Method and apparatus for screening chemical compounds

Details Method and apparatus for screening chemical compounds Method and apparatus for screening chemical compounds

1999-06-15

2002-05-14

Phan, James (Department: 2872)

Optical: systems and elements

Deflection using a moving element

C359S204200, C359S212100, C359S215100, C359S226200, C359S368000, C359S391000, C250S234000

Reexamination Certificate

active

06388788

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to methods and apparatus for identifying pharmacological agents useful for the diagnosis and treatment of disease by performing a variety of assays on cell extracts, cells or tissues where the measurement of biological activity involves the use of various embodiments of a line-scan confocal imaging system and associated data processing routines.
BACKGROUND OF THE INVENTION
There is currently a need in drug discovery and development and in general biological research for methods and apparatus for accurately performing cell-based assays. Cell-based assays are advantageously employed for assessing the biological activity of chemical compounds and the mechanism-of-action of new biological targets. In a cell-based assay, the activity of interest is measured in the presence of both competing and complementary processes. As pertains to chemical compound screening, information is available as to the specific activity of the compound. For example, it is possible to assess not only whether a compound binds the target of the assay, but also whether it is an agonist or an antagonist of the normal activity of the target. Frequently, the target is a cell-surface receptor. In some signaling pathways, the member of the pathway of greatest potential therapeutic value is not the receptor but an intracellular signaling protein associated with the receptor. It is, therefore, desirable to develop methods to assay activity throughout the pathway, preferably in the cellular milieu.
In addition, there is a need to quickly and inexpensively screen large numbers of chemical compounds. This need has arisen in the pharmaceutical industry where it is common to test chemical compounds for activity against a variety of biochemical targets, for example, receptors, enzymes and nucleic acids. These chemical compounds are collected in large libraries, sometimes exceeding one million distinct compounds. The use of the term chemical compound is intended to be interpreted broadly so as to include, but not be limited to, simple organic and inorganic molecules, proteins, peptides, nucleic acids and oligonucleotides, carbohydrates, lipids, or any chemical structure of biological interest.
In the field of compound screening, cell-based assays are run on collections of cells. The measured response is usually an average over the cell population. For example, a popular instrument used for ion channel assays is disclosed in U.S. Pat. No. 5,355,215. A typical assay consists of measuring the time-dependence of the fluorescence of an ion-sensitive dye, the fluorescence being a measure of the intra-cellular concentration of the ion of interest which changes as a consequence of the addition of a chemical compound. The dye is loaded into the population of cells disposed on the bottom of the well of a multiwell plate at a time prior to the measurement. In general, the response of the cells is heterogeneous in both magnitude and time. This variability may obscure or prevent the observation of biological activity important to compound screening. The heterogeneity may arise from experimental sources, but more importantly, heterogeneity is fundamental in any population of cells. Among others, the origin of the variability may be a consequence of the life-cycle divergence among the population, or the result of the evolutionary divergence of the number of active target molecules. A method that mitigates, compensates for, or even utilizes the variations would enhance the value of cell-based assays in the characterization of the pharmacological activity of chemical compounds.
Quantification of the response of individual cells circumvents the problems posed by the non-uniformity of that response of a population of cells. Consider the case where a minor fraction of the population responds to the stimulus. A device that measures the average response will have less sensitivity than one determining individual cellular response. The latter method generates a statistical characterization of the response profile permitting one to select the subset of active cells. Additional characterization of the population will enhance the interpretation of the response profile.
Various measurement devices have been used in the prior art in an attempt to address this need. Flow-cytometer-based assays are widely practiced and measure cell properties one at a time by passing cells through a focused laser beam. Several disadvantages accompany this method. Most important to the pharmaceutical industry is that assays can not readily be performed on compounds disposed in microtiter plates. In addition, the throughput is poor, typically 10-100 seconds per sample, the observation time of each cell is <1 ms, prohibiting kinetic assays, and finally, only the cell-averaged signal can be determined.
In addition, many assays require determination of the relative locations of the fluorescence signals. Devices called scanning cytometers, as disclosed in U.S. Pat. Nos. 5,107,422 and 5,547,849, are widely used for imaging single cells. In order to gain acceptable speed, these devices operate at low (~5-10 m&mgr;m) resolution. Thus, these devices offer little advantage over flow cytometers for assays requiring spatial information on the distribution of the fluorescence signals.
An additional alternative technology is the fast-camera, full-field microscope. These devices have the ability to obtain images at a resolution and speed comparable to the present invention, on certain samples. However, they are not confocal and are consequently susceptible to fluorescence background and cannot be used to optically section the sample. In addition, simultaneous, multi-parameter data is not readily obtained.
In contrast to the prior art, the present invention can be used to perform multi-parameter fluorescence imaging on single cells and cell populations in a manner that is sufficiently rapid and versatile for use in compound screening. Methods and apparatus are provided for obtaining and analyzing both the primary response of individual cells and additional measures of the heterogeneity of the sample population. In addition, the locations of these multiple fluorophores can be determined with sub-cellular resolution. Finally, the present invention can be used to image rapidly changing events at video-rates. Together these capabilities enable new areas of research into the mechanism-of-action of drug candidates.
The present invention may also be employed in an inventive fluorescence-based biochemical assay, somewhat analogous to the surface scintillation assay (“SSA”) which is among the more widely used methods for screening chemical compounds.
FIGS.
1
(
a
)-
1
(
f
) depict the steps of a receptor-binding SSA. In FIG.
1
(
a
), soluble membranes
10
with chosen receptors
12
are added to a well
20
containing a liquid
30
. These membranes are isolated from cells expressing the receptors. In FIG.
1
(
b
), radio-labeled ligands
14
are added to the well. The ligand is known to have a high binding affinity for the membrane receptors. The most common radio labels are
3
H,
35
S,
125
I,
33
P and
32
P. In FIG.
1
(
c
), beads
16
are added to the well. The beads are coated with a material, such as wheat germ agglutinin, to which the membranes strongly adhere. The beads have a diameter of 3-8 &mgr;m and are made of plastic doped with a scintillant. Alternatively, the order of the operations depicted in FIGS.
1
(
b
) and
1
(
c
) may be interchanged.
The radiolabels decay by emitting high energy electrons, or beta particles, which travel approximately 1-100 &mgr;m before stopping, depending on the radio-isotope. If the radiolabels are bound to the membranes attached to the beads, the beta particles may travel into the beads and cause bursts of luminescence. If the radio-labels are dispersed throughout the liquid, the emitted beta particles will not generally excite luminescence in the beads. In FIG.
1
(
d
), the luminescence of the beads caused by decay of the radio labels is detected. In FIG.
1
(
e
), a test compound 18 is a

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