Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
  • G01N35/08—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a stream of discrete samples flowing along a tube system, e.g. flow injection analysis
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
  • B01D61/14—Ultrafiltration; Microfiltration
  • B01D61/145—Ultrafiltration
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
  • B01D61/14—Ultrafiltration; Microfiltration
  • B01D61/147—Microfiltration
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D63/00—Apparatus in general for separation processes using semi-permeable membranes
  • B01D63/08—Flat membrane modules
  • B01D63/087—Single membrane modules
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
  • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
  • G01N21/255—Details, e.g. use of specially adapted sources, lighting or optical systems
• • 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
  • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
  • G01N33/521—Single-layer analytical elements

Definitions

• THIS INVENTION is concerned with improvements in and relating to continuous-flow, automatic, chemical analysis systems.
• the invention is especially concerned with modifications and improvements to components of continuous-flow analysis modules to enable them to effectively function in enzyme immunoassay systems and thereby to allow the construction of continuous flow analysis systems capable of: (a) The detection and quantification of antigens for which antibodies may be developed in animals by whatever means by continuous-flow, non-homogeneous enzyme immunoassay (NHEIA) methods.
• NHEIA non-homogeneous enzyme immunoassay
• the contiguous-flow analysis module of the invention involves the following novel components or improvements, each of which individually constitutes a separate aspect of the invention: - (1) A phototransistor-based colorimeter. (2) A solid state oil heating bath. (3) A phase separating system.
• red light blocking filter which blocks light of wavelength greater than about 660 nM in front of the phototransistor allows the development of a practical colorimenter utilizing low cost phototransistors as light transducers.
• the red light blocking filter is coloured blue.
• a blue filter consisting of a copper sulphate solution of appropriate colour density provides a highly efficient filter for this purpose.
• Conventional blue glass blocking filters are not generally satisfactor as they allow leakage of light at wavelengths below about 900 nM.
• the copper sulphate filter does not leak in this way and has an additional advantage in that the optical density of the filter can be easily adjusted by varying the concentration of the solution.
• the use of the copper sulphate filter allows -3- . - construction of a very low cost colorimeter when the photo ⁇ transistor is used as part of an appropriate electronic circuit,
• the colorimeter of the invention comprises a phototransistor, a light source of appropriate wavelength, a filter comprising a solution of copper sulphate interposed in the optical path between the source and the phototransistor, and a cell or curvette adapted to receive a sample liquid to be analysed also interposed or interposable in said optical path.
• the LED may be replaced by any other suitable light source, for example a tungsten lamp which emits a continuous spectrum. In this latter case, it may be desirable or necessary to provide further light filtration in the form of an isolation filter, which may be of the narrow- or broad-band type and which is selected according to the wavelength required for the particular analysis.
• Such a colorimeter may be used, for example, as the colorimeter in a continuous-flow, automatic chemical analysis system in which a continuous stream of the sample liquid passes through a flow-through type cell . More especially, the flow system involves the continuous passage through the cell of individual slugs of sample liquids separated by gas bubbles, which also pass through the color ⁇ imeter.
• the amplified output of the phototransistor when combined with electro! ic or computerised methods allows removal of the interfering electrical signals resulting from passage of gas bubbles through the colorimeter.
• the phototransistor may be connected to an appropriately designed electronic amplification device, the output of which is fed directly to an analogue computer system, or to a second electronic device designed to function as a peak sample and hold volt-meter.
• the output from this latter device may then be fed to a suitable computer device for further processing or may be fed directly to an appropriate chart or similar recorder.
• the computer device just referred to may consi for example, of a microprocessor holding appropriate programmed instructions to provide for the conversion of the electronic signals to a voltage signal proportional 5 to the amount of colour present in the colorimeter.
• the programmed instruction set might be organised, for example, to allow direct comparison of this colour signal with the concentration of a particular analyte measured in a continuous flow automatic chemical analysis system.
