NO841835L - IMMUNOPROEVE QUICK METHOD AND APPARATUS THEREOF - Google Patents

Description

The present invention relates to immunological methods and apparatus. More particularly, the invention relates to a special technique and apparatus for carrying out an immunological test on a fluid such as a body fluid, or on a carrier fluid on which the immunological component to be detected is carried.

With the advances in immunochemistry, there has been an increasing

and in recent times accelerated need for more effective and less expensive methods and techniques for carrying out immunoassays. In many areas related to molecular biology, biochemistry, biology and genetics, it is necessary to carry out hundreds or even thousands of immunological tests to determine a single result.

In the clinical treatment of immunological diseases and deficiencies, it is highly desirable to carry out any necessary immunological test quickly, reasonably and efficiently so that you do not spend too much time for the patient and doctor in connection with large delays and repeated visits. Accordingly, there is a strong need for more accurate, less expensive,

simpler and more efficient immunological test procedures and apparatus for use in doctors' offices and in clinical laboratories. A large number of immunological detector mechanisms, techniques and materials have been developed in recent decades. The now classic radioimmuno-technique is still in widespread use, although it is being replaced by other techniques that use non-radioactive indicators. Enzyme-linked immunoassay techniques as an example and other photometric, fluorophotometric and colorimetric methods can also be applied to special types of immunological components. While these advances have made the relatively rapid and accurate testing of antigens and antibodies rather feasible,

there is still a need for even more accurate and at the same time less expensive and more efficient equipment and methods, both when it comes to research laboratories and in the clinical laboratory.

Existing immunoassay technologies are often so complicated or time-consuming that it is only possible to carry out a very limited number of tests per day. On the other hand, in a clinical laboratory, for example, the number of samples to be run during a particular day does not justify the installation and operation of an apparatus for carrying out the test. Sometimes, under certain circumstances, patient samples are allowed to be collected until there are sufficient samples to justify the laboratory performing the tests. In many clinical situations such as e.g.

if heart disease is suspected, it is extremely unfavorable for the patient if there are delays and the like in connection with the test technology. Fast and accurate testing in such a case is of essential importance for the patient's health.

Among the features of the invention are methods and apparatus

which completely or to a large extent overcomes all the shortcomings of the known technique..

According to the method of the invention, a fluid becomes

which contains the immunological component of interest, e.g. an antigen, introduced into a plunger filter apparatus, typically but not necessarily through the filter,

and in contact with immunologically binding beads located in the plunger filter. These immunological binding beads bind to the immunological component of interest, e.g. the antigen or antibody, and extracts it from the solution in which the component 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 component of interest bound to the beads. A developer is introduced into the plunger filter which binds to the immunological component of interest, e.g. the antigen or antibody. Excess developer can be removed by washing, filtering or the like. To

finally, the immunological component of interest is determined by measuring the elicitor according to the indicator that characterizes the elicitor.

As an apparatus, the invention comprises a new and improved plunger filter apparatus containing small beads which bind selected immunologically active constituents of the fluid to be sampled, a container tube into which the plunger filter fits in a movable but fluid-tight connection to allow fluid to be forced through the filter in both directions. Also covered by the apparatus is a set including a number of such elements in connection with each other in suitable containers to provide an apparatus that is total and sufficient for the operator to carry out immunotests according to the invention.

In a more specific and non-limiting scope, the invention can be described as an immunoassay apparatus comprising a cylindrical container tube with an open end and a closed end, a plunger filter which is accommodated in the open end of the container tube and which can slide in the container tube, the plunger filter element comprising a cylindrical tube which is open at both ends, a movable seal attached to the proximal end of the cylindrical tube, the distal end extending outwards towards the open end of the container tube, whereby the gasket can be moved relative to the inner walls of the container tube and form a the substantially fluid-tight, movable seal between the proximal end of the plunger filter and the container tube,

a high surface area, with characteristic dome-shaped filter inside and closing the proximal end of the plunger filter cylinder against the passage of material in and out of the proximal end except for fluids and particles that can pass through the filter, and immunologically reactive beads that in use bind selected immunologically active constituents of the fluid to be tested, as the immunologically active spheres have porous surfaces treated with an immunologically active component and whereby these are too large for

to pass through the filter.

