JPS628749B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method and apparatus for performing an assay using one of two families of specific binding substances (SBS) in an insolubilized state. The use of immunoassays to detect physiological compounds of interest has become widely applied when a disease is discovered to be related to the presence of a certain compound or the concentration of a particular compound. Radioimmunoassays were discovered and continue to be widely used for the detection and quantification of haptens and antigens because of the sensitivity afforded by the ability to measure radioactive tracers. Radioimmunoassays are performed by mixing the compound to be detected with its similar radioactive compound. These two substances competitively bind to limited amounts of antibodies. Antibody-bound radioactivity is separated from free radioactivity by various physicochemical methods, and bound and free radioactivity are measured, respectively. In one variation, solid phase antibodies are used, whereby unreacted free radioactivity can be easily removed. There are two closely related reagent overassay formats: immunoradiometric assay (IRMA) and two-site IRMA. In these schemes, an excess of labeled antibody is used to convert the unknown into a directly detectable product. 2 site IRAM
is the point where the unknown substance is insolubilized during the preliminary reaction.
It is different from IRMA. Many immunoassays have wide ranges of error. Variations in temperature and reaction time can be critical. Many systems are particularly sensitive to errors in the separation of bound and free labeled substances, and handling techniques such as reagent preparation, measurement and transfer (pipette weighing errors) are always important. Complex assay formats using continuous and/or discontinuous reactions are particularly flawed. The concentration of the test compound in the unknown sample is determined by comparing the results obtained for the unknown sample with the results obtained when several solutions (standard solutions) in which the concentration of the compound is known are tested. It is determined. Therefore, it is extremely important that all these assays are performed using the same reagents and using the same method. There remains a need for a simple and accurate technique for performing immunoassays, which minimizes any deviations from accurate performance, such as variations in reaction times, differences in transferred volumes, etc. And it is common to both standard substances and unknown substances. Furthermore, such a system should minimize the number of operations, including in particular extraction, dilution and dispensing. If necessary or desirable, the method must be sufficiently fast and must allow accurate recording of results with respect to the original sample. This method is also applicable to the evaluation of substances such as small haptens and large proteins as much as possible, and can be used in both RIA and IRMA methods to handle equilibrated or sequential non-equilibrium reactions; Furthermore, any given implementation must be able to effectively test either large numbers or small numbers. Additionally, this method must be economical and simple. Until now, no automated testing device has been able to meet all of the above requirements. Various automated methods for measuring various samples are described in U.S. Pat. No. 3,469,438;
It is described in the specification of No. 3723066. In addition to these, there are currently a number of commercially available radioimmunoassay devices. These devices include Centria, manufactured by Union Carbide; LKB Produkter, manufactured by LKB, Sweden; and Darius, manufactured by Digico Ltd., UK. Related literature includes Miles' ` ` La Ricerce Clin.Lab.'' on immunoradiometric assays.
(1957); Clin.Chem. 21 829 of Cottrell et al. on computer-controlled automated radioimmunoassays.
(1975); and Worwood, J. Clin. Path on the immunoradiometric assay for ferritin.
20 , 540 (1975). The present invention provides methods and apparatus for conducting assays using members of two groups of a pair of particularly binding substances (SBS) in insolubilized form. A particular binding agent includes a pair of ligands and a receptor for that ligand. The receptor may be any organic compound or composition that is distinguishable primarily by a significant difference in binding constant from its conjugated ligand to other compounds of similar structure, usually organic compounds. In most cases, the ligand is a hapten and an antigen, and the receptor is an antigen (antiligand).
However, other proteins and polysaccharides can also be used as receptors. When antibodies are used with conjugated haptens or antigens, these compounds are referred to as immunologically binding substances (IBS). The device employs a plurality of stationary syringes and syringe plungers connected for synchronized movement with each other. The electrically controlled power means is programmed to move the plunger according to a predetermined operation. Mechanical and electrical means are provided to compensate for the delay in movement when changing the direction of the plunger.
Preferably, the syringe is provided with a raised fluid stop in relation to fluid supply and reception. A tube with a row of complementary conical openings is provided as the tip of the syringe. The tip members are disposed, held and supported in a close stack of connected tip support frames. The syringe is inserted into the tip member by pressing the end of the syringe downwardly into the tip member in the support frame. The tip member is supported by a special insert block. When performing the assay, the tip member is activated by insolubilizing one of the SBS pairs within the tip member. The tip member is then washed to remove soluble material and stored or used directly. The solution to be assayed is introduced into the tip member from a number of sample holders. The tip is dipped into the sample and then the sample is drawn into the tip by pulling on the plunger. Reagents can be drawn into the tip member, or introduced simultaneously as part of the sample solution, or introduced from a syringe.
The solution in the tip member can be drained into a waste tray or drawn into a syringe. All plungers move in synchrony and thus draw in or expel solution from the tip member at the same time. Another option for the storage of new or used reagents is to provide an elevated reservoir, which is connected to the syringe cylinder via its outlet. The reservoir can also provide a wash solution that can be introduced into the syringe as needed, or can receive a used sample or reagent solution by gentle suction. The ends and outer walls of the tip are wiped after each operation or after various reagents have been contacted within the tip and any washing has been performed to remove unbound material. By using a radioactive tracer as a reagent and simultaneously including a standard reagent in the sample tray to be measured, the residual radioactivity in the tip can be combined with the standard sample tip for direct measurement of unknowns in the assay sample. Can be correlated. The present invention therefore provides a technique for simultaneous assays, whereby all operational steps can be carried out synchronously for the unknown and the standard, and the resulting errors are therefore constant for unknown substances. Although the invention primarily concerns multiple simultaneous measurements, the method can also be used for single measurements. By using a single syringe and tip, assays can be quickly performed by introducing reagents and wash fluids through the tip and aspirating. This allows sample concentration, eases reagent handling, and convenient preparation of radioactive samples for measurement. The tip member may be of a type that allows collection of a very small amount of sample into a very small container and provides a surface for carrying out the various reactions required for the assay. The interior surface of the tip member can also be provided with projections to increase the surface area. A spacer arm can be provided within the tip member that can be used for insolubilization of the analyte or reagent. In describing the present invention, the assay will be described generally using an illustrative immunoassay as an example, and then three different immunoassays involving radiotracer-containing reagents: radioimmunoassay (RIA); Trick assay (IRMA); and two-site immunoradiometric assay (two-site IRMA) will be described. The apparatus including auxiliary equipment will then be described. Next, the application of the immunoassay to a specific device will be shown, and the application of the device to various immunoassays will be shown, and further examples will be shown in which the device was used to measure a specific test composition. Assay Techniques The present invention uses SBS containing a ligand and a ligand acceptor.
