BACKGROUND OF THE INVENTION

This invention relates to a method for forming a calibration curve for a component of interest in a fluid for each of a plurality of optical read stations in an automated, analytical instrument. Various types of chemical tests can be performed by automated test equipment, an example of testing of considerable interest being the assay of biological substances for human health care. Automated test equipment allows large numbers of test samples to be processed rapidly. Such equipment is employed in health care institutions including hospitals and laboratories. Biological fluids, such as whole blood, plasma, serum or urine are tested to find evidence of disease, to monitor therapeutic drug levels, etc.

In the automated test instrument a sample of the test fluid is typically provided in a sample cup and all of the process steps including pipetting of the sample onto an assay test element, incubation and readout of the signal obtained are carried out automatically. The test instrument typically includes a series of work

stations each of which performs one of the steps in the test procedure. The assay element may be transported from one work station to the next by a conveyor such as a carousel to enable the test steps to be accomplished sequentially. Typically, the conveyor carries a plurality of assay modules each of which is secured to a specific location of the surface of the conveyor. In the usual arrangement, the assay modules are spaced apart from each other in berths which are located along the periphery of the conveyor to facilitate automatic insertion and extraction. In certain types of instruments such as those which are designed to carry out assays based on immunometric interactions between analytes or metabolites and their binding partners, at least the part of the conveyor carrying the assay modules is arranged within the temperature controlled chamber since it is necessary that the assay be carried out at a very precisely controlled temperature, for example at 37 ± 0.5°C. The assay cartridges are maintained in the temperature controlled chamber for a period of time sufficient to bring them to the desired temperature prior to beginning the assay procedure and are maintained at that temperature for the duration of the process.

As is known in the art typically there is stored in the control processing unit (CPU) of such automated instruments a calibration curve for each analyte which can be analyzed by the instrument. When a patient sample is analyzed by the instrument the signal obtained from the sample is automatically applied to the calibration curve and the concentration of the analyte in the sample is calculated therefrom. New calibration curves must be prepared at

various intervals due to variables such as different production lots of the assay elements which are used in the instruments, etc.

It is known in the art to provide automated analytical instruments which include more than one optical read station. Such instruments can provide a desirably higher throughput which allows a larger number of samples to be analyzed in a given time period. To allow the instrument to be operated at its maximum capacity it must have the capability to read out the signal from any assay element at any of the optical read stations. Accordingly, those skilled in the art will appreciate that a calibration curve must be prepared for each analyte at each optical read station for the instrument to be operated in this mode.

It can be seen that such instruments therefore present considerations relating to the expense and time involved in preparing the requisite calibration curves, that is, how many assay elements and what volumes of control or calibrator compositions are required and the time period needed to do so. One technique for preparing such calibration curves involves using a separate series of assay elements for each of the optical read stations. Since each calibration curve typically requires six control or calibrator compositions of different concentrations and these are usually run in duplicate the number of assay elements needed for that method increases significantly as the number of optical read stations in the instrument increases. Thus, there is a continuing need for new and advantageous calibration methods for use with instruments having a plurality of optical read stations.

SUMMARY OF THE INVENTION It is therefore the object of the present invention to provide a novel method for providing calibration curves for use with an analytical instrument having a plurality of optical read stations. It is another object of the invention to provide a method for providing calibration curves wherein each assay device used to prepare a calibration curve is read at each of the plurality of optical read stations.

It is a further object of the invention to provide such a method wherein the assay devices are read successively at each of the plurality of optical read stations at different specifically defined times.

These and other objects and advantages are accomplished in accordance with the invention by providing a method for preparing a calibration curve for an analyte of interest on an automated analytical instrument which includes a plurality of optical read stations. According to the method, each of the plurality of assay devices to which there is applied one of the control or calibrator compositions used to create the calibration curve is read successively at each of the optical read stations at different times. The times set for taking the readings at each of the optical read stations are specifically defined and are determined, in part, by the incubation time required for the particular analytical assay for which the calibration curves are being provided and the number of optical read stations in the instrument as will be described in detail below herein.

