JP2638600C - - Google Patents

Description

BACKGROUND OF THE INVENTION The devices and methods described herein provide a particulate complex, particularly one that is difficult to form and detect.
Useful for diagnostic testing of immune complexes. Traditional methods of testing particulate composites
The same process must be repeated to perform
Costly. In addition, such tests are often present in intermediate extracts and samples.
A cleaning step is required to remove the interfering substances. To perform an immunodiagnostic test system, the user must spend time completing the test.
It is important to reduce it. As a result, the number of processes required to perform inspections is reduced,
Efforts have finally been made to make it a single step. But no single process currently exists
. In addition to reducing the time spent performing diagnostic tests, another
The desired property is that it is stable at room temperature over the course of time. Generally, diagnostic equipment
Includes multiple reagents that remain stable at different temperatures. Some of these reagents are briefly at room temperature
, But some are less stable or not stable at all. Trial
The effect of the temperature of the drug reduces the sensitivity and reliability of the test and increases the drawbacks. Available now
Most commercially available diagnostic devices are used to validate tests.
It is necessary to maintain the temperature at low temperature in order to guarantee the stability of the above reagents. Table 1 shows all
Tests found in three major kits that manufacture diagnostic or hormonal pregnancy kits
Indicates that the drug must be stored at low temperatures to be effective. Long hours
Diagnostic test systems that are stable at room temperature for a while have advanced significantly in equipment. Most diagnostic equipment currently in use is a “sandwich” test.
Inspection. Here, the analyte or analyte is bound to a solid matrix
Cultured with excess antibody molecules. Next is the analysis target
A second antibody to the second determinant of the first form is formed from the first antibody attached to the solid matrix.
It is cultured by the immune complex formed. The presence of labeled antibodies on the surface of the immune complex
It is determined by appropriate means depending on the type of sign used. This type of test is performed when the antigen is in two different regions or epitopes.
In order to have two antibodies linked in a loop, a "sandwich" or "2-
Many "sandwich" tests are patented (eg, US Pat.
361,647 or 4,497,899). But widely used
Nevertheless, its operation has difficulties. As mentioned, continuous
Requires manual operation and low sensitivity. For example, "sandwich" technology
The steps commonly used to perform the immunoassays used include the following: Determination of antibody dilution. 2. Removal of excess antibody used to increase the sensitivity of the solid matrix. 3. Washing of the solid support matrix released from unbound antibody. 4. Contact between base material and test test solution. 5. Incubate for a long time so that the analyte detected in the test sample binds to the antibody.
That. 6. Cleaning of the base material to remove unreacted material. 7. Contact of the matrix with a labeled second antibody. 8. Solid matrix wash to remove unreacted second antibody. 9. Directly if the second antibody is radiolabeled using a suitable counting technique, or
The label is an enzyme label that adds a substance that produces a detectable color change upon reaction
To determine the presence of an immune complex in some cases. 10. If the second antibody has an enzyme label, after the color development has become
Stopping the reaction with rukari or acid. In addition to being time consuming and relatively insensitive, the “sandwich” test
There are two further restrictions: first, one or more antigenic substances present in the sample
Not very suitable for use with devices that detect Test multiple antigen samples
Aliquots of the sample must be individually tested. Second, test sample
Is inefficient, and thus requires a lot of testing to get reliable results.
Fees must be checked. This is due in part to the sample adhering to the high surface area solid matrix. this
As described above, a sandwich that facilitates testing of multiple antigens and makes effective use of sample flow
It is a clear advance if there is a device based on switch technology. As described above, a characteristic not present in current diagnostic devices is stability at room temperature for a long time.
You. The reason for the instability is currently unknown. However, solid support matrix
Used to detect the instability of the antibody bound to and the presence of antigen
It is clear that this is due to the composition of the aggregate of the antibody-enzyme conjugate. Solid support mother
Store the conjugate at a temperature in the range of 2-8 ° C due to the instability of the antibody bound to the material.
Even if the composition of the aggregate is controlled, satisfactory results cannot be obtained. At these temperatures,
Aggregate formation rates are reduced. However, this method is inconvenient and can be diagnosed at low temperatures
Storage of the device is expensive, and the development of antibody-enzyme conjugates that are stable at room temperature
Efforts have been made to improve ways to reduce the drawbacks resulting from agglomerates. However
These efforts have also been unsuccessful. Diagnostic tests may also involve binding reactants that do not bind the antigen, thus forming a complex.
