WO2004040306A1

WO2004040306A1 - Diagnostic device and kit

Info

Publication number: WO2004040306A1
Application number: PCT/EP2002/012206
Authority: WO
Inventors: Claudio Filippo Maria Trovato; Matthias Peter Volk
Original assignee: Labrea Holding S.A.
Priority date: 2002-10-31
Filing date: 2002-10-31
Publication date: 2004-05-13
Also published as: AU2002350661A1

Abstract

A diagnostic device (1) for detecting the presence of predetermined substances in a biological fluid, such as human blood includes a case (2) which houses a solid support medium (3), carrying reactant agents capable of binding the substances to be detected, and a removable filtering unit (4), which is selectively coupleable to the case (2) by clamping means (5). The support medium (3) can be coated with HIV antigens (e.g. GP36, GP41 and P24) or designed for qualitative and/or quantitative multiparameter screening of tumor markers (e.g. AFP, CEA, HCG, Pepsinogen I, pepsinogen II, H. Pylori and PSA).

Description

^Λ DIAGNOSTIC DEVICE AND KIT"

TECHNICAL FIELD

The present invention relates to a diagnostic device and to a kit comprising the device.

The device of the invention is particularly intended for rapid visual test of viral infections and can therefore be used for the rapid diagnosis of a number of diseases. For example, the device of the invention is suitable for the qualitative detection of antibodies specific to human immunodeficiency virus (HIV) in human serum, plasma or whole blood, as well as for the qualitative and/or quantitative multiparameter screening of tumour markers. BACKGROUND ART

It is known that some human viral infections may be diagnosticated by detecting the presence of antibodies specific to the infecting virus in blood samples. Similarly, some tumours may be diagnosticated and/or monitored by detecting the presence of specific tumour markers .

For example, human immunodeficiency virus (HIV) is the causative agent of acquired immuno-deficiency syndrome (AIDS) . Antibodies to HIV are produced in response to HIV infection so that the presence of these antibodies is indicative of the infection. A number of diagnostic test procedures are known for the detection of these antibodies, i.e. for the diagnosis of HIV. In particular, enzyme-linked immunosorbent assays (ELISA) are widely used to screen human blood and plasma samples for the presence of HIV antibodies. However, since false-positive reactions have frequently been observed with current ELISA tests, it is strongly recommended to confirm repeatedly reactive samples by use of other reliable techniques, for example by Western blotting or recombinant immunoblot assay (RIBA) . However, these confirmatory assays, even though more reliable than any enzymatic immuno-assay, especially regarding the specificity of the final results, are very expensive and time consuming in comparison with ELISA tests.

To sum up, it is not presently available a test (and a device for performing the test) which is rapid, reliable, very specific and sensitive to the substance to be investigated; moreover, known ELISA methods cannot be carried out onto whole blood samples, plasma or serum samples being required; clearly, separation of serum or plasma is an additional procedure step which make the analysis longer and more expensive. DISCLOSURE OF THE INVENTION

It is therefore an object of the present invention to provide a diagnostic device, in particular for detection of antibodies specific to viruses or tumour markers, which is free of the aforedescribed drawbacks.

A particular object of the invention is to provide a diagnostic device which allows assays to be performed with a very high reliability, so as a further confirming test is not required, being at the same time both specific and sensitive.

It is a specific object of the invention to provide a device which is simple and straightforward to be realized and used and which can operate with either whole blood samples or blood component samples (serum, plasma) and also allows multiparameter screening to be performed simply, quickly and cheaply.

Accordingly, it is provided a diagnostic device as claimed in claim 1.

Preferred aspects of the invention are defined in dependent claims 2 to 13.

Preferred embodiments of the invention especially designed to detect HIV-specific antibodies and to detect tumour markers are claimed in claims 14 and 15 respectively. According to the invention, it is also provided a diagnostic kit as claimed in claims 16 and 17.

The device and kit according to the invention overcome the aforementioned drawbacks ' of the known devices and assays on the market.

By one hand, the device of the invention is simple and straightforward to be realized and used and can operate with either whole blood samples or blood component samples (serum, plasma) . By the other hand, by the device of the invention assays can be performed which are rapid, reliable, very specific and sensitive to the substance to be investigated.

