US20040096983A1

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Info

Publication number: US20040096983A1
Application number: US10/294,876
Authority: US
Inventors: Merrit Jacobs; Ann Skrobach; Andrew Foote; Anthony Machulskis
Original assignee: Individual
Current assignee: Ortho Clinical Diagnostics Inc
Priority date: 2002-11-14
Filing date: 2002-11-14
Publication date: 2004-05-20
Also published as: ATE401563T1; US8329475B2; DE60322165D1; US20050074897A1; JP2010210643A; JP5285026B2

Abstract

A method for removing an undesired component from a bound desired component in an analysis that includes the steps of: (a) providing a container having a desired component bound thereto and an undesired component; (b) dispensing a wash fluid in the container at a first level sufficient to contact at least a portion of the bound desired component; (c) oscillating the level of the wash fluid in the container; and (d) removing at least a portion of the wash fluid from the container. Another aspect of the invention provides a method for washing an analyte bound to the walls of a surface coated container of an analyzer that includes: (a) providing a surface coated container having an analyte bound thereto; (b) dispensing a wash fluid in the container at a first level sufficient to contact at least a portion of the analyte bound thereto; (c) oscillating the level of the wash fluid in the container; and (d) removing the wash fluid from the surface coated container. Also described is a method for removing an undesired component from a bound desired component in an analysis that includes the steps of: (a) providing a container having a desired bound component bound thereto and an undesired component; (b) dispensing a wash fluid in the container at a first level sufficient to wash at least a portion of the bound desired component; (c) removing the wash fluid from the container; (d) subsequently dispensing a wash fluid in the container at a subsequent level that is lower than the first level and is sufficient to wash at least a portion of the bound desired substrate; and (e) removing the wash fluid from the container. In a preferred embodiment, the analyte being measured is Troponin I.

Description

BACKGROUND OF THE INVENTION

1. Field of the Invention[0001]

The present invention relates to a process for removing an undesired component from a bound desired component, in particular for improving the bound-free separation efficiency. In particular, the present invention relates to a process for separating or washing a bound analyte being analyzed in an automated clinical analyzer from unbound label, particularly without decreasing processing efficiency or speed.[0002]

2. Description of the Related Art[0003]

Methods and systems for washing containers that hold samples being analyzed, such as analyzers for conducting clinical assays are known, e.g., wash stations in clinical analyzer immunochemical assay systems. For example, U.S. Pat. No. 6,096,561 and U.S. application Ser. No. 09/482,599 filed Jan. 13, 2000 entitled “Failure Detection in Automatic Clinical Analyzers” describe immunoassay analyzers that include container wash stations for washing containers containing one or more analytes bound to coated sample containers that are measured, for example, by chemiluminescence. Such systems typically contain a sample wash station that may include a wash fluid dispense nozzle and an aspirating nozzle. The sample containing analyte and reagent, e.g., label, is aspirated out of the container after it has been incubated. Wash fluid is then dispensed into and aspirated out of the container one or more times to remove any excess analyte and reagent not bound to the coating, such as streptavidin, at the side of the container. Some known surface coated containers have features, such as pockets or ledges, near the top of the container that can trap unbound material, such as unbound label, analyte, etc. These features may be the result of the process used to mold the containers and/or to keep the containers separated in a stack. See, e.g., U.S. Pat. No. 5,441,895, which describes stackable containers. The incubation, reagent metering and mixing processes involved in immunochemical assay analysis move the sample in the container in a manner that leaves a film of sample containing unbound label and/or analyte on the pockets and ledges that are in the upper regions of the container.[0004]

Typically in immunochemical assay systems, an important aspect that affects the performance is bound-free separation. The bound-free separation is controlled by two primary factors:[0005]

(1) the component or material, such as the bound label, that is intended to produce signal remains behind and intact; and[0006]

(2) the unbound (i.e., free) component or material is removed as completely as possible.[0007]