There is the step of isolating immune reactants that do not form part of the immune complex. Untied
The presence of the combined reactants increases the background of the test. Therefore, in practice, immune
Washing the coalescence to remove most of the background caused by unbound reactants,
Most tests have a so-called blocking process that further reduces the background.
The blocking step involves coating the solid support with a proteinaceous substance after coating with the antibody.
Process is included. The barrier material binds to areas of the solid matrix that are not covered with fluid.
Blocks further nonspecific binding of immune reactants that are not part of the immune complex
. Generally, the shut-off step is performed before performing the inspection, but US Pat. No. 3,888,62.
Spend more time as described in No. 9 to impregnate the solid matrix with the blocking agent,
Must be lyophilized and maintained in this state until use. Block solid surfaces
Pretreatment with blocking substances or use of lyophilized filters containing blocking proteins
Verbose, time consuming and costly. If you delete both of these steps,
The desired diagnostic device is obtained. As mentioned earlier, there are many immunodiagnostic devices, but sensitivity, simplicity, diagnostic speed,
It is clear that long-term room temperature stability is desired. [Summary of the Invention] The immunodiagnostic test apparatus of the present invention has advantages over the current one, and
And can be used to perform both non-sandwich inspections. The device of the present invention
The results are instantaneous at room temperature, very sensitive and even present in the same test solution 1
It has many features such as the ability to detect the above antigens simultaneously. The diagnostic test device described here depends on the composition of the immune complex for antigen or antibody.
It is mainly used to test any, but its scope is quite wide,
It will be understood by those skilled in the art that the present invention is not limited to molecules of The device must be at least
It is sufficient if there is a first molecule that recognizes and binds two molecules. The first molecule is usually a ligand
The second molecule is called the ligand molecule and the second molecule is called the ligand. Antibody and antigen are ligand recognition
Preferred embodiments of the molecules and ligands, the device comprises a variety of ligands and ligand recognition components.
Used with children. For example, hormone receptor molecules are coordination pre-recognition molecules,
It can be attached to body matrices and is used to test for corresponding hormone ligands. Conversely
Since hormones also adhere to the base material, they are used to test hormone receptors. Immunological examination
Various combinations of ligand-recognition molecules and ligands suitable for use in cleavage devices are known to those of skill in the art.
Will be clear to you. If sandwich or non-sandwich inspection is performed with this device, one or more
Antibodies are developed using a novel printer coder technology that sprays separate antibodies directly onto the substrate.
Impregnate the base material. Using this technique, antibodies can be used to bind one or more antigens.
Can be quickly attached to a discontinuous ring, line, or other shape. Inspection
Number of antigens performed is a function of the number of each antibody attached in a distinct pattern
. Below the matrix impregnated with the antibody are two layers of absorbent material. Immediately after the base material
Underneath is an intermediate layer of a material that reduces background. Absorbent underneath
There is a second layer of material. Its function is to absorb and retain the test solution,
Is to cleanse the body and is made of many different absorbent materials. Another feature of the present invention that reduces the background described here is the use of appropriate shielding.
Achieved by impregnating the prefilter with a blocking substance, in particular a proteinaceous substance.
You. The pre-filter is located on the base material and, under certain conditions, contacts the filter with the proteinaceous substance.
Impregnate the blocking substance by touching. When testing, use an appropriate amount of test solution
Is poured into the pre-filter and passes through the pre-filter together with the blocking substance. Test liquid
And the blocking substances contained therein, the blocking substance binds to non-specific reaction sites on the base material.
To prevent it from being used for binding by excess immunochemicals.
Contact with antibody-impregnated matrix. The device according to the invention, which is sensitive and stable for a long time at room temperature, comprises a chamber having at least two compartments.
Having. The first compartment has a matrix impregnated with antibodies and the second compartment absorbs humidity.
Chemicals. The second compartment communicates with the first compartment and allows the first compartment to dry.
To maintain, increase the sensitivity and reliability of the test. This is the pre-filter blocking agent
Maintains stability and allows the antibody to adapt well to the matrix during periods of non-use. When it is convenient
Although it cannot be said, it is not possible to adapt the base material to other chemicals that absorb moisture.
The same effect can be obtained by. Another feature of the present invention is a funnel-shaped aperture on the top of the device for accessing the test material to the base material.
It is a aperture. This shape makes effective use of the test sample and reduces the small surface of the base material.
The next wash is made effective by adhering to the product. Explain that the immunodiagnostic device tests the antigen by binding the antibody to the base material.
