HK1008244B - Use of synthetic particles as reagents in agglutination reactions - Google Patents

Description

The present invention relates to a method for the detection of an analyte in a sample liquid by agglutination, whereby the sample liquid is brought into contact with an agglutination reagent and an inert matrix, the reaction mixture is subjected to gravity and the reaction between the analyte and the agglutination reagent is determined.

Methods for detecting analytes by haemagglutination and particle agglutination tests are known.These tests are mainly used in the diagnosis of infectious diseases for antigen or antibody detection.However, all these methods have the common disadvantage of being time-consuming and time-consuming and the results are often very difficult to interpret.

For haemagglutination assays using unfixed erythrocytes as agglutination reagents, gel immunoassay methods are known whereby haemagglutinates are separated from individual non-agglutinated erythrocytes by a centrifugation step through an inert matrix (see e.g. EP-A-0 194 212, EP-A-0 305 337, EP-A-0 557 546, EP-A-0 634 216, EP-A-0 485 228 and WO95/31731). These methods retain mined erythrocytes on or in the inert matrix and allow them to be clearly separated from non-reactive individual erythrocytes which penetrate the matrix and the bottom of the reagent by aggregating sediment.

Only in EP-A-0 305 337, which is a priority application from 1987, is it stated that synthetic particles such as latex or polymerized agarose can also be used as agglutination reagents. However, there is no information on the properties of such synthetic particles.

However, unfixed erythrocytes have a disadvantage: they are unstable and, under certain reaction conditions, lead to haemolysis, which often leads to unwantedly short expiration dates of products based on unfixed erythrocytes. Furthermore, the standardised and reproducible production of erythrocyte preparations, in particular antibody or antigen-coupled erythrocyte preparations, is only possible with great effort, and therefore a sufficient uniformity of the physical properties of erythrocyte preparations is practically impossible.

One of the objectives of the present invention was therefore to eliminate at least part of the above-mentioned disadvantages arising from the use of erythrocytes as agglutination reagents. In particular, the present invention is intended to provide synthetic particles which can be used as agglutination reagents and which are capable of simulating the behaviour of erythrocytes in known gel immunoassays. Furthermore, the synthetic particles are intended to allow simple coupling of biological substances and to be so designed that they are at least equivalent as agglutination reagents to the erythrocytes used so far in terms of sensitivity and specificity. These synthetic particles are intended to be used in agglutination processes which are simpler and more efficient than the methods known so far.

This problem is solved by a method of detection of an analyte in a sample liquid by agglutination, whereby the sample liquid is brought into contact with an agglutination reagent and an inert matrix, the reaction mixture is subjected to gravity and the reaction between the analyte and the agglutination reagent is determined, characterized by the use of synthetic particles as agglutination reagents, the diameter and density of which are chosen so that they behave essentially like erythrocytes in relation to the matrix.

Surprisingly, it was found that, when the density and diameter are adjusted optimally at the same time, synthetic particles can actually be produced that can easily pass through an inert matrix in a gel immunoassay, thus perfectly simulating fresh red blood cells.

In the course of the work leading to the present invention, it was shown that the passage of rigid polymer particles is more delayed than that of erythrocytes (6-8 μm in diameter) and, under given standard conditions, this meant that commercially available standard particles with a diameter of 3 - 7.5 μm only partially settled into the gel matrix, while larger (11.9 μm) and smaller synthetic particles (< 1 μm) could only penetrate the gel weakly. The system of the invention is intended to allow a particularly time-consuming process compared with comparable methods. A possible centrifugation time of 50 minutes would triple the intended test time of 20 minutes including incubation. As the expert knows, the sensitivity in gel centrifugation processes decreases with increasing centrifugation speed.

Surprisingly, it was found that synthetic particles of smaller diameter than those of red blood cells and higher density than usual can achieve a similar behaviour to that of red blood cells when passing through the inert matrix, using both spherical and asymmetrical synthetic particles.

Synthetic particles with a mean diameter ≤ 5 μm and in particular 1 to 5 μm have proved essential for the method of the invention. The mean diameter is particularly preferable between 2 and 4 μm. The specific density of the particles is greater than or equal to 1.1 g/cm3 and is preferably in the range of 1.1 to 1.8 g/cm3. The specific density is preferably in the range of 1.1 to 1.6 g/cm3 and most preferably in the range of 1.15 to 1.4 g/cm3, especially when the detection procedure is performed under standard conditions, as specified for known commercial erythrocyte immuno-gels (e.g. DiaMed).

The manufacture of uniform polymer particles in the size range suitable for the agglutination reagents of the invention is well known to the practitioner (Arshady (1992): Suspension, emulsion and dispersion polymerization: A methodological survey. Colloid & Polymer Science 270, 717-732; Okubo and Shiozaki (1992): Production of micron-sized monodispense polymers by seeded polymer polymerization method utilizing polymer swelling with seeding.