In a preferred embodiment, the filter comprises pores

as large as possible to allow free two-way passage of test fluid and its constituents, such as blood cells, while the pores are still small enough to hold the beads on one side of the filter without plugging it;

whereby the filter gasket comprises a skirt which extends circumferentially around the plunger filter tube in movable gasket contact with the inner wall of the container tube, as well as a cuff fixedly attached to the interior of the plunger filter tube, whereby the top of the cuff is so constructed and arranged as to form a generally flat annular top surface which cuts the inner wall of the plunger filter tube at an angle of no more than 90°.

Again, in a preferred form, the immunologically active beads are large enough in terms of overall diameter not to pass through or clog the filter, but otherwise

small as possible to reduce their deposition rate and thereby accelerate the sample reactions. In addition, must

the beads have pores small enough to prevent antibodies or developing agents from entering the interior of the beads;

in this way, all coupling and reaction steps will take place on the outer surface of the spheres, which increases the reaction time and reduces background interference. Within this pore size limitation, the sphere pore size should still be maximal to reduce the specific density of the spheres, because this reduces the deposition rate of the spheres and accelerates the sample reaction time.

In another definition of the invention, this describes an immunoassay process comprising bringing a liquid sample into a predetermined physical configuration, to

forcing a filter through said configuration to thereby force the fluid sample through the filter into a second configuration in contact with a plurality of beads having immunologically reactive sites; to keep the liquid in contact with the balls in a beforehand

constituents of the sample with said immunologically reactive sites on the beads; withdrawing the filter from the configuration thereby forcing the liquid sample through the filter out of the second configuration out of contact with the beads; washing residual fluid from the balls; to determine the reaction between the immunological components from the sample with the immunological sites on the beads.

In a preferred embodiment, the determination step comprises

removing immunological components from the beads to the fluid in said second configuration, reacting the components with an indicator and determining the presence of indicator in the fluid in the second configuration as a measure of the amount of immunological component in the sample.

The invention shall be explained in more detail with reference to

the accompanying drawings, there

Fig. 1 shows the principle construction of the invention

apparatus;

fig. 2A is a cross-section showing the construction in detail

of the lower part of the plunger filter including the filter, the movable gasket, the bottom of the plunger filter tube

and the container tube;

fig. 2B shows an alternative embodiment of the gasket

as shown in fig. 2A;

fig. 3 is a perspective view of a plunger filter with a

therein contained fluid during extraction of immunological

component from the fluid to the balls;

fig. 4 is the same plunger filter, but with fluid removed

the balls being retained on the filter; and

fig. 5 is a perspective view of a set that uses the concept and principle of the invention.

In the following description, the invention will be described by means of illustrations with a view to an antigen determination immunoassay. However, the discussion is only to illustrate the construction and use of the apparatus and the techniques and steps of the method. Variations will be obvious to those skilled in the art. E.g. a technique for determining a bound enzyme is to be described. Of course, immunofluorescent, RIA or other techniques as well as ELISA techniques can be used. The presumed best method described below is thus only to be considered as an example and in no way to limit the scope and scope of the invention.

With reference to fig. 1 for a general description

of the apparatus, this comprises a container tube 10 which characteristically has a conventional cylindrical shape with a round bottom, often called a test tube or test tube. The main reaction container is a cylinder 20 to which is attached a removable gasket 22 which seals the space between the tube 20 and the container tube 10 when the former is inserted into the latter. The reaction container also includes a filter 24 and spheres

26, the latter being better shown in fig. 3 and 4. The combination of the pipe 20, the gasket 22 and the filter 24

which contains the balls 26 is called a plunger filter here.

The construction of the gasket 22 and the filter 24 is not unimportant. Reference is made to fig. 2A for details regarding this construction. The gasket 22 comprises an inserted cuff 22a which is in fluid-tight connection with the interior of the pipe 20, as well as a skirt 22b which extends outwards around the circumference of the pipe 20 in contact with the inner walls of the pipe 10 and forms a movable but fluid-tight seal between the tube 20 and the tube 10. The skirt is compliant and preferably made of an elastic rubber or polymer. The sleeve 22a is characteristically made of the same elastic material and 22a and 22b are characteristically a unitary body, although this is not significant. The cuff 22a can e.g. be relatively stiff and adhesive or on

otherwise attached to the pipe 20. It is important that the upper part of the cuff 22 is arranged in such a way as to prevent the capture or retention of particles. In the particular embodiment shown, it has been found that when the top of the cuff 22b is at right angles or approximately right angles from the wall

of the pipe 20, there is no significant trapping of particles.

