EP0311615A4

EP0311615A4 - Method and apparatus for facilitating chemical, biochemical and immunological reactions and mixing, especially where microtiter volumes are involved.

Info

Publication number: EP0311615A4
Application number: EP19870903935
Authority: EP
Inventors: Linda R Watkins
Original assignee: Individual
Current assignee: Individual
Priority date: 1986-06-20
Filing date: 1987-05-15
Publication date: 1989-03-15
Also published as: AU7580887A; JPH01501358A; WO1987007954A1; EP0311615A1

Abstract

A device and method for enhancing the speed and sensitivity of biochemical and immunological reactions performed in manifold vacuum devices. Reactions are carried out in one or numerous wells (25) which are integral parts of a manifold plate (10). The internal space of each well (25) is confluent with a waste fluid collection chamber, usually located beneath the manifold, and connected to a vacuum pump (12). A piston-driven (16) or bellows (40) device repetitively interrupts a low-level vacuum applied during the reaction steps, generating a low and continuously oscillating vacuum across the base of the manifold during the reaction procedure. In turn, this causes the reactants to be continuously mixed as the vacuum repetitively draws and releases the fluid components in the wells (25).

Description

METHOD AND APPARATUS FOR FACILITATING CHEMICAL, BIOCHEMICAL AND IMMUNOLOGICAL REACTIONS AND MIXING, ESPECIALLY WHERE MICROTITER VOLUMES ARE INVOLVED

P E C I ZIC A T I M

This invention relates to method and apparatus for facilitating reactions and mixing.

