A method for assaying an analyte in a sample. The method comprising the steps of a) contacting the sample with material comprising a receptor which is present in a liposome and which liposome comprises a detectable functionality, said contact occurring under conditions resulting in binding of the receptor to analyte if present before or concomitant with step b, wherein step b) consists of contacting the sample with an immobilised ligand for the receptor said contact occurring under conditions resulting in binding of the receptor to the ligand, with steps a and b being followed by c) separating the resulting immobilised ligand-receptor fraction and the receptor fraction present in solution and d) assaying the detectable functionality of the receptor in a fraction from step c) in a manner known per se for its detection. Suitably the receptor is being present in step a) in a concentration between 1 pM-1 nM and the detectable funcitonality in step a) is present in a concentration of 1 pM-1xe2x80x21 M and the immobilised ligand in step b) having a Kd for the receptor  less than 50 nM. The immobilised ligand should be present in an amount required to capture 10-99% of the receptors in the assay in the absence of analyte at a receptor concentration below the Kd of the immobilised ligand and receptor under conditions otherwise corresponding to those of the assay. The conditions and the detectable functionalities being selected such that a 0.1-10% change in either the ligand-receptor fraction or in the free receptor fraction can be qualitatively or quantitatively detectable.

Receptor binding assays have been commonly used for the assessment of the pharmacological properties of New Chemical Entities (NCE). Due to the introduction of combinatorial chemistry in the pharmaceutical industry, in an attempt to find succesful drug candidates, an enormous increase in NCE requires a concomitant demand for high through-put screening systems. Due to their specific properties receptor assays have been considered valuable analytical tools for the quantitation of highly potent drugs that exert their pharmacological action via a receptor interaction.
The term receptor is exclusively used for proteins which can interact with hormones, neurotransmitters and drugs or poisons yielding or blocking a pharmacological response. Thus therefore antibodies, circulating or membrane bound proteins e.g. enzymes cannot be considered receptors even if they should have ligand binding properties.
The principle of receptor binding assays is based on the competition between a ligand and an analyte for binding to a certain receptor. After incubation of ligand, analyte and receptor followed by separation of the receptor bound and the free fraction of the ligand by either filtration, centrifugation or dialysis, subsequently one or both resulting fractions are quantitated. The subsequently acquired data can be used for assessment of the affinity of a NCE for the receptor or for quantitation of a particular receptor binding analyte.
Up till now, all receptor assays have been construed around the use of a (radio) labeled ligand. Furthermore generally receptors present in animal tissue or cultivated cell lines have been used after having undergone only little purification. Typical receptor densities in commonly used receptor assays range from 10-100 picomole per gram tissue. This subsequently implies that the amount of displaceble labeled ligand in such assays is limited. The use of radioactive ligands in such cases is notwithstanding this attractive because radioactivity can be detected with good sensitivity and limited back-ground signals from such receptor material. Another important reason in favor of use of radioactive labels is that development is easy once a compound has been identified that binds to a particular receptor with high affinity. Replacing 1-6 hydrogen atoms by the same number of tritium atoms yields a product that has a receptor affinity similar to that of the unlabeled ligand. However disadavantages such as the limited shelf-life and the problems encountered with the use of radioactive tracers has stimulated the search for alternatively labeled ligands. Another motivation for such search was also the expectation that alternative labels might improve the sensitivity of the assay with regard to limits of quantitation. Taken into consideration the physical half-life and the counting time of each sample it can be calculated for tritium by way of example that only 1 out of each million labeled molecules is detected.
Almost all approaches with non-radioactive labeled ligands have been with fluorescent labels. The development of fluorescent ligands with a high receptor affinity, if the ligands itself does not have sufficient native fluorescence, is quite difficult and a compromise between affinity and fluorescence properties has always been required. The aforementioned low receptor density implies that the maximal signal is limited and lies close to the limitations of available instrumentation. High amounts of receptor containing material needs to be used per assay in order to sufficiently increase the signal.
Furthermore the currently used receptor containing materials in receptor assays contain large amounts of non-receptor proteins which cause a high fluorescence background.
Time-resolved fluorescence or fluorophores that emit at wavelengths  greater than 500 nm could partially solve this problem but provides no real break-through. In patent application PCT/NL96/00418, Janssen et al. have reviewed the existing literature on assays and recognized these problems. The review is incorporated herein by reference. Janssen et al subsequently modified the procedure to quantitate the bound fraction of labeled ligand after dissociation of the labeled ligand from the receptor which took place after the separation of the bound and free fractions of the labeled ligand. Initially they developed a fluorescent labelled benzodiazepine receptor ligand attempting to obtain a homogeneous assay using polarisation fluorescence.
The adapted procedure with the dissociation step opened a new array of detection modes for the ligand. Principally if a high affinity receptor binding compound has unique physico-chemical properties e.g. halogen atoms which can be detected with gas chromatography with electron capture detection, it can be used as a ligand in such a receptor assay.
It can be concluded that the development of non-radioactive labeled ligands is difficult because the bulky groups, required to obtain e.g. highly fluorescent probes that emit at higher wavelengths at the same time reduce the receptor affinity of the product. Furthermore a further disadvantage is that per ligand-receptor interaction only one detectable functionality is present.
Benzodiazepine receptors in particular remain important in clinical pharmacology because of their widespread use. Benzodiazepines are extensively metabolized yielding a range of active and inactive metabolites. The overall effect of a benzodiazepine can therefore not be properly related to the concentration of only the parent drug. Quantitation of the individual compounds, parent drug and metabolites on the other hand is highly impractical and assays that could measure the sum of all active compounds could be beneficial.
To solve such a problem an immuno assay could be used, However the affinity of the individual compounds towards the applied antibody does not correlate with the affinity of these individual compounds towards the receptor. This thus precludes the measured response being a proper reflection of the total pharmacological effect. It is for this reason that the use of the receptor which mediates the pharmacological effect in an assay forms a logical step.
Traditional receptor assays using radioactive or non-radioactive ligands require a separation step enabling quantitation of bound and or free fractions of the labeled ligand. Procedures used for the separation are dialysis, centrifugation and filtration. The selection depends on available instrumentation and equilibrium dissociation constants of the labeled ligand and of the analyte. It is a requirement that the separation step may not alter the amount of receptor bound ligand.
Dialysis is a slow process, up to 24 hours. Automation is cumbersome because collection of the fractions containing the bound plus free and the free fraction of label and analyte is hardly possible. In addition miniaturisation is precluded.
Centrifugation with or without precipitation of the labeled ligand or the soluble or solubilized receptor has limited applicability for receptor assays because the separation process can change the bound fraction of the labeled ligand. Moreover it is difficult to quantitatively collect sedimented receptors. Automation of centrifugation steps also remains problematic. Filtration is the method of choice albeit that due to the amounts of receptor containing tissue per sample minimal filter surface areas are required. This is to avoid clogging of these filters and to warrant accurate quantitation of the bound fraction of the labeled ligand. There are a number of filtration devices (Skatron, Brandell, Millipore) that allow parallel filtration of up to 96 samples. It should be noted however that filtration capacity per filter is limited and that the precision of the methods is strongly dependent on the differences in filtration rate between the individual filters. With the exception of the Millipore system it is not possible to collect either the free fraction of the labeled ligand or the bound fraction of the labeled ligand after dissociation from the collected receptor fraction.
It is an objective of the subject invention to provide a highly sensitive receptor-ligand assay that allows miniaturisation and automation to be suitable for high through-put purposes. The assay should preferably be amenable for non-radioactive ligands. The specific materials of the assay, being the main ingredients of a kit, should be available in adequate quantities and show controllable batch to batch variations. Such an assay must be simple to execute and economically feasible enabling routine laboratory application.
The subject invention provides a highly sensitive receptor assay that does not require ligands with a detectable label but provides a test with at least the sensitivity and specificity of radioactive receptor assays. Such an assay according to the invention is simple to execute and economically feasible enabling routine application. Also a combination of immobilised ligand and a labeled receptor preparation suitable for use in an assay according to the invention is provided. Quite specifically an assay for benzodiazepine detection and quantitation is provided. Suitably a benzodiazepine sensitive receptor will be used.