An alternative embodiment is shown in fig. 2B where the cuff 22c has an upper edge which slopes downwards and inwards towards the filter 24. In a similar way, there is no capture of particles by this device. With particular reference to fig.

2B is thus the angle between the inner wall of the cylinder 20 and the top surface of the shoulder of the gasket 22 90° or less.

In addition, it is essential that the upper edge of the cuff

22 has a fixed physical contact with the filter. This allows downward suction to be applied through the filter, which can quantitatively remove all fluid in the filter plunger.

Without this fixed contact between the cuff 22 and the filter

24 fluid pockets will be retained on the cuff 22 despite all suction attempts and the washing steps will be ineffective.

In practice, it has been shown that the right-angled relationship

between the inner wall of the cylinder 20 and the top of the sleeve of the gasket 22 has been found to be most advantageous, although there are certain theoretical advantages to having an angle of less than 90°. In practice, the 90° angle provides a strong seal, it can be mounted easily and favorably prevents the entrapment of particles.

The antibody that is covalently linked to the beads 26 is placed

in the plunger filter and is held in this even if suction is applied downwards 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 has a high surface area, characteristically a domed surface to allow maximum suction efficiency even when the balls are pulled down strongly against the filter.

The apparatus in kit form comprises the same essential components with additional components that are appropriate for carrying out the immunotest. The set must be described

The principle immunological reaction in an exemplary embodiment will be described below. An antibody with specificity for a "test antigen" is covalently linked to the beads. The beads to which the antibody is attached are held in the plunger filter. These "antibody beads" are resuspended during use in the plunger filter in the presence of the biological fluids or other fluids to be tested for "test antigen". At the same time or subsequently, 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 is additionally covalently linked to an enzyme. If the sample antigen is present in the sampled biological or any other fluid, it will bind to both antibodies. Some of the test antigen will simultaneously bind to the antibody bead and to the other antibody enzyme.

In this way, the second antibody enzyme will be bound to the beads and held on the beads through the very specific immunological reaction. The antibody beads with the antigen and antibody enzyme bound thereto are then washed using suction applied to the filter base by introducing washing solution to the plunger filter and by pulling the plunger filter up as shown in the figures, into the cylindrical container tube 10 to force the washing solution through the filter. Finally, the beads are resuspended in a "developing substrate solution". The enzyme, linked to the second antibody, changes the substrate and results in a color change or in another detectable phenomenon. In the colorimetric method, the developing solution will change color visibly or possibly in the ultra-violet or infrared range, and this color change can be detected either visually or by using suitable ultra-violet or infrared photometric instruments. The change per se constitutes a quantitative indication of the presence of the test antigen. The degree of change in the developing solution, e.g. the color intensity, the irradiation intensity, etc., is a quantitative indicator of the amount of sample antigen present in the tested fluid.

It should be clear that the description given above

is an adaptation of a known antibody enzyme-linked immunoassay to the plunger filter system according to the invention and is only given as an example.

By having a "first antibody" present on the suspendable

small spheres instead of on flat surfaces, a much larger available interface between the antibody and the test antigen is quickly achieved. Because the antibody will more frequently "see" the test antigen, the reaction time is shorter and the reaction takes longer to complete. It has been shown that if the beads are kept in suspension during the incubation, incubation times of less than 15 minutes can result in immunoassays with a sensitivity of at least nanograms/milliliter. It can be compared to immunoassays that require several hours if the first antibody is only bound to a flat surface.

Another important feature is that centrifugation is completely avoided. The liquid can be evacuated from the plunger by suction, as the antibody beads are held in the plunger filter.

Filter plungers are manufactured today for a single clinical use, collection of serum or plasma from patient blood. When the blood is centrifuged, 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, stopped short of the blood cells, which enables the serum-plasma to pass into the plunger. The filter prevents coagulated lumps and other particulate matter from entering the plungers. However, none of the filter plungers manufactured today are suitable for testing according to the invention. In order to obtain a sample with high sensitivity, short reaction time and low color background, conditions for sphere size and sphere porosity, filter pore size, plunger geometry and fluid density during the antibody-antigen reaction are very important. In addition to the present invention direct immunoassay of whole blood as opposed to conventionally tested serum or plasma. These various aspects of the invention will be discussed in more detail below.