Background of the Invention In all chemical, biochemical, and immunological interactions, the molecular entities need to come in close physical proximity for binding to occur, whether the binding is by van der Waals, ionic, covalent, or other forces. Over the years, a wide variety of methods has been used to facilitate interactions between molecular entities of interest. These methods include (1) changing the phase of the compounds (i.e., forming fluid phases from solid or gaseous phases) to enhance the ease of molecular interactions, (2) heating the reactants, both to reach the activation energy required for binding and to increase the motion of the molecules, so as to increase the chance that they will come in close proximity with other molecules, (3) using various agents such as low-ionic-strength solutions designed to decrease the repelling ionic fields between molecules, and (4) using inorganic catalysts and biological catalysts such as enzymes, to enhance the likelihood and speed of reactions. Additionally, many techniques have been developed for the mixing of reagents, including bubbling inert or nonreactive gases through the reaction solution, vortexing, mechanical stirring, and using electrically driven platforms which repetitively swirl or tilt the reaction mixture. These mixing techniques are especially useful in reactions enhanced by catalysts and enzymes, since accumulation of the end-products in the vicinity of the catalyst or enzyme decreases the likelihood of further substrate-catalyst or enzyme interactions, unless mixing techniques are employed to move the end-products away from and to move fresh substrate into the vicinity of the catalyst or enzyme reactive site. With the advent of micromethods in biochemistry and immunology comes the need to enhance molecular interactions within the constraints imposed by microliter volumes of reaction fluids. Atop this challenge, which already negates use of virtually all presently available mixing techniques, are the additional constraints imposed by recent innovations in biochemical and immunological micromethods. That is, manifold devices having a matrix of numerous small reaction wells (typically 96 wells in a 8 x 12 matrix, each able to maximally hold 350 microliters of fluid) are used for simultaneously performing numerous complex reaction procedures. Given the microtiter reaction volumes involved (typically on the order of 25 to 100 microliters) , the number of simultaneously occurring reactions (up to 96 reactions at a time) , the frequent use of enzymes (with the associated problem of end-product build-up at the enzyme reactive site) , and the desire for very rapid, very replicable reaction times, it is apparent that new methods are required to achieve effective mixing of reagents in such biochemical and immunological micromethods. ^To date, micromethods in biochemistry and immunology have either relied simply on passive diffusion f reagents or have attempted to use centrifugal forces (See U.S. Patent No. 4,515,889) or mechanical agitation induced by tilt plates or swirl plates (See U.S. Patent Nos. 4,133,639 and 4,200,613). Use of the latter devices is a hold-over from earlier technologies where tilting and/or swirling of larger volumes of reagents was effective in enhancing the efficacy of the reactions (for example, see U.S. Patent No. 3,488,156; 3,876,379,* and 4,457,894). Such devices are minimally if at all effective when combined with manifold devices, due to the small diameters of the reaction wells and the small volumes of the reaction fluids. Instead, recent methodologies have changed tack and have attempted to increase the surface area available for reactions to occur. These attempts have relied on binding at least one reactant to moieties with large total surface area, such as frosted glass (U.S. Patent No. 4,280,992), waffle-like filter surfaces (U.S. Patent No. 4,317,810), beads (U.S. Patent No. 4,133,639; 4,166,102; 4,200,613; 4,217,338), porous membranes (U.S. Patent No. 4,407,943), or the walls of the reaction chamber (U.S. Patent No. 3,646,346; 3,790,663; 4,225,784), all in hopes that this increased surface area will in part compensate for the lack of mixing. A new technology which has arisen within the last five years, and which was introduced for a purpose unrelated to the enhancement of mixing of reagents, set the stage for the present invention. This technology resulted in the invention of so-called manifold vacuum devices. Briefly, this advance in biochemical and immunological micromethods is a modification of commercially available, 96-well matrices (manifolds) . The luid-impermeable base of each well is modified by various means, so that fluid is retained in the wells (thereby allowing biochemical and immunological reactions to occur within the wells) under normal atmospheric pressures. Yet ^tne fluid is drawn out through the modified base of the wells through a fluid interface when vacuum is applied across the base of the manifold. Although various modifications have been introduced to attain this goal, they have the common feature of employing a relatively low resistance port in the base of each reaction well of the manifold, such that vacuum applied across the base of the manifold draws fluid out of the wells. As will be described below in detail, the present invention utilizes such a low-resistance port to impose an oscillating vacuum across the base of the manifold. This oscillating vacuum serves to churn and mix the fluid contents of all 96 wells of the manifold simultaneously, thereby enhancing the speed and efficiency of the reactions. Although older methodologies exist which effectively disturb fluid-filter interfaces by imposing oscillating jets of fluid (U.S. Patent No. 4,201,672), asymmetric microporous membranes (U.S. Patent No. 4,376,046), oscillating pressure waves (U.S. Patent Nos. 4,518,499; 4,526,677; British Patent No. 485,553), or mechanical vibrations (French Patent No. 1,145,263), none of these were intended to enhance small-volume, biochemical reactions but rather were intended for use in high-volume industrial procedures explicitly to unclog industrial filters (U.S. Patent Nos. 4,201,672; 4,376,046; 4,518,499; 4,526,677; British patent No. 485,553; French Patent No. 1,145,263) or to create a forward diffusional bias for enhancing the circulation of heat-exchange fluids (U.S. Patent No. 4,376,046). The present invention thus represents a novel departure from previous technologies and advances the art for biochemical and immunological micromethods.

Objects of the Invention One object of the present invention is to provide a mixing device for chemical, biochemical, and immunological tests in which the fluid contents of a single sample or a large number of samples may be mixed simultaneously. Another object of the invention is to provide a device for mixing icroliter or larger volumes by using varying vacuum pressures, thereby providing effective mixing without introducing mechanical, magnetic, or like entities into the reaction medium. Yet another object of the invention is to provide an efficient system for mixing fluid-phase components alone or combined with entities such as, but not restricted to, microspheres, beads, and whole cells. ■j Another object is to enable several sequential

2 test steps to be mixed without removal of the components

3 from the reaction wells between test operations.

4 A further object of the invention is to provide a mixing method in which large numbers of test samples may 5 be processed without the delays occasioned by introduction 7 of mechanical, magnetic, or like stirring implements or g transference of the reaction mixtures to other vessels for 9 mixing, thereby minimizing the handling and the chance of 0 contamination of reaction mixtures. Another object is to provide a simple, - inexpensive, and effective device in place of costly, ^• _|_3 ineffective, and cumbersome laboratory equipment currently ■_j_4 required to perform the tasks described above.