However, it will be understood by those skilled in the art that the use is not limited. Antibody
Antibodies present in the test solution are used for testing by binding the corresponding antigen to the matrix
Also suitable for doing. This feature of the invention is useful for detecting and diagnosing autoimmunity. Currently used by combining the characteristics of the diagnostic devices described here
More sensitive, reliable, convenient to use and more versatile than
Further, there is provided a system which can be stored at room temperature for a long time without impairing the function. As will be apparent to those skilled in the art, the essence of the invention is the impregnation with a blocking agent.
Filter, matrix properly impregnated with antibodies, and excess fluid
Adsorbent layer and capacity to support and combine all of the above for performing immunoassays
It is a vessel. Therefore, although this invention is described in detail below, this description has been published.
It is intended to be a limiting example of the invention.
must not. FIG. 1 and FIG. 2 show a suitable immunodiagnostic test apparatus which can be used in the present invention.
It is a table example. Fig. 1 shows the assembled device, and Fig. 2 shows the three-dimensional exploded view of the device.
. As shown in FIG. 1, it comprises a separable top 12, and a bottom 14 and a bottom.
It comprises a container 10 having a filtering device 28. FIG. 2 also shows the top 12 and
And bottom 14 are shown. In a preferred embodiment of the invention, the bottom 14 comprises two chambers 16 and 1
8 are separated. The chamber 16 has an absorbent material 20 for receiving the fluid from the intermediate layer 22.
Is placed. The intermediate layer 22 receives the fluid from the base material 24 in turn. In Figure 2
Also, when the top portion 12 and the bottom portion 14 are joined, they serve as exhaust ports for pressure equalization.
A moving cut 40 is shown. The latter reduces the time required to perform the analysis
Is not required to perform the analysis. Inside the top 12 of the container 10 is an opening 26. Top that fits into bottom hole 15
When the top 12 is mated with the bottom 14 by the post 13 attached to the
Is located on the base material 24. In addition, the top 12 corresponds to a filtration device 28. Flow
When the body is applied to the filtration device 28, the filtrate passes through the opening 26 and contacts the base material 24.
As such, the filtration device 28 is located above the opening 26. The filtration device 28 has many
To the top 12 by any one of the following means: In the corner of device 28
By providing a post 30 which fits into the receiving hole 32 located at the top 12.
It is convenient to accomplish Both the opening 26 and the filtration device 28 preferably have a funnel-shaped profile. This
This allows large amounts of sample fluid to pass through the low surface area matrix 24. Open
The dimensions of both the mouth 11 and the filtration device 11 do not affect the performance of the device
Although it is possible to vary considerably, we have found that the following dimensions are generally satisfactory:
Was found to be possible; the bottom diameter was 1.0 cm and the top diameter was 2.
0 cm, and 0.3 cm deep. The reagent in the filtration device 28 and the base material 24 are not deteriorated and can be stored for a long time.
A feature of the present invention that enables it is a moisture absorbing chemical located in the chamber 18. That's it
Such chemicals prevent moisture from contacting the reagents and causing a loss of activity. this
Various chemicals well known to those skilled in the art are useful for this purpose.
An advantage of the present invention is an apparatus having a chamber 18 for holding moisture absorbing chemicals.
It is clear that we are not completely dependent on. Even in a single chamber, chemicals adhere to other methods, for example, to the outer wall
It will work well if it works. FIG. 3 shows an enlarged sectional view of the present invention. The filtration device 28 is inserted into the hole 32
It is attached to the top 12 by a pillar 30. Top 12 and bottom 14 are also top
It is joined by a column 13 which fits into the hole 15 in the bottom and at the bottom. Figures 1 through
Although the container forming the diagnostic device shown in FIG. 3 has a flat shape, the invention
Not limited to form will be recognized by those skilled in the art.
In fact, any form will work well if it is assembled with the elements described above.
Will be. This diagnostic device is useful for detecting ligand-ligand discriminating molecular complexes on solid surfaces.
It is. Either the ligand or the ligand identifying molecule can bind to the base material 24.
Note that it is used to detect the corresponding components of the complex.
Is important. That is, once the ligand discriminating molecule has bound to the matrix,
The ligand can be analyzed; once the ligand has bound to the matrix 24, the ligand-identifying molecule can be analyzed.
