EP0533772A1

EP0533772A1 - Detection process

Info

Publication number: EP0533772A1
Application number: EP91911125A
Authority: EP
Inventors: Lester John Farr; Vincent Habib Atrache; Geoffrey Harold Braid; David Ernest Forester Harrison
Original assignee: Biotech Australia Pty Ltd
Current assignee: Biotech Australia Pty Ltd
Priority date: 1990-06-08
Filing date: 1991-06-07
Publication date: 1993-03-31
Also published as: NZ238444A; WO1991019003A1; EP0533772A4

Abstract

Procédés de détection et/ou de dénombrement des contaminants se trouvant dans des échantillons tels que des échantillons pharmaceutiques et des échantillons de denrées alimentaires, de boissons, et d'eau. Les procédés associent la spécificité de l'immunodétection à la vitesse des systèmes automatisés afin d'obtenir des procédés rapides de détection des contaminants spécifiques dans les échantillons.Methods of detecting and / or counting contaminants found in samples such as pharmaceutical samples and samples of food, beverages, and water. The methods combine the specificity of immunodetection with the speed of automated systems to obtain rapid methods of detecting specific contaminants in samples.

Description

DETECTION PROCESS

Technical Field

The present invention relates to processes for the detection of contaminants in samples, such as bacteria, yeasts or fungi, in, for example, food, beverage, water, dairy, cosmetic or pharmaceutical samples.

Background Art

In quality control applications it is important to be able to detect and in some cases quantify, a wide range of contaminants in the particular product undergoing analysis. In some cases evidence of a contaminant of a particular class will be sufficient, whereas in other cases knowledge of the specific contaminant is required.

Methods for the detection of microorganisms in samples include use of devices which detect the metabolic activities of microorganisms by electrical, chemical, or biochemical means, for example those which use a cell connected to an electrical circuit with a means for the measurement of variations in impedance or conductivity in the cell to give an indication of growth within the cell.

Examples of electrical detection devices for use in these methods include devices such as the Bactometer TM

(Vitek, Systems Inc., USA), the Malthus (Radiometer, Denmark) and the Rabit (Don Whitley Scientific, UK). Other examples of indirect detection systems include those which detect changes in oxygen concentration or production of gaseous or soluble metabolites or pH changes or redox or enzymatic reactions.

Electrical detection devices for the direct enumeration of microorganisms based on the electrical resistance produced by non-conductive particles suspended in an electrolyte are also known. An example of such a device is the Coulter counter. At present such instruments cannot be used for the detection of specific microorganisms present in a mixed population.

Although instruments for electrical detection have found widespread use for major industrial applications a drawback is that these techniques, if and when used for the selective detection of bacteria such as Salmonella species which is performed using selective medium, produce an unacceptably high false positive rate of approximately 10%. This unacceptably high false positive rate leads to the need to confirm positive results by an alternative technique.

Furthermore such systems cannot at present be used for the routine screening detection of other food poisoning bacteria such as Listeria species which are also of interest in quality control of food products.

Detection methods using immunoenrichment steps have been demonstrated (International Patent Application No. PCT/AU88/00274) . These methods rely on the specific binding of a particular microorganism to an antibody-coated surface to enable detection of low levels of the microorganism in a mixed population. The bound microorganisms are either grown to a detectable level followed by immunoassay or are released from the surface followed by growth on a non-selective medium and observation of the resultant colonies. This technique, however, requires a relatively high operator involvement per test sample. Whereas this is acceptable for low to medium throughputs, for laboratories with high to very high numbers of samples, the hands-on time per sample is often deemed unacceptable. For such laboratories, it is desirable to provide an accurate, automated detection method with the advantages of speed coupled with a low false positive rate. Description of the Invention Definition

The term "cell" is used herein to describe a receptacle which may include a lid and may include baffle(s) depending into the receptacle from the lid or from interior surface(s) of the receptacle.

Description

The present invention provides rapid and accurate detection processes with the benefits of immunoenrichment and with the low operator involvement and automation of detection systems such as the Bactometer TM or Coulter count .erTM

According to a first embodiment of this invention there is provided a process for the detection and/or quantification of at least one contaminant having metabolic activity, in a sample, which process comprises: providing a cell adapted to detect the metabolic activity of the at least one contaminant within the cell, the cell having a surface to which the sample is exposed carrying antibodies capable of binding the at least one contaminant which antibodies are specific for the at least one contaminant; inoculating the cell with the sample; and detecting the metabolic activity in the cell caused by the at least one contaminant bound to the antibodies.

Preferably the process for the detection and/or quantification of at least one contaminant having metabolic activity, in a sample, comprises: providing a cell adapted to detect the metabolic activity of the at least one contaminant within the cell, the cell having a surface to which the sample is exposed carrying antibodies capable of binding the at least one contaminant which antibodies are specific for the at least one contaminant and are capable of binding the at least one contaminant without adversely affecting its ability to replicate; inoculating the cell with the sample for a sufficient time to allow binding of the at least one contaminant to the antibodies; removing unbound sample; introducing a medium for the growth of the at least one contaminant into the cell; incubating the cell under conditions suitable for the growth of the at least one contaminant; and detecting the metabolic activity in the cell caused by the at least one contaminant bound to the antibodies.

