AU651617B2 - Disposable reactor vessel for solid-phase immune assays and process for measuring components detectable by immune reactions - Google Patents

Abstract

A disposable reactor vessel for solid-phase immune assays is easy to handle and can be universally used in the framework of known processes for quantifying immunologically reactive components. The disposable reactor vessel is ready to use, without the need for previous calibration or regeneration of the substrate it contains with the immunologically reactive components. The disposable reactor vessels can be used to carry out sample assays manually or entirely automatically.

Description

OPI DATE 15/04/92 APPLN. TD P'4237 91 AOJP DATE 28/05/92 PCT NUMBER PCT/DE91/00701 INTI BER DIE (51) Internationale Pateptklassiflkation 5 (11) Internationale Ver~iffentlichungsnummer: WO 92/05442 G01N 33/543, 36/00O Al (43) Internationales Veriiffentlichungsdatum: 2. April 1992 (02.04.92) (21) Internationales Aktenzeichen: PCT/DE9I/00701 (81) Bestimmungsstaaten: AT (europgisches Patent), AU, BE (europaisches Patent), CH (europ~isches Patent), DE (22) Internationales Anmeldedatumn: (europaisches Patent), DK (europaisches Patent), ES 2. September 1991 (02.09.91) (europiiisches Patent), FR (europiiisches Patent), GB (europllsches Patent), GR (europaisches Patent), IT (europ~isches Patent), JP, LU (europaisches Patent), NL Priori titsdaten: (europdisches Patent), SE (europgisches Patent), US.

P 40 29460.9 17. September 1990 (17.09.90) DE P 4126 436.3 9. August 1991 (09.08.9 1) DE VeriiffentP; Mit internationalem Reclzerchenbericht.

(71) Anmeloer (fidr alle Bestimmungsstaaten ausser US): ABION ORG [DE/DE]; Schtitzenweg 2, D-8 174 Benediktbeuern

(DE).

(72) Erfinder; und6 51 (75) Erfinder/Anmelder (nur fur US): ERHARDT, Ursula [DE/l5 DEJ; Schtitzenweg 2, D-8 174 Benediktbeuern (DE).

(74) Anwfijte: BEZOLD, Gunter usw. Maximilianstr. 58, D- 8000 Mfinchen 22 (DE).

(54) Title: DISPOSABLE REACTOR VESSEL FOR SOLID-PHASE IMMUNE ASSAYS AND PROCESS FOR MEASU- RING COMPONENTS DETECTABLE BY IMMUNE REACTIONS (54) Bezeichnung: EINWEGREAKTIONSGEFXSS FOR DIE FESTPHASENIMMUNANALYTIK UND VERFARREN ZUR MESSUNG VON OBER IMMUNREAKTIONEN BESTIMMMBARE KOMPONENTEN 7 (57) Abstract A disposable reactor vessel for solid-phase immune assays is easy to handle and can be 5,8 MM universally used in the framework of known processes for quantifying immunologically reactive components. The disposable reactor vessel is ready to use, without the need for previous calibration or regeneration of the substrate it contains with the immunologically reactive components. The disposable reactor vessels can be used to carry out sample assays manually or en- -4 tirely automatically.

E

(57) Zusammenfassung c .m I1,0 tmm PDie vorliegende Erfindung betrifft emn Einwegreaktionsgefdg ffor die Festphasenimmun- 2o.

aayidas eine einfache Handhabung erlaubt und universell anwendbar ist im Rabmen von E 0 Enltk 0 a bekannten Verfahren zur quantitativen Bestimmung von immunologisch reaktiven Komponen- 3 8 ten. Das Einwegreaktionsgefggl ist zu sofortigemn Gebrauch ohne vorherige Kalibrierung oder .0V Regenerierung des darin befindlichen Tr~germaterials mit der immunologischen Reaktivkom- -2b ponente geeignet. Das Einwegreaktionsgefagj eignet sich zur Durchfiihrung manueller wie auch 4,mm vollautomatisierter Probenanalysen.

mm 17,0 mm c2~ZE mm Disposable Reactor Vessel for Solid-Phase Immunoassays and Process for Measuring Components Detectable by Immunoreactions Abstract A disposable reactor vessel for solid-phase immunoassays is easy to handle and can be universally used in the framework of known processes for quantifying immunologically reactive components. The disposable reactor vessel is ready to use, without the need for previous calibration or regeneration of the substrate it contains with the immunologically reactive components. The disposable reactor vessels can be used to carry out sample assays manually or entirely automatically.

