WO2014154336A1

WO2014154336A1 - Microtiter plate-based microarray

Info

Publication number: WO2014154336A1
Application number: PCT/EP2014/000757
Authority: WO
Inventors: Andreas IFFLAND; Hartmut HAMPL
Original assignee: Iffmedic Gmbh
Priority date: 2013-03-26
Filing date: 2014-03-20
Publication date: 2014-10-02

Abstract

The technology pertains to microtiter plate-based microarrays suitable for simultaneous detection of a plurality of different selected targets in a biological sample obtained from a subject. In particular, in-vitro methods are disclosed for simultaneous detection of a plurality of different selected targets in a biological sample obtained from a subject, in particular in a blood sample from a human in a blood bank setting.

Description

MICROTITER PLATE -BASED MICROARRAY

FIELD OF THE INVENTION

The present disclosure pertains to microtiter plate-based microarrays suitable for simultaneous detection of a plurality of different selected targets in a biological sample obtained from a subject. In particular, in-vitro methods are disclosed for simultaneous detection of a plurality of different selected targets in a biological sample obtained from a subject, in particular in a blood sample from a human in a blood bank setting.

BACKGROUND OF THE INVENTION

Blood banks are committed to ensuring a safe blood supply for everyone who may need transfusions. An important step in ensuring safety is the screening of donated blood for infectious diseases. Today, up to nine tests for infectious diseases are conducted on each unit of donated blood. Tests for hepatitis B and syphilis were in place before 1985. Since then, tests for human immunodeficiency virus (HIV-1 and HIV-2), human T-lymphotropic virus (HTLV-I and -II) and the hepatitis C virus (HCV) have been added. Depending on the specific epidemiological situation, the following tests are performed on each unit of blood:

Hepatitis B Surface Antigen (HBsAg)

The hepatitis B virus (HBV), which replicates in liver cells, has an inner core and an outer envelope (the surface). The HBsAg test is directed against HBV-specific proteins (antigens) of the outer envelope, identifying an individual infected with the hepatitis B virus. Hepatitis B can cause inflammation of the liver, and in the earliest stage of the disease, infected people may not feel ill or even have yellow discoloration of the skin or eyes, a condition known as jaundice and thus, infected donors may be eligible for blood donation. Fortunately, most patients recover completely and test negative for HBsAg within a few months after the illness. A small percentage of people become chronic carriers of the virus, and in these cases, the test may remain positive for years. Chronically infected people can develop severe liver disease as time passes, and need to be followed carefully by an experienced doctor.

CONFIRMATION COPY Antibodies to the Hepatitis B Core (Anti-HBc)

The anti-HBc test detects an antibody to the hepatitis B virus that is produced during and after infection and may persist life-long. If an individual has a positive anti-HBc test> but the HBsAg test is negative, it may indicate that the person has recovered from the disease. However, chronic occult infection may persist. This kind of test is prone to false positives, and needs to be confirmed by similar tests from alternative providers. (Note: This antibody is not produced following vaccination against hepatitis B. Hepatitis B vaccination, by itself, will rarely cause the HBsAg test to be positive for a few days after the shots.)

Antibodies to the Hepatitis C Virus (Anti-HCV)

This test is used to screen donors for the hepatitis C virus (HCV). HCV causes inflammation of the liver, and up to 80 percent of those exposed to the virus develop chronic infection. Eventually, up to 20 percent of people with HCV may develop cirrhosis of the liver or other severe liver diseases. As in other forms of hepatitis, individuals may be infected with the virus, but may not realize they are carriers since they do not have any symptoms. Because of the risk of serious illness, people with HCV need to be followed closely by a physician with experience evaluating this infection.

Antibodies to the Human Immunodeficiency Virus, Types 1 and 2 (Anti-HIV-1, -2)

This test is designed to detect antibodies directed against antigens of the HIV-1, HIV-2 and HIV-1 subtype 0 and N viruses. HIV-1 is much more common around the world, while HIV-2 is mainly prevalent in Western Africa. Donors are tested for both viruses because both are transmitted by infected blood, and a few cases of HIV-2 have been identified in US residents as well. Both of these viruses can cause acquired immunodeficiency syndrome, or AIDS.

Antibodies to Human T-Lymphotropic Virus, Types I and II (Anti-HTLV-I, -II)

This test screens for antibodies directed against portions of the HTLV-I and HTLV-II viruses. HTLV-I is more common in Japan and the Caribbean. The infection can persist for a lifetime, but rarely causes major illnesses in most people who are infected. In rare instances, the virus may, after many years of infection, cause nervous system disease or an unusual type of leukemia. In western countries HTLV-II infections are usually associated with intravenous drug usage, especially among people who share needles or syringes. Disease associations with HTLV-II have been hard to confirm.

Syphilis

This test is done to detect evidence of infection with the spirochete Treponema pallidum that causes syphilis. Blood centers began testing for this shortly after World War II, when syphilis rates in the general population were much higher. The risk of transmitting syphilis through a blood transfusion is exceedingly small (no cases have been recognized in Germany for many years) because the infection is very rare in blood donors, and because the spirochete is fragile and unlikely to survive blood storage conditions.

All of the above tests are referred to as screening tests, and are designed to detect as many infections as possible. Because these tests are so sensitive, some donors may have a false positive result, even when the donor was never exposed to the particular infection. In order to sort out true infections from false positive test results, screening tests that are reactive may be followed up with more specific tests called confirmatory tests. Thus, confirmatory tests help determine whether a donor is truly infected. If the test result from a donated unit of blood is abnormal for any of these disease markers, the unit is discarded and the donor is notified.