Preferably the unbound sample is removed by washing.

In one preferred form the process additionally comprises incubating the sample in a suitable medium to resuscitate the at least one contaminant prior to inoculating the sample into the cell.

In another preferred form the process additionally comprises a preenrichment step which comprises: exposing the sample to a solid substrate having a surface carrying antibodies capable of binding the at least one contaminant, which antibodies are specific for the at least one contaminant, to allow binding of the at least one contaminant to the antibodies; removing unbound sample from the substrate ; followed by releasing the bound at least one contaminant from the substrate with a. releasing agent; and introducing the released at least one contaminant into the cell, for the detection process.

Alternatively, the preenrichment step may comprise incubating the sample in a medium specific for the growth of the at least one contaminant to specifically increase the numbers of the at least one contaminant with respect to other contaminants which may be present in the sample, prior to inoculating the sample into the cell. The process of the first embodiment may alternatively comprise: providing a solid substrate having a surface carrying antibodies capable of binding the at least one contaminant 5 which antibodies are specific for the at least one contaminant; exposing the sample to the solid substrate for a

^"«r sufficient time to allow binding of the at least one contaminant to the antibodies; 10 introducing the substrate into a cell adapted to detect metabolic activity of the at least one contaminant within the cell; and detecting the metabolic activity in the cell caused by the at least one contaminant. 15

Preferably, the substrate is washed to remove unbound sample prior to introducing the substrate into the cell.

Preferably, the process comprises: 20 introducing a medium for the growth of the at least one contaminant; and incubating the cell under conditions suitable for the growth of the at least one contaminant on introducing the substrate into the cell, and the antibodies are capable of 25 binding the at least one contaminant without adversely affecting its ability to replicate.

In this version of the process, resuscitation and/or preenrichment steps could also be included, prior to 30 exposure of the sample to the solid substrate.

The metabolic activity may be detected by a variation in impedance or conductivity in the cell, a change in oxygen concentration or the production of gaseous soluble 35 metabolites, or changes in hydrogen ion concentration or changes in redox potential or enzymatic activity or other means of measuring metabolic activity. As an alternative to detecting variation in impedance or conductivity the presence of the at least one contaminant could be detected by measurement of the polarization which occurs as the at least one contaminant is bound by the antibodies.

Where a process of the first embodiment is to be used for the quantification of the at least one contaminant, growth curves and detection times obtained are compared with standard curves for known concentrations of the at least one contaminant, to provide a measure of contaminant numbers.

According to a second embodiment of this invention there is provided a process for the direct detection and/or enumeration of at least one contaminant in a sample which process comprises: providing a cell having a surface to which the sample is exposed carrying antibodies specific to the at least one contaminant; inoculating the cell with the sample for an appropriate period to allow binding of the at least one contaminant to the antibodies; removing unbound material from the cell; adding an electrolyte solution to the cell; adding a releasing agent to release the bound at least one contaminant; and detecting the at least one contaminant by measuring the change in electrical resistance of the electrolyte solution on release of the bound at least one contaminant from the cell surface.

Preferably the process comprises washing the cell to remove unbound material from the cell.

Preferably the process comprises: adding a growth medium for the at least one contaminant to the cell; incubating the cell for a sufficient time to allow the bound at least one contaminant to replicate and multiply to a detectable level; and removing the growth medium prior to adding the electrolyte solution to the cell and the antibodies are capable of binding the at least one contaminant without adversely affecting its ability to replicate.

Preferably the growth medium is removed by washing.

Again, preenrichment and resuscitation steps could be incorporated, prior to inoculating the sample into the cell.

The process of the second embodiment may alternatively comprise: providing a solid substrate having a surface carrying antibodies capable of binding the at least one contaminant which antibodies are specific for the at least one contaminant; exposing the sample to the solid substrate for a sufficient time to allow binding of the at least one contaminant to the antibodies, or binding to the antibodies and growth; inserting the substrate into a cell; adding an electrolyte solution to the cell; adding a releasing agent to the cell to release the bound at least one contaminant; and detecting the at least one contaminant by measuring the change in electrical resistance of the electrolyte solution in the cell on release of the bound at least one contaminant from the substrate.

The process may additionally comprise introducing the substrate with bound at least one contaminant into a cell containing a growth medium for the at least one contaminant for a sufficient time to allow the bound at least one contaminant to replicate and multiply to a detectable level; and removing the growth medium prior to exposure to the electrolyte solution, and in this form the antibodies should be capable of binding the at least one contaminant without adversely affecting its ability to replicate. In this version also, pre-enrichment and/or resuscitation steps may be used prior to exposure of the sample to the solid substrate.

