CA2149537A1 - Method for preparing calibration curves - Google Patents
Method for preparing calibration curvesInfo
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- CA2149537A1 CA2149537A1 CA 2149537 CA2149537A CA2149537A1 CA 2149537 A1 CA2149537 A1 CA 2149537A1 CA 2149537 CA2149537 CA 2149537 CA 2149537 A CA2149537 A CA 2149537A CA 2149537 A1 CA2149537 A1 CA 2149537A1
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- optical
- optical read
- calibrator
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/27—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration
- G01N21/274—Calibration, base line adjustment, drift correction
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/96—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood or serum control standard
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- Mathematical Physics (AREA)
- Theoretical Computer Science (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Investigating Or Analysing Materials By Optical Means (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Analysing Materials By The Use Of Radiation (AREA)
- Measurement Of Radiation (AREA)
Abstract
2149537 9428021 PCTABS00034 A purified antigen having a molecular weight of about 64 to 66 kDa as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Purified endometrial proteins, free of albumin and having molecular weights of about 64 kDa and isoelectric focusing points of 3.5, 4.0, 6.0, 6.5 and 8.0 are provided. A purified endometrial protein, free of albumin and having molecular weight of 94-97 kDa and iseolectric focusing point of 3.5 is also provided. The presence of antigen composition or endometriosis-associated endometrial protein is a marker for endometriosis in a subject. A purified monoclonal antibody specifically reactive with the antigen is also provided. A method of diagnosing endometriosis in a subject is also provided. The method comprises the steps of: (a) contacting an antibody-containing sample from the subject with the antigen composition or purified endometrial proteins of the invention; and (b) detecting the reaction of the antigen with an antibody from the sample, the reaction indicating endometriosis in the subject. Having provided an endometriosis associated antigen and monoclonal antibody, a method of diagnosing endometriosis in a subject, using the MAb is also provided.
Description
~` - 21~37 ,.
94/01758 Pcr/u~s3/o~8 METHOD FC)R PREPARING CALlBRATlON CUP~VES
BACKGROUND OF TWE 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 r~ad stations in an automated, analytical instrumen~.
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 iarge 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 ~he 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 Wo 94/0175~ 1 4 ~ S ~ 7 PCr/US93/06081 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.
tO In certain types of instrumen~s such as those which are designed to carry out assays based on immunornetric 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 + 0.5C. 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 -`` 214~537 ~
~094/01758 Pcr/uss3/o6o8 vari~us 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 5 instruments can provide a desirably higher throughput which allows a larger number of sampies 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 elernent at any of the optical read stations. Accordingly, those skilled in the art 10 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 15 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 învolves using a separate series of assay elements for each of the optical read stations. Since each calibration 20 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 25 advantageous calibration methods for use with instruments having a plurality of optical read stations.
214!~S37 wo 94~n1758 ji , Pcr/uss3/o6o8 SUMMARY OF TtlE 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 whersin 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 ~o provide such a method wherein the assay devices are read successively at each of the plurality of opticai 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 - ~ cornpositions used to create the calibration curve is read successively 20 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 ` 211~7 -~0 94/017~i8 P~US93/0608 a control or calibrator composition and any other required reagent(s~
to the desired temperature in the temperature-con$rolled chamber as Tor the first optical reading is taken at optical read station R1 at a time Tl which may be the same as To or a time close to To~ The second 5 optical reading is taken at optical read station R2 at time T2 which can be defined as T1 + ~tl and a third optical reading, if required, at - optical read station R3 at time T3 which can be defined as T2 + I~t2 (where At2 may be the same as ~tl or different).
The method of the invention can be practiced with an 10 analytical instrument which has two or more optical read stations.