Many of the above mentioned common blood screening tests and methods are unspecific, slow or imprecise. For example, a virus infection can be detected with a number of conventional approaches, such as Enzyme-Linked Immunosorbent Assay (ELISA), Enzyme-linked immunoassay (EIA), which typically detect the presence of a viral antigen or an antibody to a viral antigen. The limitations of conventional assays include low throughput, low automation, and consumption of large amount of samples and reagents, and high assay cost.

Therefore, it is an object of the present disclosure, to provide improved test devices and methods for a faster and more exact screening of infectious diseases in biological samples for transfusion or transplantation.

SUMMARY OF THE DISCLOSURE

The present disclosure relates to microtiter plate-based microarrays suitable for simultaneous detection of a plurality of different selected targets in a biological sample obtained from a subject comprising: a) a plurality of separated subarrays in a microtiter well, wherein each subarray comprises a set of probes capable of selectively capturing a plurality of target ligands assign to one selected target, wherein b) each probe is coated on a predetermined location on the surface of the microtiter well and interacts with one specific target ligand associated with the selected target, and wherein the plurality of the probes in a subarray interact with different target ligands associated with one selected target.

In a further aspect, the present disclosure relates to in-vitro methods for simultaneous detection of a plurality of different selected targets in a biological sample obtained from a subject, in particular in a blood sample from a human in a blood bank setting, the method comprising: i) Applying a biological sample obtained from a subject to a microtiter well of a microtiter plate- based microarray according to the present disclosure,

ii) Incubating the sample in the microtiter well for sufficient time at a suitable temperature for binding reaction between the target ligands and the probes;

iii) Detecting the interactions between the target ligands and the probes; and

iv) Analyzing interactions to determine the presence of a selected target in the sample.

A blood bank according to the present disclosure may be a cache or bank of blood or blood components, gathered as a result of blood donation, stored and preserved for later use in blood transfusion. The term "blood bank" typically refers to a division of a hospital laboratory where the storage of blood product occurs and where proper testing is performed to reduce the risk of transfusion related events. Blood bank settings according to the present disclosure also includes plasmapheres is centers and transplantation diagnostic.

Furthermore, aspects of the present disclosure relates to multi-well microtiter plates for the use of simultaneous detection of a plurality of different selected targets in a biological sample obtained from a plurality of subjects comprising: a) a plurality of microtiter wells, wherein a plurality or each of the microtiter wells comprise a microarray according to the present disclosure, and b) the set of probes in the separate subarrays are coated in each well on the same predetermined location, whereby the wells of the microtiter plate have the identical segmented coating for different selected targets and within the segmented subarrays the different probes capable of selectively capturing a selected target ligands assigned to one selected target is present in identical locations in the subarray, and c) a plurality of each of the microtiter wells comprises the identical microarray, and d) at least one microtiter well each is employed as a positive or negative control for the verification of the functionality of the assay to detect the selected targets.

A further aspect pertains to uses of the microtiter plate-based microarrays, the microtiter plate- based assays and/or the multi-well microtiter plates according to the present disclosure for simultaneous screening of a blood sample obtained from a subject for blood transfusion for the presence of a plurality of different selected targets associated with an infectious disease, wherein a) a method according to the present disclosure is performed, and b) the results from analyzing the interactions to determine the presence of the selected target is used as a first screen to determine the presence of the specific target and at the same time as a confirmation assay to confirm the screened presence of the specific target in the same biological sample.

A further aspect relates to processes for manufacturing microtiter plate-based microarrays according to the present disclosure or to the multi-well microtiter plate-based microarray according to the present disclosure, comprising the steps of: a) Spotting each probe or set of probes on predetermined locations on the surface of a membrane, and

b) Placing the spotted membrane in the microtiter well or wells.

BRIEF DESCRIPTION OF THE DRAWING

Figure 1 is a picture of microtiter plate wells each containing membranes spotted with multiparametric probe arrays

Figure 2 is a schematic drawing of spotting of probes on membrane in micro titer plate well. Figure 3 shows a spot verification with Sypro Ruby staining.

Figure 4 shows a spot verification with fluorescence labelled anti-human detection antibodies.

Figure 5 is a diagram showing the detection limit of HIV p-24 by immobilized human anti-p24 antibody on PamChip^®.

Figure 6 is a diagram showing the detection limit of HIV- p24 by proteinA/G immobilized human anti- p24 antibody on PamChip^®. DESCRIPTION OF THE DISCLOSURE

The object of the present disclosure is to provide novel and improved microarrays suitable for simultaneous detection of a plurality of different selected targets in a biological sample obtained from a subject and simultaneous confirmation of reactive samples as positive, negative or indeterminate. In particular, it is an object of the present disclosure to provide an in-vitro method for simultaneous detection of a plurality of different selected targets in a biological sample obtained from a subject, in particular in a blood sample from a human in a blood bank setting.

In an advantageous embodiment, the present disclosure provides multiplexed approaches for detection of virus infection using a microtiter plate-based microarray. One aspect of the present disclosure relates to methods for high-throughput detection of viral infection using multiplexing approaches in microtiter plate-based microarrays. The microarrays and methods according to the present disclosure can be used for screening of donated blood, tissue or organ and/or for infectious diseases. For proof of principle see Figures 3-6. It could be shown that antibodies to HIV-1 p24 antigen could be spotted to a membrane in a microtiter plate well and that p24 antigen could be detected with high sensitivity. Further improvement of the sensitivity by a factor of 100 is feasible.