Thus, for any particular analyte, and defining the time necessary to bring the assay device to which there has been applied

a control or calibrator composition and any other required reagent(s) to the desired temperature in the temperature-controlled chamber as T ₀ , the first optical reading is taken at optical read station R*, at a time T, which may be the same as T ₀ or a time close to T ₀ . The second optical reading is taken at optical read station R ₂ at time T ₂ which can be defined as T-, + At-, and a third optical reading, if required, at optical read station R ₃ at time T ₃ which can be defined as T ₂ + Δt ₂ (where Δt ₂ may be the same as At*, or different).

The method of the invention can be practiced with an analytical instrument which has two or more optical read stations. Accordingly, where the instrument has N read stations (where N is an integer equal to or greater than 2), the method comprises the steps of taking, on an assay element, N successive optical readings at each of the N optical read stations, each reading being taken at a time which is different for each optical read station.

As noted previously, in analytical instruments which have multiple optical read stations it is preferred to provide, for each optical read station, a calibration curve for each analyte which can be analyzed by the instrument. By doing so any assay device which is used for the analysis of a patient sample fluid for any analyte within the instrument assay menu can be read at any of the plurality of optical read stations thus allowing the instrument to be operated most efficiently and maximizing the assay throughput rate as much as is possible. As will be appreciated by those skilled in the art all the optical readings taken on a patient sample fluid have to be obtained at the same optical read station and are taken at the times established

for such readings at the respective optical read stations by the calibration method of the invention.

By using only one assay device for each of the control or calibrator compositions used to provide the calibration curves for a plurality of optical read stations in accordance with the method of the invention there is provided a significant decrease in the time required to obtain the curves and a significant reduction in the number of assay devices necessary to do so. In addition, lesser volumes of calibrator and/or control compositions are needed in view of the decreased number of assay devices. It can be appreciated therefore that the method provides significant advantages.

BRIEF DESCRIPTION OF THE DRAWINGS

For a better understanding of the invention as well as other objects and further features thereof, reference is made to the following detailed description of various preferred embodiments thereof taken in conjunction with the accompanying drawings wherein:

Fig. 1 is a partially schematic, cross-sectional view of an assay device which may be utilized in the method of the invention; Fig. 2 is a partially schematic, perspective view of another assay device which may be utilized in the method of the invention;

Fig. 3 is a graphical illustration showing the calibration curves prepared for an analyte for three optical read stations utilizing an assay device of the type shown in Fig. 1 ; and

Fig. 4 is a graphical illustration showing the calibration curves prepared for another analyte for three optical read stations utilizing an assay device of the type shown in Fig. 2

DESCRIPTION OF THE PREFERRED EMBODIMENTS The method of the invention may be practiced with any assay device which includes, or to which there may be applied, one or more reagents which will provide, in the presence of a fluid sample including an analyte of interest, an optical signal which is a function of the concentration of analyte in the fluid. The optical signal may be provided by any suitable signal-generating system. Any light radiation - emitting or absorbing species, including those which react with a reagent which provides an optical signal can be utilized as the signal- generating species.

The desired optical signal may be generated as a result of chemical reactions and/or immunometric interactions. The preferred assays with which the method is practiced are those based on immunometric interactions between an analyte of interest and binding partners for the analyte, particularly specific binding partners. Particularly preferred assays are based on antigen-antibody interactions. It is known in the art to use in immunometric assays conjugates which are made up of a light radiation- emitting or absorbing label covalently bound to one of the members of the signal generating system. Any of the labels known for use in immunometric assays may be utilized including those which are directly detectable, for example, a fluorophore, a chemiluminophor, a radioactive material or a light absorbing material, and those which are indirectly detectable

such as an enzyme. Any change in fluorescence, chemilumiscence, radioactivity or other change in visible or near visible radiation can be detected. Where the label is an enzyme it can be one which interacts with a substrate to cause a change in absorption where the substrate is a chromogen, in fluorescence where the substrate is a fluorophore, in chemiluminescence where the substrate is a chemiluminescent precursor or in phosphorescence where the substrate is a phosphor. Enzyme labels are preferred in instances where it is desirable to amplify the signal which is obtained. Any suitable assay device may be utilized in the practice of the method of the invention. Suitable assay devices include containers such as test tubes and cups to which the fluid reagent(s) and calibrator or control composition are added, self-contained assay elements which include the necessary reagent(s) for the assay and to which only the fluid calibrator or control composition is added and assay elements which may include a reagent and to which one or more additional reagents as well as the calibrator or control composition are added.