For the visual detection of the agglutination reaction in the method of the invention, dyes, especially water-insoluble dyes, can be incorporated into the particles. Thus, blue, yellow, green, black or red synthetic particles are very suitable for visual evaluation.

The robustness of the synthetic particles allows the immobilization of a large number of different ligands on their surface using known industrial standard methods. It is convenient to immobilize ligand molecules that are able to bind to the analyte to be determined. The ligand molecules can be immobilized via adsorptive, covalent or high-affinity interactions. The covalent coupling can be done, for example, via chemically reactive groups exposed to the surface. Examples of such groups are carboxylic acid-amino, aldehyde and expoxy ligands. Immobilization via high-affinity interactions is achieved by two partners of a high-affinity pair of analogues, such as polypeptide/avidin, lipoproteins/lipoproteins, polypeptide/sugar, polypeptide, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, and glycoproteins, and glycoproteins, and glycoproteins,

It is also possible to immobilise ligand molecules by purely adsorptive processes, for example for cell membranes such as erythrocyte, platelet or leukocyte membranes, cell membrane fragments or lysate of cells or pathogens such as viruses, bacteria or parasites.

The method of the invention relates to the detection of an analyte in a sample fluid. The sample fluid is preferably body fluids, which may be diluted, e.g. blood, serum, plasma, saliva or urine. The volume of the sample fluid for the method of the invention may vary over a wide range, preferably volumes of 1 to 200 μl are used for microtests.

The method of the invention uses the synthetic particles described as agglutination reagents, which preferably contain on their surface a specific ligand molecule capable of binding to the analyte with high affinity. The agglutination reagent usually contains several binding sites for the analyte, which allows the formation of networked agglutination complexes from the analyte and the agglutination reagent.

The analyte detectable by the method of the invention is a substance that can interact specifically and with high affinity with the agglutination reagent, e.g. antigen or antibody, which can be determined by an immune reaction, or also nucleic acid, which can be determined by a hybridization reaction. A first preferred embodiment of the present invention concerns the detection of antibodies as analyte in the sample fluid, e.g. antibodies against pathogens such as viruses (HIV, hepatitis V), bacteria or protozoa, antibodies against autoantigen, antibodies against tumour or antibodies against allergen.

Furthermore, the method of the invention also allows class-specific detection of antibodies, e.g. a distinction between IgG and IgM antibodies, which is often of considerable importance in the diagnosis of e.g. infectious diseases and autoimmune diseases.

On the other hand, the method of the invention can also be used to determine antigens, e.g. free antigens such as serum proteins, metabolites, hormones, mediators, etc. or carrier-bound antigens such as cellular blood group antigens, etc.

The method of the present invention uses a reaction vessel with a matrix, so that after the application of gravitational forces, a qualitative or semi-quantitative determination of the agglutination reaction between the analyte to be determined and the agglutination reagent is possible. Although the application of gravitational forces can also be caused by prolonged sedimentation, it is advantageous to use centrifugation, since the desired sedimentation can be achieved after a short time. The optimum conditions for centrifugation time and g-number can be determined by the expert without difficulty for each system. These conditions are determined in particular by the nature of the agglutination complex between the agglutination matrix and the agglutination reagent, the component used in the analysis and the unrelated reaction conditions.

The method of the invention preferably uses an inert particle matrix, but the compact matrix described in EP 96 10 428.3 may also be used.

Err1:Expecting ',' delimiter: line 1 column 123 (char 122)

The results of the method of the invention are interpreted as follows: (a) at high agglutination, the reaction product cannot penetrate the matrix from the analyte to be determined and the agglutination reagent, or only very slightly; (b) at low agglutination, the reaction product penetrates the matrix but cannot penetrate it completely; and (c) at low agglutination, the components contained in the reaction vessel can penetrate the matrix substantially completely.

In a preferred embodiment of the method of the present invention, two or more different types of synthetic particles are used as agglutination reagents. There are several diseases which are associated with each other and for which conventional testing methods require a so-called screening procedure to establish whether the person under investigation carries antibodies to a disease belonging to that group. If the tested body fluid of the person under investigation is positive in the screening test, it must be subjected to a second step, by conventional methods, called a differentiation test, in which it is then determined which of the diseases in the antibody group are targeted. An example of this is the so-called HIV-specific screening.

The method of the present invention now allows two different types of synthetic particles to be used, which preferably do not differ in size, shape, density and flexibility, but have different colouration, e.g. red and blue, and different surface ligands. Since these different species of synthetic particles form a homogeneous suspension with equal proportions of each species, of course this suspension can be used for a single test. The resulting results can then be interpreted as follows: If both analyte to be analyzed are present simultaneously in the upper profile matrix, then after centrifugation a red and a leafy matrix or a number of different matrix bands are found in the upper profile matrix.

