GB1573627A

GB1573627A - Process and device for the determination and detection of a reaction component

Info

Publication number: GB1573627A
Application number: GB3901876A
Authority: GB
Original assignee: West P M Goldie D J
Current assignee: West P M Goldie D J
Priority date: 1976-09-18
Filing date: 1976-09-18
Publication date: 1980-08-28
Also published as: DE2736527A1; BE858790A

Description

(54) PROCESS AND DEVICE FOR THE DETERMINATION AND DETECTION OF A REACTION COMPONENT (71) We, PETER MICHAEL WEST, DAVID JOHN GOLDIE, AND ADEL ABBAS AHMED ISMAIL of 6 Yorks Gardens, Winterbourne, Bristol, 1 Radnor Road, Westburyon-Trym, Bristol, Bristol BS9 4DX, and 43 Old Sneed Road, Stoke Bishop, Bristol BS9 lES respectively, of British, British and Dual Egyptian/British nationality respectively, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:: The present invention is concerned with a process and device for the detection and determination of a component of the reaction between a specific binding protein and the corresponding bindable substance, utilising the binding affinity of these components for one another, in an automated system with a continuously flowing current of liquid, and is especially concerned with an immune test process.

The immune test processes, i.e. the radio-immune test (RIA), the enzyme-immune test (EIA) and the fluorescence-immune test (FIA) are used worldwide in clinical laboratories.

Although originally used in the field of endocrinology, these techniques are also employed, inter alia, in a wide variety of different fields, including immunology, oncology, clinical pharmacology, microbiology and haematology. Since antibodies can be produced with a strong affinity not only for immunogenic but also for non-immunogenic substances, determinations of practically any desired compounds are possible in very small amounts. Furthermore, the determination can be carried out on a relatively impure sample which, prior to analysis, has only been slightly purified or has not been purified at all.

Hitherto, the precision of such immune tests, for example the radio-immune test and similar analytical processes, was relatively low in comparison with other methods employed in clinical chemical laboratories. The reason for this is, apparently, that most immune tests are, at present, carried out manually, whereby problems arise, such as a relatively low degree of precision, a smaller throughput, fatigue of the personnel and the tediousness of the determination. Automation has long been needed in this field; hitherto, the commercially available systems, such as LKB, Baird and Tatlock, Micromedic and Centria, representdiscrete analysis systems which include the use of sample diluters, a series of samples being supplied to the sample diluter.Furthermore, in certain stages, manual intervention is necessary, such as removal of the sample holder from the incubation device, centrifuging, separation of the supernatant liquids and transferal to a counter device. In general, these systems are expensive, mechanically not dependable, of relatively low flexibility and, in general, only provide slight advantages.

Therefore, attempts have been made to provide a relatively simple, flexible and fully automatic process which can be used in all laboratories. The attractiveness of such a process would also be that it would be possible to make use of the devices and the skills which are to be found in a reasonably modern clinical chemical laboratory. The process should be flexible and versatile so that all analyses usually carried out can be performed with a minimum of difficulty. Finally, the process should also be inexpensive to perform and, in comparison with the conventional, manually performed, semi-automatic or automatic radio-immune tests and similar tests, should require low operational costs.

A fully automated process with a continuously flowing current of fluid would certainly fulfil the above-mentioned requirements. The most critical parts of such a process are, on the one hand, the device which separates free residues from labeled residues present in the insoluble phase and, on the other hand, in the case of the radio-immune test, but not in the case of the other tests, a counter system for the determination of the radioactivity. In Anal. Biochem., 65, 355-361/1975, there is described an automatic system with continuous flow which employs antibodies bound to red blood corpuscles. For the separation of the labeled antigen bound to the antibodies from the free residues, the erythrocytes are flocculated or agglutinated with the help of polybrene (hexa-dimethrine bromide) and separated off by decanting from the supernatant liquid. In Clin. Chim.Acta, 63, 69/1975, there is described a solid phase system which depends upon the use of antibodies which are covalently bonded to iron oxide coated with polymers ("Enzacryl"). ("Enzacryl" is a Registered Trade Mark). An electromagnet is used not only for mixing the reaction components but also for separating the tracers bound to the antibodies from the free fraction. This principle represents the basis for a fully automated system.