One preferred type of assay device is the "dry" assay element including those which are made up of only one reagent layer carried by a support layer and multilayer assay elements which have at least one reagent layer and one or more other layers which may be reagent layers, a light-blocking layer, a layer for receiving a signal- generating species formed in, or liberated from, another layer, etc. Another preferred type of assay device is that wherein the reactions and/or interactions which are utilized to provide the desired optical signal are carried out on a solid carrier material.

The assay method practiced with the assay devices may be an end point assay, i.e., one where an optical reading is taken at a defined period of time after the sample fluid and any other required reagent(s) are applied to the assay device and the signal applied to a calibration curve to determine the concentration of analyte in the fluid. The assay method may also be a rate assay in which a plurality of optical readings are taken at specific times after applying the sample fluid and any other required reagent(s) to the assay device to obtain a linear reaction rate relationship. The slope of the linear rate relationship is taken and applied to a calibration curve to determine the analyte concentration.

As noted previously the calibration method of the invention can be practiced generally with analytical instruments having a plurality of optical read stations. The maximum number of optical read stations with which the method can be practiced in any instance is determined in part by the incubation period required for the assay. This is so because the shape of the calibration curve which is obtained at any optical read station may begin to change when the time the assay device remains in the temperature-controlled chamber after the last required fluid, i.e. the control or calibrator composition or another reagent, has been applied to the assay device exceeds some maximum period which is a function of the particular assay method. This consideration places constraints on the amount of time which can be allowed to pass between the end of the required incubation period and the first optical reading taken at the first optical read station and also on the time period(s) between the successive optical readings taken at the plurality of read stations. Accordingly,

it is preferred to obtain the first optical signal at the first optical read staion at a time which is very close to the required incubation time, for example, within a time which is approximately one percent (based on the incubation time) after the expiration of the incubation period. It is further preferred to complete the successive optical readings taken on an assay device at the plurality of optical read stations within a time period which is approximately ten percent (based on the incubation time) after the expiration of the incubation period. The calibration method is preferably practiced with an analytical instrument having from two to six optical read stations.

As will be described in detail below herein the method by which the calibration curves are formed may involve only the application of the control or calibrator composition to the assay device and therefore only one incubation period is required. However, the method may require, in addition to applying the control or calibrator composition to the assay device, the application of one or more reagents. In such assay methods there may be required two or more separate incubation periods. It should be understood that the incubation period to which reference has been made as being controlling with respect to setting the times for the optical readings is that which occurs after the last (which may be the only) fluid reagent is applied to the assay element.

The method of the invention will now be described with respect to a specific preferred embodiment wherein there is utilized a multilayer assay element as illustrated in Fig. 1 with which there is carried out an end point assay. Referring now to Fig. 1 there is seen an assay element 10 which is a thin film multilayer element typically

having a thickness of about 0.1 mm and comprised of a transparent support 12 which carries in succession a reagent layer 14, a light- blocking layer 16 and an optical top coat layer 18 which may serve as a reagent layer, a filter layer such as for proteins, an antiabrasion layer, etc. The reagent layer 14 is very thin, typically having a thickness of about 0.025 mm and includes a immunocomplex of a binding partner for the analyte of interest and a conjugate of a labeled analyte (the same as the sample analyte, an analogue thereof or a structurally similar material which will bind to the binding partner). The binding partner, an antibody when the sample analyte is an antigen, is immobilized in the reagent layer 14 by being covalently bound to the surface of the support layer 12, which may be of any appropriate material such as a polyester or a polystyrene, or to a matrix material or by being physically held by the matrix material. The matrix material may be hydrophilic gel material such as gelatin, a polysaccharide, e.g. agarose, a derivatized polysaccharide, mixtures thereof, and the like. Light-blocking layer 16 may comprise any suitable material such as, for example, iron oxide, titanium dioxide or the like dispersed in a binder material such as a polysaccharide. The optional topcoat layer 18 may comprise an antiabrasion layer of a material such as a polysaccharide or preferably may include buffers, blocking and displacing agents, etc.