Electrolyte solutions suitable for use in the processes of the second embodiment include physiological saline and phosphate buffered saline.

Typically, the sample to be analysed in a process of the invention is a food, beverage, water, dairy, cosmetic or pharmaceutical sample, which may be prepared for analysis by standard methods of sample preparation.

Typically, the at least one contaminant to be detected in a process of the invention is a microorganism, such as a bacterium, yeast or fungus. Other contaminants include other prokaryotic and eukaryotic single celled organisms.

Preferably, the contaminant is a bacterium selected from Salmonella species and Listeria species, particularly Salmonella typhimurium or Listeria monocvtoαenes.

Where growth media are used it is preferred that they are selective media. A selective growth medium may enhance the specificity of the process.

Where the contaminant is a microorganism, it is preferred that the antibody is raised against a surface antigen of the microorganism. More preferably, the surface antigen is a flagellar protein or lipopolysaccharide.

Where the removal of unbound material, or sample, or growth medium is required in a process of the invention, the removal step is preferably one which includes washing. The invention provides for processes wherein the antibody is coated onto the interior walls of the cell, and those where the cell is provided with a separate antibody support such as a cap having dependent baffles coated with 5 antibody.

In one preferred form for use in the detection of

<ff microorganisms the "ImmunoCap" is used. This can be designed in a number of ways as indicated in Figure 1 10 (la,le,lf).

In one form of using the "ImmunoCap" following a pre-enrichment step a volume of the pre-enrichment broth is removed and placed in a receptacle, which may be purpose 15 made. To this is added the "ImmunoCap" which seals the receptacle and captures specific microorganism species using specific antibodies immobilised on the suspended support system.

20 The "ImmunoCap" is then transferred to the well of an electrical detection device, eg. a Bactometer TM or Coulter counter TM. This is then inserted into the standard incubation system for the detection device and variations in iimmppeeddaannccee oorr ccoonndduuccttiivviittyy ccaauusseedd bbyy ggeenneerraattiioonn ooff cchhaarreged 5 metabolites are measured in the case of the Bactometer .r

2 T'M oorr rreelleeaassee ooff oorrggaann:isms into an electolyte in the case of Coulter counter -r the T'M

If one or more of the "ImmunoCap" supports is 30 removable and coated with a specific antibody to eg.

Listeria monocytoσenes. then following a positive reaction in the detection device, the removable support can be taken from the assembly. This can then be used to perform an ELISA type reaction or other form of secondary analysis to 35 confirm or otherwise the presence or absence of the specific organism or organisms. Secondary analysis may be used at the end of any version of the process.

In another form, for use in the detection of microorganisms, the sample is added to a cell coated with antibody and incubated so that microcolonies of bound microorganism form. After washing to remove unbound material the antibody-bound microcolonies are detected by measuring variations in impedance or conductivity in the cell caused by generation of charged metabolites or by changes in electrical resistance caused by release of the microcolonies from the cell surface. The microcolonies may alternatively be detected directly.

The sample to be analysed may be subjected to preenrichment and/or resuscitation steps.

Some versions of the processes of the invention contemplate the release of bound contaminants. Contaminants bound to the antibodies may be released from the antibodies, or the antibody-contaminant complex may be liberated from the cell surface.

Releasing agents suitable for dissociating the antibody-contaminant bond include:

1) heat

2) chaotropic agents such as 4.5 M MgCl₂ pH 7.5 or 2.5M Nal pH 7.5 3) polarity reducing agents such as ethylene glycol in solutions of up to 50% 4) pH change inducing agents such as glycine/HCl pH 2.5, aqueous H₃ pH 11 or 0.5% KOH pH 12.5.

Preferably the releasing agent is 0.5% KOH pH12.5. To this solution protein may be added to provide a carrier for the released contaminant. Releasing agents suitable for dissociating the antibody-contaminant complex from the cell or substrate surface include papain and similar proteolytic enzymes.

The released contaminant can be measured directly by Coulter Counter. Release is also desirable in those processes which include a step whereby the released contaminants are plated out for colony counts or are subjected to other analysis such as ELISA or fluorescent immunoassay. Contaminants may be released from the

ImmunoCap baffle or dipstick for plating if desired or for adding to an ELISA well, or other secondary analysis, as well as for direct detection.

Brief escription of the Drawings

FIGURE 1

Figure la: shows a single ImmunoCap to fit a Bactometer type of well.

Figure lb: shows a bank of ImmunoCaps.

Figure lc: shows a well designed to hold 5 to 10 mL of enrichment broth.

Figure Id: shows a bank of wells, each designed to hold 5 to 10 mL of enrichment broth.

Figure le: shows a further enhancement whereby a removable baffle can be an integral part of the ImmunoCap. This baffle can be coated with antibodies to a specific species/strain which can be removed for confirmation.

Figure If: shows a further enhancement of the ImmunoCap shown in Figure le with a different antibody coated baffle configuration. FI^GURE i : shows an inverted plan (undersi_de₎ view of the ImmunoCap as shown in Figure if.

