Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/5302—Apparatus specially adapted for immunological test procedures
  • G01N33/5304—Reaction vessels, e.g. agglutination plates
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form

Definitions

• This invention relates to immunological methods and apparatus. More specifically, this invention relates to a specific technique and apparatus for conducting an immunological assay procedure upon a fluid, such as a body fluid, or a carrier fluid, in which the immunological component to be detected is carried.
• a fluid such as a body fluid, or a carrier fluid, in which the immunological component to be detected is carried.
• a fluid which contains the immunological constituent of interest e.g. an antigen
• a fluid which contains the immunological constituent of interest is introduced into a plunger filter apparatus, typically though not necessarily through the filter, and into contact with immunologically binding beads which are in the plunger filter.
• immunologically binding beads bind to the immunological constituent of interest, e.g. the antigen or antibody, and extract it from the solution in which it is carried.
• the solution is then forced to flow out of the plunger filter, through the filter, leaving the beads in the plunger filter.
• the beads are washed to remove excess fluid and to retain only the immunological constituent of interest
• O PI which is bound to the beads.
• a developer is introduced' into the plunger filter which binds to the immunological constituent of interest, e.g. the antigen or antibody. Excess developer may be removed by washing, filtration, etc. Finally, the immunological constituent of interest is determined by measuring the developer according to the indicator which characterizes the developer.
• the invention comprises a novel improved plunger filter assembly containing beads which bind selected immunologically active constituents of the fluid to be tested, a container tube in which the plunger filter fits in a movable, fluid-tight relationship to permit fluid to be forced through the filter in either direction.
• a kit including a number of such elements associated one with another in appropriate container structures to provide an apparatus complete and sufficient for the technician to run im unoassays according to this invention.
• the invention may be described as an immunoassay apparatus comprising a cylindrical container tube having an open end and a closed end, and a plunger filter assembly received in the open end of the container tube and being slidable in the container tube, the plunger filter assembly comprising a cylindrical tube open at both ends, a movable seal fixedly secured at the proximal end of the cylindrical tube, the distal end extending outwardly toward the open end of the container tube, the seal being movable with respect to the interior walls of the container tube and forming a substantially fluid tight movable seal between the proximal end of the plunger filter and the container tube, a high surface area, typically dome-shaped filter inside and closing the proximal end of the plunger filter cylinder to the passage of material in and out of said proximal end except for fluids and particles which can pass through the filter. and immunologically reactive beads which, in use, bind selected immunologically active constituents, of the fluid to be assayed, the immunological
• the filter includes pores as large as possible to permit free two-way passage of the assayed fluid and its constituents, such as blood cells, while still being small enough pored to retain the beads on one side of the filter without clogging;
• the filter seal comprises a skirt extending circumferentially around the plunger filter tube into movable sealing contact with the internal wall of the container tube and a sleeve securely received inside the plunger filter tube, the top of the sleeve being so constructed and arranged as to form a generally flat annular top surface which intersects with the internal wall of the plunger filter tube at an angle of not more than 90 * .
• the immunologically active beads are large enough overall diameter not to pass or clog the filter, but otherwise as small as possible to slow their rate of settling, thus speeding the assay reactions.
• the beads should be small enough pored to prevent any antibody or -developing agent from entering the interior of the beads; in this way, all coupling and reaction steps occur on the outer surface of the beads, speeding reaction time and decreasing background interference.
• the bead pore size should still be maximal to decrease the specific gravity of the beads, hence slow the rate of bead settling, and further speed assay reaction times.
• the invention is an immunoassay process comprising the steps of containing a liquid assay sample in a predetermined physical configuration; forcing a filter through said configuration thereby forcing liquid sample through said filter into a second configuration into contact with a multiplicity of beads having immunologically reactive sites thereupon; maintaining the liquid in contact with said beads for a predetermined time to permit reaction of immunological constituents in the sample to react with said immunologically reactive sites on said beads; withdrawing the filter from the configuration thereby forcing liquid sample through the filter out of . the second configuration out of contact with te beads; washing residual liquid from the beads; determining the reaction of i ⁇ nunological constituents from the sample with the immunologically reactive sites on the beads.
