METHOD AND MEANS FOR SEPARATION OF A PARTICULATE PHASE IN ANALYSIS

Abstract: Method for the separation by centrifugation of a particulate phase from a liquid phase in which the particulate phase is insoluble. The separation is part in a method of analysis which, besides the separation step, comprises at least one washing or incubation step. The invention is characterized in microorganisms being added to the liquid phase prior to the centrifugation. These microorganisms are suspensible in the liquid phase prior to the centrifugation but are sedimented therein during the centrifugation. All the particulate phase together with the microorganisms then will adhere to the centrifugation vessel. The invention also relates to means for decantation which contains microorganisms having the properties given above.

Description: METHOD ANDMEANSFORSEPARATIONOFAPARTICULEPHASEINANALYSIS The present invention relates to a method for the separation by centrifugation of a particulate phase from a liquid phase in which the particulate phase is insoluble; said separation being part of a method of analysis comprising, besides the separation step, at least one washing or incubation step. In the claims and in the descriptive part, separation by centrifugation refers to the centrifugation as such as well as to the removal of the supernatant after the centrifugation. Primarily, the invention is related to methods of analysis which involve biospecific affinity reactions, e g immunological ones, and in which a labelled reactant is partitioned between a liquid phase and a particulate phase. The partition should be related uniquely to the entity or substance to be determined. After separation a measurement of the labelled reactant is then performed in either the liquid phase or in the particulate phase or separately in both of them. The invention also relates to means for decantion of a liquid phase from a particulate phase, said decantion being preceded by a centrifugation and being part of methods of analysis comprising at least one washing step or at least one incubation step. The invention is an improved method for separation by centrifugation in that the particulate phase is completely retained to the centrifugation vessel during the removal of the liquid phase from above. Earlier this has been a problem. At certain test methods which are based on the so called biospecific affinity reactions, suspensible particulate phases were used which by the aid of centrifugation and decantation can be separated from a liquid phase. After centrifugation it can be difficult to remove the supernatant without also removing the particulate phase. This is a large problem, especially when one wants to measure a property which is dependent on the total amount of particulate phase. If one wants to perform the measurement in the centrifugation vessel, problems may also arise for systems utilizing non-suspensible phases such as discs and strips made of paper. Even these ones may disappear from the vessel during the decantation. The liquid phase is aqueous and contains salts and a buffer. These components are adjusted to the ion strength and pH of the method of analysis to be performed. A similar problem has been solved for the separation by centrifugation of whole blood. In this case, a "clot"-fraction, i.e. the coagel, is separated from a serum fraction. In the US patent specification No. 3780035, a viscous silicone oil of suitable density has been added prior to the centrifugation. At the centrifugation, the oil will settle like a cover in the interphase on top of the "clof'-fraction. Thereafter, decantion can easily be carried out, without the serum fraction becoming contaminated by the "clot"-fraction. In practice, this silicone oil method has the drawback that the oil is difficult to add. The silicone oil is water-insoluble and will place itself around a particulate phase, whereby a supposed further treatment, e g repeated washing by water, is complicated. At the methods of analysis mentioned above the same problem will of course also arise . However, it has now surprisingly been found that all these difficulties can be overcome. According to the invention this is achieved by a separation, characterised in an addition of microorganisms to the liquid phase before or during the centrifugation; said microorganisms being suspensible in the liquid phase before the centrifugation but being sedimented therein during the centrifugation. After centrifugation, all the particulate phase together with the microorganisms will, according to" the invention, adhere to the centrifugation vessel. The means for decantation of a liquid phase from a particulate phase after centrifugation are characterized in containing microorganisms which are suspensible in the liquid phase prior to the centrifugation but which are sedimented therein during the centrifugation. Thereby the means adhere the particulate