Reports have recently been made of an automatic oneline system with the designation "Gammaflow" in which the separation of the free ligands from the bound, labeled ligands is achieved with the help of a mixed bed separation column of active charcoal and "Dowex" resin. ("Dowex" is a Registered Trade Mark).

However, all known fully automatic systems with continual flow suffer from various disadvantages or limitations. The system employing antibodies bound to red blood corpuscles is not suitable because of the low throughput (10 samples per hour) and has a low sensitivity and a low degree of precision. The system involving the use of magnetic particles necessitates an extremely laborious magnetic separation, which makes the device complicated and expensive. The use of a mixed bed column for the separation of the bound residues from the free residues also has its limitations since the system cannot be universally employed for the effective separation of all kinds of antigenic materials. For example, immunoglobulins and also high molecular weight antigens cannot be separated with the help of such a column.The system is also not suitable for the enzyme-immune test in which the charge and mass difference between the bound residues and the free residues is very small.

Therefore, it is an object of the present invention to provide a simple, flexible and fully automatic process which is particularly suitable for carrying out the radio-immune test, the enzyme-immune test and the fluorescence-immune test.

Thus, according to the present invention, there is provided a process for the detection and determination of a component of the reaction between a specific binding protein and the corresponding bindable substance with the utilisation of the binding affinity of these compo nents for one another in an automated system with a continuously flowing fluid stream, wherein successive mixtures of the sample material with a definite amount of one component of the reaction in labeled form and of a further component, which comprises a carrier, in an insoluble, particulate state or in a state easily converted into an insoluble particulate state are introduced into the liquid stream in such a manner that the individual mixtures remain separate, incubated and continuously passed into a separation device in which, if necessary after previous insolubilisation of the carrier, at least a part of the liquid phase is separated from the solid phase and the amount of the labeled components contained in one of the separated phases is measured.

The present invention also provides a device comprising means for providing a continuously flowing stream with successive samples, incubation means, means for passing the stream through the incubation means to a separating device for the separation of at least a part of the liquid phase from the solid phase and a measurement device for one of the separated phases.

According to a preferred embodiment of the present invention, there is provided a device comprising a block divided into two halves, having oppositely lying recesses in the block halves, the halves being separated by a filter, an inlet tube for a sample stream and an inlet tube for a dilution agent, which tubes combine upstream of one recess, and an outlet tube from that recess and an outlet tube from the other recess.

The specific binding proteins which can be used according to the present invention include antibodies, anti-antibodies, hormone receptors and other proteins capable of hormone binding, such as thyroxin-binding globulin TBG, intrinsic factor and the like.

The corresponding bindable substances are to be understood to be compounds which are able to form a bond, generally of a complex nature, with the specific binding protein. Typical examples thereof include antigens, haptens, steroids, antibodies and other substances capable of bonding with a specific binding protein.

At least one component of the bindable substances, i.e. the specific binding protein or the substance capable of binding therewith, is employed in labeled form. Thus, it can be not only the specific binding protein but also the corresponding bindable substance. As labelling, there can be used, for example, the introduction of radioactive atoms or groups, enzyme, coenzyme or substrate labelings or the introduction of fluorescing or light-absorbing substances. Other labelings can also be used in which the residue is excited by some other physical method, for example electron spin resonance.

Typical examples of radioactive atoms which can be used according to the present invention include iodine, tritium, sulphur, selenium, chromium, carbon and phosphorus. Enzymes frequently employed for labeling include, for example, peroxidase, glucose oxidase, alkaline phosphatase and p-glucosidase. Typical examples of fluorescing substituents which can be used include rhodamine, fluorescein isothiocyanate (FITC) and umbelliferryl derivatives.

The labeled components of the reaction (tracer) can be the same as the substance to be determined in the process, for example, in the case of the determination of an antigen, can be labeled antigen. However, the tracer can also be some other substance bindable with the component to be determined, for example a further labeled antibody.