Concerning performing tests using pairs. An open-ended tube is used, which allows for the introduction and removal of reagents and the cleaning of the tube to remove unwanted or unwanted substances, which is simple, reproducible, and allows for simultaneous operation. ,
And accurate measurements can be made. In the broadest sense, one substance of a specific binding substance pair (SBS) (including heptene, antigen, antibody, enzyme, enzyme substrate, serum protein, etc.)
The insolubilized tube is used during the assay. The reagent binds to the insolubilized substance of one of the SBS pairs, and is therefore itself a detectably modified SBS beforehand. In certification, as a means of making measurements,
A variety of atoms and compounds have been used to provide the desired signals, such as radioactive atoms such as iodine, tritium, carbon; stabilized free radicals; enzymes; coenzymes; bacterial phages; and fluorophores. The reagent used for assay measurements is called a assay agent (detechtent). The distribution of the assay agent between the solid and liquid phases influences the amount of the compound to be measured (analyte) present in the sample. The amount of analyte provided by providing competition between the detection agent and the analyte for the insolubilized substance of the SBS pair, or by sequentially introducing the analyte and detection agent into an open-ended tube. influences the amount of insolubilized detection agent. Due to the nature of the test,
The amount of detection agent present in the tube or in the liquid phase or both can be measured. The tube or tip member, which plays a critical role in the present invention, can be constructed from a variety of materials. The tube need not be of a single material; the key to the tube is the internal surface or packing within the tube. Surfaces can be provided in which one material of the SBS pair is insolubilized on either the internal surface or the packing, or both. The tip member is made of polyethylene; polypropylene;
It can be derived from a wide variety of polymers such as polyvinyl compounds such as polyvinyl chloride, polyacrylonitrile, polymethacrylates, polyacrylates and copolymers thereof; polystyrene; nylon; polyester; cellulose. Thus, naturally occurring polymeric materials, particularly modified natural polymeric materials, and synthetic addition and condensation polymeric materials can be used. Inorganic materials such as glass can also be used. Although it is convenient to provide only the internal surface of the tip member with an active surface for insolubilizing one substance of the SBS pair, the tip member may be conically shaped or partially closed to form small particles or beads of said material. and can provide an active area for insolubilization independently of or in conjunction with the interior surface of the tip member. Alternatively, a non-smooth surface,
For example, protrusions may be provided on the inner surface in the form of ridges or hills to increase the surface area. Insolubilization can be accomplished in a variety of ways. Depending on the nature of the specific surface and the substance of one of the SBS pairs to be insolubilized, absorption or adsorption should be satisfactory. After a sufficient period of time, the solution is removed and the interior surface of the tip member is washed with a suitable medium, usually a buffered medium. In a preferred situation, the analyte from the unknown sample can be the insolubilized material of one of the SBS pairs that has been absorbed onto the tip surface from the sample. Labeled receptors can be used to detect absorbed analyte. Another technique is to covalently bond one of the SBS pairs to a surface. The spacer arm can be covalently attached to the surface by various means known in the art while leaving a functionality active or activatable to react with one of the SBS pairs. for example,
The nitrile is modified to form an imidoester, which reacts with available amino groups on the antibody or antigen. There is extensive literature on attaching proteins to such surfaces by forming amide, amidine or amino bonds using activated carboxylic acids, carbodiimides, imidoesters, activated alkyl halides, and the like. Alternatively, immunospacer arms can be used. The method consists of binding or combining one of the SBS pairs to a surface and then binding the complement substance of the SBS pair to the surface. Other constituents of the SBS pair for binding include plasma binding proteins,
It includes any substance that exhibits specificity for the analyte, such as tissue receptors, enzymes, coenzymes, substrates, and inhibitors. A commonly used small conical tip member is approximately
It has an inferior opening diameter (ID) of 0.25 to 2 mm, more commonly 0.4 to 1 mm. The height of the tip member is not critical and can vary depending on the desired volume of the tip member. Normally, the height of the tip member is approximately 1
8 cm to 8 cm, more commonly 2 to 5 cm. The upper opening attached to the syringe is sized to fit snugly over the syringe barrel and generally has a diameter (ID) of about 2.5 to 6 mm. Wall thickness is not critical to the invention and can vary. Conveniently, the wall thickness can be varied such that it decreases as the inner diameter of the cone decreases. The thickness range to provide the desired dimensional stability is about 0.1 to 1.5 mm, more usually 0.2 to 1 mm. Here, the ratio of the top to bottom thickness of the tip member can vary from 1:1 to 10:1. Conveniently, the volume of the tip member ranges from about 50 to 700 μl, more usually from 75 to 500 μl. In most cases, the assay will involve a combination of SBS pairs, but in situations where the target analyte has specificity for a substance other than the other member of the SBS pair, it may be preferable to use related binding substances. . For example, if the enzyme substrate is what is being measured,
Enzymes and substrates could be used.
More specifically, if measurement of NAD is desired, a tip member could be provided that has NAD bound to its surface. By combining excess enzyme with the sample using NAD as a substrate and then introducing the composition into the tip member, only the enzyme that bound NAD in the sample will be bound to the tip member surface. By expelling the solution from the tip, the amount of uninsolubilized enzyme can be determined. The tip member can be activated immediately before performing the assay, or it can be prepared and stored in advance. For storage of the tip, generally mild temperatures can be used, typically temperatures of about -40 to 25°C, and the tip can be stored under controlled conditions to prevent deactivation of the insolubilized material. Retained.