Can be analyzed. Ligands are generally, but not necessarily, drugs, peptide hormones, and
It is a low molecular weight molecule like other bioactive molecules. On the other hand, ligand recognition molecules are generally
, But also, but not necessarily, high molecular weight molecules, often proteins,
It is a body molecule. Therefore, this diagnostic device provides a specific example
Antigens and antibodies (mono- or polyclonal) as ligands and ligand-identifying molecules, respectively
However, the invention is not limited to the use of this ligand and ligand discriminating molecule.
It will be appreciated by those skilled in the art. However, this
Since they are often used in diagnostic assays, the present invention has been described accordingly.
Is done. The diagnostic devices described herein often use "antibodies" formed of antibodies and antigens.
It is used to detect the "sandwich" immune complex and therefore is labeled
A support material suitable for binding the first antibody not used is used as the matrix 24. However,
It is recognized that the instrument can also perform non-sandwich analysis, especially competitive binding analysis.
Let's go. The latter have a single antibody binding site or
Because of this, they are often used for the analysis of any low molecular weight molecule that would hinder the binding of one or more antibodies due to steric hindrance. So
Therefore, the present invention can be used for drugs, steroids and the like. The attachment of the antibody to the solid matrix is directly or well known to those skilled in the art.
This is done by absorption or covalent bonding, using a linker of the type indicated. Many
Suitable types of performing these steps, within the class of, for example, Biochemical Jo
urnal (1972), vol. 129; p. 255, Iman and Ho.
Hornby, Biochemical Biophysical Acta (1975), Volume 384;
Campbell, Hornby, and Morris at page 07
, And FEBS. Matisson in Letters (1977), vol. 104; p. 78
Mattisson) and Nilsson. Besides, anti
Chemically pretreated materials suitable for association with the body can also be developed commercially. Many materials are available for assembling the support. Such materials are generally synthetic or
Or a natural polymer. Examples of useful synthetic polymers include polyethylene and polyacrylic.
Luamide, nylon, resin, polyvinyl chloride, and polystyrene. Typical
Natural polymers commonly used are cellulose, polysaccharides, sepharose, agar
Loin, and various dextrans. Additives that can be used to assemble supports
Materials are silica, especially glass, collagen, and polynucleotide. Up
Although many of the materials described above work well in this invention, a preferred embodiment is nylon.
It is a material consisting of An important aspect of the diagnostic device is a method of applying an antibody to the solid base material 24. Current
In most methods being performed, antibodies are applied non-selectively to the entire surface of the matrix 24.
To wear. This wastes antibodies, which are often expensive and difficult to obtain.
And the elimination of tests for more than one antigen present in the same sample.
You. The present inventors spray-transfer the antibody into a dilute fluid and attach it to the base material.
As a result, it has been found that both of the above problems can be avoided. This technique is
The solution containing the antibody is passed through the stoma nozzle, whereupon the solution is sonicated or otherwise manipulated.
Works best by fragmenting into individual droplets using columns
be able to. The droplet is then charged by passing it through an electric field,
To deflect. A method for accomplishing this method is described in U.S. Pat. No. 3,281,86.
No. 0 and 4,121,222. The above method is manufactured by Videojet Systems International
This can be most easily performed by using a commercially available printing device. This equipment
The device is called a video jet coder / printer, and under various conditions and seeds
Provides a flow of antibodies at various flow widths. With this device, different antigen characteristics
Formation of a series of lines and other patterns on the base material, each containing antibodies of opposite sex
can do. Spraying the antibody to the matrix will bind the antibody to the matrix simply by absorption
Or covalently bonded to a chemically pretreated matrix.
doing. FIG. 2 shows that an intermediate layer 22 is provided below the solid base material.
The intermediate layer is located between the absorbent material 20 and the base material 24, and is used for the background of the test fluid.
It acts to reduce undulation. After the test fluid passes through the base material 24, the intermediate layer 22
And is filtered through it. The middle layer should reduce the backflow of unreacted reagents.
This reduces the background of the test fluid, and the unreacted reagents
And contact. Various materials are used to form the intermediate layer 22
be able to. Particularly preferred materials are U.S. Patent Nos. 3,860,003 and 4,08.
Disposable diapers described in 1,301 and 4,515,595
It is a commonly used non-woven polypropylene material. In addition to the middle layer 22, the main diagnosis has a low background and improves the convenience of use.
Another feature of the disconnect device is a filtration device 28 located on the top 12. This
This device has a funnel-shaped central area that can contain the test fluid necessary to perform the test at one time.