Accordingiy, 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 15 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 assa~ device which is 20 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 25 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 214~37 WO g4/01758 ~ ! ~` PCI /US93/06081 -6- ~.
for such readings at the respective optical read stations by the calibration method of ~he 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 ~VO 94/01758 21~ 7 pcr/uss3/o6o8l t 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 Fiy. 2 DESCRIPTION OF THE PREFERRED EMBC)DIMENTS
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 us~ in immunornetric 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 detectabte, - 25 for example, a fluorophore, a chemiluminophor, a radioactive material or a light absorbing material, and those which are indirectiy detectable 2149S37 ,--WO 94/01758 PC~/US93~06081 1': ~' . ! ., " ~: .
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 5 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 signai 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 1~ which only the fluid calibrator or control composition is added and assay elements which may inGlude 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 20 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.
25 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.
.
~ , ," ~,, " ", ,~ " ,~,, , ", ~ , ", ~` !`
`; ~0 94/01758 2 1 4 9 ~ ~ 7 PCr/VSs3/06081 g The assay me~hod 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 ftuid and any other required reagent(s) are applied to the assay device and the signal applied to a 5 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 ar~ taken at specific times after applying ~he sampie fiuid and any other required reagent~s) to the assay device to ob~ain a linear reaction rate relationship. The slope of the linear rate 10 relationship is taken and applied to a calibration curve to determine the analyte concentration.
As noted pr~viously the calibration method of the ~ invention can be practiced generally with analytical instrumentsi~ having a plurality of optical read stations. The maximum number of 15 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 20 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 25 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, 21~!3537- ~
i WO 94/017~8 PCI`/US93/06081 `. '-10 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.
- 5 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 frorn 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 on!y the application of the control or calibrator ~omposition to the assay device and ~herefore only one incubation period is required. However, the method rnay 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 - ~ 20 controlling with respect to setting the times for the optical readings is that which occurs after t'ne last (which rnay be the only) fluid rea~ent 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 `,~0 94/01758 PCI'/US93/06081 -1 '1 -having a thickness of about 0.1 mm and cornprised of a transparent - support 12 which carries in succession a reagent layer 14, a light-blocking layer 1~ and an optical ~op coat laycr 18 which may serve as a reagent layer, a filter layer such as for proteins, an an~iabrasion 5 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~.
10 The binding partner, an antibody when the sample analyte is an antigen, is immobilized in the reagent layer 14 by being covaiently 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.
15 The matrix rnaterial 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 20 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 tnot shown) for distributing the sample fluid uniformly across 25 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 214~ 5 37 ~ `
WO 94/01758 PCI`/US93tO~081 ---12- `-transport systems which have been disclosed in the art as being suitable for this purpose. Many such fluid distribution systerns 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 5 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.
10One of the series of control or calibrator compositions is distributed across the surface of the assay element 1 () 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 15 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 20 controt, 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 25 of control analyte.
The assay element 10 is then allowed to remain in a temperature - controlled chamber for the appropriate incubation time 214~5~7 `- ~ ~o 94/01758 Pcr/US93/o6081 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 iayer 14 through support layer 12 with the appropriate electromagnetic 5 radiation. After a brief period of time a readout signal is obtained at the seoond 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 1 0 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 interes~. The number of15 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 20 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 25 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 214~53~
'.''' 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 5 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 10 the assay element. In a preferred embodiment the ratio of the thickness of reagent layer 14 to the combined thickness of the light-blocking iayer 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 15 optical signals obtained with assay elemen~ 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 firs~, or "dry", reading is carried out by irradiating the assay element in the same manner described above to obtain the 20 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 25 in layer thickness frorn 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, 214~S37 - ~0 94/01758 P~r/uss3/o6o8 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 oorrected for relative 5 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 10 utilizes an assay element as illustrated in Fig. 2 with which there is carried out a rate assay. Referrin~ 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 15 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 20 shown). An optional third chamber serves as a mixing bowl 28 for the mixing of reagents and a fourth charnber 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 25 material for absorbing fluid removed from the porous member such as by a wash fluid as it propagates through the porous member 32.
21~S~7 - i WO 94/01758 PCI`/US93/0~i081 ., .
