Patent Application
20070264718
Turning now to the drawings, a preferred analyzer 20 in accordance with the invention is depicted in
In more detail, the mounting block 22 has rear wall 28 and forwardly extending, spaced apart sidewalls 30, 32. The primary function of the block 22 is to provide a stable base for tumble block 24 and therefore the precise shape and dimensions of block 22 are variable. As an alternative design the mounting block 22 may contain the light sources and sensors presently shown in
The tumble block 24 includes an upper, primary section 34 as well as a lower connection portion 36. The section 34 has a central, vertically-extending, substantially square in cross-section opening 38 formed therein which is designed to receive the lower end of cuvette 26. In addition, the section 34 is equipped with one or more sources 40 of electromagnetic radiation (e.g., LEDs or lasers) adjacent one face of the opening 38, along with three photo sensors 42, 44, 46 disposed adjacent the remaining three faces of opening 38. Each of the photo sensors 42-46 may optionally have a long pass filter 42a, 44a, and 46a located between the respective sensor and the cuvette opening 38. Additionally each of the sources of electromagnetic radiation and sensors may have an aperture to control the direction of the rays of the electromagnetic radiation. Appropriate electrical leads 39 extend from block 24, in order to operatively connect the internal components 40-46 to a computer or other digital processor (not shown). Specific design and combination of sources of electromagnetic radiation, sensors, filters and apertures are dependent on specific applications. In an alternate configuration all the optical components are disposed in the support block 22.
The lower connection portion 36 is pivotally coupled to the sidewalls 30, 32, as best seen in
The cuvette 26 includes an upper, radially enlarged segment 52 equipped with a stopper or sealing cap 54, as well as a lower, substantially square in cross-section segment 56 complemental with opening 38. Preferably, at least the segment 56 is formed of glass or synthetic resin material which is capable of transmitting electromagnetic radiation of desired wavelength for analyses of samples using analyzer 20; most typically, the cuvette body is formed of essentially transparent material.
It will be appreciated that the apparatus illustrated in
The optical components presented in
As described, the analyzer 20 is used for two-phase assays carried out in a single container or cuvette having walls formed of electromagnetic radiation-transmissible material. The assay mixture normally includes a liquid buffer, a known amount of solid particles or beads, a sample which may or may not contain analyte(s) of interest, an affinity agent (typically bound to the beads) operable to bind the analyte(s) to the beads, and a radiation-activated marker such as a fluorescent dye which activates upon incident electromagnetic radiation of known wavelength.
In the assays of the invention, the assay mixture is mixed and incubated for a period of time sufficient to effect affinity binding of analyte(s) to the beads, and to ensure essential homogeneity of the beads within the buffer. Thereafter, the mixture is allowed or caused to settle and separate into a bead-rich lower phase and a substantially bead-free upper or supernatant phase. Preferably, when using an analyzer such as that illustrated in
After incubation and tumbling, the cuvette is returned to an upright position and allowed to settle and fractionate under the influence of gravity. Before, during and after separation into the upper and lower phases is achieved, measurements are taken by passage of electromagnetic radiation through the mixture to obtain sensed assay data giving information about the analyte(s) and about completion of the separation of phases. Such data may be derived using a two-sensor arrangement wherein respective sensors are located at vertically spaced locations adjacent the upper phase zone and the lower phase zone. In this type of analyzer, incident radiation is directed through the container at the two spaced locations, and such is sensed by the individual sensors. In a more preferred embodiment, a single sensor arrangement is employed wherein only a single sensor is located adjacent either the upper phase zone or lower phase zone, and most preferably adjacent the latter.
The present invention includes a number of improvements over the conventional two-phase assays known in the art and described in U.S. Pat. No. 5,674,699. For example, the '699 patent preferably employs a centrifugation step in order to effect fractionation of the assay mixture into a particle-rich fraction and a particle-free fraction. In the preferred embodiment of the present invention, however, such centrifugation is eliminated and replaced by gravity-induced settling and instrumental monitoring of fractionation during the course of settling. When this settling reaches a steady state as verified by the instrumental monitoring, final assay measurements may be taken with assurance that adequate separation has been achieved.
In order to monitor the settling process on a consistent basis, measurements should begin essentially immediately upon the termination of the mixing or tumbling step. A standard profile of settling is produced if the sample is successfully settled and fractionated. The preferred profile takes into account both the disappearance of micro beads from the upper supernatant phase, and full packing of micro beads into the lower phase.