15

15 Summary of the Invention _j The device and method described herein were

•_jo developed to overcome the problems inherent in attaining in efficient biochemical and immunological reactions using

2o small reaction volumes, though the device and method are

2i also applicable to procedures using larger volumes.

22 The device and method modify the function of

23 prior-art single reaction wells or manifold vacuum devices

24 which incorporate a relatively low-resistance port. Such a

25 port enables the reaction fluid to remain within the 2 reaction wells under normal atmospheric conditions. The 27 fluid within the wells can be drawn out of the wells 23 through the ports when vacuum is applied across the port. 2g Such prior-art devices include, but are not limited to, 30 those disclosed in Cleveland U.S. Patent 4,427,415 of 2i January 1984; Fernwood and Burd, U.S. Patent 4,493,815, of

32 January 1985; Klovsky and Hendriσk, U.S. Patent 4,526,690,

3 of July 1985; and Watkins and Wertz U.S. Patent

3 Application Ser. No. 856,647, filed April 25, 1986.

3 The present invention cyclically modifies the 3g strength of the vacuum applied across the base of the 37 single reaction well or manifold. Thereby, the fluids are

38 not voided from the reaction wells but are, rather, repetitively and gently drawn and released by the applied vacuum. The result is a churning and mixing effect of the fluids. This mixing effect can be attained by a variety of means including, but not limited to, (1) a piston-driven or similar device which cyclically decreases or occludes . the effective bore diameter of the tubing connecting the vacuum pump to the vacuum chamber underlying the single well or the manifold plate, or (2) a piston-driven or similar device which has the capability of providing both vacuum and positive pressure, in an alternating fashion, to the tubing connecting the device to the vacuum chamber underlying the single well or manifold plate. This invention is applicable whenever reagents are in a fluid phase or are suspended in or are in contact with a fluid phase. It includes applications such as immunoassays employing enzyme-labeled antibodies, direct radioimmunoassays, indirect radioimmunoassays, competitive inhibition immunoassays, immunoassays employing fluorescently labeled antibodies, other binding agents (such as staphylococcus aureus protein A) or antigens, and reagents bound to entities such as filters, bioaffinity membranes, beads, microspheres, or the walls of a reaction well.

Brief Description of the Drawings Fig. 1 is a schematic representation of a system embodying the principles of the invention, by which a motor-driven piston is used cyclically to interrupt the effect of a vacuum pump on a manifold plate by physically compressing or occluding the tubing forming the vacuum line. 1 Fig. 2 is a schematic representation of the

2 sequential' events which occur during each cycle of the

3 piston in Fig. 1, relating to the compression of the

4 vacuum line and the corresponding position of the piston

5 in its stroke-cycle.

6

7 Fig. 3 is a graph vertically aligned with Fig. 2,

Q providing a representation of the cyclical change in g vacuum across the stroke cycle in Figs. 1 and 2.

10

11 Fig. 4 is a schematic representation of a modified

12 form of a system embodying the principles of the invention T wherein a motor-drive piston interrupts the vacuum path.

14 ιr Fig. 5 is a view like Fig. 2 and vertically ig aligned with Fig. 3, but related to_^Fig. -4.

17

•ro Fig. 6 is a schematic representation, vertically

■jo aligned with Fig. 3, illustrating the cyclic fluid

2o movement which occurs in the reaction wells in Figs. 2 and

2τ_ 5. The view shows a well in cross-section throughout each

22 stroke-cycle.

23

24 Fig. 7. is a schematic representation of another

25 system embodying the principles of the invention in which 2g a motor-driven piston provides alternating pumping and 2 vacuum action. When the one-way valve is set in the closed 2g position, the motor-driven piston provides the vacuum 2_Q required to void the reaction wells as well as an n alternating vacuum and pumping action to mix the fluid in 3-. the wells. In contrast, when the one-way valve is allowed 32 to function, it provides a low-resistance pathway for 3 pumped air, thereby effectively providing only vacuum to

34 the manifold. 35 36 37

38 Fig. 8 is a schematic representation illustrating the pattern of air flow n the system of Fig. 7, where the pathway is not blocked, at various stages of the stroke-cycle.