The interior surface of the tip member can be coated with various liquids to maintain a moist polar environment. Exemplary substances are polyhydric alcohols such as glycerol. Depending on the nature of the assay, the unknown sample may also be combined with other reagents. In many cases, the sample is suitably buffered to enhance binding, using common buffers and providing a pH within or near the physiological range. For assays that require the analyte to bind to the detection substance and thereby influence the number of available binding sites that can be insolubilized, the assay sample is combined with the detection reagent under conditions such that binding can occur. Usually combined. When the detection agent is insolubilized to the extent that the analyte is insolubilized, the analyte is usually introduced into the tip member first and then the detection agent is introduced.
Each time a solution is introduced into the tip, the tip
Any material that is not specifically bound to the surface is washed away. The method of the present invention can include various storage steps in which the tip member is stored under normal or controlled conditions, typically at temperatures of 15 to 40° C. and humidity conditions to prevent drying out. Typically, the tip member is left in place after the introduction of the analyte and, if the detection agent is introduced separately, after the introduction of the detection agent. The storage period depends on the specific composition involved, the concentration to be detected, etc.
It can be varied from 0.5 to 12 hours. The present invention has particular benefits for dilute analyte solutions. By appropriate selection of the assay method, large excesses of insolubilized SBS pair constituents can be used. The analyte solution is drawn through the tip member such that the analyte solution is insolubilized by the excess insolubilizing component affixed or bound to the surface. The controlled aspiration rate of the solution drawn into the syringe through the tip member allows relatively fast quantitative expulsion of the analyte to be achieved. To further explain the methods of the invention, three different assays using radioactive tracers are generally described. The first test to consider is the RIA. In RIA, assuming that the antigen is the test substance, competition occurs between the radioactively labeled antigen (detection agent) and the antigen test substance. A limited amount of antibody directed against this antigen is bound to the interior surface of the tip member. An unknown antigen solution is introduced into the tip member simultaneously with or prior to the detection agent. The advantage of introducing the two solutions one after the other, rather than as a single mixed solution, is that any harmful substances in the unknown sample can be washed away from the tip before introducing the detection agent. In addition, if the ligand and receptor are in rapid equilibrium, the insolubilized constituents of the SBS pair and the detection agent can be pre-situated before being combined with the analyte. This allows the reagents to be prepared easily and reproducibly, and further reduces changes due to serum. The second verification technique is called IRMA. In IRMA, an unknown test substance is combined with a reagent consisting of a soluble purified radioactive antibody (detection agent). The tip member has an insolubilized analyte or a cognate analyte. A solution of the permeated analyte and antibody is drawn into the tip member, where all free sites of the antibody are bound to the insolubilized constituents of one of the SBS pairs. A variation of the IRMA method is to use radiolabeled (detection agent) antiglobulin (anti (anti-ligand)). In this method, antibodies against the test substance are not labeled. After the test antibody is insolubilized in the tip member, radioactively labeled antiglobulin is introduced into the tip member, thereby binding the labeled antiglobulin to any test antibody that has been insolubilized. The third method is 2-site IRMA. In this method, antibodies against the test substance are insolubilized in the tip member. Next, the test substance is introduced into the tip member and allowed to bind to the antibody. It is necessary that the test substance be polyepitopic, that is, it must have at least two sites capable of simultaneously binding at least two antibody molecules. After the analyte is bound to the insolubilized antibody, unreacted antibodies and other solutes are washed away, and a radiolabeled antibody to the analyte is introduced into the tip, which binds all the insolubilized analyte. be combined. Alternatively, as described at IRMA, a labeled antiglobulin (detection agent) can be used instead of a labeled anti (analyte), thereby making the labeled antiglobulin independent of the individual analyte. This method can be used for In all assays, after the labeled detection agent is introduced into the tip and insolubilized, the solution is drained or withdrawn from the tip and the interior of the tip is washed with an appropriate solution, usually a buffer, to ensure specificity. Remove any unbound material and wipe and blot the end of the tip. This removes any residual liquid at the end of the tip and any material on the outer surface of the tip. Device The device can be divided into three basic parts: a syringe device; 2 electronically controlled drive force; and 3 auxiliary instruments. Consider the syringe device first. Syringe device The syringe device has a plurality of syringes arranged vertically and horizontally uniformly in a chamber, where the syringe cylinder (or syringe body) is fixed between two tables,
The end of the syringe cylinder extends through the lower platform. The upper platform includes a hollow part, which serves as a liquid reservoir. Each syringe has one or more inlets connected to a reservoir. A tip member for receiving a syringe end extends through a plate movably attached to the frame and located below the base. This plate can be pulled downwardly to remove and remove the tip from the tip receiving end of the syringe, thereby allowing a new tip to be installed on the receiving end of the syringe. Connection means are provided for connecting a number of plungers, the plungers being arranged to match the syringe array and extending into the syringe cylinder. The plunger is long enough to advance the length of the syringe cylinder and expel all material from the cylinder. The plunger may be hollow or solid with a spring valve that allows solution to be introduced into the syringe through the plunger. Electronically Controlled Drive The syringe device is attached to an electronically controlled drive,
The device is shaped to receive the syringe device and has an upper platform for enclosing the syringe plunger plate. This upper stage is mounted for vertical movement in synchronization with the movement of the syringe plunger. The lower support member is mounted to support the sample tray or reagent tray;
These trays can also be raised to the tip member and removed. Alternatively, the syringe device can be lowered towards the sample stage, although this is not a convenient method. Restraint and support means are provided to secure the lower plate in position during various operations. Drive means are provided for raising and lowering the upper platform and the support member and are electronically controlled and programmed, so that the verification can be performed substantially automatically. During the assay, it may be necessary to change the sample tray. Auxiliary Equipment A tip rack is provided, which is a plate with a number of holes that correspond to the array of syringes. Preferably, in addition to the holes for the tip, there are a number of smaller holes similarly spaced between these holes. In this method, the tip member can be overlapped with the smaller end of the tip member placed in the smaller hole;
This allows multiple tip racks to be mounted one above the other in a staggered configuration. Therefore, a large number of tip members can be stored within a relatively small area. The second device is called the insertion block. The block has a number of chambers positioned to receive and hold the smaller end of the tip by providing mechanical support for the tip. The insertion block is used to attach the tip member to the receiving end of the syringe by forcing the syringe into the tip member supported by the block. The third device is called a holding box and provides a humidity controlled environment that can be temperature adjusted as desired. For a better understanding of the device, it will be explained with reference to the drawings. As shown in FIG. 1, multiple assay device 10 has a motor drive chamber 12 and a syringe device 14. As shown in FIG. The motor drive chamber 12 has a support frame 16 under which a motor 20 is mounted, and the motor is electronically controlled by a programmer 22. Four threaded bolts 24 attached to a gear 26 are rotatably attached to the base 12. Gear 30 in the center is motor 2
0, and gear 26 is also driven at the same time. The upper stand 32 is threadedly attached to the bolt 24 and is vertically movable by rotation of the bolt. The upper platform 32 has symmetrically disposed L-shaped arms 34 and 36 for receiving and holding the syringe plunger plate 40 in place. The side wall 42 is the base 12
It is mounted on the opposite side from the syringe device 14. A shoulder 44 extends inwardly from the sidewall 42 to support the syringe device and secure it in place. A guide 46 extends downwardly from shoulder 44;
A lower stand is movably attached to this guide. The lower platform is secured to a cam (not shown) having cam arms 52 for raising and lowering the lower platform. Lower platform 5
A spring is attached to the guide 46 to push down the 0. A stop member 56 is provided on each guide to hold the lower platform on the central gear 30. Safety switches 60 and 62 are provided to control the range of vertical movement of upper platform 32.
