Background of the Invention Cancer is the second overall leading cause of death, after ischemic heart disease, in the United States and Western Europe and despite recent advances in its treatment, there is, for most cancer types, no miracle cure on the horizon. Cancer causes approximately 25 % of all deaths. The incidence continues to rise, probably reflecting the increasing average age of the population. The key to survival is early diagnosis and treatment.

Lung cancer has a high incidence and mortality. Early detection programs with conventional methods such as X-ray and sputum cytology have failed to improve mortality.

Lung carcinomas are now considered a genetic disease. Many regions in the genome have been thought to contain candidate genes related to the development of lung cancer.

Compared to deletions in chromosome 3p and 9, which occur in non-malignant appaering cells, mutations in p53 and Kirsten ras (K-ras) genes occur late in the development of lung cancer. Therefore, new approaches that use genetic alterations such as K-ras as potential biomarkers may be beneficial for early detection of lung cancer (Somers V. A. M. C., Thesis Maastricht University (1998), Netherlands).

Point mutations in the human genome play a central role in tumorigenesis (Bishop M. H., Science (1987) 235: 305-311). Several methods for detection of known point mutations have been disclosed, which to a variable extent are time-consuming, technically complex, or hazardous due to the use of radioactive materials. See, e. g., Caskey C. T., Science (1987) 236: 1223-1229; Landegren U., et a/., Science (1988) 242: 229-237; and Sommer S. S., et al., BioTechniques (1992) 12: 82-87.

Holloway B., et al., Nucl. Acids Res. (1993) 21: 3905-3906, disclose an exonuclease-amplification. coupled capture technique (AEXACCT_), which improved detection of PCR product.

Based on this technique, Thunnissen and Murtagh developed a simple, highly specific non-radioactive microtiter plate format for detection of PCR products offering a high sensitivity towards the detection of known point mutations, which was illustrated for the detection of human K-ras oncogene. See, e. g., Thunnissen F. B. J. M., Murtagh Jr., J. J., Somers V. A. M. C., et aL, Lung (1994) 11 (Suppl. 1): 19, U. S. Patent No. 5,518,901, U. S.

Patent No. 5,744,306, Somers V. A. M. C., et al., Nucl. Acids Res. (1994) 22 : 4840-4841, and Somers V. A. M. C., Thesis ibid.

Briefly, this method is based on the following principle : after exonuclease digestion, polymerase chain reaction fragments are determined by simultaneous hybridization with a capture probe and a detection probe complementary to sequences near the 3= end of the antisense fragment. The capture probe bears a biotin residue and the detection probe digoxigenin. After hybridization, the PCR product hybrids are captured in streptavidin-coated microtiter plates and detected with labeled anti-digoxigenin antibody.

For the detection of known point mutations this procedure was extended by using after the capture step the ligation of a mutation-specific capture probe with adjacent detection probe (APoint-EXACCT_).

Essential in the Point-EXACCT technique is that only molecules will be detected by this format which have been hybridized with two probes and subsequently ligated, resulting in a very high degree of specificity. In addition, it has been found that Point-EXACCT requires considerably less time and effort as compared to other techniques used for the detection of known point mutations. The method can be easily automated, permitting rapid screening of tissue banks with multiple probes to individual base substitutions, deletions or additions.

Various attempts have been made to further improve and optimize Point- EXACCT and other point mutation detection methods. Somers V. A. M. C., et al., Biochimica et Biophysica Acta (1998) 1379: 42-52, disclose an improvement of solution hybridization after exonuclease pretreatment of amplification products for fluorescent cycle sequencing and point mutation detection. Digestion of a double-stranded amplification product to single strands by T7 gene 6 exonuclease increases hybridization efficiency and confers increased sensitivity and specificity of detection. The use of single-stranded amplification products gave by far the best results and is therefore almost required, especially in particular cases. A prominent example is that with this approach DNA of one mutated cell can be detected in a DNA background of 15,000 wild type cells. This surprising finding may to a large extent be due to careful moving of the hybridisation fluid across the solid

support. This procedure looks in fact like the roling of the waves over the sand on the beach, and takes care of frequent mixing of molecules to be bound with the substrate on the solid support.