Moreover, the device allows several parameters to be detected at the same time. Therefore this test may be regarded as a ^ΛΛbiochip" without having the need to use expensive equipment.

BRIEF DESCRIPTION OF THE DRAWINGS

Further objects and advantages of the present invention will be apparent from the following description of non-limiting examples of the invention, with reference to the accompanying drawings, wherein:

- Figure 1 is a schematic partial representation of a diagnostic device according to the invention; - Figure 2 is a longitudinal section view of the device of Figure 1;

- Figure 3 is a schematic view of a detail of the device of Figure 1 especially designed for detecting HIV- specific antibodies;

- Figure 4 is a schematic view of a detail of the device of Figure 1 especially designed for the qualitative and/or quantitative multiparameter screening of tumour markers. BEST MODE FOR CARRYING OUT THE INVENTION

With reference to figures 1 and 2, a diagnostic device 1 comprises a case 2 which houses a solid support medium 3, a removable filtering unit 4 selectively coupleable to case 2, and clamping means 5 for connecting filtering unit 4 to case 2.

Case 2 comprises a base body 6 and a cover 7 positioned one above the other to delimit an inner reaction chamber 8; base body 6 is provided with a substantially rectangular tank 9 delimited by a lateral wall 10 and having a substantially plane bottom wall 11; a peripheral edge 12 of tank 9 is provided with coupling members 13 (for example projecting pins) fitted to respective seats 14 formed in cover 7 for connecting base body 6 and cover 7. Reaction chamber 8 is divided into a plurality of wells 15 by partitions 16; in the non limiting example shown, wells 15 are defined by respective slits 17 which are formed through an upper- wall 18, opp'osite to bottom wall 11, of case 2 and are separated from one the other by partitions 16; partitions 16 perpendicularly project towards bottom wall 11; slits 17 are delimited by respective pair of parallel lateral flanks 19.

Bottom wall 11 of tank 9 carries an absorbent layer 20, for example made of spongy material, over which support medium 3 is applied; support medium 3 is defined by a membrane 21 made of, for example, nitrocellulose or suitable polymer material (PVDF, PA, etc.).

Advantageously, membrane 21 is positioned at the end of slits 17 only, i.e. at the bottom of each well 15 and comprises therefore a plurality of parallel strips 22 housed in respective wells 15.

Filtering unit 4 comprises a plate 23 provided with a plurality of seats 24: seats 24 are defined by respective grooves 25 delimited by respective lateral edges 26 and closed by respective lower filter elements 27 and upper covering slabs 28. Slabs 28 are provided with respective intake ducts 29 communicating with seats 24 and comprising respective tubes 30 vertically projecting from slabs 28 and parallel to each .other. Filter elements 27 are made of a filter material of any known type capable to separate the liquid part of blood (i.e. serum and/or plasma, which pass through the filter material) from the blood corpuscle part (in particular from erythrocytes) .

A dispenser 31 for dispensing a fluid to be tested is associated to inlet ducts 29 and comprises a central channel 32, provided with a handle 33, and a plurality of outlet ducts 34 fittable to respective open ends 35 of inlet ducts 29 and connected to central channel 32 by a connector channel 36.

Plate 23 is positioned, in use, over upper wall 18 so as seats 24 are placed inside respective slits 17 and, therefore, inside respective wells 15.

Plate 23 (and consequently filtering unit 4 too) is applied in a removable manner to case 2 by clamping means 5: in particular, clamping means 5 comprise two clips 37 positioned at respective opposite ends of case 2; each clip 37 comprises an elastically deformable U-shaped body 38 having two substantially parallel arms 39 projecting from a central root portion 40 and provided with respective teeth 41 for engaging blocking seats 42 formed in plate 23 and in bottom wall 11 respectively; when device 1 is assembled, plate 23 is clamped to case 2 in such a way that filter elements 27 are pressed to contact support medium 3.

Support medium 3 carries reactant '"agents suitable for the specific test to be performed, as described in the following non limiting examples.

EXAMPLE 1 - HIV infection diagnosis A device 1 as previously described is especially designed for detecting HIV-specific antibodies in human sera, plasma or, most preferably, whole blood.