The test may be carried out in any test vessel suitable for use in a gel agglutination test. The volume of the test vessels should preferably be between 50 μl and 2 ml. The test vessels should preferably be equipped with a funnel to receive the reagents. A preferred embodiment is an arrangement of several test vessels arranged together on a card or disc. These test vessels on a card or disc may be designed to detect the same or different analyte.

For example, the blood group analysis cards distributed by Fa. DiaMed, which contain six micro-reaction vessels, can be used to perform the procedure of the present invention.

The reaction mixture is preferably centrifuged by centrifuging the reaction vessels in a suitable centrifuge, so that the commercially available DiaMed ID 12 S II centrifuge can be used in a preferred embodiment of the method of the invention with the fixed parameters (t = 10 min, v = 85 g).

However, in another preferred embodiment of the present invention, modified centrifugation methods can be used which take particular account of the kinetics of the underlying reactions, reflected in a reaction amplification of especially weak reactions.

Furthermore, for certain embodiments of the method according to the present invention, it is preferable to work with matrices containing a second antibody, for example a so-called group-specific antibody, such as anti-IgG. Here, a two-phase reaction takes place in the reaction vessels. The first reaction takes place in the reaction chamber when in a positive reaction the ligand molecule on the synthetic particles, e.g. an antigen, reacts with the analyte, e.g. an antigen-specific antibody, in the sample.

With a total test time of only 20 minutes, the test system described in the invention allows a significant time saving compared to known systems. Compared to test systems based on fixed or unfixed erythrocytes as agglutination reagents, an additional advantage is the much longer shelf life of the synthetic particles and the better uniformity of the starting material.

The test system is designed to be simple to perform and the results clearly readable, so that the test can be performed by medical assistants. The application is conceivably simple, since it involves only two pipette steps. Moreover, with a battery-operated centrifuge and a similar vortex device, the tests can be easily performed outside fixed laboratory facilities without electricity. The small sample size, the absence of washing steps and the possibility of closing the test vessels after use ensure the shortest possible exposure of the person to potentially infectious material and the greatest possible control of potentially infectious waste.

Another subject of the present invention is synthetic particles with a mean diameter ≤ 5 μm, preferably 1-5 μm, preferably 2-4 μm and a specific density ≥ 1.1 g/cm3, preferably 1.1 - 1.8 g/cm3, preferably 1.15 - 1.4 g/cm3, on the surface of which ligand molecules are immobilized, for example, by covalent, adsorptive or high affinity interactions.

The present invention also relates to a reagent kit for detecting an analyte in a sample liquid, comprising: (b) at least one type of synthetic particles on the surface of which ligand molecules are immobilized.

For certain types of determination method according to the invention, it is preferable to add a detergent to the reaction mixture. Examples of suitable detergents are ionic detergents such as SDS or non-ionic detergents such as Triton X-100 and Tween 20. Furthermore, the addition of mucin may be preferred. Addition of detergents and/or mucin can improve the uniformity of the particles in the suspension or during the gel pass. This results in more complete sedimentation in the case of a negative reaction.

It may also be appropriate to conduct the reaction with the addition of a reducing agent, e.g. a sulphydryl reagent, such as 2-mercaptoethanol, diithiothritol or sodium dithionite, or another reducing agent, such as tributylphosphine.

Furthermore, in certain tests, an improvement in test results may be achieved by modifying the centrifugation conditions. This is particularly true of tests using a matrix coated with a receptor, e.g. a second antibody. A preferred modification of the centrifugation conditions involves an interval centrifugation, i.e. a first centrifugation step, then a break and then a second centrifugation step. If necessary, centrifugation may be carried out in several interval steps. The first centrifugation step is preferably only a short centrifugation step of up to 1 minute, preferably 30 s.

The following examples and figures illustrate certain aspects of the invention. Fig. 1 :the results obtained in the positive, weak positive and negative reaction in the method of the invention,Fig. 2:a schematic representation of the test execution,Fig. 3 :the reactions taking place in a micro-reaction vessel in which the matrix contains anti-IgG antibodies: (a) in the reaction chamber and (b) in the buffer layer of the matrix or matrix,Fig. 4: the schematic result of a differential diagnosis on two different analyte (A1 and A2) in a reaction vessel: (a) Patient sample 1, positive antibody for A1; (b) Patient sample 2, positive antibody for A2; (c) Sample 3 (mixture of serums from Patient 1 and 2), positive antibody for A1 and A2; (d) Patient sample 4, negative antibody for A1 and A2; andFig. 5:the schematic representation of the reaction zones referred to in Table 1.