• the determination step comprises removing immunological constituents from the beads into liquid in said second configuration, reacting said constituents with an indicator, and determining the presence of indicator in said liquid in the second configuration as a measure of the amount of immunological constituent in the sample.
• Figure 1 is a perspective view of the basic structure of the apparatus of this invention.
• Figure 2A is a cross-sectional view showing in detail the structure of the lower part of the plunger filter, including the filter, the movable seal, the bottom of the plunger filter tube and the container tube.
• Figure 2B depicts an alternative embodiment of the seal as shown in Figure 2A.
• Figure 3 is a perspective view of a plunger filter with a fluid contained therein during extraction of the immunological component from the fluid onto the beads.
• Figure 4 is the same plunger filter but with the fluid removed, the beads being retained on the filter.
• Figure 5 is a perspective view of a kit embodying th ' e concept and principle of the invention.
• the inventive apparatus comprises a container tube 10 which typically is in the form of a conventional cylindrical tube with a round bottom, often referred to as a test tube.
• the essential reaction vessel is a plastic cylinder 20, to which a removable seal 22 is affixed which seals the space between the tube 20 and the container tube 10 when the former is inserted in the latter.
• the reaction vessel also comprises a filter 24 and beads 26, the latter being shown better in Figures 3 and 4.
• the combination of the tube 20, the seal 22 and the filter 24, which contains the beads 26, is referred to as a plunger-filter.
• the seal 22 comprises an insert sleeve 22a, which is in fluid-tight relationship on the interior of the tube 20 and a skirt 22b which extends outwardly around the circumference of the tube 20 into contact with the inner walls of the tube 10 forming a movable, fluid-tight seal between the tube 20 and the tube 10.
• the skirt is resilient, and preferrably, is made of a resilient rubber or polymer.
• the sleeve 22a is typically also made of the same resilient material and 22a and 22b are typically a unitary body, although this is not essential.
• the sleeve 22a may, for example, be comparatively rigid and be adhesively, or otherwise, sealed or secured in the tube 20. It is important that the upper portion of the sleeve 22 be constructed in such a manner as to preclude the entrapping or retention of any particles. In the particular embodiment shown, it has been found that when the top of the sleeve 22b extends at right angles, or approximately right angles, from the wall of tube 20, there is no significant entrapment of particles.
• FIG. 2B An alternative embodiment is shown in Figure 2B, in which the sleeve 22c has an upper edge which slopes downward inwardly toward the filter 24. Similarly, there is no entrapment of particles in this arrangement. Thus, with particular reference to Figure 2B, a single angle between the interior wall of the cylinder 20 and the top surface of the shoulder of the seal 22 is 90 * or less.
• sleeve 22 make firm physical contact with the filter. This permits downward suction to be applied through the filter which can quantitatively remove all fluid in the filter-plunger. Without this firm contact of sleeve 22 to filter 24, pockets of fluid are retained on sleeve 22 despite all suction efforts and wash steps are poorly efficient.
• OMPI most advantageous, notwithstanding that there are some theoretical advantages to having the angle less than 90°.
• the 90° angle provides a strong sealing relationship, is conveniently assembled, and quite adequately prevents the entrapment of particles.
• Antibody covalently coupled to beads 26 are placed in the plunger-filter and will be retained in the plunger- filter even if suction is applied downwardly through the filter, because the size of the particles is such that they cannot pass through the filter.
• An important filter characteristic is that it possess a high surface area, typically a domed surface, so as to maximize suction efficiency even while beads are pulled down snugly upon it.
• the apparatus in kit form, comprises the same essential components with additional components for convenience in carrying out the immunoassay.
• the kit will be described hereinafter.
• the basic immunological reaction in an exemplary form, is described as follows.
• An antibody with specificity for a "test antigen” is covalently coupled to the beads.
• the beads, to which the antibody is coupled are retained in the plunger-filter.
• These "antibody- beads” are resuspended, during use, within the plunger- filter in the presence of the biological fluids or other fluids to be assayed for the "test antigen”.
• the antibody-beads are incubated in a solution containing a "second antibody-enzyme”.
