JPH0792160A - Sampling device, testing sampling device, testing method and measuring method - Google Patents

Abstract

PURPOSE: To detect a cancer antigen and the like from an organismic fluid in a short time easily with a good sensitivity, and at the same time, to filter and collect various cellular components from the fluid. CONSTITUTION: A fluid inlet and outlet are formed in a container 10 in which a filtering film 17 is arranged to divide a chamber in the container 10 into two chamber 13, 15. Cellular components in an organismic fluid can not pass through the film 17 so that they are concentrated in the chamber 15. In the opposite chamber 13 to the chamber 15, chromatography beads 70 are contained. A cellular component collecting cup 20 having an inlet and an outlet is set to the housing of the container 10. The cap 20 is in a circulation relation with the chamber 13 on the side of the beads 70 and the opposite chamber 15. By the aid of a cellular film 27 arranged in the cap 20, the passing of celullar components is prevented in disposing the fluid from the cap 20.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION This invention relates to an apparatus for collecting and testing medical and laboratory fluid samples. More particularly, it relates to a device for filtering, partially purifying, concentrating and detecting specific antigens in biological fluids such as urine and collecting urine precipitates from the urine.

[0002]

[Prior art]

Urine Cytology Under normal conditions, urine contains a small number of cells and other particulate matter that is shed from the entire length of the urinary tract. These substances are commonly known as urine sediments. Usual urine sediments consist of red blood cells, white blood cells, epithelial cells, casts, mucus and crystals. Furthermore, in patients suffering from various diseases or engaged in specific activities, sporadic urine sediments such as bacteria, yeast, parasites and sperm are produced. Examination of urine sediments is a routine procedure in urine analysis. When sick, these cells and other biologically formed elements often increase, helping to locate and type the wound. For example, an excess number of red blood cells may indicate the presence of tumors, stones or inflammation. Excessive white blood cell counts may indicate infection or other inflammatory disorders. In contrast to the low cell nature of normal urine, in the bladder epithelium under malignant conditions, malignant cells (eg, transitions, flattened and columnar cells) are often shed.

Biological Separation Method In order to analyze a complex mixture, it is often necessary to first separate it into its constituents. Even if the analytes in a complex mixture are chemically similar and the analytes in this sample are scarce, the characteristics of these analytes overlap with each other, resulting in a method or tester with sufficient selectivity. Is not present and separation may be required.
In these cases, the separation step is an important part of the analytical process. There may be reasons, for example, for the rate of analysis, that force separation to be performed as part of the overall analytical process, even when the mixture contains chemically significantly different species. If one or more of the various separation measurements were time consuming, then the total time to analyze one sample could be significantly longer. On the other hand, if the preliminary separation can be carried out sufficiently quickly, it opens the possibility to apply non-selective measurement techniques to each component after this separation. If the analytical process is compatible with the separation rate, combining the two gives an effective solution to the time problem. In modern chromatographic techniques, there have been successful examples of combining separation and measuring the components after this separation almost instantaneously.

The bioseparation method is used to purify organic compounds (ie hydrocarbons, proteins, lipids, hormones and vitamins) and inorganic compounds (ie metals and ions) and is based on chromatographic techniques. Resin-based gel chromatography utilizes ion exchange, ion exclusion, ligand exchange, reverse phase, normal phase and high affinity mechanisms. Since these interactions consist of a number of aspects, charge (ie positive, negative and neutral charge), solubility (ie hydrophobic and hydrophilic), specific ligand or chemical group interaction (eg enzyme) and There is a unique possibility of separating compounds based on antigen-antibody reaction.

The following table shows commercially available column support materials. Table 1: Ion exchange HPLC of proteins Table 2: Reversed phase HPLC of proteins and peptides Table 3: Hydrophobic interaction HPLC of proteins Table 4: Immunoglobulin HPLC Table 5: Separation of oligonucleotides and related products Table 6: Oligos Cation exchange polymerization column for sugar

[0006]

[Table 1]

[0007]

[Table 2]

[0008]

[Table 3]

[0009]

[Table 4]

[0010]

[Table 5]

[0011]

[Table 6]

Chromatography Today's chromatography exists in many forms, so it is almost impossible to define it in a concise and comprehensive manner. The so-called mobile phase in chromatography is a gas,
It has to be known that it may be a liquid or a critical fluid and may contain organic molecules, inorganic molecules or other modifiers required in the separation process. The definition of chromatography should not include all manifold morphologies of the stationary phase, solids, gels, liquids fixed in the solids, coatings on the walls of the capillaries and even when there appears to be no stationary phase at all. I won't. Furthermore, a sufficient definition should include the idea of the various ways in which the two phases can exist together. For example, in a column, the two phases meet each other, as a thin layer on a plate, as a paper strip hanging in a solvent reservoir. Evolutionary gas chromatography is a rapid, selective and sensitive method for analyzing complex mixtures of very similar volatile organic compounds. Recently, significant advances have been made in the use of liquid mobile phases, extending chromatography to the full range of organic materials, whether they are volatile or not. In this regard, solute partitioning between phases is of great interest and is one of the main concepts of chromatography. The success of performing chromatography depends largely on the basic knowledge of the partition equilibrium of solutes and their ability to influence this parallelism.

Analytical chemistry of inorganic species is largely the analysis of electrolytes in aqueous solutions, to which ion exchange has provided significant benefits. Many ion exchange chromatography methods have been devised to cleanly separate organic and inorganic charged molecules, both positive and negative. However, although this separation was effective,
They were used exclusively as an adjunct to existing wet chemical methods to solve interference and matrix problems. As a result, analysis rates were often determined by relatively slow wet chemical methods. Today, chromatographic analysis of ionic substances is widely applied and rapidly developing. The number of measurable species continues to grow, and LC plays an important role in many areas of science and technology. Table 7 below shows the breadth of chromatographic applications at this time.