Examples: 1. experiments with state-of-the-art synthetic particles which do not work in the gel test (comparison) The test procedure:

The suspensions of commercially available coloured synthetic particles, which are normally present at concentrations of 10% solids (w/v), were washed with a 10 mM PBS solution, pH 7.4 (Sigma P 4417), 0.1% Tween-20 (Sigma P 7949) (v/v), and adjusted in the same solution to a concentration of 0.15% solids.

Each 25 μl of this suspension was pipetted into a micro-reaction tube and sedimented under the conditions given in Table 1.

The production of suitable synthetic particles

The synthetic particles used in the following examples were produced by dispersion polymerisation, as this method is generally best suited to the production of particles in the 1-5 μm range. In a first step, base particles were synthesized from polystyrene (2a).

2a. Polymerization of the base particles Implementation of the programme:

60 g 1-hexadecanol (Aldrich 25,874-1), 216 g polyvinylpyrrolidone 40000 (PVP) (Aldrich 85,656-8), 1744 g dest. styrene (Aldrich S497-2) and 10261 g industrial spirit (Banners IMS 99) were weighed in a 20 1 round-piston reactor (equipped with stirrer, water condenser and nitrogen gas) The stirring rate was set to 35 - 40 U/min The mixture was tested at a temperature of 16 hours, then the room was raised to 70 °C. 17.4 g azoisobutyronitrile (Fis A/9050/50) and 10261 g industrial spirit (Banners IMS 99) were weighed in a 20 1 round-piston reactor (equipped with stirrer, water condenser and nitrogen gas) The stirring rate was set to 35 - 40 U/min The mixture was tested at a temperature of 16 hours, then the room was raised to 70 °C. 17.4 g azoisobutyronitrile (Fis A/9050/50) were now passed through a monodic reaction, resulting in a further 0.1 μm of the reaction.

Err1:Expecting ',' delimiter: line 1 column 63 (char 62) Implementation of the programme:

1.5 g benzoyl peroxide (Aldrich 22.887-7) was dissolved in 21.6 g 4-bromo styrene (Avocado 17896) and 250 g of polyvinyl alcohol (Harco 26-88) (PVA) (5% w/v) solution were weighed in a 11 1 round-piston reactor (equipped with agitator, water condenser and nitrogen aeration) and the stirring rate was set to 250 U/min. 1.5 g benzoyl peroxide (Aldrich 22.887-7) was further dissolved in 21.6 g 4-bromo styrene (Avocado 17896) solution and this solution was recovered in 250 hours of a 0.5 % (w/v) SDS solution. The polymeric polymeric polymers were then further recovered in a round-piston reactor.

This method polymerized different particles of the same diameter but with different bromestyrene/styrene ratios and thus with different densities (Table 2).

2c. Colouring of high density particles Implementation of the programme:

The following is added: 200 g of a 10 per cent (w/v) suspension of synthetic high density particles (made as described in 2a, 2b) with a diameter of 3.1 μm, 11.6 g of a 5 per cent (w/v) solution of polyvinyl alcohol (Harco 26-88), 40 g of methanol (Hammond) and 240 g of water were weighed in a 1 l round-piston reactor (equipped with PTFE agitator, water condenser). 0.4 g of dye Sudan IV (Kodak 112 6150) were dissolved in 30 g of dichloromethane (Fisons D/1852/25) ml and the solution emulsified in 250 ml of a 0.5 per cent (w/v) SDS solution. The dichloromethane emulsion was then given at room temperature in the reactor. After 4 hours the resulting emulsion was given in a 2 l glass measuring vessel.To evaporate the dichloromethane as quickly as possible, the suspension surface was sprayed with nitrogen under stirring. To maximize evaporation, the suspension was additionally slightly heated. The resulting red particles were washed in water by repeated centrifugation (1000 rpm, 5 min). They yielded 19.8 g. 50 g of a 10 % (w/v) suspension of synthetic high density particles (made as described in 2a, 2b) with a diameter of 3.1 mm, 2.9 g of a 5 % (w/v) solution of polyvinyl alcohol (Harco 26-88) and 60 g of water were weighed in a 1 l round-piston reactor (equipped with PTFE agitator, water condenser).The dichloromethane emulsion was then introduced into the reactor at room temperature. After 2 hours, the resulting blue particle emulsion was introduced into a 500 ml glass. To evaporate the dichloromethane as quickly as possible, the emulsion was placed in a glass containing a small amount of blue particles.

The resulting blue particles were washed by repeated centrifugation in water (1000 rpm, 5 min) and yielded a yield of 4.8 g.

The first is the use of a new methodology for the analysis of the particles.