In particular, there are several assays that have clinically significant performance very close to the background of the assay. This means that small amounts of unbound material present during the measurement portion of the process, particularly signal producing material, can produce a substantial adverse impact on performance.[0008]

To remove unbound material as completely as possible, a container wash process typically includes multiple wash cycles, such as filling a surface coated container to a first height on the container with a wash fluid and aspirating the wash fluid after a predetermined amount of time. For example, in one known process, a dispensing nozzle fills the container, which has a 300 μl capacity up to a height of 270 μl and then sets the soak height to 230 μl so the well can be transported during the wash incubation step (≅37-40 seconds). These steps, including the incubation step, are repeated multiple times (e.g., four times) using the same fluid heights. Unbound material that can be present in the upper regions of the container is only removed by inadvertent exposure to the wash fluid. Considerable erroneous signal can be generated when the wash fluid makes contact with unbound material in the upper regions of the container where the material is re-hydrated but not removed. The unbound material can then drop into the signal reagent during the last processing step that is intended to detect the amount of label bound to the container surface.[0009]

Another problem with the known art that removes fluid by aspiration is that the outer surface of an aspirating nozzle can become contaminated with the wash fluid containing unbound material. This can lead to contamination of the container with unbound material in subsequent wash cycles (in that particular test or across subsequent tests). All of the problems described above lead to the unbound material remaining in the test container and possibly interfering with the subsequent analysis of the analyte, leading to tests that need to be repeated at considerable inconvenience and expense due to inaccurate results.[0010]

It is known in the art that the unbound material in the upper regions of the container can be removed more completely if the fluid were to be filled higher in the container and remain there for the soak cycle (i.e., container wash incubation). This is not practical for a random access analyzer system since the test element needs to be transported during the soak cycle so other tests elements can be processed. Batch analyzers leave the test element static during this process step, which allows the fluid level moved to the very top of the container (positive meniscus). Even analyzers that fill the test element to the very top of the container may still have issues with not completely removing unbound label at the very top, if there are features in this region that can trap or retain unbound material. This is especially true when each wash processing cycle raises the fluid to the same height. In this process, the last processing step can cause any residual unbound material to flow into a region of the test element where it can interact with the signal generating reagents, thus producing erroneous results.[0011]

SUMMARY OF THE INVENTION

One object of the invention is to overcome the disadvantages of the known art described above. Another object of the invention is to provide an improved wash process for washing a container holding a sample being analyzed; in particular, a wash process that allows separation of undesired unbound material from desired bound material. Still another object of the invention is to provide an improved wash process for an immunochemical assay system that removes unbound material without substantially reducing the amount of signal from the bound fraction. Yet another object of the invention is to provide an improved process for analyzing an analyte having a signal strength close to the background noise. Another object of the invention is to provide for improved removal of unbound material in a time frame of ˜2.5 seconds (excluding wash incubation time), which enables the test elements to be processed without any significant and preferably no degradation in the system throughput (efficiency or number or tests per hour).[0012]

The foregoing and further objects of the invention are accomplished according to one aspect of the invention that provides a method for removing an undesired component from a bound desired component in an analysis that includes the steps of: (a) providing a container having a desired component bound thereto and an undesired component; (b) dispensing a wash fluid in the container at a first level sufficient to contact at least a portion of the bound desired component; (c) oscillating the level of the wash fluid in the container; and (d) removing at least a portion of the wash fluid from the container. Another aspect of the invention provides a method for washing an analyte bound to the walls of a surface coated container of an analyzer that includes: (a) providing a surface coated container having an analyte bound thereto; (b) dispensing a wash fluid in the container at a first level sufficient to contact at least a portion of the analyte bound thereto; (c) oscillating the level of the wash fluid in the container; and (d) removing the wash fluid from the surface coated container.[0013]

Still another aspect of the invention provides a method for removing an undesired component from a bound desired component in an analysis that includes the steps of: (a) providing a container having a desired bound component bound thereto and an undesired component; (b) dispensing a wash fluid in the container at a first level sufficient to wash at least a portion of the bound desired component; (c) removing the wash fluid from the container; (d) subsequently dispensing a wash fluid in the container at a subsequent level that is lower than the first level and is sufficient to wash at least a portion of the bound desired substrate; and (e) removing the wash fluid from the container.[0014]