FIGURE 2

FI^GURE 2a: shows a schematic representation of a process of the invention using an ImmunoCap as shown in Figure la. The process involves a first step of resuscitation/pre-enrichment. This is followed by the specific immunocapture of a putative contaminant using the Immuno^Cap of Figure la. The final step in the process involves placing the ImmunoCap into the well of an electrical detection device and measuring changes in impedance^' or conductivity.

FI^GURE 2b: shows a typical positive result for the process of Figure 2a.

FI^GURE 2c: shows a typical negative result for the process of Figure 2a.

FIGURE 3

This is a schematic representation of a confirmation of a presumptive positive using an ImmunoCap as shown in Figure le. The removable dipstick is removed from the

Immuno^Cap shown in Figure le and is placed into a container to which conjugate is added. The dipstick is then washed an^d substrate added. Colour development indicates a positive result.

FIGURE 4

FI^GURE 4a: this shows a schematic representation of another process of the present invention, wherein the a^bsence or presence of micro-colonies are detected by changes in impedance or conductivity.

SUBSTITUTE SHEET - 12/ - _, - -

FIGURE 4b: shows a typical positive result for the process of Figure 4a.

FIGURE 4c: Shows a typical negative result for the process of Figure 4a.

FIGURE $

Shows a graph representation of the results of Example 3 at 10 Salmonella per ml. The graph shows typical conductance curves demonstrating comparative detection time for Salmonella following immunoenrichment using antibody coated dipstick (— ) and casein control coated dipstick (...).

FIGURE 6

Shows a graph representation of the results of 5 Example 3 at 10 Salmonella per ml. The graph shows typical conductance curves demonstrating comparative detection time for Salmonella following immunoenrichment using antibody coated dipstick ( ) and casein control coated dipstick (...).

FIGURE 7

Shows a graph representation of the results of ₇ Example 3 at 10 Salmonella per ml. The graph shows typical conductance curves demonstrating comparative detection time for Salmonella following immunoenrichment using antibody coated dipstick ( ) and casein control coated dipstick (...).

SUBSTITUTE SHEET - 13 - Best Method of Carrying Out the Invention

Standard devices are available for the automated detection of microbial contaminants in samples.

The processes of the first embodiment of the present invention can use automated detection devices such as the Bactometer , the Malthus or the Rabit, or pH detection units, or units for the detection of oxygen depletion, or for detection of production of gaseous or soluble

■metabolites or for measuring redox potential or enzymatic activity.

The antibodies for use in the processes of the invention can be either obtained commercially or prepared using standard techniques for the raising of antibodies. The use of polyclonal and monoclonal antibodies is included. For some applications mixtures of antibodies may be desirable, for instance, where different species or serotypes are to be detected, but not distinguished between in the sample being tested. Alternatively, it may be desirable for some applications to use single species or serotype specific antibodies but to check different aliquots of the same sample using the different antibodies to distinguish the species and/or serotypes present in the sample. Antibodies can be tested for their specificity and/or their effect on the replication of bound contaminants by standard techniques.

The binding of the antibodies to the cell surfaces or substrate surfaces can be performed by standard techniques.

Resuscitation, pre-enrichment and growth of the samples can be performed by standard techniques of microbiology for the processes of the invention. - 14 - The samples or cells or baffles may be exposed to subsequent detection procedures such as ELISA techniques, or plating techniques. Alternative methods which may be used include immunofluorescent techniques using whole fluorescent labelled antibodies or fluorescent labelled Fab fragments, or colorimetric detection processes. Each of the secondary analysis techniques can be performed using methods and reagents standard in the appropriate arts.

Secondary detection methods can be used for confirming the presence and/or quantity of the contaminants identified, in any of the processes of the invention.

Release of contaminants or contaminant-antibody complexes from cells, substrates or baffles can be performed using standard techniques employing suitable dissociating agents.

Agents suitable for dissociating the antibody- contaminant bond include: heat; chaotropic agents such as 4.5M MgCl_ pH 7.5 or 2.5M Nal pH 7.5; polarity reducing agents such as ethylene glycol in solutions of up to 50%; and pH change inducing agents such as glycine/HCl pH 2.5, aqueous H₃ pH 11 or 0.5% KOH pH 12.5.

Agents suitable for dissociating the antibody-contaminant complex from the cell or substrate surface include papain and similar proteolytic enzymes.

The process of the second embodiment of the present invention can use automated detection devices such as the Coulter Counter. These processes involve the direct . detection of the contaminant rather than detecting a product or an effect generated by the contaminant. - 15 - Since the initial capture is carried out using specific antibodies for the contaminant of interest then any change in electrical measurement will be due to that contaminant. In other words, the risk of another type of contaminant giving an interfering response is virtually eliminated, and hence the false positive rate is substantially reduced.