(a) Manufacture of aldehyde particles A 1 ml aliquot of red 3.1 μm high density particle (17.1 %) (w/v) (produced as described in examples 2 a to c) was placed in a 15 ml centrifuge tube and washed 3 times by centrifugation with ultra-pure (18 MegOhm) water (1000 rpm, 5 min). To the washed particles, 15 ml of bovine serum albumin solution (2.67 mg/ml) (Sigma A9647) were added in 10 mM PBS at pH 7.4.0.05 % NaN3 and stirred for 4 hours at room temperature. The particles were washed 4 times by centrifugation with 15 ml water (1000 rpm, 5 min).To obtain a concentration of 1.7% (w/v), 3.75 ml of water were added to one 0.5 ml aliquota of this suspension to obtain a 0.2% (w/v) suspension. To this end, 4.25 ml of a 2 % glutaraldehyde solution were added to water and the reaction mixture stirred for one hour at room temperature. The particles were washed 4 times by centrifugation with 15 ml of water (1000 rpm, 5 min). A suspension of 8 mg red 3.1 μm high density particles (produced as described in 2a-c and functionalised as described in 3a) was resuspended in 4 ml of water and immersed for 1 min in an ultrasonic water bath (Sonomatic by Langford Ultrasonics,After another 2 min in the ultrasonic water bath, 1 ml of 50 mM sodium cyanborohydride (Aldrich 15,615-9) solution was immediately added to the acetate buffer. This mixture was incubated overnight at room temperature by light shaking. The particles were washed 3 times by centrifugation with 15 ml (1000 rpm, 5 min) and resuspended in 5.33 ml water at pH 10 mM PBS, 7.4, bringing the particle concentration to 0.15 % (w/v) (wysincyl) to produce polypyridine. A 500 mg suspension of red 3.1 μm high density particles in 1 ml water (produced as in 2a-c and functionalised as in 3a) was obtained from 4 ml 50 mM Na acetate,The reaction mixture was incubated in an ultrasonic water bath for 4 min before adding 1 ml 50 mM sodium cyanborohydride in acetate buffer. The reaction mixture was incubated by light shaking overnight at room temperature. The particles were washed with water 3 times by centrifugation (1000 rpm, 5 min) and resuspended in 333 ml 10 mM PBS at pH 7.4 to bring the particle concentration in the suspension to 0.15 % (w/v).

4. coupling of ligands to the functionalised particles

(a) Covalent coupling 0.1 ml solutions of biotinated antigens were administered to 1 ml of a 0.15 % % (w/v) suspension of red 3.1 μm high density particle (produced as in 2a - c, functionalized and streptavidinised as described in examples 3a, 3b) in an Eppendorf micro-tube and incubated for 30 min under shaking. 10 μl of 20 μg/ ml ds DNA (Sigma D 1501) solution was added to 500 μl of a 0.15% suspension of red 3.1 μm high density polylysin particles (produced as described in 2 a-c and 3a, 3c) in 10 mM PBS, pH 7.4. After a vortex step (Bender & Hobein, Vortex Genie 2) at step 8, the suspension was incubated for 5 sec at room temperature under light shaking for 12 hours. The suspension was centrifuged twice with 1 ml 10 mM PBS, pH 7.4, washed (1000 rpm, 5 min) and resuspended again in 500 μl 10 mM PBS, pH 7.4.

The buffer composition of the gel matrix

The gel matrix in the micro-reaction vessels was in the following aqueous buffer medium: 5 mM KH2PO4 (Merck 4873) / Na2HPO4 (Merck 6580), 150 mM NaCl (Fluka 71381), 0.024 % (w/ v) NaN3 (Fluka 71290), 1.875 % (v/ v) albumin (Miles 81-177), 0.05 % (w/ v) EDTA (Fluka 03685), 1.3 mM Tris (Merck 8382), 1.25 mM N-acetyl-L-cysteine (Merck 12422), 0.025 % (w/ v) Mucin (MMA 1778).

The test is performed on a single test chemical (see paragraphs 6.