• This second antibody-enzyme also has specificity for the test antigen and, in addition, is covalently coupled to an enzyme. If the test antigen is present in the assayed biological or other fluid, it will bind to both antibodies. Some of the test antigen will be bound simultaneously to the antibody bead and to the second antibody enzyme. In this way, the second antibody enzyme
• O PI is bound to the beads and is retained on the beads through the highly specific immunological reaction.
• the antibody beads, with the antigen and the antibody-enzyme bound thereto, are then washed by vacuum suction applied at the filter base, by introducing the wash solution into the plunger-filter and pulling the plunger-filter up, as shown in the Figures, in the cylindrical container tube 10 to force the wash solution through the filter. Finally, the beads are re-suspended in a "developing substrate solution".
• the enzyme coupled to the second antibody, alters the substrate and results in a color change, or some other detectable phenomena.
• the developing solution will, in the colorimetric method, change color visibly, or in the ultraviolet or infrared range, and this color change can be detected either visually or by use of appropriate ultraviolet or infrared photometric instruments.
• the change per se, constitutes a quantitative indication of the presence of the test antigen.
• the amount of the change in the developing solution e.g. the intensity of the color, the intensity of the radiation, etc., is a quantitative indicator of the amount of the test antigen in the assayed fluid.
• OIvlPI immunoassays which require several hours if the first antibody is attached only to a flat surface.
• the liquid can be evacuated from the plunger by suction, with the antibody beads being retained within the plunger filter.
• Filter plungers are currently manufactured for a single clinical use, the collection of serum or plasma from patient blood. When blood is cent ifuged, the blood cells are spun down to the bottom of the tube, leaving the plasma-serum at the top. Filter plungers are inserted into the tubes, stopping short of the blood cells, enabling serum-plasma to pass into the plunger. The filter prevents clots and other particulate debris from entering the plunger. None of the currently manufactured filter-plungers are, however, suitable for assay according to this invention. To achieve an assay of high sensitivity, short reaction time, and low color background, the conditions of bead size and bead porosity, filter pore size, plunger geometry, and fluid density during the antibody-antigen reaction are very significant. In addition, the present invention permits direct immunoassay of whole blood, as opposed to conventionally assayed serum or plasma. These various facets of the invention are now discussed.
• plungers are fitted with filters of effective 15-17 micron pore size. Since normal blood cells are all smaller than 15 microns in diameter, they can pass freely through the filter into the plunger-filter which defines the reaction chamber to begin the antibody-antigen reaction, then. pass freely out through the filter at the end of the reaction, leaving the antibody beads behind. Hemolysis is minimal.
• OMPI antibody beads are chosen to be sufficiently larger than the filter pores so that they are retained by and do not clog the filter. It has been found that crosslinked dextran beads of 20-30 micron swollen diameter, with swollen bead pore size excluding proteins larger than 30,000-70,000 Daltons, are quite suitable for this purpose. Such beads are small and porous enough to remain largely in suspension without mechanical means during the reaction steps, aa opposed to beads 35 microns or larger. Smiliar sized beads of polyacrylamide are also suitable for this type of assay. The bead types defined here are exemplary only; other bead sources are also suitable.
• the beads should be porous enough to slow ' bead settling, thereby speeding assay reactions, but still be small enough pored to prevent antibody and developing agent molecules from entering the beads.
• Crosslinked dextran beads with swollen pore size excluding proteins larger than 30,000-70,000 Daltons are suitable for these purposes, showing good buoyancy while still excluding larger proteins such as antibodies from their interiors.
• Similar sized polyacrylamide beads are also suitable, and these bead types are exemplary only. This carefully defined bead geometry in regards to diameter and pore size is critical to this assay's rapidity and sensitivity.
• the bead pores are small enough to ensure that all immunological reagents coupled to the beads are present on the outer surface of the beads. This speeds the assay reactions since the test antigen does not have to migrate into the beads to interface with antibody bound to the beads.
• the invention's defined bead geometry shows much less bead settling over time than larger diameter or small pored beads, this feature greatly enhancing assay rapidity.
• the pore size excludes second antibody-enzyme or other developing agents from entering the beads. Larger pored beads such as Sepharose (Trademark) permit second antibody-enzyme to enter the beads, to remain nonspecifically trapped during wash steps, and finally to cause unwanted high background interference during development.