The ratio of the two final measurements is taken as representing the proportion of analyte distributed between the supernatant phase and the settled micro bead phase. In an unknown sample, this ratio may be related by comparison with ratios of at least two known standards to give the percentage of bound and unbound analyte.
A further analysis of
A further benefit of using a single sensor for the two-phase assay is the realization of a faster end point. The sensing area at the bottom sensor is in a steady state earlier in the settling process, because some of the smaller micro beads have not settled from the top sensor region. This observation is also illustrated in
The foregoing description involves embodiments making use of fluorescence quenching. Another mode of measurement is direct fluorescence of a fluorescent analyte, or fluorescence-labeled analyte that may be specifically adsorbed onto the micro beads. For example, the proportion of glycosylated proteins may be determined in a mixture also containing non-glycosylated proteins by brief exposure of the mixture to fluorescamine, which attaches to free amino groups on both types of the protein. The non-reacted fluorescamine becomes non-fluorescent and does not further react within a few minutes of exposure to aqueous solution. The mixture is subsequently transferred to a tube containing buffer, and micro beads that attract carbohydrates and other cis-glycols, such as micro beads conjugated to aminophenyl boronic acid, as described in U.S. Pat. No. 4,269,605, incorporated by reference herein. The glycosylated proteins are attracted to the micro beads and contribute fluorescence to the micro beads, whereas non-glycosylated proteins remain in solution and contribute fluorescence to both the solution and as a background in the region of the micro beads. By the same principles of measurement, fluorescent-labeled antibodies may be used to measure the presence and/or quantity of a non-labeled analyte.
Many other assay applications are made possible by the two-phase assay of the present invention. An antibody or lectin, or indeed any other ligand, may be substituted for boronic acid affinity reagents attached to micro beads. Antibody to a hemoglobin variant, such as fetal hemoglobin, sickle cell hemoglobin, and hemoglobin C, may be used to screen for the presence of the variant. Moreover, antibodies to multiple variants of hemoglobin may be attached to micro beads all in a single container, for the general purpose of screening. Variants attached to micro beads may be further distinguished from those not attached and remaining in the solution phase. Thus, a general approximation may be made between presence only of variants in a patient and a heterozygous patient in whom some hemoglobin is normal and some is variant. Assays for the same variant hemoglobins may also be performed by use of ion exchange micro particles and varying the ionic concentration of the buffer.
The following examples set forth presently preferred equipment and process steps useful in carrying out the assays of the present invention. It is to be understood, however, that these examples are presented by way of illustration only, and nothing therein should be taken as a limitation upon the overall scope of the invention.
In this assay, a transparent polystyrene cuvette 26 was used having a lower segment 56 of 4 mm square cross-section. An assay mixture was placed within the cuvette, comprising 700 micro liters of buffer and 6% agarose micro beads affinity labeled with amino phenyl boronic acid, the latter in an amount totaling precisely 10% of the total volume of the mixture. The labeled beads were prepared by the method described in U.S. Pat. No. 4,269,605, incorporated by reference herein. The buffer was 50 mM glycine buffer at pH 9.2, containing 50 mM magnesium chloride, 0.1% Triton X100 surfactant, 10% reagent grade ethanol, and distilled water. The mixture also contained 0.11 mM of fluorescent dye, 8-hydroxy-3,6,9-pyrene trisulfonate (HPT).
Calibrated samples commercialized by Primus Corporation of Kansas City, Mo., containing 5.5 and 14% glycated hemoglobin were mixed together in such proportions that five samples of known glycated hemoglobin content were prepared. These knowns are referred to as the “linearity series.”
Four micro liters of one of the linearity series samples was placed in a cuvette containing the mixture described above. The cuvette was then tumbled for seven minutes by repeatedly inverting the cuvette. The cuvette was then immediately placed within a measuring device using light emitting diodes (405 nm) applied in an alternating on/off fashion to the bottom of the cuvette tube and at a position 12.5 mm above the bottom measurement point. Digitized data was then collected for a period of 3.5 minutes, until a steady state was achieved, thus establishing essentially complete fractionation of the sample into a bead-rich lower phase and a substantially bead-free upper phase. This data was then plotted to develop a normalized profile of the type illustrated in
Next, all five of the linearity series were processed in duplicate, and data recorded individually for each sample. The results are set forth in
In this example, all of the reagents and instrumentation of Example 1 were used, but with a different method of data analysis. Specifically, for this example, only data from the bottom sensor was evaluated, and the ratios developed were that of the initial fluorescence intensities observed at the bottom sensor at the conclusion of tumbling and before any substantial settling occurred, versus the final fluorescence intensities observed at the bottom sensor after complete settling. Data from the same linearity series described in Example 1 was recalculated using only the single bottom sensor data, and the result is set forth in
In this example, the two data analysis methods of Examples 1 and 2 were compared in the course of running a large set (166) of clinical glycated hemoglobin samples.
