other test solutions.

Typical examples of specific biological particles detected by the methods and devices described above include: the first biological particle being hepatitis B antigen (HBAg); the second biological particle being hepatitis B antigen (HBAg); There is an antibody (HBAb). In many cases,
The first particle specimen is a specimen containing a specific antigen, such as HBAg.

In this case, the test solution is a drop of human serum from a patient suspected of having hepatitis B, and in a direct test on it, the antibody ( HBAb) is detected. Alternatively, the particles in the first sample may be antibodies against a particular disease, and in a direct test the presence of antigens against such antibodies in a serum sample is determined by the detection test of the present invention. be done. The device of the invention can also be used to perform indirect or suppressive tests to detect specific biological particles that are immunoreactive. The principle of the inhibition test is that the first particles, if present in sufficient quantity, will neutralize the free second particles in the solution. That is, in the suppression test, H
BAg particles, if present in sufficient quantities, neutralize free antibodies against hepatitis B in solution. This reaction results in
When the test specimen is deposited on top of the fixed moisture layer, the antibodies
Formation of observable bell bodies with Ag is prevented. Inhibition tests against antigens, particularly HBAg, are carried out as follows.

A sample of a known solution of HBAg is deposited onto a fixed moisture layer, and the HBAg and other particles present in the solution are deposited as a monolayer on the metallized side of the substrate, as in the direct test described above. Let it absorb. A sample of the human serum to be tested is added to the HBA in a vial or other suitable container.
Prepare the test solution by adding to the solution of b. Next, if the antigen is present in the human serum sample, HBA
The vial is stored for a sufficient period of time for b to form rust bodies with HBAg in human serum. It is preferable to increase the rate at which the vial is formed to form a rust body. lastly,
A sample of the test solution is piled onto the fixed moisture layer, and after a suitable period of time for the sample to diffuse, the fixed moisture layer (e.g. gel 12
Alternatively, remove the membrane 11) from the substrate and visually inspect the metal surface. The results of the inhibition test are opposite to the direct test. That is, if HBAg is present in the human serum sample, no sedimentation line will be formed, but if such a sedimentation line is formed, it means that there are not a few HBAs in the human serum sample. The inhibition test for detecting HBAb is carried out in the same way as the inhibition test for HBAg, but in each step an antigen is used instead of the anti-Z antibody,
It goes without saying that antibodies can be used instead of antigens. In the hepatitis test described above, HBAb can be obtained from the serum of a patient known to have hepatitis B.

Alternatively, HBAg in goats, rabbits or other suitable animals2.
It can be produced by injecting it with a protein, allowing a suitable incubation period, such as two weeks, and then removing blood containing the specific antibody from the animal and separating the antibody from other particles in the blood. If the first solution is known to contain the first biological particles, place a sample of the first solution in the center of the settled moisture layer and suspect that the second solution contains the biological particles. Samples of various test solutions can then be deposited on top of the settled moisture layer surrounding this first deposit. After this, if there is a sufficient concentration of second particles in the (various) test solution, the initially deposited core solution will diffuse outward and intersect with the various test solutions deposited subsequently. Where you do
Straight or curved sedimentation lines are formed. This allows testing for the presence of second biological particles in the test solution. If the first center solution is known to contain first biological particles and one of the surrounding test solutions contains a known concentration of second particles, then the first center solution and the concentration are The relative position of the sedimentation line formed on the metallized surface with the known surrounding test solution is used as a reference, and the second
The relative positions of any other sedimentation lines formed as a result of the reaction of test solutions suspected of containing biological particles with the first center solution are compared to this criterion and The concentration of certain second particles can be determined.

That is, if the position of the sedimentation line between the first central sample and the unknown test solution is closer to the central solution than the reference sedimentation line, this means that the concentration of the second particles in this test solution is Indicates that the concentration is higher than the standard concentration. In this (concentration) test, the peripheral portions of the test solution are equidistant from the first central portion. From what has been said above, it can be seen that precipitation of the protein as a result of an immunoreaction between a first biological particle and a second biological particle of interest specific to this first particle. By directly visualizing the metallized surface of a solid member on which objects are formed, the present invention provides an improved dual-stage method for detecting immunoreactive biological particles in test solutions. It will be appreciated that the diffusion methods and equipment utilized may be utilized.

The method and apparatus of this invention include:
It is very simple in that only a fixed moisture layer needs to be placed on the surface of the solid member. Additionally, due to the unique sensitive nature of the metallized elements used in this invention, there is no need to stain the fixed moisture layer or substrate to detect sedimentation lines by direct visual observation. The fixed moisture layer used in the device of this invention can be much thinner than previously used. This is because in the present invention, this is used only for spreading the specimen, whereas in the conventional case, a considerable thickness is required to form a sedimentation line therein. (However, some sedimentation lines are formed within the settled moisture layer of the present invention.) The detection method of the present invention is similar to other simple methods of detecting immunoreactive biological particles.
Much more sensitive than heavy diffusion methods, in one example 10-
A quantity of 9 grams of BSA antibody was detected. Even higher sensitivities are possible. As a result, the present invention provides a simple method for making the metallized elements described in the above-referenced U.S. patent application usable for dual diffusion of specimens within a fixed moisture layer for the detection of biological particles. That's what I did. Since metallized components, such as slides, can be made repeatedly with the same properties, the detection results of biological particles according to the present invention are consistent from beginning to end.
It can serve many useful purposes, especially the analysis of human serum in the field of medical diagnostics, such as the detection of various antibodies and antigens therein. The visual contrast between the sedimentation line and the monolayer of biological particles is so sharp when using the metallized solid surfaces used in this invention that detection can be performed directly with the naked eye. It is done by observing,
Therefore, large-scale testing equipment is not required. lastly,
It should also be noted that the sedimentation lines formed with this invention are durable. In FIGS. 7A and 7B, a fourth embodiment of a device according to the invention for determining the concentration of immunoreactive biological particles in a test solution is shown in top view and side view, respectively.