The subject invention provides a method for quantitatively and/or qualitatively assaying an analyte in a sample, said analyte being a receptor binding compound. In an assay according to the invention the receptors of interest can originate from receptor containing tissue of animal source such as in vitro cultures of animal cells or tissues or from microorganism cultures. The method has low detection limits equivalent to those of traditional receptor assays that employ labeled ligands. The method comprises the steps of
a) contacting the sample with material comprising a receptor which is present in a liposome and which liposome comprises a detectable functionality, said contact occurring under conditions resulting in binding of the receptor to analyte if present before or concomitant with step b, wherein step
b) consists of contacting the sample with an immobilised ligand for the receptor said contact occurring under conditions resulting in binding of the receptor to the ligand, with steps a and b being followed by
c) separating the resulting immobilised ligand-receptor fraction and the receptor fraction present in solution and
d) assaying the detectable functionality of the receptor in a fraction from step c) in a manner known per se for its detection. In a suitable embodiment the receptor is present in step a) in a concentration between 1 pM-1 nM. The detectable functionality in step a) is suitable present in a concentration of 1 pM-1 xcexcM. The said immobilised ligand in step b) suitably has a Kd for the receptor  less than 50 nM.
The method according to the invention is suitable for automation allowing high through-put of samples.
Liposome is an art recognised term and refers to particles capable of maintaining their basic structural integrity in an aqueous environment. They are formed from amphipathic molecules with hydrophilic surfaces that are exposed to an inner aqueous space and also to surrounding aqueous medium. They can also be called multilamellar vesicles which are comprised of multiple concentric layers of lipid. Numerous methods are described in the art for making liposomes. In summary a known process for example as disclosed in U.S. Pat. No. 4,235,871 comprises preparing and mixing a solution of lipid to be deposited in an organic solvent, followed by evaporating the solution to dryness to produce a thin lipid film on a glass vessel followed by hydration and liposome formation by vortexing or in any other mechanical means in an appropriate buffer. The resulting structure is such that the hydrophilic heads are orientated outwards toward the aqueous medium and the hydrophobic structure is oriented inwards. Further purification is possible for example by column chromatography. Alternative methods are disclosed in U.S. Pat. Nos. 5,017,501 and 5,094,785. The cited documents are incorporated by reference and serve merely as examples of possible ways to manufacture liposomes. Alternatives are available to the person skilled in the art and such methods are also to be considered incorporated.
Suitably the immobilised ligand will be used in the method in an amount required to capture 10-99% of the receptors in the assay. This amount is the amount required in the absence of analyte and at a receptor concentration below the Kd of the immobilised ligand and receptor under conditions otherwise corresponding to those of the assay. A method according to any of the embodiments of the invention can suitably use an immobilised ligand that is a structurally related analog of a compound with affinity towards the receptor of interest which is coupled directly or coupled via a spacer arm to the surface of a carrier material. In the event of a spacer being applied this will usually have a length of between 5-20 carbon atoms preferably of 10-15.
The conditions of the assay and the detectable functionality are selected such that a 0.1-10% change in either the ligand-receptor fraction or in the free receptor fraction can be qualitatively or quantitatively detected. A method according to the invention thus suitably uses an amount of immobilised ligand per assay, between 0.5 femtomole and 0.5 nanomole. When the ligand bound fraction of the receptors is quantitated, dissociation of the receptor from the immobilised ligand may be required. The selection of the detectable functionality and its quantitation in either the free or ligand-bound fraction determines whether the functionality can be detected itself, or whether the functionality is such that it is degraded completely and uniformly thereby yielding a detectable product, or whether the functionality converts a further molecule into a detectable product. Suitably detection can occur after mixing reagents with the liposomes to enable the incorporated detectable functionalities (e.g. enzyme) to produce a readily detectable product. Alternatively the free fraction of liposomes containing the receptor of interest and the detectable functionalities are transferred into a container or detection cell. There they can either be readily detected or be subjected to further treatment leading to production of detectable product. In a method according to the invention it is preferable the receptor material and the immobilised ligand release no interfering compounds during the incubation or during the quantitation of the detectable functionalities. Also in a method according to any of the preceding claims the conditions are preferably chosen to ensure the stability of the detectable functionality and dependent on the detection mode also the stability of the detectable product.
The steps a and b can be carried out concomitantly. The sensitivity of the method can however be improved by carrying out step a) prior to step b) in order to enable establishment of controlled non-equilibrium thus providing greater sensitivity. Low concentrations of immobilised ligand and labeled receptor can also improve the sensitivity, particularly for high-affinity ligands with Kd below 10 nM. In a suitable embodiment of the method acording to the invention the number of detectable functionalities is 1-10000 times the number of receptors. Also in a suitable embodiment of the invention the detectable functionality for example an enzyme is able to produce a detectable product in a number between 10-1000000 times the number of receptors.
In particular the method according to the invention is directed at carrying out assays wherein the analyte is to be detected at a level of 1 xcexcM, preferably below 0,5 micromolar level. The assay can also even be carried out successfully on analyte levels below 100 nM, even below 50 nM. Preferably the invention is carried out to ascertain levels as low as possible. With a view to developing potent drugs and analysis of their activity levels analyte concentations as low as 1 picomolar but also as low as 100 femtomolar are of interest, The method is applicable to use on analyte containing samples of less than 1 ml, preferably even less than 250 xcexcl. In a suitable embodiment of the invention the method takes place in assay volumes between 1 and 250 xcexcl. Preferably the method according to the invention is carried out in volumes as small as 10 xcexcl but also samples as low as 1 xcexcl are of interest.
In a method according to the invention a suitable embodiment comprises adding labeled receptor in an amount equivalent to a concentration within the range 0.001-1*Kd of the receptor and the immobilised ligand. Preferably the receptor is added in a concentration between 0.01 and 0.5*Kd. Quite suitably a concentration of approximately 0.05*Kd is applied. For a large number of ligands Kd values are already available from handbooks. A person skilled in the art can also ascertain in a manner known per se what the Kd value is for a particular ligand and receptor. In general an immobilised ligand with a Kd below 50 nM, preferably below 10 nM, is an excellent candidate for use in the assay according to the invention. Such a Kd ensures the required sensitivity for detecting and quantifying trace amounts of analytes or receptors.
The constitution of the receptor preparation is adapted to this alternative method set-up. Most receptors in their native form are imbedded in cell membranes in a manner which warrants their ligand and analyte binding ability. A receptor preparation as used in traditional receptor assays consists of homogenized tissue, rich in the receptor of interest, which is fractionated by centrifugation to remove cell nuclei, soluble proteins and unhomogenized fragments. This preparation consists of membrane fragments in size ranging from 0.1-5 xcexcm and further containing a highly variable number of receptors per fragment. For the method according to the invention more uniform material labeled with a detectable functionality is required. To arrive at such material proteins including the receptor protein in membrane fragments are solubilized for example by addition of detergents. The resulting solubilized receptors can then be purified by affinity chromatography which results in fairly unstable product. Addition of an excess of selected lipids to solubilised receptor will lead to the formation of liposomes and reconstitution of the solubilized (receptor) proteins. This means the receptor protein is incorporated in liposomes like the receptor protein is incorporated in membrane fragments. This procedure thus restores binding properties of the receptor protein and also warrants receptor stability. Functionalities required for the detection of the receptor must also be incorporated in the liposomes. The functionalities can be modified substituents of the liposome itself, e.g. lipids or lipid like molecules to which a detectable functionality is coupled. Alternatively the functionalities can be proteins or protein like, molecules that are themselves reconstituted in the liposome like the receptor protein.
The simplest way to introduce detectable functionalities into the receptor comprising liposome is by adding them to the solubilized receptor preparation prior to the addition of the lipids required for the reconstitution. Alternatively liposomes containing the reconstituted receptor can be mixed with liposomes containing the detectable functionalities. Fusion by freeze-thawing cycles and/or sonication of the different types of liposomes can subsequently yield the labeled receptor containing liposomes. (Ref.: MacDonald).
The solubilisation of receptors and the reconstitution of the solubilized receptor into liposomes and the labeling of the receptor containing liposomes can be carried our in a number of manners. Each receptor will require its own set of reagents and procedures to maintain or restore the native binding properties of the receptor.
In the method according to the invention step c) can be carried out in a number of manners which will be apparent to a person skilled in the art It will depend on the, nature of the carrier material with the immobilised ligand whether additional pretreatment steps prior to separation are required. The simplest way of carrying out step c) comprises emptying and optionally rinsing containers, i.e. tubes or microtitre plates to which the ligand is coupled e.g to the inside wall. Either the free fraction of labeled receptors in the solution or the bound fraction of the labeled receptor remaining in the container can be measured.
The carrier material on which the ligand is immobilised can include the walls of test tubes or microtitre plates, beads, nitrocellulose, membranes, latex microparticles, glass metal or gel. Polystyrene and polypropylene are suitable materials. The exact material is not critical. The requirement is that the ligand can be immobilised on such carrier material. The immobilisation encompasses all mechanisms for binding the ligand to the solid carrier such that during the assay the ligand remains fixed to the carrier. Such mechanisms can comprise covalent or non covalent binding, absorption by ionic interactions, hydrophobic/hydrophobic or hydrophilic/hydrophylic, interactions.