In this preferred embodiment the porous member 32 is - a thin p,orous member possessing an intercommunicating ne~work of openings throughout such that a fluid deposited on the member will propa~ate 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 1 û 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 - 15 bottom surfaces of th~ guide may be in the range of from about 0.30 mm to about 0.60 m,m; the preferred spacing is about 0.40 mm.
The porous member 32 extends from the dispenser 30 to the Ghamber 34 which holds the absorbing material. The dispenser chamber 30 is configured as a well for hold,ing a fluid, the ;~ 20 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 materia!,, 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 ` ~o ~4/017~8 2 1 ~ ~ 5 3 7 P~r/USg3/0~081 (as illustrated) is formed c~nveniently 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 5 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) positloned immediately below the bottom horizontal surfar e of porous rnember 32 to provide access for the illumination used to measure any 10 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 15 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 20 solid carrier 32 to which there are immobilized antibodies to HCG and the assay element is incubated. Next the enzyme oonjugate 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 carrier through chamber 38. The assay 2~ 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 214~5~7 WO 94/~1758 ~ Pcr/uS93/o6081 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 enzyrne 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 elemen~ 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 charnber 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, 150.00 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 ~o 94/017~8 Pcr/u~93/06~81 obtain four readings on each assay element a~ each optical read station, the kinetic reading of the liberated detectable species may require a different number for optical readings, for examplet only two or as many as five or more.
As noted previously, when a patient sample fluid is analyzed with the instrurnent 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 1~ of the three optical read stations at times of 350, 36Q 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 WO 94/01758 2 1 4 ~ 5 3 7 .~ ~ PCI`/US~3/06~81 ~
-ZO-having, respectively, .l 2.50, 5.00, 1().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 charnber and an optical reading 5 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 10 divided by the "dry" reading at that station to obtain a normalized optical signal.
- The procedure was repeated for each calibrator composi~ion and duplicates o~ each composition were run. The results are shown in Table 1. Each signal is the average of the two 15 signals obtained from the duplicates.
~`
, _ _ _ TABLF I
. _ , CALIBRATOR OPTICAL READ OPTICAL FIEAD ¦ OPTICAL READ
ug/mL) STATION #1 STATION ~2 I STATION ~3 _ _ SIGNAL (NORMALIZED) O 00 ¦ 14.54 ¦ 14.79 114.72 I i 20 2.50 1 1 .36 1 1 .57 1 1 .50 l .1 5 00 10.02 10.21 10.09 I . i _ 10.00 8.09 8.20 8.10 ~
_ l _ 3 20.00 1 6.22 1 6.33 1 6.24 40 00 4.31 4.39 4.32 I . _ .
- 21~5~7 `
--- ,iO 94/01758 Pcr/US93/o6081 The calibration curves for the three optical read stations are shown in Fig. 3.
~1!
Calibration curves for an HCG assay for three optical 5 read stations in an automated analytical instrument were obtained according to the method of the invention. Six calibrator composil:ions having, respectively, 0.00, 5.00, 1 5.00, 50.00, 150.00 and 500.00 mlU/mL of HCG were used.
An assay etement of the ty,~e illustrated in Fig. 2 was 10 inserted in a temperature controlled charnber and the calibrator composition dispensed thereto. After a ~hree minute incubation period the enzyme conjugate solution comprising alkaline phosphatase bound to an antibody to HCG was applied to the assay element follovved by a nine minute incubation period. The substrate solution 15 comprising methyl um~elliferyl 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 secorld kinetic 20 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 25 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 ll. Each 214~37 ~
WO 94/017~;8 ;; - r ~ PCI /US93/06081 signal (slope) is the average of the two signals obtained from the duplicates.
_ , -- - _ _ -- ~
Calibrator OPTICAL READ OPTICAL READ OPTICAL FiEAD
(mlU/mL) STATION #1 STATION #2 STATION #3 SIGNAL (mV/sec) _ 0.00 9.70 10.32 9.6 I . . .