In this example, an LED emitting light at 650 NM was used to obtain light-scattering data. This data comprised two components, namely light loss as a result of the transmission mode where light passing from the LED to the opposite photo sensor was diminished by the presence of the beads within the sample, and by light scattering onto the photo sensors located at right angles to the LED, where the micro beads scatter light proportional to the amount of beads present. It should be noted that light from the 650 nm LED is not absorbed by the hemoglobin analyte that may be present in the sample, or by the dye, HPT, that is used to generate contrast in the fluorescent mode of evaluation descried in Example 1 and Example 2. Therefore the measurement here described may be performed in the presence of sample and the full chemical complement of the assay for analytes.
Specifically, five suspensions having from 8.3 to 12.5% micro beads in distilled water were prepared, and transmission and light scattering measurements were taken in duplicate and averaged. The collected data is set forth in
Samples of bovine serum albumen (BSA) and bovine gamma globulin (BGG) were prepared at concentrations of 10 mg/ml in distilled water. Twenty micro liters of lisamine rhodamine sulphonyl chloride (1 mg/ml) was added to 2 ml of each protein sample, and labeling was allowed to continue for two hours at room temperature. Measurement cuvettes were prepared containing 700 micro liters of 10% agarose gel labeled with Protein G (Amersham Biosciences HiTrap Protein G HP), a known protein ligand which absorbs BGG, but not BSA. One measurement cuvette was supplemented with only BSA, a second was supplemented only with BGG, and a third was supplemented with equal volumes of BSA and BGG. Tumbling, settling, and measurement of fluorescence were carried out as described in Example 1, and the ratios of fluorescence observed from the substantially bead-free supernatant phase and the affinity-labeled, bead-rich agarose phase were calculated. These ratios are set forth below.
This data demonstrates that labeled proteins with different ligand characteristics may be distinguished by the assays of the present invention, so long as the label characteristics are known.
In this example, beads having the property of cation exchange are employed in a plurality of different containers in order to screen for hemoglobin variants. Specifically, carboxymethyl agarose is prepared using 4% cross-linked agarose. A 50 mM glycine buffer at pH 6.85 is also prepared, containing 0.1% Triton X100 and 11 μM 8-hydroxypyrene trisulphonic acid. The buffer is divided into three parts which are adjusted to sodium chloride concentrations of 0.011, 0.016, and 0.026 M, respectively. The prepared micro beads are washed in these buffers and suspensions are made of the beads at a level of 10% in the individual buffers.
Next, 10 micro liters of whole blood are added to each suspension. These samples may contain hemoglobin variants having different elutions from chromatography columns at different retention times, using gradients similar to the buffers described herein. As a screening test, the different suspensions would either retain a hemoglobin variant in the micro bead after tumbling and settling, or would permit the hemoglobin variant to remain in solution, according to Table 2:
The position of a particular hemoglobin variant in the micro beads or in solution is determined by the amount of hemoglobin measured by the fluorescence method of Examples 1 or 2. Thus, according to a logic scheme such as that set forth in Table 2, it is possible to determine variant types through the screening process of this Example.
In greater detail, if there is a hemoglobin variant in solution in the 0.011 M NaCl suspension, then it is a “fast moving” variant, such as HbF. If in the 0.011 M NaCl suspension there is a hemoglobin variant in the micro beads, then this cannot be distinguished as being normal HbA0, HbS, or HbC. However, if in combination with such 0.011 M NaCl suspension results, there is no hemoglobin variant in the 0.016 and 0.026 M NaCl suspensions, then the presence of HbS and HbC may be ruled out. On the other hand, if all hemoglobin variants are retained in the micro beads until the highest molarity, then the presence of HbC is confirmed. Thus, both homozygous and heterozygous patients with variant hemoglobins may be distinguished.
This application claims the benefit of Provisional Application Ser. No. 60/799,590 filed May 12, 2006. This Provisional Application is incorporated by reference herein.
| Number | Date | Country | |
|---|---|---|---|
| 60799590 | May 2006 | US |