• This device comprised_ the combination of a meta preferably aluminium, containing vessel adapted to contain oil or any other suitable heat-transfer fluid, a heating circuit comprising one or more power transistors
• the transistors and/or resistors ar recessed into the external wall or walls of the vessel.
• Control of the temperature of the block and the oil is achieved with a thermistor or other like thermal electric sensor appropriately placed in the fluid bath which controls the energy supply to the heating circuit by means of suitably designed electrical or electronic
• Stirring to ensure even temperature in the fluid bath may be provided, for example, by means of a small electric motor driving a paddle wheel stirrer positioned to provide efficient stirring.
• tube of the required length is positioned in the path so as to be immersed in the heat-transfer fluid and thereby to allow heating of a continuously flowing liquid stream which is passed through the tube, e.g. by action of an appropriate pumping device.
• phase separating system. -5- The phase separating system according to this invention, is designed especially with a view to providing continuous flow separation of a non-homogeneous enzyme immunoassay solid phase from its liquid phase while still maintaining the integrity of the flowing streams necessary to allow the efficient performance of such immunoassays.
• the system is based on the continuous- flow dialyser modules known per se in the art. Briefly, such a module comprises a dialysis membrane clamped between two flat plates each having surface grooves in the opposed faces thereof which, with the membrane, constitute ducts for the passage of liquid. The ducts are thus superimposed and separated by the membrane to provide a dialysis cell.
• the dialysis membrane is replaced by a membrane filter, e.g. of the "Millipore" type, having a pore size appropriate to the particular application.
• a standard sterilizing filter having a pore size of about 0.2 microns is often appropriate.
• the usual arrangement is to pump the sample stream from the preceding stage of the system through one of the ducts and thence to waste.
• the recipient stream is pumped through the other duct from which it passes to the next stage, the two flows being synchronised.
• the sample stream after passage through the module and before discharge, is passed back through the pumping device with a pumping -6- volume selected by the user, thereby to maintain a closed system.
• Variation of the volume is achieved by, for example, varying the diameter of the pumping tube used in a peristaltic pump.
• a greater or lesser (as desired) amount of unbound enzyme-linked component from the non-homogeneous enzyme assay incubation mixture is passed across the filter to the recipient stream, in which the enzyme activity may then be subsequently measured.
• the sample stream rate is 1.0 ml/min and the return stream pumped at 0.5 ml/min. and the recipien stream entry flow rate is 0.5 ml/min
• the above mentioned closed system will ensure that the recipient stream exit flow rate is 1.0 ml/min thus ensuring passage of 0.5 ml/min of filtered fluid from the sample to the recipient stream.
• such a module may comprise a suitable container, typically a box, which houses one or more of the solid state heating devices and are more of the phototransistor colorimeters just described, together with their associated electronic circuitry, which may include an integral micro-processor. Also associated with these components will be the necessary plumbing for the hydraulic system which typically consists of one or more coiled glass tubes which in use are immersed in the heating bath(s) and one or more dialysis or phase separation modules, together with appropriate interconnecting tubing. Conveniently, these components may be mounted on or suspende from (as appropriate) the lid of the box for ease of access, removal or replacement.
• Such analysis module may then be utilized, in conjunction with the pumps and other anciliary equipment known per se in automatic analysis.
• the module may be used for a wide range of chemical analysis and the "bubble-through-the- colori eter" capabilities of the integral colorimeter provides for enhanced sample number throughput capability with this module.
• the following description shows the use of the module in a continuous-flow, non-homogeneous, enzyme immunoassay, specifically the analysis of dilantin in serum.
• An appropriate aliquot of serum is mixed with 1 ml of incubation mixture consisting of a drTantin-
• the continuously flowing stream at 1 ml per munute is segmented with air by an appropriate pumping device and is mixed on the surface of the module in a glass coil before entering a first heating bath at 37° for the time of passage of five minutes.