Using carefully defined filter and bead dimensions, it is possible to sample anticoagulated whole blood. This eliminates the time-consuming preparation of plasma or serum from blood. To sample whole blood, plungers are fitted with filters

with an effective pore size of 15-17 unt. Because normal blood cells are less than 15 µm in diameter, they can pass freely through filters into plunger filters that define reaction chambers to begin the antibody-antigen reaction and then pass freely through the filter at the end of the reaction, leaving behind the antibody beads. Hemolysis is minimal. The 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 cross-linked dextran beads with a swollen diameter of 20-30 µm, with a swollen bead pore size that excludes proteins of over 30,000-70,000 Daltons, are rather suitable for this purpose. Such spheres are small and porous enough to remain substantially in suspension without mechanical aids during the reaction steps, unlike spheres of 35 µm or greater.

Balls of similar size made of polyacrylamide are also suitable for this type of testing. The bullet type defined here is only mentioned as an example, other bullet sources are also suitable.

To carry out a reaction optimally on whole blood, is

the need for a filter with a pore size just large enough to allow the blood cells to pass freely, but small enough to hold back the spheres which are not so small that they clog the filter, but are themselves as small as possible to still be able to retained in suspension during incubation. Of course, less than optimal results can be obtained using smaller balls although some clogging may occur or with larger balls which tend to settle over time, requiring agitation, shaking or other aids to overcome particular defects encountered.

The beads should be porous enough to reduce bead deposition to thereby accelerate the sample reaction, yet be small enough in terms of pore size to prevent antibody and developer molecules from entering the beads. Cross-linked dextran beads with a swollen pore size

which exclude proteins above 30,000-70,000 Dalton are suitable for these purposes, showing good usability while still excluding larger proteins such as antibodies from their interior. Correspondingly sized polyacrylamide balls are also suitable and these ball types are only mentioned as examples. This carefully defined spherical geometry in terms of diameter

and pore size is essential for the speed and sensitivity of the sample. The bead pore size is small enough to ensure that all immunological reagents that bind to the beads are present on the outer surface of the beads. This increases the speed of the sample reaction, because the sample antigen does not need to migrate into the beads to the interface with antibody bound to the beads. In the same way, the defined ball geometry of the invention shows much less ball deposition over time than would be the case with balls with a larger diameter or smaller pores, which significantly increases the test speed. Finally, the pore size precludes other antibody-enzyme or developer agents from entering the beads. Larger porous beads such as "Sepharose" allow other antibody enzymes to enter the beads and remain non-specifically captured during the washing step, ultimately causing undesired high background interference during development. The defined sphere geometry according to the invention excludes this source of sample background interference.

In today's manufactured filter plungers, the filter is held in place

using a rubber ring device. If the rubber ring twists around the filter inside the plunger, this causes unwanted fluid retention, even after significant suction is applied. It has been shown that by reshaping the rubber rings so that the filter freely protrudes well above the rubber into the plunger filter, the antibody beads or other immunological reagent-bound beads can be sucked completely dry during the washing step. It is essential that the balls are sucked completely dry

during each wash if one wishes to achieve a quantitative washout of the enzyme that is not specifically bound to the beads. According to the invention, the plungers are designed for

to achieve this. This is a significant change in the plunger construction. In addition, by increasing the exposed surface area of the filter in the plunger filter construction, a much more efficient suction can be achieved. Without this design change, it is for all practical purposes impossible to effectively wash small balls.

It has also been found important that the rubber ring has contact with

the interior of the plunger filter cylinder at an angle of 90° or less, this with reference to the angle <t> in fig. 2A and 2B. If the contact angle is more than 90°, a cavity is created in which blood cells can be trapped. These blood cells cannot be completely removed during washing and hemolysis can cause a very strong increased background in the final development step in the sample. Reshaping the rubber to make it perpendicular to the contact with the interior of the tube 20 eliminates blood cell entrapment and transfer. An alternative, albeit somewhat less preferred, is to make the contact angle less than 90°.