The determination of suitable incubation conditions, selection of growth media and electrolytes can all be performed by standard techniques. The choice of incubation conditions and growth media will, of course, be influenced by the nature of the contaminant(s) being detected.

Washing steps are performed with standard washing buffers compatible with the contaminants and antibodies.

Similarly, the choice of suitable releasing agents can be performed by standard techniques. This choice may be influenced by whether it is desirable to keep the contaminant alive for secondary analysis.

The cells for use in the processes of the invention may comprise commercially available well units such as those provided for use with a Bactometer or Coulter Counter.

Alternatively, the cells may be purpose made from materials suitable for binding antibodies and holding samples for analysis.

Similarly, substrates for carrying antibodies could comprise commercially available dipsticks either with or without preformed antibody coatings or baffles or paddles made from suitable materials for antibody coating.

Materials suitable for use include polystyrene, polyvinyl chloride, nylon, nitrocellulose, agarose and titanous hydroxide. - 16 -

Example 1

Figure 2 shows a process of the invention in schematic form. The contaminant in this case is a bacterium.

In this case, the sample to be tested is subjected to a resuscitation/pre-enrichment step which allows damaged cells to become revived prior to analysis. The-- pre-enrichment step improves the sensitivity of the assay, concentrating the organism of interest in the sample. The next step incorporates the use of the ImmunoCap to selectively capture organisms of interest using the specific antibodies on the baffles. This takes place by transferring 5 to 10 mL of the pre-enrichment broth into the ImmunoCap well and incubating for a short period of time.

The ImmunoCap is now removed and washed if necessary, before placing in the Bactometer or equivalent well containing electrodes and a suitable broth medium. The wells are next placed in the incubator unit and the impedance of the broth measured as normally carried out in the Bactometer. During this time, microcolonies will grow on the surface of the baffles and their metabolites will cause a change in the impedance or conductance of the broth medium.

Example 2

Figure 3 shows how a presumptive positive result from the Bactometer can be confirmed using the ImmunoCap with removable baffle (Figure le,lf).

In this version, following a positive reaction in the instrument, the removable baffle coated with specific antibodies to a particular species of organism, eg. Listeria monocytogenes or Salmonella typhimurium is placed into a tube (or the Bactometer well could be reused if washed).

During the incubation, the baffle has specifically captured this species and microcolonies grow. The baffle is now - 17 - washed, incubated with a specific antibody-conjugate, washed and further incubated with a substrate. Generation of a colour on the baffle acts as confirmation of the presence of the specific species of microorganism etc.

Alternatively, the bound cells can be released from the baffle and added to an ELISA well if the Optical Density (OD) is required to be measured, or released onto an agar plate etc if formation of colonies is required.

Esample 3

Figure 4 shows a process of the invention in schematic form. The contaminant is a microorganism such as a bacterium.

In this case, the sample to be tested is subjected to a resuscitation step or a preenrichment step whereby a baffle carrying antibodies specific for the microorganism of interest is inserted into the sample to bind the microorganism of interest.

The baffle may be washed and then the microorganism is released into the cell which in this case is a Bactometer well carrying antibodies, specific to the microorganism, which do not adversely affect the ability of the microorganism to replicate, on the interior side walls of the well. The cell is incubated to allow binding of the microorganism to the antibodies and growth, the cell containing a medium which may be selective or non-selective for the growth of the microorganism.

The Bactometer unit is used to monitor growth. As the sample is added the conductivity rises and plateaus as microorganisms are bound by antibodies. The broth is removed, the cell washed and fresh medium added. The conductivity declines at first but as growth of bound organisms occurs, the conductivity increases. - 18 - If the contaminant is not present the increase in conductivity after washing does not occur.

E-sample 4 Salmonella ImmunoCap dipsticks: Automated detection using a

Bactometer following capture.

A Bactometer impedance/conductance detection system (Bactometer M128 - Vitek Systems 595, Anglum Drive, Hazelwood, Missouri, USA) was evaluated for the automated detection of Salmonella, following initial capture on ImmunoCap dipsticks.

Methoflolgy Coating of dipsticks with anti Salmonella flaoella antibodies

Nunc Maxisorp Immunosticks (A/S Nunc-Postbox 280-Kamstrup-DK4000 Roskilde-Denmark.) were incubated in a solution containing 30μg/mL sheep anti-Salmonella flagella antibodies in lOmM Phosphate buffer pH8.1, for 24 hours at room temperature ( 22°C) . The sticks were then washed once with water and then incubated in a solution containing 0.1% gelatine in lOμm phosphate buffer, pH8.1 overnight at room temperature to block any remaining sites on the stick surface. The coated and blocked immunosticks were washed once with water and dried. The dried antibody coated sticks were used directly in the experiments described below. Anti-Salmonella flagella antibody coated dipsticks are also commercially available through Bioenterprises Pty Ltd., 28 Barcoo Street, Roseville NSW 2069, Australia.