A post 64 is provided in bracket 66 and engages safety switch 60 to control the extent to which upper platform 32 can be lowered. bracket 6
The post 70 attached to the safety switch 62
The arm 72 that engages with the upper table 32 is held to control the upward movement range of the upper table 32. Safety switches 60 and 62, which are normally closed, are connected to control mechanism (programmer) 22 and isolate motor 20 when switched. With reference to FIGS. 2 and 4, the syringe device 1
4 will be considered in more detail. The syringe device 14 is connected to an upper guide plate 72 provided on the ceiling 74 of the chamber.
It has a rectangular chamber 70. The guide plate has multiple passages for guiding the plunger 76. The ceiling 74 has a conformal passageway or is hollow and serves as a reservoir for solutions, especially cleaning solutions. This will be explained in more detail later. Support posts 80 support ceiling 74 and are mounted on floor 82 . Floor 80, along with ceiling 72, holds syringe 84 in position. Floor 82 is held firmly in place by side walls 42 and shoulders 44. The tip member receiving end 86 of the syringe 84 extends through and beyond the floor 82 to a sufficient length such that the tip member 90 is attached to the syringe end 8.
It can be attached to 6. Syringe plunger plate 40 has a hollow center portion for receiving plunger head 94 and holding it in a fixed position. The removal plate 96 has rods 1 at the four corners.
It is fixed at 00. Each edge of the syringe device 14 is a rod 102. Spring 104 connects rod 102 and floor 8
2 and maintains upward pressure on the rod 102. Cam 106 is rotatably mounted within frame 112 by pin 110 and has arm 1 of the cam.
14. By rotating the cam 106, the removal plate 96 moves along the dashed line 116.
, thereby forcing the tip member 90 to be removed from the tip receiving end 86 of the syringe. Rod 100 has ceiling 7 upwards.
4, thereby ensuring proper positioning without binding during exercise. Tip member 90 is arranged in rack 116 during storage and use within assay device 10. Rack 116 allows all tip members to be simultaneously attached to and removed from the end of the syringe. During this time, they are maintained in the originally planned order. The sample or reagent tray 120 includes auxiliary cups for supplying reagents to the tip member or for receiving reagents expelled from the tip member. It is preferable to minimize manual intervention and attention by the operator during the assay. Therefore, the solution,
It is preferable that reagents and the like can be automatically supplied to the tip member 90. Referring to FIG. 3, ceiling 74 with reservoir 118 is shown as hollow. Reservoir 118 is fluidly connected to syringe 104 through opening 122. When the plunger 76 moves to the top of the opening, the position pressure necessary to force liquid into the syringe is applied to the reservoir, fluid from the reservoir enters the syringe 104, and then by moving the plunger downwards, the fluid is removed. Syringe 76 can be ejected into tip member 90 . Alternatively, a hollow plunger having a center post 124 with a resiliently attached leg 126 can be used. An O-ring 130 is compressed between the leg 126 and a protruding member 136 that is rigidly attached to the plunger 76. The leg 126 is shaped to leave a gap between the leg 126 and the inner wall of the syringe 84. O-ring 130 provides airtightness of the inner wall of the syringe. By pressing on the center post, the legs 12
6 is lowered to provide a fluid connection between the central hollow of the plunger and the syringe 84. The fluid can thus be dispensed by plunger means or drawn from the syringe by suction. Plungers of this type are commercially available and are available from Eppendorfer.
It's called a syringe. Auxiliary Equipment Rack 116 was previously shown as a means for aligning tip member 90 with syringe 84. Racks are also useful for storing and transporting tip members. Additionally, as shown in FIGS. 5 and 6, the rack may have multiple small holes 134 arranged symmetrically as well as large holes 136 for retaining the tip. By providing these two types of holes, the tip member rack can be
The stoma aligns and supports the tip members in adjacent upper racks so that they can be stacked upwardly. A tip insertion block 140, as depicted in FIG. 7, is also preferred. When the tip member 90 is attached to the tip receiving end 86 of a syringe, the tip member is received in the conical bore 142 that mechanically supports the tip member. This applies sufficient pressure to the tip member to securely attach the tip member to the end of the syringe. Tip member 90 is compact and has a wall thickness that provides sufficient mechanical stability to the tip member. Examples of Immunoassays To further illustrate the invention, multiple assays were performed using a multiple assay device. For illustrative purposes, only a 2-site IRMA assay was performed. A large number of tip members 90 are placed in the tip member rack 116.