Table 7: Types of Samples Analyzed by Chromatography Acid Rain Analgesics Chemicals Detergents Drinking Water Fermentation Broth Fertilizers Foods and Beverages High Purity Waters Ore Insecticides Drugs Physiological Fluids Plating Baths Protein Water Sediments Pulped Liquor Soil And plant extract wastewater

(Immunoassay) The immunoassay works based on the simple principle that an antibody specifically recognizes an antigen. Here, in order to detect and quantify a specific antigen, an antibody that recognizes the uniqueness of the antigen is required. The antigen-binding site of an antibody recognizes about 6 amino acids or their equivalent in the substance. One unique binding site
Serves as a marker to identify the protein.

If a definite antibody is obtained for a given antigen, it is used to identify the antigen in the sample mixture. Once the antibody binds to the antigen, a means of recognizing the complex thus obtained is needed. If no measurable amount of antigen is present in a particular fluid volume, it is necessary to concentrate the antigen from that volume of fluid.

The diagnosis and testing of many diseases generally requires the collection of biofluid from a patient. Serum, urine and cerebrospinal fluid are the most common samples taken for diagnosis. However, other fluids such as semen, synovial fluid, pleura, pericardium, peritoneum, amniotic membrane and sweat are also associated with particular conditions and diseases. During the collection and processing of biofluidic samples, it is important to minimize sample degradation, contamination and spread of infection from the sample. Usually, the sample is taken in a large container, but usually the actual test is a relatively small sample,
Use a volume of about 0.2 to 0.5 ml. Here, because the amount tested is small, the cancer-producing antigen can only be identified after the cancer has entered the aggravating or late tumor stage. The remainder of this body fluid sample is used for another test, frozen or abandoned. New storage problems arise when processing urine samples. This is because a relatively large amount of fluid is involved in taking a test sample. The sample may also deteriorate. This is because the shelf life of the urine sample shelf is relatively short unless kept under specific temperature conditions and / or treated with various preservatives. In addition, the sample may be damaged or spilled during the collection and / or storage process, and certain molecular components in the sample, such as antigens and cellular materials such as urine sediment, may be destroyed. There is a possibility. Packaging does not protect urine from the possibility of chemical conversion of urine to different chemical constituents, and when testing this sample, this chemistry could invalidate or result in false test results. Can bring data.

As is apparent here, in order to diagnose the existence of a disease process at an early stage of its progress, it is necessary to concentrate the molecular components of urine and to separate and concentrate the urine precipitate.

A typical sampling device is shown in US Pat. No. 4,741,346. The device includes a base stand that supports the sample bottle in an upright position. A funnel is loaded into the open end of the sample bottle and surrounds the top of the bottle. The base stand has an upwardly extending cylindrical wall that is connected to the lid and at least partially surrounds the bottle so that the user does not touch the surface of the bottle and contacts the sample. You will be able to remove the bottle without. Various types of liquid containers for collecting and transporting urine are described in U.S. Pat.No. 3,777,739,
No. 3,881,465, No. 4,042,337, No. 4,084,937, No. 4,
No. 244,920, No. 4,492,258 and No. 4,700,714.

In another sampling device shown in US Pat. No. 4,040,791, a sampling tap receiver having a nipple is disclosed and a sample container is mounted on the nipple, which receives a predetermined amount of sample under sealed conditions. . The sample container is provided with an integrally formed lid, the lid is arranged beyond the opening, and the sampling nipple is loaded in the opening. The midstream urine collector disclosed in US Pat. No. 4,557,274 has a funnel for transferring urine into the cup member, which is covered by a membrane cover.

A combination of a strip testing device and a sampling device is shown in US Pat. No. 4,473,530. It is intended for a device that combines test and collection by providing a strip of chemical reagent test placed in a tube with a specific gravity decoding means to enable rapid urine testing. is there. The liquid collecting device targeted by US Pat. No. 4,573,983 has an antiseptic member on the erasing part, and the erasing part filters urine using a filter made of a material that is impermeable to air and bacteria.

[0022]

Therefore, transporting a fluid sample such as urine through a specific immobilized bead bed,
(EN) Provided is a method for capturing a concentrated amount of antigen, separating a urine precipitate from this urine, enabling more sensitive detection of both soluble antigen and cells, and a simple method for handling a disposable device. Is desired. It would also be desirable to be able to store the test sample in a concentrated form in a compact form for a considerable period of time to allow for rapid and accurate disease marker testing at the terminal testing facility.

[0023]

SUMMARY OF THE INVENTION The present invention is directed to an apparatus for collecting urinary antigens and cellular components based on filtration and chromatography. The delivery device of the present invention provides a relatively simple method for capturing or immobilizing soluble solutes in a body fluid sample (eg, urine) and separating the particulates from the solution. Can interfere with the antigen-antibody reaction,
Filtration is essential to remove particulate matter in urine samples. Chromatographic beads allow the concentration and partial purification of soluble antigens in urine, help to increase antigen concentration and help remove other substances that may interfere with these analytical assay measurements.