It has an area 38 and a tab 34 from which the device 28 can be grasped and removed. Filtration equipment
A filter 36 is provided at the bottom of the device 28. This filter checks
And the test fluid is required when the test fluid is applied to the filtration device 28.
Together with one or more reagents carried on the matrix. Various materials can be used to make the filter for the filtration device 28.
You. Indeed, in most cases, the above materials that make up the base material are also suitable for filters.
Can be used. We have found that glass fibers are particularly suitable.
Was. An example is Ultipol GF filter U6-40, commercially available from Paul. The filter 36 can be impregnated with various reagents. Especially protein shielding
It is convenient to impregnate the cleavage reagent. The type of blocking reagent is not important. Various
Protein substances, amino acids, and peptides can be used. However, the inventors
Found that milk protein was satisfactory, and Carnation
Milk powder was used regularly. If the solid matrix 24 is chemically pretreated to covalently bind to the antibody
When used, it is preferable to use a low molecular weight amino reagent such as glycine.
New In order for the blocking reagent to fit well into the filter 36, the dried reagent material must be evenly distributed.
Preferably, the contact is effected for a sufficient time to be applied to the filter. this is
After removing material that does not adhere well to the filter, keep the blocking reagent
This can be achieved by keeping the ingredients in contact. Instead, a method of adapting the blocking reagent to the filter 36 is to block the filter.
The filter is not lyophilized after impregnation with the solution containing the cleavage reagent.
It will be evaluated once. This method uses low molecular weight blocking reagents (eg glycine)
This is useful when using. The filter processed in this way is
Works well, but lyophilization hardens the filter so that the solution
It takes time to penetrate and is not preferred. In addition, a blocking reagent is added to the base material 24.
Non-uniform adhesion causes an increase in background. In addition to adapting the blocking reagent to the filter 36 of the filter device 28,
It is also preferable to impregnate the components used in the test with the above reagent. by this
This is because there is no need to add these reagents in the separation step. For example, anti
Reagents used to determine the presence of a body-antigen complex, ie, antibody enzyme conjugates
Alternatively, the enzyme substrate can be similarly adapted to the filter 36. A second feature of the invention is the use of a suitable desiccant in the chamber at the bottom 14.
. This is important in maintaining the stability of the diagnostic device at room temperature over a long period of time. Base material 24
Or, the stability or useful life of the filter 36 is a function of the humidity surrounding the device.
The useful life of the currently used diagnostic equipment at room temperature is less than 6 months,
That of the invention is one year. The present inventors have applied the desiccant to the diagnostic device to stabilize the reagent,
It has been found that it can be used for a long time. Various known drying agents can be used.
You. To detect the presence of the immune complex in the matrix 24, a labeled second test is performed.
It is necessary to use exit molecules. If the complex is an immune complex, the first
A section that is specific to the antigen bound to the antibody and that is separate from the place where the first antibody bound
At this point, a second antibody that binds to the antigen is used as the detection molecule. Detect presence of antigen
Traditional methods use a labeled second antibody. Labels are usually radioactive
Racers and more recently hydrolyze colorless substrates to produce a detectable color change
Enzymes are used. This can reveal the presence of the immune complex. This
In this case, various enzymes can be used in combination with an appropriate substrate. An example
For example, horseradish peroxidase, beta-galactosidase, glue
Cosmic oxidase, alkaline phosphatase, etc. well known to those skilled in the art
Can be used. Suitable substrates for most of these enzymes are diazonium or
It is a tetrazolium salt. As an example of the former used for alkaline phosphatase
Are naphthol AS-MX-phosphate and diazo-2-amino-5-chloro-
There is anisole. Methods for coupling an enzyme to a second antibody are well known to those skilled in the art, but mainly
And coupling the enzyme to the antibody. Coupling by cross-linking antibodies
Osullivan and Marks, "Methods in Enzymology," 1981, (7
3: 147) Academic Press, New York. Horstra
A suitable coupling method when using dish peroxidase is Naka
Ne and Kanaoi, "Histology and Cell Chemistry", 1974 (82: 1084). this
The method effectively and directly conjugates the enzyme to the antibody. But other methods are well known
ing. For example, the biotin-avidin bridge is a hose cross-linked to avidin
Formed on a second antibody with radish peroxidase. Instead of coupling the enzyme to a second antibody-enzyme conjugate, the enzyme
Can also be incorporated into This includes, for example, both the antibody binding site and the enzyme active site.