The carrier used can be an insoluble, particulate or cellular material, for example, porous "Sephadex", porous "Sepharose", microcrystalline cellulose, latex, glass spheroids or cells, such as erythrocytes, to which material the antibody or antigen is firmly bound. ("Sephadex" and "Sepharose" are Registered Trade Marks). The bulk or volume of the carrier does not impair the antigen-antibody reaction and permits an adequate separation of the bound residues from the free residues. Alternatively, use can be made of porous, particulate, ion-exchangeable carrier materials, such as an ion exchanger gel, "Sephadex" or a resin, to separate the non-bound marked component, for example the antigen, from that on the other specific binding component, for example the antibody-bound part of the labeled component.

This embodiment can be used for those components which behave like anionic and cationic residues, for example some haptens. In the case of this embodiment, for example the antibodies are not bound to the particulate carriers but rather are used as such after an appropriate dilution of the antiserum.

As a rule, the process according to the present invention makes use of a carrier which, ab initio, is in solid, particulate form. However, it is also possible to use a liquid but precipitatable carrier and preferably one which is reversibly precipitatable. In other words, the carrier is not continuously present as a solid material. As an example of such a material, there maybe mentioned a latex based on polyacrylamide which, depending upon the pH, is present in liquid or solid form. This embodiment of the present invention has the advantage that the actual immune reaction can be carried out in a homogeneous liquid phase but, before the phase separation, the carrier is converted into solid form, for example by altering the pH.In a preferred embodiment, the precipitated carrier can subsequently again be reversibly liquefied which has advantages for the exactitude of the measurement reaction if that phase is subjected to measurement in which the solid parts are present.

The precipitation of the carrier can be carried out especially favourably by means of the current of dilution agent which, in the case of a preferred embodiment of the present invention, is carried out after dilution forced filtration (explained hereinafter) shortly before reaching the filter membrane. Thus, for example, the dilution agent can be an organic liquid, a salt solution or a buffer solution which brings about the precipitation by alteration of the ion concentration, of the pH value or the like.

The separation of the bound labeled component from the free labeled component is preferably achieved by the use of a highly porous membrane. The current of liquid is allowed to flow past on one side of the membrane and diluted upstream in order to increase the pressure. On the other side of the membrane (filtrate side), it is also possible to apply a negative pressure. Therefore, the first phase is obtained on this filter side, whereas the second phase containing the insoluble components is obtained downstream of the retentate side. The amount and the nature of the dilution agent and/or the amount of the liquid filtered through the membrane can be adjusted as desired. This technique is referred to as filtration brought about by dilution or dilution forced filtration.

Although the use of a system with continuous flow for the immune test was to have been expected to give rise to a number of problems, these have, surprisingly, not arisen to any marked extent. These problems include the difficulty of separating the bound and the free labeled components at the end of the determination, insufficient incubation time, which prevents the reaction from proceeding to such an extent that a satisfactory degree of precision and sensitivity is obtained, the difficulty of pumping small amounts of labeled components since, in particular, there is a tendency for adsorption losses to arise in the tubes of the system, and problems of entrainment.However, preliminary investigations have shown that small amounts of labeled and unlabeled components can be pumped in a system with continuous flow, for example with the use of air segments or of a substantially immiscible liquid, without serious problems arising with regard to the adsorption losses or an entrainment of the materials. Furthermore, the sample mixtures can be introduced block-like, without substan tial change of the flow velocity, into the continuously flowing fluid stream.

For the further explanation of the present invention, some embodiments thereof will now be described in more detail, with reference to the accompanying drawings, in which: Figure 1 shows a schematic sectional view of a separation device used according to the present invention; Figure 2 shows a schematic vertical sectional view of a radioactivity counter head; Figure 3 is a block diagram of a radio-immune test system according to the present invention; Figure 4 is a block diagram of an enzyme-immune test system according to the present invention; and Figure 5 shows a standard curve produced with the system according to Figure 4.