Yet another aspect of the invention provides a method for washing an analyte bound to the walls of a surface coated container of an analyzer that includes the steps of: (a) providing a surface coated container having an analyte bound thereto; (b) dispensing a wash fluid in the container at a first level sufficient to wash at least a portion of the analyte bound thereto; (c) removing the wash fluid from the surface coated container; (d) subsequently dispensing a wash fluid in the sample container at a second level that is lower than the first level and sufficient to wash at least a portion of the analyte bound thereto; and (e) removing the wash fluid from the sample container.[0015]

Still another aspect of the invention provides a method of determining the amount of an analyte in a sample, that includes the steps of: (a) providing a sample containing an analyte in a coated container; (b) providing a reagent in the container; (c) optionally incubating the combined sample and reagent; (d) performing a wash as described above; (e) optionally adding a signal reagent; and (f) analyzing the sample for an analyte. Preferably, the analyte being measured is Troponin I.[0016]

Another aspect of the invention provides the methods described above implemented by a computer program interfacing with a computer, and an article of manufacture that includes a computer usable medium having computer readable program code configured to conduct the methods described above.[0017]

Another aspect of the invention provides for improved removal of unbound material in a time frame of ˜2.5 seconds (excluding wash incubation time), which enables the test elements to be processed without any significant and preferably no degradation in the system throughput (efficiency or number or tests per hour).[0018]

Further objects, features and advantages of the present invention will be apparent to those skilled in the art from detailed consideration of the preferred embodiments that follow.[0019]

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a cup-shaped container according to one embodiment of the present invention.[0020]

FIG. 2 shows a container wash dispenser according to one embodiment of the present invention.[0021]

FIG. 3 shows a graph comparing assay performance using a wash process according to a preferred embodiment of the present invention and a conventional wash process.[0022]

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention provides a method for washing a container containing a sample being analyzed, in order to assure that components (such as analyte and bound label) in the sample bound to the substrate remain, while unbound components, such as unbound label, are removed from the container. While much of the foregoing and following description is related to automated immunochemical assay analyzers, the present invention is not so limited. In particular, the present process can be applied to any analysis or separation, chemical, immunological or otherwise. The coated container being washed includes those components (single or multiple) of the sample that are bound and those components (single or multiple) of the sample that need to be removed, such as excess analyte (e.g., Troponin I), excess reagents, such as biotin or unbound label (e.g., HRP), the liquid phase, undesired materials/interferents (e.g., hemoglobin), etc. The coated container can also include streptavidin coated containers that have not been further modified or treated, such as biotinylation. In this instance, the bound material is the streptavidin coating and the unbound material is any impurity to be removed before further treatment in the process.[0023]

According to a first preferred embodiment, a container suitable for containing a wash fluid and sample is provided. The container can include materials such as plastic, glass, metal, etc. and can be configured as a cup, well, cuvette, test tube, etc. As noted above, the container can include features from an injection molding process and/or features from stacking the containers. Prior to the wash process, the container and the sample may have been through previous processes, such as reagent addition or incubation as will be described more fully below. As used herein, a “wash cycle” is the dispensing and aspirating of wash fluid into and out of the container and does not include the incubation time, which is generally on the order of ≅37.5 seconds.[0024]

Prior to the first dispense of wash fluid, the container may have any liquid phase and/or solids not containing the portion of the sample, e.g., the analyte, not being washed, first removed, such as by aspiration. The wash fluid is then dispensed into the container. It should be understood that in some cases, it is possible for the wash fluid to be first dispensed before the sample is present in the container. However, in a preferred aspect of the invention, the sample, including the analyte and label, are present in the container prior to the first dispense. Upon dispensing, the wash fluid contacts at least a portion of the container containing the bound desired material or component to ensure removal of the undesired and unbound material or component into the wash fluid. The dispense can be accomplished with a dispensing nozzle, or any other satisfactory fluid dispensing apparatus. If a dispense nozzle is used, it can be the same or different than the aspirate nozzle, described below. In those embodiments where a pre-wash is performed before a sample is added to the container, the wash fluid may be of a different composition than the wash fluid typically used. For example, the wash fluid for a pre-wash cycle may include a protein, such as bovine serum albumin (BSA) to act as a blocking agent.[0025]