After a first monolayer of biological particles with specific immunoreactivity for the particles in the test solution is adsorbed onto the metallized surface of the substrate, the biological particles in the test solution are A second monolayer is formed on the metallized solid substrate. Basically, the device is a metallized group base turtle 0
and a fixed moisture barrier formed by a volume of moisture localized by member 11 disposed on substrate 10, as shown in FIGS. 7A and 7B. The spacing between the base 10 and the backing 11 has been exaggerated in Figures IB, 3B and 4B to make it easier to see the separate parts of the device. The substrate has a generally flat upper surface 10a and is made of a suitable material, which may be metal, glass, plastic or similar material. The substrate 1 is preferably in the form of a glass slide, such as a cover glass.
The flat top surface 10a of 0 is metallized as described in the above-cited US patent application. As an example of this, the metallized portion may include {1} a discontinuous layer, i.e. the typical metal is indium, but metal particles or beads, or {2} a thin film of gold on top of the first layer of indium beads. A thin film of gold alloyed with indium is provided on the first layer of indium beads (or a continuous layer of indium with a certain thickness), and then a thin film of indium oxide is provided. or {4) a metal such as nickel and its oxide film. Metallizing with indium particles is often preferred when the particles have approximately the same size because of its high sensitivity. When metallizing with a discontinuous layer of indium particles, it is necessary to use a transparent substrate material such as glass or plastic, and the indium particles deposited on the surface of the substrate have a diameter of about 1000A. There is no particular requirement on the size of the particles, as long as the diameter of the particles is within a large fraction of the wavelength of visible light. When metallized with indium particles, the color is light brown. As described in U.S. Patent Application Ser. This is because the contrast between single and double monolayers of immunoreactive biological particles, typically hepatitis antigens and antibodies, is optimal and visible to the naked eye. As mentioned earlier, the thickness of indium is roughly twice the thickness of gold when first deposited (indium thickness is about 2000△ and gold is about 1000△), and the indium oxide coating is approximately twice as thick as the gold. Make it 100 angstroms thick so that you can get this bronze color of the film. As set forth in the above-referenced U.S. patent application:
The degree of oxidation of the first metal determines the color of the oxide film, so by varying the degree of oxidation different colored slides with different sensitivities can be obtained for layers of different thickness of biological particles. It will be done. When the metallized coating 10b on the top surface of the substrate 10 is formed of beads of a first metal, such as indium or an indium-gold alloy, and a coating of indium oxide, "the top surface of this metallized coating is somewhat irregular.

Alternatively, if the metallized coating is formed with a continuous layer of indium, a gold coating and an indium oxide coating of constant thickness, the top surface will be substantially flat. Either type of metallized substrate 10 can be used in any of the embodiments of the invention described in section 3). Member 11 may typically be a clear glass slide with the same or different dimensions as substrate 10. In typical applications, and for convenience in describing the apparatus of the present invention, the acoustic B material 11 is illustrated in all figures as being smaller in length and width than the substrate 10. One or more holes 11a, 11b, 11c, 11d, 11e are formed in the member 11. If there are more than one holes, the holes are spaced apart from each other and are preferably, but not necessarily, arranged symmetrically, as shown in a particular arrangement with five holes in Figure 7A.

These holes are of equal small size, each with a diameter ranging from 1 mm to 1 cm. Base 1
Depending on the number of holes 11a to 11e formed in the member 11, the diameter is approximately 10 square inches or less.

For this reason, in the case of the member 11 having only one hole, the base 1
0 is said to be 0.5 inches square, and it can be as small as 0. The toughness may be square or less. After selecting the metallized base 10 and the backing material 11, the ninth
In all embodiments other than FIGS. A and 9B, a monolayer of immunoreactive first biological particles Z is adsorbed onto the coated surface 10b of the substrate 10.

To adsorb the first biological particles, simply immerse the substrate in a solution of the first biological particles so that the metallized surface is completely covered. Alternatively, a few drops of the solution may be placed on the metallized surface Z and the substrate kept in a wet chamber. The first biological particle is selected on the basis of being specific for a certain second biological particle with which it is immunoreactive. This second biological particle, if present in the test solution, immunologically binds to the first particle 2 to form a second, smaller monolayer on the surface of the substrate. The first particles may be made in a laboratory culture medium or, as previously mentioned, may be obtained from a living higher biological system, and may be commercially available in highly pure form; If it is not available, it can be chemically purified. The solution of the first biological particles is typically a sample salt solution of water or other liquid that is suitable for and does not react with the first biological particles or human serum. The first biological particles in solution (as well as any other non-specific biological particles that may be present therein) are present in the substrate 1.
After submerging the substrate in the solution for a period sufficient to cause the substrate to be adsorbed onto the surface of the metallized coating 10b to form a substantially complete monolayer 10c along the entire top surface of the metallized coating 10b, the substrate is removed from the solution. The time period required to form a monolayer 10c on the substrate 10 (typically up to 1 hour) is an inverse function of the concentration of the first particles in the solution. Base 1
After the monomolecular layer 10c is formed on the substrate 10, it is often advisable to wash the coated surface of the substrate 10 with bran in order to remove foreign substances.

The monolayer coated substrate 10 is dried, preferably by blowing room temperature air onto the substrate to speed up the drying process. The monolayer 10c on the coated side of the substrate typically has a thickness in the range of 30 to 100 Å, although this dimension will of course depend on the particular biological particles forming this first layer. This monolayer is visible to the naked eye as a dark brown shade on slides coated with indium particles, but is very faint, if visible, on slides coated with indium-gold alloys and indium oxide. After adsorbing the first monomolecular layer 10c over substantially the entire upper surface of the member 10, the member 11 is placed on the upper surface of the substrate coated with the monomolecular layer.