In one embodiment, the present disclosure provides a microarray system capable of simultaneous detection of a plurality of target ligands in a plurality of test samples. The microarray system comprises anti-ligands (probes) immobilized on a solid support in the form of microtiter plate-based microarrays and membrane-based microarrays. The anti-ligands (probes) are capable of specific binding to target ligands, such as viral antigens and anti-viral antibodies.

In an advantageous embodiment of the present disclosure, specific probes (e.g. antigens, antibodies) for a defined target (HCV, HBV, HIV, etc.) are coated to a predetermined location on the surface of a microtiter well. Within this predetermined location (subarray or segment) of the microtiter well for a target, selected antigens of this one selected target (i.e HIV) are coated as defined antigen spots (i.e. p24,gp41, gpl20,...), each spot representing one pure antigen, and the same and identical location of this antigen spot will be found in all different microtiter wells of the assay, that are intended to be used as positive or negative control or as well for testing unknown samples. Different targets (HCV, HBV, HIV, etc.) have different segments of the surface of the microtiter well and that segment will be always in the very same location as in all the other 96 wells of the microtiter plate, as well. In summary; every single of the 96 wells of the microtiter plate of this assay has the identical segmented coating for different target and within the segment the different antigens of the target in identical locations in the segment. Antigens of a target are in the conventional ELISA (enzyme linked immuno sorbent assay) randomly coated to the surface of all microtiter wells. No preselected areas within the surface of a microtiter well are selected for defined targets or antigens of the targets. If a reactivity of a tested sample happens with the coated antigens, a change in colour of the liquid sample in the well is induced, d ue to a conjugated secondary antibody and additional reagents. The change in colour is determined by a reading system, reading through the well of the microtiter plate. In our method, different targets (HCV, H BV, H IV, etc.) have known segments of the known surface of the microtiter well and within the segments there are on defined spots respective antigens of the target coated to the surface of the microtiter well.

Surprisingly, the inventors found that with the microarrays and methods according to the present disclosure a reactivity of the tested sample happens with coated antigen spots and a change in colour in this antigen spot is induced. The supernatant of the well (the sample) remains unstained. As all targets and of the targets all antigens are coated to a defined segment and within the segment coated to defined spots within the segment, only the reactive spots show a detectable change. This change can be detected via a camera monitoring the whole well and registering the changes and the location of the altered spot. Because of the detection system and the defined location of the antigen spots of the targets it is possible to determine two results for one target with the one assay in a single microtiter well, that means two results for a target with one sample.

Firstly, the sample will be defined as reactive with one or more segments, each segment covering one target (i.e. HCV, H BV, HIV, etc.) and within one segment (i.e. HIV-segment) there are only respective HIV-antigen spots in their defined location. If one or more of the antigen spots in the H IV- segment are reactive, this sample is to be called reactive. It is the same situation, as you will find in a conventional HIV-ELISA after reading the colour change in the liquid sample in the microtiter well after reactivity of the well with the coated antigens. In a conventional H IV-ELISA microtiter well no additional result to determining positive, negative or indeterminate is possible.

In the microarrays and methods according to the present disclosure, a result is generated to determine the sam ple used in the microtiter well as either positive, negative or indeterminate for as many targets as segments are defined for the respective targets in the microtiter well.

In addition, it is possible with the microarrays and methods according to the present disclosure to not only see the reactivity, but with the same result a confirmation of a reactive sample can be shown simultaneously in the identical microtiter well. In other words, one well can show up to two results (reactivity and confirmation). This is achieved with the detection of reactivity of single antigen spots within a segment or target. For example in a possible H IV-Segment, if the system determines two or more antigen spots as reactive, it is only a question of definition to call the sample generating the reactivity with different antigen spots as sufficient in number and correlating to the type of antigen as positive, negative or indeterminate. This is done i.e. for the conventional HIV-ELISA subsequent with an additional test for confirmation, i.e. by applying the Western-Blot-Technology.

The microarrays and methods according to the present disclosure are able to determine by applying a single sample in one microtiter well as positive, negative or indeterminate for one or more targets/viruses in the first place and in addition by further interpretation of the whole data generated by the method, even confirming the positivity, negativity or indeterminate status for the respective target/segment of initial reactivity. Therefore, no confirmatory tests for each target screening are needed any more. Only for confirmation of the identity of the sample to a defined i.e. blood donor, a second sample from the donor is required and needs to be investigated again in the described method.

In an advantageous embodiment, the disclosure pertains to microtiter plate-based microarrays suitable for simultaneous detection of a plurality of different selected targets in a biological sample obtained from a subject comprising: a) a plurality of separated subarrays in a microtiter well, wherein each subarray comprises a set of probes capable of selectively capturing a plurality of target ligands assign to one selected target, wherein b) each probe is coated on a predetermined location on the surface of the microtiter well and interacts with one specific target ligand associated with the selected target, and wherein the plurality of the probes in a subarray interact with different target ligands associated with one selected target.

In an advantageous embodiment, each subarray comprises at least two probes interacting with the same target ligand to increase the specificity of the array.