Fig. 9 is a similar view illustrating the pattern of pressure changes occurring during the stroke-cycle of Fig. 7 where the pathway is blocked.

Fig. 10 is a partly diagrammatic perspective view of a modified form of the invention employing a bellows instead of a cylinder and piston.

Detailed Description of the Preferred Embodiment. Referring to the drawings. Figs. 1 and 4 illustrate two related but different systems in which the fluid in a single well or numerous wells of a manifold can be mixed by motor-driven piston devices. In Fig. 1, a manifold plate and vacuum chamber 10 — having a large number of individual wells — is connected by a vacuum line 11 to a vacuum pump 12. The line 11, which comprises an elastomeric tube, passes through a cylinder 13 that may be denominated a piston uide cylinder. For this purpose, the cylinder 13 may have diametrically opposite openings 14 and 15 through its cylindrical walls. A piston 16 is reciprocated in the cylinder 13 by a motor 17. ^BY such a system, when the piston motor 17 drives the piston 16 through its cycle, the piston 16 cyclically compresses and thereby occludes the vacuum line 11. This cyclical compression serves to vary continuously the strength of the vacuum produced by the vacuum pump 12 and applied across the base of a manifold plate through the vacuum chamber 10. Fig. 2 illustrates the effect of the piston 16 on the diameter of the vacuum line 11 and consequently on the strength of the vacuum drawn through this line. Fig. 3 shows how the pattern of pressure on the vacuum line, illustrated in Fig. 2 , varies the pressure within the vacuum line. Fig. 4 schematically illustrates a modified form of design wherein the piston 16 driven by the motor 17 cyclically interrupts the vacuum generated by the vacuum pump 12 for application to the base of the manifold plate through the vacuum chamber 10. In this version, a vacuum line 20 leads by a sealed end through an opening 21 into a piston guide cylinder 22. Another opening 23 is sealed to a conduit 24 that goes to the vacuum pump 12. In this manner, the air within the piston guide cylinder 22 actually becomes functionally part of the vacuum path. As the piston 16 cyclically moves between the ends of the two vacuum lines 20 and 24, it thereby effectively interrupts the air flow and then reinstates it. Fig. 5 illustrates, the effect of the piston 16 of Fig. 4 on the vacuum being carried through the vacuum line 24 at various points in the stroke cycle. Fig. 6 illustrates the effect of either of these two devices, — that of Figs. 1-3 and that of Figs. 4 and 5, — on the fluid volume of wells 25 within the manifold. Although a cross-section of only a single well 25 is shown, the effect upon all the wells is the same. As can be seen from this illustration, liquid 26 is cyclically drawn down and then released as the piston 16 follows through its stroke cycle. Fig. 7 shows an alternative approach to mixing fluids in a single well or in a manifold tray. Here, a piston guide cylinder 30 includ-es two ports shown as being through the cylinder head 31: one port 32 (with no valve) which leads via tubing 33 to the manifold 10, while a second port 34 with a one-way valve 35 is connected to a vent 36 provides a low resistance exit for pumped air, relative the resistance of the first port 32. Although 1 there are multiple ways to design the details of the valve

2 systems, all such designs basically are the same way. The

3 piston 16 and motor 17 act as a pump.

4 Whether the pump 16, 17 acts as a vacuum pump

5 (that is, where no positive pressure is delivered to the manifold 10) or whether the pump acts to oscillate the 7 pressure delivered to the manifold 10 (that is, positive g and negative pressure are alternately delivered) , depends a upon the patency of the air flow through the one-way valve

1 35. If this pathway is blocked (by clamping, valving, or

1 otherwise reversibly stopping air flow) , then oscillating

12 pressure is delivered to the manifold 10 as in Fig. 9. If 3 this pathway is not blocked, then only vacuum is applied •_jA across the manifold 10, as in Fig. 8.