I fitted it. A hole in the rack corresponded to each position of the syringe in the syringe device 14. It was not necessary to use all holes. This is because the method and apparatus do not necessarily require the use of all syringes in the syringe device. Typically, standard solutions and samples of test substances are
It may be diluted with any diluent as necessary. Various standard solutions and test samples were introduced into cups in assay tray 120. Various sample preparations, such as diluting the sample and adding reagents, are conveniently performed using the syringe device 14. By using the syringe device 14,
All operations will be identical to those used for standard solutions. Tip member 90 supported by rack 116 is inserted into insertion block 140 and syringe end 86
was pushed into the tip member and the syringe device 14 was lowered to tightly fit the syringe end into the tip member. The syringe device was then attached to the motor drive chamber by inserting the syringe plunger plate 40 between the L-shaped arms 34 and 36 of the upper platform 32. A sample with the appropriate antibody was placed on the lower stage, with the upper stage in its movable lower position so that the plunger extended far enough down into the syringe. After partially filling the syringe 84 with the wash solution, thereby drawing the antibody into the tip member, after ejecting the antibody solution by lowering the plunger and expelling the wash solution through the tip member; The tip member can be cleaned directly. After a sufficient period of time, approximately 1 to 16 hours, the contents in the tip member are drained into a waste tray and the tip is flushed with wash solution in syringe 84 supplied from upper reservoir 118 or from wash solution in the sample tray. The parts were cleaned twice. After each operation, the liquid was sucked up around the tip member. As shown in FIG. 4, the tip member 90 can be removed by means of a removal plate and frozen or stored in a simple humidity storage container 144. The conditioned support container 144 is an open top rectangular box 146 that supports a syringe device. The syringe device serves to close the box. A damp sponge 150 is placed within the box to provide the desired humidity so that the tip does not dry out. Assuming that the tip member 90 is retained on the syringe end 86, the tray containing the samples and standards has been raised and placed on the upper stage so that the tip member rests on the bottom of the cup 152. reach substantially. (See Figure 4) The appropriate amount of solution was drawn into the tip and the device was then left in the motorized chamber 12 or transferred into a supporting storage box. Sample aging was performed under controlled temperature and humidity conditions. The tip member is then cleaned as previously described and the sample tray containing the radioactive antibody is placed on the lower stage 5.
0 and raise the tray so that the tip extends far enough to the bottom of the cup 152. Reagents are sucked into the tip by raising the upper stage 40, and after sufficient antibody binding time, the labeled antibody solution is drained into a waste tray and the tip is washed, wiped and blotted dry. Make it. Multiple reagent solutions can be contained in a syringe, with air bubbles separating a series of different solutions. The amount of isolation air must be sufficient to draw the solution away from the tip. These used solutions can be disposed of by suctioning into a reservoir. The tip member remained attached to the tip member rack 116 and was removed again into the insertion block. Then,
The tip members were inserted one by one into the counter tube in order to measure the radioactivity attached to the inner wall. The amount of radioactivity on the tip is directly correlated to the amount of analyte present in the standard and unknown solutions. By creating a standard curve, the results of the unknown sample can be compared to the standard curve and the amount of analyte in the unknown sample can be calculated. By the same method based on applicable technology, RIA,
Other immunoassays such as IRMA, enzyme immunoassay, etc. can also be performed. Although the device of the invention is described for use in immunoassays, the device can be applied in any case where the simultaneous transfer of multiple liquids is desired, and thus can be used for automated pipetting, sampling and aspiration, etc. can. Experimental Unless otherwise indicated, all temperatures in this experiment are in °C and all percentages are by weight. Example 1 Two-site immunoradiometric assay for human plasma growth hormone (HGH)
How to use the assay device at IRMA) 1 1 Preparation of standard solution and unknown sample HGH standard solution was prepared by diluting hormone-free human plasma with a diluent in a ratio of 1:5. Diluent: 0.05M sodium barbitan, 0.1M
NaCl, 0.1% bovine serum albumin (BSA) and
It consists of 0.01% sodium azide, has a pH of 8.0, and is called BSA-buffer. Four replicates of each of the 10 standard solutions (0-10 ng/ml) were 96
Cooke Microtiter
Stored in the company. Six replicates of each of three quality-controlled human plasmas, also diluted 1:5, were placed in this tray. These human plasmas (Q ₁ , Q ₂ and Q ₃ ) were selected to represent low, intermediate and high levels of circulating HGH and were frozen in multiple sets of samples. A human plasma sample with unknown HGH content was diluted 1:5 and assayed in duplicate. Any samples assayed at a dilution of 1:5 or higher were further diluted with hormone-free human plasma diluted 1:5. For each of the 44 unknown tips, a replicate zero-content standard and _two replicate Q samples were included. These assay samples were prepared and diluted in trays using the assay device of the present invention as a sampling-diluter. The samples were stored frozen in trays after being sealed with a Cookes dispenser and tape. 2. Preparation of tip members Each rack for the tip member was inserted into a device using an insertion block. The tip member remained attached to this tip member support rack. Diluted 1:4000 with sodium bicarbonate 0.2M solution (PH: 9.2)
The inner wall of the tip member was coated with anti-HGH by drawing 120 μl of guinea pig anti-HGH into the tip member. The antiserum was aspirated from a raised tray under the tip. After 1-16 hours, the contents within the tip were drained into a waste tray and the tip was washed twice with wash solution from the reservoir. each cleaning solution
120 μl was discarded into a waste tray. The cleaning solution
0.05M phosphate buffer (PH8.0) 0.1% BSA,
It contained 0.45% NaCl and 0.01% _NaN3 .