The device of the present invention is in the form of two removable, sealable units. The first unit is
A urinary sediment / antigen container having two chambers separated by a porous support partition. A porous support partition sandwiches the filter membrane and allows fluid to flow bi-directionally therethrough. A gel chromatography resin (beads) is placed in the upper chamber across the membrane and a bottom chamber designed to hold the urine sediment of the urine sample is placed under the separation membrane. The filtration membrane allows the soluble material to pass through to the upper chamber for mixing with the beads while retaining the urine sediment in the bottom chamber. The second unit is a cell collection cup, which is attached to the bottom chamber of the first unit after the sample has been drawn through the first unit.

The cell cup is composed of two screw-fastenable parts, has an open inlet (upper part) and outlet (lower part), and contains a cell membrane. A cell membrane having a pore size dimension greater than 2.0 microns is laid over the porous membrane supporting septum as part of the lower part of the cell cup. The cell cup is configured to receive the urine sediment retained in the bottom chamber of the first unit on the upper surface of its cell membrane.

These two units can be separated after processing the sample and both the urine antigen and / or the precipitate can be carried or processed separately.

It is an object of the present invention, in particular, when ligands such as antigens are removed from body fluids for testing and precipitates such as cells are removed from body fluids for pathological examinations. The collection and concentration of specific antigens from body fluids and the collection of cells and other particulates onto membranes for further testing. In the past, the above-mentioned test was conducted by a series of tests using a large number of different containers and expensive laboratory equipment with limited sensitivity.

The accompanying drawings illustrate embodiments of the present invention. From these, the objects, new features and benefits of the present invention will be readily apparent.

[0029]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT A preferred example and best mode of the invention is shown in FIG.
~ 6. Usually, the first urine collected is contained in a 100 ml container with a gradient as shown in FIG.
Currently, such vessels are manufactured under the designated 4013 sample vessel by Becton Dickerson Labware. The collection container holds 4.5 ounces (approximately 133 ml), is graded,
Equipped with a polyethylene snap lid. The antigen precipitate transport container 10 has a dome portion 12 formed with an outwardly extending flange 14 and a flange outwardly extending in the form of a corresponding mirror image.
14 'consists of a funnel portion 19 also formed. Both flanges 14 and 14 'are joined together to hold the filtration membrane 17 in position between the porous membrane supports 18, which are joined to the inner surface of each of the two parts 12 and 19. There is. Desirably, the diaphragm support 18 can be cut so that it can be flatly mounted on the inner wall of each container portion, or can be fitted into a groove cut in the inner wall of each container portion. it can. The flanges 14 and 14 'can be sonic welded to each other or can be joined by an adhesive or a fastener. The filtration membrane 17 is placed in the porous membrane support 18 and the container is divided into separate chambers 13 and 15. A bed of beads 70 with immobilized antibody bound to the beads is deposited in chamber 13 on the syringe side of filter 17. The filtration membrane 17 preferably has a filtration particle size of 5 microns, but may vary from 0.5 to 5 microns, or may allow the fluid to flow through and allow the antigen to pass through, and the beads 70 And may vary within suitable dimensions to prevent passage of urine sediment 56.
Xydex (Xy), a branch of Genex
Any suitable filtration membrane 17 can be used in the container housing, such as aqueous glass microfiber filters manufactured by dex) or membrane members manufactured by Millpore. One end 11 of the dome portion 12 of this container is attached to the protrusion, and Becton Dickinson (Becton Dicki
nson) 30 cc manufactured by And Corporation
Remove the threaded luer lock on the syringe 40.
ck) 44 is configured to be screwed around and attached. It should be noted that all pump-type devices could be used in place of syringe 40, such as the autovial fiberglass filters manufactured by Genex. The syringe 40 has a cylinder 41 to which a luer lock 44 is connected, a piston 42, and a piston head 43. Although the present invention is applicable to all body fluids, it initially tested various cancers in the body,
It was designed for use in collecting concentrated urine antigens and urine sediments to determine the presence and stage of this cancer.

As shown in FIGS. 1-4, the bead transport (shuttle) container is made of polystyrene. The container housing has an outer cylindrical wall with end walls 42 and 44, where the end walls 42 and 44 have a urine inlet 46 outlined by the end conduit 21 and an end conduit 20. Contoured exit 48
And are provided respectively.

The cell pellet cup 20 is screwed onto the end conduit 21 and the bottom wall 28 of this cup acts as the final screen filter.

The beads 70 are preferably visible (greater than 10 microns in diameter) so that they can be visually observed as they flow into the syringe barrel 41 and back into the container 10. Can keep maximum contact with urine. It should be noted that the volume of the beads 70 should not be larger than the volume of the chamber 15 of the container so that the neck of the syringe does not begin to become blocked. Monoclonal antibodies and other suitable ligands have been immobilized in urine (covalently bound) on beads 70 as is well known in the art and complexed with prelabeled polyclonal antibodies as described below. It is designed to have a binding site with high affinity for the epitope of the cancer antigen being carried.

As an example of such beads, “Protein G Sepharose” manufactured by Pharmacia, Bioprobe International (Bio Probe internatio)
"Hydrazide Avid Gel Ax" manufactured by nal) and "Actigel-ALD" manufactured by Sterogene BioSeparation Inc.

A good reason for using "Actigel-ALD" is that it does not crosslink with the protein and therefore retains the protein's high biological activity after immobilization of the protein. Also available from Sterogeen Bioseparation, Inc. "Actigel-
According to "ALO SUPERFLOW", up to 3000 cm per hour
The linear flow velocity of the
It will fit well with 10-100 cm).

The resin beads 70 are provided with a matrix and a primary ligand (in this case, an immobilized monoclonal antibody). However, other suitable ligands may be used. Urine after filtration in a buffered form by adding 200 mM Tris-HCl buffer together with NaN ₃ manufactured by Pharmacia and the resin beads are brought into contact with each other while flowing, and the resin beads are allowed to react with the antigen / antibody reaction. Or by an immune response
Captures specific ligand components carried by urine, ie complexed antigen / labeled antibody. Note that the labeled polyclonal antibody in solution had already been added to the buffered urine.