Point mutations in the K-ras oncogene are one of the most common genetic alterations involved in various types of human cancer. In lung cancer, K-ras mutations occur predominantly in codon 12. The frequency of those alterations varies within different histological subtypes. K-ras point mutations are found in approximately 15-56% of the adenocarcinomas and to a lesser extent in other types of non-small cell lung carcinomas (NSCLC). See Somers V. A. M. C., Thesis ibid., and references mentioned therein.

In conclusion, the Point-EXACCT has been designed for analysis of single base substitutions, where the exact place of said substutions in the nucleotide sequence of the gene to be analyzed is known beforehand. Validation of the method for the detection of known point mutations in a large group of patients with NSCLC has confirmed' its high sensitivity. Importantly, with this technique a relatively low amount of target cells is required before a signal is obtained.

Recently,"chip"or"microarray"technology has been developed, which is disclosed in, e. g., U. S. Patent No. 5,445,934. According to this technique analysis of many small spots is performed to facilitate large scale nucleic acid analysis, thus enabling simultaneous analysis of thousands of DNA sequences. This technique is considered an improvement on existing methods, which are largely based on gel-electrophoresis. For a review, see Nature Gen. (1999) 21 Suppl. 1. There are microarrays of different densities.

High density microarrays usually have a density up to about 106 spots per cm2. Low density microarrays contain at least about 5 spots per cm2. The microarray technique allows large scale nucleic acid analysis, but require a large amount of target cells, since the detection mechanism is based on hybridization. Hence there is a need for optimization. When the hybridisation is improved low target samples may be examined with greater efficiency i. e. lower concentrations in shorter time.

In another field of clinical diagnosis and biological research incubation of antibodies is used for various purposes. This was initially performed by adding on top of a microscopic slide a small amount of waterbased test fluid containing the antibody.

Incubation was performed for several minutes usually 45-60 minutes in order to bind the antibody to the epitope present on the solid support. The immunohistochemical or cytochemical staining procedure may take several steps such as blocking, incubation with one antibody or several antibodies, washing and staining steps. These procedures were initially performed by hand. Later, instruments were developed which facilitate the

immunohistochemistry staining procedures. In some of the automatic staining systems the microscope slides are in horizontal position with test fluid on top. Whereas in others the microscope slide is attached to an incubation chamber and the test fluid is present in a thin layer between chamber walls and glass surface. The advantage of the incubation chamber is that the amount of testfluid necessary is much less than for the horizontally incubated slides. The disadvantage of both procedures is that the fluid is not moved across the slide. During incubation diffusion and binding to target takes place. Binding to target is only a matter of a fraction of a second. Therefore, diffusion time is the important parameter determining the efficiency of the incubation. Although the total thickness of the fluid column above the epitopes in the incubation chamber can be reduced to around 80 microns, the antibodies present at a longer distance in the other two directions can take quite a while before they are diffused to the epitopes. In theory this depends strongly on the distance and may take more than 24 hours for larger distances. In daily practice incubation time varies about 30-60 minutes. It is conceivable that not all antibodies in the test fluid are used for binding to their target. Thus incubation of antibodies may be improved if diffusion time is reduced.

Recently, a three dimensional flow trough microarray system was developed using thin membranes with nanometer wide channels. Above and below the plate are reservoirs for test fluid. The channels and surfaces around the channels may be bound with nucleic acids as target for hybridisation. A controlled pump mechanism takes care of transport of fluid through the channels. In this setup the diffusion time is reduced and hybridisation time is only a matter of a few minutes.

The present invention provides both an improvement of the static two- dimensional hybridisation procedure of nucleic acids on solid supports, for example as used in microarrays, and an improvement of the incubation procedure of antibodies to epitopes on solid supports, for example as used in immunohistochemistry, in immunocyto- chemistry and ELISAs.