In an embodiment whereby the bound fraction of the labeled receptor is retained by immobilised ligands on the surface of a well of a container e.g. a microtitre plate the amount of receptor material must be such that in the absence of analyte between 10 and 99% of the labeled receptor can be bound to the surface. By way of example the amount of labeled receptor material can be less than 0.1 pmole when the assay volume equals 250 xcexcl as is the case when using 96 well microtitre plates. Quite suitably in general the amount of receptors used per assay can lie between 1 femtomole and 1 picomole. The detection can occur for example by adding reagents to the liposomes bound by the immobilised ligands to enable the incorporated detectable functionalities (e.g. enzyme) to produce a readily detectable product. Where bound fraction is to be detected the free fraction can be suitably transferred to waste. This can ocur with or without additional washing steps of the container with the liposomes bound to the immobilised ligands. Suitably in any case the liposomes with the detectable functionality are dissolved in a medium allowing quantitation of the functionality in a manner known per se for said functionality.
The method according to the invention can be carried out using membrane bound receptors as illustrated for the benzodiazepine receptor, which are solubilized and reconstituted in liposomes. Alternatively a receptor of interest can be a soluble receptor which is isolated and subjected to a labelling process. Suitably the soluble receptor is selected from the following group: steroid hormone, androgen, estrogen and progesteron receptors. Suitably liposomes according to the invention also contain labeled lipids (e.g. fluorescent) as detectable functionality. Any of the embodiments described for the invention can be applicable for a benzodiazepine receptor based assay. A benzodiazepine based assay is not restricted to the parameters and variables as disclosed merely in the example. In such an assay the ligand can suitably be a benzodiazepine derivative. A large number of such compounds are available for use in the assay and are well known to the person skilled in the art. Suitable examples of the receptor of interest being a membrane bound receptor can be selected from the following group: adenosine, adrenergic, dopamine, histamine, muscarinic (or acetylcholinergic), nicotinic, opiate, serotonin, benzodiazepine, GABA, glycine, calcium channel, sodium channel, chemotactic peptide, epidermal growth factor, glucocorticoid, cannabinoid, cholecystokinin, cytokines, leuktriene and neurokinin receptors. Receptors that are soluble by nature can be incorporated directly in liposomes.
It is commonly known that ligand receptor binding can be very sensitive to structural modifications of the ligand as was observed for the development of fluorescent labeled ligands for e.g. the benzodiazepine receptor as reported by Janssen et al Generally the label is linked via a spacer to a compound with affinity for that receptor or a closely related analog thereof. The impact of the size of the fluorophore being used as label on the binding is much larger than the impact of the spacer alone. In the present approach the ligand can be coupled via a spacer to a rather inert carrier material e.g. the modified surface of a container e.g. a microtitre plate. The most favorable location for such linkage and also places impossible for such linkage have been assayed in the prior art for numerous ligands. Naturally for application in the subject method the ligands should only be considered with a spacer in such a favorable location. In general when using a spacer linking the ligand with the carrier material, the spacer will comprise a carbon chain of between 5-20, preferably 10-15 carbon atoms. The general requirements for the detectable ligand have been summarized elsewhere in the description of the method according to the invention.
The suitable ligands and spacers can be determined by the person skilled in the art using details readily available concerning affinities and receptor structures. We illustrate this for the immobilised ligand for the benzodiazepine receptor. A number of suitable immobilised ligands already exist and naturally it is possible to prepare numerous immobilised ligands on the basis of the information presented in the subject application which can be suitably applied in the subject invention. Preferred immobilised ligands will be those described in literature. We provide an example of an immobilised ligand that can be readily produced. We also refer to publications of Sigel and Taguchi which illustrate the preparation of immobilised ligands.
In the method according to the invention a ligand was linked to a spacer and subsequently to a reactive group attached to the surface of the container e.g. microtitreplate. The spacer used comprised an amino group The receptor preparation used was prepared by first subjecting a purified and lyophilized membrane fraction to a treatment with deoxycholate under well-defined conditions to minimize loss of active receptor binding sites. By centrifugation or membrane filtration the solubilized material could then be separated from the remainder of the membrane fraction. The next step was the reconstitution of the solubilized receptor by the addition of asolectin. Other lipids, alone or in combination can also be be used to yield comparable products. In a method according to the invention the receptor material, used for solubilisation can be a P2-pellet derived from receptor containing tissue. Such a P2-pellet may suitably have a receptor concentration of 0.5-5 picomole/mg protein. The P2-pellet can be solubilized with a detergent or a mixture of detergents, said detergent suitably being selected from the following group: salts of anionic detergents, cationic detergents, zwitterionic detergents and nonionic detergents. The reconstitution of solubilised receptor material can occur with lipids suitably selected from the following group: phospholipids, glycolipids and cholesterol.
Theoretically a large variety of detectable functionalities can be used in combination with a technique or procedure that can be used for selective and sensitive quantitation. Considering one particular intended application of the method the quantitation technique or procedure should be fast, rugged, cost-effective and applicable to small sample volumes and be amenable to automation.
Radiolabels, preferably 3H or 125I, attached to the constituents of the liposomes e.g. the lipids, remain attractive as a form of label to be used in the method according to the invention. This is because a large number of labels per liposome or per receptor binding site can be obtained. Moreover quantitation of bound or free fractions of radioactively labeled liposomes is simple and reliable. In addition considering the availability of multi-channel detectors high through-put can be warranted. However, wide-spread use of this radioactive variation is limited because of legal limitations in the use of radioactive substances and the disposal of radioactive waste. Thus non radioactive functionalities are also of eminent importance as they have more potential in practical terms.
Functionalities that can readily be detected with spectroscopic, preferably fluorescent or chemi-luminescent, techniques thus have a better practical potential. In contrast to traditional receptor assays in which fluorescent labeled ligands are used, the selection of the fluorophore in the method according to the invention is not affected by a possible effect on the receptor binding properties. Thus any fluorophore with favorable spectroscopic properties can be used. Favorable spectroscopic properties are an emission wavelength and preferably an excitation wavelength of the fluorophore longer than 500 nm in order to limit background fluorescence caused by membrane proteins that are also reconstituted in the liposomes. Selected fluorescent labeled lipids, as depicted in FIG. 1 were mixed with the lipids used for the reconstitution process. Between 0.1 and 2% of all lipids contained a fluorescent label and provided good results. With increasing label concentrations some quenching was observed however the highest labeling density yielded the highest fluorescent signal.
A different type of detectable functionality that offers new opportunities for reduction in sample size and improvement of method sensitivity is formed by the group consisting of enzymes. Enzymes that can be incorporated in liposomes containing the receptor of interest can be used to amplify the signal by converting a precursor into a spectroscopically detectable product, preferably of fluorescent or chemiluminescent nature. An assay based on luciferase appears to have high potential in that only an increase in signal and virtually no back-ground signal is detected. Alternatives that are suitable as well are by way of example detectable functionalities selected from alkaline phosphatase and monoamine oxidase. Illustrative examples of the labels mentioned are known in the art and can also be selected from pigments, dyes, stable free radical luminescents etc.
The selection of lipids for the reconstitution of solubilized receptors is not very critical and can be done by a person skilled in the art. The selection of detectable functionalities can be done by a person skilled in the applied detection technique implying knowledge of the physico-chemical characteristics of the functionality and tuning of the detection to optimize signal to noise ratios. The same detectable functionality can be used for any receptor under the condition that the receptor and the detection to optimize signal to noise ratios. The same detectable functionality can be used for any receptor under the condition that the receptor and the detectable functionality can be both reconstituted in the same lipid mixture or that liposomes containing either the receptor or the detectable functionality can be fused e.g. by freeze-thawing cycles and/or sonication, yielding liposomes containing both entities.
Where different receptor types are simultaneously used in one test, liposomes containing different receptors and different labels are to be used. Thus each liposome type comprises only one receptor type and different immobilized ligands.
