The present invention concerns improved methods for detecting micro-organisms particularly yeast and bacteria in colloidal mixtures such as beer. It is also concerned with detecting micro-organisms in air and solid samples (such as food or bacterial spores) which can be placed in a liquid phase suspension or dissolved.
The production of foodstuff and beverages such as beer is accompanied by testing for the presence of certain micro-organisms in order to ensure the quality of the end-product. The brewing process may for example require in-line testing every few hours of a sample having a volume of at least 25 ml, and preferably sample volumes of for example 250 ml. Particulate matter which may include micro-organisms, namely yeast and bacteria, must then be separated from the sample and then tested to determine the presence or absence of specific micro-organisms. Devices used to achieve this include the Bibby disposable vacuum filter unit having a flat filter with an average pore diameter of 0.45 um and the Nalgene filter holders with receivers, having a flat filter with an average pore diameter of 0.45 um or 0.2 um (see for example Merck Laboratory Supplies Catalogue 1998, p. 482). Such devices allow the filtration of maximum sample volumes of only 100 ml, have a flat surface area of 50 cm2 and can take up to 30 minutes to test a sample due to their complexity of use. Once their maximum volume has been filtered, they become blocked by particulate matter such as proteins present in the sample fluid (e.g. lager, ales and other colloidal solutions) and any subsequent filtration would require pressures so high as to cause cell lysis, preventing the detection of the micro-organisms and giving false results.
Prior art devices take substantially more time to separate and detect micro-organisms from a sample than is required using the devices and methods of the present invention. WO 01/11006 discloses improvements to the prior art allowing more rapid separation and detection of micro-organisms, as well as relatively simple and easy subsequent recovery of, and thus testing for, micro-organisms. The devices and methods of the present invention are distinct from those of WO 01/11006, and in particular (as detailed below) the methods involve a washing step rather than just a resuspension step.
The devices and methods of the present invention further improve upon WO 01/11006 and the other prior art by providing a yet more rapid and simple method for the separation and detection of micro-organisms from a sample.
In particular it has been found that the devices of the present invention can rapidly detect all of the bacteria in e.g. a 100 ml volume of test solution containing as few as 1-3 bacteria. Similar results can also be achieved with larger volumes e.g. 1000 ml of lager containing 1-3 bacteria.
According to the present invention there is provided a method for detecting the presence of a micro-organism in a fluid sample, comprising the steps of:
i) passing said sample through the sample inlet of a filter device comprising a plurality of hollow fibre filter membranes which have attached to them a first member of a specific binding pair, said micro-organism displaying the second member of said specific binding pair, said membranes having first and second ends, an outer surface and an inner surface defining a lumen, said first end of each of said membranes being open and communicating with said sample inlet and flow through said second end of each of said membranes being restricted such that said flow occurs only through said first end and the pores of said membranes, the sample mixture being filtered through the pores of said membranes, leaving a filtrand in said lumen of said membranes;
ii) washing the unbound part of said filtrand from said lumen of said membranes;
iii) detecting the presence of any of said specific binding pairs attached to said membranes; and
iv) correlating the results of detection step (iii) with the presence of said micro-organism in said fluid sample.
The sample may for example have a volume of at least 25, 50, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900 or 1000 ml.
A “member of a specific binding pair” is one of two different molecules, having an area on the surface or in a cavity which specifically binds to and is thereby defined as complementary with a particular spatial and polar organization of the other molecule. The members of the specific binding pair are commonly referred to as ligand and receptor (antiligand), sbp member and sbp partner, sbp members or the like. These are usually members of an immunological pair such as antigen-antibody, although the term does have a broader meaning encompassing other specific binding pairs.
The term “antibody” in its various grammatical forms is used herein to refer to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antibody combining site or paratope. Such molecules are also referred to as “antigen binding fragments” of immunoglobulin molecules.
By “attached” is meant that the first member of the specific binding pair is physically constrained and prevented or hindered from separating from the membrane. This may be by means of covalent binding to the membrane, for example of an antibody to the membrane, or it may be by other means, for example van der Waals'forces. Thus a first member of a specific binding pair, such as an antibody, may simply be trapped by the fibres of a membrane, and need not be covalently bound to it.