Summary of the invention In accordance with the present invention two devices are provided for dynamic flow through systems for incubation and/or hybridisation on two-dimensional solid supports. These approaches may also be combined in one assembly.

The first device comprises the following parts (see Figures 1-4): a. a solid support comprising a target for binding;

b. a support holder i) comprising the solid support as one of the walls including an incubation space, ii) two reservoirs one on each side of the incubation space where the first one allows test fluid to be entered into the incubation space, and the second one allowing collection of the test fluid; c. means for providing pressure, for example a pump, allowing transport of the test fluid from one reservoir to the other; d. a control system for moving the fluid from one reservoir along the incubation chamber to the other reservoir and back, allowing repeated exposure of test fluid components to the component present on the solid support.

In a preferred embodiment of the invention, the solid support comprises microscope slides containing nucleic acids as targets for hybridisation to other nucleic acids or binding to proteins suitable for microarray analysis.

In another aspect of the invention, the solid support comprises microscope slides with proteins as targets for antibodies, other proteins or nucleic acids, suitable for microarray analysis.

In still another aspect of the invention, the solid support comprises microscope slides with histologic sections or cytologic material suitable for immunohistochemistry or microscope slides.

In yet another aspect of the invention, a flow through device is provided for rapid immunohistochemistry of a single or a few microscopic slides.

In still a further embodiment the two approaches for dynamic two dimensional hybridisation are combined into one assembly. This assembly conveniently comprises a pump as well as a rotatable disc for moving the test fluid.

In still another aspect of the invention, a flow through device is provided which is suitable for carrying out the hybridisation and/or incubation methods in an automatic fashion with a plurality of support holders, suitable for analysis of multiple supports.

In still another aspect of the invention the reservoir may be very small having hardly any reservoir function left.

In still another aspect of the invention the rotatable disc space may be optionally designed such that hardly any fluid will occupy the disc area.

These and other aspects of the invention will be outlined in more detail in the following description.

Figure 2 depicts a possible vertical option where the solid support is attached to the support holder, leaving variable spaces between surface area of solid support and support holder. Note the reservoirs on both sides of the incubation space.

Figure 3 depicts also the vertical option but now with front view and possible place for pressure regulating channels and fluid inflow (pipetting spot). Optional are sensors for detecting the fluid level.

Figure 4 depicts a horizontal option of the flow through system with similar features as in Figure 3.

Figure 5 depicts schematic front and side views of the holder with a totatable disc in the wall of the holder opposite to the solid support. The arrow points to the cylinder where rotating force can be applied.

Figure 6 depicts front and side view of the holder with two totatable discs in the wall of the holder opposite to the solid support.

Figure 7 depicts the front and side view of the holder with rotatable disc against the wall of the holder opposite the solid support. The disc is rotated e. g. by magnetic forces created by the device outside/on top of the support holder.

Detailed description of the invention The present invention provides a significant improvement of the hybridisation of nucleic acids and incubation of antibodies by reducing the diffusion time. This is obtained by a flow through system.

The terms"incubation of proteins"and"hybridisation of nucleic acids"have similar aspects i. e. diffusion and binding of molecules to their specific targets. With "diffusion", as used herein, similar processes are meant for nucleic acids, proteins and other molecules interacting with a target on the solid support.

The target on the solid support consists of a solid part with attached i. e. immobilized thereon one or more different kinds of molecules, such as nucleic acids, proteins, whole cells, sections of cells or tissues.

The term"incubation chamber"and"hybridisation chamber", as used herein, are synonyms and are meant to indicate the three dimensional space above the target present on the solid support, where the solid support is an integral part of the incubation and/or hybridisation chamber.

The terms"microarray"or"chip"technique or technology, as used herein, are synonyms and are meant to indicate analysis of a plurality of small spots of nucleic acids distributed on a small surface area to facilitate large scale nucleic acid analysis enabling

the simultaneous analysis of thousands of DNA and/or RNA sequences. The terms are likewise applicable to the analysis of peptides or proteins in a similar way.