Preparation of cg ein coated Con rol dipsticks

Nunc Maxisorp Immunosticks were incubated in a solution of 1.75% casein pH7.0 for 24 hours at room temperature. The coated sticks were washed once with water then dried. - 19 - Salmonella tvphimurium (ATCC7823) and E _L. coli (ATCC27325) were grown overnight at 37C, in buffered peptone water.

Serial dilutions of S. typhimurium were prepared in buffered peptone water and added to a stock solution of J _L cali to give final concentration of (organisms per ml) :

S. tvphimurium £_*. coli 10³ 10⁸ 10⁵ 10⁸

10⁷ 10⁸

Dipsticks were added to each of the dilutions for 20 min at room temperature ( 22C) .

The dipsticks were then washed 3 times with 4.5mL of buffered peptone water, before being fixed in position in the Bactometer wells, one to each well. Each dipstick was immersed in a modified lysine decarboxylase media (Vitek Systems) and incubated at 37C during which time, continual conductance measurements were taken and the data captured.

Standard sofware version R02.1 (Vitek Systems 595, Anglum Drive, Hazelwood, Missouri, USA) was used to determine the detection times for the various concentrations of Salmonella on both control and antibody coated dipsticks.

The results obtained are shown as follows: SALMONELLA CONCENTRATION DETECTION TIMES

ANTIBODY CONTROL

DIPSTICKS DIPSTICKS

10 per mL 7.8hrs 10.3hrs

5 10 per mL 6.0hrs 9.3hrs

7 10 per mL 3.5hrs 4.9hrs - 20 - See also Figures 5-7 which show percentage change in conductance vs. time in hours. The generated curves show that the antibody coated dipsticks are introducing selectivity in conjunction with the selective media which together reduce the detection time as compared to using selective media alone.

Example 5

USE OF COULTER COUNTER FOR THE DIRECT ESTIMATION OF SALMONELLA FOLLOWING SPECIFIC IMMUN CAPTURE AND ENRICHMENT, A Multisizer II Coulter Counter (Coulter Electronics, Luton, England) was used for estimation of bacterial number of Salmonella post capture using dipsticks coated with anti-flagella antibodies.

Methodology

Coating of dipsticks with anti-Salmonella flagella antibodies

Nunc Maxisorp Immunosticks (A/S Nunc-Postbox 280-Kamstrup-DK4000, Roskilde-Denmark.) were incubated in a solution containing 30μg/mL sheep anti-Salmonella flagella antibodies in lOmM Phosphate buffer pH8.1, for 24 hours at room temperature ( 22°C). The sticks were then washed once with water and then incubated in a solution containing 1.75% casein in pH7.0 overnight at room temperature to block any remaining sites on the stick surface. The coated and blocked immunosticks were washed once with water and dried. The dried antibody coated sticks were used directly in the experiments described below. Anti-Salmonella flagella antibody coated dipsticks are also commercially available through Bioenterprises Pty Ltd., 28 Barcoo Street, Roseville NSW 2069, Australia.

Preparation of casein coated Control dipsticks.

Nunc Maxisorp Immunosticks were incubated in a solution of 1.75% casein pH7.0 for 24 hours at room temperature. The coated sticks were washed once with water then dried. - 21 - Salmonella tvphimurium (ATCC7823) and E-- COli (ATCC27325) were incubated overnight at 37°C, in Buffered Peptone Water.

Antibody-coated and casein control dipsticks were added in triplicate to each of the following dilutions and incubated for 20 min. at room temperature ( 22°C).

Dilutions of S. tvphimurium (ATCC7823) in E. coli (ATCC27325) were prepared in saline:

10 cells/mL S. tvphimurium in 10⁸ cells/mL E. coli

10 3 cells/mL S. typhimurium in 108 cells/mL E. coli

10 4 cells/mL S. typhimurium in 108 cells/mL E. coli 10 5 cells/rnL S. typhimurium in 108 cells/mL E. coli o

10 cells/mL E.coli

The dipsticks were then removed and placed into tubes containing M-broth (DIFCO) (a non-specific growth medium) and incubated for 4 hours at 37°C. The dipsticks were removed from the growth medium then washed to displace any non-specifically bound organisms. The washed dipsticks were placed in an electrolyte solution and estimation of bacterial numbers in solution, before and after release of the organisms from the dipstick surface, made using the

Coulter Counter. The Salmonella bacteria were released from the dipstick surface into the electrolyte solution either by heating the dipstick in electrolyte at 100°C for 10 minutes or by treatment of the dipstick with papain lOμg/mL in 0.5M Acetate buffer pH5.2.

Results

The results are tabulated as follows (Tables 1-3). - 22 -

TABLE 1 ;

Particle Count Determined bv Coulter Counter Standard Curve of Salmonella tvphimurium

&- .

2.31 x 10' 1.02 x 10⁽

10 2.03 X

¹⁰! 3.12 x ιo^:

Aperture of 30μm diameter was used, at 0.88-2.00μm window using Isoton II electolyte as the diluent.