The detectable functionality in the example provided of the method according to the invention is fluorescein labeled-DHPE. This was added in an amount of 0.3% relative to the amount of asolectin used for the reconstitution. In the method the liposomes containing the benzodiazepine receptor and fluorescein labeled DHPE preferably bind to the immobilised ligand and do not bind to the surface of the microtitre plate. Suitable detectable functionaliuties are well known in the art. They can by way of example be selected from the group consisting of dansyl DHPE (dansyl-dihexadecanoylphosphatidylethanolamine), fluorescein DHPE and bodipy FLC5HPC (beta-BODIPY labeled pentanoyl-1-hexadecanoylphosphatidylcholine). The measured signal of the detectable functionalities in the liposomes changes with the concentration of analyte present in the sample. It should be noted that the analyte will only affect the fraction of the liposomes bound to the immobilised ligand and not the liposomes that are non-specifically bound to the surface of the microtitre plate. The latter fraction in general should be substantially smaller than the first fraction to warrant substantially the precision of the method. In the method in the example no additional measures were take to reduce non-specific binding of the liposomes to the surface. The procedure can however be improved by reducing nonspecific binding. Different procedures to reduce non-specific binding are known to a scientist, skilled in the art The selection of the microtitre plate material, coating of the surface, addition of competing reagent in the incubation medium, washing procedures, or combinations thereof are factors that can help to reduce non-specific binding. As an alternative to the exemplified procedure the unbound fraction of labeled liposomes can be quantitated.
A kit for carrying out a method according to the invention is also envisaged as falling within the scope of the invention, said kit comprising a combination of components for carrying out the method or for making the components required for carrying out the method as detailed in the above. Thus a kit according to the invention can comprise a ligand optionally immobilised on carrier material e.g. a reaction container in which the method is to be carried out and a liposome containing receptor for binding the ligand incorporated therein. The kit can further comprise the detectable functionality either incorporated in the receptor containing liposome or in another liposome. A kit according to the invention may further comprise any of the following components reagent for the assay and/or for quantitation of the detectable functionality, calibration standard and quality control sample.

Screening for analytes using labeled receptors


The present invention provides a reference standard for calibration of an analysis instrument. The reference standard comprises a solid body formed of a number of compounds and a substrate having scattering properties similar to a product to be analyzed with said analysis instrument and being spectrally neutral in a wavelength range to be used in the analysis instrument. The substrate and the compounds in combination with respect to intensity, wavelength and scattering properties imitate the spectral response of the product to be analyzed with said analysis instrument. The present invention also provides a method for calibration of an analysis instrument, which method comprises recording, by means of said analysis instrument, the spectral response of a reference standard comprising a solid body, which with respect to intensity, wavelength and scattering properties imitates the spectral response of a product to be analyzed with said analysis instrument; evaluating the differences between the response from said analysis instrument and an expected spectral response; and calibrating said analysis instrument according to the result of the evaluation.

Reference standards are widely used in visible and near infrared spectroscopy to ascertain proper performance of analysis instruments. The accuracy of the instruments in analyzing and identifying materials depends upon the accuracy with which the wavelengths and intensity of the radiation within the wavelength range to be used in the analysis instrument are known. The very capability of the instrument to measure properties of different products is dependent upon this accuracy. For this reason, there is a need to verify with precision that the calibration of the instrument is correct for the wavelength range to be used in the instrument.
Typically, two types of reference standards are used. Intensity standards are used to set and check that the detector accurately detects the intensity of the radiation and wavelength standards are used to check that the wavelength scale of the instrument is accurate. An example of a wavelength standard is shown in U.S. Pat. No. 5,936,727.
Analysis instruments can be calibrated along the intensity scale with transmittance screens or filters and reflectors of neutral density. The instrument is then calibrated to correct discrepancies between the detected intensity and an expected intensity result. In U.S. Pat. No. 4,761,552 a reference standard that is used for reflectance calibration of an instrument is disclosed.
Calibration along the wavelength scale is made by using emission lines from arc sources such as deuterium or xenon, or absorption bands from polymer films such as polystyrene or from rare earth oxide doped materials. The wavelength of the emission line is well-defined. Therefore, a wavelength scale may suitably be calibrated using these emission lines as adjusters of the wavelength scale.
However, the current technology for ensuring calibration with respect to intensity and wavelength is not satisfactory to ensure adequate matching between instruments, quality control of the instruments or performance validation of the instruments. Calibration is impaired by the fact that measurements of a product to be analyzed are not well characterized by intensity measurements that take no account of the scattering properties or physical nature of the product.
One attempt to remedy this inadequacy is to use natural products as reference standards, i.e. each reference standard is made of the respective product to be analyzed on the analysis instrument. However, these reference standards lack temporal and mechanical stability and therefore have a short lifetime, requiring great care in the handling of the reference standards to minimize spectral changes between two sets of measurements. Consequently, these reference standards are inadequate in use, especially across large geographical areas. The usage of such reference standard is shown in U.S. Pat. No. 4,866,644.

Reference standard and method for calibration


The invention is relating to device for the controlled heating and cooling and conservation of eggs (18) that is provided with an oven space (13) with a carrier plate (7) with holders for the positioning of the eggs (18) substantially upright and a spray device (12) for the cooling of same with a coolant, and which is further provided with a heating element (11) for the generation of steam, whereby means (14) are provided that allow said steam to be subjected to a forced flow whereby this steam flows over the eggs (18) in order to heat the eggs, whereby said carrier plate (7) has openings (16) such that at least part of said steam is guided along the side of the eggs (18) to the underside of the carrier plate (7) so that the underside of the eggs (18) is heated.

The devices known according to the current state of the art for heating and/or cooling eggs have the disadvantage that they do not allow the precise control of the heating or cooling of eggs. It appears that the shape of the temperature curve of the heating and subsequent cooling or quenching of the eggs is critical to the characteristics of the treated eggs.
When eggs are boiled with the aid of the devices known according to the current state of the art, it appears that it is virtually impossible repeatedly to boil soft-boiled eggs in which the white is fully set and the yolk of the egg is not set, and in which the yolk has reached a sufficiently high temperature to destroy any bacteria that it may contain.
Furthermore, according to the state of the art, the shell of boiled eggs has a very variable attachment to the white of the egg. As a result it is a common occurrence when shelling traditionally boiled eggs that part of the set white of the egg is removed together with the eggshell.
It has also been observed that the yolk of an egg boiled according to the state of the art, does not lie in the centre of the egg. During the boiling of the egg, the yolk in fact moves up towards the eggshell so that a boiled egg is obtained in which the white is not uniformly set.
Furthermore the device and the method according to the state of the art do not offer any certainty about the bacterial safety of these soft-boiled eggs.
The invention attempts to remedy these disadvantages by proposing a device and method for heating and cooling eggs that produces virtually perfectly boiled eggs that are easy to shell and which have the yolk located in the centre of the egg. Furthermore the invention makes it possible to obtain eggs with a desired degree of setting of the white and/or the yolk.
The device and the method, according to the invention, also make it possible to pasteurise eggs without causing the white or the yolk to set, whereby the processing characteristics of the egg are markedly improved compared to those of unpasteurised eggs.
To this end means are provided that permit aforesaid steam to be subjected to a forced flow, whereby this steam flows over the carrier plate from a first extremity to the opposing second extremity of same so that the upper side of the eggs are heated, whereby said carrier plate is provided with openings so that at least a part of said steam is guided along the side of the eggs to the underside of the carrier plate.
According to the invention the device is purposely fitted with a preferably removable oven plate mounted between the carrier plate and the bottom of the oven space so that a free space is created between the latter and the oven plate.
According to the invention aforementioned spray device comprises in an advantageous way at least one sprayer that allows aforementioned coolant to be sprayed as a cylindrical jet of coolant with an annular section.
Said sprayer has in a particularly advantageous fashion a cone-shaped hole that opens into a spray opening whereby said liquid is squirted via a canal adjacent to the circumference of this opening so that this liquid rotates in the opening and is thus guided into the oven space via aforesaid spray opening.
In a specific embodiment said spray opening has a diameter of 1 to 2.5 mm and this is by preference substantially equal to 2 mm.
Said spray device has liquid lines that connect said sprayer to the source of the liquid, whereby the length of these lines is substantially the same for all sprayers.
According to a preferred embodiment of the device, according to the invention, this is provided with a preferably removable liquid reserve comprising two compartments, where a first compartment has at least one valve that admits liquid to said spray device and/or the heating element, and where a second compartment is provided with at least one inlet opening so that liquid can flow from the oven space into this second compartment.
According to the method according to the invention for heating or cooling eggs so that they set at a predetermined point or for pasteurising them, the eggs, which may be at differing initial temperatures, are placed next to one another on a carrier plate placed in an oven space, whereby the latter is preheated in accordance with a predetermined temperature curve by means of steam to a temperature of between substantially 95° C. and 110° C. and by preference to 98° C. The eggs are then cooled with the aid of a coolant for a period of 10 to 50 seconds, by preference from 15 to 30 seconds, before the eggs have reached such a high temperature in their cores that bacteria are destroyed. After cooling, the eggs are maintained in the oven space at a temperature that lies below the setting temperature of the yolk while the core temperature of the eggs rises further as a result of heat transmission from the surrounding white to a temperature at which bacteria are destroyed.