The first stage of incubation, during which the antibody beads are reacted with the sample antigen, occurs more quickly if the beads are evenly dispersed in the incubation fluid. It is therefore highly desirable to avoid depositing the balls. This can be achieved by shaking, stirring or agitating the plunger filter apparatus during the reaction. This can be done by hand or automatically using suitable equipment. According to the invention, however, this shortcoming is also avoided by carefully selecting the ball size and porosity in advance in order to minimize deposition, but still allow ball retention in the filter. In addition to this, certain carrier substances have sufficient density and have suitable charge properties to further prevent the deposition of antibody beads in this way. Carriers such as casein and albumin are suitable for many samples. Other density-increasing carriers can also be used. It should be noted that in whole blood samples there is little or no need for additional carrier because the density of the blood itself is usually sufficiently high to prevent deposition or at least to provide very low deposition of the beads.

A further important feature according to the invention is the use of cleavable cross-linking agents to link the second antibody to the enzyme. This allows rapid release of the enzyme from the beads upon introduction of a reducing agent, during development, to cleave the enzyme from the antibody, leaving the enzyme in solution and able to react with the developing solution to provide a color or other indicator result. Releasing the enzyme to the liquid phase greatly increases the reaction rate of the color reaction and allows the enzyme to pass through the plunger filter to a new reading tube. According to this embodiment, the developed solution in the plunger filter tube can thus be quantitatively transferred from the plunger filter tube to a container tube, simply by applying a vacuum through the filter, and then drawing the developed solution into the container tube.

There are other important features of the invention as well. The

It has been shown that non-ionic detergents provide a more total washing of unbound enzyme from the plunger filter tube, resulting in a lower background. Non-ionic detergents do not produce enzyme activity or antigen-antibody reactions during the washing steps. In addition, they promote light and removal of blood cells retained in the plunger. Foaming sometimes occurs when non-ionic detergents are used. Excessive foaming can be prevented by adding small amounts of antifoaming agents. Antifoam agents have not been shown to affect the enzyme activity during the washing step.

Coated plunger filter surfaces have also been found to be too partial. Coating the surface with polytetrafluoroethylene, "Teflon", silicone lubricants, acrylic lubricants or other various coatings, changes the properties of the inner surface of the plunger filter and can result in less adhesion of the balls to the inner walls of the plunger filter. Such treatments also reduce unwanted adhesion of the second antibody-enzyme, which then reduces the background.

The foregoing technique is generally applicable to biological fluids of essentially any type. The technique can also be applied to other fluids and to other types of bio-samples, which will be clear to the person skilled in the art. E.g. if it is desired to exudates, secretions, faecal substances, etc., it is easily possible to adapt the technique to this. A soft, inert, easily compressible material such as silicone foam or a re-dissolvable calcium alginate material, e.g. placed■on a removable plastic or cardboard "test" stick, stroked over the area to be tested such as pharynx, rectum, after which the stick is then detached from the sponge which is then dropped into a suitably sized container. The plunger containing antibody beads is then pressed against the test tube as far as possible, while the sponge bead is forcefully pressed down. When the fluid covers the sponge ball, it can be "pumped" a few times. By strong compression one or more times, the biological fluid is pressed out of the ball and then led with the solution through the filter to the plunger filter. When the sample is run on such type of materials instead of whole blood, the filter porosity can be much smaller than 20 µm and the sphere sizes correspondingly smaller, which results in a lower sphere deposition.

As mentioned above, the technique according to the invention can be applied to essentially any type of immunoassay. E.g. can it be used to carry out immunocomparison tests.

While antibody beads have been used for some time in immunological assays (see, e.g., Richman, Douglas D, et al., "The Binding of Staphylococcal Protein A By the Sera of Different Animal Species", Journal of Immunology, vol. 12 8, no. 5, 1982), the plunger filter construction and the technique described above are believed to be completely new and very advantageous compared to all known techniques. The plunger filter apparatus according to the invention is completely new and its use as a system for testing without radioactivity and in a simple way eliminating several steps is also new. In particular, the test method's usability with a view to reducing reaction time by hours is a new concept and new in this technique. As described above, the existing plunger technology as well as reagents and sample processing have been greatly improved to achieve the special results of high sensitivity, fast reaction time, applicability, low cost and low background development.