After each operation, the tip member was wiped off. 3 Preservation of Activated Tips Alternatively, freeze the tip containing 120 μl of coating antiserum or wash solution and fill the tip support rack with the removed tip using a tip removal device. They were stored frozen in racks stacked in plastic bags. 4 Assay Method a Unknown and Standard Samples Standing (Reaction 1) The assay was initiated when the standard and unknown samples were simultaneously drawn into the coated and cleaned tip member. 120 μl of solution was aspirated under programmed electronic control using optimal syringe air space. The equipment was either left in the motor mounting area or removed and placed in a storage support box. In all cases, storage was carried out under controlled temperature and humidity. b First cleaning The tip member is cleaned by the same method as above,
I wiped off the tip. c Second reaction 120 μl of purified radioactive anti-HGH was aspirated from the reagent tray. This reagent was diluted with BSA buffer to 120 μl to 20,000-40,000 cpm. The device was again left as described above. d Final Wash After 24-28 hours, the labeled antibody was drained into a waste tray and the tip was washed and wiped down again. 5 Radioactivity measurement on the tip member The tip member was removed into the insertion block while attached to the support plate using the tip member removal mechanism. Next, the tip members were inserted one by one into a counter tube in order to measure the radioactivity attached to the inner wall of the tip member using an automatic gamma ray counter. 6 Data Analysis The amount of radioactivity on the tip was a direct function of the amount of HGH present in the standard sample and unknown solution. Create a standard curve from the results of the HGH standard sample and compare the measured radioactivity of the unknown sample with the measured radioactivity of the standard sample.
HGH concentration was calculated. Method 2 Use of the Assay Device for Two-Site IRMA Plasma HGH with Washing from the Syringe This method is the same as Method 1 except that the syringe is filled with solution. The first and second reactions were performed with unknown or labeled antibody separated from the wash solution by an air bubble. All solutions were formulated with a small amount of propene glycol to reduce surface tension. At the end of each reaction, the solid phase was washed with 200 μl of wash solution, which drained the solution in the tip into a waste tray, then expelled from the syringe and passed through the tip. Fresh buffer can be introduced into the syringe through a syringe plunger from a chamber located near the syringe device, or by inhalation from below. Method 3 Use of the assay device for two-site IRMA of plasma HGH where all the solution is introduced into the syringe by suction At the end of each reaction, the solution in the tip is aspirated into the syringe through the tip. This method was otherwise the same as Method 1. A bubble space was required during reaction 2 to separate the waste fluid in the syringe and the labeled antibody in the tip. All solutions contained a small amount of polypropylene glycol to reduce surface tension. The waste liquid in the syringe was disposed of through the plunger or through a chamber near the syringe device by gentle suction. Method 4 Perform assay reaction 1 by repeatedly taking unknown samples (e.g. at 1 hour intervals).
The assay was the same as Example 1, except that 120 μl). When a new sample was taken, the previous sample was drawn into the syringe. Alternatively, the previous sample was drained into a waste tray. This method increased the sensitivity of the assay. Method 5 HGH by slow continuous suction of unknown substance
Use of the Assay Device for the 2-Site IRMA was the same as Method 1 except that Assay Reaction 1 was performed by reacting a very large amount of the unknown. This method was performed by slowly dispensing the unknown sample into the syringe through the tip and continuously aspirating it. This method concentrated a large amount of unknown material for reaction with a labeled antibody (reaction 2). Example 2 Use of Assay Device for IRMA In the IRMA method, an unknown antigen was reacted with a soluble purified radioactive antibody. The radioactive complex remained in solution during the second reaction with solid phase antigen to remove unused antibody. The amount of radioactivity remaining in solution was a direct function of antigen concentration.
IRMA is another universal reagent labeled anti-
It is also suitable for use with IgG, making the preparation of radioactive antibodies specific to unknown substances unnecessary. HGH assays using IRMA are RIA and 2-
This was done in a manner similar to that described for the site IRMA. In IRMA, excess antigen was immobilized in a solid phase (tip member) and the standing mixture containing unknown antigen and purified labeled specific antibody was aspirated into the tip member. Any labeled antibody that did not bind soluble antigen was bound to the tip member. coupled to the tip member. The tip member was washed and the label bound to the tip member was inversely proportional to the amount of antigen present in the unknown solution. Example 3 Use of Assay Devices in RIAs In RIAs and similar competitive binding assay techniques, a limited amount of specific binding reagent (usually an antibody) is used.
competitive binding occurs between the unknown antigen and the labeled antigen derivative. Free labeled antigen was separated from labeled antigen bound to antibodies (or other specific binding agents) by various methods. If the binding reagent is a solid phase, unreacted labeled antigen can be easily washed away. The amount of labeled antigen bound to the binding agent was an inverse function of unknown antigen concentration. The assay can be performed in a single reaction, or the labeled antigen can be added later as a lag or second reaction. This latter procedure is often referred to as tracer post-addition and has often given superior assay sensitivity. If the solid phase is washed to remove unreacted and unknown solution components before adding the tracer (labeled antigen), the labeled antigen will not be directly lost and will not interfere with the components of the unknown substance. Will. Alternatively, the labeled derivative (detection agent) can be reacted with a solid phase antibody (insolubilized component) prior to reaction with the analyte. Hapten assays allow for reproducible pre-manufacturing without degradation in dose response and prevent assay confounding due to the presence of serum. Example 4 Method 1 1 Preparation of standard, unknown and quality control samples Nine samples were prepared in a solution containing 0.05M phosphate buffer (PH = 8.0), 0.1% bovine serum albumin and 0.01% _NaN3 . (0-5000 ng/ml) were prepared in four replicates each. The above solution is called BSA buffer. Four replicate quality control samples (Q ₁ , Q ₂ and Q ₃ ) each were selected to represent low, medium and high levels of circulating plasma cortisol. Unknown plasma samples were assayed in duplicate. ¹²⁵ I cortisol was diluted with BSA buffer. Standards, quality control plasma and unknown plasma samples were diluted 1:21 with 95% ethanol. The resulting precipitate was centrifuged, and the supernatant portion was diluted with ¹²⁵ I cortisol. The final sample dilution was 1:399 and contained 6000 cpm/100 μl of radioactivity.