According to a preferred embodiment of the method, according to the invention, to obtain eggs of which only the white is set, the oven space is heated by means of steam for 6.2 to 6.8 minutes whereby, when the core of the eggs has reached a temperature of between 45° and 50° C., the oven space is subsequently cooled to a temperature of between 55° C. and 65° C. and by preference to about 60° C.
The same reference numbers in the various figures relate to the same or similar elements in the various figures.
The device according to the invention, as represented in FIGS. 1 and 2, shows two sidewalls located opposite each other, which are not represented in the figures and which join to back wall 1 and a plate 2 that constitutes the bottom plate of the device. The upper side of the device consists of a plate which, for the clarity of the figures, is likewise not shown. A rectangular frame 3 is provided on the front of the device on which a swing-down closing door 4 is installed in order to shut the device. A water reservoir 5 that can be removed from the device so that it can be emptied or filled is provided below this door 4.
In addition the device is provided with an oven plate 6 which more or less extends between the sidewalls of the device and on which a carrier plate 7 rests for the positioning of the eggs, which are not shown in FIGS. 1 and 2. This carrier plate 7 stands by means of two parallel extended vertical ribs 8 and 9 that are perpendicular to the frame 3 on said oven plate 6 such that a free space 10 is formed between the latter and the carrier plate 7.
Above the oven plate 6 and under the plate which forms the upper side of the device a so-called spray plate 12 extends which is connected via a pump 46 to a water reservoir 5. In this way an oven space 13 is formed under the spray plate 12 in which said carrier plate 7 and the oven plate 6 are located.
The oven space 13 is bounded on the underside by a bottom plate of which the edges connect to the vertical sides of the oven 50, 51 and 52. These oven walls 50, 51, and 52 connect on their upper side to aforementioned spray plate 12.
On the front side the oven wall is formed by aforesaid closing door 4.
On the rear wall 1 a heating element 11 is provided that consists of an electrical boiler for the generation of steam. The heating element 11 is connected to a ventilator 14 which is located between this heating element 11 and the oven plate 6 in order to blow steam into the oven space 13 above the carrier plate 7.
Next to the heating plate 11 and the oven space 13 an electronic control unit 15 is also provided for the device.
The carrier plate 7 is provided with square openings 16 with rounded corners that constitute holders for the vertical positioning of eggs. To this end these openings 16 are provided with projections 17 that lie in the plane of carrier plate 7. When an egg is positioned in such a holder the egg will thus partly project under the carrier plate 7, whereby it bears on said projections 17 in such a way that the opening 16 is not entirely filled with the corresponding egg. In the embodiments of the carrier plate shown in the figures each opening 16 is provided with four such projections 17.
In the longitudinal section of the device according to the invention shown in FIG. 3, eggs 18 are shown that are positioned more or less upright. That is to say the eggs 18 rest with their most rounded ends on the carrier plate 6 against the projections 17. Furthermore this figure shows the closing door 4 in a vertical position so that it shuts said oven space 13.
The closing door 4 is provided with an opening 58 level with the oven plate 6. This opening 58 is semi-cylindrical in shape and extends over the entire width of the oven plate 6 in such a way that steam in the space below the carrier plate 7 and above the bottom plate 6 can flow via this opening 58 over the underside of the oven space to fan 14 and which can return this steam back over the eggs.
Said fan 14 is by preference of the elongated type and extends over virtually the entire width of oven space 13. The presence of said opening 58 in the closing door 4 which thus works in conjunction with fan 14 means that the steam is evenly distributed over the eggs placed in the device independently of the number of eggs located in the oven space 13.
When the carrier plate 7 is not entirely filled with eggs, a uniform heating of the eggs is thus nonetheless obtained.
Furthermore a removal duct 59 is provided that connects the oven space 13 with an exhaust pipe 60. This exhaust pipe 60 leads via one extremity to an opening 61 under the closing door 4. The other end of the exhaust pipe 60 works in conjunction with a fan 62 that draws in air from the surroundings so that this ambient air is mixed with the steam in the exhaust pipe 60. In this way there is no danger that a person standing in front of opening 61 might be burnt by the extracted steam.
As is apparent from this figure the water reservoir 5 has an oblong shape and is provided with a handle 19 that allows the water reservoir to be removed from the device in a simple way.
The water reservoir 5 has two compartments 20 and 21 separated from one another by a bulkhead 22. This bulkhead 22 extends between the sides of the water reservoir 5 and is inclined, where the higher end 23 is located on the side of the handle 19, while the lower end 24 is located against the side wall opposite this handle 19. In this way liquid in the upper compartment 20 will always flow to the lower part 24.
Furthermore a valve 25 is provided in the side wall of compartment 20 near said lower part 24 of the bulkhead 22. This valve works in conjunction with a water pipe not shown in the figures that is connected to a heating element 11 via a pump 26. In this way this valve 25 ensures that water from the upper compartment 20 can flow to the heating element 11 with the aid of the pump 26.
The lower compartment 21 has two inlet openings 55 that connect to two tubular connecting pieces 27 that extend through said upper compartment 20 to an opening 29 provided in the upper surface 28 of water reservoir 5.
Further there is a tubular drain channel 30 provided that extends from the bottom plate 31 of the oven space 13 to aforesaid opening 20 in the upper surface 28 of water reservoir 5. In this way water on the bottom plate 31 can flow via the drain channel 30 and the connector piece 27 to said lower compartment 21.
In order to allow this lower compartment 21 to drain in a simple fashion when it is removed from the device, said connector pieces 27 are located near to the two opposing edges of the upper surface 28. It is thus possible to allow the liquid to flow from this compartment 21 along a first connector piece 27 when the water reservoir 5 is placed vertically on one side, while air is sucked in in compartment 21 via the second connector piece 27.
To fill the upper compartment 20 with water the upper surface 28 is provided with at least one opening not shown in the figures.
FIG. 4 shows a schematic plan view of the device according to the invention. As can be clearly seen in this figure two spray plates 12 are provided next to one another which each have a connection opening 32 for a water line not shown in the figures.
Each of the spray plates 12 are formed by three plates installed one above the other, 33, 34 and 35.
A first plate 33 forms the upper side of the oven space and consists by preference of a hard plastic that can withstand temperatures of up to 110° C. In this plate 33 conical sprayers 36 and liquid channels have been milled out. FIG. 6 schematically shows a section of such a sprayer 36 with a channel 37. The sprayer 36 opens at its smallest diameter via a spray opening 47 into the oven space 13 and is positioned in such a way that it is located almost centrally above a corresponding opening 16 in carrier plate 6. The spray opening 47 has by preference a diameter between 1 and 2.5 mm, and is usually virtually equal to 2 mm.
Furthermore the liquid channel 37 connects tangentially to the largest diameter of the sprayer 36 so that a liquid, flowing under sufficiently high pressure from channel 37 into the sprayer 36 undergoes a rotational motion.
In order to limit the transfer of heat from the oven space 13 through the spray plate 12, the material of the plate 33 is partly milled away in the vicinity of the channels 37 so that recesses 38 are obtained in countersunk relief separated by strengthening ribs 39. In this way the plate 33 has upstanding strengthening ribs 39 and channel walls 40.
This first plate 33, represented in FIG. 5, is provided with several sprayers 36 that are connected via a distribution chamber 41 to an equally long liquid channel 37.
A second plate 34 is mounted against this first plate 33 in which a central cylindrical distribution space 42 is milled. Multiple liquid channels 43 of equal length depart from this latter space 42 and issue in drill holes that extend perpendicularly through plate 34. Each drill hole 44 operates in conjunction with a corresponding distribution space 41 of the first plate 33 in such a way that liquid brought into the distribution space 42 via liquid channels 43 and drill holes 44 can flow to the distribution spaces 41 and ultimately reach the sprayers 36 via the liquid channels 37.
Each distribution chamber 41 is made cylindrical so that the liquid entering them via the drill holes 44 is subjected to a rotational motion. As a result of this rotational motion of the liquid there is a virtually uniform pressure on the walls of the distribution chamber 41 and in this way the same rate of flow arises in the liquid channels 37 that issue into the distribution chamber on the circumference of same.
Furthermore material is also milled away in this second plate 34 such that the walls 45 of the liquid channels 43 are raised in comparison with this plate 34.
A third plate 35 is mounted against the second plate 34 such that the channels 43 of this second plate are closed off on their upper side by means of plate 35. Said connection opening is provided in this third plate 35 which in consequence is located virtually centrally above the distribution space 42.
In this way liquid that reaches the cylindrical distribution space via connection opening 32 is entirely distributed in the latter, so that the same liquid flow rate is pushed through each of the channels 43.