In operation, the cell cup 20 is removed from the container 10 prior to the procedure. The cup 20 consists of two threaded parts 22 and 25. This upper part 22 is provided with a neck 21, which forms the inlet, the neck 21
An external thread formed on the can engages with the thread 16 of the container 10. A threaded skirt 23 is formed at the other end of this portion 22 and the skirt 23 is adapted to mate with the screw 24 of the skirt of the portion 25. The outer diameter of the skirt and the screw 24 is smaller than the inner diameter of the skirt 23. A porous support member 28 is placed inside the skirt of portion 25. The porous support member 28 may be placed in a groove cut in the inner wall surface of the portion 25, may be sonic welded to the inner wall surface, or may be adhesively coupled to the inner wall surface. A cell membrane 27 having a cell diameter dimension exceeding 2.0 microns is laid on the support member 28. A cell membrane that can be preferably used is a polycarbonate membrane filter manufactured by Nuclepore, and is identified as follows.

Storage number description 110104 PC MEMB 13MM 3.0 μm 110410 PC MEMB 13MM 5.0 μm 110414 PC MEMB 13MM 8.0 μm 110612 PC MEMB 25MM 3.0 μm 110613 PC MEMB 25MM 5.0 μm 110614 PC MEMB 25MM 8.0 μm 111112 PC MEMB 47MM 3.0 μm 111113 PC MEMB 47MM 5.0 μm 111114 PC MEMB 47MM 8.0 μm 113313 PC MEMB 19 × 42MM 3.0 μm 113313 PC MEMB 19 × 42MM 5.0 μm 113314 PC MEMB 19 × 42MM 8.0 μm

The end of section 25 is funnel-shaped downwards towards outlet tube 26. The cell cup is designed to contain urine sediment 56 in a chamber delineated by section 22.

Two types of membrane filters are mainly used in cytodiagnosis. Cellulose and polycarbonate. Cellulose membrane is composed of cellulose nitrate,
Manufactured from cellulose acetate or a combination of both. The method for producing a cellulose membrane involves blending a casting solution consisting of several solvents and additives to give the membrane strength, specific pore size and pore volume (porosity). . The casting solution is then cast on a conveyor belt,
Passing the conveyor belt through a controlled heating tunnel,
Here the solvent is evaporated. Evaporation of the solvent in this manner will deposit a thin film of the desired thickness, consisting of a porous material having a defined structure that is considered as a precise and precise film stacked on top of each other. The point of attachment of these polymer threads is
It may be referred to as the nucleation site. Here, the pore diameter and the porosity can be designated by increasing or decreasing the nucleation site. Pore size measurements are made by the "bubble point" test which includes the surface tension of the material at hand, the viscosity of the fluid filling the capillaries and the diameter of the capillaries. Almost all samples of diagnostic importance (urine, cerebrospinal fluid, etc.) contain measurable amounts of protein, which can bind to the cellulose membrane and cause background staining. Most staining protocols for cellulosic membranes provide staining tailored to minimize this protein staining. The more protein present, the more membranes will absorb and stain it. Moreover, the diagnostic information becomes unclear.

The composition and manufacture of polycarbonate membranes are quite different from cellulose membranes. Polycarbonate membranes are produced by bombarding a thin film (10 microns) of polycarbonate material in a nuclear reactor. Particles of atoms pass through the material, weakening the chemical bonds in the polycarbonate. Subsequent etching baths create precision pores whose dimensions can be controlled very precisely as a result of how long the material remains in the bath. Unlike cellulose membranes, the exact dimensions of Nuclepore polycarbonate membranes can be physically measured by scanning electron microscopy to ensure proper pore size.

Urine 55 is removed from container 100 until the total volume of urine / buffer pre-labeled antibody solution is 10 cc at pH 8.8. The cell cup 20 is attached to the container end 16 and the urine buffer mixture is applied by the piston 42 to the container 10 and the cell cup.
Push through 20 filters 27 into a clean container.
The cell cup 20 is then removed from this transport (shuttle).

A 3 cc syringe is used to inject 1 cc of the cell fixative into the uncoupled cell cup 20, and the cup is covered with a lid member 30. The cell sample is then stabilized until the next step. Return the urine buffer mixture into syringe 40 and set aside. The fluid is then released after transport through the shipping container into a waste container. At this point, the cell sample was reattached to container 10 for cell testing.
It can be placed in the box to be returned and cell tests may be performed by the relevant laboratory.

When a specific cancer antigen is present in the urine test sample 55, which is preferably the urine excreted first thing in the morning, this cancer antigen reacts with the labeled antibody to form the antigen / antibody complex. To generate. This complexed antigen / antibody is a bead
It is captured by the immobilized antibody carried by 70, where it remains in the housing chamber 15 as clearly shown in FIG. If no antigen is present in sample 55, beads
The immobilized antibody on 70 will remain unoccupied.

Draw the buffered sample into a syringe and blow it out, preferably 3-5 times, or back flush;
Maximize the flow of liquid over the beads. The shuttle is then washed with the coloring reagent. Flush the urine over the beads 70 and deposit the conjugated antibody on the immobilized antibody,
It is preferred to soak this bead bed in ABTS solution. When using OPD or TMB or other dual substrate devices, a hydrogen peroxide (H ₂ O ₂ ) solution can be placed on the bead bed instead.