The assay element 10 may also include a layer or other means (not shown) for distributing the sample fluid uniformly across the surface of the top layer of the element. Any suitable fluid distribution technique may be used including, for example, particulate layers, polymeric layers, fibrous layers, woven fabric layers and liquid

transport systems which have been disclosed in the art as being suitable for this purpose. Many such fluid distribution systems and materials for providing a uniform distribution of a fluid sample across the surface of an assay element are known in the art and therefore extensive discussion of such materials and systems is not required here. A particularly preferred fluid transport system is that described in United States Patent 5,051 ,237. The distribution means, whether a layer of fibrous material, etc. or a liquid transport system is preferably relatively thick in comparison to reagent layer 14. One of the series of control or calibrator compositions is distributed across the surface of the assay element 10 and the fluid diffuses throughout layers 14, 16 and 18 as well as any fluid distribution layer or liquid transport system present and an equilibrium is established. The analyte present in the control will compete with the labeled analyte in reagent layer 14 for the available binding sites on the antibodies immobilized in layer 14, the labeled analyte being dissociated therefrom and replaced by the control analyte in a ratio approximately equal to the relative amounts of control analyte and labeled analyte. Thus, depending upon the amount of analyte in the control, some percentage of the labeled analyte initially bound to the immobilized antibodies in layer 14 will be displaced therefrom and distributed throughout the remainder of the assay element. The amount of labeled analyte bound to the immobilized antibodies in reagent layer 14 at any time in inversely proportional to the amount of control analyte.

The assay element 10 is then allowed to remain in a temperature - controlled chamber for the appropriate incubation time

and at the end of the period, or as close as possible thereto, for example, within approximately one or two percent, a readout signal is obtained at the first optical read station by irradiating reagent layer 14 through support layer 12 with the appropriate electromagnetic radiation. After a brief period of time a readout signal is obtained at the second optical station in the same manner. Optical readings are then obtained at any additional optical read stations with a brief period of time between each. The time between each of the successive readings obtained with the assay element is preferably the same.

The procedure is repeated for each of the series of control or calibrator compositions required to provide the calibration curve, each composition being applied to a different assay element which is specific for the same analyte of interest. The number of control or calibrator compositions required in any instance is dependent in part on the assay concentration range. To illustrate, in an assay for theophylline it is preferred to utilize six calibrator compositions, respectively containing 0.00, 2.50, 5.00, 10.00, 20.00 and 40.00 ug/mL of theophylline to create the calibration curve. In a preferred embodiment wherein calibration curves are prepared for a theophylline assay and the analytical instrument includes three optical read stations, each assay element to which there is applied one of the calibrator compositions is preferably read at the three optical read stations after residing in the temperature controlled chamber for 350, 360 and 370 seconds, respectively.

The optical signal obtained from assay element 10 is inversely proportional to the amount of analyte in the calibrator

composition, that is the signal decreases as the amount of analyte increases. Since reagent layer 14 is relatively thin in comparison to the combined thickness of layers 18 and 18 together with that of any fluid distribution layer or liquid transport system present and because light blocking layer 16 prevents any of the readout electromagnetic radiation from entering layer 18 or anything above it, the second signal obtained will be a function of the labeled analyte which is bound to the immobilized antibodies and a small percentage of the free labeled analyte which is distributed throughout the remainder of the assay element. In a preferred embodiment the ratio of the thickness of reagent layer 14 to the combined thickness of the light- blocking layer and the remainder of the assay element is from about 1 :20 to about 1 :100 or more.

In a particularly preferred embodiment of the method, the optical signals obtained with assay element 10 are normalized by obtaining for each assay element used, prior to any calibrator composition being applied, an optical reading at each of the optical read stations. This first, or "dry", reading is carried out by irradiating the assay element in the same manner described above to obtain the optical readings after the calibrator composition has been applied to the assay element and the appropriate times have elapsed. The second, or "wet", reading at each optical station is divided by the first, or "dry" optical reading at that station to normalize the signal so as to compensate for variations in reagent levels because of variations in layer thickness from assay element to assay element and also for variations in the analytical instrument position response. Copending, commonly assigned application serial no. 382,555, filed July 19,

1989 discloses and claims this method for determining the concentration of analyte in a sample fluid.