• the defined bead geometry of this invention eliminates this source of assay background interference.
• the filter In currently manufactured filter plungers, the filter is held in- place by a rubber ring entrapment. If the rubber ring bulges around the filter on the inside of the plunger, it causes unwanted fluid retention even after exhaustive suction has been applied. It has been shown that by reshaping the rubber rings so that the filter protrudes freely well above the rubber into the plunger- filter, the antibody beads, or other immunological reagent-bound beads, can be sucked completely dry during the wash steps. It is essential that the beads be sucked fully dry during each wash, if one is to achieve a quantitative washout of the enzyme which is not specifically bound to the beads. According to the present invention, plungers are designed to achieve this effect. This is an important change in the plunger design. In addition, by increasing the exposed surface area of the filter inside the plunger-filter assembly, a very much more effective suction can be applied. Without this design alteration, it is, for all practical purposes, virtually impossible to wash small-mesh beads effectively.
• the first stage incubation proceeds faster if the beads are evenly dispersed in the incubation fluid. It is, therefore, highly desirable to avoid the settling of the beads. This can be achieved by rocking, stirring or agitating the plunger-filter assemblies during the reaction. This may be done by hand or automatically by appropriate rocking equipment. However, according to this invention, even this inconvenience is avoided by carefully preselecting bead size and porosity to minimize settling while still permitting bead retention by the filter.
• certain carrier substances are of sufficient density and possess appropriate charge properties further to prevent the settling of antibody beads. Carriers such as casein and albumin are suitable for many assays. Other densifying carriers may also be used.
• cleavable cross-linking agents to couple the second antibody to the enzyme. This permits rapid liberation of the enzyme from the beads by introduction of a reducing agent, during development, to cleave the enzyme from the antibody, leaving the enzyme in solution and capable of reacting with the developing solution to provide the color or other indicator result. Liberation of the enzyme into the liquid phase greatly speeds the color reaction, and permits the enzyme to pass through the plunger-filter into a fresh reading tube.
• the developed solution in the plunger-filter tube may be quantitatively transferred from the plunger-filter tube into a container tube, simply by drawing a vacuum suction through:—the—filter drawing tHe developed solution into the container tube.
• nonionic detergents provide for more complete washing of the unbound enzyme from the plunger-filter tube, resulting in a lower background.
• Nonionic detergents do not impair enzyme activity or antigen-antibody reactions present during the wash stages.
• they promote lysis and removal of any blood cells retained in the plunger.
• foaming occurs when nonionic detergents are used. Excessive foaming may be prevented by adding minute amounts of anti-foaming agents. Anti-foaming agents have been shown not to impair the enzyme activity during the wash steps.
• Coated plunger-filter surfaces have also been found to be advantageous. Coating the surface with polytetrafluroethylene (Teflon: a trademark of DuPont), silicone lubricants, acrylic lubricants, or other various coatings, alters the inner surface properties of the plunger-filter, and may result in less sticking of beads to the inner walls of the plunger-filter. Such treatments also reduce unwanted sticking of the second antibody- enzyme, thus reducing background.
• Teflon a trademark of DuPont
• a soft, inert, easily compressible material such as silicone foam or a resolubilizable calcium alginate swab, etc., mounted on a detachable plastic or cardboard "swab" stick, is wiped on the area to be tested, such as the pharynx, rectum, and the stick is then detached from the sponge ball, which is dropped into an appropriately sized container tube.
• the plunger, containing antibody beads, is then pressed against the test tube as far as possible, bearing down forcefully on a sponge ball.
• the sponge ball With fluid covering the sponge ball, the sponge ball may be "pumped" a number of times. When strongly compressed one time or repeatedly in a pumping approach, the biological fluid is expressed out of the ball and then is carried with the solution through the filter into the plunger-filter assembly.
• the filter porosity can be very much smaller than 20 microns, and the bead size may be commensurately smaller, thus resulting in a slower bead settling.
• the technique of this invention may, as earlier indicated, be applied to virtually any type of immunoassay.
• it may be used to carry out i munocompetition assays.
• antibody beads have been used for some time in immunological assays (see, for example, Richman, Douglas D., et al, "The Binding of Staphylococcal Protein A By the Sera of Different Animal Species", Journal of Immunology, Vol. 128, No. 5, 1982)
• the plunger-filter construction and technique described hereinbefore is believed to be entirely novel and to be highly advantageous over all