In the initial stages, the process according to the present invention uses conventional continuous flow systems and devices, which do not need to be described in more detail. The samples of tracer, for example the labeled antigen, and the component present in the solid phase, for example the antibody, are introduced in precisely measured amounts which are determined by the tube size of the tube distributor. Between the samples, there is present a wash liquid which can be characteristically identified, for example by colouring. This is only of importance in the case of the radio-immune test but not in the case of the other immune test systems since the counter device can be controlled in such a manner that it selects the amount to be determined on the basis of this characteristic property.The stream divided into segments by sections of air is passed through a tube at a controlled temperature, good mixing of the reagents being achieved in the course of the incubation time. The incubation time can be 25 minutes or less. The current of liquid is then transferred to the separation device, as is illustrated in Figure 1 of the accompanying drawings. In this, a dilution forced filtration is carried out. The separation device comprises a divided block, which preferably consists of transparent synthetic resin, which, between the halves 1 and 2 thereof, contains a filter 3 of porous material made, for example, of synthetic resin, nylon "Teflon" or some other organic or inorganic synthetic material ("Teflon" is a Registered Trade Mark).The pore size of this membrane should be uniform and large (for example 10 ttm) but smaller than the carrier particles or the materials which are used to absorb the non-bound ligands. The half block 1 has an inlet tube 4 for the segment-form current and a further inlet tube 5 for the dilution agent. These tubes join upstream to a separation chamber 6 which is formed by semicylindrical oppositely-lying recesses in the half blocks 1 and 2, the chamber 6 being divided by a membrane 3. On the opposite end of the chamber 6, downstream of the inlet pipes, there is provided, in the half block 1, an outlet tube 7 with an adjustable cross-section (not shown) and in the half block 2 there is an outlet tube 8.The volume of the liquid/air mixture which emerges from the outlet tube 7 is adjusted and coordinated with the magnitude of the underpressure which is applied to the outlet tube 8.

During operation, the segmented fluid stream is diluted by the dilution agent before it reaches the chamber 6 and is passed in segmented form under the membrane 3. The pressure produced by the addition of the dilution agent and the restriction of the outflow through the outlet tube 7 forces excess liquid and particles which are smaller than the pore size of the filter through the membrane. The filtration is also promoted by an appropriate negative pressure which is applied to the outlet opening 8.The filtrates, which contain non-bound tracer, for example antigen, can be used for the determination of the free residues by counting the radio-activity in the case of the radio-immune test or, in the case of the fluorescence-immune test or of the enzyme-immune test, measuring the intensity of the fluorescence or the changes of the enzyme reaction, for example the absorption. The tracer (antigen) bound to the solid phase remains, because of its large particle size (which is usually above 20 jLtm) in the segmented current and is collected at the outlet 7. The material can then be passed to a detection device, such as described hereinafter, for the counting of the radioactivity.

In this way, it is possible to count either the bound or the non-bound tracer in dependence upon the selected outflow current. In any case, the strength of the negative pressure and the amount of supplied dilution agent can be used to control the volumes filtered through the membrane within very narrow and precise limits.

Many highly porous membranes very easily take up an electrostatic negative charge and retain this so that the operation of the membrane can be influenced in a large number of ways.

For example, particles can be retained which are smaller than the size of the pores. However, the electrostatic charge can also be utilised to minimise blocking by larger particles present in the liquid, as well as particles introduced into the system by the air. The intensity of the electrostatic charge depends upon the pH value and can be modified by the use of a diluting buffer with the desired pH value. When the membrane consists of a conductive material, the charge can be influenced by external means. The dilution agent can also be a solution which exercises a special function. Thus, for example, it can contain a substance, such as polyvinyl alcohol, which coats the membrane and, in this way, reduces, if desired, the electrostatic charge. As dilution agent, there can also be used a liquid which brings about precipitation of the carrier, for example a salt solution which has a precipitating action upon the carrier by increasing the ion concentration.

The radioactivity can be counted in a device, such as is illustrated in Figure 2 of the accompanying drawings, which comprises a conventional "'-counter head 21 which contains a closed cuvette 22. This is provided with an inlet tube 23, which cooperates with an electric sensor 24, and with an outlet tube 25 to which an underpressure is applied. The outlet tube 25 is connected via tube 26 and valve 27 with a peristaltic pump or some other suction device, the valve 27 being controlled by the sensor 24 and a time measurement device (not illustrated).

Alternatively, the tube 26 can be omitted and the valve 27 attached directly to the outlet tube 25.

