Liquid purification or separation – Processes – Liquid/liquid solvent or colloidal extraction or diffusing...
Reexamination Certificate
1997-12-16
2001-03-20
Savage, Matthew O. (Department: 1723)
Liquid purification or separation
Processes
Liquid/liquid solvent or colloidal extraction or diffusing...
C210S782000, C530S815000, C436S528000, C436S823000
Reexamination Certificate
active
06203706
ABSTRACT:
DESCRIPTION
The present invention concerns a method for the detection of an analyte in a sample liquid by agglutination wherein the sample liquid is contacted with an agglutination reagent and an inert matrix, the reaction mixture is subjected to the action of gravitation and the reaction between the analyte and the agglutination reagent is determined. Furthermore new reagents are disclosed for carrying out the method according to the invention.
Methods for the detection of analytes by haemagglutination and particle agglutination tests are known. These tests are used above all to detect antigens and antibodies for the diagnosis of infectious diseases. However, a common disadvantage of all these methods is that they are time consuming and labour-intensive and that the results are often very difficult to interpret.
Gel immunoassay methods in which haemagglutinates are separated from individual non-agglutinated erythrocytes by a centrifugation step through an inert matrix (cf 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 WO 95/31731) are known for haemagglutination tests in which unfixed erythrocytes are used as agglutination reagents. In these methods agglutinated erythrocytes are retained on or in the inert matrix and can thus be distinctly separated from non-reacting single erythrocytes which can penetrate through the matrix and sediment to the bottom of the reaction vessel.
Only in EP-A-0 305 337 which goes back to a priority application from the year 1987 is there a statement that synthetic particles such as latex or polymerized agarose can also be used as agglutination reagents. However, there is no information about the properties of such synthetic particles. Furthermore this proposal was not taken up in the following period either in patents or in other publications. On the contrary recent publications such as EP-A-0 557 546 emphasise unfixed erythrocytes as agglutination reagents.
However, a disadvantage of unfixed erythrocytes is their instability which, under certain reaction conditions, leads to haemolysis. This instability often results in undesirably short expiry dates of products based on unfixed erythrocytes. Moreover a standardized and reproducible production of erythrocyte preparations, in particular of antibody-coupled or antigen-coupled erythrocyte preparations, can only be achieved in a very labour-intensive manner. This practically rules out an adequate uniformity of the physical properties of erythrocyte preparations.
Hence an object of the present invention was to at least partially eliminate the aforementioned disadvantages which result from the use of erythrocytes as agglutination reagents. In particular the present invention intends to provide synthetic particles which can be used as agglutination reagents and are capable of simulating the behaviour of erythrocytes in known gel immunoassays. Furthermore the synthetic particles should allow a simple coupling of biological substances and should be structured so as to be at least equivalent agglutination reagents to the previously used erythrocytes with regard to sensitivity and specificity. It should be possible to use these synthetic particles in agglutination methods that are easier to carry out and evaluate than the previously known methods.
This object is achieved by a method for the detection of an analyte in a sample liquid by agglutination, wherein the sample liquid is contacted with an agglutination reagent and an inert matrix, the reaction mixture is subjected to the influence of gravitation and the reaction between the analyte and the agglutination reagent is determined, characterized in that synthetic particles are used as the agglutination reagent whose diameter and density are selected such that their behaviour towards the matrix is essentially the same as that of erythrocytes.
It was surprisingly found that, if both the density and the diameter are optimally adjusted, synthetic particles can in fact be produced which can pass through an inert matrix without difficulties in a gel immunoassay and hence perfectly simulate fresh erythrocytes.
In the experiments that led up to the present invention it was observed that there was a greater delay in the passage of rigid polymer particles compared to erythrocytes (diameter 6-8 &mgr;m). Under given standard conditions this meant specifically that commercially available standard particles with a diameter of 3-7.5 &mgr;m only sediment incompletely in the gel matrix whereas larger (11.9 &mgr;m) or smaller synthetic particles (<1 &mgr;m) are only able to penetrate slightly into the gel. Although an increase in the parameters centrifugation period (from 10 min up to 50 min) and centrifugation speed (from 1030 to 1300 rpm) improved the sedimentation it was still not complete. Even if both these measures would have been able to achieve an optimal sedimentation, a change/increase in the centrifugation period and speed is, however, very disadvantageous for the following reasons:
1. The system according to the invention should allow a particularly non-time-consuming procedure relative to comparable methods. Thus a possible centrifugation period of 50 min would have tripled the intended test period of 20 min including incubation.
2. As is well known to a person skilled in the art the sensitivity in the gel centrifugation method decreases with increasing centrifugation speeds.
3. If the specified parameters can be adhered to, a commercially available centrifuge would be available for the system according to the invention.
It was surprisingly found that during passage through the inert matrix a comparable behaviour to erythrocytes can be achieved with synthetic particles of a smaller diameter than that of erythrocytes and a higher density than usual. In this connection it is possible to use spherical as well as asymmetric synthetic particles.
Synthetic particles with an average diameter of ≦5 &mgr;m and in particular of 1 to 5 &mgr;m have proven to be preferable for the method according to the invention. The average diameter is particularly preferably between 2 and 4 &mgr;m. The specific density of the particles is preferably ≧1.1 and is more favourably in the range of 1.1 to 1.8. The specific density is preferably in the range of 1.1 to 1.6 and most preferably in the range of 1.15 to 1.4 especially when the detection method is carried out under standard conditions as defined for known commercial erythrocyte gel immunoassays (e.g. DiaMed).
The synthetic particles used as an agglutination reagent in the method according to the invention are preferably organic polymer or copolymer particles. Particularly preferred materials are styrene and styrene derivatives such as bromostyrenes and in particular copolymers thereof. The manufacture of uniform polymer particles in a size range suitable for the agglutination reagents according to the invention is adequately well-known to a person skilled in the art (Arshady (1992): Suspension, emulsion and dispersion polymerization: A methodological Survey. Colloid & Polymer Science 270, 717-732; Okubo and Shiozaki (1992): Production of micron-size monodisperse polymer particles by seeded polymerization utilizing dynamic swelling method with cooling process. Polymer International 30, 469-474). Such methods usually utilize a combination of emulsion and dispersion polymerization in order to produce particles with exactly defined physical properties.
For the visual detection of the agglutination reaction in the method according to the invention it is possible to incorporate dyes, in particular water-insoluble dyes, into the particles. Thus synthetic particles dyed blue, yellow, green, black or red have proven to be particularly well suited for a visual evaluation. In a particularly preferred embodiment of the present invention red particles are used due to their especially good detectability.
The robustness of the synthetic particles enables a large number of different ligands to be immobilized on their surface by known standard industrial methods. In this process it is expedient to
Bashforth David
Hobbs Roderick N.
Margetts Graham
Marshall Michael J.
Roberts Mark J. J.
Arent Fox Kintner Plotkin & Kahn
Cecil Terry K.
Savage Matthew O.
Stiftung fur Diagnostische Furschung
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