The invention as described above includes a number of advantageous features. The first is the application of an enzyme-linked sample setup in a plunger filter system, which provides extremely high sensitivity compared to low costs and high efficiency in a short time. The intentional use of antibody beads instead of an antibody on

a flat phase to increase the reaction surface area accelerates the reaction time. The combination of the use of spheres with the plunger filter system further reduces the time required in a sample. A balancing of the necessary properties for the filter and the beads to reduce bead deposition during incubation in the sample greatly shortens the incubation time with large and unexpected advantages compared to the known technique. The deliberate choice of slow-depositing spheres with pores small enough to prevent penetration of a developer, e.g. a second antibody-enzyme, and to promote the encounter between antigen and developer, has also been shown to be a very big advantage, reducing time and increasing the sensitivity of the sample. The construction of the plunger filter to eliminate fluid and self-retention is also considered a very important feature of the invention. Effective washing suction cannot be applied to the plunger filter by

the known techniques.

These features include the possible use of a specific densifying agent, such as albumin, to keep the beads in suspension. The reducible cross-linking enzyme in the second antibody-enzyme conjugate is also very advantageous, so that the enzyme can be released from the beads, and color development or other development reaction can take place in a clean tube,

something that is also important. The use of non-ionic detergents and anti-foaming agents is a further advantage of using a coating to prevent sticking of the beads to the inner walls of the plunger filter.

A very distinct and important advantage of the present invention is that it can be used to test whole blood, uncoagulated blood, other blood and carrier solutions, as well as biological substances. Another advantage is that the technique and apparatus according to the invention can be used to carry out essentially any type of immunological test.

For certain tests such as testing the presence of a certain type of bacterial antigen in the throat, the doctor is not interested in any quantitative measurement, a qualitative result is sufficient. In other situations, e.g. in the case of heart defects where an indicative protein will be found in the blood as a measure of the severity of the attack, the doctor is interested in both the presence and quantity of the protein, and thus quantitative results are important. In the latter case, time is also an extremely important factor and the doctor simply cannot wait for many days or hours before starting the treatment.

The set shown in fig. 5 is particularly advantageous for

to be able to provide the doctor or the clinical laboratory with a very fast and efficient apparatus to carry out small numbers of tests at low costs in a very short time.

A base 30 is generally provided for holding the kit held by a holder 32 which may be of any construction and which holds a number of reservoir tubes 10 and plunger filter elements 20. In a particular embodiment, the holder 32 includes four reservoir tubes, two for use for standards or references and two for carrying out the testing of the biological fluid. The set typically also includes two plunger filters, one for a standard or reference and one for the sample. In a preferred embodiment, the reference containers and plunger filters are color coded as shown at 12a, 12b and 12c, e.g. in green, while the antigen-sample components are color-coded in red as shown at 14a,

14b and 14c in fig. 5. A number, usually two or more, of reagent dispensers 34a and 34b, usually equipped with a reagent pump 36a and 36b, also present. These are color coded in the same way with a green code at 12d and a red code at 14d and 14e.

A pair of vacuum containers 38a and 38b, connected through a connecting pipe to 40, in use connected to a vacuum device, are provided for convenient washing and removal of liquids from the plunger filter.

The base 30 can either be permanent or temporary. E.g. it can consist of wood or metal or it can be temporary, made of cardboard or styrofoam. The base has a number of receiving holes into which either individual canister tubes and plunger filter elements can be inserted or into which a set such as 32 containing the canister tubes and plunger filters can be inserted. This base will also characteristically provide for a number of reagent dispensers and, of course, for the vacuum container. The base may be constructed to include a number of sets, e.g. the base can include a number of receivers to receive two, three or more sets as desired.

In use, by way of example and not in any way limiting, each set contains a number of holders, each e.g.

with a set of six tubes, such as e.g. shown in fig. 5. The container 32 will contain all the tubes necessary to carry out a single patient test.

With reference to fig. 5 where the kit 32 includes pipes

and plunger filters are a tube 10a color coded e.g. green,

and is a clean test tube in which the standard control is carried out. The second tube 10b, color-coded with a different color, e.g. red, is a clean test tube where the patient's blood or another fluid is tested. In the case of whole blood, the tube 10d can be a blood sample collection tube of the "vacutainer" type, the type conventionally used for blood sampling.

20a is a plunger filter containing antibody beads in a buffer with a suitable preservative. It is filled to the top and closed tightly.

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-coded test tube where the standard control is finally read. The tube 10d is identical to the tube 10c, except that it is color coded red for reading the fluid sample. In addition to the previous part of the set, it will also contain reagent dispensers. These dispensers will contain the necessary stock solutions such as the second antibody-enzyme or control standards.