One hybrid molecule may be genetically engineered. For example, antibodies can be
(Neuberger et al. “Having recombinant antibodies
New Effector Function ", Nature, 1984 (312: 604). This type of enzyme conjugate is a direct filter
-36, or added to subsequent steps to form the immune complex on the matrix 24.
Reveal the existence. An antibody attached to the base material 24 or an antibody comprising an antibody-enzyme conjugate
Or polyclonal can be known to those skilled in the art. Antibody-enzyme conjugation
Enough to allow the body to penetrate the matrix 24 and maximize the binding of the antibody enzyme conjugate to the antigen
After a certain period of time, the solution containing the appropriate enzyme substrate is added to
See if it is colored as an indicator of presence. In most cases, after penetrating the conjugate,
A washing step is required before adding the substrate. This is because the shape of the funnel in the opening 26 is large.
This allows the substrate solution to pass through a small surface area of the base material. Substrate solution
The addition of serves as a wash. Also remove unnecessary background
Therefore, a washing step may be required. Hereinafter, the present invention will be described by way of examples. Example 1 Detection of chorionic gonadotropin (hCG) This embodiment will be described with reference to FIGS. hCG25mIU / mL
About 0.5 mL of urine containing was placed in the filtration device 28. Urine is filtered by filtration device 28
With the protein blocker milk protein impregnated in the filter.
Pass through The filtrate containing the blocking agent passes through the opening 26 in the upper surface 12 and passes through the base material.
Touch 24. The base material 24 is impregnated with an antibody against hCG. Base material is our business
Made of nylon that is well known to the elderly. The impregnation of the base material 24 is described in U.S. Patent Nos. 3,281,860 and 4,121,2.
No. 22, using a printer / coder machine, the hCG α chain
By passing a small stream through the matrix 24 containing the mouse monoclonal fluid
Done. The antibody was in a broad stream of approximately 1.5 mL. For end-users of equipment
The antibody is perpendicular to the horizontal direction of the goat anti-mouse antibody of the immunoglobulin class.
Is printed in the opposite direction. This will be described in detail later. 1 mL of mouse monoclonal antibody
A solution containing 4 mg per h is sprayed onto the hCGα chain in a suitable buffer. Rin
Saline-containing saline is suitable as a buffer. This buffer contains 150 mM sodium chloride and 10 mM sodium phosphate.
H is 7.1. In addition, this solution contained 10 μg of fluorescein and
Bacteriostatic agents, such as 0.1% sodium azide. Fluorescein
The solution is placed in a solution so that the pattern of the antibody on the base material 24 can be visually evaluated.
The filtrate contacts the base material 24 after passing through the filter 36. HCG in the filtrate is the base material
24 binds to the hCG antibody impregnated. At the same time,
The blocking agent binds at a location different from the location of the matrix 24 to which the monoclonal antibody has bound
. Then, the portion does not react with the reaction reagent added later. Filtrate is the base material
Shortly after contact with C.24, the second monoclonal antibody-enzyme conjugate of the mouse
Two drops of the solution containing the coalescence are dropped. The second antibody is directed against the beta subunit of hCG.
Different from the epitope bound by the first antibody attached to the matrix
Binds to an epitope. The enzyme component of the conjugate is alkaline phosphatase. Anti
The body-enzyme conjugate was passed through filter 36 and the conjugate was converted to hC bound to the first antibody.
Contact with base material 24 for a time sufficient to react and bond with G. Usually this takes about one minute
is there. To visually identify the complex formed on the base material 24, an alkaline phosphine was used.
A solution containing a substrate for indoxyl phosphate, which is a vatase, was directly poured into the base material 24.
. In about 1 minute, the blue color appears on the base material 24 as a “+” mark and the test sample contains hCG.
Was shown. If a "-" mark appears, the sample does not contain significant amounts of hCG
I can not say it. For a substrate solution containing 4 mM indoxyl phosphate, about 0
. 5L is enough. Finally, an appropriate static reaction solution is added to the base material 24. It requires 0.1% acetic acid
0.5 L of the liquid is sufficient. As described above, the “+” mark indicates that the first anti-hCG antibody in the vertical direction is the second anti-hCG antibody in the horizontal direction.
Obtained by enrichment with either antibody-enzyme conjugate, enzyme alone or goat anti-mouse antibody
Can be Of these, goat anti-mouse antibodies are preferred. Because the "+" sign is vertical
This is because it goes well with the reagents (ie, protein antibodies) used to make the components.