A "microarray" as used herein generally refers to an array in which detection requires the use of microscopic detection to detect complexes formed with agents on the substrate. A "location" or "spot" on an array refers to a localized area on the array surface that includes agents, each defined so that it can be distinguished from adjacent locations (e. g., being positioned on the overa ll array, or having some detectable characteristic, that allows the location to be distinguished from other locations). Typically, each location includes a single type of agent but this is not required. The location can have any convenient shape (e.g., circular, rectangular, elliptical or wedge-shaped). The size or area of a location can vary significantly. In some embodiments, the area of the location is less than 1 cm, in other instances less than 1 mm, in still other instances less than 0.5 mm, in yet still other instances less than 10,000 urn, or less than 100 urn.

A "microtiter plate-based microarray" as used herein, refers to a microarray fabricated on the surface of a well of a microtiter plate, such as a 96-well microplate.

In another advantageous embodiment, the probes each probe or set of probes can be coated on a membrane and the coated membrane is placed in the microtiter well or wells. In other words, the microarray can be fabricated on a supporting membrane, such as a nitrocellulose membrane and then placed in the microtiter well.

An "array" broadly refers to an arrangement of agents (e. g., proteins, antibodies) in positionally distinct locations on a substrate. In some instances the agents on the array are spatially encoded such that the identity of an agent can be determined from its location on the array. A„subarray" refers to an array for detection of a selected target, for example for one specific virus comprising a set of probes capable of selectively capturing a plurality of target ligands assign to one selected target.

The microtiter plate-based microarrays according to the present disclosure is suitable for simultaneous detection of a plurality of different selected targets, wherein the targets may be associated with an infectious disease caused by a virus, a viroid, a prion, a protozoa, a fungus or a bacterium. In an advantageous embodiment, the target is a virus selected from the group consisting of Hepatitis B Virus, Hepatitis C Virus, Human Immunodeficiency Virus and Human T-Lymphotropic Virus. In another embodiment, the target may be a bacterium like caused by the spirochete bacterium Treponema pallidum.

According to the disclosure, the selected targets are contained in a biological sample obtained from a subject. Examples for typical biological samples are a blood, cerebrospinal fluid, cell culture, urine, sweat, buccal swab, tissue biopsy, or aspiration sample. In an advantageous embodiment, the biological sample is a blood sample like plasma or serum or components thereof selected from the group consisting of red blood cells, white blood cells, clotting factors, and platelets. Preferably, the blood sample is obtained from a subject for blood transfusion, wherein the subject is a mammalian, in particular a human. In one embodiment, the microtiter plate-based microarray comprises a plurality of separated subarrays in a microtiter well, wherein each subarray comprises a set of probes capable of selectively capturing a plurality of target ligands assign to one selected target.

As used herein, the term "ligand" refers to one member of a ligand/probe binding pair. The ligand may be, for example an antigen or antibody in an antigen/antibody binding pair.

As used herein, the term "probe" refers to the opposite member of a ligand/probe binding pair. The probe may be the other of an antigen or antibody in an antigen/antibody binding pair. In particular, the probes in the subarrays are polypeptides like antibodies, antigens, enzymes, zinc-finger binding proteins, minor-groove binders, transcriptional factors, combinations thereof, or chimeras thereof.

In particular, the target ligands are target antigens or anti-target antibodies assigned to the selected target like, wherein the target antigens are viral antigens and the anti-target antibodies are anti-viral antibodies.

In general the term "antibody" includes, but is not limited to, polyclonal antibodies, monoclonal antibodies, single chain antibodies, humanized antibodies, minibodies, dibodies, tribodies as well as antibody fragments, such as Fab', Fab, F(ab')2, single domain antibodies and any mixture thereof. Antibodies and antibody fragments can be produced by techniques well known in the art which include those described in Harlow and Lane (Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. (1989) ) and Kohler et al. , (Nature 256: 495-97 (1975) ) and U. S. Patent Nos. 5,545,806,5,569,825 and 5,625,126, incorporated herein by reference. Correspondingly, antibodies, as defined herein, also include single chain antibodies (ScFv), comprising linked VH and VL domains and which retain the conformation and specific binding activity of the native idiotype of the antibody. Such single chain antibodies are well known in the art and can be produced by standard methods.

An "antigen" as used herein, includes substances that upon administration to a vertebrate are capable of eliciting an immune response, thereby stimulating the production and release of antibodies that bind specifically to the antigen. Antigen, as defined herein, includes molecules and/or moieties that are bound specifically by an antibody to form an antigen/antibody complex. In accordance with the invention, antigens may be, but are not limited to, peptides, polypeptides, proteins, nucleic acids, DNA, RNA, saccharides, combinations thereof, fractions thereof, or mimetics thereof. A "viral antigen" as used herein, include an antigen derived from a virus, and any antigenic substance that is capable of eliciting an immune response to an antigen derived from a virus, thereby stimulating the production and release of antibodies that bind specifically to the antigen derived from a virus.

In an advantageous embodiment, each probe is coated on a solid support at a predetermined location on the surface of the microtiter well. A "solid support, " as used herein, means any support capable of binding an antigen or an antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody. Preferred supports include nitrocellulose membrane, nitrocellulose-coated slides, 96-well microtiter plates, and polystyrene or carboxyl beads. Those skilled in the art would understand that many other carriers are suitable for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation. In a advantageous embodiment, the solid support may be various types of microtiter plates made of polystyrene with or without coating, polymer surfaces, porous filters and/or the probes or set of probes are coated on a membrane in the microtiter well like membranes made from polyvinylidene fluoride (PVDF), nitrocellulose, nylon and/or other suitable materials, such as Biotrans (ICN), Zeta- probe (Bio-Rad), Colony/Plaque Screen (NEN), Hybond-N+ (Amersham), Magnacharge (MSI), Magnagraph (MSI) and Hybond ECL (Amersham).