This solution was stored in a sample plate and covered with a special film, ready for application in the assay. 2. Preparation of the tip member The tip member was activated by the method described above, except that multiple layers of IgG were used. normal rabbit serum
A solution diluted in 0.2M NaHCO ₃ (PH=9.2) was first coated, followed by guinea pig anti-(rabbit IgG) serum diluted 1:1000 in BSA buffer. After 4 hours, drain the solution and replace the tip.
Washed twice with BSA phosphate buffer. Any insolubilized antiserum can be bound to this tip, provided it has been previously prepared for rabbits. In this cortisol assay, rabbit anti-cortisol diluted 1:25,000 in BSA buffer was used.
Serum was aspirated into the tip and left for 16 hours before washing with BSA buffer. All storage was done at room temperature. The tip member can be stored for later use by freezing in a rack containing antibody solution or BSA buffer. The tip, saved before reacting with the final specific antibody, can be used in any assay using rabbit antiserum. 3 Assay Method A test solution containing also ¹²⁵ I cortisol was aspirated from the sample tray into the tip member. Deposition was either at room temperature in the area where the motor was installed or in a storage support box under controlled temperature and humidity.
After 16 hours, the tip was emptied, washed, wiped dry, and the radioactivity bound to the solid phase (tip) was counted. (Alternatively, ^{the 125} I cortisol was added late in the second reaction, without mixing with the unknown, i.e. as a post-addition of the tracer or as a pre-incubation of the tracer.) 4 Dose Interpolation Tips The amount of radioactivity bound to was an inversely proportional function of the amount of cortisol present in the standard or unknown solution. Method 2 for RIA of cortisol with post-addition of tracer and repeated use of known small amounts of unknown. Use of the Assay Device The assay was performed as described above, except that known small amounts of the unknown were taken repeatedly and the unknown was not mixed with ^{the 125} I cortisol. ^{The 125} I cortisol was aspirated as a second (delayed) reaction. This increased the sensitivity of the assay. Method 3: Use of the assay device for RIA of cortisol with subsequent addition of tracer and slow continuous aspiration of the unknown. The assay was performed as described in Method 2, except that the assay was performed as described in Method 2 (as described in the 2-site IRMA). Therefore, the following results were obtained.

Table: Sensitivity was significantly increased when using the 2-site IRMA technique method and 5 described above. 10 to 100 pg/ml for method 1 in standard curve
The difference in measured radioactivity (cpm) between the two was 300 cpm. For method 4, the difference is about 1200 cpm,
In method 5, it was about 1250 cpm. Additionally, the curves for Methods 4 and 5 are 10 to 10% lower than Method 1.
The slope was steeper in the 50 pg/ml region.
Thus, depending on the need for accuracy and sensitivity,
Testing was possible even at extremely low concentrations. The method of the invention provides an extremely simple and sensitive technique for measuring a wide range of immunologically binding substrates or other compounds for which receptors are known. Reduce manual operations to a minimum, especially in areas where errors are likely to occur. Furthermore, by performing all transfers simultaneously with carefully scaled identical syringes, time-varying conditions do not affect unknown samples any differently than standard samples. Therefore, standard measurements and standard curves are obtained under the same conditions under which the unknown substance is measured. In the present invention, the assay method is carried out in an open-ended tube, so that solutions can be easily introduced and aspirated without moving or disturbing the tube. In contrast to ordinary test tubes, open-ended tubes have no mechanical turbulence during solution removal. Furthermore, for open pipes, a column effect can be applied,
Rather than thereby providing an equilibrium between the insolubilized SBS and the analyte, a solution of the unknown substance can be introduced into the new insolubilized SBS by sucking the unknown solution into the tip member. Reagents and/or wash solutions can be introduced from the syringe into the tip member and then disposed of into the wash tray. Reagents and/or wash solutions can be introduced into the syringe from a reservoir near the syringe device. Alternatively, the reagents, wash solution and test sample can be introduced from the tray into the tip member and then drawn into the syringe. Spent samples, reagents and/or wash solutions can be discarded from the syringe by suction through the reservoir. If desired, continuous solutions can be separated by bubble spacers. The device of the present invention provides a convenient method by which multiple samples can be prepared in trays, the samples introduced into the tip member, and multiple assays performed simultaneously under the same conditions. By using carefully standardized and uniform syringes and plungers, highly accurate and reproducible results can be obtained. Due to the large volume of the tip member, large or small amounts of sample can be used with different insolubilization of the analyte. The tip member is extremely versatile in its role. By using a conical tip with a small opening and a small opening outer diameter, the tip can collect and aspirate extremely small amounts of sample, thereby allowing effective transfer from the sample tray to the tip. I can do it. The tip member can be inserted into the sample without spilling the sample. The volume of the tip member is sufficient to carry out the various reactions required for the assay and therefore can be used directly for measuring the detection agent. Prior to the present invention, devices used a single sample. Extraneous substances present in the sample may precipitate into the sample, creating a common difference for all subsequent samples. In the present invention, such a situation can be prevented. Furthermore, the initial combination of sample and standard is maintained throughout the assay, so that each sample is kept in a specific configuration that facilitates recording of results, especially by automated means. Finally, the tip member can prepare all the samples and reagents within it, thereby eliminating any measurement of reagent availability by the researcher and substantially preventing the occurrence of human error. It should be noted that the detailed description and examples for the present invention are intended to be illustrative of the present invention and are not intended to be limiting.

[Brief explanation of the drawing]

FIG. 1 is a perspective view of a syringe device attached to a motor drive control mechanism. FIG. 2 is a front view, partially in section, of the syringe device in contact with a sample plate. FIG. 3 is a front view showing a cross section of the hollow plunger. FIG. 4 is a side view of the syringe device during storage. FIG. 5 is a front view of the tip member attached to the stacked racks. 6 is a partial perspective view of the tip member shown in FIG. 5. FIG. FIG. 7 is a front view of the tip mounted in the rack and supported by the insertion block.