(a) Antigenic: Synthetic peptides Ag-2, TcD and TcE are obtained from Alta Bioscience (Birmingham, UK). 1 ml of 0.15% (w/v) suspension of red 3.1 μm high density particles (produced as in 2a - c, functionalized and streptavidinised as described in examples 3a, 3b) were coupled with 2 ng TcD, 35 ng Ag-2 and 282 ng TcE, synthesised by standard techniques using a biotinyl lysine in addition to the antigen sequence and purified by RP-HPLC to > 90% purity by standard methods, with the peptides being prepared in a 10 x stock solution in H2O (20 TcD, 350 ng Ag-2, 2820 TcE per ml).The reaction mixture was then incubated for 30 min at room temperature under light shaking, then the particle suspension was centrifuged for 5 min at 1000 rpm, the residue decanted and washed twice with 10 mM PBS at pH 7.4. The final concentration of the particles was again 0.15% (w/v) in the suspension. The sensitized particles were then stored at 4 °C. A 0.15% (w/v) suspension of the sensitized synthetic particles as described in 4a was treated in the ultrasonic water bath for 5 min.After 10 min incubation at room temperature, the patient's serum or plasma was centrifuged for 10 min at 85 g in a specially designed centrifuge (ID centrifuge 12 S II, DiaMed, Cressier sur Morat). The results can be evaluated immediately after centrifugation. Positive results are detectable as distinct bands on the gel or 1 to 2 mm of penetration into the gel.Negative results are detected by a significant sediment of the synthetic particles deposited on the bottom of the vessel, in which case there is no band in or on the gel.

The test is performed on a single test tube.

(a) Antigenic: The following is the list of active substances in the active substance: 1 ml of recombinant antigen rK39 (0.6 mg/ml) in 10 mM Tris, pH 8.0, was dialyzed against 10 mM PBS, pH 7.4 (Sigma P 4417) at 4°C and then filtered by a Puradisc 25 AS membrane filter (Whatman). Biotinylation was performed with sulpho-NHS biotin (Pierce 21217). A 500 μl aliquot of rK39 in 10 mM PBS, pH 7.4 (300 μg) was dissolved with 17.5 μl of an aqueous 1 mg/ml solution of sulpho-NHS biotin. The reaction mixture was incubated for 150 min at room temperature. The biotinyl rK39 was then separated from free biotin by a QuarkSep salt solution (Enceule).Elution was performed with 10 mM PBS, pH 7.4, 0.05 % NaN3. Each ml of a 0.15% (w/v) suspension of red 3.1 μm high density particles (produced as in 2a - c, functionalized and streptavidinised as described in example 3a, 3b) was then coupled with 160 ng biotinyllated rK39, to produce a 10 x stock solution in H2O (1600 ng rK39 per ml). The test procedure is identical to that in Example 6, except that in this case a suspension of synthetic particles sensitized with rK39 is used.

Hepatitis B surface antigen test (covalent binding of monoclonal antibodies) is used to test the presence of hepatitis B in the liver.

(a) Antibodies: The monoclonal antibody (MAb) against hepatitis B surface antigen MIH9701 detects both ad and ay subtypes and was obtained from Medix Biotech, Walchwil, Switzerland. 200 μl of 3 mg/ ml solution MIH9701 were diluted to 1 ml with 0.1 M sodium acetate buffer, pH 5.5, and dialyzed overnight at 4°C against the same buffer. 900 μl of an ice cold 20 mM sodium metaperiodate solution were added to 900 μl of the resulting MAb solution and the reaction mixture incubated in the dark for 20 min at 0°C. The reaction was stopped by adding 11 μl of a 10% (w/ v) glycerol solution. The MAb were separated from the remaining reaction components by a chromatography step via a decomposition column.The volume of MAb after elution was 2.7 ml. 270 μl of a 50 mL solution of EZ-Link Biotin-LC hydrazide (Pierce 21340) was added to dimethyl sulfoxide (Sigma D 8418) and incubated under light shaking for 2 hours at room temperature. The reaction mixture was then dialyzed at 4°C against 10 mM PBS, pH 7.4.0.05 % NaN3, and then filtered by a 0.2 μm Puradisc AS filter (Whatman). A final desalination was repeated with a cyclic encapsulation set at 10 mM PBS, pH 7.4.05 0.05 % NaN3. The MAb thus biotinised was adjusted to 35 μg/ml in the same PBS buffer.100 μl of diluted MAb solution were added to 1 ml of 0.15% (w/v) suspension of red 3.1 μm high density particles (produced as in 2a-c, functionalized and streptavidinised as in 3a, 3b). This reaction mixture was incubated at room temperature under light shaking for 30 min. The particles were then washed 3 times by centrifugation with the same buffer (1000 rpm, 5 min). The test was performed as in Example 6 with the following differences: (i) A suspension of synthetic particles sensitized with MAb MIH9701 was used. (ii) The gel matrix did not contain anti-human globulinid.

The test is performed on a single test chemical (i.e. a single test chemical) and a single test chemical (i.e. a single test chemical).

(a) Antigenic: The following information is provided for the purpose of the analysis: After a vortex step (Bender & Hobein, Vortex Genie 2) for 5 sec at stage 8, the suspension was incubated under light shaking for 12 hours at room temperature. The suspension was washed twice by centrifugation with 1 ml of 10 mM PBS, pH 7.4, and resuspended in 500 μl of 10 mM PBS, pH 7.4. The final concentration of the particles was again 0.15% (w/v), which was maintained at 4°C.