The connection opening 32 is connected via a water line not shown in the figures to valve 25 of said upper compartment 20 of water reservoir 5. In this way water can be pumped with the aid of a pump 46 provided for this purpose from this upper compartment to the sprayers 36. This pump 46 is by preference realised in such a way that this water is sent to the sprayers at a pressure of 0.3 to 0.5 bar.
As the length of the water line and the channels 37 and 43 is equally long for each sprayer 36 a virtually identical jet of water 48 is obtained at each spray hole 47 at virtually the same moment in time.
This liquid jet 48 has as schematically shown in FIG. 6 an annular section, whereby the jet 48 changes from a conical form to a cylindrical form as the distance from the spray hole 47 becomes greater. In this way according to the invention a spray jet with an annular section is by means of a spray device, which comprises spray plate 12 with sprayers 36, obtained above each opening 16 in the carrier plate 7.
The control unit 15 of the device according to the invention works in conjunction with a temperature meter that measures the temperature in the oven space 13, such as for example a thermocouple. Furthermore this control unit also works in conjunction with a time counter and with said spray device and this controls the admission of steam to the oven space 13.
The embodiment of the device according to the invention as illustrated in FIG. 7 shows a spray device that is different from the embodiment of the device described here above.
In this way the embodiment of the device shown in FIG. 7 shows two sprayers 56 and 57 located in two opposing sidewalls 50 and 51 of the oven space 13. Each of these sprayers 56 and 57 allow the entire oven space to be sprayed with liquid from reservoir 5. In order to spray all eggs 18 to the same extent during the cooling, these sprayers 56 and 57 are by preference installed in such a way that they spray liquid into the oven space 13 in turns.
It is of course possible to provide more than two sprayers in the device. For an embodiment of the device according to the invention in which large numbers of eggs can be placed, it may be necessary to install several sprayers in order to cool all the eggs in accordance with the same temperature curve.
In an advantageous embodiment of the device according to the invention it is furthermore provided with a vibratory source that ensures that the liquid that is present in the oven space 13 or in the boiler 11 is evaporated or atomised. This vibratory source is by preference made of a piezo-electrical material such that ultrasonic vibration with a frequency of between 1 and 3 MHZ can be achieved. Vibration of this kind permits water to be evaporated to a large extent at relatively low temperatures, such as room temperature. For example when heating the liquid in the boiler 11 this is subjected to vibration during a period of 3 minutes at a temperature of 25° to 30° C.
In this way a relatively high level of humidity of at least 50% to virtually 100% is achieved in the oven space 13. This evaporated water condenses on the eggs that are located in the oven space 13 and moistens them. In this way it is ensured that the heat absorption of the eggs is considerably improved and made uniform when these eggs are subsequently heated with steam.
Furthermore said vibratory source ensures that the eggs 18 are vibrated at a frequency of between 1 and 3 MHZ. This vibrating causes the yolk of the eggs 18 to rise so that it is substantially in the centre of the eggs.
It is a matter of fact that eggs are usually stored standing vertically, whereby the most rounded end and the air chamber of the egg points upwards. It appears that the egg yolk in eggs kept in such a way rises so that it is virtually next to this air chamber. When such eggs are placed in the device for the preparation of soft-boiled or hard-boiled eggs this takes place by preference by placing them vertically in the device whereby they rest with their most rounded end and the air chamber on said carrier plate.
By subjecting the eggs to vibration by means of said vibratory source for a period of more or less 2.5 to 3.5 minutes, and preferably for 3 minutes, during the heating of the eggs, the yolk rises so that it lies centrally in the eggs in the so-called natural position.
The length of time during which the eggs are vibrated depends on the temperature curve in the oven space 13 and is experimentally determined for each type of egg and then programmed with the aid of the control unit 15.
When the yolk has reached this position, the yolk has set to such an extent by reason of the heating of the oven space that the yolk takes up a fixed position vis a vis the white of the egg.
In an interesting embodiment of the device according to the invention it is furthermore provided with an electromagnetic source of radiation to heat the eggs. This electromagnetic source of radiation produces in particular so-called microwaves, which are generally known.
This radiation source is by preference used in combination with said heating element 11, which produces steam, in order to prevent the eggs from bursting by the application of excessively high power to the radiation source.
When, however, the eggs are only heated with the aid of said radiation source, the radiation source required for heating 60 "medium" eggs with a total weight of between 3.18 kg to 3.78 kg from 5° C. to 58° C. has a power of 1.2 kW. When power of this magnitude is applied the eggs reach a temperature of 58° C. after roughly 8 minutes. Of course this is likewise possible for eggs in other weight classes. This embodiment is highly interesting for the pasteurisation of eggs.
In order to determine what power is required to heat the eggs, a temperature meter is provided in the oven space that for example consists of an infra-red sensor that measures the temperature curve of a randomly selected egg in the oven space for a particular period of time. This temperature curve is for example measured for a period of 2 minutes.
The device described above makes it possible to apply the method according to the invention for the boiling of eggs.
Pursuant to this method uncooked eggs in their shells 18 are placed in the oven space 13. These eggs 18 are positioned vertically in the openings 16 in the carrier plate 17. That is to say the most rounded end 49 of the eggs protrudes to some extent below the carrier plate 7. Subsequently the closing door 4 is closed so that the oven space is virtually entirely sealed off from the surroundings.
In a following step the oven space 13 is heated with steam from the heating element 11 to a temperature between 95° and 110° C. Good results are obtained when the oven space 13 is heated to a temperature of more or less 98° C. After a period of time of 6 to 7 minutes the eggs are cooled using the said spray plate by water at room temperature during 10 to 50 seconds, in particular during 15 to 30 seconds and the oven space 13 is then maintained at a temperature of between 50° C. and 65° C.
When the oven space 13 is thus maintained at this consumption temperature of between 50° C. and 65° it is possible to keep the eggs a long time without further setting of the yolk or the white occurring. This makes it possible among other things to use the device or the method according to the invention in self-service counters so that consumers can always be sure of having a virtually perfect soft-boiled egg and the certainty that harmful bacteria in the egg have been destroyed.
When heating the eggs the outer layer of the egg is first heated, while the heating of the core of the eggs takes much longer by reason of the relatively slow diffusion of heat in the egg. As a result it is important that the eggs 18 are cooled before they have reached the desired temperature in their centre as it has been determined that after the cooling of the eggs and the oven space there will be a further rise of the core temperature as a result of the continued diffusion of the heat still in the egg.
When the eggs are cooled too late, the continued diffusion of heat in the eggs will cause the entire egg to set after a certain period of time.
The temperature ultimately desired in the core of the eggs is thus dependent on the extent to which the eggs are to set.
In order to avoid possible bacterial infection in eggs care must be taken to ensure that the temperature reached in the core is sufficiently high to ensure that all bacteria possibly present in the egg are destroyed. The temperature required is known and for a period of heating of 2 to 3 minutes is virtually equal to 60° C.
In FIG. 8 two curves are shown which show the course of the temperature as a function of time for the preparation of soft-boiled eggs in which only the egg white has set. A first curve 53 shows the course of the temperature in the oven space 13, while a second curve 54 shows the course of the temperature measured in the core of the eggs.
In this way the oven space is heated for about 6.5 minutes with steam from a temperature of 35° C. to 97° C. During this heating of the oven space the core temperature of the eggs rises from about 15° C. to about 48° C. When the eggs are sprayed with a coolant, namely water at room temperature, for 20 seconds the core temperature of the eggs continues to rise to about 62°, while the temperature in the oven space is maintained at a virtually constant 60° C. When said closing door 4 is opened, the temperature in the oven space 13 falls almost immediately to room temperature, which is about 23° C.
However it is also possible to maintain the temperature of the oven space 13 at more or less 60° C. without there being any further setting of the eggs.
By using the device and the procedure according to the invention it is for example possible to boil a number of eggs in the oven space so that they are hard-boiled, while the remaining eggs are soft-boiled. To achieve this the eggs that are to be hard-boiled are cooled for a shorter period of time than the eggs which are to be soft-boiled.
Furthermore soft-boiled eggs can after being for example cooled outside the device to a temperature between 2° C. and 22° C. be again placed in the device and be again heated and cooled perhaps together with raw eggs in accordance with the method according to the invention so that these eggs are once again brought to consumption temperature.