Although this arrangement of monolayer-coated substrate 10 and component 11 may be satisfactory in some cases, such as when the metallized coating 10b of the substrate is formed of indium beads,
In many cases, pores are caused by capillary action caused by the irregular spacing between the lower surface of member 11 and the upper surface of monolayer 10c, which is irregular due to the irregularity of the upper surface of substrate coating 10b. It was found that the test solutions deposited in 11a through 11e formed second monolayers of unreproducible and excessively large dimensions. For this reason, if the covered surface 10b of the base 10 is irregular, apply a slight tightening force to the bottom surface of the backing material 11 on the base 1.
It is preferable to make stronger contact with the monomolecular layer 10c of 0 to obtain reproducible results. If the metallized coating 10b is made of layers of indium and gold (and indium oxide) of constant thickness, such clamping forces are generally not necessary. If necessary, 10 materials
This tightening is accomplished by any suitable swaging device. The device of all embodiments of the invention can be sold commercially as a device consisting only of a metallized substrate 10 and a section 11. Alternatively, it may also include a monolayer 10c. As shown in FIGS. IA and IB and other embodiments, a member 11 is placed on a substrate 10 coated with a monolayer (
and optionally cotton), moistens the area between the slides 10 and 11 used for concentration determination to complete the fixed moisture layer and contain the second biological particles. A reproducible diffusion of the possible solution is then performed.

Typically, one or more are used for a test solution suspected of containing second biological particles that have specific immunoreactivity with the first particles forming the monolayer 10c. A fixed moisture layer is set by pouring one drop of water into each of the holes 1 1a to 11e. A drop of water is sucked in by capillary action between the lower surface of the section 11 and the free surface of the monomolecular layer 10c.

When tightening members 10 and 11, this tightening minimizes the amount of water required. The apparatus shown in Figures 7A and 7B, as well as other embodiments, is placed in the greenhouse as described above, either immediately before or after the established moisture layer is created.

This greenhouse is used to keep the settled moisture layer moist. This takes many hours to diffuse one drop of the test solution.)
During this period, the wetted areas may dry out and prevent the test solution from spreading along the monolayer 10c. A device consisting of two slides is placed in a greenhouse, and after a drop of water is sucked between the two slides by capillary action, a first layer is formed to form a monolayer 10c.
A drop of solution is deposited into one of the holes 11a to 11e in which the presence and concentration of second biological particles with specific immunoreactivity for the particles are to be tested.

If several different test solutions are used, one drop of each is deposited in the various holes, so that each hole receives one drop of only one solution. After this, if a second particle is present in the test solution, the diffusion of the test solution between the two slides will cause the immunoreaction between the first and second particles to cause a second biological reaction. The assembly of two slides is kept in a greenhouse for a predetermined amount of time to allow a monolayer of chemical particles to form. After this predetermined amount of time (which may be several hours) has elapsed, member 11 is removed, substrate member 10 is rinsed, typically with distilled water, dried, and the substrate 10 is then coated. Visual inspection is performed by observing with the naked eye the light reflected from or transmitted through the coated surface. Slides made of indium particles use the transmission of light, while slides made of indium-gold alloys and indium oxide use reflection of light. Without the second particles in the test solution, no second monolayer is formed on the first monolayer 10c in the area of the pores where the drops of test solution were subsequently deposited. However, the presence of second particles in the test solution causes the formation of a second monolayer radially outward along the free surface of monolayer 10c from the corresponding area of the pores in material 11. . The diameter or area of this second layer is related to the concentration of second biological particles present in the test solution. Therefore, by measuring the diameter of the second layer, the concentration of the second particles in the test solution can be determined. Of course, the diameter of the second layer is related to both the duration of the diffusion step as well as the concentration of the second particles in the test solution. For this reason, ``One method is to set the duration of the diffusion step constant for all measurements and to determine the concentration of the test solution by placing one drop of solution containing a known concentration of second particles into one hole as a reference. It is easy to decide.Instead of this,
Concentration can be assessed by simply observing the area or diameter and comparing it mentally to previous experience. As previously mentioned, by utilizing other holes for other test solutions, any number of different test solutions can be quantitatively evaluated simultaneously in the same device. 8th A
The figure is a plan view of the coated surface of the substrate 10 when the quantitative measurement described above is completed, and FIG. 8B is a side view thereof.

A drop of test solution (i.e., concentration is known) is placed in the central hole 1.
1c and thus forms a reference second monolayer layer 10c below the pores 11c.
Assume that c' is at the center of the substrate 10. As a result of sinking one drop of the first test solution into the hole 11a, a second layer was not formed on the coated surface of the substrate 10 below the hole 11a, and therefore this first test solution It does not contain any particles or does not contain any noticeable number of particles. A drop of the second test solution was subsequently deposited in hole 11b, resulting in a maximum diameter (layer 11c).
A second layer 11 was obtained, which was twice as large as 1. From this diameter measurement, comparison with the diameter of layer 11c' shows that the concentration is much higher than in the reference solution. 1 of the third test solution
As a result of the deposition in the droplet hole 11d, a second layer 11d' having a slightly smaller diameter than the reference layer 11c' was formed. This measurement shows that the concentration of the third test solution is slightly lower than the reference concentration. Finally, when a drop of the fourth test solution was deposited into the hole 11e, a second monolayer 11e' was formed with a diameter of about 1.73 mm of the reference diameter. The first monolayer 10c of particles contains hepatitis B antigen (BH
specific antigens such as Ag). In this case, the test solution is human serum taken from a patient suspected of having hepatitis B, and in the case of a direct test on which the diameter of the second monolayer of the test as well as the reference is measured. , the presence and concentration of antibodies to hepatitis B (HBAb) are determined. Instead of this, the first monolayer 10
The particles in c may be antibodies against a specific disease;
In the immediate test, the presence and concentration of antigen for this antibody in the serum sample is determined. Indirect or Z-inhibition tests for detecting and determining the concentration of immunoreactive specific biological particles, as previously described, can also be performed with the device of the present invention. The principle of the inhibition test is that the first particles, if present in sufficient quantity, will neutralize the second particles in the solution. Therefore, in inhibition tests, free HBAb in solution is
Neutralize particles. This reaction prevents the antibodies from forming observable collar bodies (ie, bilayers) when the slide with the antigen layer (first monolayer) is exposed to the test solution. Inhibition tests against antigens, particularly HBAg, are carried out as follows.