• the plunger-filter apparatus of this invention is entirely new and its use as systems for assays not involving radioactivity or simply to eliminate more steps is novel. Particularly, its usefulness in reducing reaction time by hours is a new concept and approach in the art. As described above, the existing plunger technology as well as reagent and sample handling, have been greatly improved to achieve the particular ends of high sensitivity, rapid reaction time, convenience, low cost, and low background development.
• the invention includes a number of advantageous features.
• First is the use of enzyme-linked assay in a plunger-filter system, which provides extremely high sensitivity coupled with low cost and high efficiency and a short time delay.
• the deliberate use of antibody-beads rather than antibody on a flat phase to increase the reaction surface area accelerates reaction time.
• Combining the use of beads with the plunger-filter system further reduces the time in running an assay.
• the balancing of the necessary properties of the filter and the beads to reduce bead settling during incubation of the assay very much shorten incubation time with great and unexpected advantages over the prior art.
• the deliberate choice of slow settling beads still small pored enough to prevent the entrance of a developing reagent, e.g.
• the features include the optional use of a specific gravity increasing carrier, such as albumin, to keep the -17- beads in suspension.
• a specific gravity increasing carrier such as albumin
• the reducible cross-linking enzyme on the second antibody-enzyme conjugate is also highly advantageous so that the enzyme can be liberated from the beads and color development, or other development reaction, can proceed in a clean tube, is also significant.
• the use of nonionic detergents and anti-foam agents is a further advantage in the use of a coating to prevent sticking of the beads to the interior wall of the plunger-filter.
• a very distinct and important advantage of the present invention is that it can be used to assay whole, uncoagulated, blood and carrier solutions, as well as biological materials. Another advantage is that the technique and apparatus of this invention can be used to run virtually any kind of immunological assay.
• the physician is not interested in any quantitative measure, and only a qualitative result is significant.
• the physician is interested in both the presence and the amount of the protein and, thus, quantitative results are important. In the latter instance, for example, time is also extremely important and the physician simply cannot wait for many hours or days before beginning treatment.
• the kit depicted in Figure 5 is particularly advantageous in providing the physician or the clinical laboratory with a very quick, efficient apparatus for conducting small numbers of assays at low cost and with very short time delay.
• a base 30 is usually provided, in a convenient form of the kit invention, to hold the kit which a base receives a holder 32, which may be of any design, which holds a number of container tubes 10 and plunger-filter assemblies 20.
• the holder 32 includes four container tubes, two for use as a standard or reference, and two for running the assay on the biological fluid.
• the kit typically also includes two plunger-filters, one for the standard or reference and one for the assay.
• the reference containers and plunger-filter assemblies, the reference containers and plunger-filter assemblies are color coded as shown at 12a, 12b, and 12c in, for example, green, whereas the antigen test components are color coded in red as shown at 14a, 14b, and 14c, in Figure 5.
• a number, usually two or more, reagent dispensers 34a and 34b, typically equipped with a reagent pump 36a and 36b, are also provided. These are color coded in a like manner with a green code at 12d and a red at 14d and 14e.
• the base 30 may be either permanent or temporary. For example, it may be made of wood or metal, or it may be temporary, made of cardboard or styrofoam.
• the base has a number of receptacles into which either individual container tubes and plunger-filter apparatus may be inserted or into which a kit component such as 32, which contains the container tubes and the plunger-filter assemblies, may be inserted.
• the base also, typically, would provide for a number of reagent dispensers and, of course, would provide for the vacuum cups.
• the base may be designed to include a number of kits, for example, the base may include a number of receptacles for including two, three or more kits, as desired.
• each kit would include a number of holders, each with a set of, for exa ple, six tubes in their proper relationship, shown in Figure 5.
• the holder 32 would contain all of the tubes necessary to run a single patient assay.
• one tube 10a is color coded, e.g. green, and is a clean test tube in which the standard control will be assayed.
• the second tube, 10b color coded another color, e.g. red, is a clean test tube in which the patient's blood or other fluid is to be assayed. If whole blood, tube 10b may be a vacutainer type blood collection tube, the type conventionally used in the collection of blood samples.