After a selected time of contact, the wash fluid is removed from the container, such as by aspirating with a nozzle, resulting in the completion of a wash cycle. Alternatively, the fluid removal can be accomplished with any other known fluid removal devices, such as inversion of the container, etc.[0026]

According to a particularly preferred embodiment, the wash cycle can include an oscillating process. Upon dispensing the wash fluid into the container, small amounts (i.e., a less than complete emptying of the container, for example,) of wash fluid are dispensed and removed from the container. The oscillating action of the wash fluid creates a moving meniscus. The moving meniscus reduces the concentration gradient at the boundary layer of the container wall by constantly refreshing the wash fluid at the surface on the container wall in contact with the moving meniscus. The meniscus moving along the surface of the container forces the wash fluid to drain from the container surface providing convective transport along the surface. That is, the moving meniscus made possible by the oscillation, enhances the fluid velocity as close as possible to the boundary layer to maximize the concentration gradient at the boundary. While moving meniscus are known to enhance diffusion at boundary layers, the present inventors believe that this is the first time such a concept has been applied to the present invention. In a preferred embodiment, two complete oscillations may be provided, one oscillation being up and down nozzle travel, or vice versa. Applicants have found that, even with the oscillation, it is possible to provide a wash cycle that takes approximately the same time as known wash processes, even though there are several additional steps that are required by the oscillation. Preferably, the wash cycle takes approximately 3 seconds, more preferably approximately 2.5 seconds.[0027]

According to another preferred aspect, after removal in the first wash cycle, a wash incubation step of approximately 37.5 seconds follows. After the wash incubation step, a further dispense and removal wash cycle may be provided. The dispense step in the further wash cycle or cycles dispenses fluid into the container at a level which is lower than a previous wash cycle. This ensures that once the upper portion of the container has been cleaned and removed of sufficient undesired material, it will no longer contact subsequent washes and not allow for the possibility of recontamination of the upper reaches of the container in subsequent wash cycles.[0028]

As many wash cycles as required can be used according to the present invention. A preferred number of wash cycles is four. Also, other steps during the wash cycle can also be performed, if desired. Moreover, the oscillating process embodiment described above can be carried out during one or all of the wash cycles.[0029]

In systems where an aspirating nozzle is used to remove the wash fluid, the rate of the nozzle descent is generally balanced with the amount of wash liquid being removed from the container. That is, the aspirating nozzle relative to the surface of the fluid will remain substantially constant. In the present invention, the inventors have found that reducing the rate of the aspirate nozzle descent and elimination of any fluid dispense during the aspirate nozzle descent, particularly in the final wash, preferably the fourth wash, reduces the likelihood that the aspirate nozzle will be submerged in the wash fluid. Submerging the nozzle in the wash fluid on the final wash cycle makes it likely that any unbound material on the outside of the nozzle will be washed off the nozzle and leave residual undesired or unbound material in the container that can give an incorrect result, such as an elevated signal. In a preferred embodiment, the rate of descent is ⅓ slower relative to the rate of descent in previous wash cycles (where the previous wash cycle rate of descent is where the fluid aspirate rate and rate of descent are balanced as described above).[0030]

In some embodiments, particularly in systems where the wash fluid is temperature controlled, the wash fluid dispense may be on during the aspirate nozzle descent. This is done to further improve the control of the wash fluid temperature during the soak cycle where the temperature control device is in the wash nozzle head[0031]50 (FIG. 2). The nozzle contains fluid (˜80 μl) that is retained by the nozzle after the temperature control element in the wash head so this amount of fluid quickly reaches room temperature. By dispensing and quickly aspirating during nozzle descent, the colder fluid in the nozzle not at wash temperature is quickly removed. In this embodiment, the present inventors have found it particularly advantageous to turn off the wash fluid dispense during the final wash aspirate nozzle descent in order to reduce the curvature of the fluid meniscus in the container. This further reduces the likelihood that the aspirate nozzle will be submerged and become contaminated.