Description A disposable reactor vessel for solid-phase immunoanalysis is described as well as methods for its use.

Affinity chromatography is a technology which is broadly applied in preparative purification of bio-molecules. Therein, advantage is taken of a specific interaction between the molecule to be determined and a complementary binding partner. In the general practice of such methods, a sample containing the bio-molecule to be purified is applied to a chromatographic column, which contains one of the mutually complementary binding partners bound to a solid substrate. Examples of pairs of complementary binding partners are enzymes and their substrates, antibodies and hapten resp. antigen, and mutually complementary DNA or RNA single chains.

In immunoaffinity chromatography, for quantification use is made of the interaction between an antigen resp. a hapten and the complementary antibody.

In immunoassays using affinity chromatography up to now reusable columns are generally used which are packed with the antibody or antigen (named ligand in the following) bound to a solid substrate. The sample to be analyzed is pressed through the chromatographic column by high pressure to guarantee a rapid chromatographic processing. The application of high-pressure liquid chromatography (HPLC) for immunoassays is described e.g. by de Elvis, and Wilson, in "Analytical Chemistry", 1985, Vol. 57, pp. 2754-2756. and Sportsman. J.R. et al. in "Analytical Chemistry", 1983, Vol. 55, pp. 771-775. However, the use of HPLC for immunoassays has several drawbacks. On the one hand, the use of p. costly instruments is. necessary for this techniaue, and on the other hand the chromatographic columns for the analysis must meet high technical demands. The columns must withstand the high pressures applied in HPLC as well as they must be reusable to allow a rational performance of this technique. However, the reuse of the columns requires additional measures before a new sample can be analyzed on the same column. As published in DE-OS 37 11 894, reusable columns are regenerated prior to every analysis to prevent effects of displaced and incompletely eluted Substances of the previous test.

c i i- 2 in the DE-OS 24 48 411 a reaction vessel is described for solidphase immunoanalysis. However, prior to a quantitative determination, this vessel for multiple use requires a calibration with standards of the component to be determined. Besides long incubation times are necessary in this reaction vessel. 6 hours to 2 days are recommended, to guarantee a sufficient binding of the component to be determined to the immunoloqicallv complementary bindinq partner on the substrate.'However, the binding reaction is always terminated before che binding equilibrium is reached, which takes 1-2 days, to guarantee rational performance. The requirement of calibration with standard solutions is also a consequence of this early termination of the binding reaction.

The object of the present invention is therefore to provide a disposable reaction vessel for the solid-phase immunoanalysis which allows the rapid performance of an immunoassay, which is easy to handle, and which is ready to use without prior calibration or regeneration.

This is accomplished in this invention by a disposable reaction vessel for the solid-phase immunoanalysis which is open at the upper and lower end, containing two porous separating devices permeable for liquids, between which at least one immunologically reactive component is bound to a substrate, characterized by a substrate bed volume of about 600 and by determination of the liquid flow velocity by the permeability of the upper and/or lower separating device in combination with the substrate bed.

Besides, a method is described for the rapid performance of determinations of components which can be determined by immunoreactions.

This method is characterized by the application of the sample tu be analyzed to the described disposable reaction vessel, and by the retention of the component to be determined in the sample by the cu:mplementary reactive component in the disposable reaction vessel, whereby a binding equilibrium is achieved, The component to be determined is then a3) aluted and determined in a wellknown manner or b) marked with common immunological markers in the disposable reaction vessel in a wellknown manner or c) determined via a marked compound in reactive competition to the component to be determined.

1 -3 The invention is described in detail with relation to figures (1) and which show: Fig. la a longitudinal section of a disposable reaction vessel according to the invention containing two separating devices (2a) and between which the substrate with the immunologically reactive component is located, Fig. Ib a top view of the disposable reaction vessel and Fig. 2 a scheme of an online-system for antibody production control.

The dimensions given in figures (la) and (Ib) are for example only.

The described disposable reaction vessel and the described method, in which the disposable reaction vessel is used, exhibit several considerable advantages compared to knot'n reaction vessels resp. methods.

The described disposable reaction vessel allows a rapid performance of the quantitative determination of components which can be determined by immunoreactions. The handling of the reaction vessel is very simple and does not require the use of costly instruments such as HPLC apparatus. Besides excellent results are obtained with the aforesaid disposable reaction vessel at extremely low consumption of substrate with bound immunologically reactive component (named substrate bed in the following). A disposable reac+-'n vessel with a substrate bed volume i.e. the volum- etween the upper and lower separating device (2a) and of _J ,1l is preferably used. The small substrate bed volume allows'the use of small quantities of elution liquid with the consequence that the dilution effect of the elution remains small, and thus the detection limit is not decreased in contrast to other methods in which larger elution volumes are necessary.