• the stream flows to the top side of a phase separation module containing a 0.2 micron illipore type filter and is then returned to the pumping device where 1 ml per minute of liquid continues to be pumped.
• An air segmented 1 ml per minute recipient stream of buffer is pumped on to the lower side of the phase separating device and on exit from this device is joined by a 1 ml per minute continuous stream of para-nitrophenyl galactoside in buffer..
• the stream is heated at 37° for five minutes in a second heating bath before the colour developed measured by passage through a colorimeter with a 405 nm filter.
• the electronic signal disturbances from passage of the air bubble through the colorimeter are removed by appropriate means and the resulting signal displayed on a chart recorder or alternatively handled by a computer system.
• A(i) An antigen to which has been covalently coupled an appropriate enzyme suitable for use in an NHEIA.
• A(ii) An antibody directed against the antigen in
• A(i) above which may be used directly, chemically modified for example to a Fab fragment, or covalently coupled to an appropriate solid phase e.g. a Sepharose particle.
• A(iii) An appropriately designed continuous flow NHEIA system module together with appropriate continuous flow analysis components.
• a suitable aliquot of the biological or other material containing the antigen required to be measured (or alternatively an appropriate standard containing a graduated known level of this same antigen) is mixed in a continuously flowing buffer stream containing suitable aliquots of A(i) and A(ii) above.
• a suitable incubation period under appropriate conditions during which the 1iqand-enzyme complex compete for binding sites on the antibody against possible ligand molecules derived from the sample, a portion of the unboun enzyme-1igand complex is separated on-line from the antibody. This separation is effected preferably by passa of unbound enzyme-1 igand complex across a porous membrane into a recipient stream.
• a solid phase of the coupled antibody enzyme-ligand derivatives is required. This may be obtained directly by previous coupling of the antibody to a solid phase, e.g. a Sepharose particle, or by subsequent addition to th flowing stream of a second antibody or binding material wh
• OMPI W1PO is directed against the first antibody and which is bound to a solid phase.
• the enzy e-ligand complex separated from the antibody-bound enzyme-ligand is then subjected to enzyme measurment techniques and this may include the use of the enzy ic cyclicising methods described in our copending Australian Patent Application no.
• the analysis of dilantin in serum may be carried out by mixing an appropriate aliquot of serum with 1 ml of an incubation mixture consisting of a dilantin- ⁇ -galactosidase derivative and Sepharose- bound anti-dilantin antibodies.
• the continuously flowing sample stream segmented with air by an appropriate pumping device is mixed, e.g. in a glass coil, before entering a heating coil at 37 for the time of passage of five minutes.
• the stream flows to a phase separation module containing a 0.2 micron millipore type filter and is then returned to the pumping device where 1 ml per minute, of liquid is discharged to waste.
• An air segmented 1 ml per minute recipient stream of buffer is pumped to the lower side of the phase separating device and on exit from this device is joined by a 1 ml per minute continuous stream of para-nitrophenyl galactoside in buffer.
• the stream is heated at 37° for five minutes before the colour developed measured by passage through a colorimeter with a 405 nm filter.
• the essential requirements are the following: B(i) A purified antibody directed against a particular ligand from whatever source to which is covalently
• B(ii) Appropriate components capable of binding to the antibody enzyme in B(i) above.
• This product must be capable of removal in a separation phase of a continuous flow non-homogeneous enzyme immunoassay syste and to this end the product may be linked chemically to a solid phase, for example, a Sepharose particle.
• a suitable aliquot of the biological o other material containing the suspected antibody or alternatively appropriate standard containing graduated known levels of this same antibody are mixed in a • continuously flowing buffer stream containing suitable aliquots of B(i) and B(ii) above, with for example, covalently bound Sepharose particles.