The coloring solution used on the bead matrix is composed of several acronyms, namely ABTS (2,2'-azino-di- [3-ethylbenzthiazoline sulfonate.
(6)]: OPD (ortho-phenylene diamine): or
Under one of the TMB (tetramethylbenzidine),
It is preferably a substrate manufactured by Kirkegaard and Perry Laboratories. To select the substrate, the sensitivity of the immunoassay is measured by fractionating the antibody reagents. When this happens, using a more sensitive substrate can only increase the signal and background in a stoichiometric manner. The result is more coloring, but the signal to noise ratio is the same. The more sensitive substrates push absorption beyond the cutoff of the decoder, while the faster substrates may actually reduce the signal to noise ratio.

The preferred coloring solution according to the invention is ABTS. The preferred ABTS substrate is a one-component substrate. The HRP label on the pre-labeled antibody was turned blue-green by ABTS, and when the reaction was stopped with SDS (sodium dodecyl sulfate), while reading the expressed color using a reflectometer,
No change in color or absorption. If optimizing the assay indicates that the sensitivity of the immunoassay is limited by the color produced by the HRR substrate, the longer sensitivity
The TMB substrate can develop more color without a corresponding increase in background. Another benefit of TMB substrates is that TMB substrates often reduce the amount of reagents required for immunoassays.
The TMB substrate is a binary liquid substrate and requires hydrogen peroxide. HRP turns TMB into a blue product. When the reaction is stopped by acidification, the TMB product turns yellow. Generally, ODP is provided as tablets and dissolves in buffer at the time of use. HRP converts OPD to a yellow product, which continues to oxidize to a brown precipitate. Upon acidification, this OPD product turns orange.

The bead bed matrix and the immobilized ligand (immobilized antibody in this case) capture the antigen / antibody complex by an antigen / antibody reaction or an immune reaction.
The antibody in the complex as described was given labeled with the coloring enzyme HRP. The antibody labeling enzyme reacts with ABTS poured onto the bead surface, turning the bead surface into a blue-green color. If there is no specific antigen in sample 55,
This labeled antibody will remain unoccupied and will not bind to the immobilized antibody. The degree of color developed should be related to the amount of antigen present in sample 55. The color developed on the beads is then read using a reflectometer, as is well known in the art.

The current high affinity beads 70 are capable of capturing complexed antigen / antibody present in 100 ml or more samples depending on the number of times the syringe is filled and emptied.
This would result in a 500-fold increase in the amount of antigen captured by the beads. Preferably, the syringe is filled with urine and the beads are free to move into the barrel of the syringe to maximize fluid contact and mixing. This syringe 3-5
By emptying and refilling, the concentration is maximized, and an antigen concentration 1000 times higher than previously possible is obtained.

The sample life of the buffered sample is more than 6 months under normal storage conditions after washing the beads with a preservative solution, for example 0.01% sodium azide (bacteriostatic).

Precipitate / antigen container 10 with urine in syringe 40
Through, where the urine is drawn to the filter 17,
pass. Filter 17 has an average pore size of greater than 0.2 micron. The filter 17 blocks cells and cell debris into the urine sediment chamber (bottom chamber) 15, while passing filtered urine fluid and associated antigens through the bead chamber (top chamber) 13. The beads are moved into the syringe by passing the filtered urine and the beads leaving the bead bed are allowed to mix freely with the solution. The chromatography beads are capable of capturing specific antigens carried by buffered urine fluid. Prior to placing the urine in a syringe, the urine is buffered to physiological pH so that its cellular constituents remain stable. While the urine passes through the filter 17,
Urine cells and / or precipitates are retained under the filter in the bottom chamber 15 of the precipitate / antigen container. After a short incubation time of the syringe / precipitate vessel, the inlet 21 of the cell cup 20 is then attached to the cylindrical outlet member 16 of the syringe / precipitate vessel and the syringe is emptied. Upon emptying the syringe, the beads 70 return from the syringe to their container chamber 13, while the urine sediment retained in the bottom chamber 15 of the bead / precipitate container is transferred to the cell cup by the urine of the processing liquid. It is pushed inside. Although the syringe is emptied at the same time with both units attached, the cell membrane 27 in the cell cup can trap urine deposits on its upper surface. After emptying the syringe completely, the cell cup is removed from the bead container, which in turn is removed from the syringe. Cells trapped on the cell membrane are fixed and / or stained with a suitable reagent that can be injected through the entrance of the cell cup. Both the urine antigen container and / or the cell cup can be treated in the field like a screening test or transported to the laboratory as a transport device for further testing (analytical test)
You can also do it.

The method and apparatus of the present invention will be further described by the following examples, which are not intended to limit the invention.

Example 1 Improvement of Sensitivity of Enzyme Immunoassay (EIA) Detection Device Using CDI-Shuttle Device Purpose: Pair of antigen concentration in the eluted sample after using the device of the present invention which is a CDI shuttle device To determine the sensitivity of measurement of urinary antigen concentration.