•te The effectiveness of the pulse pump was- tested ig studying the rate of alkaline phosphatase activity on a iη para-nitrophenol substrate. The following demonstrates a

-to significant increase of the optical density with the pulse

1 pump and an essentially flat response without it.

20

21 P.P. at 30 sec. P.P. at 1.5 in.

22 With Pulse Pump .474 .547

2 Without Pulse Pump .066 .065

24

25 Fig. 10 shows a modified form of the invention

2g using a bellows 40 instead of a cylinder and piston. The

27 bellows 40 is sealed to a fixed upper end plate 41 with

2 one-way valve 42 and is seated to a movable lower end

2Q plate 43. The lower end plate 43 is connected to a piston

3Q rod 44, which is reciprocated by a pump 45. The airtight i interior of the bellows 40 is connected by a conduit 46 to

22 a waste fluid trap bottle 47, which, in turn, is connected n by a conduit 48 to a vacuum chamber 49 below a microtiter

₃, plate 50.

3₅ The stroke length of the rod 44 is adjusted by a g control 51; the stroke rate is adjusted by a control 52,

27 and there is an on-off switch 53. The stroke rate and

38 volume are dependent upon the amount of dead space contained in the conduits 46 and 48, the volume of the waste fluid trap bottle 47, and the volume of the vacuum chamber 49, as well as upon the size of the bellows 40 and its contained chamber.

Examples of applications of the oscillating vacuum pump Studies using the oscillating vacuum pump for microtube immunoassays preferably involve first binding cellular antigen to glass microfibers in the following manner.