Besides the aforesaid reaction vessel allows the quantitative determination at binding equilibrium (end-point determination) between the component to be determined and the immunologically reactive component. The binding equilibrium is reached within an incubation time of minute already in the aforesaid disposa.le reaction vessel The rapid equilibration is rendered possible by the upper and/or lower separating device (2a) resp. the small substrate bed volume and the high loading of the substrate with the immunologically reactive componenc. The upper and/or lower separating device causes, in connection with the selected substrate bed volume, a controlled flow. and the consequence of the high loading of the substrate with the reactive component is that the component to be determined is bound nearly qunatitatively to the reactive component within about 1 minute, i.e. before the flowing liquid leaves the substrate bed volume.

Therefore a comparative analysis with standard references is spared.

rr -v .i i i 4 Besides, a rapid analysis of samples is possible, because the disposable reaction vessel 1 allows immediate use, because the substrate 3 with the immunologically reactive component is standardizable and confectionable, and the disposable reaction vessel lymus. no be calibrated before use.

cdoes no+ne\ee C\Ce not- ne.A +c The substrate 3 mus-tRAtebe stable against pressure, because the disposable reaction vessel 1 allows the rapid analysis of samples without application of HPLC. As substrate, all common materials for affinity chromatography can be considered. Prefered substrate materials are polymeric sugars,Plastics, or silicates, an especially prefered material is agarose.

The immunologically reactive component applied to the substrate 3 can be bound to the substrate covalently as well as adsorptively.

The immunologically reactive component can be selected from the group including haptens, antigens, and .ntibodies. Polyclonal as well as monoclonal antibodies can be used.

In a prefered design of the disposable reaction vessel 1, the substrate 3 with the immunologically reactive component is located between two separating devices, and the upper and/or lower separating device 2a resp. 2b is a porous frit. The porosity of the upper and/or lower frit 2a resp. 2b is about 0.05 Im up to about 100 Am, a porosity of about 0.2/ 4m to about 100/m is prefered, a porosity of 2 /m is especially prefered. The materials of the frits 2a resp. 2b are selected from the group containing plastics, metals, and glass, plastics are prefered, polyethylene and teflon are especially prefered. In another prefered design the upper and the lower separating device 2a and 2b are two equal porous frits.

In another prefered design the layer thickness of the upper and/or lower frit is about 0.05 mm to about 2 mm, about 0.2 to about 2 mm are prefered, a layer thickness of about 1 mm is especially prefered.

coes no ree Vo The vessel material 4 must GRe-tbe stable against pressure, because the disposable reaction.vesse. is not subjected to hiqh pressures in the present process. Therefore besides pressureresistant materials such as metals, other not pressure-resistant materials can be considered for the production of the disposable reaction vessel 1 such as glass, natural materials, and plastics.

Prefered plastics are polyethylene, polypropylene, and/or polystyrene, and prefered metals are porous aluminum and stainless steel.

For practical purposes, the disposable reaction vessel 1 has a swelling 5 at one end to allow adaption of the reaction vessel into a holding and moving device for automatic transport. This is necessary for the use in automated sample processing devices.

9 -i -L 1 Besides for practical purposes. the disposable reaction vessel 1 has a smaller-diameter joining at the end opposite to the swelling, preferentially a joining for a socket-switch connection with another disposable reaction vessel. This facilitates e.g.

joining in series of several disposable reaction vessels 1, whereby the outlet 6 of the first vessel is connected with the inlet of the second vessel bya simple male-female connection.

Besides the disposable reaction vessel 1 is closable by caps 7 and 9.

The present 'procedure using the said disposable reaction vessel 1 is* strongly simplified comparea to common procedures, and allows a rapid quantitative and qualitative determination. This is enabled partly because the determination of the component to be determined does not require a preceding calibration or regeneration of the used substrate in the disposable reaction vessel I, and because no series reference measurements of standard solutions are necessary.

The easy handling is a consequence of the possibility, to apply samples and other solutions by usual laboratory equipment or with a pipetting automate.

Besidesmanual procedures, automated procedures are improved by application of the invented reaction vessel 1. In such procedures, the application of the sample and the further steps of the procedure are performed by an automated sample processing device.

Such a sample processing device is used for example in the online production process control of antibodies produced in a fermenter.