A monomolecular layer 10c of HBAg is adsorbed onto the coated surface 10b of the substrate 10, as in the direct test described above.
By adding a sample of the serum of the person to be tested to a solution of HBAb in a vial or other suitable container,
Prepare the test solution. The vial is then stored for a period sufficient for the HBAb to complex with the HBAg in the human serum sample, if the antigen is present therein. Preferably, the vial is rotated to increase the speed of curing.
Finally, a drop of test solution is placed into one hole in member 11, and after a suitable period of time, member 11 is removed and the coated surface of substrate 10 is polished, dried and visually inspected. The results of this suppression test are opposite to the direct test. That is, the presence of HBAg in the human serum sample does not result in the formation of a second monolayer, whereas the presence of this layer indicates the absence of HBAg in the human serum sample. The inhibition test for detecting HBAb is carried out in the same manner as the inhibition test for HBAg, but of course the antibody is replaced with the antigen and the antigen is replaced with the antibody in each step.

In the hepatitis test described above, HBAbs can be obtained from the serum of patients known to have hepatitis B, or goats, rabbits, or other suitable animals can be injected with HBAgs for a two-week period. After a suitable incubation period, blood containing specific antibodies is taken from the animal and the antibodies are separated from other blood particles. Thus, the spot of the second monolayer on the coated surface of the substrate 10 is formed when the first and second biological particles have very different dimensions.
In a preferred embodiment, coated substrate 10 is made according to the method described in U.S. Patent Application Serial No. 445,204, cited above, and is represented by a purple spot. However, as previously mentioned, other colored backgrounds of the oxide coating as well as other colors of the spots produced by the second monolayer are possible. For slides using indium particles, which is preferred when the biological particles are of approximately the same size, the spots in the second layer will be noticeably darker. Figures 9A and 9
FIG. B shows a fifth embodiment of the invention.

The apparatus of FIGS. 9A and 9B is similar to that of FIGS. 7A and 7B, except that the metallized substrate 10 is coated with a monolayer of first biological particles on the free surface of the metallized surface 10b. The difference is that it is not covered. That is, the coated substrate 1
The top surface of 0 is simply a free metallized surface before testing, as in the case of the gel and cellulose membranes discussed earlier. Two holes 11a, 11b are shown formed in the section 11, such as a slide (but not necessarily a slide), these holes being along the centerline of the member 11, each Preferably, the hole is spaced from the opposite green of the slide by a distance greater than about 1'4 of the width of the member.

In fact, the holes 11 and 11b are brought much closer together. The advantage of this device is that the distance between the holes can be made as small as 1 mm between the green holes 11a and 11b, and for this reason, the diffusion time for forming the monomolecular layer, which will be explained below, is reduced. This is a significant shortening. The dimensions of the holes 11a and 11b are:
This is the same range as the hole in the fourth embodiment of the invention.
A fixed moisture barrier was established by moistening the area between the two slides in the area surrounding the hole, as described above for detecting a second biological particle in the test solution. Thereafter, a drop of a test solution known to contain immunoreactive first biological particles is deposited into hole 11a.

Immediately thereafter, a drop of a test solution suspected of containing second biological particles specific immunoreactive with respect to the first particles is applied to hole 11b. The first particles as well as any non-specific biological particles present in the droplet of the first solution form a first monolayer 11a', which extends along a circular area around the pores 11a of the substrate. It is attracted to the metallized free surface 10b. A drop of the test solution that may contain the second biological particles as well as other biological particles than the
9A and 9B.
As shown in Figure B, the amount of the metallized free surface lob' of the substrate is adsorbed along a circular area centered on the center of the hole 11b. One or both biological particles are present in the first and second solutions in sufficient concentration such that when the diffusion of the first and second particles intersects, no visible particles are visible on the metallized surface of the substrate. A clearly visible sedimentation line 30 was formed, indicating the formation of a straight or curved protein sedimentation line.
The sedimentation line 30' is attached to the metallized substrate, and therefore,
When member 11 is removed (with associated moisture), substrate 1
0 leaves a clearly visible permanent record of the immunoreaction without the need for any staining. Assuming equal diffusion rates of biological particles, the position of the female meridian 30 between the two holes 11a, 11b represents the relative concentration of biological particles in the two solutions. That is, FIGS. 9A and 9
In the case shown in Figure B, it is assumed that the concentration of the second biological particles in the drop of solution placed in hole 11b is significantly higher than the concentration of the first particles in the solution placed in hole 11a. ing. This means that the intersecting sedimentation lines 30 are at a distance of 1 from the hole 11a.
This is because it is located at No.3. Of course, if the diffusion rates of the two types of biological particles are different and their concentrations are equal, the calculation for determining the concentration of the test solution from the position of the sedimentation line 30 requires the following:
These points must be taken into consideration. Also as previously mentioned, the two solution drops are allowed to diffuse within the settled moisture barrier for a predetermined period of time. In the present embodiment, this time is sufficient for the first and second biological particles to intersect and form a sedimentation line on the metallized surface of the substrate. The absence of a bell sedimentation line 30 indicates that the test solution does not contain any second particles, or if it does, the concentration is so low that no noticeable immune reaction can occur. Again, no staining is required to observe the sedimentation line. If it is known that the first solution contains the first biological particles, more than two pores can be formed in the member 11. It is convenient to arrange the holes as in FIG. 7A. Hole 11c centered on the first solution sample
sample of various test solutions suspected of containing a second biological particle into the surrounding holes 11a, 11b, 11d, 11.
It settles into e. If the second particles are present in a sufficient concentration in the corresponding test solution, a sedimentation line will form at the intersection of the diffusing first particles and the corresponding specimen of the diffusing second particles, is a detection test to confirm the presence of a second biological particle. If the first solution is known to contain the first biological particles and one of the other solutions contains a known concentration of the second particles, then A sample of the reference solution (with a known concentration of second particles) is poured into one of the surrounding holes.
For example, the next card is inserted into the hole 11a. At this time, the relative position of the sedimentation line formed between the holes 11c and 11a is used as a reference, and the test solution suspected of containing the second biological particles deposited in the other surrounding holes 11b, 11d, and 11e. The relative positions of any other sedimentation lines formed by these specimens are compared to this reference position and the second
Determine the concentration of particles. In this (concentration) test, the surrounding holes are at equal distances from the central hole 11c. 1st
A sixth embodiment of the device of the invention is shown in FIGS. 0A and 10B. In particular, the substrate 10 is metallized;
As in Figures 7A and 7B, a monolayer of first biological particles 10c is adsorbed thereon. The difference in structure between the two embodiments is that the member 11 does not have a through hole.