Required Materials 1 Urine Sample 2 10cc Becton and Dickinson Ruhr (Becton
and Dickenson Luer) Lock syringe 3 100 mM Tris-HCl buffer (pH7.8) 4 50 mM Tris-HCl buffer (pH7.8) 5 50 mM Tris-HCl buffer (pH7.8) containing 1M NaCl 6 CDI shuttle device 7 Sigma from human fetus (SIGMA) α-fetoprotein (AFP) (F-8004) 8 DAKO anti-α-1-fetoprotein antibody (immunoglobin for human rabbit) 9 KPL Affinity purified goat peroxidase
Antibodies to (0.1 mg) labeled rabbit IgG (H + L) 10 "KPL ELISA MATE" Peroxidase ELISA (ELISA) device and reagents 11 KPL-TMB peroxidase substrate system

Procedure: 1. For the purposes of this study, 15 urine samples from patients with various medical conditions were selected as representative of patients in the clinical setting shown below. (1) Bladder cancer (2) Bladder cancer, PO (3) History of bladder cancer (4) Testicular cancer (mild) (5) Urethral stones (6) Prostatic hyperplasia (7) Prostate cancer, stage IV (8) Prostate Hypertrophy (9) Bladder tumor (superficial) (10) Bladder cancer, PO (11) Rt. Epidemo-orciti
s) (12) Urethral stones (13) Standard control (14) Standard control (15) Standard control

2. Urine pH was adjusted as follows: 18 ml of urine was added to 18 ml of 100 mM Tris buffer (pH 7.8) at a 1: 1 dilution. 3. The following preparations were performed using a pH adjusted urine / buffer mixture. (a) 5.0 ml of urine / buffer mixture was used as a background control. (b) 5 ml of urine / buffer mixture was added to AFP antigen in various amounts as indicated below: 1.2 μg 2.0.4 μg 3.0.08 μg

4. Each urine preparation was dispensed into a syringe + CDI shuttle device. This mixture is then allowed to stand at room temperature for 15
Placed on the rotator for minutes. After a 15 minute incubation, the solution was removed from the shuttle and discarded. 50 ml of 10 ml for each shuttle
Wash by filling the syringe with mM Tris buffer (pH 7.8) and emptying.

6. The protein bound to the beads in the shuttle was treated with 2 ml of 50 mM Tris buffer containing 1 M NaCl.
The buffer was eluted at (pH 7.8) by aspirating through a shuttle into a syringe. After leaving for a few minutes to mix thoroughly, the syringe was emptied and the solution was collected in a test tube and used for the ELISA test. 7. Each solution was tested by the Bradford assay with the BIO-RAD protein assay and the results reported in μg / ml.

8. Elisa test (a) Dynatech Laborato
Immulon I plate sold by ries) was coated with a mixture of 50 μl of each sample and 50 μl of coating buffer. AFP standard curve
Prepared on the plate using AFP antigen. All plates were then incubated overnight at 4 ° C. (b) All plates were blocked with 1% BSA blocking solution. 300 μl was added to each well and incubated at 37 ° C. for 30 minutes. (c) Primary antibody: octopus anti-α-1-fetoprotein 0.02
Diluted 1: 300 in% Tween PBS solution. Ten
0 μl was added to each well and the plates were incubated at 37 ° C for 1 hour. (d) Secondary antibody: KPL goat rabbit antibody was diluted 1: 400 with 0.02% Tween PBS solution. 100 μl was added to each well and the plates were incubated at 37 ° C for 1 hour. (e) Substrate: 100 μl of TMB peroxidase substrate was added to each well and the plate was left to develop for 15 minutes. (f) Stop: After 15 minutes of incubation, in each well
The color reaction was stopped by adding 100 μl of 1 MH ₃ PO ₄ . The plate was then read at a wavelength of 450 nm.

Direct EIA measurements of antigen concentration in urine samples showed poor correlation (R ² >
0.4) was shown (Fig. 7). However, some samples eluted from the same urea showed a very high correlation (R ² > 0.9) to the actual concentration increase after using the shuttle method. Urine samples (body fluid samples) appear to contain various substances that can interfere with the antigen detection device. The shuttle method appears to reduce these interfering substances and allow more accurate detection of the antigens present in urine.

Example 2 Comparison of CDI Shuttle Instrument with Standard Column Chromatography Objective: To demonstrate the advantages of the CDI shuttle over standard column chromatography procedures.

Procedure: 1. For comparison, the following shuttles and columns were prepared by the following method: (a) 6 shuttles with Q-Sepharose beads (b) Column 6 with 300 μl Q-Sepharose particles.
Seed (C) 6 columns with 1 ml Q-Sepharose beads

2. The following solutions were prepared to test the shuttle-column comparison: (a) 1 mg of BSA was dissolved in 10 ml of 50 mM Tris solution (pH 7.8).
BSA stock solution (b) Urine # 1-100 mM Tris buffer (pH7.8) diluted 1: 1 (c) Urine # 2-100 mM Tris buffer (pH7.8) diluted 1: 1 The volume of the solution was 70 ml.

3. For shuttle testing: (a) Using a replication shuttle for each sample, place 10 ml of solution into each shuttle device and incubate on the rotator for 15 minutes to ensure that the beads are homogeneously mixed with the sample. did. (b) After 15 minutes, each shuttle was thoroughly washed with 50 mM Tris solution (pH 7.8). (c) Each sample was treated with 50 mM Tris buffer containing 1 M NaCl (p
H7.8) was eluted with 2 ml and the sample was saved for protein analysis.

4. For column test: (a) 10 ml of each solution was put into the corresponding column. Duplicate columns were performed for each sample. (b) After the solution has completely passed through the column, 50
It was thoroughly washed with mM Tris buffer (pH 7.8). All washes were left for a sufficient time to pass through the column. (c) Each column was eluted by passing 2 ml of 50 mM Tris buffer (pH 7.8) containing 1 M NaCl, and the whole solution was collected. 5. Protein analysis was performed on all samples using Bradford analysis. The proteins recovered from each sample were compared using various methods of preparing elution samples.

Advantages of CDI Shuttle over Column Method 1. Time efficiency The entire process on the CDI shuttle can be completed within 20 minutes. However, in the column method, time depends on the condition of the sample. If the sample is unusually thick, the time for testing will be longer compared to a standard urine sample.