The sensor 24 is adapted to the properties of the wash liquid, for example its colour, whereby, in the case of recognition of this characteristic property, the valve 27 is closed, the cuvette is filled and counting then commences. After expiry of a predetermined period of time, the counter is stopped and adjusted to zero, the valve 27 is opened and the cuvette is emptied via the reduced pressure which is continuously applied to the outlet 25. The counting result can be printed out or indicated or shown in some other manner. It is of importance that the counting method depends upon a new concept, namely, a "simultaneous feed-in counting". Preliminary investigations have shown that the relationship between the number of the counting impulses and the time follows a curve which can be reproduced by a quadratic equation.This counting method can be advantageous since, in the case of the use of a minicomputer, marked irregularities within a sample segment can lead to marked deviations from a calculated curve. This can be ascertained and the error determined.

Referring now to Figure 3 of the accompanying drawings, the further developed fundamental system is further explained, by way of example, on the basis of an antigen determination. In this case, the reagents, which contain the antigen in serum or urine, are passed, as described above, via a coil 31, which is kept at a constant temperature, into a current divided by air sections into the separation device 32 illustrated in Figure 1 of the accompanying drawings, in which the dilution forced filtration takes place. In this way, the antigen bound to the solid phase antibody is separated from the free and non-bound antigen, as well as from the other serum components. The latter are discarded, whereas the segmented stream is mixed with another specific antibody (which acts against the same antigen) but is marked with 125 I.

There takes place a second antibody reaction during the course of passing through another coil 33 maintained at a constant pressure, which leads to the formation of a labeled complex in the solid phase in that the antigen is present between two antibodies, one of which is bound to the solid phase (immobilised antibody) and the other of which is labeled. After this reaction, the complex bound to the solid phase is separated in a second separation device 34 from the labeled free antibody. The free fraction is then counted and the bound fraction is discarded or vice versa.

The present invention can also be used to automate a conventional radio-immune test (i.e.

a test in which the antibody is used as such, without it being changed, for example bound to a solid phase). The automation of this test can be achieved, for example, by mixing the segmented stream, which contains the incubated analysis material (e.g. the antibody, the labeled antigen (tracer) and the non-labeled antigen), with a particulate material which is able to bind either with the free or with the bound components. An example of a material of the first type is a porous ion exchange gel or resin which is able to capture anionic or cationic particles. whereas an example of a material of the latter type is a second antibody which is bound covalently to a porous gel matrix, such as "Sephadex" or "Sepharose".

The process according to the present invention can also be used for automating the determination of steroids and pharmaceuticals or drugs in which an extraction with an organic solvent is not necessary. Furthermore, the process can be used for the automation of methods of investigation other than immune-tests. For example, the determination of the total iron binding ability includes the addition of excess iron salt in order to saturate the carrier protein transferrin. The separation of the excess iron can take place in a flow system by mixing it with a porous cation exchanger and subsequently carrying out the dilution forced filtration. The iron in the filtrate can be determined by conventional methods.

Thus, to summarise, the process according to the present invention can be used for automating the radio-immune test, the enzyme-immune test or the fluorescence-immune test in which a particulate material, such as a porous gel or resin, is used as analytical reagent. For the achievement of the best results, the particles should have an attrition-free matrix which is of spherical shape and has a uniform particle size.

The following Examples are given for the purpose of illustrating the present invention: Esantple 1 Automatic determination of thyroxin with the use of a device according to Figure 4, the enzyme reaction being omitted and the free fraction counted in the counter after the separation device. The determination can be carried out in an automatic analyser of the Auto-Analyzer type of the firm Technicon.

Switching in of the counter and of the interface 1. membrane change 2. pumping of the buffer for achieving a good bubble pattern through the filter (this is important) 3. pumping of the tracer (l25I-T4) for the determination of the total count rate (which should be 20 to 25,000) 4. addition of antibody and pumping of the dyestuff through the sample tube 5. when the dyestuff emerges, test the binding by activation of the counter - adjustment of the level 6. commencement of the sample taking Sample plate = 5 x 'O' standard curve 2 control sera at 39 and 40 8 samples 2 control sera etc 7. calculation in the usual way Reagents 1. AB (insoluble antibody) 10 ml."Sepharose" -antibody to T4 are diluted to 100 ml. with tris buffer with a pH value of 8.5.

2. Tracer 1.0 ml (10 iLl).

40 ml. tris buffer 4 ml.5 % albumin 250 mg. A.N.S.