Glass fiber binding Preparation of the antigen bound glass microfibers involves 4 stages: (a) preparation of the glass microfibers, (b) preparation of the isolated cell membranes, (c) coupling of the isolated cell membranes to the glass microfibers followed by blocking of any remaining binding sites, and (d) dispensing and storage of the bound glass. (a) The glass microfibers are prepared by first exposing them to 50% (v/v) HCl at room temperature for an hour and then rinsing them with distilled water to clean the glass surfaces. The cleaning exposes surface hydroxyls (that is, silanols) and negatively charged groups resulting from the presence of boron in the glass (Lewis acid sites) ; the silanols and Lewis acid sites are the entities allowing the coupling process to occur. Following acid cleaning, the glass microfibers are preferably broken into short lengths, as by using an electric blender. (b) In preparation of the isolated cell membranes, the primary considerations focus upon maintenance of the integrity and reactivity of the antigen sites, as well as removal of cell components (such as hemoglobin and σytoplasmic proteins) which would interfere with the coupling of the cell membranes to the prepared glass microfibers. For red blood cells, the procedure may comprise lysing with a 1% LAS-10 mM PBS (pH 7.2) solution followed by a series of incubations in 10 mM PBS (pH 7.2) at room temperature. Between successive incubations, the solutions are centrifuged at 12,000 g for 30 min. , and the supernatant is decanted and discarded. (c) Coupling and blocking. It is desirable to form a negative-positive-negative "sandwich", wherein positively charged polyvalent amino acids serve to link the negatively charged glass microfibers to the negatively charged cell membrane fragments. To achieve this, acid cleaned glass may be exposed to 0.1 mg/ml poly-lysine (30,000-70,000 MW) at room temperature for 15 minutes. Following distilled water rinses, isolated cell membranes are then added and bound by centrifugation. Any remaining unbound sites on the glass are bound to prevent nonspecific binding of reagents during immunological testing. Blocking may be readily accomplished by addition of 5% nonfat dried milk dissolved in 10 mM PBS (pH 7.2) followed by centrifugation. Milk is an excellent blocker, due to its cost effectiveness, stability, and high content of small proteins (primarily caseins and lactoglobulin with MW of 20-30,000 daltons) which are known to be good blockers of nonspecific binding by reagents such as antibodies. (d) Upon completion of the three preceding steps, the bound glass readily lends itself to being dispensed by pipetting. A desired amount of glass microfibers is simply added to a specially modified microtiter tray, as described above and allowed to air dry, to form filters in the bases of each reaction well. To date, these antigen-bound filters exhibit no noticeable loss of antigenicity following six months storage at room temperature and humidity. Once the antigen-bound filters are formed, they may be reacted with patients' sera to detect the presence of immunologic binding. This may be performed as an enzyme immunoassay as follows: Enzyme immunoassay The dried antigen-bound glass microfiber filters are preferably first rehydrated by addition of a drop of a wash buffer (10 mM diethanolamine, DEA; pH 7.3 containing both 10 μl Antifoam A (Sigma) and 1 gm nonfat dried milk/ 100 ml DEA; 0.01% thimerosal may be added to the wash solution if long term storage is desired) . The rehydrated filters may then be exposed to 5 μl of serum in 45 μl 1% nonfat dried milk-10 mM PBS (pH 7.3) for 3-5 minutes. Following a wash cycle, 50 μl of a 1:100 dilution of the appropriate enzyme-linked secondary antibody (either anti-human IgG and/or IgM conjugated to alkaline phosphatase) in 1% nonfat dried milk - 10 mM DEA (pH 7.3; with MgCl added to stabilize the enzyme) may be added and incubated for 3-5 min. Following a wash cycle, a unique combination of substrate reagents is preferably added; unlike other presently available reagents, this unique combination rapidly forms a highly visible purple precipitate upon contact with the alkaline phosphatase conjugate. The substrate may comprise 50 μl nitro blue tetrazolium (NBT) salt solution (Kirkegarrd & Perry Labs) containing MgCl followed immediately by 50 μ 1 of equal parts diethanolamine (1M, pH 12) and pnpp solution. Using this substrate with oscillating vacuum/pump pressures the reaction product is visible in the fluid within 15-30 sec and yields a precipitate of large enough granular size to be readily trapped by the glass microfiber filter within 1-3 min. The reaction is stopped by addition of pH 7.3 DEA which both washes out the unreacted substrate and shifts the pH away from the reaction optimum. Upon completion of the reaction, the filters can be easily removed from the reaction wells and stored as a permanent record of the reaction result. Extremely small volumes are used so to enhance the speed and sensitivity of the reactions the oscillating vacuum/pump device described herein is employed. It serves as the vacuum source for evacuating the reaction wells to terminate each step of the enzyme-linked immunoassay procedure. It also provides efficient washing of the intermediate reaction products. Additionally, the prototype device provides an oscillating pull (vacuum) and push (pump) force across permeable bases of the reaction chambers. These oscillating forces act to repetitively draw and release the reaction fluids contained within the chambers, thereby effectively churning and mixing the contents. By this system, a single electronically driven piston device provides all of the advantages of the prior vacuum pumps and also enhances the speed and sensitivity of the reactions, as compared to other currently available techniques. The reactants are repetitively drawn through the antigen-bound glass microfiber filter, thereby providing intimate contact between the reactants and obviating the prior problems of end-product build-up at enzyme reactive sites. The bellows or piston and cylinder system may operate at a stroke frequency of between a half and two seconds, and the stroke volume may be 15 to 20 cubic inches. However, the stroke frequency and volume are very dependent on the total air volume in the system. The above methods have been applied to the detection of both red blood cell (RBC) and white blood cell (WBC) antibodies, using donor cells as sources of cell membrane antigens and both commercial and donor/patient sera with confirmed antibodies as the primary antibodies in the enzyme-linked immunoassay. The complete system employing the oscillating pump/vacuum has been used for many things. 1. It has been used to: (a) bind Anti-A to A and AB cell membranes, without binding the Anti-A to B or 0. (b) bind Anti-B to B and AB cell membranes, without binding to A or .0. (σ) bind Anti-AB to A, B, and AB cell membranes, without binding to 0. 2. in using the system, (a) Anti-human IgM, but not IgG, recognizes donor/patient sera containing antibodies directed against the ABO system. (b) Anti-human IgG, but not IgM, recognizes sera containing antibodies directed against the D antigen (other Rh antigens/antibodies remain to be tested) . 3. It has been found that Anti-A, Anti-B, Anti-AB and Anti-human IgM do not bind to control filters (bound with milk proteins but not cell membranes) , the microtiter plate walls or the spun-bounded polyester base filter. 4. Glass microfibers bound with cell membrane A or B antigens, have been air-dried, and stored at room temperature and humidity maintains its reactivity with no noticeable loss of sensitivity for at least 6 months. Parallel studies relate to the Rh system. 5. For the ABO system (and preliminary studies of the Rh system concur) , between 1,200 and 1,500 antigen-bound glass microfiber filters can be produced from 1 ml of cell membrane fragments without loss of immunoassay sensitivity. 6. For the ABO system, the sensitivity of the immunoassay appears to be at least 8 times greater than standard test tube agglutination procedures. 7. For the ABO and Rh systems, icrofiber-bound antigens are correctly detected/identified following a 3 minute exposure to primary antibody, a 3 minute exposure to second antibody and a 15-30 seconds exposure to the alkaline phosphatase substrates. The speed attained is directly due to the use of the novel oscillating vacuum/pump device which brings the antibodies into intimate contact with the bound antigen sites. 8. All Rh antibodies tested have been correctly identified. Anti-D, Anti-C, and Anti-E commercial antisera correctly detect homozygous and heterozygous corresponding antigens compared to homozygous negatives and milk-bound control filters. Anti-D and Anti-E donor sera also bind to their corresponding antigens relative to homozygous negatives and milk-bound control filters. 9. Initial studies using human leukocyte antigen (HLA) A2 on white blood cells bound to the glass microfibers versus anti-HLA A2 antibody have yielded appropriate positive results, while control serum devoid of these antibodies have produced the expected negative results. Application of the system for quickly detecting HLA antibodies is particularly significant, since the current prior-art procedure takes two days. To those skilled in the art to which this invention relates, many changes in construction and widely differing embodiments and applications of the invention will suggest themselves without departing from the spirit and scope of the invention. The disclosures and the descriptions herein are purely illustrative and are not intended to be in any sense limiting.