The invention furthermore also concerns a method for the pasteurisation of unshelled eggs. Pursuant to this method the eggs are heated until the core, in particular the centre of the yolk, has reached a temperature of between 53° C. and 65° C., and preferably between 57° C. and 61° C., and maintained at this temperature during a sufficiently long period of time for the bacteria present in the eggs to be destroyed. The temperature required for destroying bacteria, such as for example salmonella, is sufficiently known. American patent documents U.S. Pat. No. 5,843,505 and 6,004,603 give in both their FIG. 1 a chart that illustrates the relationship between temperature and the period of time during which this temperature must be maintained in order to destroy bacteria.
In order to achieve a sufficiently high temperature in the eggs as quickly as possible, the eggs are by preference warmed simultaneously by steam and microwave radiation. It has in fact been determined that when microwaves are used the yolk of the eggs is heated more quickly than the white of the egg.
Furthermore it appears that the appearance of the white, namely its colour among other things, changes when it is maintained for a certain period of time to the pasteurisation temperature of between 53° C. and 65° C. Consequently it is important after heating up the eggs, to cool them as quickly as possible to a temperature below 45° C. The eggs are thus cooled for example to a temperature between 5° and 25° C.
By cooling in this way the desired temperature of the eggs is reached within 15 to 120 seconds thus avoiding the occurrence of this colour change in the white of the egg.
The eggs are cooled by preference by irrigating them or spraying them with water at a temperature of between 5° C. and 25° C.
When the eggs are cooled down after the core has been exposed for virtually 3.5 minutes to a pasteurisation temperature of 60° C., it appears that the appearance of the egg white and the yolk of the eggs can hardly be distinguished from that of unpasteurised eggs.
In another example the core of the eggs, namely the centre of the yolk, is maintained at a temperature of 57° C. and the eggs are then cooled. Here too it was determined that as a consequence of the cooling of the eggs there was virtually no change in the white or the yolk of the eggs.
It is thus in this way possible pursuant to this procedure to pasteurise eggs very quickly without causing significant changes in the properties of same. By heating eggs which are at room temperature using both microwave radiation and steam the eggs can be fully pasteurised in less than 30 minutes when a pasteurisation temperature of 60° in the core of the eggs is applied.
The temperature curve for another example of egg pasteurisation is given in the graph of FIG. 12. This figure shows two curves. The first curve 82 shows the temperature evolution in the oven space while the second curve 83 represents the temperature evolution in the core of the eggs. In this example, the oven space that contains the eggs has been heated with steam from substantially 23° C. to 61° C. in 60 seconds, whereby subsequently, the temperature in the oven space has been maintained during ca. 940 seconds at a temperature of about 62.5° C., and, finally, the oven space was cooled for about 40 seconds to about 25° C. by spraying the eggs with water. During this process the temperature at the core of the eggs is between 59° C. and 61° C. for about 4 minutes.
In the case of such rapid heating of the oven space, and therefore also of the eggs, pasteurised eggs are obtained, whereby the egg white near the shell has already more or less set while the egg white around the yolk is still transparent. The contents of such a rapidly pasteurised egg is slightly more viscous than a non-pasteurised fresh egg but it can however be used without any problem for processed food stuffs, such as for preparing fried or scrambled eggs.
The eggs are moistened in an advantageous fashion before heating with the help of water vapour at a relatively low temperature formed by the ultrasonic vibration of the water in said heating element. In this way the heat transfer between the eggs and the steam is considerably improved.
The dimensions of the device according to the invention as represented in the figures are chosen such that it can be installed in a kitchen in the same way as a conventional oven. By adjusting the dimensions and shape of the device according to the invention, it can be made suitable for handling only a few eggs or a very large number of eggs.
Furthermore the upper compartment of the water reservoir can be replaced by a connection to a conventional water supply, while the lower compartment can be replaced by a drain pipe leading to a connection to the drains.
A third embodiment of the device, according to the invention, is shown in FIGS. 9 and 10. This device has a protective cover 63 which can be removed from a housing 64. The protective cover 63 is in the shape of a semi-ellipsoid and its circle-shaped edge 65 fits onto the housing 64. This protective cover 63 surrounds an oven space 13 which is closed at the bottom by a ring-shaped oven plate 6 which has a central circle-shaped opening 6′.
Besides this oven plate 6, the housing 64 also comprises a first compartment 66 which contains an electronic control unit for the device and a water reservoir that consists of two separate compartments 20 and 21. The latter two compartments 20 and 21 are U-shaped such that they partly surround the aforementioned first compartment 66.
On the aforementioned oven plate 6 rests a ring-shaped carrier plate 7 which has holes in order to hold the eggs 18 upright on this carrier plate 7 in the oven space 13 as shown in FIGS. 10 and 11. This carrier plate 7 is made preferably from a wired structure in order to ensure adequate circulation of steam around the eggs 18.
The device further is provided with a ventilator 14, in a co-axial position with the aforementioned oven plate 6 and the carrier plate 7, which extends above the circle-shaped opening 6′. This ventilator 14 has short blades 69 which run practically parallel to its shaft. In addition, the ventilator 14 has a ventilator housing 67 that separates the ventilator 14 on the sides and at the top from the oven space 13 and that has openings 68 along its entire circumference at the height of the ventilator 14, in order to let the steam flow over the eggs 18.
The ventilator 14 is driven via its shaft 70 by means of an electrical motor 71 which is mounted in the aforementioned first compartment 66.
The shaft 70 of the ventilator 14 is partly situated in a cylindrical co-axial sleeve 72 with respectively on top of each other a layer of thermal insulation 73, a cooking plate 74 surrounded by a sealing ring 75 and a connecting piece 76.
The boiling plate 74 comprises a heating resistor so that water on the plate can be heated to produce steam. In this respect, the connecting piece 76 is connected to the boiling plate 74 so that a cylindrical boiling vessel is produced. The steam that is produced by means of the boiling plate 74 leaves the boiling vessel via the aforementioned opening 6′ in the oven plate 6 and as such the steam is extracted by the ventilator 14 that distributes the steam via the aforementioned openings 68 of the ventilator housing 67 over the eggs 18.
Further, a pump 77 is mounted in the compartment 66 of the housing 64 that works in conjunction with the bottom compartment 21 of said reservoir in order to distribute the water from this compartment 21 via a water pipe 78 over the eggs 18.
The water pipe 78 leads, in particular, to the centre and above the ventilator 69 via an opening 84 which is provided in the spherical top side 79 of the ventilator compartment 67. When the ventilator 69 is working, the water that comes out of the water pipe 78 is attracted by the ventilator and squirted through the openings 68 of the ventilator housing 67 and the water is spread over the eggs 18.
This makes it possible to moisten the eggs 18 before they are subject to thermal treatment or to cool the eggs rapidly when the thermal treatment has finished. By varying the rotation speed of the ventilator 14, the speed of the squirted water varies and the water is distributed over the eggs 18 substantially evenly and over substantially the entire oven space 13.
In order to regulate the temperature of the steam in the oven space 13, the said oven plate 6 has a temperature sensor 80 that co-operates with the said electrical control unit and that measures the temperature of the oven space 13.
The water that is thus transferred from the bottom compartment 21 into the oven space 13 ends up on the oven plate 6 which has a conical surface so that the water flows to the said central opening 6′ and to the said boiling plate 74.
The connecting piece 76 that connects to said opening 6′ also has a so-called overflow, which is not shown in the figures, which evacuates the water when the latter exceeds a pre-set level in the said boiling vessel to the top compartment 21.
Furthermore, the oven plate 6 along the inside of the said opening 6′ has a series of upright teeth 81 at a small distance from each other. In the case of an egg 18 breaking, these teeth 81 prevent egg white or yolk or even egg shell from going through the said opening 6′ with the water. Since any egg white or yolk that is deposited on the oven plate 6 will set immediately, this can then be stopped by the said teeth 81.
So in order to boil eggs hard or soft or to pasteurise them, the protective cover 63 is removed from the housing 64 and the eggs 18 are placed on the said carrier plate 7 in the oven space 13. Subsequently, the protective cover 63 is placed back on the housing 64 in order to close the oven space 13.
The bottom compartment 21 of the reservoir is filled with water and placed in the housing 64. This water is then used during the cooking of the eggs 18, to generate steam by means of the boiling plate 74 on the one band, and on the other hand as cooling water to cool the eggs 18 rapidly after they have been heated by the steam.
By means of a control panel which is not shown in the figures, it is possible to select the desired thermal treatment for the eggs.
Notwithstanding that in the aforementioned embodiment of the device, according to the invention, the water pipe 78 constitutes a simple spraying device, it is however possible to provide a different spraying device.
It is possible, for example, that each egg 18 is irrigated with water by means of individual sprays. It is also possible to squirt water against the inner walls of the protective cover 63 from the oven plate 6, whereby the inner wall above the eggs 18 is uneven so that water would be guided onto the eggs 18 from these uneven parts.
