Abstract:
Devices, systems, and methods for conducting chemiluminescent immunoassay testing and, more particularly, to initiating and monitoring a chemiluminescent reaction in a plurality of such assays, of different types, on a single immunoassay instrument, in a single procedure, using a plurality of labels and a triggering reagent combination are disclosed. Moreover, by including a base reagent injector assembly having an “e-channel” to provide a swirling turbulence to the base reagent immediately before it is introduced into the well of a cuvette containing a sample and an acid reagent. The added turbulence addresses the phenomenon referred to as “RLU shift,” in which the luminescence output can increase or decrease between assays.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims the benefit of U.S. provisional patent application No. 60/737,650 filed on Nov. 17, 2005, which is incorporated herein in its entirety by reference. 
     
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
       [0002]    (Not Applicable) 
       BACKGROUND OF THE INVENTION 
       [0003]    The present invention relates to means and methods for conducting chemiluminescent immunoassay testing and, more particularly, to initiating and monitoring a chemiluminescent reaction in a plurality of such assays, of different types, on a single immunoassay instrument, in a single procedure, using a plurality of analyte-specific, labeled reagents and a triggering reagent combination, wherein the phenomenon referred to as “RLU shift,” in which the luminescence output can increase or decrease, is reduced or eliminated. 
         [0004]    Assays are available to test a variety of body functions, including, for example, anemia, growth, thyroid, hypertension, tumor markers, neonatal conditions, the adrenal/pituitary system, bone and mineral metabolism, and the reproductive system. Immunoassay analyzers, such as chemiluminescent immunoassay analyzers, which automatically assay specimens, are one such device. 
         [0005]    Chemiluminescence is a chemical reaction that emits energy in the form of light. The intensity of the luminescence when compared to a control intensity provides a measure of the concentration of the sample being tested. For example, during a chemiluminescent assay, a test sample and an analyte-specific, labeled reagent, such as an acridinium ester antigen or an acridinium ester antibody, are dispensed in a cuvette. Next, a solid phase reagent, such as paramagnetic particles having a binding substance coupled thereto, is added to the sample-containing cuvette. The sample-containing cuvette is then incubated. 
         [0006]    During incubation, acridinium ester-labeled antibodies bind specifically to the antigen of interest in the sample. Alternatively, acridinium ester-labeled antigens compete with the antigen or interest already in the sample, to bind to the available antibody in the sample. Unbound labeled reagent is washed away. 
         [0000]    Chemical reagents, often referred to as “trigger” reagents, are then added to the sample-containing cuvette to oxidize the analyte-specific, labeled reagent, or, more specifically, to initiate the chemiluminescent reaction. The trigger reagents, conventionally, include an acid reagent and a base reagent. First, an acid reagent, e.g., hydrogen peroxide, is dispensed into the sample-containing cuvette, to initiate oxidation. Then a base reagent is dispensed into the sample-containing cuvette, to alter the state of the environment from acidic to basic, which accelerates oxidation. 
         [0007]    After the chemiluminescent reaction has been initiated, oxidation of the label to its excited state and its subsequent return to the ground state generates a flash, which typically lasts a few seconds. Conventionally, this flash is detected by a system-integrated luminometer. By examining the luminescence output resulting from mixing the test sample and trigger reagents, the automated analyzer can determine the concentration of the specific chemical constituent, for which the testing is being performed, in the patient&#39;s specimen. 
         [0008]    More specifically, the luminescence output of the chemiluminescent reaction, i.e., the intensity of the flash, which is measured and expressed in relative light units (RLU), can be compared to a calibrated test standard, to determine the amount, for example, of bound, labeled antibody or antigen in the patient&#39;s blood. 
         [0009]    Addition of a surfactant, such as a cationic detergent, to the base reagent is common. Advantageously, addition of a surfactant increases the intensity of light. However, when a surfactant is added, analyzers become susceptible to a phenomenon referred to as “RLU shift”. During an “RLU shift”, the luminescence output of the test sample can increase or decrease, which is to say “shift”, by as much as fifteen or more percent. As a result, the measurements of specific chemical constituent concentration can be over- or under-estimated. This causes unacceptable total imprecision. 
         [0010]    Therefore, it would be advantageous to be able to perform a plurality of such assays, of different types, on a single immunoassay instrument, in a single procedure, using a plurality of labels, and triggering reagents to initiate a chemiluminescent reaction for each of the labels sequentially. 