In particular antibodies and other first members of specific binding pairs may be covalently bound to polypropylene membranes by cross linking them with glutaraldehyde. Methods of cross-linking will be readily apparent to a person skilled in the art and can be achieved by e.g. oxidation or halogenation of the polypropylene groups to a reactive monomer.
In order to test samples which are not in the liquid phase, for example air samples or particulate matter or other solids such as foodstuffs, they must first be placed in a fluid suspension. There are two basic situations in which this is done: testing air or other gaseous samples for the presence of micro-organisms, and testing solids such as powders and foodstuffs for the presence of micro-organisms.
1. Testing of Gaseous Samples:
In order to test a gaseous sample such as air for the presence of a particular micro-organism, an air (i.e. gas) filtration device is employed having a pore diameter sufficient to entrap the chosen micro-organism. For example, in the case of anthrax (Bacillus anthracis) which has a diameter of 1 urn, a pore size of e.g. 0.2 pm may be used. The air filtration device may also comprise a fan to encourage the flow of air through the filter, and a large volume of air can be passed through the filter in a relatively small period of time, all particulate matter of a greater size than the pore diameter being retained. The filter is then contacted with a sample fluid (e.g. a buffer or another solution, fluid or mixture into which the given micro-organism can be suspended or solubilised) in which the micro-organism can be suspended or solubilised, and any entrapped micro-organisms are placed into suspension or solubilised as appropriate (i.e. fluid-phase). Once this initial stage of capturing micro-organisms and placing them in suspension/solubilising them has been completed, the rest of the method of the present invention can then be worked (i.e. steps (i)-(iv) as defined above).
The exact nature of the sample fluid such as a buffer or other solution, fluid or mixture in which the given micro-organism can be solubilised or suspended is dependent upon the nature of the micro-organism itself and the first member of the specific binding pair used in step (i) above. The function of the sample fluid is to enable the given micro-organism to interact with the first member of the specific binding pair such that the specific binding pair can be formed as appropriate to allow the detection of the given micro-organism. For example, the first member of the specific binding pair may form a specific binding pair with the or a given micro-organism, and thus its detection can be effected, the formation of a specific pair being correlated with the presence of the given micro-organism.
2. Testing of Solids:
In order to test a solid such as a foodstuff (e.g. cake or bread) for the presence of a given micro-organism, the solid is contacted with a sample fluid in which the micro-organism can be suspended or solubilised, and any entrapped micro-organisms are placed into suspension or solubilised as appropriate. As before, once this initial stage of capturing micro-organisms and placing them in suspension/solubilising them has been completed, the rest of the method of the present invention can then be worked (i.e. steps (i)-(iv) as defined above).
Thus according to the present invention there is also provided a method for detecting the presence of a micro-organism in a solid sample, comprising the steps of: a) contacting said solid sample with a sample fluid such that said micro-organism is placed in fluid-phase, to give a fluid sample; and b) performing the method of steps (i)-(iv) upon said fluid sample.
Another example of the testing of solids is the testing of the contents of a package such as a letter. The above method is performed, namely contacting the solid with a sample fluid and then performing steps (i)-(iv) to determine the presence of the given micro-organism. However, the contents of the package must first be removed. In order to achieve that, the present invention provides apparatus comprising a first chamber into which said package may be placed, a second chamber containing sample fluid, means (such as a pump or syringe) to alter the relative pressure in said first and second chambers, said second chamber communicating with said first chamber by means of a piercing implement having a lumen through which flow may occur.
The present invention also provides a method for testing the contents of a package for the presence of a given micro-organism, comprising placing said package in said first chamber, altering the relative air pressure in said first and second chambers, and contacting said package with said piercing implement such that said package is pierced and solid matter in said package, particularly particulate matter, passes from said package to said sample fluid and is suspended or solubilised in said sample fluid to give a fluid sample. The method of steps (i)-(iv) is then performed with the fluid sample and any micro-organisms therein detected.