Since there are no pores, the surface 10 coated with a monolayer of the substrate 10
Deposit a drop or more of water along one or more greens of slide 11 on top of c, so that the drop of water flows from this green (or greens) of the slide into a capillary along one dimension. The action causes it to be sucked in between the two slides to form a fixed moisture layer. In Figures 10A and 10B,
The case is shown in which only one edge of the member 11, typically a slide, is used. After the specific area between the two slides has been moistened, a drop of the test solution is placed on the monolayer-coated side of the substrate 10 at the moistened green of the member 11 (or at the edge of the member 11). (two or more drops) spaced apart) are then deposited and the drops of this solution are allowed to diffuse into the fixed moisture layer for a predetermined length of time.

In the case shown in FIGS. 10A and 10B, the presence of the second biological particles in the test solution causes a second monolayer to form in a direction perpendicular to the edges of the impregnated member 11. 11
a' is formed. The width of the second monolayer 11a' (measured between the two long sides shown) is the second monolayer in the test solution.
is a measure of the concentration of biological particles. Figures 7A to 8
As in the embodiment of Figure B, a drop of the reference solution (i.e., a solution containing a known concentration of second biological particles) is deposited along the second edge of the material 11 and the appropriate If a standard has not yet been established, it may be possible to compare it with a test solution. It goes without saying that other edges of the material 11 may be used to test the concentration of other test solutions containing or suspected of containing second biological particles. 11th A
A seventh embodiment of the device of the invention for determining the concentration of immunoreactive specific biological particles in a test solution is shown in the figures and in FIG. 11B.

In the embodiments previously described with respect to FIGS. 7A-10B, when a drop of test solution diffuses through a layer of settled moisture,
The second biological particles formed a second monolayer. This process is of course relatively slow, taking up to two hours depending on the concentration of particles in the test solution. The process of creating the second monolayer can be speeded up by utilizing an effective mechanism such that the second biological particles are distributed further. A first effective mechanism for increasing this rate of particle distribution is shown in FIGS. 11A and 11B, and is shown in FIGS. By applying pressure, a forced flow of the second solution is utilized. This forced flow of test solution is achieved by connecting the input end of tube 50 (not shown) to a pressurized source of test solution and suitably connecting the output end to the upper end of hole 11a in member 11. achieved by Although in the embodiments described so far, member 11 can be made as thin as desired, in this embodiment it is preferred that member 11 be thicker than in the previous embodiments for connection to the tube. . In all other respects, the structure shown in FIGS. 11A and 11B is the same as that of FIGS. 7A and 7B. As the test solution passes through tube 50 and hole 11a, it flows between the two slides as indicated by the arrows in Figure 11b. This forced flow of test solution allows the second biological particle, if present, to enter into an immunoreaction with the monolayer 10c of the first particle in a much shorter period of time than if simple diffusion were used. cause. For this reason, a monolayer 11a' of the second biological particles of the test solution is formed within a shorter period on top of the monolayer 10c of the first particles, also radially outward below the pores 11a. Formed in the direction. In the case of fluid flow at a constant pressure, the diameter of the second monolayer 11 is again related to the concentration of the second biological particles present in the test solution. As in the related embodiments described so far, by comparison with the diameter of the second layer formed in the reference solution on the second slide device, or by ensuring that the second monolayer does not intersect. Hole 1
By providing a second hole in member 11 (sufficiently spaced from 1a), the concentration can be determined. Figures 12A and 12B show an eighth embodiment of the device of the invention.

This is similar to the device shown in FIGS. 11A and 11B, but the pressurized fluid flow does not flow out in all directions, but rather through a narrow channel 6 between the two villages 10, 11.
The difference is that it is limited to within 0. In this particular example:
The metallized coating 10b on the substrate 10 is formed of a layer of indium with a constant thickness to provide a generally flat top surface of the indium oxide coating, as opposed to the irregular surface that would result if indium beads were used. It is preferable to Channel 60
may have a height of about 5 microns, and is formed in the lower surface of the backing material 11 from the output end of the hole 11a to the furthest edge of the member 11. The width of the channel 60 is equal to or somewhat larger than the diameter of the hole 11a, so that the pressurized fluid can easily pass through the channel and between the two members, as shown by the arrow in FIG. 12b. to be able to leave. The length of the second monomolecular layer 11a' formed on the first monomolecular layer 10c within the channel 60 is also the same as that of the tube 50, the hole 1
1a and the concentration of second biological particles in the solution flowing through channel 60. A second mechanism useful for speeding up the process of forming a second monolayer of biological particles is to apply an electric field to the particles to speed up the process electrophoretically.