What is claimed is:

^•

Claims

- 17 -

1 1. A method for facilitating mixing and reactions

2 of chemicals, comprising the steps of

3 inserting chemicals to be mixed in a liquid in a

4 well having a lower end which is liquid permeable,

5 retaining said well generally vertically with its g lower end in a vacuum chamber, and

7 alternating the pressure in said chamber between vacuum and non-vacuum conditions.

9 1_Q 2. The method of claim 1 wherein said alternating

21 step comprises

12 periodically connecting and disconnecting said

^■ _j_3 vacuum chamber to a vacuum pump.

14

■_j_ 3. The method of claim 2 whereby said connecting

1_Q and disconnecting step comprises

•_j_7 connecting said chamber to said pump by a conduit

1_Q and blocking off said conduit to do said disconnecting.

19

20 ⁴« ^τhe method of claim 3 wherein said conduit is

2 flexible and said blocking off is done by pinching said

22 conduit closed.

23

24 5. The method of claim 4 wherein said pinching is

25 done by moving a piston cyclically against and away from 2 the exterior of said conduit.

27

28 ⁶« The method of claim 3 wherein said blocking off

2<j is done by interposing a chamber in said conduit so as to

3Q provide two conduit portions connected by said chamber and

32 moving a piston cyclically to isolate said conduit

32 portions and reconnect them.

33 34 35 36 37 38 2 7. The method of claim 2 wherein said connecting

2 and disconnecting is done by driving a piston type vacuum

3 pump via a pump chamber and a conduit leading from said

4 vacuum chamber to said pump chamber and cyclically venting said pump chamber and reclosing it.