Thus according to the present invention there is provided a method for testing the contents of a package for the presence of a given micro-organism, comprising the steps of:
a) placing said package in a first chamber which communicates by way of a piercing implement with a second chamber containing a sample fluid;
b) contacting said package with said piercing implement such that any solid material in said package is able to pass via said piercing implement to said sample fluid in said second chamber, to give a fluid sample; and
c) performing the method of steps (i)- (iv) upon said fluid sample.
The method may additionally comprise the step of effecting a relative gaseous pressure change in said first and second chambers such that the pressure in said first chamber is greater than that in said second chamber. The movement of any solid matter, such as particulate matter, from the package in the first chamber to the second chamber can thereby be encouraged or effected.
In order to allow the package to be safely removed from said apparatus, it may be provided with a self-sealing pad which is contacted with the piercing implement-upon its removal from the piercing implement the pad will self-seal, preventing any further egress of solid from the package and making it safe to remove from the apparatus.
The piercing implement may be sealed such that the seal is broken upon piercing of the package-in this way it is possible to maintain a pressure difference between the first and second chambers, the pressure being reduced in the second chamber relative to that in the first. Alternatively, the pressure in the chambers can be altered after piercing has occurred. The piercing implement may be self-sealing. Thus also provided according to the present invention is a method for testing the contents of a package for the presence of a given micro-organism, comprising placing said package in said first chamber, contacting said package with said piercing implement such that said package is pierced and solid matter in said package, particularly particulate matter, is able to pass from said package to said sample buffer (or said another solution, fluid or mixture into which the given micro-organism can be suspended or solubilised), and reducing the air pressure in said second chamber relative to that in said first chamber, such that said solid matter passes from said package to said sample buffer and is suspended or solubilised in said sample buffer (or said other solution, fluid or mixture into which the given micro-organism can be suspended or solubilised).
Alternatively of course it is possible not to effect any pressure difference between the first and second chambers and to effect the transfer of solid matter between the two by e.g. gravity.
In all of these cases of testing gases and solids for the presence of a given micro-organism the present invention provides the distinct advantage of giving rapid and accurate results. The time taken for a sample assay can be many times less than other comparable assays such as ELISA and PCR. A typical assay time using the present invention is 15 minutes.
In the case of sampling air, extremely large volumes of air may be passed through the filter (for example 10, 25, 50, 100, 250, 500, 750, 1000, 2500, 5000, 7500 or 10000 litres of air) and the retained particulate matter tested in an extremely short period of time-again an assay time of 15 minutes is typical. Thus these testing methods for micro-organisms allow for testing in previously un-suggested ways, and allow for the extremely rapid generation of results, which it has not been previously possible to achieve.
The present invention with its testing of fluid samples for the presence of micro-organisms can of course also be used for the testing of fluid samples for the presence of e.g. anthrax with the use of e.g. an anti-anthrax antibody as the first member of a specific binding pair.
As well as testing for anthrax, the present invention can also be used to test for any other micro-organisms which may be used in biological warfare.
The sample is passed through the filter membranes under conditions which allow for binding of the first and second members of the specific binding pair. If it is desired or necessary to reduce the possibility of binding of moieties other than the second member of the specific binding pair to the first member of the specific binding pair, more stringent binding (also referred to as “stringent hybridisation”) conditions may be used. For example, the temperature or pH may be varied to provide stringent hybridisation conditions. Appropriate hybridisation conditions will depend upon the nature of the members of the specific binding pair and will be readily apparent to one skilled in the art.
At wash step (ii), it is obviously essential that bound specific binding pairs are not washed from the lumen of the membranes, and so the washing step removes the filtrand other than any second members of the specific binding pair which have bound to the first members of the specific binding pair attached to the membranes. It is of course possible that, occasionally, a specific binding pair could become detached from the membranes and washed, together with the unbound filtrand, from the lumen of the membranes. Although this may occasionally happen, substantially all (i.e. essentially all) of the first members of the specific binding pair having bound to them second members of the specific binding pair remain attached to the membranes.
The detection step (iii) may take one of many forms. For example, the specific binding pair may be detected by the binding to the pair or to the micro-organism of a labelled probe, for example a detection antibody which incorporates an enzyme (the classical enzyme immunosorbent assay, EIA). Alternatively the probe may be a radiolabelled antibody or a fluorescently labelled antibody. Other probes will be readily apparent to a person skilled in the art.