Other examples of solid support material include silicon, silica, quartz, glass, controlled pore glass, carbon, alumina, titania, tantalum oxide, germanium, silicon nitride, zeolites, and gallium arsenide. Many metals such as gold, platinum, aluminum, copper, titanium, and their alloys are also options for solid support material of the array. In addition, many ceramics and polymers may also be used as solid support material. Polymers which may be used as solid support material include, but are not limited to, the following: polystyrene; poly(tetra)fluoroethylene (PTFE) ; polyvinylidenedifluoride; polycarbonate; polymethylmethacrylate; polyvinylethylene; polyethyleneimine; poly (etheretherjketone; (POM) ; polyvinylphenol; polylactides; polymethacrylimide (PMI); polyalkenesulfone (PAS) ; polypropylethylene, polyethylene; polyhydroxyethylmethacrylate (HEMA) ; polydimethylsiloxane; polyacrylamide; polyimide; and block-copolymers. Preferred solid support material for the array include silicon, silica, glass, and polymers. The solid support material on which the binding molecules reside may also be a combination of any of the aforementioned substrate materials. Geometry of solid support includes 2-dimensional planar or flat surface materials, 3-dimensional flow-through silicon and glass wafers, as well as porous filters or hydrogels. All those solid support can be used with or without additional supportive materials for multiplexed detection of viral infection. The solid support may be fabricated inside a cartridge for applications and operations in manual, semi-automatic or automatic modes.

In an advantageous embodiment, the probes or set of probes are arranged and coated on a planar membrane in a microtiter well.

In a further embodiment, a probe interacting with one specific target ligand is a mixture of antigens with different lengths in the amino acid sequence.

The probes or set of probes may be immobilized on the solid support through covalent or non- covalent interactions between various functional groups. A detailed discussion on immobilization technologies can be found in, for example, U.S. Patent No. 6,329,209 and 6,305,418, which are hereby incorporated by reference in their entirety.

An array of the present disclosure may optionally further comprise a coating between the solid support material and the binding molecules (i. e., the probe). This coating may either be formed on the solid support material or applied to the solid support material. The solid support material can be modified with a coating by using thin-film technology based, for instance, on physical vapor deposition (PVD), plasma-enhanced chemical vapor deposition (PECVD), or thermal processing. Alternatively, plasma exposure can be used to directly activate or alter the solid support material and create a coating. For instance, plasma etch procedures can be used to oxidize a polymeric surface (for example, polystyrene or polyethylene to expose polar functionalities such as hydroxyls, carboxylic acids, aldehydes and the like) which then acts as a coating.

Microspotting encompasses deposition technologies that enable automated microarray production by printing small quantities of pre-made biochemical substances onto solid surfaces. Printing is accomplished by direct surface contact between the surface of a solid support and a delivery mechanism, such as a pin or a capillary. Robotic control systems and multiplexed printheads allow automated microarray fabrication. The anti-ligands can also be deposited on the solid support by non-contact dispensing with piezo-electric ceramics or microsolenoid valves. Other technologies for microarray production include photolithography and ink jet technologies. Methods for fabricating microarrays are described in, for example, U. S. Patent No. 5,807,522 to Brown et al. , U. S. Patent No. 6,110,426 to Shalan et al. , and U.S. Patent No. 6,139,831 to Shivashankar et al., which are hereby incorporated by reference in their entirety.

Depending on the application, a single microarray may be immobilized with antibodies to, or antigens from a plurality of selected targets, and multiple target-specific antibodies/antigens may be used for each target. The probes in the subarray for each selected target may each represent a different species of protein. Different antibodies to, or antigens from, the same target are spotted individually. As is well-known to one skilled in the art, positive and negative controls are also included in each microarray. Examples for a positive control to verify the presence of the biological sample in the microtiter well comprising the microarray is spotted human IgG antibodies or antigens as probes, for example in a separate subarray. Concentrations of antibodies/antigens deposited on microarrays will be optimized in terms of dynamic ranges, assay sensitivity and specificity. Immobilization efficiency of proteins on a solid support depends on various factors including concentration of the proteins.

In a further aspect the present disclosure pertains to microtiter plate-based assays comprising a plurality of microtiter wells, wherein a plurality or each of the microtiter wells comprise a microarray according to the present disclosure.

In an advantageous embodiment the plurality or each of the microtiter wells comprise the identical microarray, in particular that each probe is coated on the same kind of solid support at the same predetermined location on the surface of the respective microtiter well for interacting with one specific target ligand associated with the selected target, and wherein the plurality of the probes in a subarray interact with different target ligands associated with one selected target.

Furthermore, the assay may employ at least one microtiter well each as a positive or negative control for the verification of the functionality of the assay to detect the selected targets. For example, a positive control may be a biological sample from a patient known to be positive for a selected target, in particular for having an infectious disease. Furthermore, a positive control may be also a mixture of biological samples from different patients known to be positive for different selected target. The microtiter well being employed as positive control comprise the same microarray according to the present disclosure as the other microarrays of the plurality or of each of the other microtiter wells. A further advantageous embodiment according to the present disclosure pertains to multi-well microtiter plate-based microarrays suitable for simultaneous detection of a plurality of different selected targets in a biological sample obtained from a plurality of subjects comprising: a) a plurality of microtiter plate-based microarrays according to the present disclosure and microtiter wells being employed as positive or negative controls, wherein b) the set of probes in the separate subarrays are coated in each well on the same predetermined location, whereby the wells of the microtiter plate has the identical segmented coating for different selected targets and within the segmented subarrays the different probes capable of selectively capturing a selected target ligands assign to one selected target is present in identical locations in the subarray.