The term"incubation fluid"is meant to indicate the fluid containing the substrate to be bound on the solid support.

The term"test fluid"is meant to indicate the volume of any fluid component necessary for the experiment test to be carried out with the flow through system.

The terms"immunohistochemistry"and"immunocytochemistry", as used herein, are meant as synonyms, indicating the binding of antibodies to parts of tissues or cells present on the solid support.

It has now surprisingly been found that compared to the static hybridisation system the hybridisation time can be dramatically reduced in the devices for flow through dynamic hybridisation. In one device the test fluid is transported from one reservoir to the other and vice versa (Figures 1-4). In another device or combined with the previous device a rotatable disc is part of one of the walls of the holder (Figures 5-6). This rotating disc takes care of transport of test fluid. In either way the test fluid repeatedly passes along the incubation space with binding target. The dynamic flow procedure reduces the distance of diffusion by repeated short exposures to the target on the solid support. The binding time of the molecules is the restricting factor which is only a matter of a split second. The incubation time for each individual step can be reduced from 30-60 minutes to 15 minutes, preferably 10 or even more preferably 5 minutes or less.

The dynamic flow through system consists of a holder for the solid support.

The flow through system has an integrated device where two different approaches for fluid transport may be combined or one of both approaches. A simple device has the following parts: i) a place where the solid supports fits in and is an integral part of one of the walls of the incubation chambers, ii) a place for input of test fluid, iii) two reservoirs, one on each side of the incubation chamber, iv) a pump related to one incubation chamber, v) sensors close to the inlet outlet side of incubation chamber, vi) output for overflow/waste of fluids.

In another simple device,, contains in the holder one or more rotatable disc (s)' Still another device combines the two approaches: thus fluid transport is performed by the combination of pressure changes and by rotatable disc (s).

In one embodiment of the invention, a vertical slide holder is used comprising a small fluid-containing incubation >chamber-, where the slide is in vertical position in the holder. One of the broad vertical walls of the incubation chamber consists of the solid support with the microarrays (3x1 inches, about 5x2.5 cm). The distance between solid

support wall of teh incubation chamber and the opposite parallel wall may vary from several hundred microns to one tenth of a micron. The minimal distance is dependent of the size of the objects on the solid support. A distance of 80 microns is sufficient for arrays of nucleic acids, proteins, individual cells and tissue sections. But a distance of 1 or 0,1 micron, which is not working for cells and most tissue sections, is sufficient for analysis of proteins and nucleic acids.

Test fluid can be added on top of the upper wedge shaped and wider part of the incubation chamber and the incubation space is filled with test fluid by capillary and gravity force. Excess fluid on the upper level will in due time flow through the incubation space by gravity. The incubation space will remain filled with fluid due to the capillary force in a static fashion. The invention in effect is the possibility of two dimensional dynamic incubation, which is achieved by pressure on the level of one of the reservoirs (e. g-1), containing excess test fluid. The pressure will speed up the passage of fluid from that reservoir along the incubation chamber to the other reservoir. The sensor detects signs of fluid level passage and is used for the stopping of the overpressure. The other reservoir now contains the excess fluid. Reduction of pressure (creating underpressure) on reservoir 1 leads to transport of the fluid back along the incubation chamber to reservoir 1.

This procedure can be repeated untill no improvement in signal detection is achieved.

In another embodiment of the invention, a slide holder contains the slide not in vertical, but in another position, e. g. skewed or horizontal.

The invention can be performed without sensor (s), using e. g. visual inspection as a way of controlling the fluid transport.

In still another part of the invention the pressure channels can be located on the other reservoir or alternatively on both reservoirs.

The size of the incubation chamber can vary from the complete size of the solid support, preferably the size of a microscope glass slide or more preferably the size of a paraffin embedded tissue block or even smaller i. e. the size of cross section of one or a few tissue biopsies.