3. Wash solution 1.8% sodium chloride + 1 ml. "Bri"/ 1., for use filter through a 1 ,um filter 4. Dyestuff 15% bovine serum = 100 ml + 8 ml green dyestuff 5. tris buffer 12 g/l tris buffer adjusted to a pH value of 8.5 with hydrochloric acid. 2.5 g.

azide are added thereto and filtered, before use, through a 1 iLm filter.

("tri;" is a Registered Trade Mark) Standard Solutions Standard solution T4 'A' : 57.21 mg. pure T4 in 6 ml. 0.1N aqueous sodium hydroxide solution made up to 50 ml. with absolute ethanol and freeze dried.

Standard solution T4 'B' : 0.1 ml. of Standard solution A is diluted with 1.9 ml. tris buffer, which contains 0.5% albumin, and then freeze dried.

Working standard solution: 19.9 ml. of 5%bovine albumin + 0.1 ml. of Standard solution B = 325 nMol/ 1. Double dilution with 5% human albumin (which has been treated with resin), with the formation of 163.82, 41.20 and 10 nMol/1.

6. Antibody: 1 ml. Beneden T4 antibody is bound to 5 g. "Sepharose", diluted with tris buffer to 100 ml., 0.1 g. azide added and then freeze dried in aliquots of 10 ml. each.

Adjustments: sample time: 75 seconds washing time: 15 seconds delay: 5 seconds counting: 55 seconds recording: 2V Example 2 Automated determination of thyroxin-(T4) by means of the enzyme-immune test according to Figure 4 in which is illustrated one scheme of carrying out the determination. The determination can be carried out in an automatic analyser of the Auto-Analyzer type of the firm Technicon.

Preparations as in the case of the radio-immune test of Example 1.

1. Reagents: 1. T4 antibody It was covalently bound, according to the instructions of the manufacturer (Pharmacia), on to "Sepharose" activated with cyanogen bromide and suspended, by means of a magnetic stirrer, in 0.1M tris hydrochloride buffer (pH 8.5) containing 0.1aJo"BriJ" 2. Tracer T4 was marked with peroxidase (EC 1.11.1.7, Boehringer Mannheim, Cat. No. 15629) and diluted to an end volume of 50 ml. with tris hydrochloride buffer (pH 8.5) with 0.5i: albumin, 250 mg. A.N.S. (ammonium 8-anilinonaphthalene-1 -sulphonate) and O.1c/ir 3. Dilution agent .2 M Phosphate buffer, pH 8.0, with 0.1% "Bri 4. Enzyme reaction mixture 0.1 M phosphate buffer (pH = 8.0), with 11 mM 2,4-dichlorophenol, 1.2 mM 4-aminophenazone and 1.5 mM sodium perborate.

5. Wash solution: 1.8% sodium chloride with 0.1% "Brij".

II. Carrying out: 1. Rinsing with wash solution and adjustment of a constant bubble throughput through the separator.

2. Dosing of the tracer, addition of the enzyme reaction mixture, balancing of the photometer and adjustment of the recorder: determination of the total activity of the tracer.

3. Addition of the T4 antibody, testing of the tracer binding, again balancing the photometer and the recorder.

4. Commencement of sample taking sample plate 5 ml '0' standard (=cho) standard curve 2 control sera 8 samples 2 control sera 8 samples, etc.

5. Evalution of the results in the usual manner according to the recorder trace.

In an analogous manner, by replacing the enzyme tracer by a fluoroescent tracer and using a fluorimeter, the FIA test can be carried out, for example with the use of a Perkin-Elmer spectrofluorimeter, Mod. 1000, or of a Kontron spectrofluorimeter S FM 22.

WHAT WE CLAIM IS: 1. Process for the detection and determination of a component of the reaction between a specific binding protein and the corresponding bindable substance with the utilisation of the binding affinity of these components for one another in an automated system with a continuously flowing fluid stream, wherein successive mixtures of the sample material with a definite amount of one component of the reaction in labeled form and of a further component, which comprises a carrier, in an insoluble particulate state or in a state easily converted into an insoluble particulate state are introduced into the liquid stream in such a manner that the individual mixtures remain separate, incubated and continuously passed into a separation device in which, if necessary after previous insolubilisation of the carrier, at least a part of the liquid phase is separated from the solid phase and the amount of labeled components contained in one of the separated phases is measured.