In some embodiments, the mentioned multi-well microtiter plate may comprise: a) a plurality of microtiter wells, wherein a plurality or each of the microtiter wells comprise a microarray according to the present disclosure, and

b) the set of probes in the separate subarrays are coated in each well on the same predetermined location, whereby the wells of the microtiter plate has the identical segmented coating for different selected targets and within the segmented subarrays the different probes capable of selectively capturing a selected target ligands assign to one selected target is present in identical locations in the subarray, and

c) a plurality or each of the microtiter wells comprises the identical microarray, and

d) at least one microtiter well each is employed as a positive and negative control for the verification of the functionality of the assay to detect the selected targets.

A further advantageous embodiment according to the present disclosure pertains to in-vitro methods for simultaneous detection of a plurality of different selected targets in a biological sample obtained from a subject, in particular in a blood sample from a human in a blood bank setting, the method comprising: i) Applying a biological sample obtained from a subject to a microtiter well of a microtiter plate- based microarray according to the present disclosure,

iii) Detecting the interactions between the target ligands and the probes; and iv) Analyzing interactions to determine the presence of a selected target in the sample.

Depending on the nature of the detection technique utilized, labeled target protein or various labeled detection reagents (e.g., labeled antibodies or labeled packages) can be used to detect the formation of a complex between a target protein and package/antibody reagent. A variety of different labels can be utilized in these detection schemes. The proteins, antibodies or packages can be labeled with any of a variety of different types of labels, provided the label does not interfere with the formation of a complex between a package/antibody reagent and a target protein and can generates a detectable signal once such a complex is formed. Suitable labels include, but are not limited to, radiolabels, chemiluminescence labels, chromophores, electron dense agents, NMR spin labels, a chemical tag suitable for detection in a mass spectrometer, agents detectable by infrared spectroscopy or NMR spectroscopy, and enzyme substrates or cofactors for example. Radiolabels, particularly for spatially resolved proteins, can be detected using phosphor imagers and photochemical techniques.

Certain methods utilize fluorophores since various commercial detectors for detecting fluorescence from labeled proteins are available. A variety of fluorescent molecules can be used as labels including, for example, fluorescein and fluorescein naphthylamine derivatives, benzamidizoles, ethidiums, propidiums, anthracyclines, mith ramycins, acridines, actinomycins, merocyanines, coumarins, pyrenes, chrysenes, stilbenes, anthracenes, naphthalenes, salicyclic acids, benz-2-oxa-l- diazoles (also called benzofurazans), fluorescamines and Bodipy dyes.

While fluorescence labeling has been widely used in microarray technologies owing to the benefits from stable and inexpensive fluorophores, high sensitivity and environmental safety, fluorescence detection requires expensive instrumentation. Moreover, the auto-fluorescence on chip surface, which causes higher background and lower signal to noise ratio, also limits the application of fluorescence in microarray technologies. Novel detection strategies such as colorimetric imaging provide an alternative detection method on multiplexed microarray-based systems. One of the attractive features of colorimetric detection is the low background. In fluorescence imaging, an excitation source is needed and certain amount of signals is generated in areas of the microarray where the fluorophore is not present. In colorimetric imaging, however, photons are generated only where the reactants such as labeling enzymes are present. Consequently, nonspecific radiation is significantly reduced. Moreover, colorimetric imaging does not have the excitation source-related problems, such as warm-up and drift of the light source, and interference from light scattering.

Another attractive feature of colorimetric detection is its low cost (a CCD- based colorimetric scanner with associated software for image capture and analysis costs at a fraction of a comparable fluorescent scanning equipment). Complementing fluorescent scanning technology, the colorimetric system allows horseradish peroxidase (HRP), alkaline phosphatase (AP), gold-silver developers, and any other labeling system that produces a pigmented reaction precipitate.

Typically, a multiplexed assay contains multiple incubation steps, including incubation with the samples and incubation with various reagents (e.g., primary antibodies, secondary antibodies, reporting reagents, etc.). Repeated washes are also needed between the incubation steps. The multiplexed assays of the present disclosure may be performed in a fast assay mode that requires only one or two incubations. For example, an antigen microarray may be first incubated with the samples, and then with a mixture of the primary and secondary antibodies, staining reagents. It is also conceivable that the formation of a detectable immune complex (e. g., a captured antivirus antibody/anti-lg/label complex) may be achieved in a single incubation step by exposing the protein microarray to a mixture of the sample and all the necessary reagents.

In an advantageous embodiment, the samples are agitated during incubation.

In yet another embodiment, the present disclosure provides a competitive immunoassay using an antibody microarray. Briefly, a microarray comprising immobilized antivirus antibodies is incubated with a test sample in the presence of a labeled viral antigen standard. The labeled viral antigen competes with the unlabeled viral antigen in the test sample for the binding to the immobilized antigen-specific antibody. In such a competitive setting, an increased concentration of the specific viral antigen in the test sample would lead to a decreased binding of the labeled viral antigen standard to the immobilized antibody and hence a reduced signal intensity from the label.