5.00 35.43 38.60 37.82 I . _ _ ..
15.00 86.00 94.31 94.23 I , _ 50.00 256.26 269.72 263.42 _ _ _ . .
150.00 _ 623 .00 643 .95 63 1.48 - 1 C 500.00 1262.92 1310.24 1297.37 ~,, 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 lirnited 15 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.
, j . .
94/01758 Pcr/u~s3/o~8 METHOD FC)R PREPARING CALlBRATlON CUP~VES
BACKGROUND OF TWE 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 r~ad stations in an automated, analytical instrumen~.
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 iarge 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 ~he 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 Wo 94/0175~ 1 4 ~ S ~ 7 PCr/US93/06081 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.
tO In certain types of instrumen~s such as those which are designed to carry out assays based on immunornetric 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 + 0.5C. 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 -`` 214~537 ~
~094/01758 Pcr/uss3/o6o8 vari~us 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 5 instruments can provide a desirably higher throughput which allows a larger number of sampies 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 elernent at any of the optical read stations. Accordingly, those skilled in the art 10 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 15 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 învolves using a separate series of assay elements for each of the optical read stations. Since each calibration 20 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 25 advantageous calibration methods for use with instruments having a plurality of optical read stations.
214!~S37 wo 94~n1758 ji , Pcr/uss3/o6o8 SUMMARY OF TtlE 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 whersin 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 ~o provide such a method wherein the assay devices are read successively at each of the plurality of opticai 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 - ~ cornpositions used to create the calibration curve is read successively 20 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 ` 211~7 -~0 94/017~i8 P~US93/0608 a control or calibrator composition and any other required reagent(s~
to the desired temperature in the temperature-con$rolled chamber as Tor the first optical reading is taken at optical read station R1 at a time Tl which may be the same as To or a time close to To~ The second 5 optical reading is taken at optical read station R2 at time T2 which can be defined as T1 + ~tl and a third optical reading, if required, at - optical read station R3 at time T3 which can be defined as T2 + I~t2 (where At2 may be the same as ~tl or different).
The method of the invention can be practiced with an 10 analytical instrument which has two or more optical read stations.
Accordingiy, 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 15 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 assa~ device which is 20 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 25 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 214~37 WO g4/01758 ~ ! ~` PCI /US93/06081 -6- ~.
for such readings at the respective optical read stations by the calibration method of ~he 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 ~VO 94/01758 21~ 7 pcr/uss3/o6o8l t 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 Fiy. 2 DESCRIPTION OF THE PREFERRED EMBC)DIMENTS
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 us~ in immunornetric 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 detectabte, - 25 for example, a fluorophore, a chemiluminophor, a radioactive material or a light absorbing material, and those which are indirectiy detectable 2149S37 ,--WO 94/01758 PC~/US93~06081 1': ~' . ! ., " ~: .
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 5 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 signai 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 1~ which only the fluid calibrator or control composition is added and assay elements which may inGlude 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 20 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.
25 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.
.
~ , ," ~,, " ", ,~ " ,~,, , ", ~ , ", ~` !`
`; ~0 94/01758 2 1 4 9 ~ ~ 7 PCr/VSs3/06081 g The assay me~hod 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 ftuid and any other required reagent(s) are applied to the assay device and the signal applied to a 5 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 ar~ taken at specific times after applying ~he sampie fiuid and any other required reagent~s) to the assay device to ob~ain a linear reaction rate relationship. The slope of the linear rate 10 relationship is taken and applied to a calibration curve to determine the analyte concentration.
As noted pr~viously the calibration method of the ~ invention can be practiced generally with analytical instrumentsi~ having a plurality of optical read stations. The maximum number of 15 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 20 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 25 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, 21~!3537- ~
i WO 94/017~8 PCI`/US93/06081 `. '-10 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.