The minimal size of the reservoir is related to the size of the incubation chamber i. e. a larger incubation chamber requires a larger reservoir. The whole flow trough system can be larger than the size of a microscope slide, for larger supports the size of the flow trough system can be adjusted.

In another embodiment the wall of the holder contains two or three rotatable discs.

The size of the disc may depend on the size of the binding area on the solid support. For example, in microarray analysis and immunohistochemistry on small biopsie specimen on area of 1x1 cm square or less may be sufficient. Whereas for larger sections the area may be 5x2 cm square or even larger.

The surface of the rotatable disc may be flat or have some channels or other irregularities on the surface, facilitating mixing of test fluid and reduction of the diffusion distance.

The present invention of dynamic incubation and/or hybridisation has the advantage that at least for most specimen in routine pathology a reduction of the amount of incubation and other fluids can be achieved. This effect may even be more impressive for smaller incubation chambers, leading to lower material costs in the test/experiment. In addition to a reduction in amount of test fluid the concentration can be reduced of primary antibodies in immunohistochemistry, since a higher chance of binding may be present due to repeated exposure from the components to be bound.

Another important aspect of the invention may be that for samples with a low amount of binding targets in a mixture of other (non-binding) components the chance of binding is dramatically increased. The dynamic incubation not only results in reduction of material costs as mentioned above, but also improves the efficiency low target specimen to a large extent. This is useful e. g. in the field of research for early detection.

! In another embodiment the device may be used for immuno linked assays other than immunohistochemistry or immunocytochemistry well known to a person skilled in the art.

In either device the temperature may be raised as part of optimisation of the hybridisation or incubation conditions.

In either device it is important to keep the fluid flow rate below the speed that may possibly damage the binding target on the solid support.

In another part of the invention the dynamic flow through system is designed to examine the binding efficiency during flow with conventional fluorescence microscopy and other modes, such as laser scanning microscopy, which will be evident to a person skilled in the art.

The detection mode after hybridisation/incubation on the solid support may vary but the invention can be suitably carried out with absorption microscopy and other modes, such as fluoresence and laser scanning microscopy, which will be evident to a person skilled in the art.

The label selected for detection may vary depending on the purpose. As indicated above, this may be a label for fluorescence or chemiluminescence optical systems. Other detection systems may be adapted to the purpose, e. g. IR microscopy, atomic force microscopy, electrical conductance, image plate transfer, and interference microscopy (e. g. Jamin-Lebedief). Such variations which are entirely clear to the man skilled in the art, are all encompassed within the scope of the present invention.

Another embodiment of the invention is an instrument having the parallel setup of an array of flow through systems. This allows several solid supports to be analysed at the same time. This system can be automated.

Summarizing, the new two dimensional flow through system has the advantage of more efficient hybridisation and/or incubation due to a reduction of diffusion time and test fluids compared to present two dimensional systems, leading to reduction of test time, material costs, better results with low target specimen.

General applications using the two dimensional flow through system invention: -Immunohistochemistry - immunocytochemistry -microarray analysis -protein analysis Specific applications -daily immunohistopathology for diagnostic or research purpose -daily immunocytopathology for diagnostic or research purpose -daily histochemical analysis in pathology -detection of abnormal ODNA/RNA in human specimen - (early) detection of abnormal DNA/RNA in body fluids, such as blood, urine, sputum, bile, lavage fluids, cerebrospinal fluid, stool - (early) detection from cancer cells in blood/bone marrow -prediction of treatment/chemosensitivity of tumor cells -detection of specific microorganisms e. g. for clinical and food applications, such as human papilloma virus, legionella, tuberculosis, etc.

-comparison of levels of gene expression.

-detection of parent-child relationship

-comparison of levels of gene expression.

-detection of parent-child relationship -analysis of genetic predisposition for specific diseases, e. g. lung cancer, COPD, atherosclerosis.

Although the present invention is herein described in certain typical embodiments, it will be understood that variations may be made without departing from the spirit of the invention. Such variations are all encompassed within the scope of the present invention.