The microarrays of the present disclosure can be processed in manual, semi- automatic or automatic modes. Manual mode refers to manual operations for all assay steps including reagent and sample delivery onto microarrays, sample incubation and microarray washing. Semi-automatic modes refer to manual operation for sample and reagent delivery onto microarray, while incubation and washing steps operate automatically. In an automatic mode, three steps (sample/reagent delivery, incubation and washing) can be controlled by a computer or an integrated breadboard unit with a keypad. For example, an antigen microarray can be processed with a ProteinArray Workstation (PerkinElmer Life Sciences, Boston, MA) or Assay 1200# Workstation (Zyomyx, Hayward, CA). Scanners by fluorescence, colorimetric and chemilluminescence, can be used to detect microarray signals and capture microarray images. Quantitation of microarray-based assays can also be achieved by other means, such as mass spectrometry and surface plasma resonance. Captured microarray images can be analyzed by stand-alone image analysis software or with image acquisition and analysis software package. For example, quantification of an antigen microarray can be achieved with a fluorescent PMT-based scanner - ScanArray 3000 (General Scanning, Watertown, MA) or colorimetric CCD-based scanner - VisionSpot (Allied Biotech, Ijamsville, MD). Typically, the image analysis would include data acquisition and preparation of assay report with separate software packages.

To speed up whole assay process from capturing an image to generating an assay report, all the analytical steps including image capture, image analysis, and report generation, can be confined in and/or controlled by one software package. Such an unified control system would provide the image analysis and the generation of assay report in a user-friendly manner.

Furthermore, the present disclosure relates to uses of the microtiter plate-based microarrays according to the present disclosure for simultaneous screening of a blood sample obtained from a subject for blood transfusion for the presence of a plurality of different selected targets associate with an infectious disease, wherein a) the method according to the present disclosure is performed, and

b) the results from analyzing the interactions to determine the presence of the selected target is used as a first screen to determine the presence of the specific target and as the same time as a confirmation screen to confirm the screened presence of the specific target.

A further embodiment pertains to processes for manufacturing a microtiter plate-based microarray according to the present disclosure or the multi-well microtiter plate-based microarray according to the present disclosure, comprising the steps of: a) Precoating each probe or set of probes on predetermined locations on the surface of a membrane, and

b) Placing the precoated membrane in the microtiter well or wells. The inventions described and claimed herein are not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control. Various references are cited herein, the disclosures of which are incorporated by reference in their entireties. The present invention is further described by the following examples, which should not be construed as limiting the scope of the invention.

Embodiments

The following example shows that spotting and immobilization of capture antibodies onto a small area of the selected aluminium oxide membrane, mounted to a microtiter well (PamChip), was feasible with standard spotting devices (compare Fig. 3). Verification of immobilization was performed by Sypro Ruby staining and by anti-human antibody conjugated with FITC and/or Alexa Fluoro 647. Non-modified and SPDP-modified human anti-p24 MAbl were provided by Q-Biologicals.

Different buffers and molarities were applied:

5.9μΜ SPDP modified human anti-p24 with ΙΟΟΟμΜ TCEP in PBS (lx, pH 7.8)

5.9μΜ SPDP modified human anti-p24 with ΙΟΟΟμΜ TCEP in Na₂.HC0₃ (0.1M, pH 8.3)

3.2μΜ SPDP non-modified human anti-p24 with ΙΟΟΟμΜ TCEP in Na₂.HC0₃ (0.1M, pH 9)

The solutions were diluted 8 times by a factor of 2 and spotted onto PamChip arrays including control peptides having high signal (EPHB1) on Sypro Ruby staining and a phosphorylated peptide (ART 003) from PamGenes PamChip PTK microarray product.

Spot verification with Sypro Ruby staining see Fig. 3.: There is an increasing concentration dependent signal for modified as well as unmodified MAbl, spotted in duplicate, indicating successful spotting and immobilization.

Spot verification was also shown with various detection antibodies, an example of which (staining with goat anti-human IgG-AlexaFlour 647) is shown in Fig. 4. For sample incubation, washing steps and detection, standard methods for ELSA testing were applied.

After confirming immobilization, incubation with HIV-1 p24 antigen was shown with sufficient sensitivity down to <400 pg/ml for two different capture antibody concentrations (Fig.5)

In an alternative approach, immobilization was performed by binding of antibody to protein A/G coated chips. This method worked with similar sensitivity in detection of HIV-1 p24 antigen. The results are shown in Fig. 6

In summary, proof of principle could be shown. A multiple of spots of capture antibodies could be placed on a membrane in a microtiter well format with sufficient sensitivity for HIV-1 p24 antigen detection, an infectious disease marker which is used for screening as well as for confirmation.

Claims

1. A microtiter plate-based microarray suitable for simultaneous detection of a plurality of different selected targets in a biological sample obtained from a subject comprising: a) a plurality of separated subarrays in a microtiter well, wherein each subarray comprises a set of probes capable of selectively capturing a plurality of target ligands assign to one selected target, wherein b) each probe is coated on a solid support at a predetermined location on the surface of the microtiter well and interacts with one specific target ligand associated with the selected target, and wherein the plurality of the probes in a subarray interact with different target ligands associated with one selected target.

2. The microarray according to claim 1, wherein the selected target is associated with an infectious disease caused by a virus, a viroid, a prion, a protozoa, a fungus or a bacterium.

3. The microarray according to claim 2, wherein the virus is selected from the group consisting of Hepatitis B Virus, Hepatitis C Virus, Human Immunodeficiency Virus and Human T-Lymphotropic Virus.

4. The microarray according to any one of claims 1 to 3, wherein the biological sample is a blood, cerebrospinal fluid, cell culture, urine, sweat, buccal swab, tissue biopsy, or aspiration sample.