In a particularly preferred embodiment, the wash process is employed in an immunodiagnostic assay analyzer, such as those described in U.S. Pat. No. 6,069,561 and copending U.S. application Ser. No. 09/482,599 filed Jan. 13, 2000 entitled “Failure Detection in Automated Clinical Analyzers,” both of which are incorporated by reference in their entireties. In preferred immunodiagnostic analyzers, the container is cup-shaped. Preferred containers are 0.35 ml, conical containers coated with a material complementary to the reagents. Container coatings can comprise materials such as streptavidin and/or other materials useful for immunochemical analysis as is well known in the art to facilitate binding by a biotinylated antigen or antibody to which an analyte binds as part of the assay chemistry. An[0032]

exemplary container

10 is shown in FIG. 1. Also preferred are separate wash dispense and aspirating probes, such as the wash dispense20 andaspiration nozzles30 inwash unit40 shown in FIG. 2.

In a typical immunodiagnostic analyzer, the analyzer is categorized into systems and subsystems of components that perform different processes in the sequence of measuring a sample for an analyte, such as those described in the '599 application. A typical process involves a sample being dispensed into a container that may or may not already have a reagent present in the container that is dispensed by a reagent metering system. After the reagent is added, the sample is diluted, if necessary, and then incubated. After incubation, the container is washed, in this instance according to the inventive wash. After washing, a signal reagent is added, followed by further incubation, if necessary. The signal produced by the combination bound analyte/signal reagent is read by the appropriate detector, e.g., a luminometer.[0033]

The wash process according to the present invention can be implemented by a computer program, having computer readable program code, interfacing with the computer controller of the analyzer as is known in the art.[0034]

A particularly preferred wash sequence (not including the wash incubation) is as follows (with a typical wash sequence shown for comparison).[0035]



			Process
			step
	Pro-		(preferred
	cess		embodi-
	step		ment) of	Present invention
Wash	(kno-	Known wash	present	preferred wash
#	wn)	process	invention	process