• a suitable incubation period in an appropriately designed module during which enzyme antibody competes to bind the solid phase ligand with possible antibodies in an aliquot of the biological or other material a portion of the unbou enzyme antibody is separated on-line from the enzyme antibody bound to the solid phase ligand by whatever means, for example by passage across a millipore-1ike membrane, into a recipient stream.
• the enzyme antibody so separated is measured by usual means, and this may include the use of the aforementioned enzymatic cyclic- i.zing methods.
• By comparison of the unknown enzyme rate with that resulting from known levels of antibodies quantification of the test levels of antibodies is achieved.
• the following components would be used in the above system.
• An appropriate subststrate for B-glactosidase together with cyclicizing enzymes for example, ⁇ -glactoside, p-catechol , laccase, and NADH.
• the essential require ⁇ ments of the performance of this system are the following: (i) A highly purified specific antibody directed against an antigen on the organism of interest and this may be covalently bound to a solid phase are directly orbound indirectly by a solid phase second antibody. (ii) An enzyme antigen complex which is specifically bound at high affinity to the antibody in (i) above. (iii) An appropriately designed continuous flow NHEIA system module together with appropriate continuous flow analyses components.
• a suitable aliquot of the biological or other material to be tested for the presence of the organism is sampled directly or following an initial pre-incubation ' step which may include incubation of -12- - :. _ - — aliquots of the test material in the presence of anti ⁇ biotics or other anti-growth factors directed against the organism in question.
• the aliquot or alternatively appropriate standards containing graduated known levels of the antigen are mixed in a continuously flowing buffer stream containing suitable aliquots of (i) and (ii) above. Following this, there is a suitable incubation period in an appropriately designed module during which ligand enzyme competes for binding sites on the antibody against possible ligand molecules derived from the sample portion.
• ligand molecules may be intact micro ⁇ organism particles or could be particles resulting from the on-line disruption of intact organism by whatever means.
• portion of the unbound enzyme ligand is separated across a porous membrane in an appropriate separation module and into a continuous-flow recipient stream.
• the unbound enzyme activity is then subsequently measured by established methods which may include the aforementioned enzyme cyclizing techniques.
• FIGURE 1 is an exploded view of the colorimeter of the invention
• FIGURE 2 shows the heating bath
• FIGURE 3 is the electronic heating circuit for the bath of FIGURE 3;
• FIGURE 4 shows the phase separation module
• FIGURE 5 shows the hydraulic circuit for the module of FIGURE 4 and the complete analysis module.
• the colorimeter shown diagram atically in FIGURE 1 comprises a block 1 of "Perspex" (or like material) 12 mm wide, 18 mm high, 15 mm long and is drilled horizontally through the centre with a hole 2 of 0.75 mm diameter. At angles from the top of the block 1 through to within 2 ram of this transverse hole 2, connecting holes 3, 4 are drilled at each end of the transverse hole. Connecting grooves 6, 7 are drilled on each surface to a depth of 0.45 mm to all connections between the horizontal and vertical hole.
• Grooves 8, 9 are milled outside the resulting holes at each end of the transverse hole to accept appropriate "0" rings 11, 12 and suitably shaped 1 mm thick glass end plates 13, 14 are compressed against the "0" rings by restraining blocks (not shown) to provide a leak-proof assembly.
• Suitable nipples (not shown) are fixed to the openings of the transverse holes (3, 4) on the top of the block.
• the assembly thus comprises a modified u-tube assembly.
• a suitable phototransistor 16 is positioned in an approp ⁇ riately aligned holder 17.
• a light emitting diode 18 or a tungsten bulb in a holder 19 together with a copper sulphate blocking filter 20 and, if required, an appropriately sized narrow band isolation filter 21 are positioned.
• the copper sulphate filter 20 consists of a hollow glass or plastic cell containing a copper sulphate solution.
• a stabilized electrical supply (not shown) is provided for the phototransistor or lamp 18 and the phototransistor 16 is connected to a suitable amplifier (not shown) the signal from which may be fed to a- recording or indicating device or to a computer for signal processing.