       SUMMARY OF THE INVENTION 
       [0011]    The present invention provides for prevention of the RLU shift phenomenon by introducing various features into the fluidic subassembly that delivers base reagent (i.e., the base trigger reagent) to a reaction receptacle, such as a cuvette. More specifically, the present invention discloses a base reagent injector assembly that includes a channel in the shape of the letter “e” (hereinafter an “e-channel”) and/or that is subject to sonic vibration. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0012]    The invention will be better understood by reference to the following more detailed description and accompanying drawings where like reference numbers refer to like parts: 
           [0013]      FIG. 1  shows a cut-out base injector assembly in accordance with the present invention; 
           [0014]      FIG. 2A  shows a lower injector portion of a base injector assembly in accordance with the present invention; 
           [0015]      FIG. 2B  shows a cut-out of the lower injector portion of a base injector assembly in accordance with the present invention; 
           [0016]      FIG. 3A  shows an upper injector portion of a base injector assembly in accordance with the present invention; 
           [0017]      FIG. 3B  shows a cut-out of the upper injector portion of a base injector assembly in accordance with the present invention; 
           [0018]      FIG. 4  shows an injector tubing in accordance with the present invention; and 
           [0019]      FIG. 5  shows a base injector assembly and measurement chamber in accordance with the present invention. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0020]    As discussed above, automated chemiluminescent immunoassay analyzers are susceptible to a phenomenon known as “RLU shift”, whereby the luminescence output from the flash, which is measured in RLUs, can increase or decrease by 15% or more from one assay run to the next. The mechanism(s) or factor(s) causing RLU shift, however, are not fully understood. Hence, the present inventor conducted empirical testing. The results and observations of the empirical testing are summarized below: 
         [0021]    “RLU shift” is related to and caused by a base reagent pump transitioning from a “standby state” to a “ready state” or transitioning from a “ready state” to a “standby state”, which transitions can be reproduced under controlled experimental conditions. In the “ready state”, a stepper motor that drives the base reagent pump is energized. In the “standby state”, the stepper motor is not energized. Thus, of all the factors potentially involved, the mechanism of the “RLU shift” involves the base reagent pump. 
         [0022]    The magnitude of the luminescence signal is very sensitive to pump speed variations over a narrow range of pump speeds. For example, increasing the base reagent pump dispense speed from 950 to 1400 micro-liters per second (μl/sec) caused the luminescence to increase by about 20%. At a pump dispense speed above about 1400 μl/sec there was no increase in luminescence observed. Likewise, reducing the base reagent dispense speed from 950 to 700 μl/sec had no observable effect on luminescence. 
         [0023]    Accordingly, in this narrow range of pump dispense speeds, which is to say, between about 950 and about 1400 μl/sec, the system is more sensitive to “RLU shift”. In other words, within this narrow range of pump dispense speeds, the precision of repeated measurements is more susceptible to “RLU shift” than at pump dispense speeds above or below this narrow range. Thus, subtle variations in the dispense speed of the base reagent pump can cause substantial shifts in luminescence. For example, a 20% change in dispense speed may result in a 10% change in luminescence. The prior art does not suggest any correlation between pump dispense speed and luminescence and/or sensitivity when using, for example, a piston pump, driven by a stepper motor. 
         [0024]    Evidence, therefore, suggests that the mechanism of an “RLU shift” involves the base reagent dispense step. This result, also, was unexpected, because, during a chemiluminescent immunoassay, the base reagent dispense step is tightly controlled and gross variations in the pattern or timing of the base reagent dispense, e.g., the speed and angle of dispensing the base reagent into the reaction liquid, were not observed. Nor does the prior art suggest that the base reagent dispense step is critical to achieve a reproducible luminescence signal. 
         [0025]    Further experimentation revealed that the “RLU shift” can be mediated by adding a surfactant, e.g., a cationic detergent, to the base reagent solution, for the purpose of enhancing the luminescence signal. This result suggests that disruption of the micellar structure of the surfactant during the base reagent dispense step mediates the “RLU shift”. The prior art is silent about micellar disruption affecting luminescence intensity. 
         [0026]    Experimentation also demonstrated that an external source of vibration, when applied to base line, can mediate “RLU shifts”. This result is consistent with a mechanism in which turbulence of the liquid flow in the base line—whether the turbulence is caused by the base reagent pump or by external vibration—causes disruption of the micelles, which enhances the chemi-luminescence. 
         [0027]    The results of this testing suggest the following system model:
       Between assay runs, when the system transitions to or from a standby state, the base reagent pump is switched from a smooth motion profile to a rough motion profile (or vice versa).   The change in pump state causes some change in the flow characteristics within the base line.   The altered flow of base reagent in the base line changes the structure of the micellar surfactant in a way that makes it more effective at enhancing the luminescence reaction.       