The water pipe 78 in an alternative version of this embodiment of the device could be co-axial with the said ventilator shaft 70 and, for example, coincide with said shaft 72.
It is clear that although the parts in the aforementioned version of the device mainly have an axial symmetry, the device can be executed in a variety of geometrical shapes.
During the pasteurising or the preparing of eggs, the oven space 13 is heated with only part of the power that is available for the heating of the oven space. For example, this means that only 80% of the available power is used to heat the oven space from about 35° C. to 50° C. For this process, the time necessary to reach the 50° C. in the oven space is measured. From this data, which is a measure for the thermal heat capacity, the amount of power that is necessary in the other part of the pasteurising process in order to obtain the desired temperature curve, is calculated.
In this description steam is understood as every form of water in the gas phase. This may be both water vapour resulting from the boiling of water in said heating element or water that has been atomised in the oven space using said vibratory source.
Furthermore aforementioned coolant and the liquid that is heated in the heating element is by preference water.
The invention is of course not limited to the embodiment described here above of the device and the method according to the invention, for example the carrier plate for the eggs may take various forms and this may for example have a wire structure so that the steam can flow freely over the entire surface of the eggs. Furthermore the oven space may be heated in other ways, or this may be heated both by means of steam and by means of electrical resistances.
Notwithstanding the fact that the device according to the invention is highly suited to using water as coolant, it is not impossible that other coolants or cooling gases could be used.

This patent is subject to a terminal disclaimer.

Device for the thermal treatment of unshelled eggs


A method of determining a respiratory parameter for a subject using an indirect calorimeter is provided. The indirect calorimeter includes a respiratory connector for passing inhaled and exhaled gases, a flow pathway operable to receive and pass inhaled and exhaled gases having a flow tube within the flow pathway through which the inhaled and exhaled gases pass, a flow meter for determining an instantaneous flow volume of the inhaled and exhaled gases, a component gas concentration sensor for determining an instantaneous fraction of a predetermined component gas and a computation unit having a processor and a memory. The method includes the steps of initializing the indirect calorimeter and the subject breathing into the respiratory connector if the indirect calorimeter is initialized, sensing the flow volume of the inhaled and exhaled gases passing through the flow pathway using the flow meter and transmitting a signal representing the sensed flow volume to the computation unit. The method also includes the steps of sensing a concentration of a predetermined component gas as the inhaled and exhaled gases pass through the flow pathway using the component gas sensor, and transmitting a signal representing the sensed concentration of the predetermined component gas to the computation unit. The method further includes the steps of calculating at least one respiratory parameter for the subject as the subject breathes through the calorimeter using the sensed flow volume and the sensed concentration of the predetermined component gas, and providing the subject with the at least one respiratory parameter.

U.S. Pat. Nos. 4,917,108; 5,038,792; 5,178,155; 5,179,958; 5,836,300; and 6,309,360, all to Mault, a coinventor of the present application, are incorporated herein by reference. These patents disclose systems for measuring metabolism and related respiratory parameters through indirect calorimetry. These instruments generally employ flow meters which pass both the inhalations and the exhalations of a user breathing through the instrument and integrate the resulting instantaneous flow signals to determine total full flow volumes. In one embodiment, the exhaled gases generated by the user are passed through a carbon dioxide scrubber before passing through the flow meter so that the differences between the inhaled and exhaled volumes is essentially a measurement of the oxygen consumed by the lungs. In an alternative embodiment, the concentration of carbon dioxide exhaled by the user is determined by passing the exhaled volume through a capnometer and integrating that signal with the exhaled flow volume. The oxygen consumption can then be calculated as the difference between the inhaled and exhaled volumes minus the exhaled carbon dioxide volume.
The scrubber used with certain of these systems was relatively bulky and required replenishment after extended usage. The capnometers used with the instruments to measure carbon dioxide concentration had to be highly precise and accordingly expensive because any error in measurement of the carbon dioxide content of the exhalation produces a substantially higher error in the resulting determination of the oxygen content of the exhalation.
Additional approaches to indirect calorimetry and cardiac output monitoring are disclosed in Mault's co-pending application Ser. No. 10/090,984; PCT/US02/06764; Ser. No. 09/191,782; PCT/US99/02448; PCT/US99/17553; PCT/US99/27297; and PCT/US00/12745, each of which are incorporated herein by reference. Thus, there is a need in the art for a method of respiratory gas analysis using an indirect calorimeter.

Method of respiratory gas analysis using a metabolic calorimeter


An anti-tipping device for a shopping cart having a pair of non-pivotal wheels and a pair of pivotal wheels. The anti-tipping device comprises a pair of concave downward extending anti-tipping members clamped to the outer surface of the lower frame adjacent to the pivotal wheels. The lower end of the anti-tipping members extend close to but not touching the cart support surface. When the cart is tipped sideways at least one of the anti-tipping members contacts the cart support surface preventing any further tipping of the cart.

The present invention is directed to shopping carts and particularly to the prevention of four wheeled shopping carts having two fixed direction wheels and two swivel wheels from tipping over forward or sideways when the swivel wheels are swivelled toward each other.
In most retail stores, such as super markets or the like, shopping carts are provided for use so that customers can conveniently transport their selected merchandise through the store, past the checkout stand and eventually to the customer""s auto or other transportation means.
In the event that the cart is loaded and the greater mass of the merchandise is positioned in the front of the cart or children are allowed to ride in the cart while shopping or transporting the merchandise and they move around within the cart to a position that renders the cart front end or side heavy the cart could tip dumping the merchandise or a child in the cart to the ground or cart support surface.
Obviously if this were to occur the merchandise would be damaged and even more seriously a child could be severely injured.
U.S. Pat. No. 3,051,936 issued to A. E. Finger et Al teaches principally an alarm system to detect merchandise positioned on a bottom shelf of a four wheel shopping cart. The patent does utilize a pair of legs on the back sides of the cart that could prevent the cart from tipping sideways but would not prevent the cart from tipping forward.
U.S. Pat. No. 5,090,724 to inventor Deborah A. Flore teaches a pair of anti-tip legs, one in the front of the stroller and one in the back of the stroller. The legs can be deployed manually to prevent the stroller from tipping either backwards or forward but not sideways. Unlike the present invention the anti-tipping feature of this invention does not teach sideways tipping and is not always in place to prevent front tipping while the cart is moveable and must be manually deployed if the cart is to be unattended and stowed in a non-deployed position to again move the stroller.
U.S. Pat. Nos. 4,610,454 and 5,630,600 respectively to William H. Gill and Norbert Q. Pasillas both teach stops that must be manually deployed for use as brakes that would could possibly prevent backward and forward tipping respectively when deployed for rendering the cart stationery but would not prevent sideways tipping even when deployed.
As such, it is very advantages to provide both an anti-sideways tipping structure for a shopping cart as described that is always fixed is a deployed position to prevent tipping either forward or sideways and yet allows the shopping cart to function as intended at all times.
The invention is directed to preventing the forward end sideway tipping of shopping carts to prevent injury to a child riding in the cart or the damage to merchandise in the cart. A typical shopping cart on which the anti-tipping device is intended can be found, by way of example, in U.S. Pat. No. 3,051,936 which teaches a shopping cart in with the two front support wheels pivot relative to the rear to the non-pivotal rear wheels. It should be understood that this invention works equally as well when applied to carts with pivotal rear wheels and non-pivotal front wheels.
One anti-tipping member is attached to the frame adjacent to and on the outside of each of the pivotal wheels. The anti-tipping member is formed with a concave side adjacent to the wheel so that the wheels can pivot relative thereto in a normal manner. The anti-tipping members are clamped to the bottom support frame of the cart and can be added to existing carts as well as new carts.
The manufacturing of the carts does not have to be changed to utilize the anti-tipping members of this invention.
The clamping of the anti-tipping members can be with clamps formed on anti-tipping member on the cart frame attachment end or can be clamped to the cart frame by any manner suitable for the purpose intended. The anti-tipping members can be constructed of plastic, fibreglass, metal or any other material suitable for the purpose intended.
The principal object of this invention is to provide a fail safe means for preventing shopping carts from tipping forward or sideways normal use.
Another object of this invention is to prevent shopping carts from front end and sideways tipping when the pivotal wheels become directed toward each other.
Still another object of the invention is to add anti-tipping members that do not interfere with normal wheel pivoting.
Yet another object of this invention is to provide an inexpensive means for preventing the forward and sideways tipping of shopping carts that is easily attachable to existing or newly manufactured shopping carts of the type defined herein.
Other objects and advantages of the invention will be apparent during the reading of the following description along with the accompanying drawing Figures in which like numerals are used to designate like parts or elements.