They will be color coded to indicate whether the solution should be delivered to red tubes or to green tubes or to both. In the particular exemplary embodiment only, the reagent dispenser 34a is designed to dispense to both the green and red tubes, while the reagent dispenser 34b only dispenses to the red tubes. It should be understood that any number of color codes may be used to satisfy the particular needs of any particular sample.

The kit may also include other dispensers such as an eye drop bottle containing a stop solution. Extra bottles, e.g. a washing bottle, may also be included in the set.

When carrying out a special immunological test, this

by way of example only, the six tubes of the set holder 32 are inserted into the cavity of the base 30. If the base is adapted to receive more than one set, any number of sets that can be received in the base may be inserted therein.

The patient sample or another biological fluid to be sampled is added to the tube 10b up to a level that can be determined in advance. If, alternatively, the tube 10b is a blood sampling tube of the vacuum type, the vacuum in this will be such that the exact amount of blood consumed in the sample is collected, to which a suitable volume of an anticoagulant, such as EDTA or heparin, is present or added.

Reagents are then injected into the tube as indicated by the color coding 10a and 10b. The tube 10 receives both a second antibody-enzyme and a standard. Tube 10c receives only the second anti-substance f enzyme. The two plunger filters 20a and 20b are opened, placed in the vacuum receivers and the balls are sucked dry. The plungers are then introduced into the correctly colored tubes 10a and 10b. With this, the blood or other biological solution in one tube and the standard in the other are forced up through the filter in the plunger filter, the antibody beads are resuspended and the reaction initiated. The beads are kept in suspension by any suitable means including the use of high density carriers such as albumin, or simply by mechanical shaking or stirring.

At the end of the reaction time, which is usually only approx.

15 or 20 minutes, the plungers are withdrawn from the tubes and placed in the vacuum containers 30a and 30b, coupled in fluid communication with any vacuum source. The tubes

10a and 10b can then be discarded. The balls in the plungers 20a and 20b are then washed the desired number of times with a suitable washing solution which may be provided as part of the kit or simply be a standard stock solution available in the laboratory. The washing removes blood cells and enzymes that are not specifically bound to the beads. The imposed vacuum suction washing solutions through the balls and through the filter. Then the plunger filters are then inserted into the correct color coded tubes 10c and 10d and the plungers are inserted down to the predetermined position. The developing solution, in a usually predetermined amount,

although this is not necessary, is then introduced through the plunger filter according to the particular immunoassay to be carried out. If a reducing agent is used, the reducing agent in the solution releases the enzyme from the beads and allows the enzyme to pass through the filter in the plunger filter. After a certain period of time, the plungers were pulled out of these tubes, which creates a vacuum which sucks the developing solution carrying the enzyme into the respective tubes 10c and 10d. The plungers can then be discarded. The tubes 10c and 10d are then measured, either by visual color change or by spectrophotometry, or by any other suitable means. Qualitative colorimetric differences can be measured visually or with a suitable apparatus. Quantitative differences will usually be determined using any suitable instrument such as e.g. a photometer,

a b or y counter, etc. The tubes can be of any size and configuration for recording directly in the cell chambers of commercially available photometers or colorimeters. Depending on the particular immunoassay carried out, the color change or another detection reaction can be retained by a stop solution.

In large laboratories it is quite possible to construct a quantitative reading device such as a colorimeter or photometer which is designed to accept canister tubes of any particular or desired configuration. This is appropriate in certain laboratories, but of course not part of the invention.

The apparatus and methods according to the invention are adapted for use in industrial, research, scientific and clinical laboratories in general where any type of immunological tests are carried out. The invention provides very efficient, very sensitive and very reasonably good immunotechniques and apparatus.