15

TABLE 2;

Particle Count Determined bv Coulter Counter after release of bound Salmonella from the dipstick by use of Heat

Aperture Count/dipstick * 10

20 size 0.88-2.00μm Control Antibody Specific Count Dipstick, coated, Antibody-coated Heat Heat minus control, x 10⁶/dipstick

25 10 cells/mL

Salmonella in 1 100⁸ cceel. ls/mL 0.45 0.23 -0.22

30 10* cells/mL

Salmonella in 1 100⁸⁸ ccee!lls/mL 0.17 0.33 0.16 E.coli

35 10 cells/mL Salmonella in 10⁸ cells/mL 0.49 0.50 0.01

E.coli - 23 -

10 cells/mL

Salmonella in 1 100⁸⁸ ccee:lls/mL 0.23 2.28 2.05 E.coli

10" cells/mL

Salmonella in

10⁸ cells/mL 0.15 12.00 11.85

E.coli

10⁸ cells/mL E_^-Cfili (non¬ specific binding) 0.16 0.20 Electrolyte blank 0.04 Each value is the mean of duplicates Aperture of 30μm was used, at 0.88-2.00μm window.

TABLE 3; Particle Count Determined bv Coulter Counter after Release of bound Salmonella from the dipstick by use of Papain

Aperture Count/dipstick x 10 size 0.88-2.00μrn Control Antibody Specific Count Dipstick, coated, Antibody-coated Papain Papain minus control, x 10 /dipstick

10 cells/mL Salmonella in 1 100⁸⁸ ccee:lls/mL N.T. N.T. E.coli

10 cells/mL Salmonella in 10⁸ cells/mL N.T. N.T E.COli - 24 -

10³ cells/mL Salmonella in

10⁸ cells/mL 0.13 0.04 -0.09

E.COli

10⁴ cells/mL Salmonella in

10⁸ cells/mL 0.20 2.19 1.99

10⁵ cells/mL Salmonella in

10⁸ cells/mL 0.33 8.13 7.80

E.COli 10⁸ cells/mL

E.coli (non- 0.24 0.15 specific binding)

Electrolyte blank 0.05

Each value is the mean of duplicates

Conclusion

The direct detection of low numbers of Salmonella in a mixed culture following specific antibody capture, their multiplication to a detectable level and subsequent release has been demonstrated using a Coulter Counter. This method

4 ccaann ddeetteecctt aapppproximately 10 cells/mL initial concentration.

- 25 - Industrial Applications

The present invention is of use in the field of quality control in the food, beverage, water supply, dairy, cosmetic and pharmaceutical industries.

Claims

- 26 -Claims

1. A process for the detection and/or quantification of at least one contaminant having metabolic activity, in a sample, which process comprises: providing a cell adapted to detect the metabolic activity of the at least one contaminant within the cell, the cell having a surface to which the sample is exposed carrying antibodies which specifically bind the at least one contaminant; inoculating the cell with the sample; and detecting the metabolic activity in the cell caused by the at least one contaminant bound to the antibodies.

2. A process according to claim 1 which process comprises: providing a cell adapted to detect the metabolic activity of the at least one contaminant within the cell, the cell having a surface to which the sample is exposed carrying antibodies capable of binding the at least one contaminant which antibodies are specific for the at least one contaminant and are capable of binding the at least one contaminant without adversely affecting its ability to replicate; inoculating the cell with the sample for a sufficient time to allow binding of the at least one contaminant to the antibodies; removing unbound sample; introducing a medium for the growth of the at least one contaminant into the cell; incubating the cell under conditions suitable to allow for growth of the at least one contaminant; and detecting the metabolic activity in the cell caused by the at least one contaminant bound to the antibodies.

3. A process for the detection of at least one contaminant having metabolic activity in a sample which process comprises: - 27 - providing a solid substrate having a surface carrying antibodies capable of binding the at least one contaminant which antibodies are specific for the at least one contaminant exposing the sample to the solid substrate for a sufficient time to allow binding of the at least one contaminant to the antibodies; introducing the substrate into a cell adapted to detect metabolic activity of the at least one contaminant within the cell; and detecting the metabolic activity in the cell caused by the at least one contaminant.

4. A process according to claim 3, which process comprises: introducing a medium for the growth of the at least one contaminant; and incubating the cell under conditions suitable for the growth of the at least one contaminant, on introducing the substrate into the cell, and wherein the antibodies are capable of binding the at least one contaminant without adversely affecting its ability to replicate.

5. A process for the direct detection and/or enumeration of at least one contaminant in a sample which process comprises: providing a cell having a surface to which the sample is exposed carrying antibodies specific to the at least one contaminant; inoculating the cell with the sample for an appropriate period to allow binding of the at least one contaminant to the antibodies; removing unbound material from the cell; adding an electrolyte solution to the cell; adding a releasing agent to release the bound at least one contaminant; and detecting the contaminant by measuring the change in electrical resistance of the electrolyte solution on release of the bound at least one contaminant from the cell surface. - 28 -

6. A process according to claim 5 which process comprises: adding a growth medium for the at least one contaminant to the cell; incubating the cell for a sufficient time to allow the bound at least one contaminant to replicate and multiply to a detectable level; and removing the growth medium, prior to adding the elctrolyte solution to the cell, and wherein the antibodies are capable of binding the at least one contaminant without adversely affecting its ability to replicate.