Alternatively, the micro-organism may be eluted (i.e. separated) from the first member of the specific binding pair and a separate detection step employed. For example, eluted micro-organism can be lysed and any ATP released detected using a luciferase assay.
Alternatively, micro-organism specific antibodies may be used, or the eluate can be plated out on a general (or micro-organism specific) nutrient culture and the growth of any micro-organism colonies detected. The eluate may also be tested using oligonucleotide probes specific to the micro-organism in a conventional PCR test.
The range of detection steps available for use in the present invention also means that the membranes may have attached to them first members of a plurality of specific binding pairs. The different first members may be mixed together and attached throughout the membranes, or first members of a given specific binding pair may be attached to the membranes at a specific position. The detection step employed may allow the general detection and/or quantification of the specific binding pairs, or it may allow the detection and/or quantification of a chosen specific binding pair or pairs. For example, the presence of a first specific binding pair could be detected using a first fluorophore, and the presence of a second specific binding pair detected using a radiolabel, or by the use of a second fluorophore having an excitation and/or emission spectrum distinguishable from that of the first fluorophore.
Prior art devices typically present filtered particulate matter as a hard “biscuit” (a relatively highly compressed high density block of particulate matter) on a membrane surface, micro-organisms and other particulate matter blocking and being trapped in membrane interstices. This biscuit is difficult to remove and difficult to process to enable it to be tested for the presence of micro-organisms.
The configuration of the devices of the present invention results in the formation of a resuspendable “cake” which can be subsequently washed away, and allows the use of lower pressures during filtration, which in turn prevents the formation of a dense biscuit and the need for higher pressures. If operated at higher pressures, lysis of bacteria can occur, in turn giving incorrect results. High pressure can also cause distortion of bacteria, allowing them to pass through the membrane and giving incorrect results.
Prior art filtration device and methods include those of GB 2135902, EP 302949, WO 94/00222, WO 84/00015, U.S. Pat. No. 5,863,501, U.S. Pat. No. 5,814,179, U.S. Pat. No. 4,501,793, JP 4-135478 (WPI Abstract 1992-205001), JP 63-104615 (WPI Abstract 1988-165566), JP 63-088007 (WPI Abstract 1988-145060) and JP 61-133105 (WPI Abstract 1986-200908). However, none of them disclose or suggest the methods of the present invention including each of the steps necessary to obtain the results which they are capable of providing. In particular, the prior art does not suggest producing a filtrand in the form of a re-suspendable “cake” rather than a more solid “biscuit”, nor does the prior art suggest washing the filtrand from the membranes as part of a subsequent processing step.
For example, JP 63-104615 discloses a device for separating e.g. viruses from fluids, comprising a plurality of porous hollow cellulose fibres, one end of them being embedded in a filler material and open to the atmosphere, and the other end being sealed. However, it does not suggest the specific methods of the present invention, nor their advantages. Other filtration devices are also known from e.g. the “CultureGard Hollow Fiber Filter” from Cole Parmer (www. coleparmer. com), product code EW-295 10-50.
Advantageously, it has been found that polypropylene fibre membranes may be used (the Cole Parmer product above uses cellulose hollow fibre membranes).
In particular, membranes can be treated with a wetting agent such as iso-propanol and/or a detergent such as Tween-20 to make them more hydrophilic and susceptible to the attachment of the first member of the specific binding pair. The surprisingly good results achieved by treating membranes with iso-propanol prior to attaching antibodies to them are shown below. For example, a membrane can be soaked in iso-propanol and then allowed to dry, prior to treatment with Tween 20 (typically, 0.1%) and antibody.
By pre-treating the membranes with a wetting agent such as an alcohol it has been found that the rate of flow of the sample mixture through the membranes is increased massively. This is particularly true when comparing dried treated membranes with dry untreated membranes. This increased flow rate ensures that micro-organisms are collected without causing their lysis or forcing them through the membranes.
Useful detergents include non-ionic detergents, particularly Tween 20, more particularly a solution of 5% Tween 20.
