Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-07-28
2001-05-29
Chin, Christopher L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007230, C435S007240, C435S007300, C435S029000, C436S063000, C436S514000, C436S064000, C436S172000, C436S809000
Reexamination Certificate
active
06238874
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to an assay for determining the motility of cells. In particular, the invention provides an apparatus, and method of use, for determining the motile response of a selected type of cell to a given chemotactic agent.
BACKGROUND OF THE INVENTION
Chemotaxis is broadly defined as the orientation or movement of a cell or organism in relation to a chemical factor (Harris, 1954; Armstrong, 1985). Certain cells are capable of sensing a particular chemical factor and, in response, migrating toward or away from higher concentrations of the substance. In recent years, researchers and clinicians have expended great effort in studying cell motility for a number of cell types. Oftentimes, for example, it is useful for a clinician to determine the motility (e.g., ascertain whether the response is normal or depressed) of immune cells from patients suffering from disease, or to measure the motility of sperm for infertility patients.
Traditionally, the most popular assays for measuring cellular chemotaxis have utilized a so-called “Boyden chamber” (Boyden, 1962), or similar apparatus, in which the cells migrate through a filter that has pore openings that are smaller than the cell diameter. Typically, cells of a particular type are placed in a chamber on one side of the filter and a chemotactic agent is placed in a chamber on the other side. Results are usually quantified by counting the number of cells that have migrated through the filter. While such techniques are reasonably simple and widely available, they are associated with certain disadvantages. For example, they typically require a large number of highly purified cells. Isolating the cells, setting up the apparatus, and counting the cells which have migrated can be very labor intensive. Frequently, the process of isolating the cells can require several hours. Moreover, the cells are sometimes damaged during the process. For rare cell types, of course, the task of isolating a sufficiently large number of cells for use in the assay can be especially difficult and time consuming.
With most conventional chemotaxis assays, the process of examining and counting the cells that have moved has also been tedious and time-consuming. This is particularly true for assays that have relied upon manual examination, counting and analysis. Although some chemotaxis assays have automated, to varying degrees, these processes, they have not been entirely satisfactory either. For example, certain chemotaxis assays have employed automated readers of the type that detect the bulk amount of radiation emitted from a sample, e.g., a densitometer, fluorimeter, or spectrophotometer. While useful for determining the overall number of labeled cells in a region, such devices are not able to provide information regarding the positions of individual cells. Thus, it is not possible to image or map individual cells using such devices.
As another disadvantage, conventional cell-motility assay devices are not capable of simultaneously processing a large number of separate samples. In fact, most of the known assays accommodate only a single sample at a time. Of course, in situations where it is desirable to assay numerous samples in a relatively short time period, e.g., certain commercial research and clinical labs, the limited capacity of the known assay devices, which places a ceiling on throughput and productivity, can be a serious problem. Although a few multiple-site assays have been developed that can accommodate a plurality of samples at once, the total number of samples that can be simultaneously assayed is typically quite limited (e.g., less than 100). Moreover, the known multiple-site assays continue to suffer the disadvantages discussed above, e.g., their set-up and operation are tedious and labor-intensive, and they are not capable of providing positional information on individual cells.
SUMMARY OF THE INVENTION
One aspect of the invention provides an apparatus for assaying motility in response to a selected chemotactic agent. According to one embodiment, the apparatus includes a chamber having first and second side regions. Structure is provided facilitating the establishment of a concentration gradient of the chemotactic agent which increases on progressing from the first side region to the second side region. An optical system is provided for detecting selected individual cells in an interrogation region of the chamber. The apparatus further includes means for assigning positions to individual detected cells in the interrogation region in electronic readable form (e.g., by a digitization means). A programmed computing device is provided for comparing the distribution of detected, position-assigned cells in the interrogation region at two or more spaced time intervals.
In one embodiment, the computer means functions to compare the total number of detected cells whose positions are within the interrogation region at each of the spaced time intervals. In another embodiment, the computer means functions to (i) calculate a motion vector in the direction of the concentration gradient for each of the detected cells in the interrogation region, over the spaced time interval, and (ii) determine an average motion vector from the calculated individual vectors of the detected cells in the interrogation region.
According to one embodiment of the invention, the optical system includes an illumination source for directing a beam of light toward the chamber. Structure is provided to permit relative motion between the chamber and the beam of light for causing the beam to sequentially scan the area forming the interrogation region.
According to another embodiment, wherein the apparatus of the invention is used to detect fluorescent-labeled cells, the chamber is adapted to hold a cell-suspension liquid to a selected depth. Further, the optical system has a fluorescence excitation light source whose beam is adapted to be focused at a depth in the chamber that is intermediate the selected depth.
In one embodiment of the invention, the chamber is a capillary of substantially rectangular cross-section, having a first inlet proximate the first side region and a second inlet proximate the second side region.
In another embodiment, the chamber includes a plurality of separate sample wells positioned along the first side region.
Another aspect of the invention provides an apparatus for determining the motile response to a given chemotactic agent of a selected type of cell contained in a sample of mixed cell types. According to one embodiment, the apparatus includes a chamber having a first side region adapted to receive the mixed-cell sample and an opposed, second side region. Structure is provided facilitating the establishment of a concentration gradient of the chemotactic agent which increases on progressing from the first side region to the second side region. The apparatus further includes means for discriminating individual cells of the selected type from other components contained in the mixed-cell sample, as well as means for detecting and mapping the positions of individual cells of the selected type in a region between the first and second side regions.
According to one embodiment, the means for discriminating individual cells of the selected type includes a fluorescent reporter capable of selectively binding to the individual cells. In this embodiment, the fluorescent reporter is of a type having wavelengths of excitation and emission that minimize optical interference in the sample. In a related embodiment, the means for detecting and mapping the positions of individual cells includes an illumination source for directing an excitation beam of light toward the chamber. In this embodiment, the excitation beam has a wavelength that minimizes optical interference from components in the unpurified sample other than the selected type of cell.
According to one embodiment, the means for detecting and mapping the positions of individual cells further includes a focusing lens positioned proximate the chamber. The focusing lens is co
Jarnagin Kurt R.
Kunitake Steven T.
Manian Bala S.
Biometric Imaging Inc.
Chin Christopher L.
Nguyen Bao-Thuy L.
Schneck David M.
Schneck Thomas
LandOfFree
Cell motility assay does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Cell motility assay, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Cell motility assay will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2500123