13A and 113B, the second monolayer 11
The apparatus of the present invention is shown to utilize aerophoresis to accelerate the formation of a'. This device is shown in Figure 12A and Figure 1.
It is similar to the device shown in Figure 28, but the narrow rare road 70 is
It differs in that it is formed on the lower surface of the hole 11a and extends from the output end of the hole 11a toward the far green side of the member 11, and does not require the tube 50. Figures 12A, 128 and 13A, 13
In order to obtain a passage of sufficient length in the embodiment of the invention shown in Figure B, the hole 11a in member 11 is located at the edge of member 11 opposite the edge containing the output end of the narrow passage. It is preferable to form them nearby. In the embodiment of FIGS. 13A and 13B, the output end of the flow path 70 is surrounded by a first electrode 71. In the embodiment shown in FIGS. This electrode is connected by an electrical conductor to the first
Appropriately connected to an output with polarity. Similarly, “hole 11
The end of the channel 70 at the output end of a is surrounded by a second electrode 72, which is connected by a second conductor to the output of the voltage source of opposite polarity. The apparatus of Figures 13A and 13B is used as follows. A drop of the test solution suspected of containing second biological particles is deposited in the hole 11a, and the DC voltage source is energized to apply a suitable DC electric field between the electrodes 71,72. By suitably choosing the polarity and magnitude of the voltage applied to the electrodes, a particular second biological particle can be driven with a velocity determined by the product of its electrokinetic mobility and the magnitude of the electric field.
Forced to move along electric lines of force. Therefore, the velocity of the second biological particles significantly reduces the time required to form a second monolayer on top of the first layer. In this case as well, as in the embodiment of FIGS. 12A and 12B, the second monolayer 11a is formed in a manner similar to the embodiment of FIGS. 12A and 12B. It has a length that is related to the concentration of particles. From the above explanation, this invention
A method for determining the concentration of immunoreactive specific biological particles in a test solution by measuring the dimensions of a second monolayer of specific particles formed on a metallized substrate; and It will be understood that the device has been made available.

The dimensions of the second monolayer are measured by direct viewing with the naked eye, and this second monolayer can be formed by diffusion of the solution or by using its forced flow. 4, or can be formed more quickly by an effective mechanism such as applying an electric field using an open air electrophoresis method. The dimensions of the second monolayer can be compared to known standards or to tables or graphs generated from test results using standard solutions with known concentrations. As a result, the present invention provides a simple method for making metallized slides, such as those described above, available for determining the concentration of biological particles in a test solution. Because metallized slides can be made repeatedly with the same properties, the quantitative measurements obtained with this invention are highly accurate and useful for many useful purposes, especially in the field of medical diagnostics. It can be useful for analyzing serum, for example for detecting various antibodies as well as antigens and measuring their concentrations. When using the metalized slides of this invention, the visual contrast between the monolayer and the bilayer of immunoreactive biological particles is very sharp, making it possible to Measurement of the concentration of target particles is carried out by direct observation with the naked eye and therefore does not require extensive testing equipment.
Although the invention has been described in terms of eight specific embodiments, it will be apparent from the foregoing that various modifications of the invention are possible.

That is, the shapes and dimensions of the various substrates, associated components, and fixed moisture layers may be varied, and the apparatus of the present invention may be applied to virtually any set of immunoreactive biological particles that are immunoreactive with each other. You can use That is, in this specification, the first
and the biological particles referred to as second particles include antigens, antibodies,
Viruses, bacteria, hormones, enzymes, and other particles that can be easily grown or isolated and collected or present in human serum or other solutions to be tested. . Furthermore, metallization methods other than those specifically described in section 2) may be utilized and may have superior sensitivity to certain biological particles. As an example, when metallizing with a metal and a metal oxide, nickel may be used.

At least some embodiments of the invention, namely FIG. 7A;
Figures 7B, 9A, 9B and 13A, 13
In the embodiment shown in FIG.
By forming the hole 11a (in the orientation shown in FIGS. 13A and 13B), it can be made without using the member 11. In that case, the slide is inverted and water and test solution are applied sequentially into the holes to moisten and then diffuse the second biological particles along the first monolayer. It is therefore to be understood that various modifications may be made to the embodiments of the invention described above without departing from the scope of the invention. This invention can take the following embodiments in relation to the description of claim 1.

Depositing a test solution suspected of containing a second biological particle onto the settled moisture layer deposits the test specimen at close intervals relative to the deposit of the first specimen. stacked so that the first and second areas on the fixing moisture layer are spaced from each other on the order of a few millimeters.

(b) adsorbing a first monolayer of biological particles onto the metallized surface before applying the anchoring moisture layer to form the test structure, then contacting the anchoring moisture layer with the monolayer; One or more specimens suspected of containing second biological particles are fixed onto selected areas on the moisture layer and then mirrored onto the first monolayer of first biological particles. form one or more monolayers of the second biological particles in the layer, and after this observe the dimensions of the one or more layers of the second particles, if present, and The dimensions of the one or more layers are related to the concentration of second particles in the one or more specimens.