Claims (1)

[Scope of Claims] 1. The test substance is a constituent of one of two groups consisting of a pair of substances capable of specifically binding, and in the assay, the substance of the pair is insolubilized. detection of an analyte in a sample using one constituent and a detection agent which emits a physically measurable signal proportional to the number of detection agent molecules present and which is a constituent of one of the two aforementioned groups. In an assay method for determining quantity, a test substance solution is aspirated by syringe means into an open tip removably attached to the syringe;
the tip member contains the insolubilizing component (provided that the analyte can be the insolubilizing component if the analyte can be bound to the tip member); a solution of a detection agent or other reagent; and introducing a wash solution from an external source or from the syringe into the tip member by syringe means; removing the solution from the tip member by the syringe means; an amount of detection agent insolubilized in the tip member; Alternatively, the amount of the test substance in the sample is measured by measuring the amount of the detection agent removed from the tip member using a physically measurable signal. 2. The assay is an immunoassay. 2. The method according to claim 1, wherein the pair of specific binding substances are a ligand and an antiligand. 3 the analyte is a ligand, the insolubilized constituent is an antiligand, the antiligand is in an insufficient amount to bind all of the ligand and the detection agent; and the detection agent is radioactive. 3. The method according to claim 2, wherein the ligand is a labeled ligand. 4. said ligand is a polyepitopic ligand; said insolubilizing constituent is an anti-ligand and is in excess of the amount of ligand present;
3. The method according to claim 2, wherein the detection agent is a radioactively labeled anti-ligand. 5 The test substance is a polyepitopic ligand; the insolubilized constituent is a first type of antiligand; the detection agent is a second type of antiligand and an anti(antiligand) to the antiligand; The method according to claim 2, characterized in that the method is a combination of: 6 the insolubilized component is a ligand and is present in an amount in excess of the amount of detection agent present; the detection agent is an anti-ligand and is introduced into the tip member together with the analyte; A method according to claim 2, characterized in that: 7. the insolubilized component is a ligand present in excess of the detection agent; the analyte is a polyepitopic ligand; and the detection agent is a first type of antiligand and a radioactive substance for the antiligand. 3. The method according to claim 2, which is a combination with a labeled anti (anti-ligand). 8. the insolubilized component is present in excess relative to the analyte, and both the analyte solution and the detection agent solution are drawn into the syringe through the tip, thereby causing the unbound insolubilized component to be absorbed. 3. The method according to claim 2, characterized in that the test substance solution is exposed. 9. In the method in which the insolubilized component is an antiligand and is present in an excess amount relative to the analyte, the analyte solution is removed; the tip member is washed with a cleaning solution before introduction of the detection agent. drawing the detection agent solution into or through the tip member by syringe means; and rinsing the tip member to remove any non-specifically binding detection agent. The method described in Section 2. 10 A method of performing multiple assays on multiple samples, in which the analyte to be measured is a member of one of two groups consisting of a pair of specific binding substances, and at least one sample is The assay contains a known amount of the analyte; furthermore, the assay provides an insolubilized constituent of one of the two groups, giving a physically measurable signal proportional to the number of detection agent molecules present. and a detection agent that is a member of one of the two groups, and a synchronously moving plunger and removably attached to the end of the syringe for receiving the solution and retaining the insolubilized member. a method using a syringe device having a tip member, wherein each of said samples is simultaneously drawn into each tip member containing said insolubilized constituent; a detection agent solution or other reagent solution and a wash solution from an external source or syringe; into the tip member by the syringe means; removing the solution from the tip member by the syringe means; and determining the amount of detection agent insolubilized in the tip member or removed from the tip member. An assay method characterized in that the amount of a detection agent is measured by a physically measurable signal to determine the amount of a test substance in each sample. 11. Aspirating a solution containing the constituent to be insolubilized in a soluble state into the tip member by means of the injection means and binding the insolubilized constituent to a surface within the tip member. 11. The method of claim 10, comprising the step of insolubilizing the insolubilized constituent. 12. The amount of the insolubilized substance is insufficient to bind all of the detection agent and the test substance,
11. The method of claim 10, wherein said detection agent is radiolabeled and capable of competing with said analyte for said insolubilized constituent. 13. The insolubilized component is present in excess of the analyte, the detection agent is radioactively labeled, and the insolubilized component is capable of binding to the analyte. The method according to claim 10. 14. The insolubilized constituent is a first type of antibody, and the detection agent is a second type of antiligand and a radiolabeled anti(antiligand) against the second type of antiligand. 14. The method according to claim 13. 15 the insolubilized component is capable of competing with the analyte for the detection agent, and the detection agent is radiolabeled; Claim 10 characterized in that the invention is combined before introduction into the
The method described in section. 16 The test substance and the insolubilized substance are members of the same group, and are ligands selected from heptone or antigen; the detection agent is an antiligand and a radioactively labeled anti(antiligand). 16. The method according to claim 15, characterized in that: 17. A device for the simultaneous transfer of multiple liquids, including the suction and discharge of solutions from a plurality of small tip members attached to the tip member receiving end of a plurality of syringes, the device comprising: an upper stage; a chamber having a lower pedestal; a plurality of syringe cylinders held between the upper pedestal and the lower pedestal disposed at predetermined intervals, the tip receiving end extending from the cylinder through the lower pedestal; a plurality of syringe plungers inserted into the syringe cylinder, each of the syringe cylinders having at least one port in the top of the cylinder; a fluid reservoir communicating with the port; a rod extending above the upper platform; means connected to the upper part of the plunger for simultaneously moving the plunger in a vertical direction;
and means for simultaneously removing a plurality of the tip members from the syringe end, the means being provided between a lower base and an upper edge of the tip member attached to the syringe end. 18. The apparatus of claim 17, wherein each syringe cylinder has at least one or more inlets thereon, each inlet including a fluid reservoir fluidly connected thereto. 19. The device of claim 17, wherein the syringe plunger has a hollow center. 20 the means for removing said tip member being a plate having a number of holes through which the syringe end extends; a cam operatively connected to said plate for lowering said plate to release said tip member; 18. The apparatus of claim 17, comprising: means; and resilient means for returning said plate to its rest position.