2. Binding capacity As is evident from the results shown in Table 8 below, the binding capacity with the CDI shuttle is at least 50% higher than with a column with the same amount of Q-Sepharose. This binding is achieved by adding the sample to the beads by 1 in the column method.
Even a 15 minute suspension, rather than a single pass, can be adjusted and improved.

3. Ease of use This CDI shuttle requires little technical expertise. All that is required is the ability to place the solution in a syringe-like device. However, in the column method, depending on the operator's failure, the beads may be disturbed and lost, or bubbles may enter the bead suspension. The CDI shuttle keeps the beads in a restricted location so that the risk of failure is extremely low.

Table 8-Comparison of CDI shuttle and conventional LC technology Compact packaging LC column Membrane LC CDI LC shuttle 1 Particle size 70.22 μ Membrane 70.22 μ 2 Filter pore size 70.22 μ 70.22 μ 70.22 μ 3 Column support Bead Membrane beads And / or Membrane 4 Solute phase Mobile phase Mobile phase Mobile phase 5 Ligand phase Stationary phase Stationary phase Mobile phase 6 ^* B / M ratio Very high 71 Very high 7 Flow rate Low Low High 8 Functional groups Unlimited Finite Unlimited 9 Solute And repetitive phase of ligand phase N / A finite unrestricted mixing 10 Sample pre-filtration No Yes 11 Retention time High High Low Low Eluent concentration Rare Rare concentration 13 Purification Yes Yes Yes 14 Particle size exclusion chroma Yes No No Tography 15 Ion-exchange chromatography Yes Yes Yes Matography 16 Reversed-phase chromatography Yes Yes Yes Yes Raffy 17 Hydrophobic reaction chroma Yes Yes Yes Yes Tomatograph 18 Affinity chroma Yes Yes Yes Yes Romanography 19 Separation of granules / No No Separation * Surface area available for ligand binding / membrane surface area

Example 3 Effect of Time on Cell Preparation Objective: To determine the effect of time on the morphology of cells in a cell cup.

Procedure: 1. Two fresh urine samples to be used were obtained. An empty shuttle (no particles) was used to add 9 ml urine + 1 ml 200 mM Tris buffer (pH 7.8). 2. The solution was left in the cylinder for the following times: 0, 5,
10, 15 and 60 minutes. 3. For each sample, after the appropriate amount of time, the cell cup was attached to the end of the shuttle and pushed as much urine as possible. 4. The cell fixative was poured into the cell cup until it was found that the fixative reached the bottom of the cup. The fixative in the cup was incubated for 2 minutes. 5. Diffquick solution A was poured into the cell cup until the fixative was found to reach the bottom of the cup. Incubated for 2 minutes. 6. Diffquick's Solution B was poured into the cell cup until the strain was found to reach the bottom of the cup. Incubated for 3 minutes. 7. After the staining was completed, the cell cup was opened, the membrane was placed on the slide and left to dry. After drying, the membrane was removed and the cells were observed under a microscope to compare any differences in cell morphology.

Conclusion: Time did not seem to have any effect on cell morphology. 15 minutes was sufficient for the obtained dyeing, and color development was best at 60 minutes.

Example 4 Stability of Q-Sepharose Immunizing Antigens (eg Actin) Objective: To determine the ability of Q-Sepharose beads to retain protein activity after immunization in the shuttle. To test the stability of this activity by comparing incubation at room temperature with cooling (+ 4 ° C).

Procedure: 1.4 g of actin was placed in 12 ml of Q-Sepharose beads and incubated for about 1 hour at room temperature on a shaker. 2. The beads were then washed several times with 20 mM Tris solution (pH 7.8). 3. Actin beads were used to set up the shuttle device (about 35). The 4.5 shuttles were eluted with 2 ml of 20 mM Tris solution (pH 7.8) containing 1 M NaCl. The eluate was tested on an Eliza plate to determine the activity of actin on the beads. 5. The remaining shuttles were divided for incubation at 4 ° C. and room temperature, and two shuttles were tested under each condition once a week to observe the retention of actin activity from the beginning.

Elisa Test Procedure for Eluates: 1. The eluates were tested in duplicate with 90 μl of each sample eluate + 10 μl of concentrated coating buffer (10x) and incubated for 1 hour at 37 ° C. It also contains 1 mg actin for the standard curve with an initial dilution of 1: 160 and serial dilutions with 100 μl of 1x coating buffer. 2.1% BSA blocking solution was added and incubated at 37 ° C for 30 minutes. 3. Primary antibody: Actin antibody was diluted 1: 200 with Tween PBS solution (Sigmalot No. # 97F-4828) and incubated at 37 ℃.
And incubated for 1 hour. 4. Secondary antibody: goat rabbit antibody was diluted 1: 400 in Tween PBS solution and incubated for 1 hour at 37 ° C. 5. Substrate: ABTS was applied and the reaction read after 5 minutes. 6. The average true absorbance and μg of actin recovered from each shuttle were calculated.

Conclusion: 1. Actin was
It seems to be stable in the shuttle for about 3 weeks. All expansions during this time indicate the potential for drying of the beads on the shuttle and / or a loss of 1/2 of the initial activity.

2. Incubation at room temperature retained most of the activity for 3 weeks, but the loss of activity was still significant. Therefore, the recommended storage method is to properly seal the shuttle at the refrigerator temperature for accurate results. The storage container must contain a small amount of water to prevent the beads from drying out on the shuttle.