In such procedures the samples are automatically applied in time intervalls of about 6 to 8 hours, and the antibody concentration in the fermentation medium can be continuously determined rapidly and easily over a long time period using the disposable reaction vessel 1. A system for antibody production control using the invented disposable reaction vessel 1 is schematically described in fig. 2. Therein the disposable reaction vessel I is included in the automated sample processing device.

The present procedure can be performed rapidly without application of high pressures to the disposable reaction vessel I after sample application. However, low suction or pressure forces can be used for quicker performance. e.g. by centrifugation or pumping.

If necessary, in the present procedures several disposable reaction vessels 1 can be connected in series or parallel. This allows e.g. the simultaneous determination of several parameters in one single sample resp. the simultaneous analysis of several samples.

The connection of several disposable reaction vessels in series is advantageous e.g. for the performance of allergy tests. Therein, a different antigen (allergen) is applied to each different column, then the columns are connected in series, and the sample containing the antibody of the class IgE which mediates the allergic reaction is applied to the first reaction vessel.

The sample flows successively through the different disposable reaction vessels L containing the different allergens. The IgE of the sample will be bound in the reaction vessel which contains the allergy-evoking antigen, and can be detected e.g. via fluorescence detection. In this way, one single sample can be tested simultaneously with many different potential allergens.

The following examples describe the invention.

Example 1 Manual Performance of the Invented Procedure A disposable reaction vessel 1 made of polypropylene as shown in fig. 1 contained the following components: Between two frits 2a and 2b made of teflon (pore size about 2,Am, layer thickness about 1 mm) there are about 100/Al of a suspension containing commercialcyanobromide-activated cross-linked agarose with covalently bound polyclonal mouse anti-human IgG in 0.5 M NaCl. 100 /Al of the suspension give a final substrate bed volume 8 of 50 Al. The binding capacity per ml substrate bed is about 700 /Ag human IgG.

lOmM phosphate buffer pH 7.2, 150 mM NaCl was used as equilibration buffer 1 M propionic acid as elution buffer RPMI medium with 10% fetal calf serum (FCS) as cell culture medium (CM).

Firstly, 50 ,4l sample containing 0.5 "g to 50 /4g human IgG in CM were applied to the reaction vessel. It was washed with 1 ml EB, the eluate was discarded, and afterwards it was eluted with ml EL. The eluate was collected in a cuvette for fluorescence detection and measured in the detector. For the -quantitative determination of the sample content of human IgG to-be determined a calibration curve has been set up in advance. IgG concentrations from 0 /g/ml up to 1000 g/ml, preferentially from O0 g/ml to 100 /,Ag/ml, have been used for the set-up of the calibration curve, and there have been measured a) either the extinction in the UV detector in the range 250-300 nm, preferentially at 280 nm, b) or the relative fluorescence intensity in the fluorescence detector at an excitation wave length of 250-290 nm and an emission wave length larger than 320 nm.

If marker molecules are used the detection wave lengthswill have to be tuned to the marker. The measured value for the cell culture medium alone has been substracted from the value of the sample.

In the practical performance of the determination, the sample resp. eluate flow through the reaction vessel 1 can be caused by gravitation in the easiest case. For flow acceleration the following alternatives can be considered: 7 a) Pressure: This can be produced in the easiest case by use of a disposable syringe which is connected to the reaction vessel 1 by a luer-lock connection.

Besides, the pressure may be produced with other instruments such as a manually operated dispenser.

b) Vacuum: th._ an be produced by connection of a usual suction pump to the outlet of the reaction vessel.

c) Centrifugal Force: In this variation, the sample, washing, and elution liquid can be brought e.g. into S' Iseparated chambers having predetermined breaking points, and upon application of an appropriate centrifugal force, the liquids flow through the predetermined breaking points into the reaction vessel at a given centrifugal force.

Example 2 Fully Automated Performance of the Invented Procedures The used apparatus was made up of the following components: an HPLC sampler (A]BIMED ASPEC system), disposable reaction vessels 1, a fluorescence or a UV detector with a flow cell and a computer for data processing.