• the heating bath shown diagrammatically in -14- - V- " " ⁇ FIGURE 2 comprises an aluminium (or other suitable) metal cylinder (30) 75 mm in diameter and 100 mm in height having a milled central cavity 31 of 63 mm in diam ⁇ eter to a depth of 94 mm. In the outside wall of the cylinder at regular intervals are milled grooves 32, 33 to allow placement respectively of two ' power transistors 34 and three heating resistors 36.
• the bath is provided with a lid 37 which supports a small electric motor 38 driving a central paddle wheel stirrer 39.
• the lid is provided with a small hole to allow a thermistor (not shown) to dip into the oil as well as two holes to allow placement of tubed glass coils (e.g., 41) of 50 mm diameter and of varying total length, in the bath.
• Temperature regulation is provided by a solid state temperature controlling device which is capable of controlling the temperature in the oil contained in the bath over the range of ambient to 100° to an accuracy of 0.1°.
• the circuit of this device is shown in FIGURE 3.
• the phase separation module is shown in FIGURE 4. It consists of two 50 mm square blocks 50,51 of "Perspex” or other suitable material, between which is sandwiched a sheet 52 of membrane filter material, e.g., a "Millipore” filter of 0.2 micron pore size. Other membrane filters of dialysis membranes may of course be used.
• the inner faces 53, 54 of the blocks 51, 52 i.e. those faces which contact the membranes are provided with matching surface grooves 56, 57 which are basically U-shaped in plan. These grooves, which act as fluid passages in use, terminate at ports of passageways 58, 59, 61, 62 drilled between the opposed faces 53, 54 of the blocks and their respective end faces 63, 64. Fluid connections to the passageways 58 to 62 and hence -15- the grooves 56, 57 may be made by suitable nipples (not shown)
• FIGURE 5 The hydraulic circuit of the phase separation system and the configuration of the whole automatic analysis module is shown schematically in FIGURE 5. The operation of the module will be explained by reference to the dilantin assay already described.
• the module consists of all of the integers enclosed by the dashed line.
• the other integer is a peristatic pump 70 of standard type, which as shown here has 7 pumping lines A to G. Operation of the system is as follows:
• the incoming serum sample is pumped in through line B and is mixed with 1 ml of incubation mixture con- sisting of a dilantin-B-galactosidase derivative and
• Sepharose-bound, anti-dilantin antibodies pumped in through line C.
• the continuously flowing stream at 1 ml per minute is segmented with air pumped in at line D and is allowed to mix in a glass coil 71 before entering a first heating bath 72 at 37° for the time of passage of five minutes.
• the stream flows through to line H to the top of the phase separation module 73 and is then returned through line A to the pumping device where 1 ml per minute of liquid is discharged to waste.
• the stream is passed through a second mixing coil 74 and then to a second heating bath 76 where it is heated at 37° for five minutes.
• the stream then passes through the colorimeter 77 where the colour developed is measured using a 405 n filter.
• the electronic signal disturbances from passage of the air ⁇ - bubble through the colorimeter are removed (as already described) by appropriate means and the resulting signa displayed on a chart recorder or alternatively handled by a computer system.
• automati chemical analysis module may include any conventional colorimeter or water bath in place of the colorimeter a solid state heating bath specifically described above.

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• 1980
  • 1980-06-02 AT AT0903180A patent/AT377493B/de not_active IP Right Cessation
  • 1980-09-11 WO PCT/AU1980/000066 patent/WO1981000911A1/en not_active Application Discontinuation
  • 1980-09-11 JP JP50199780A patent/JPS56501383A/ja active Pending
  • 1980-09-11 AU AU63936/80A patent/AU522965B2/en not_active Ceased
• 1981
  • 1981-04-08 EP EP19800901707 patent/EP0036000A4/de not_active Withdrawn

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