1	1	Turn on vacuum	1	Starts downward
		before starting		travel with vacuum
		nozzle downward		off
		travel
1	2	Starts to dispense	2	Finds sample + reagent
		fluid when aspirate		fluid
		nozzle is at the top		height
		of the incubator
		ring. Start of 80 μl
		predispense
1	3	Travels to bottom	3	Turns on vacuum
		of container and		and turns on start
		waits until for 80 μl		of 80 μl predispense
		dispense to be
		complete
1	4	Reverses direction	4	Travels to bottom
		with vacuum on (no		of container and
		delay) and starts to		waits until for 80 μl
		dispense 270 μl		dispense to be
		volume		complete
1	5	Waits for 270 μl of	5	Waits 30 ms at
		fluid to be dispense		bottom of container
				with vacuum on
				and dispense off
1	6	Lowers nozzle to	6	Reverses direction
		the 230 μl position		with vacuum on
		with the vacuum on		and starts to
		and then		dispense 270 μl
		immediately		volume waiting at
		reverses direction		the 270 μl position
		lifting nozzle to
		home with vacuum
		on for 800 ms to
		clear fluid from
		nozzle and line
1	7		7	With dispense on
				and vacuum on
				nozzle raises to 320 μl
				position and waits
				60 ms (sufficient time
				dispense of >60 μl)
1	8		8	With dispense and
				vacuum on nozzle
				lowers to 250 μl
				position
1	9		9	With dispense on
				and vacuum on
				nozzle raises to 320 μl
				position and waits
				60 ms (sufficient time
				dispense of >60 μl)
1	10		10	Nozzle moves down
				to 270 μl position
				remains steady for
				50 ms and then
				dispense is turned off
				and waits an other 30 ms
1			11	Nozzle lowers to
				230 μl height with
				vacuum on and stays
				there for 30 ms before
				lifting
1			12	Nozzle lifts to home
				position and
				incubator starts to
				turn with nozzle
				vacuum still on for
				500 ms to evaluate
				nozzle and line of
				fluid
2	1	Turn on vacuum	1	Starts downward
		before starting		travel with vacuum
		nozzle downward		off
		travel
2	2	Starts to dispense	2	Finds soak volume
		fluid when aspirate		from wash #1 (should
		nozzle is at the top		be at 230 μl)
		of the incubator
		ring. Start of 80 μl
		predispense
2	3	Travels to bottom	3	Turns on vacuum
		of container and		and turns on start of
		waits until for 80 μl		80 μl predispense
		dispense to be
		complete
2	4	Reverses direction	4	Travels to bottom of
		with vacuum on (no		container and waits
		delay) and starts to		until for 80 μl
		dispense 270 μl		dispense to be
		volume		complete
2	5	Waits for 270 μl of	5	Waits 30 ms at
		fluid to be dispense		bottom of container
				with vacuum on and
				dispense off
2	6	Lowers nozzle to	6	Reverses direction
		the 230 μl position		with vacuum on and
		with the vacuum on		starts to dispense
		and then		270 μl volume waiting
		immediately		at the 270 μl position
		reverses direction
		lifting nozzle to
		home with vacuum
		on for 800 ms to
		clear fluid from
		nozzle and line
2	7		7	With dispense on
				and vacuum on
				nozzle raises to 320 μl
				position and waits
				60 ms (sufficient time
				dispense of >60 μl)
2	8		8	With dispense and
				vacuum on nozzle
				lowers to 250 μl
				position
2	9		9	With dispense on
				and vacuum on
				nozzle raises to 320 μl
				position and waits
				60 ms (sufficient time
				dispense of >60 μl)
2	10		10	Nozzle moves down
				to 270 μl position
				remains steady for
				50 ms and then
				dispense is turned off
				and waits another 30 ms
2			11	Nozzle lowers to
				230 μl height with
				vacuum on and stays
				there for 30 ms before
				lifting
2			12	Nozzle lifts to home
				position and
				incubator starts to
				turn with nozzle
				vacuum still on for
				500 ms to evaluate
				nozzle and line of
				fluid
3	1	Turn on vacuum	1	Starts downward
		before starting		travel with vacuum
		nozzle downward		off
		travel
3	2	Starts to dispense	2	Finds soak volume
		fluid when aspirate		from wash #2 (should
		nozzle is at the top		be at 230 μl)
		of the incubator
		ring. Start of 80 μl
		predispense
3	3	Travels to bottom	3	Turns on vacuum
		of container and		and turns on start of
		waits until for 80 μl		80 μl predispense
		dispense to be
		complete
3	4	Reverses direction	4	Travels to bottom of
		with vacuum on (no		container and waits
		delay) and starts to		for 80 μl dispense to
		dispense 270 μl		be complete
		volume
3	5	Waits for 270 μl of	5	Waits 30 ms at
		fluid to be dispense		bottom of container
				with vacuum on and
				dispense off
3	6	Lowers nozzle to	6	Reverses direction
		the 230 μl position		with vacuum on and
		with the vacuum on		starts to dispense
		and then		270 μl volume waiting
		immediately		at the 270 μl position
		reverses direction
		lifting nozzle to
		home with vacuum
		on for 800 ms to
		clear fluid from
		nozzle and line
3	7		7	With dispense off
				and vacuum on
				nozzle lowers to
				220 μl position and
				waits 30 ms
3	8		8	With dispense and
				vacuum on nozzle
				raises to 270 μl
				position waits there
				for the dispense to
				complete the 40 μl
				dispense (40 ms)
3	9		9	The dispense is
				turned off and the
				nozzle lowers to
				220 μl position and
				waits there 30 ms to
				top control the fluid
				height
3	10		10	The dispense is
				turned on and the
				nozzle raises to
				250 μl position waiting
				for the 30 μl dispense
				to be completed
3			11	The dispense is
				turned off and the
				nozzle lowers to
				230 μl height with
				vacuum on and stays
				there for 30 ms before
				lifting (which is the
				soak height)
3			12	Nozzle lifts to home
				position and
				incubator starts to
				turn with nozzle
				vacuum still on for
				500 ms to clear
				nozzle and line of
				fluid
	1	Turn on vacuum	1	Starts downward
		before starting		travel with vacuum
		nozzle downward		off and finds soak
		travel		height of wash #3
				with level sensing
4	2	Starts to dispense	2	Turn on vacuum
		fluid when aspirate		before starting nozzle
		nozzle is at the top		downward travel
		of the incubator		dropping at a speed
		ring. Start of 80 μl		that is 1/3 slower
		predispense		than the baseline
				wash rate of nozzle
				descent (goes to the
				bottom of the
				container).
				Note that there is no
				80 μl dispense
4	3	Travels to bottom	3	Waits at the bottom
		of container and		of the container for
		waits until for 80 μl		30 ms before
		dispense to be		reversing direction
		complete
4	4	Reverses direction	4	Turns on the
		with vacuum on (no		dispense and lifts the
		delay) and starts to		nozzle to the 230 μl
		dispense 270 μl		height waiting for
		volume		230 μl of fluid to be
				dispensed
4	5	Waits for 270 μl of	5	Turns off the
		fluid to be dispense		dispense and the
				nozzle drops to the
				bottom of the
				container with the
				vacuum on at the
				slower rate of decline
				(same as the first
				part of wash #4)
4	6	Lowers nozzle to	6	The nozzle waits in
		the bottom of the		the bottom of the
		container with the		container for 500 ms
		vacuum on		with the vacuum on
				(the total amount of
				time that the
				container is being
				evacuated is the
				same since the
				slower rate of decline
				took an additional
				300 ms)
4	7	Waits at the bottom	7	The nozzle is lifted
		of the container for		with the vacuum on
		800 ms with the
		vacuum on to
		reduce the amount
		of wash residual
4	8	Lifts the nozzle to	8	Nozzle lifts to home
		home with the		position and
		vacuum on and		incubator starts to
		leaves the vacuum		turn with nozzle
		on for 800 ms		vacuum still on for
		before the ring		500 ms to clear
		moves		nozzle and line of
				fluid