The use of a plurality of hollow fibre filter membranes also provides a relatively large surface area (typically at least three times as much) across which filtration may take place, when compared to the surface area provided by a single device of similar overall dimensions (i.e. size) having a single flat membrane. This also allows for the filtering of a relatively large volume of sample prior to any blockage of pores occurring. This is particularly useful with turbid samples (e.g. stout) which contain large amounts of particulate matter which can rapidly block flat filter membranes.
The exact nature of the filter membrane material has also been found to be important—commercially available polypropylene hollow fibre membranes having an average pore diameter of 0.2 um pre-treated with iso-propanol have been found to allow much greater flow rates than e.g. polysulfone membranes having an average pore diameter of 0.2 m, even when identically pre-treated. Thus in a preferred embodiment of the present invention, the hollow fibre membrane is a polypropylene membrane. Naturally, other membranes may also be used, particularly those having similar physical characteristics e.g. a similar average pore diameter and area of pores per unit area of membrane surface, and these include the likes of polysulfone, cellulose acetate and nylon membranes.
Hollow fibre membranes used in the present invention may have an average pore diameter of 0.2 urn.
Also provided according to the present invention is a device having a sample inlet and a plurality of hollow fibre filter membranes which have attached to them a first member of a specific binding pair, the second member of said specific binding pair being displayed by a given micro-organism, said membranes having first and second ends, an outer surface and an inner surface defining a lumen, said first end of each of said membranes being open and communicating with said sample inlet and flow through said second end of each of said membranes being restricted such that said flow occurs only through said first end and the pores of said membranes, such that a sample mixture passed into said device through said sample inlet is filtered through the pores of said membranes, leaving a filtrand in said lumen of said membranes.
The ease of testing for micro-organisms using the methods and devices is supplemented by the speed of filtration-as is seen from experimental results, the present invention allows for the detection of specific micro-organisms in a given volume of sample fluid in a fraction of the time required by other devices, and is frequently at least ten times as fast.
The present invention also provides the important advantage of providing consistent results for a given sample, even when a highly turbid mixture is being filtered-at least 99% consistency between different sets of results is readily achievable. This compares favourably to results obtained using flat membranes, which can be relatively inconsistent.
In various embodiments of the present invention, the hollow fibre membranes consist of polypropylene, which is used in many biomedical applications due to its low capacity for absorbing proteins. It has not been previously suggested that polypropylene membranes should have antibody or other members of specific binding pairs attached to them. In particular the present invention shows that by treating the polypropylene membranes with iso-propanol as described herein, the specific binding pair members remain attached to the membranes even when washed with detergents such as SDS and Tween-20. This has not previously been suggested.
The invention will be further apparent from the following description, with reference to the several figures of the accompanying drawings, which show, by way of example only, one form of filter device.
Unless stated otherwise, all procedures were performed using standard protocols and following manufacturer's instructions where applicable. Standard protocols for various techniques including PCR, molecular cloning, manipulation and sequencing, the manufacture of antibodies, epitope mapping and mimotope design, cell culturing and phage display, are described in texts such as McPherson, M. J. et al. (1991, PCR: A practical approach, Oxford University Press, Oxford), Sambrook, J. et al. (1989, Molecular cloning: a laboratory manual, Cold Spring Harbour Laboratory, New York), Huynh and Davies (1985, “DNA Cloning Vol I-A Practical Approach”, IRL Press, Oxford, Ed. D. M. Glover), Sanger, F. et al. (1977, PNAS USA 74 (12): 5463-5467), Harlow, E. and Lane, D. (“Using Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratory Press, New York, 1998), Jung, G. and Beck-Sickinger, A. G. (1992, Angew. Chem. Int. Ed. Eng., 31: 367-486), Harris, M. A. and Rae, I. F. (“General Techniques of Cell Culture”, 1997, Cambridge University Press, ISBN 0521 573645), “Phage Display of Peptides and Proteins: A Laboratory Manual” (Eds. Kay, B. K., Winter, J., and McCafferty, J., Academic Press Inc., 1996, ISBN 0-12-402380-0).