 
       Base Reagent Injector Assembly 
       [0031]    Experimentation suggests that the “RLU shift” phenomenon, which occurs during a chemiluminescent reaction associated with an immunoassay system, can be prevented by “activating” the base reagent during the base reagent dispense step and/or, alternatively, by sonically vibrating the base fluid tubing during the base reagent dispense step. In short, prior to or at the time the base reagent is dispensed into the well of the sample-containing cuvette, the base reagent should be subject to turbulent flow. 
         [0032]    The altered, i.e., turbulent, flow of the base reagent changes the structure of the micellar surfactant, which enhances the luminescent reaction and prevents “RLU shift”. 
         [0033]    A base reagent injector assembly  10  in accordance with the present invention is shown in  FIG. 1 . The base reagent injector assembly  10  includes a lower injector portion  20  and an upper injector portion  30 . The lower injector portion  20  is structured and arranged to be mechanically- and fluidly-coupled to a measurement unit (not shown). The upper injector portion  30  is structured and arranged to be mechanically- and fluidly-coupled to injector tubing  40 . 
         [0034]    Referring to  FIG. 2A  and  FIG. 2B , the lower injector portion  20  will be described. The lower injector portion  20  includes a hollow, cylindrical body  29 , a measurement unit interface  26 , and a nozzle  28 . The hollow, cylindrical body  29  includes a chamber cavity  24  that is structured and arranged to accommodate the protrusion portion  36  of the upper injector portion  30  (described below). More specifically, the chamber cavity  24  of the hollow, cylindrical body  29  is structured and arranged to have an inner, peripheral circumference that provides a tight, interference (press) fit with a plurality of seal rings  37  disposed on the outer periphery of the protrusion portion  36  of the upper injector portion  30 . 
         [0035]    A channel opening  25  is provided at or near the center of the chamber cavity  24 . The channel opening  25  is fluidly-coupled to the outlet  27  of the nozzle  28  via an injection channel  22 . An exemplary length of the injector channel  22  for a prototype used in testing was about 0.33 inches (8 mm). The inner diameter (ID) of the injector channel  22  includes a step-down transition  23  from about 0.043 inches (about 1.1 mm) to about 0.033 inches (about 0.8 mm). The diameter step-down transition  23  introduces more turbulence to the base reagent fluid passing through the injector channel  22 . 
         [0036]    Referring to  FIG. 3A  and  FIG. 3B , an upper injector portion  30  will be described. The upper injector portion  30  includes a cylindrical protrusion portion  36  and a cylindrical body portion  38 . The cylindrical protrusion portion  36  is structured and arranged to mate with, i.e., to provide a tight interference (press) fit with, the lower injector portion  20 . The cylindrical body portion  38  is structured and arranged to mate with injector tubing  40 . 
         [0037]    The cylindrical protrusion portion  36  is concentric with and fixedly attached to a proximal end of the body portion  38 . The distal end of the body portion  38  includes an injector tubing interface  34  for releasably attaching injector tubing  40  to the body portion  38  of the upper injector portion  30 . More specifically, spiral threadings  39  are provided on the peripheral surface of the injector tubing interface  34  to accommodate associated threadings  46  disposed on a first end  41  of the injector tubing  40  (shown in  FIG. 4 ). The injector tubing interface  34  is, preferably, not coaxial with the cylindrical body portion  38 . 
         [0038]    An opening  32  is provided at or near the center of the injector tubing interface  34 . Because the injector tubing interface  34  is not coaxial with the cylindrical body portion  38 , the opening  32  is also off-set from the center of the cylindrical body portion  38 . The opening  32  is fluidly-coupled to an outlet  31  disposed on the face  33  of the protrusion portion  36  via an e-shaped channel, or “e-channel”  35 . 
         [0039]    The “e-channel”  35  traverses through the protrusion portion  36 , from the opening  32  to the outlet  31 . An exemplary inner diameter (ID) of the opening  32  and the “e-channel”  35  is about 0.06 inches (about 1.5 mm). The outlet  31  is structured and arranged to be in registration with the channel opening  25  in the lower injector portion  20  when the upper and lower injector portions  30  and  20  are press-fitted together. 
         [0040]    The “e-channel”  35  is adapted to provide swirling turbulence to the base reagent fluid passing through the upper injector portion  30 . Although this disclosure refers to the channel providing swirling turbulence as an “e-channel”, the channel can take on any shape so long as it provides swirling turbulence. For example, in lieu of an e-shaped channel, the channel can be structured and arranged to include at least two rather abrupt, 90 degree changes in direction. By undergoing abrupt, orthogonal changes in flow direction, the base reagent fluid is “activated”, i.e., is in a turbulent flow state, prior to entering the injection channel  22  of the lower injector portion  20 . 