phase to the centrifugation vessel. This method for separation by centrifugation gives advantages in those methods of analysis, in which working sequences consisting of at least one centrifugation in order to separate a particulate phase from a liquid phase is carried out. Other steps in such methods of analysis may be i.a. at least one incubation in order to perform some type of reaction, e g a biospecific affinity reaction. Examples of other steps are washing of the particulate phase and measurement of the labelled reactant. During the centrifugation, the microorganisms added will adhere the particulate phase to the bottom of the centrifugation vessel. The particulate phase then will adhere to the bottom better than if the microorganisms had not been used. After centrifugation the supernatant can easily be removed for instance by decantation. At a supposed and subsequent washing and/or incubation step the added microorganism is resuspended together with the particulate phase. It is known that microorganisms can be sedimented by centrifugation and that they then will become adhered to the bottom of the centrifugation vessel. However, this fact has previously not been used according to the invention in methods of analysis. Different microorganisms may be used such as e g gramnegative or grampositive bacterias, for instance Staphylococcus aureus or E. coli respectively, and fungi such as baker's yeast, e g Saccharomyces cerevisiae. In most cases, microorganisms have a density which is close to that of water. This is essential for the invention, as they are not allowed to sediment by themselves. If they have such an inherited ability to sediment rapidly, the particulate phase will adhere poorly to the bottom during the centrifuga¬ tion. Of course, the microorganism must " sediment during the centrifugation. It is also essential that the density of the microorganism is larger than that of the liquid phase used, as the micro¬ organism otherwise would be able to migrate in an improper direction. By microorganisms are meant living organisms which are smaller than 2 x 10 mm (J jellander- and D Danielsson, Klinisk ikrobiologi, Almqvist & iksell, Stockholm 1973) . Microorganisms especially convenient for use according to the invention have a size of between 0,2 7 um and 15 um, but can of course also be larger and thread-like, such as e g a mycelium from a fungus. However, it is most convenient to use microorganisms having a size of between 0,5 urn and 11 um. Of course, the microorganisms are not allowed to autolyze prior to or during the separation having been performed according to the invention. In order to prevent autolysis the microorganisms may be treated by heat, e g by boiling, or be treated by some low-molecular aldehyde, e g formaldehyde, or by both of them. The treatment must not remove the general adhesive capacity of the microorganisms used. Performed in a proper way, these two types of pretreatments will lead to inert and killed micro¬ organisms, which is of the greatest importance for the stability of the means for decantation. - For certain methods of analysis it is essential that the microorganisms added are inert towards the chemicals used in the analysis. The amount of microorganisms added may be varied within wide limits. The exact amount differ from case to case depending on i.a. the amount and kind of p particulate phase. As an ultimate lower limit 10 cells per ml of the mixture consisting of liquid and particulate phase may be given. There is no upper limit, except that practical and economical reasons are of importance. This means e.g. that the supernatant to be decanted, has to contain the major part of the labelled reactant which has been partitioned into the liquid phase. This is easily realized by the man ordinary skilled in the art. The centrifugation can take place at different g-values. At a g-value of between 2 000 and 3 000 a centrifugation for 10 minutes is enough, g-values down to 200 may be used too, but then of course a prolonged time for the centrifugation is required. Upwards, the g-value is limited by the construction of the centrifuge. The centrifugation time is also dependent on the distance for sedimentation, which is related to the length of the tube among other things. The shape of the particulate phase may vary within wide limits. Physically, the particulate phase may be solid in the form of grains of different sizes such as beads, plates, discs and rods or in the form of one or more strips, rods or slips. Chemically, it consists either of an organic or an inorganic material or a mixture of both of them. In the method of analysis involving biospecific affinity methods previously mentioned, the particles may consist of porous glass or different organic polymers being insoluble in the liquid phase. Examples of such insoluble polymers are poly (acryl amide), pol (hydroxyacrylates) , cellulose, agarose, dextran, starch