6

7 8. The method of claim 1 wherein said alternating

8 stop comprises: compressing and expanding a bellows type of pump 0 that is connected to said vacuum chamber by a conduit. 1 2 ⁹« Apparatus for mixing chemicals in a liquid, 3 including in combination: 4 a vacuum-tight chamber, 5 a well for said chemicals and liquid, having a 5 lower fluid permeable end retained in said chamber, and 7 pump means for applying positive pressure and 8 vacuum alternately and rapidly to said chamber, so as to g mix the chemicals together. 0 i 10. The apparatus of claim 9 wherein said pump 2 means comprises a vacuum pump, a conduit connecting said vacuum pump to said 5 chamber, 5 a piston guide cylinder, through which said tube passes, 8 ^a piston movable in said cylinder to close off and 9 reopen said conduit, and 0 motor means moving said piston cyclically. 1 2 11* The apparatus of claim 10 wherein said conduit 3 is flexible and said piston acts to close off and reopen 4 said conduit by pinching said conduit closed and releasing 5 ^{i t}' 6 7 8 12. The apparatus of claim 10 wherein said conduit -has two portions and said chamber is interposed between them, said piston cyclically moving to isolate said conduit portions and reconnect them.

13. The apparatus of claim 10 wherein said conduit is located in said cylinder head and leads from said vacuum chamber to said cylinder, said pump compressing said piston, motor and cylinder and said cylinder head has valve means for cylindrically venting said cylinder head to atmosphere.

14. The apparatus of claim 10 having a series of said wells retained in said chamber.

15. The apparatus of claim 9 wherein said pump means comprises: a sealed bellows, conduit means connected to said sealed bellows and to said chamber, bellows actuating means for compressing and expanding said bellows, and motor means moving said bellows cyclically.

16. The apparatus of claim 15 wherein said conduit means includes a waste fluid trap bottle.

17. The apparatus of claim 15 having means for adjusting the stroke length of said bellows compression air expansion and means for adjusting the stroke rate.

18• The apparatus of claim 15 having a series of said wells sharing a common said vacuum chamber. 19. A method of preparing a special microtiter filter apparatus from a microtiter tray having wells with fluid permeable lower ends, comprising preparing glass microfibers by treating them with an aqueous solution of acid in water for about one hour and rinsing them with H₂0 to produce negatively charged glass microfibers, breaking the prepared glass microfibers into short lengths, preparing negatively charged isolated cell membrane fragments by removing from cell membranes having antigen sites those cell components that tend to interfere with coupling of the cell membranes to said prepared glass microfibers while maintaining the integrity and reactivity of the antigen sites of said cell membranes, binding the negatively charged glass microfibers to the negatively charged cell membrane fragments through positively charged polyvalent amino acids, blocking unbound sites on the glass microfibers to prevent non-specific binding of latet-to-be added reagents, drying the resulting said • glass microfibers ^ι situ in the wells of the microtiter tray to form filters at the lower end of each said fluid-permeable well and storing the resulting dry microtiter trays.

20. The method of claim 19 wherein the preparation of the isolated cell membranes relates to the preparation of red blood cells and is done by lysing the blood cells and following that by room temperature incubation.

21. The method of claim 20 in which there is a series of room temperature incubations, comprising centrifuging the solutions between successive incubations and discarding the supernatant. 22. The method of claim 19 wherein said binding is done by exposing the prepared glass fibers to a dilute poly-lysine solution in which the poly-lysine has a molecular weight between 30,000 and 70,000, rinsing the material with distilled water, then adding the isolated cell membranes, and then centrifuging the materials to provide the binding.

23. The method of claim 22 wherein the blocking is accomplished by adding 5% nonfat dried milk dissolved in 10 PBS and then centrifuging the mixture.

24. A method for enzyme immunoassay employing the microtiter tray resulting from the method of claim 19, comprising rehydrating the dried antigen-bound glass fiber microfilters of said trays, exposing said rehydrated filters to patient's serum dilution for about three to five minutes, rinsing the filters, adding an appropriate enzyme linked secondary antibody and exposing the filters to it for about three to five minutes, rinsing the filters, reacting a substrate reagent with the material in said filter, alternating the pressure in the well between vacuum and non-vacuum conditions to assure good mixing, and drying the resultant filters.

25. The method of claim 24 wherein said substrate reagent comprises equal parts of (1) nitro blue tetrazolium salt solution containing magnesium chloride and (2) equal parts of diethanolamine and a pnpp substrate solution.