• 20a is a plunger-filter containing antibody beads in a buffer with a suitable preservative. It is filled to the top and capped securely.
• Tube 20b is identical to tube 20a, except that tube 20a is color coded green as shown at 12c and tube 20b is color coded red as shown at 14c.
• the container tube 10c is a clean, green color coded test tube in which the standard control will finally be read.
• Tube lOd is identical to tube 10c, except that it. is color coded red for reading the fluid assay.
• the kit will include reagent dispensers. These dispensers will contain the necessary stock solutions, such as the second antibody-enzyme or control standards. They will have color codings to indicate whether the solution therein is to be dispensed into the red tubes, or the green tubes, or into both.
• the reagent dispenser 34a is designed to dispense into both the green and the red tubes whereas the reagent dispenser 34b dispenses only into the red tubes. Quite obviously, any number of color codings to meet the particular needs of the particular assay may be provided.
• the kit may also include other dispensers, such as an eyedrop bottle which will contain a stop solution.
• Extra bottles e.g. a wash bottle, etc., may also be included in the kit.
• the six tubes in the kit holder 32 are inserted in the cavity in the base 30. If the base is adapted to receive more than one kit, any number of kits receivable within the base may be inserted therein.
• tube 10b The patient's sample, or other biological fluid to be assayed, is added to tube 10b up to a level which may be predetermined.
• tube 10b is a vacutainer type blood collection tube, the vacuum in it will be such as to collect the exact amount of blood used in the assay, to which an appropriate volume of an anti-coagulant, such as EDTA or heparin, would be added or present.
• an anti-coagulant such as EDTA or heparin
• Reagents would be squirted, as indicated by the color coding, into tubes indicated by the color coding into tubes 10a and 10b.
• Tube 10b would receive both a second antibody-enzyme and a standard.
• Tube 10c would receive only the second antibody-enzyme.
• the two plunger-filters 20a and 20b would be uncapped, placed on the vacuum receptacles, and the beads sucked dry. The plungers then would be inserted into the appropriately colored tubes 10a and 10b. In so doing, the blood or other biological solution in one tube and the standard in the other is forced up through the filter in the plunger-filter, resuspending the antibody beads and initiating the reaction.
• the beads are maintained in suspension by any of a number of different means, including the use of high density carrier substances such as albumin, or simply by mechanically rocking or agitating.
• the plungers are withdrawn from the tubes and placed in the vacuum receptacles 30a and 30b, which are attached in fluid communication relationship with any source of vacuum.
• the tubes 10a and 10b may then be discarded.
• the beads within the plungers 20a and 20b are then washed the desired number of times with ' .
• a suitable wash solution which may be provided as part of the kit or may be simply a standard stock solution available in the laboratory. The washing removes blood cells and enzymes which are not specifically bound to the beads.
• the vacuum sucks the wash solutions through the beads and through the filter.
• the plunger-filters ace then inserted in the appropriately colored tubes 10c and lOd and the plungers are inserted down to the predetermined position.
• Developing solution in a predetermined quantity usually, though not necessarily, is then introduced into the plunger-filters according to the particular immunoassay that has to be done. If a reducing agent is used, the reducing agent in the solution liberates the enzyme from the beads, permitting the enzyme to pass through the filter of the plunger-filter. After an appropriate period of time, the plungers are pulled out of these tubes, creating a vacuum which sucks the developing solution which is carrying the enzyme into the respective tubes 10c and lOd. The plungers may then be discarded.
• the tubes 10c and lOd are then measured, either by visual color change, or spectrophotometrically, or by any other desired detecting means.
• Qualitative differences, in the colorametric may be assessed by eye or by colorimeter. Quantitative differences will normally be determined by any suitable instrument such as a photometer, a beta or gamma counter, etc.
• the tubes 10 may be of a size and configuration to be received directly into the cell chambers of commercially available photometers or colorimeters. Depending upon the particular immunoassay being run, the color change, or other detecting reaction, can be arrested by a stop solution.
• a quantitative reading device such as a colorimeter or photometer which is designed to accept
• OMPI container tubes of any particular or desired configuration. This is convenient in certain laboratories but, of course, is not a part of the present invention.
• the apparatus and methods of this invention are adapted for use in industrial, research, scientific and clinical laboratories generally, wherever any type of immunological assay is run.
• the invention provides highly efficient, high sensitivity, low cost immunoassay techniques and apparatus.