Claims (12)

1. Immunotesting equipment, characterized in that it includes: a cylindrical container tube (10) with an open end and a closed end; a plunger filter element (20, 22, 24 and 26) received in the open end of the container tube (10) and slidable therein; while the plunger filter element (20, 22, 24 and 26), comprising a cylindrical tube (20) which is open at both ends, a movable gasket (22) fixedly attached to the proximal end of the cylindrical tube (20), the distal end projecting outwards towards the open end of the container tube (10), whereby the gasket ( 22) is movable with respect to the inner walls of the container tube (10) forming a substantially fluid-tight, movable seal between the proximal end of the plunger filter (20, 22, 24 and 26) and the container tube (10), a dome-shaped filter (24) inside and sealing off the proximal end of the plunger filter cylinder (20) against the passage of material in and out of said proximal end except for fluids and particles that can pass through the filter (24), and immunologically reactive beads (26) as in use binds selected immunologically active components of the fluid to be tested, the immunologically active spheres (26) having porous surfaces treated with an immunologically active component, the spheres not being too large to pass through the filter (24). 2. Apparatus according to claim 1, characterized in that the filter (24) has pores large enough to allow free two-way passage of tested fluid components such as blood cells, but pores small enough to retain slowly settling balls (26) on one side of the filter (24). 3. Apparatus according to claim 1 or 2, characterized in that the gasket (22) comprises a skirt (22b) which extends circumferentially around the plunger filter tube (20) in movable gasket contact with the inner wall of the container tube (10) and a cuff (22a) firmly engaged in the plunger filter tube (20), whereby the top of the sleeve (22a) is so constructed and arranged that it forms a substantially flat ring top surface which intersects the inner wall of the plunger filter tube (20) at an angle of not more than 90°. 4. Apparatus according to claim 3, characterized in that the angle is approx. 90°. 5. Apparatus according to claims 1 to 4, characterized in that the spheres (26) have a pore size which excludes immunological and development molecules, but on the other hand ensures maximum porosity and minimal total sphere size to promote buoyancy and low sphere (26) deposition, with simultaneous retention on one side of the filter (24). 6. Set for carrying out an immunological test, characterized in that it includes: a number of container tubes (10), each of which is generally cylindrical; a number of plunger filter elements (20, 22, 24 and 26), each of which comprises a cylindrical tube (20) open at both ends, a movable gasket (22) fixedly attached to the proximal end of the cylindrical tube (20), with the distal end extending outwards towards the open end of the container tube (10), whereby the seal (22) is movable in relation to the inner walls of the container tube (10) and forms an essentially fluid-tight, movable seal between the proximal end of plunger filter (20, 22, 24 and 26) and the reservoir tube (10), a high surface area filter (24) within and enclosing the proximal end of the plunger filter cylinder (20) for passage of material into and out of the proximal end except fluids and particles that can pass through the filter (24), and immunologically reactive spheres (26) which in use bind selected immunologically active constituents from the fluid to be tested, the immunologically active spheres (26) having porous surfaces that have been treated with an immunological ouch tive component and are too large to pass through the filter (24); and a number of dispensers (34a and 34b) for reagent and washing solutions. 7. Apparatus according to claim 6, characterized in that the filter (24) includes pores large enough to allow two-way passage of sampled fluid components such as blood cells, but pores still small enough to retain slowly settling beads (26) on one side of the filter ( 24). 8. Apparatus according to claim 6 or 7. characterized in that the gasket (22) comprises a skirt (22b) which extends circumferentially around the plunger filter tube (20) in movable gasket contact with the inner wall of the container tube (10), and a cuff (22a) ) fixed inside the plunger filter tube (20), the top of the cuff (22a) being so constructed and arranged that it has a generally flat annular top surface which intersects the inner wall of the plunger filter tube (20) at an angle of no more than 90°. 9. Apparatus according to claim 8, characterized in that the angle is approx. 90°. 10. Apparatus according to claims 6 to 9, characterized in that the spheres (26) have a pore size that excludes immunological and eliciting molecules, but otherwise manifests maximum porosity and minimal total sphere size to enforce buoyancy and slow sphere (26) deposition, still retained on one side of the filter (24) . 11. Immunoassay process, characterized in that it includes: containing a liquid sample in a predetermined physical configuration; forcing a filter (24) through said configuration to thereby force the liquid sample through the filter (24) into a second configuration in contact with a plurality of beads (26) having immunologically reactive sites; keeping the liquid in contact with said beads (26) for a predetermined period of time to allow reaction between immunological constituents in the sample and said immunologically reactive sites on the beads (26); withdrawing the filter (24) from the configuration thereby forcing liquid sample through the filter (24) out of the second configuration, out of contact with the beads (26); washing residual liquid from the balls (26); determination of the reaction of immunological components from the sample with the immunologically reactive sites on the beads (26) . 12. Method according to claim 11, characterized in that the determination step includes: 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 constituents in the sample.