Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/5302—Apparatus specially adapted for immunological test procedures
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
  • B01F31/00—Mixers with shaking, oscillating, or vibrating mechanisms
  • B01F31/29—Mixing by periodically deforming flexible tubular members through which the material is flowing
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
  • B01F31/00—Mixers with shaking, oscillating, or vibrating mechanisms
  • B01F31/65—Mixers with shaking, oscillating, or vibrating mechanisms the materials to be mixed being directly submitted to a pulsating movement, e.g. by means of an oscillating piston or air column

Definitions

• This invention relates to method and apparatus for facilitating reactions and mixing.
• 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.
• 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.
• microtiter reaction volumes 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
• new methods are required to achieve effective mixing of reagents in such biochemical and immunological micromethods.
• 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.
• 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
• 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, •
• 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
• 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.
• 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.
• Fig. 2 is a schematic representation of the
• Fig. 3 is a graph vertically aligned with Fig. 2,
• Fig. 4 is a schematic representation of a modified
• ⁇ r Fig. 5 is a view like Fig. 2 and vertically ig aligned with Fig. 3, but related to ⁇ Fig. -4.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• the air within the piston guide cylinder 22 actually becomes functionally part of the vacuum path.
• 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.
• 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.
• piston 16 and motor 17 act as a pump.
• Fig. 10 shows a modified form of the invention
• one-way valve 42 is seated to a movable lower end
• 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
• a waste fluid trap bottle 47 which, in turn, is connected n by a conduit 48 to a vacuum chamber 49 below a microtiter
• 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.
• 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.
• the glass microfibers are preferably broken into short lengths, as by using an electric blender.
• 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 ⁇ ytoplasmic proteins) which would interfere with the coupling of the cell membranes to the prepared glass microfibers.
• 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.
• 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.
• 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.
• 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 ⁇ l 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 ⁇ l of serum in 45 ⁇ l 1% nonfat dried milk-10 mM PBS (pH 7.3) for 3-5 minutes.
• a wash cycle 50 ⁇ l 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.
• 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 ⁇ l nitro blue tetrazolium (NBT) salt solution (Kirkegarrd & Perry Labs) containing MgCl followed immediately by 50 ⁇ 1 of equal parts diethanolamine (1M, pH 12) and pnpp solution.
• NBT nitro blue tetrazolium
• 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.
• the filters can be easily removed from the reaction wells and stored as a permanent record of the reaction result.
• the oscillating vacuum/pump device described herein 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.
• 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.
• 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.
• the sensitivity of the immunoassay appears to be at least 8 times greater than standard test tube agglutination procedures. 7.
• 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.
• 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.
• HLA human leukocyte antigen
• 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.

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• 1987
  • 1987-05-15 EP EP87903935A patent/EP0311615A1/de not_active Withdrawn
  • 1987-05-15 JP JP62503634A patent/JPH01501358A/ja active Pending
  • 1987-05-15 AU AU75808/87A patent/AU7580887A/en not_active Abandoned
  • 1987-05-15 WO PCT/US1987/001119 patent/WO1987007954A1/en not_active Application Discontinuation

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