Landscapes

• Health & Medical Sciences (AREA)
• Immunology (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Chemical & Material Sciences (AREA)
• Urology & Nephrology (AREA)
• Hematology (AREA)
• Biomedical Technology (AREA)
• Molecular Biology (AREA)
• Analytical Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Cell Biology (AREA)
• Pathology (AREA)
• Food Science & Technology (AREA)
• Medicinal Chemistry (AREA)
• Physics & Mathematics (AREA)
• Biotechnology (AREA)
• Biochemistry (AREA)
• Microbiology (AREA)
• General Physics & Mathematics (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Automatic Analysis And Handling Materials Therefor (AREA)
• Steroid Compounds (AREA)
• Sampling And Sample Adjustment (AREA)
• Compounds Of Unknown Constitution (AREA)

Applications Claiming Priority (1)

Publications (2)

Family

ID=22175563

Family Applications (1)

Country Status (6)

Families Citing this family (3)

Family Cites Families (12)

• 1983
  • 1983-11-08 WO PCT/US1983/001757 patent/WO1985002260A1/fr not_active Application Discontinuation
  • 1983-11-08 JP JP84500095A patent/JPS60501570A/ja active Pending
  • 1983-11-08 AU AU23412/84A patent/AU556193B2/en not_active Ceased
  • 1983-11-08 EP EP19830903878 patent/EP0161247A4/fr not_active Withdrawn
• 1984
  • 1984-05-08 NO NO841835A patent/NO841835L/no unknown
  • 1984-11-05 DK DK525584A patent/DK525584A/da not_active Application Discontinuation

Non-Patent Citations (2)

Also Published As

Similar Documents

Legal Events