Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
2000-12-29
2004-08-03
Gitomer, Ralph (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C422S051000
Reexamination Certificate
active
06770434
ABSTRACT:
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to a biological assay and a biological assay apparatus.
Biochemical, microbiological, chemical and many other assays are being performed every day in laboratories. While a considerable amount of attention has naturally been placed on such biological cell assaying for humans, this is also be becoming more important in the field of animal welfare and indeed plant production generally.
A rapidly advancing research area in biology is the study of cell receptor-ligand interactions resulting in cell-substratum and cell-cell adhesion followed by subsequent cell migration. The pre-requisite to transendothelial migration of certain cell lines into sites of infection is paramount to the study of inflammatory diseases. This can be briefly summarised as cell flow and rolling, tethering and activation of integrin receptors which is a key recognition step, attachment to the endothelial ligands via activated integrins and finally transendothelial migration or diapedesis. Unfortunately, to date, most of the assay techniques are not particularly successful for the study of these mechanisms. Currently, the majority of studies involving cell rolling and chemokine induced cellular arrest have utilised capillary systems wherein cell flow and shear stress are controlled utilising syringe pumps. Such observations are constrained by a number of factors. Firstly, the relative large (>100 &mgr;m) size of the standard glass capillaries limits the physiological analogies to the proximal microvascular regions. Secondly, such studies can only be utilised to study single end-points and cannot be utilised to examine cell choices in migration Thirdly, optical aberrations related to the spherical geometry of the glass capillary sections limit stage-related in situ (post-fixation) analysis of the intracellular structures (cytoskeleton and signalling molecules). Finally and most importantly, the usual observation periods lie between 5-30 minutes for rolling experiments. Longer studies are required to study subsequent crawling steps on endothelial and extracellular matrix ligands. In this regard, studies relating to the effects of chemokines have largely been limited to cellular arrest on adhesion receptor ligands and have not been extended to the study of cell crawling. For example, specific chemokines have been shown to induce rolling arrest with enhanced binding of lymphocytes to ICAM-1, otherwise known as CD54.
Presently accepted techniques for cell adhesion or binding assays involve the initial coating of a surface of a device with a substrate, typically a protein. Cells are deposited onto the substrate and allowed to settle. Following the settling of the cells, the device is placed on a heating stage at 37° C., which is attached to an inverted microscope for visual analysis, or alternatively to a stand-alone heating stage and progression of cell binding can be checked at intervals with the inverted microscope. The duration of these assays may be varied depending on the cell line and choice of substratum. Following cell adhesion, free cells may be washed away and a subsequent cell count may be carried out.
Although these methods provide us with semi-quantitative information regarding a cell type's affinity for a particular substratum, there is no simple method for quantitative characterisation of binding or methods enabling a prolonged study of cell rolling, the ensuing capture by the substratum and subsequent attachment. Furthermore, direct studies of changes in cell morphology, cell growth and biochemical changes cannot be provided easily with these techniques since, determining the kinetics of attachment and resulting morphological changes requires multiple replicated experiments being analysed at different times.
DESCRIPTION OF PRIOR ART
U.S. Pat. No. 5,998,160 (Berens et al) describes a static assay which unfortunately does not have any consideration of cell flow and rolling.
The ability of T-cells circulating in the bloodstream to adhere to the endothelium, switch to a motile phenotype and penetrate through the endothelial layer is recognised as a necessary requirement for the effective in vivo movement or as it is sometimes referred to, trafficking of specific lymphocyte sub-populations. Motility assays are done in combination with attachment assays since following adhesion; cells are expected to switch to the motile phenotype. Motility assays are assessed by estimating the ratio of cells undergoing cytoskeletal rearrangements and the formation of uropods (extension of the trailing tail). One of the major disadvantages of this and the previous adhesion assays is the geometrical design (microscope slides and multiple well chambers), which does not at all resemble the in vivo situation.
The most commonly used cell transmigration assay is a modified “Boyden chamber” assay such as described in U.S. Pat. No. 5,578,492 (Fedun et al). This involves assessing the crossing of a quantity of cells through a microporous membrane under the influence of a chemoattractant, recombinant or cell-derived. Here the diameter of the micropores are less than the diameter of the cells under investigation, such that the cells must deform themselves in order to squeeze through the pores thereby constructing an analogy to the transendothelial migration of cells in physiological circumstances. Once the cells are deposited onto the membrane, the chamber can be incubated for intervals over time at a suitable temperature, usually 37°. Following this, the bottom chamber or opposite side of the top chamber may be analyzed for cells that have squeezed through the microporous membrane.
U.S. Pat. No. 4,912,057 (Guirguis et al), U.S. Pat. No. 5284753 (Goodwin et al), U.S. Pat. No. 5,302,515 (Goodwin et al), U.S. Pat. No. 5,514,555 (Springer et al) and U.S. Pat. No. 5,601,997 (Tchao) are typical examples of these assays. The main disadvantage of the assays described in those specifications is that the biological process of transmigration through the micropores is difficult to observe due to the geometrical configuration of the apparatus involved. The lens of the optically inverted microscope must be able to focus through the lower chamber and the microporous membrane. This obviously leads to difficulties due to optical aberrations. In effect, the study of the cells morphology changes while transmigrating across the membrane and their subsequent cytoskeletal changes reverting to their former state is a process which is difficult to monitor and record due to limitations with current techniques. In addition, although it is possible to alter such an experiments parameters following the initiation of the experiment, such as the introduction of a second chemoattractant, recombinant or cell-derived, at some specified time after commencing the experiment, it is not possible to distinguish separate effects from each said chemoattractant.
In addition to cell biology studies, the pharmaceutical industry has major problems in the drug screening process and while high throughput screening (HTS) has been extremely successful in the elimination of the large majority of unsuitable drugs, it has not progressed beyond that and usually, after a successful HTS assay, a pharmaceutical company may still have 7,000 possible drugs requiring assessment. This requires animal trials and anything that can be done to reduce the amount of animal trials is to be desired. Thus, there is a need for new techniques for drug testing in the pharmaceutical industry. The current proposals are to screen the physiological response of cells to biologically active compounds such as described in U.S. Pat. No. 6,103,479 (Taylor). This again, unfortunately, is still a static test. Since the cells are spatially confined with the drug, there may be a reaction but it may not necessarily take place when the cells are free to flow relative to the drug as in, for example, the microcapillaries of the body. There are other disadvantages such as the transport and subsequent reaction of the drug following
Kashanin Dmitriy
Kelleher Dermot
Shvets Igor
Volkov Yuri
Williams Vivienne
Birch & Stewart Kolasch & Birch, LLP
Gitomer Ralph
The Provost, Fellows and Scholars of the College of the Holy & U
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