While the present invention has been described with reference to particular preferred embodiments, the specific details shown are merely examples and the invention is not to depart from the spirit and scope of the appended claims. It should be understood that this can be done by

[Brief description of drawings]

FIG. 1 is a sectional view of a shuttle device according to the present invention.

2 is a cross-sectional view of the antigen / precipitate assembly with the shuttle device of FIG. 1 mounted on a syringe.

FIG. 3 The antigen / precipitate assembly of the shuttle device of FIG. 2 is submerged in buffered urine, which moves in the direction indicated by arrow A into the syringe. It is sectional drawing which shows the state which is.

4 is an illustration of the shuttle device antigen / precipitate assembly of FIG. 2 wherein urine precipitate is retained underneath the membrane of the sediment chamber while buffered urine and beads are mixed inside the syringe. It is sectional drawing which shows a state.

FIG. 5: Shuttle device attached to syringe, cell cup attached to this antigen / precipitate assembly,
The syringe plunger moves in the direction opposite to that shown in FIG. 3, the buffered urine is discarded from the syringe in the direction of movement indicated by arrow B, beads are deposited in one chamber of the shuttle container, and the upper surface of the cell membrane. It is sectional drawing which shows the state which the urine cell precipitate was captured on.

6 is a cross-sectional view showing a state in which the antigen / precipitate assembly is removed from the syringe and the cell cup is removed from the antigen / precipitate assembly in the shuttle device shown in FIG.

FIG. 7 is a cross-sectional view showing another example of the antigen / precipitate assembly and the cell cup removed from each other.

[Explanation of symbols]

 10 Antigen precipitate transport container 11 One end of dome part 12 Dome part 14, 14 'Flange 13 Chamber, bead chamber 15 chamber, urine sediment chamber 16 Screw of container 10, container end, outlet member 17 Filtration membrane, filter 18 Porous Membrane support, diaphragm support 19 Funnel 20 Cell pellet cup, cell cup 21 End conduit, neck, inlet 22 Upper part 23 Skirt 25 Lower part 26 Outlet tube 27 Filter 28 Cup bottom, porous support member 30 Lid member 40 Syringe 41 Cylinder 42 Piston 43 Piston head 44 Luer lock 55 Urine, Urine test sample 56 Urine sediment 70 Beads 100 Container

Claims (6)

[Claims] 1. An apparatus for collecting a molecular sample and a urine sediment from a urine sample, comprising a cylindrical container, and a sample collecting unit having a housing having a filtration membrane supporting means, on the supporting means. A specific antigen to be carried in urine, comprising a filtration membrane laid in the housing to divide the housing into a plurality of chambers, and a bead means arranged with an immobilized antibody means in one of the housing chambers. The bead means is biologically configured to capture, and the filtration membrane mounted within the housing of the sampling unit prevents the bead means from flowing into the urine sample with the immobilized antibody means. On the other hand, the bead means is allowed to flow into the cylindrical container, and a cell collection cup detachably attached to the housing of the collection unit is provided. The filtration means attached to the intake cup
A collection device that allows urine to pass through the cell collection cup while retaining the urine sediment in the cell collection cup to prevent the urine precipitate from being discarded with the urine from the cell collection cup. 2. An apparatus for testing a molecular sample in a biological fluid and collecting cellular components from the fluid, the apparatus comprising a container housing with fluid inlet and outlet means and contouring a chamber, the housing comprising: The chamber is equipped with a filtering means which is installed in the chamber and divides the chamber into two chambers, through which the flow of the biological fluid and the antigen carried in the flow flow through the filtering means into one chamber. On the other hand, it is possible to concentrate the cell component in the other chamber by preventing the cell component from passing through the filtration means, and the chromatography beads contained in the chamber opposite to the cell component side chamber. Means for removably mounting to the container housing with inlet and outlet means and for communicating with the chamber opposite the chamber containing the chromatographic bead means. A test and collection device comprising a cell component collection cup in general communication, wherein the cell membrane means mounted in the cell cup prevents passage of cell components when discarding biological fluid from said cell cup. 3. The cellular component collection cup has an upper portion,
The lower part detachably connected to the upper part, a porous membrane supporting partition mounted on the lower part, and a membrane member laid on the porous membrane supporting partition. Testing and sampling equipment. 4. The membrane member has a pore size dimension that allows urine to pass through the membrane member while urine sediment from the urine is retained in the cell collection cup. Three
Test and sampling equipment as described. 5. A method of testing a given molecular entity in a urine sample, comprising: a. Collecting urine in a container; b. Buffering the urine to the desired pH; c. Adding to this urine a labeled antibody that complexes with a specific antigen in buffered urine; d. Urine after buffering with the complexed antibody / antigen is passed through a urine treatment container holding immobilized antibody bead means, whereby the urine is brought into contact with the bead means and the desired complexed sample is placed on the bead means. Capturing e .; e. Washing the processing vessel containing the captured complexed sample with a buffer; and f. A test method comprising the step of eluting a desired ligand for analysis from an immobilized antibody bead means. 6. A method for measuring the sensitivity of measurement of antigen concentration in urine, comprising: (a) withdrawing a urine sample from a patient with cancer disease; (b) adding TRIs buffer to the pH of the urine.
And (c) adding 0.08 microgram to 2 micrograms of antigen to the urine buffer to prepare a mixture; (d) immobilizing ligand Filtering the mixture by a filtration device comprising a bead means provided; (e) placing the filtered mixture on a rotary shaker at room temperature; (f) capture bound to the beads in the filtration device. A method for measuring, comprising the steps of eluting a protein into a solution; and (g) testing this solution using a Bradford assay.