- 5 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 frorn 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 on!y the application of the control or calibrator ~omposition to the assay device and ~herefore only one incubation period is required. However, the method rnay 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 - ~ 20 controlling with respect to setting the times for the optical readings is that which occurs after t'ne last (which rnay be the only) fluid rea~ent 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 `,~0 94/01758 PCI'/US93/06081 -1 '1 -having a thickness of about 0.1 mm and cornprised of a transparent - support 12 which carries in succession a reagent layer 14, a light-blocking layer 1~ and an optical ~op coat laycr 18 which may serve as a reagent layer, a filter layer such as for proteins, an an~iabrasion 5 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~.
10 The binding partner, an antibody when the sample analyte is an antigen, is immobilized in the reagent layer 14 by being covaiently 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.
15 The matrix rnaterial 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 20 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 tnot shown) for distributing the sample fluid uniformly across 25 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 214~ 5 37 ~ `
WO 94/01758 PCI`/US93tO~081 ---12- `-transport systems which have been disclosed in the art as being suitable for this purpose. Many such fluid distribution systerns 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 5 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.
10One of the series of control or calibrator compositions is distributed across the surface of the assay element 1 () 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 15 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 20 controt, 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 25 of control analyte.
The assay element 10 is then allowed to remain in a temperature - controlled chamber for the appropriate incubation time 214~5~7 `- ~ ~o 94/01758 Pcr/US93/o6081 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 iayer 14 through support layer 12 with the appropriate electromagnetic 5 radiation. After a brief period of time a readout signal is obtained at the seoond 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 1 0 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 interes~. The number of15 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 20 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 25 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 214~53~
'.''' 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 5 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 10 the assay element. In a preferred embodiment the ratio of the thickness of reagent layer 14 to the combined thickness of the light-blocking iayer 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 15 optical signals obtained with assay elemen~ 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 firs~, or "dry", reading is carried out by irradiating the assay element in the same manner described above to obtain the 20 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 25 in layer thickness frorn 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, 214~S37 - ~0 94/01758 P~r/uss3/o6o8 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 oorrected for relative 5 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 10 utilizes an assay element as illustrated in Fig. 2 with which there is carried out a rate assay. Referrin~ 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 15 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 20 shown). An optional third chamber serves as a mixing bowl 28 for the mixing of reagents and a fourth charnber 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 25 material for absorbing fluid removed from the porous member such as by a wash fluid as it propagates through the porous member 32.
21~S~7 - i WO 94/01758 PCI`/US93/0~i081 ., .
In this preferred embodiment the porous member 32 is - a thin p,orous member possessing an intercommunicating ne~work of openings throughout such that a fluid deposited on the member will propa~ate 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 1 û 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 - 15 bottom surfaces of th~ guide may be in the range of from about 0.30 mm to about 0.60 m,m; the preferred spacing is about 0.40 mm.
The porous member 32 extends from the dispenser 30 to the Ghamber 34 which holds the absorbing material. The dispenser chamber 30 is configured as a well for hold,ing a fluid, the ;~ 20 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 materia!,, 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 ` ~o ~4/017~8 2 1 ~ ~ 5 3 7 P~r/USg3/0~081 (as illustrated) is formed c~nveniently 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 5 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) positloned immediately below the bottom horizontal surfar e of porous rnember 32 to provide access for the illumination used to measure any 10 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 15 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 20 solid carrier 32 to which there are immobilized antibodies to HCG and the assay element is incubated. Next the enzyme oonjugate 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 carrier through chamber 38. The assay 2~ 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 214~5~7 WO 94/~1758 ~ Pcr/uS93/o6081 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 enzyrne 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 elemen~ 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 charnber 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, 150.00 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 ~o 94/017~8 Pcr/u~93/06~81 obtain four readings on each assay element a~ each optical read station, the kinetic reading of the liberated detectable species may require a different number for optical readings, for examplet only two or as many as five or more.