(c) In paragraph (b) above, a separate specimen containing a known concentration of second biological particles is placed in a selected area on the settled moisture layer (separated from the area in which the previous one or more specimens were deposited). forming another layer of biological particles separated from said one or more layers of second particles to a known concentration of second particles in said another specimen; Examine the metallization surface by noting the dimensions of the further layers of the second grains concerned, and compare the dimensions of the one or more layers of the second grains with the dimensions of the further layers of the second grains. comparing and then being able to determine the concentration of a second particle present in said one or more specimens.

D. The first specimen contains a specific antigen, the second specimen suspected of containing a second biological particle contains a specific antibody against said specific antigen, and the antibody is detected by examining the metallized surface. To detect.

(e) The first specimen contains a specific antibody, the second specimen suspected of containing a second biological particle contains an antigen for which said specific antibody is specific, and the metallized surface is investigated. , consisting of observing the light reflected from a metallized surface.

In paragraph B above, adsorbing a monolayer of the first biological particle along the metallized surface comprises adsorbing a monolayer of a specific antigen thereon, and the test sample is The step of depositing onto the fixed moisture layer comprises the subsequent deposition of a test specimen suspected of containing antibodies that are specific for a particular antigen adsorbed to the metallized surface member, such that the test specimen is deposited on the fixed moisture layer. By allowing time for the antibody to diffuse through the inside, if there is an antibody present in the inside, this antibody will form a monomolecular layer on the antigen layer due to an immune reaction with the antigen, and the antibody will be released. Note the diameter of the monolayer and from that be able to determine the concentration of antibody present in the test specimen.

G. A second test specimen containing a known concentration of antibody is then deposited onto a selected area on the established moisture layer several millimeters from the first area, and the second test specimen is allowed to pass through the established moisture layer. With sufficient time to diffuse, the antibodies in the second test specimen separate from the previous layer of antibody and form another single molecule of antibody on top of the antigen as a result of an immune reaction with the antigen. Form the layers, observe the metallized surface and compare the diameter of each layer of antibody so that from this it is easy to determine the concentration of antibody present in the previous specimen.

(h) In the above item (b), the step of introducing the second biological particle sample into the test device consists of creating a forced flow of the second biological particles.

IJ In paragraph (b) above, the step of introducing the second biological particle sample into the test device deposits one drop of the second biological particle solution into the test device, and the second biological particle sample is deposited into the test device. comprising applying a direct current voltage between a first and a second electrode arranged in the device such that the layer is formed by electrophoretic methods. This invention may take the following embodiments in relation to the description of claim 2.

n. Including a monolayer of immunoreactive first biological particles between the metallized surface of the substrate and the fixed moisture layer.

The base body is formed as a flat thin plate.

(o) The base body is formed as a plate. (iii) The base is made of a translucent material such as plastic or glass.

Force The base body is made of metal.

(e) The moisture fixing means is a gelling material.

(t) In the above item, one or more wells are provided within the layer of gel material to introduce one or more specimens for analysis. In the above item ① or ②, ``The gel is agar or agarose.''

(e) The moisture fixing means is made of cellulose material.

In the above item, the cellulose material is composed of cellulose acetate, porous paper, or tissue paper.

(e) The moisture fixing means must be a solid member.

(n) In the above item (n), one or more through-holes are provided in the solid member as a means for introducing the specimen.

In the preceding paragraph, the specimen introducing means has a tube, a first end of which is connected to a source of pressure on the specimen, a second end of which is threaded into a hole in the solid member, and which is connected to a settled moisture layer. to obtain a forced flow of specimens.

M In the above item A, a narrow channel is formed on the lower surface of the solid member, the first end overlaps the hole in the solid member, and the second end of the solid member is significantly separated from the through-hole. Provide along the edges.

C. In the above item (N), a narrow channel is formed on the lower surface of the solid member, the first end is overlapped with the hole in the solid member, and the second end is formed on the edge of the solid member that is significantly separated from the through-hole. placed close together,
connecting the first electrode to the first end of the narrow channel, connecting the second electrode to the second end of the channel, and applying a DC voltage between the first and second electrodes; By electrophoresis, it is possible to form a second layer of particles much further away than if the specimen were simply diffused through the area.

ヰ The thickness of the fixed moisture layer is less than 1 mm.

(g) The metallized surface of the base member is a discontinuous film composed of metal particles. E The metallized surface of the base member is 2
Formed of an alloy of different metals.

(h) The metallized surface of the base member is formed of an alloy of two types of metals, and has an outer coating consisting of an oxide of one of the two types of metals.

Y In the above item H, the two types of metals are indium and gold, and the oxide of the coating is indium oxide.

M. The major surface of the substrate member is flat, and the indium is initially deposited as a discontinuous coating of indium beads onto the flat major surface of the substrate member, so that the metallized surface is somewhat irregular. .

(i) The metallized surface of the base member shall be formed of a film of metal and its oxide.

(f) The metallized surface of the base member is formed of a metal and an outer coating of an oxide of this metal.

[Brief explanation of drawings]