The present invention has proved to be particularly useful in improving the analysis performance of Troponin I (cTnI), a protein detectable in the bloodstream 4 to 6 hours after an acute myocardial infraction. Using the above wash process (both known and the present invention), several runs were carried out to determine levels of Troponin I according to the procedure set out below.[0036]

Biotin reagent was added to streptavidin-coated containers containing samples to initiate a reaction between biotinylated anti-cTnI antibody, the streptavidin coated container and the cTnI present in the sample. HRP conjugate reagent was also added to initiate a reaction between HRP-conjugated anti-cTnI antibody and the cTnI in the sample. The sample was then incubated for 8 minutes and 37° C. After incubation, the containers containing the samples were washed according to the present invention and according to the known wash process. Following washing, signal reagent containing a luminol derivative, a peracid salt and a substituted acetanilide electron transfer agent was added to produce luminescence that was read using a luminometer. The results are shown in the graph set forth in FIG. 3. As the graph shows, the results using the wash process of the present invention are much more consistent and have fewer outliers than the results using the known wash process.[0037]

It will be apparent to those skilled in the art that various modifications and variations can be made to the compounds, compositions and processes of this invention. Thus, it is intended that the present invention cover such modifications and variations, provided they come within the scope of the appended claims and their equivalents.[0038]

The disclosure of all publications cited above are expressly incorporated herein by reference in their entireties to the same extent as if each were incorporated by reference individually.[0039]