As noted previously, when a patient sample fluid is analyzed with the instrurnent 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 1~ of the three optical read stations at times of 350, 36Q 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 WO 94/01758 2 1 4 ~ 5 3 7 .~ ~ PCI`/US~3/06~81 ~
-ZO-having, respectively, .l 2.50, 5.00, 1().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 charnber and an optical reading 5 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 10 divided by the "dry" reading at that station to obtain a normalized optical signal.
- The procedure was repeated for each calibrator composi~ion and duplicates o~ each composition were run. The results are shown in Table 1. Each signal is the average of the two 15 signals obtained from the duplicates.
~`
, _ _ _ TABLF I
. _ , CALIBRATOR OPTICAL READ OPTICAL FIEAD ¦ OPTICAL READ
ug/mL) STATION #1 STATION ~2 I STATION ~3 _ _ SIGNAL (NORMALIZED) O 00 ¦ 14.54 ¦ 14.79 114.72 I i 20 2.50 1 1 .36 1 1 .57 1 1 .50 l .1 5 00 10.02 10.21 10.09 I . i _ 10.00 8.09 8.20 8.10 ~
_ l _ 3 20.00 1 6.22 1 6.33 1 6.24 40 00 4.31 4.39 4.32 I . _ .
- 21~5~7 `
--- ,iO 94/01758 Pcr/US93/o6081 The calibration curves for the three optical read stations are shown in Fig. 3.
~1!
Calibration curves for an HCG assay for three optical 5 read stations in an automated analytical instrument were obtained according to the method of the invention. Six calibrator composil:ions having, respectively, 0.00, 5.00, 1 5.00, 50.00, 150.00 and 500.00 mlU/mL of HCG were used.
An assay etement of the ty,~e illustrated in Fig. 2 was 10 inserted in a temperature controlled charnber and the calibrator composition dispensed thereto. After a ~hree minute incubation period the enzyme conjugate solution comprising alkaline phosphatase bound to an antibody to HCG was applied to the assay element follovved by a nine minute incubation period. The substrate solution 15 comprising methyl um~elliferyl 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 secorld kinetic 20 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 25 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 ll. Each 214~37 ~
WO 94/017~;8 ;; - r ~ PCI /US93/06081 signal (slope) is the average of the two signals obtained from the duplicates.
_ , -- - _ _ -- ~
Calibrator OPTICAL READ OPTICAL READ OPTICAL FiEAD
(mlU/mL) STATION #1 STATION #2 STATION #3 SIGNAL (mV/sec) _ 0.00 9.70 10.32 9.6 I . . .
5.00 35.43 38.60 37.82 I . _ _ ..
15.00 86.00 94.31 94.23 I , _ 50.00 256.26 269.72 263.42 _ _ _ . .
150.00 _ 623 .00 643 .95 63 1.48 - 1 C 500.00 1262.92 1310.24 1297.37 ~,, 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 lirnited 15 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.
, j . .
Claims (13)
1. A method for creating a calibration curve for a component of interest in a fluid for each of a plurality of optical read stations included in an analytical instrument, the method comprising:
(a) applying a calibrator or control composition for a component of interest to an assay device which is adapted to provide, in the presence of at least a fluid containing a component of interest, an optical signal which is a function of the concentration of the component of interest in said fluid;
(b) incubating said assay device;
(c) obtaining successively an optical signal with said assay device at each of a plurality of optical read stations, each optical signal at each optical read station being obtained at a specifically defined time for each optical read station;
(d) repeating steps (a), (b) and (c) for the remainder of the plurality of control or calibrator compositions utilized to prepare the calibration curve, wherein for each said calibrator or control composition the optical signal at each optical read station is obtained at the same specifically defined times as those obtained in step (c);
and (e) preparing a calibration curve for said component of interest for each optical read station from the plurality of optical signals obtained from said plurality of calibrator or control compositions at each optical read station.