FIG. 1A is a plan view of an apparatus using metallized blood and a gel layer according to one embodiment of the present invention, before the specimen is submerged in a well formed in the gel layer. Figure IB is a side sectional view taken along line IB--IB of the apparatus shown in Figure IA, and Figure 2A is a plan view of the apparatus shown in Figure IA after the specimen has been diffused to form a sedimentation line. Figure 2B is the second
FIG. 3A is a side cross-sectional view of the device shown in FIG. The state before the next accumulation of specimens in the formed well is shown. Figure 3B is a side sectional view taken along line 3B-3B of the apparatus shown in Figure 3A, and Figure 4A is a plane view of the apparatus shown in Figure 3A after the specimen has been diffused and a sedimentation line has been formed. 4B is a side sectional view taken along line 4B-4B of the apparatus shown in FIG. 4A;
FIG. 5A is a plan view of a device according to yet another embodiment of the invention, in which two liquids of a solution that may contain biological particles are collected.
The point at which the drop was deposited on the cellulose acetate membrane is shown. Figure 5B is a side cross-sectional view of the apparatus shown in Figure 5A taken along line 5B--5B, and Figure 6A shows the apparatus of Figure 5A in its state after the two particles have been diffused to form a precipitate. 6B is a cross-sectional view taken along line 6B-6B of the apparatus shown in FIG. 6A, and FIG. 7A is a top view of the present invention for determining the concentration of immunoreactive biological particles in a specimen. The first device of
FIG. 7B is a side sectional view taken along line 7B-7B of the apparatus shown in FIG. 7A; FIG.
After exposing the metallized slide shown in the figure to the specimen,
8B is a plan view with the upper slide removed.
a side cross-sectional view of the device shown in Figure A taken along line 8B-8B;
Fig. 9A is a plan view of a second embodiment of the device of the present invention, which is mainly used to detect the presence of biological particles, but can also measure their concentration to some extent, and Fig. 9B is a plan view of Fig. 9A. FIG. 10A is a plan view of a third embodiment of the device of the present invention that utilizes specimen diffusion; FIG. 10B is a side cross-sectional view taken along line 9B-9B; FIG. Connect the device to line 10B
11A is a plan view of a fourth embodiment of the apparatus of the present invention utilizing forced flow of specimens; FIG. 11B shows the apparatus shown in FIG. 11A along line 11B-
11B is a side cross-sectional view taken along line I2B--12B; FIG. 12A is a plan view of a fifth embodiment of the device of the present invention utilizing forced flow; and FIG. 12B shows the device shown in FIG.
13A is a plan view of a sixth embodiment of the device of the present invention utilizing fin pneumophoresis, and FIG. 13B is a side sectional view of the device shown in FIG. 13A taken along line 138-13B. FIG. Explanation of main symbols, 10; Base, 10
b, 1 1, 31: metallized surface, 10c: monomolecular layer of first particle, 11a, 11b, 11c, 11d, 11e
, 13a, 13b, 13c, 13d, 13e: Upper slide hole, 11, 11th, 11c', 11d', 1
3e': Monomolecular layer of second particles, 12: Fixed moisture layer. Mago glo Taku glb Magotane Chikugo 2b Jita 30 Magota 3b Shio Sosogi 4o Magota 4b 2 horses/SA Mason 9.5B Ogi, 86A Mason 8.6B Autumn competition 'ZA Mononofuki Hiiragi Monoson Kan 22 Many grandsons Shio grandson statue Matsu grandson Matsu l Yuwonu A Isongochu 8 Ta wo〃Yushio moxibustion statue Yuson Kyouyoyu 3/2a Monono. Shioyu Yuko. 〇8

Claims (1)

[Scope of Claims] 1. A fixing moisture layer is placed in direct contact on the metallized surface covering the main surface of the substrate member, and an antibody or antigen that immunologically specifically binds to a particular antigen or antibody is attached. placing an amount of a solution containing the fixing moisture layer in contact with one location of the fixing moisture layer; placing a quantity of a biological specimen in contact with the fixing moisture layer at another location apart from the location; maintaining a moisture layer to diffuse the solution and biological specimen through the settled moisture layer into contact with the metallized surface, removing the settled moisture layer, and inspecting the exposed metalized surface. A method for examining the presence or absence of a specific antigen or antibody in a biological specimen, comprising the steps of examining the presence or absence of a sedimentation line attached to the metallized surface between the two parallel positions. 2 depositing a first monolayer of an antibody or antigen that immunologically specifically binds to a particular antigen or antibody onto a metallized surface covering a major surface of the substrate member; a fixed moisture layer is placed in contact with the fixed moisture layer, a quantity of the biological specimen is placed in contact with the fixed moisture layer, and the fixed moisture layer is maintained to allow the biological specimen to pass through the fixed moisture layer. diffuse into contact with the first monolayer, remove the fixed moisture layer, and inspect the exposed metallized surface for the presence of a second monolayer attached to the first monolayer. A method of determining the presence or absence of a specific antigen or antibody in a biological specimen and determining its concentration when present, which consists of several steps of determining the area. 3. A gel layer having a plurality of spaced through holes and substantially free of biological particles is disposed in direct contact on the metallized surface covering the major surface of the substrate member, and is coated with a specific antigen or antibody. A volume of a solution containing an antibody or antigen that specifically binds immunologically to one of the holes is placed in one of the holes, a volume of biological specimen is placed in another hole, and the welding and biological Preventing the gel layer from drying while the specimen diffuses through the gel layer adjacent to the metallized surface, removing the gel layer and inspecting the exposed metallized surface at the bottom of the two holes. A method for testing a biological specimen for the presence or absence of a specific antigen or antibody, comprising the steps of testing for the presence or absence of a sedimentation line attached to the metallized surface between positions corresponding to . 4. A porous membrane made of a material selected from the group consisting of cellulose and cellulose derivatives is brought into direct contact over its entire lower surface with the metallized surface covering the main surface of the base member, and a layer of liquid is placed in the membrane. A fixed amount of a solution containing an antibody or antigen that is immobilized and that immunologically specifically binds to a particular antigen or antibody is placed in contact with a selected area of the membrane; A biological specimen is placed in contact with another selected area of the membrane spaced apart from the selected area, and the solution and biological specimen are passed through the immobilized liquid into the membrane. Preventing drying of the liquid layer during diffusion to the metallized surface on the underside of the membrane, removing the porous membrane and inspecting the exposed metallized surface to form the two selected areas. A method for determining the presence or absence of a specific antigen or antibody in a biological specimen, comprising the steps of determining the presence or absence of a complex sedimentation line attached to the metallized surface between juxtaposed positions.