That is, the loss of activity over time in one reagent is the corresponding loss in the other reagent.
Balance. The reagents used to make the horizontal component are
Regardless of the type, it is sprayed on the base material 24. Example 2 Room temperature stability The materials and methods used in Example 1 were also used here. Diagnostic device stored at room temperature for one year
After that, the device was used to test a sample containing 25 mIU / L of hCG. Example 3 Impregnation of base material with antigen It will be apparent to those skilled in the art that the device of the present invention is not limited to the detection of antigen.
There will be. Detect antibodies circulating in the body fluids of patients examining the immune system
You can also. To do this, first attach a source that causes an immune response to the base material, and then
Then, the presence of the antibody may be examined. This makes the diagnostic device autoimmune or
It can be used to monitor patients with rugie constitution. Inactive rubella to demonstrate this use
The virus is applied to the base material 24 in FIG. 2 by the printer / coder machine described in the first embodiment.
Attached. Next, the solution containing the antiviral antibody to be detected is poured into the filtration device 28.
Then, this solution passes through the filter 36 and the filtrate passing through the opening 26 is obtained.
. The filtrate comes into contact with the base material 24 to which the virus has adhered. Then the anti-rubella antibody is on the base material
Binds virus. Then, detection of anti-rubella antibody in the filtrate was carried out using the antibody-enzyme conjugate.
By passing through the solution. Here, the antibody reacts with the bound anti-rubella antibody.
Is what you do. The antibody component of the conjugate can confirm the epitope of the anti-rubella antibody
Should be fine. If the anti-rubella antibody tested is human, the antibody component of the conjugate is
Must be an anti-human antibody. Subsequent steps are similar to those in Example 1.
. Eventually, the color of the base material 24 in which the presence of the anti-rubella antibody is recognized in the test sample is not sufficient.
Was. Example 4 Impregnation of assay reagents into filter material One of the ultimate goals of a diagnostic device is to develop a device that requires little manual operation
That is. By allowing the test reagent to mix with the raw material of the filter 36,
Can be omitted separately from the base material 24. Milk protein obtained by removing large grains from skim milk powder (Carnation)
Grind the material and impregnate the filter with a proteinaceous blocking agent. Then the glass fiber
Filter (Perth prefilter grade Ultipol GF filter U6-
40) is cut into 2 cm squares and stored in a sealed container with a suitable desiccant.
. The filter is then combined with the crushed skim milk for a sufficient time, usually 3 minutes.
Impregnate the filter with skim milk for about an hour. Milk protein is evenly filtered
Stir well with the filter and milk protein
. Excess milk protein is removed from the horny filter by sieving. Next
Transfer the filter into the container with a sieve to sift off unfamiliar milk proteins
Take enough time to rock. This time is usually one hour. After this step
Once again, subject to the process of removing milk proteins that have not been removed. filter
Is stored in a container with a suitable desiccant. The filter thus obtained is directly
It can be used for a diagnostic device. Example 5 Detection of multiple antigens The materials and methods in this example are the same as in Example 1 except as described below.
You. The matrix 24 is treated with two antibodies having significant specificity for the antigen. 1
One is an antibody against the beta subunit of luteinizing hormone (LH),
One is for the beta subunit of follicle stimulating hormone (FSH). Implementation
Antibodies fly over matrix 24 using the printer / coder machine described in Example 1.
On the skin. An antibody-enzyme conjugate that can detect either LH or FSH
A second antibody is a single monoclonal antibody that can identify a common epitope on LH and FSH.
Lonal antibody and two monoclonals that bind to different epitopes on LH and FSH
Any of the nal antibodies may be used. In the latter case, there are two
Different enzymes bind the monoclonal antibody and express a prominent "+" sign. This
For example, alkaline phosphatase and β-galactosidase can be used.
it can. The former exhibits a red color when combined with a suitable substrate, and the latter exhibits a blue color. The horizontal component of the “ten” mark of LH and FSH is formed as described in the first embodiment.
. Example 6 Diagnostic device sensitivity Using the materials and methods described in Example 1, the sensitivity and
The time required for diagnosis was compared. For commercially available equipment, follow the manufacturer's procedures.
Followed. Solutions containing various amounts of hCG were diagnosed. Table 2 shows the detection limits of the equipment and
Indicates sensitivity. The table also lists the other prerequisite methods, antibody types, and
The time when it was done is also shown.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a perspective view of a core portion of a diagnostic apparatus of the present invention, FIG. 2 is an exploded view of the same portion, and FIG. 3 is an enlarged view along line 3-3 in FIG. It is sectional drawing. 24 ... base material, 28 ... filtration device, 36 ... filter.