2. Process according to claim 1, wherein the phases are separated by membrane filtration.

3. Process according to claim 2, wherein the pressure in the continuously flowing fluid stream is increased before reaching the filter membrane by passing in a further liquid stream.

4. Process according to claim 2 or 3, wherein the pressure on the filtrate side of the filter membrane is lowered.

5. Process according to any of the preceding claims, wherein, for the separation of the sample mixtures, there is used a continuously flowing stream of liquid segmented by gas bubbles.

6. Process according to any of claims 1 to 4, wherein, for the separation of the sample mixture, the continuously flowing fluid stream is segmented by a liquid which is substantially immiscible therewith.

7. Process according to any of claims 1 to 4, wherein the sample mixtures are introduced block-like, without substantial change of the flow velocity, into the continuously flowing fluid stream.

8. Process according to any of the preceding claims, wherein there is used a radioactive component or a component labeled by an enzyme or by a fluorescing residue.

9. Process according to any of the preceding claims, wherein the specific binding protein is an antibody or anti-antibody or a hormone receptor and the corresponding bindable substance is an antigen, hapten, antibody or hormone.

10. Process according to any of the preceding claims, wherein a precipitatable soluble carrier is used.

11. Process according to claim 10, wherein a reversibly precipitatable carrier is used.

12. Process according to claim 11, wherein precipitation takes place by means of a further stream of fluid introduced before the filter membrane is reached.

13. Process according to claim 1 for the detection and determination of a component of the reaction between a specific binding protein and of the corresponding bindable substance, substantially as hereinbefore described and exemplified.

14. Device for carrying out the process according to any of claims 1 to 13, comprising means for providing a continuously flowing stream with successive samples, incubation means, means for passing the stream through the incubation means to a separating device for the separation of at least a part of the liquid phase from the solid phase and a measurement device for one of the separated phases.

**WARNING** end of DESC field may overlap start of CLMS **.

Claims

**WARNING** start of CLMS field may overlap end of DESC **. 5. Wash solution: 1.8% sodium chloride with 0.1% "Brij". II. Carrying out: 1. Rinsing with wash solution and adjustment of a constant bubble throughput through the separator. 2. Dosing of the tracer, addition of the enzyme reaction mixture, balancing of the photometer and adjustment of the recorder: determination of the total activity of the tracer. 3. Addition of the T4 antibody, testing of the tracer binding, again balancing the photometer and the recorder. 4. Commencement of sample taking sample plate 5 ml '0' standard (=cho) standard curve 2 control sera 8 samples 2 control sera 8 samples, etc. 5. Evalution of the results in the usual manner according to the recorder trace. In an analogous manner, by replacing the enzyme tracer by a fluoroescent tracer and using a fluorimeter, the FIA test can be carried out, for example with the use of a Perkin-Elmer spectrofluorimeter, Mod. 1000, or of a Kontron spectrofluorimeter S FM 22. WHAT WE CLAIM IS:

1. Process for the detection and determination of a component of the reaction between a specific binding protein and the corresponding bindable substance with the utilisation of the binding affinity of these components for one another in an automated system with a continuously flowing fluid stream, wherein successive mixtures of the sample material with a definite amount of one component of the reaction in labeled form and of a further component, which comprises a carrier, in an insoluble particulate state or in a state easily converted into an insoluble particulate state are introduced into the liquid stream in such a manner that the individual mixtures remain separate, incubated and continuously passed into a separation device in which, if necessary after previous insolubilisation of the carrier, at least a part of the liquid phase is separated from the solid phase and the amount of labeled components contained in one of the separated phases is measured.

15. Device according to claim 14, comprising a block divided into two halves, having

oppositely lying recesses in the block halves, the halves being separated by a filter, an inlet tube for a sample stream and an inlet tube for a dilution agent, which tubes combine upstream of one recess, and an outlet tube from that recess and an outlet tube from the other recess.

16. Device according to claim 14 or 15, comprising a measurement device which contains a counting head in which is arranged a closed cuvette which has an inlet tube with a sensor and an outlet tube adapted to be connected with a suction device via a pipe and a valve, which valve is controlled by the sensor.

17. Device for carrying out the process according to any of claims 1 to 13, substantially as hereinbefore described and with reference to the accompanying drawings.