In another particularly preferred embodiment of the method the first, or "dry" optical reading can be corrected for relative humidity and/or temperature variations. Copending, commonly assigned application serial no. 533,163, filed June 4, 1990 discloses and claims this method for determining the concentration of analyte in a sample fluid.

Another specific preferred embodiment of the invention utilizes an assay element as illustrated in Fig. 2 with which there is carried out a rate assay. Referring now to Fig. 2 there is seen a self- contained, capillary assay module 20 which carries all the test reagents except for the sample fluid necessary for a particular assay. This preferred assay element includes a plurality of chambers in a housing 22 wherein a first chamber serves as a front reservoir 24 for the storage of a labeled conjugate solution. The solution is covered with a frangible or puncturable foil layer (not shown). A second of the chambers serves as a back reservoir 26 for the storage of a substrate solution which is also covered with a similar foil layer (not shown). An optional third chamber serves as a mixing bowl 28 for the mixing of reagents and a fourth chamber forms part of a dispenser 30 which is utilized to dispense the substrate solution to one end of the porous member 32. There is also shown a chamber 34 within the housing 22 wherein there is arranged an absorbing material for absorbing fluid removed from the porous member such as by a wash fluid as it propagates through the porous member 32.

In this preferred embodiment the porous member 32 is a thin porous member possessing an intercommunicating network of openings throughout such that a fluid deposited on the member will propagate throughout the member because the capillary action. The thin porous member 32 may be any suitable element such as a porous membrane, a fibrous mesh pad or the like and may be of any suitable material such as glass, polymeric materials, paper, etc. In a particularly preferred embodiment porous member 32 comprises a nonwoven glass fiber mesh having very thin fibers such as on the order of about 1 micrometer.

The porous member 32 is mounted within a guide (not shown) formed within the housing 22 and having top and bottom surfaces which are spaced apart a distance sufficient to support the member 32. By way of example, the spacing between the top and bottom surfaces of the guide may be in the range of from about 0.30 mm to about 0.60 mm; the preferred spacing is about 0.40 mm.

The porous member 32 extends from the dispenser 30 to the chamber 34 which holds the absorbing material. The dispenser chamber 30 is configured as a well for holding a fluid, the dispenser 30 including a port at the bottom of the well and means for allowing communication of fluid from the bottom of the well into the porous member 32. Liquid absorbing material 36, which may be any suitable material, is located within chamber 34 and forms a part of the chamber 34 for taking up fluid expelled from the porous member 32 and the guide area, or reaction zone. Absorbing material 36 is located contiguous porous member 32 and in a preferred embodiment

(as illustrated) is formed conveniently as an extension of the porous material folded back and forth on itself.

The housing 22 also preferably includes a chamber 38 which is positioned immediately above the top horizontal surface of porous member 32 and has a port at the bottom periphery thereof to allow fluid to be delivered to the porus member 32. The housing 22 may include a transparent window area (now shown) positioned immediately below the bottom horizontal surface of porous member 32 to provide access for the illumination used to measure any detectable change effected in the porous member as a result of the assay method or preferably an opening in the housing to permit readout illumination to be directed onto the porous member without having to pass through the material of which the housing is comprised. A preferred assay module of the type illustrated in Fig. 2 is disclosed and claimed in copending, commonly assigned application serial no. 354,026, filed May 19, 1989.

For purposes of illustration the method will be described with respect to an assay for human chronic gonadotropin (HCG) . Initially, the control or calibrator composition is deposited onto the solid carrier 32 to which there are immobilized antibodies to HCG and the assay element is incubated. Next the enzyme conjugate solution comprising an antibody to HCG covalently bound to an enzyme such as alkaline phosphatase is aspirated by a pipette from reservoir 24 and deposited on the solid earner through chamber 38. The assay element is then allowed to incubate for a second time. Subsequently, a substrate solution for the particular enzyme label, for example, methyl imbelliferyl phosphate when the enzyme is alkaline

phosphatase, is aspirated by a pipette from reservoir 26 and deposited in dispenser 30 where it is allowed to enter one end of the porous member 32 and propagate through the member. The substrate solution functions as a wash solution to remove free labeled conjugate from the reaction zone and also to react with the enzyme label to provide a detectable species.