Claims (1)

Claims: (1) An apparatus which can store at room temperature for performing a diagnostic measurement of a biological fluid for a molecule contained in the biological fluid, and which can be divided into at least two or more chambers. When,
A first chamber having an antibody or antigen-containing base material and a means connected to the base material for absorbing liquid, and for communicating with the first chamber and absorbing moisture from the first chamber. A second chamber provided with a chemical means, and an exhaust port for balancing the pressures of the first chamber and the second chamber, wherein the base material is at the top of the first chamber. A device communicating with the exterior of said first chamber through an opening. 2. The apparatus according to claim 1, wherein said absorbing means comprises an absorbent layer and an intermediate layer, wherein said intermediate layer is located between said base material and said absorbent layer . (3) The base material according to claim 2, wherein the base material further includes a reagent selected from the group consisting of hormones, hormone receptors, enzymes, and derivatives or combinations thereof.
Item. (4) A patent wherein the base material includes a solution containing the antibody or the antigen, the solution containing the antibody or antigen being supplied to the base material, and the supplied solution being deflected into a predetermined pattern to contain the antibody or the antigen absorbed by the base material. Apparatus according to claim 3. 5. The device according to claim 3, wherein said opening is funnel-shaped. (6) for the molecules contained in the biological fluid, a device for performing measurements on the diagnosis of a biological fluid, and the container, located inside the container, the base material containing an antibody or antigen and the A means for communicating with the base material to absorb the liquid, a chemical means for absorbing moisture present in the container, and a funnel-type filter holding means positioned on the base material. The base material communicates with the outside of the container through a funnel-shaped opening at the top of the container, and the filter holding means communicates with the base material through the opening at the top of the container; As a material, to remove the interfering substance present in the biological fluid, and to detect the molecule in cooperation with the matrix by providing the matrix with a blocking agent such as a protein or other reagent. Said Device is a filter retaining means having one coupled to lifting means. (7) The apparatus according to claim 6, wherein the absorbing means comprises an absorbent layer and an intermediate layer, and the intermediate layer is located between the base material and the absorbent layer . (8) The method according to claim 7, wherein the base material further comprises a reagent selected from the group consisting of hormones, hormone receptors, enzymes, and derivatives or combinations thereof.
Item. (9) The patent, wherein the matrix contains a solution containing the antibody or the antigen, and the antibody or the antigen absorbed by the matrix by deflecting the supplied solution into a predetermined pattern. An apparatus according to claim 8. (10) An apparatus for performing a diagnostic measurement of a biological fluid with respect to a molecule contained in the biological fluid, wherein the container can be divided into at least two or more chambers, and a matrix containing an antibody or an antigen. A first chamber connected to the base material and having means for absorbing liquid; and a first chamber having a chemical means communicating with the first chamber for absorbing moisture from the first chamber. A second chamber, an exhaust port for balancing the pressures of the first chamber and the second chamber, and a filter holding means located on a matrix containing the antibody or the antigen. The base material communicates with the outside of the first chamber through an opening at the top of the first chamber, and the filter holding means is configured to open the mother material through the opening at the top of the first chamber. To remove the interfering substances present in the biological fluid as a filter material. Device is a filter retaining means having one coupled to the holding means in order to detect the molecules in cooperation with the base material by providing a reagent. (11) The first aspect of the present invention, wherein the filter holding means is of a funnel type.
The apparatus according to item 0. (12) the chemical substance provided by the filter material is a protein substance;
An apparatus according to claim 11. (13) The apparatus according to claim 12, wherein the protein substance is selected from the group consisting of an antibody, an antibody-enzyme conjugate, and an enzyme substrate. 14. The apparatus according to claim 13, wherein the chemical substance adheres to the filter by being brought into contact with the filter material in a powder form. (15) by passing it and having at least one container opening for introducing a liquid sample, in said container adapted to contact by the liquid sample is made
A matrix containing the disposed liquid absorbent and at least one dry form of the immunological reagent disposed on the liquid absorbent , wherein the matrix is connected to the at least one opening;
Through which the liquid sample is received) , wherein a chemical substance for absorbing moisture is arranged in the container separately from the liquid absorbing means and the base material. Chemicals can degrade the reagents
A device that is delayed to allow room temperature storage of the immunoassay device.