         [0041]    The cylindrical protrusion portion  36  includes a plurality of seal rings  37  for providing a tight, interference (press) fit in the cavity  24  of the lower injector portion  20 . Although  FIG. 1  and  FIG. 3A  show three seal rings  37  on the protrusion portion  36 , more or fewer seal rings  37  can be included without violating the scope and spirit of this disclosure. 
       Chemiluminescent Immunoassay System 
       [0042]    Referring to  FIG. 5 , a chemiluminescent immunoassay system  50  using the base reagent injector assembly  20  will be described. The system  50  includes injector tubing  40  for providing base reagent, the base reagent injector assembly  10 , and a measurement unit  52 . A first end  41  of the injector tubing  40  is mechanically- and fluidly-coupled to the upper injector portion  38  via the injector tubing interface  34 . More specifically, the injector tubing  40  is structured and arranged so that the outlet (not shown) of the injector tubing  40  is in registration with the opening  32  in the injector tubing interface  34 . 
         [0043]    The measurement unit interface  26  of the lower injector portion  20  is mechanically- and fluidly-coupled to the measurement unit  52  via a mechanical interface  54  that is disposed on the cover  51  of the measurement unit  52 . Proximate to the mechanical interface  52  is a fitting screw interface  56  that is structured and arranged to mate with an eccentric, fitting screw or bolt  58  for further tightening the fit between the measurement unit interface  26  and the mechanical interface  54 . 
         [0044]    Referring to  FIG. 4 , injector tubing  40  for use with the chemiluminescent immunoassay system  50  will now be described. The injector tubing  40  includes a first end  41  and a second end  49 . The injector tubing  40  can be made of black Teflon® tubing  43  (manufactured by DuPont Corp. of Wilmington, Del.) having a Norprene® cover  45  (manufactured by Saint-Gobain Performance Plastics Corp. of Aurora, Ohio). The black tubing  43  protects the base reagent fluid from light and the cover  45  prevents kinked tubing. Optionally, black heat shrink material (not shown) can be included on the outside of the tubing  43  proximate to the fitting screws  46  and  48 . 
         [0045]    The ends  41  and  49  of the tubing  43  are flanged. The tubing  43  has an inner diameter of about 0.06 in. (about 1.5 mm). For mechanically-attaching the injector tubing  40  to the upper injector portion  30 , fitting screws  46  and  48  and press rings  42  and  44 , which are well known to those skilled in the art, are provided. 
         [0046]    Optionally, the system  50  can include vibration means (not show) adapted to apply sonic vibrations to the injector tubing  40 , to induce turbulence in the base reagent fluid. For example, the injector tubing  40  can be operationally-coupled to a small loudspeaker (about 200 mW)(not shown). More specifically, the injector tubing  40  can be adhesively attached to the center of the diaphragm of the loudspeaker. A pulse generator provides sonic vibrations to the injector tubing  40  via the loudspeaker. The pulse generator is adapted to generate a tone at a frequency between about 280 Hz and about 1000 Hz having an amplitude of about 5V (peak-to-peak). 
       Method of Conducting Chemiluminescent Assays 
       [0047]    Having described a chemiluminescent immunoassay system  50  and a base reagent injector assembly  20  therefor, a method of conducting chemiluminescent assays and, more particularly, for actively dispensing a base reagent into a reaction liquid so as to prevent “RLU shift” will now be described. Protocols for conducting chemiluminescent assays are well known to the art and will not be described in detail herein. 
         [0048]    According to the present invention, a cuvette, having a well that contains an acid trigger reagent and a test sample that has been labeled using, for example, luminol or acridinium, is loaded into a measurement unit. A background measurement of the luminescence of the test sample is, typically, taken to establish a base line. After the background measurement, while the test sample is still in the measurement unit, a base reagent is added to the well of the cuvette, which triggers a chemiluminescent reaction with the label. As provided above, the base reagent is added to the cuvette in a turbulent flow condition so that the structure of the micellar surfactant in the base reagent is changed. Injecting the triggering (base) reagent under turbulent flow conditions tangentially along or against the walls of the cuvette well washes down any of the sample or of the acid reagent thereon. As a result, cross-contamination caused by the triggering (base) reagent exiting the nozzle of the base reagent injector assembly is substantially eliminated. 
         [0049]    The base reagent liquid can be placed in a turbulent flow condition by one or more of the “e-channel” in the upper injector portion, the step-down diameter in the lower injector portion, and/or sonically vibrating the injector assembly. 
         [0050]    The luminescence can then be measured, e.g., using a photomultiplier tube (PMT). Typically, the reaction liquid in the well of the cuvette is aspirated and discarded and the emptied cuvette is removed from the measurement unit. 
         [0051]    The invention has been described in detail including the preferred embodiments thereof. However, those skilled in the art, upon considering the present disclosure, may make modifications and improvements within the spirit and scope of the invention.