(a) applying a calibrator or control composition for a component of interest to an assay device which is adapted to provide, in the presence of at least a fluid containing a component of interest, an optical signal which is a function of the concentration of the component of interest in said fluid;
(b) incubating said assay device;
(c) obtaining successively an optical signal with said assay device at each of a plurality of optical read stations, each optical signal at each optical read station being obtained at a specifically defined time for each optical read station;
(d) repeating steps (a), (b) and (c) for the remainder of the plurality of control or calibrator compositions utilized to prepare the calibration curve, wherein for each said calibrator or control composition the optical signal at each optical read station is obtained at the same specifically defined times as those obtained in step (c);
and (e) preparing a calibration curve for said component of interest for each optical read station from the plurality of optical signals obtained from said plurality of calibrator or control compositions at each optical read station.
2. The method as defined in claim 1 wherein a calibration curve is prepared for said component of interest at each of from two to six optical read stations.
3. The method as defined in claim 1 wherein the first optical signal at the first optical read station is obtained at a time which is within about one percent of the incubation time in step (b).
4. The method as defined in claim 1 wherein the optical signals at the plurality of optical read stations are obtained within a time period which is within about ten percent of the incubation time in step (b).
5. A method for creating a calibration curve for a component of interest in a fluid for each of a plurality of optical read stations included in an analytical instrument, the method comprising:
(a) distributing a calibrator or control composition across the surface of a multilayer assay element which comprises:
i. a light-blocking layer which is permeable to said composition; and ii. a reagent layer comprising a signal generating species.
(b) incubating said assay device;
(c) obtaining successively an optical signal with said assay element at each of a plurality of optical read stations, each optical signal at each optical read station being obtained at a specifically defined time for each optical read station;
(d) repeating steps (a), (b) and (c) for the remainder of the plurality of control or calibrator compositions utilized to prepare the calibration curve, wherein for each said calibrator or control composition the optical signal at each optical read station is obtained at the same specifically defined times as those obtained in step (c);
and (e) preparing a calibration curve for said component of interest for each optical read station from the plurality of optical signals obtained from said plurality of calibrator or control compositions at each optical read station.
(a) distributing a calibrator or control composition across the surface of a multilayer assay element which comprises:
i. a light-blocking layer which is permeable to said composition; and ii. a reagent layer comprising a signal generating species.
(b) incubating said assay device;
(c) obtaining successively an optical signal with said assay element at each of a plurality of optical read stations, each optical signal at each optical read station being obtained at a specifically defined time for each optical read station;
(d) repeating steps (a), (b) and (c) for the remainder of the plurality of control or calibrator compositions utilized to prepare the calibration curve, wherein for each said calibrator or control composition the optical signal at each optical read station is obtained at the same specifically defined times as those obtained in step (c);
and (e) preparing a calibration curve for said component of interest for each optical read station from the plurality of optical signals obtained from said plurality of calibrator or control compositions at each optical read station.
6. The method as defined in claim 5 wherein a calibration curve is prepared for said component of interest at each of from two to six optical read stations.
7. The method as defined in claim 5 wherein the first optical signal at the first optical read station is obtained at a time which is within about one percent of the incubation time in step (b).
8. The method as defined in claim 5 wherein the optical signals at the plurality of optical read stations are obtained within a time period which is within about ten percent of the incubation time in step (b).
9. A method for creating a calibration curve for a component of interest in a fluid for each of a plurality of optical read stations included in an analytical instrument, the method comprising:
(a) applying a calibrator or control composition for a component of interest to an assay device which includes a solid carrier material;
(b) incubating said assay device;
(c) obtaining successively an optical signal with said assay device at each of a plurality of optical read stations, each optical signal at each optical read station being obtained at a specifically defined time for each optical read station;
(d) repeating steps (a), (b) and (c) for the remainder of the plurality of control or calibrator compositions utilized to prepare the calibration curve, wherein for each said calibrator or control composition the optical signal at each optical read station is obtained at the same specifically defined times as those obtained in step (c);
and (e) preparing a calibration curve for said component of interest for each optical read station from the plurality of optical signals obtained from said plurality of calibrator or control compositions at each optical read station.