7. A process for the direct detection and/or enumeration of at least one contaminant in a sample which process comprises: providing a solid substrate having a surface carrying antibodies capable of binding the at least one contaminant which antibodies are specific for the at least one contaminant; exposing the sample to the solid substrate for a sufficient time to allow binding of the at least one contaminant to the antibodies, or binding to the antibodies and growth; inserting the substrate into a cell; adding an electrolyte solution to the cell; adding a releasing agent to the cell to release the bound at least one contaminant; and detecting the at least one contaminant by measuring the change in electrical resistance of the electrolyte solution in the cell on release of the bound at least one contaminant from the substrate.

8. A process according to claim 7, which process additionally comprises: introducing the substrate with bound at least one contaminant into a cell containing a growth medium for the at least one contaminant for a sufficient time to allow the bound at least one contaminant to replicate and multiply to a detectable level; - 29 - and removing the growth medium prior to exposure to the electrolyte solution, and wherein the antibodies are capable of binding the at least one contaminant without adversely affecting its ability to replicate. 5 *"^"

9. A process according to any one of claims 1 to 8 wherein the process additionally comprises incubating the sample in a suitable medium to resuscitate the at least one contaminant prior to introducing the sample into the cell, 10 or exposing the sample to the solid substrate.

10. A process according to any one of claims 1 to 8 wherein the process additionally comprises a pre-enrichment step which comprises:

15 exposing the sample to a solid substrate having a surface carrying antibodies capable of binding the at least one contaminant which antibodies are specific for the at least one contaminant to allow binding of the at least one contaminant to the antibodies;

20 then washing the substrate to remove unbound sample; followed by releasing the bound at least one contaminant from the substrate with a releasing agent; then introducing the released at least one contaminant into the cell, or exposing the released at least

25 one contaminant to the solid substrate.

11. A process according to any one of claims 1 to 8 wherein the process additionally comprises a preenrichment step which comprises incubating the sample in a medium

30 specific for the growth of the at least one contaminant to specifically increase the numbers of the at least one contaminant with respect to other contaminants which may be present in the sample, prior to incubation into the cell or exposure to the solid support.

35

12. A process according to any one of claims 1 to 8 wherein the samples is a food, beverage, water, dairy, cosmetic or pharmaceutical sample.

- 30 - 13. A process according to any one of claims 1 to 8 wherein the at least one contaminant is a bacterium, yeast or fungus.

14. A process according to claim 13 wherein the contaminant is a bacterium selected from Salmonella species and Listeria species.

15. A process according to claim 14 wherein the contaminant is Salmonella typhimurium or Listeria monocytoαenes.

16. A process according to any one of claims 1 to 4 wherein the metabolic activity detected is a variation in impedance or conductivity in the cell, a change in oxygen concentration, the production of a gaseous metabolite, a change in hydrogen ion concentration in the cell, a change in redox potential, a change in enzymatic activity, or the polarization which occurs as the at least one contaminant is bound by the antibodies.

17. A process according to any one of claims 1 to 8 wherein the contaminant is a microorganism and the antibody is raised against a surface antigen of the microorganism.

18. A process according to claim 17 wherein the surface antigen is a flagellar protein or a lipopolysaccharide.

19. A process according to claim 1, claim 2, claim 5 or claim 6 wherein the antibody is coated onto the interior walls of the cell.

20. A process according to any one of claims 1 to 8 wherein the cell is provided with an antibody support coated with antibodies. - 31 -

21. A process according to any one of claims 1 to 8 which process additionally comprises a secondary analysis to confirm the presence of the at least one contaminant detected.

22. A process according to any one of claims 1 to 4 wherein the metabolic activity is detected using a Bactometer.

23. A process according to any one of claims 1 to 8 which comprises releasing contaminants or contaminant-antibody complexes from the cell surface or substrate surface.

24. A process according to claim 23 wherein release is performed using an agent selected from: heat; chaotropic agents; polarity reducing agents; pH change inducing agents; and proteolytic enzymes.

25. A process according to claim 24 wherein the agent is 0.5% KOH pH 12.5.

26. A process according to claim 24 wherein the releasing agent is the proteolytic enzyme papain.

27. A process according to any one of claims 5 to 8 wherein the detection is performed using a Coulter Counter.

28. A process according to any one of claims 1 to 8 wherein the removal of growth medium or removal of unbound material or sample is performed by washing.

29. A process according to any one of claims 2, 4, 6 or 8 wherein the growth medium is selective for the at least one contaminant.