HIV1/HIV2 Antibody differentiation test (using two different sensitized species of synthetic particles of different colours but otherwise similar physical properties)

(a) Antigenic: (b) Coupling as in example 6 with 72 ng gp-41 peptide and 40 ng gp36 peptide per 1 ml of 0.15% (w/v) particle suspension. Red particles (produced as in 2a-c, functioned and streptavidinised as described in example 3a, 3b) were carried out with the HIV-2 specific peptide (antigen sequence from gp36) and blue particles (produced as in 2a-c, functionalised and streptavidinised as described in example 3a, 3b) with the HIV-1 specific peptide (Anquenzp41) gp4c sensitive test. As in example 6, with the following differences: 10 μl of red and blue particulate suspension were pipetted into the reaction chamber of a micro-tube.The patient's serum was added to this. HIV-1/HIV-2 positive results were detectable as a distinct red/blue band on the gel or 1 to 2 mm penetrated into the gel. In this case, there was no sediment of particles. HIV-1 antibody positive results showed a blue band on the gel or penetrated into the gel, while the red particles formed a sediment on the bottom of the reaction vessel. HIV-2 antibody positive results showed a reverse picture. HIV-1/HIV-2 negative results were clearly detectable in the red-blue sediment on the bottom of the micro-reaction vessel.

The test chemical is used to detect the presence of antibodies to the antigen.

(a) Antigenic: S. aureus Protein A was derived from Sigma (P 6650); Goat anti-human IgM antibody (μ-chain specific) Biotin conjugate F (ab) 2 Fragment was derived from Sigma (B 2641). Biotinylation of protein A was performed as described in example 8, the biotinylated anti-IgM antibodies were adjusted to a concentration of 1 μg/ml of protein A in 10 mM PBS, pH 7.4, in an approach to 40 μg/ml 3.1 μm high density particles (1.5% w/v), in a further approach to 1 μg/ml particles (0.15% w/v), the biotinylated protein A to a concentration of 1 μg/ml particles (0.15% w/v).The results of the studies were presented in the following sections (see section 4.4.1.1, 4.4.1.2, 5.4.1.2, 5.4.1.2, 5.4.1.2, 5.4.1.2, 5.4.1.2, 5.4.1.2, 5.4.1.2, 5.4.1.2, 5.4.1.2, 5.4.1.2, 5.4.1.2, 5.5.2, 5.5.2, 5.5.2, 5.5.2, 5.5.2, 5.5.2, 5.5.2, 5.5.2, 5.5.2, 5.5.2, 5.5.2, 5.5.2, 5.5.2, 5.5.2, 5.5.2, 5.5.2, 5.5.2, 5.5.2, 5.5.2, 5.5.2, 5.5.2, 5.5.2, 5.5.2, 5.5.2, 5.5.2, 5.5.2, 5.5.2, 5.5.2, 5.5.2, 5.5.2, 5.5.2, 5.5.2, 5.5.2, 5.5.2, 5.5.2, 5.5.2, 5.5.3, 5.5.3, 5.5.3, 5.5.3, 5.5.4, 5.5.5, 5.5.5, 5.5.6, 5.5.6, 5.5.6, 5.5.6, 5.5.6, 5.6.6, 5.6.6, 5.6.6, 5.6.6, 6.6.7, 6.6.7, 6.6.7, 6.6.7, 6.6.7, 6.6.7, 6.6.7, 6.6.7, 6.7.8, 6.7.8, 6.7.8, 6.7.8, 6.7.8, 6.7.8, 6.7.8, 6.7.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8.8. This reaction mixture was incubated at room temperature under light shaking for 30 min. The particles were then washed three times by centrifugation with the same buffer (1000 rpm, 5 min). 1 ml of anti-IgM sensitized particles were centrifuged for 5 min at 1000 rpm and discarded. 20 μl of a 1 in 10 dilution of serum from a healthy person was added. After 5 s vortex to stage 8, the reaction mixture was incubated for 30 min at room temperature under light shaking.The following masses were then pipetted into an anti-IgG card (as described in example 6):Position 1: 25 μl Protein A Particle (1 μg/ml)Position 2: 25 μl Protein A Particle (1 μg/ml)Position 3: 25 μl Anti-IgM Particle (1 μg/ml)Position 4: 25 μl Anti-IgM Particle (1 μg/ml).Positions 1 and 3 were given 5 μl of 1 to 10 μl of diluted serum before adsorption, and 2 and 4 5 μl of 1 to 10 μl of diluted serum after adsorption.After 10 min incubation at room temperature, the test substance was centrifuged for 10 min at 85 g in a centrifuge specially designed for the micro-tube (ID centrifuge 12 S II, DiaMed, Cressier sur Morat). Both control tubes 1 and 3 showed markedly positive responses as described in example 6. Of the test approaches 2 and 4, 2 showed a positive response that was no different from that in tube 1, while in position 4 the particles formed a sediment on the bottom of the test vessel and thus showed a negative response.