and their derivatives. Often, the derivatives are in a crosslinked form. The method decribed in the invention for separation of a particulate phase from a liquid phase has been found to be most convenient when two types of particulate phases simultaneously are present, e.g. a paper disc together with beads of agarose or crosslinked dextran (Sephadex , Pharmacia Fine Chemicals AB, Sweden) . Of course, the invention also comprises those cases when the particulate insoluble phase itself is a microorganism. In such a case the particulate phase itself adhere to the bottom of the centrifugation vessel, but, when necessary, this adhesive effect may be enhanced by the addition of microorganisms according to the invention. Below, examples are given showing how the invention may be utilized. Of course the man in the art realize easily when and how he can apply the invention to his own problem. Example 1 Preparation of reagents for the separation. Staph lococcus aureus sta Cowan 1 (ATCC 12598 alt NCTC 8530) was cultured in 1 000 ml of CCY-medium (Arvidsson et al, Acta pathol Microbiol Scand. sect B 79(1971) p 399) in shake flask culture at 37 C for 8 hours. The bacterias were separated from the culture medium by centrifugation at + 5 C and were washed once with saline of + 5°C and being buffered by phosphate to pH 7,4 (PBS). The cells were then suspended in PBS to a final concentration of 10 % (weight of wet cells/volume suspension) . Formaldehyde was added to a final concentration of 1 % and the cell suspension so formed was pumped through a heat exchanger which was kept at a temperature of 80 C. The heat exchanger consists of a glass tube 900 cm long, having an inside tube diameter of 0,5 cm, being formed like a coil and being immersed in a water bath of a thermostate. The pump rate was adjusted to keep every volume unit of the cell suspension at 80 C for 10 minutes. The cell suspension so treated by heat and formal¬ dehyde was centrifuged and the supernatant contain¬ ing no cells was decanted. The bacterial cells were once more suspended in PBS to a concentration of 10 % and may be stored in the cold (4 - 8°C) for a long time without being changed. Example 2 Method for separation At a radioimmunologic determination of Thyroidea stimulating hormone, variant Dry Spot (Phadebas TSH Dry Spot. Pharmacia Diagnostics AB, Uppsala) beads consisting of crosslinked dextran and being coated by antibodies were used as particulate phase, while whole blood adsorbed to a disc of filter paper constitutes the test sample. The final step in this method of analysis is -the separation of soluble reactants from insoluble ones. This separation is performed in the following new way: To the reaction mixture consisting-of the paper disc, the beads mentioned above and-buffer with dissolved human TSH R labelled with I ( I-hTSH from Phadebas TSH Dry Spot), 2,5 ml of a 0,3 % suspension of Staphylococcus aureus cells prepared according to the description in example 1 was added. This mixture was centrifuged at room temperature for 15 min at 2 000 x g. Thereafter the supernatant is decanted by turning the centrifugation tube upside down. The last drops of liquid may advantageously be removed from the tube by placing the tube upside down on a filter paper for 5 min. By the addition of the bacteria prior to the centrifugation, it is achieved a) that all the solid reactants are retained at the bottom of the centri.fugation tube when decanting and b) that the I-hTSH bound to the solid phase can be determined quantatively at a subsequent measurement in a gamma- counter. The standard curves being obtained were equivalent to those ones being obtained in the method for separation recommended by the manufacturer.

Claims: AMENDED CLAIMS (received by the International Bureau on 08 June 1983 (08.06.83)) Claim 1 (amended) Method for separation by centrifugation of a particulate phase from a liquid phase in which the particulate phase is insoluble, said separation being part of a method of analysis involving biospecific affinity reactions and comprising besides the separation step at least one washing or incubation step and being c h a r a c t e r i.z e d in addition of microorganisms, before or during the centrifugation, to the liquid phase containing the particulate phase, said microorganisms being suspensible in the liquid phase before the centrifugation but being sedimented therein during the centrifugation. Claim 2 (amended) Method according to claim 1 or 2, c h a r a c t e r i z e d in the microorganisms being selected from those having a size of between about 0,2 μ and 15 / urn Claim 3 (amended) Method according to claim 1 , c h a r a c t e r i z e d in the centrifugation being carried out at more than 200 x g. Claim 4 (cancelled)

Inventors: RUDIN LARS, RUDIN, LARS
Applicants: PHARMACIA DIAGNOSTICS AB
Application year: 1983
Main classification: G01N33
IPC codes: B01D21, C12Q1, G01N33