(a) applying a calibrator or control composition for a component of interest to an assay device which includes a solid carrier material;
(b) incubating said assay device;
(c) obtaining successively an optical signal with said assay device at each of a plurality of optical read stations, each optical signal at each optical read station being obtained at a specifically defined time for each optical read station;
(d) repeating steps (a), (b) and (c) for the remainder of the plurality of control or calibrator compositions utilized to prepare the calibration curve, wherein for each said calibrator or control composition the optical signal at each optical read station is obtained at the same specifically defined times as those obtained in step (c);
and (e) preparing a calibration curve for said component of interest for each optical read station from the plurality of optical signals obtained from said plurality of calibrator or control compositions at each optical read station.
10. The method as defined in claim 9 wherein a calibration curve is prepared for said component of interest at each of from two to six optical read stations.
11. The method as defined in claim 9 wherein the first optical signal is obtained at a time which is with about one percent of the incubation time in step (b).
12. The method as defined in claim 9 wherein the optical signals at the plurality of optical read stations are obtained within a time period which is with about ten percent of the incubation time in step (b).
13. A method for determining the concentration of a component of interest in a sample fluid in an analytical instrument containing a plurality of optical read stations; the method comprising:
(a) preparing a calibration curve for the component of interest at each of said optical read stations in accordance with the method defined in claim 1;
(b) applying a sample fluid to an assay device;
(c) incubating said assay device;
(d) obtaining an optical signal with said assay device at one of said plurality of optical read stations, said optical signal being obtained at said optical read station at the same specifically defined time as said optical signals for said assay devices bearing said calibrator or control compositions was obtained at said optical read station; and (e) applying said signal obtained in step (d) to said calibration curve for said optical read station utilized in step (d) to determine the concentration of the component of interest in said sample fluid.
(a) preparing a calibration curve for the component of interest at each of said optical read stations in accordance with the method defined in claim 1;
(b) applying a sample fluid to an assay device;
(c) incubating said assay device;
(d) obtaining an optical signal with said assay device at one of said plurality of optical read stations, said optical signal being obtained at said optical read station at the same specifically defined time as said optical signals for said assay devices bearing said calibrator or control compositions was obtained at said optical read station; and (e) applying said signal obtained in step (d) to said calibration curve for said optical read station utilized in step (d) to determine the concentration of the component of interest in said sample fluid.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US90782092A | 1992-07-02 | 1992-07-02 | |
| US07/907,820 | 1992-07-02 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2149537A1 true CA2149537A1 (en) | 1994-01-20 |
Family
ID=25424694
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2149537 Abandoned CA2149537A1 (en) | 1992-07-02 | 1993-06-28 | Method for preparing calibration curves |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0648326A1 (en) |
| AU (1) | AU683218B2 (en) |
| CA (1) | CA2149537A1 (en) |
| WO (1) | WO1994001758A1 (en) |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS55136958A (en) * | 1979-04-14 | 1980-10-25 | Olympus Optical Co Ltd | Automatic analyzer |
| JPS59108942A (en) * | 1982-12-14 | 1984-06-23 | Fuji Photo Film Co Ltd | Determining method using sheet analysis element |
-
1993
- 1993-06-28 EP EP93916769A patent/EP0648326A1/en not_active Ceased
- 1993-06-28 CA CA 2149537 patent/CA2149537A1/en not_active Abandoned
- 1993-06-28 AU AU46512/93A patent/AU683218B2/en not_active Ceased
- 1993-06-28 WO PCT/US1993/006081 patent/WO1994001758A1/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| EP0648326A1 (en) | 1995-04-19 |
| WO1994001758A1 (en) | 1994-01-20 |
| AU683218B2 (en) | 1997-11-06 |
| AU4651293A (en) | 1994-01-31 |
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