The following table shows the results of the analysis:

(a) Antigens and coupling: (b) Test execution: Conventional As described in example 6, 25 μl of the sensitized synthetic particles and then 5 μl of serum/plasma were pipetted into the incubation chamber of prefabricated microtubes and centrifuged for 10 min at 85 g at room temperature in a microtubes-appropriate centrifuge (ID centrifuge 12 S II, DiaMed, Cressier sur Morat) after 10 min incubation. Modified As described in example 6, 25 μl of the synthetic particles and then 5 μl of serum/plasma were pipetted into the incubation chamber of pre-fabricated microtubules. 1) 5 s centrifugation, 30 g2) 5 min rest, 0 g3) 10 min centrifugation, 85 g) Evaluation: The evaluation was as in example 6. In tests using anti-IgG gel matrices, a general reinforcement of especially weak positive results is observed.

Claims (27)

1. Process for detecting an analyte in a sample fluid by agglutination, in which the sample fluid is brought into contact with an agglutination reagent and an inert matrix, the reaction mixture is exposed to the action of gravitation and the reaction between the analyte and the agglutination reagent is determined, characterised in that synthetic particles are used as agglutination reagent, which behave substantially as erythrocytes relative to the matrix and have a mean diameter ≤ 5 µm and a specific density ≥ 1.1 g/cm³.
2. Process according to claim 1, characterised in that the synthetic particles have a mean diameter in the range of 1 to 5 µm.
3. Process according to claim 2, characterised in that the synthetic particles have a mean diameter in the range of 2 to 4 µm.
4. Process according to claim 1, characterised in that the synthetic particles have a specific density in the range of 1.1 to 1.8 g/cm³.
5. Process according to claim 4, characterised in that the synthetic particles have a specific density in the range of 1.15 to 1.4 g/cm³.
6. Process according to one of claims 1 to 5, characterised in that synthetic particles composed of organic polymers or copolymers are used.
7. Process according to claim 6, characterised in that synthetic particles composed of styrene or styrene derivatives are used.
8. Process according to one of claims 1 to 7, characterised in that coloured particles are used.
9. Process according to claim 8, characterised in that red-coloured particles are used.
10. Process according to one of claims 1 to 9, characterised in that ligand molecules capable of bonding to the analyte to be determined are immobilised on the surface of the particles.
11. Process according to claim 10, characterised in that the ligand molecules are immobilised via adsorptive, covalent or high-affinity interactions.
12. Process according to claim 10 or 11, characterised in that peptides, proteins, nucleic acids, nucleic acid analogues, saccharides, lipids, hormones or metabolites are immobilised via covalent or high-affinity interactions on the surface of the particles.
13. Process according to claim 10 or 11, characterised in that cell membranes, fragments of cell membranes or lysed cell materials or pathogens are bound by adsorptive interactions on the surface of the particles.
14. Process according to one of claims 1 to 13, characterised in that the analyte is determined by an immune reaction.
15. Process according to claim 14, characterised in that the analyte is an antibody.
16. Process according to claim 15, characterised in that class-specific detection of antibodies is carried out.
17. Process according to claim 14, characterised in that the analyte is an antigen.
18. Process according to one of claims 1 to 13, characterised in that the analyte is determined by a nucleic acid hybridisation reaction.
19. Process according to one of claims 1 to 18, characterised in that a particulate matrix is used.
20. Process according to claim 19, characterised in that the mean diameter of the matrix particles is in the range of 10 to 200 µm.
21. Process according to one of claims 1 to 20, characterised in that a plurality of analytes are determined simultaneously, a differently coloured agglutination reagent being used for each analyte.
22. Process according to one of claims 1 to 21, characterised in that the reaction mixture contains detergents or/and mucins.
23. Process according to one of claims 1 to 22, characterised in that the reaction mixture contains a reducing agent.
24. Process according to one of claims 1 to 23, characterised in that the effect of gravitation on the reaction mixture comprises centrifugation.
25. Process according to claim 24, characterised in that centrifugation is carried out at a plurality of intervals.
26. Reagents kit for detecting an analyte in a sample fluid, comprising

    (a) at least one reaction vessel containing an inert matrix and

    (b) at least one type of synthetic particles with a mean diameter ≤ 5 µm and a specific density ≥ 1.1 g/cm³ having ligand molecules immobilised on its surface.
27. Reagents kit according to claim 26, characterised in that a plurality of reaction vessels are arranged on a card or disc.