After a third period of incubation, the detectable species which is liberated by the reaction between the enzyme and the substrate material is read kinetically. It is preferred to obtain four optical readings on each assay element. In the illustrative embodiment wherein the analytical instrument includes three optical read stations, each assay element to which there is applied one of the calibrator compositions is preferably read a first time at the three optical read stations after residing in the temperature controlled chamber for 228, 240 and 252 seconds respectively. The second readings are obtained at 288, 300 and 312 seconds, respectively. The third readings are taken at 348, 360 and 372 seconds respectively and the fourth readings at 408, 420 and 432 seconds respectively. The slope of the curve for each calibrator composition at each optical read station is calculated and these values are used to form the calibration curve for each optical read station. In the illustrative assay for HCG it is preferred to utilize six calibrator compositions containing 0.00, 5.00, 15.00, 50.00, 1 50.00 and 500.00 mlU/mL HCG respectively.

It should be noted that although the rate assay has been illustrated with respect to the assay for HCG where it is preferred to

obtain four readings on each assay element at each optical read station, the kinetic reading of the liberated detectable species may require a different number for optical readings, for example, only two or as many as five or more. As noted previously, when a patient sample fluid is analyzed with the instrument the optical readings taken on the assay device can be obtained at any of the plurality of optical read stations. Of course, it will be understood that for each assay device bearing a patient sample fluid, whether an end point assay or a rate assay, the optical reading(s) must be taken at the same optical read station. Further, the readings must be taken at the times the calibrator compositions were read at the particular optical read station. For example, for the illustrative theophylline example described above, an assay device bearing a patient sample fluid would be read at any one of the three optical read stations at times of 350, 360 and 370 seconds respectively.

The invention will now be described further in detail with respect to specific preferred embodiments by way of examples, it being understood that these are intended to be illustrative only and the invention is not limited to the materials, procedures, etc. recited therein.

EXAMPLE I

Calibration curves for a theophylline assay for three optical read stations in an automated analytical instrument were obtained by the following procedure. Six calibrator compositions

having, respectively, 0.00, 2.50, 5.00, 10.00, 20.00 and 40.00 ug/mL of the theophylline were used.

An assay element of the type illustrated in Fig. 1 was inserted in a temperature controlled chamber and an optical reading obtained at each of the three optical read stations. Subsequently, a calibrator composition was dispensed to the assay element and the assay element was read at each of the three optical read stations after residing in the chamber for 350, 360 and 370 seconds respectively. The "wet" reading at each read station was then divided by the "dry" reading at that station to obtain a normalized optical signal.

The procedure was repeated for each calibrator composition and duplicates of each composition were run. The results are shown in Table I. Each signal is the average of the two signals obtained from the duplicates.

The calibration curves for the three optical read stations are shown in Fig. 3.

EXAMPLE II

Calibration curves for an HCG assay for three optical read stations in an automated analytical instrument were obtained according to the method of the invention. Six calibrator compositions having, respectively, 0.00, 5.00, 15.00, 50.00, 150.00 and 500.00 mlU/mL of HCG were used.

An assay element of the type illustrated in Fig. 2 was inserted in a temperature controlled chamber and the calibrator composition dispensed thereto. After a three minute incubation period the enzyme conjugate solution comprising alkaline phosphatase bound to an antibody to HCG was applied to the assay element followed by a nine minute incubation period. The substrate solution comprising methyl umbelliferyl phosphate was then applied and four optical readings were taken at each of the three optical read stations. The first optical readings were taken at the three optical read stations at 228, 240 and 252 seconds, respectively, after the substrate solution has been applied to the solid carrier. The second kinetic readings were obtained at 288, 300 and 312 seconds respectively. The third readings were obtained at 348, 360 and 372 seconds respectively and the fourth at 408, 420 and 432 seconds respectively.

The procedure was repeated for each calibrator composition and duplicates of each composition were run. The slope for each of the calibrator compositions at each of the optical read stations was calculated. The results are shown in Table II. Each

signal (slope) is the average of the two signals obtained from the duplicates.

The calibration curves for the three optical read stations are shown in Fig. 4.

Although the invention has been described with respect to specific preferred embodiments it is not intended to be limited thereto but rather those skilled in the art will recognize that variations and modification may be made therein which are within the spirit of the invention and the scope of the appended claims.