The analysis was performed as follows: The sampling from the cell culture was performed by common sampling systems. After separation of the cells via filters (in the ASPEC system) and positioning of the rack with the disposable reaction vessels 1 in the waste |i positico the sample was applied, the reaction vessel was washed EB, and the rack with the reaction vessels was positioned xn the elution position. The elution of the component to be determined was done in one step. The component to be determined was transferred in the elution solution to the flow detector and was measured. The used volumes correspond to the volumes in example 1.

f 4'

Claims (28)

1. Disposable reaction vessel for solid-phase immuno analysis having an open upper and lower end and containing two porous separating devices which are permeable for the liquids containing the component to be determined, between which there is at least one immunologically reactive component applied to a substrate with such a loading density that the component to be determined is bound quantitatively to the reactive component before the flowing liquid leaves the substrate bed, the substrate bed volume being up to 600[Il, and the permeability of the upper and/or lower separating device controlling the flow velocity of the liquids.

2. Disposable reaction vessel according to claim 1, characterised by standardisation and confectionation of the substrate with the immunologically reactive component to allow immediate use of the disposable reaction vessel.

3. Disposable reaction vessel according to claim 1 or 2, characterised by the selection of the substrate material from the group containing polymeric sugars, plastics, or silicates.

4. Disposable reaction vessel according to claim 3, characterised by agarose as polymeric sugar.

Disposable reaction vessel according to claim 1, characterised by covalent or adsorptive binding of the immunologically reactive component to the substrate.

6. Disposable reaction vessel according to claim characterised by the selection of the immunologically reactive component from the group containing hapten, antigen, and antibody.

7. Disposable reaction vessel according to claim 6, characterised by a polyclonal antibody.

8. Disposable reaction vessel according to claim 6, characterised by a monoclonal antibody.

9. Disposable reaction vessel according to claim 1, I' S." I--I I- I 9- characterised by the use of a porous frit of a membrane for the upper and/or lower separating device.

Disposable reaction vessel according to claim 9, characterised by a frit with a porosity of 0.2pn to 100pm.

11. Disposable reaction vessel according to claim 9, characterised by a frit with a layer thickness of 0.05 mm to 2 mm.

12. Disposable reaction vessel according to claim 11, characterised by a layer thickness of 1 mm.

13. Disposable reaction vessel according to any one of claims 9 to 12, characterised by a frit made of plastics, metal, or glass.

14. Disposable reaction vessel according to claim 13, characterised by the use of polyethylene or teflon as plastic material and porous aluminium or stainless steel as metal.

Disposable reaction vessel according to any one of claims 1 to 14, characterised by the selection of the vessel material from the group containing plastics, metals, and natural materials.

16. Disposable reaction vessel according to claim characterized by the use of polyethylene, polypropylene, and/or polystyrene as plastic material.

17. Disposable reaction vessel according to any one of claims 1 to 16, characterised by a swelling at one end of the reaction vessel, which allows the adaption of the reaction vessel into holding and moving devices for automatic transport.

18. Disposable reaction vessel according to any one of claims 1 to 17, characterised by a joining with a r rr l" I -miil smaller diameter than the vessel diameter at the end opposite to the swelling (sI.

19. Disposable reaction vessel according to claim 18, characterized by the possibility of connecting the opposite ends of reaction vessels by male-female connections for in series connection of several disposable reaction vessels.

Disposable reaction vessel according to any one of claims 1 to 19, characterized by a substrate bed volume Syf of

21. Procedure for the determination of components which I I are determinable via an immuno reaction, characterized by the application of a sample to be analyzed to a disposable reaction vessel according to any one of claims 1 to 20, by the binding of the component in the sample to be determined by the complementary immunologically reactive component in the disposable reaction vessel, and by the determination of the cc:,:ponent to be determined. via elution and determination, or via marking by amplifying reactions in the disposable reaction vessel with immunological markers and determination, or via a compound competing with the compound to be determined.

22. Procedure according to claim 21, characterized by the determination without proceedingcalibration or regeneration cf the substrate and of the applied reactive component.

23. Procedure according to claim 21 or 22, characterized by the application of the sample and of the other solutions with common laboratory equipment or with a pipetting* automate.

24. Procedure according to claim 21 or 22, characterized by the use of an automated sample processing device for sample application and further process steps.

Procedure according to claim 24, characterized by automated sample application in an on-line process control system.

26. Procedure according to any one of claims 21 to oc- characterized by application of Emall suction o pressure forces to the disposable reaction vessel afte. sample application for more rapid performance.

27. Procedure according to any one of claims 21 to 26, characterized by serial or parallel arrangement of several disposable reaction vessels.

28. Procedure according to claim 27, characterized by its use for the determination of allergens. DATED THIS 6TH DAY OF JULY 1993 ABION OHG By Its Patent Attorneys GRIFFITH HACK CO Fellows Institute of Patent Attorneys of Australia I