5. The microarray according to any one of claims 1 to 3, wherein the biological sample is a blood sample.

6. The microarray according to claim 5, wherein the blood sample is plasma or serum or components thereof selected from the group consisting of red blood cells, white blood cells, clotting factors, and platelets.

7. The microarray according to claims 6, wherein the blood sample is obtained from a subject for blood transfusion.

8. The microarray according to any one of claims 1 to 7, wherein the subject is an animal or human.

9. The microarray according to any one of claims 1 to 8, wherein the probes in the subarrays are polypeptides.

10. The microarray according to claim 9, wherein the probes are antibodies, antigens, enzymes, zinc- finger binding proteins, minor-groove binders, transcriptional factors, combinations thereof, or chimeras thereof.

11. The microarray according to any one of claims 1 to 10, wherein the target ligands are target antigens or anti-target antibodies assigned to the selected target.

12. The microarray according to claim 11, wherein the target antigens are viral antigens and the anti- target antibodies are anti-viral antibodies.

13. The microarray according to any one of claims 1 to 12, wherein the probes or set of probes is arranged on a substrate in the microtiter well.

14. The microarray according to claim 13, wherein the substrate is silicon, silicon dioxide, glass, polystyrene, gold, metal, metal alloy, zeolyte, polymer, or other organic or inorganic molecule.

15. The microarray according to claim 13 or 14, wherein the substrate is the surface of the microtiter well with or without coating.

16. The microarray according to claim 13 or 14, wherein the substrate is a coated membrane.

17. The microarray according to any one of claims 1 to 16, wherein a probe interacting with one specific target ligand is a mixture of antigens with different lengths in the amino acid sequence.

18. The microarray according to any one of claims 1 to 17, wherein each subarray comprises at least two probes interacting with the same target ligand.

19. The microarray according to any one of claims 1 to 18, wherein at least one subarray in the microtiter well comprises a positive control for the verification of the presence of the biological sample in the microtiter well.

20. A microtiter plate-based assay comprising a plurality of microtiter wells, wherein a plurality or each of the microtiter wells comprise a microarray according to any one of claims 1 to 19.

21. The assay according to claim 20, wherein a plurality or each of the wells comprise the identical microarray.

22. The assay according to any one of claims 20 to 21, wherein at least one microtiter well is employed as a positive or negative control each for the verification of the functionality of the assay to detect the selected targets.

23. An in-vitro method for simultaneous detection of a plurality of different selected targets in a biological sample obtained from a subject, in particular in a blood sample from a human in a blood bank setting, the method comprising: i) Applying a biological sample obtained from a subject to a microtiter well of a microtiter plate-based microarray according to any one of claims 1 to 19,

iii) Detecting the interactions between the target ligands and the probes; and

24. The method according to claim 23, wherein the selected target is associated with an infectious disease caused by a virus, a viroid, a prion, a protozoa, a fungus or a bacterium.

25. The method according to claim 24, wherein the virus is selected from the group consisting of Hepatitis B Virus, Hepatitis C Virus, Human Immunodeficiency Virus and Human T-Lymphotropic Virus.

26. The method according to any one of claims 23 to 25, wherein the biological sample is a blood, cerebrospinal fluid, cell culture, urine, sweat, buccal swab, tissue biopsy, or aspiration sample.

27. The method according to any one of claims 23 to 26, wherein the interactions between the target ligands and the probes are detected by fluorescence, chemiluminescence, or colorimetric reaction.

28. The method according to any one of claims 23 to 27, wherein a probe selectively interacts with a target ligand by specifically binding to the target ligand to form a complex.

29. The method according to any one of claims 23 to 28 wherein the results from analyzing the interactions to determine the presence of the selected target is used as a first screen to determine the presence of the specific target and at the same time as a confirmation screen to confirm the screened presence of the specific target by detection of one ore more selected targets.

30. A multi-well microtiter plate for the use of simultaneous detection of a plurality of different selected targets in a biological sample obtained from a plurality of subjects comprising: a) a plurality of microtiter wells, wherein a plurality or each of the microtiter wells comprise a microarray according to any one of claims 1 to 19, and

d) at least one microtiter well each comprises employed as a positive or negative control for the verification of the functionality of the assay to detect the selected targets.

31. Use of the microtiter plate-based microarray according to any one of claims 1 to 19 for simultaneous screening of a blood sample obtained from a subject for blood transfusion for the presence of a plurality of different selected targets associate with an infectious disease, wherein a) the method according to any one of claims 23 to 29 is performed, and

b) the results from analyzing the interactions to determine the presence of the selected target is used as a first screen to determine the presence of the specific target and at the same time as a confirmation assay to confirm the screened presence of the specific target in the same biological sample.

32. Use of the microtiter plate-based assay according to any one of claims 20 to 22 or of the multi- well microtiter plate according to claim 30 for simultaneous screening of a blood sample obtained from a subject for blood transfusion for the presence of a plurality of different selected targets associate with an infectious disease, wherein c) the method according to any one of claims 23 to 29 is performed, and

d) the results from analyzing the interactions to determine the presence of the selected target is used as a first screen to determine the presence of the specific target and at the same time as a confirmation assay to confirm the screened presence of the specific target in the same biological sample.

33. A process for manufacturing a microtiter plate-based microarray according to any one of claims 1 to 19 comprising the steps of: a) Precoating each probe or set of probes on predetermined locations on the surface of a membrane, and

b) Placing the precoated membrane in a microtiter well.