As schematically shown in figure 3, each strip 22 of membrane 21 housed in a well 15 is provided with a sequence of reactive sites 50, constituted by respective bands: a first band 51 is pre-coated with HIV antigen p24, a second band 52 is pre-coated with HIV antigen gp36, and a third band 53 is pre-coated with HIV antigen gp41. A forth band 54 is a control band coated with rabbit-anti-goat polyclonal antibody.

If whole blood is to be tested, samples are introduced through dispenser 31 in respective seats 24; after filtration by filtering elements 27, blood liquid part filtered through filtering elements 27 penetrates into wells 15 and contacts membrane 21.

When HIV specific antibodies are present in a sample, they react specifically with the antigens coated on membrane 21 in one or more reaction sites 50.

Filtering unit 4 is removed from case 2 and a washing buffer (known, for example TBS buffer) is added in each well 15; afterwards an enzyme conjugate is added; this enzyme conjugate is preferably a goat-anti-human polyclonal antibody linked to alkaline phosphatase (AP- conjugate) . The enzyme AP-conjugate reacts with antibodies (if any) bound in reaction sites 50 and always react with the rabbit-anti-goat polyclonal antibodies in control band 54. After washing away unbound conjugates, an indicating substance (a known colour substrate for alkaline phosphatase) is added to induce a colour variation and the reaction is stopped by means of an acidic solution (e.g. diluted HC1) .

The appearance of a colored stain in correspondence of a particular band reveals a reaction between the antigen of that, band and the relevant antibody, disclosing the presence of the antibody in the tested sample.

Device 1 may operate also with human serum or plasma instead of whole blood. If serum or plasma samples are used, filtering unit 4 is not required. EXAMPLE 2 - Screening of tumour markers A device 1 as previously described is especially designed for the qualitative and/or quantitative multiparameter screening - of tumour ' markers, and specifically intended to detect AFP, CEA, HCG, Pepsinogen I, Pepsinogen II, H. pylori and PSA in a single test out of whole blood samples.

AFP (alpha-phetoprotein) , CEA (carcinoembryonic antigen) , HCG, Pepsinogen I, Pepsinogen II, H. pylori and PSA are recognized tumour markers. Known monoclonal antibodies against each of the above substances have been selected and used as reactant agents arranged onto support medium 3. In particular, as schematically shown in figure 4, support medium 3 is provided with a plurality of reactive sites 50 arranged onto membrane 21 according to a predetermined pattern and constituted by respective spots: in particular, each spot 50 include a monoclonal antibody specific to a respective tumour marker and is associated to a reference spot 54 including a goat-anti-mouse polyclonal antibody.

Device 1 is still operated as previously described. Whole blood samples are introduced through dispenser 31 in respective seats 24 and, after filtration (where, in particular, erythrocytes are filtered out) by filtering elements 27, contacts membrane 21, i.e. spots 50, in respective wells 15.

Tumour markers in the samples react with the respective specific monoclonal antibodies ■' on membrane 21. Filtering unit 4 is then removed from case 2 and membrane 21 is washed using a known washing buffer (for example a TBS buffer) ; addition of a mixture of monoclonal antibodies-AP-conjugates specific to each tumour marker to be investigated causes antigen-antibody reaction for each parameter (i.e. for each tumour marker) .

After washing away unbound conjugates (again by using TBS buffer) , a known colour substrate for alkaline phosphatase as indicating substance is added; reaction is stopped after an appropriate time period by addition of an acidic solution.

Colour variations are then detected by a known detector or reader; colour intensity is directly proportional to the amount of the respective tumour marker in the tested sample.

Claims

C LA I M S

1) A diagnostic device (1) for detecting the presence of predetermined substances in a biological fluid, in particular in human blood samples, comprising a case (2) which houses a solid support medium (3) , said support medium carrying reactant agents capable of binding the substances to be detected, the device being characterized by comprising a removable filtering unit (4) selectively coupleable to said case (2) .

2) A device as claimed in claim 1, characterized by comprising clamping means (5) for connecting said filtering unit (4) to said case (2) .

3) A device as claimed in claim 2, characterized in that said filtering unit (4) comprises at least a seat (24) for receiving said biological fluid and closed by a filter element (27) .

4) A device as claimed in claim 3, characterized in that said case (2) is provided with an inner reaction chamber (8) in which said support medium (3) is housed, said filtering unit (4) being removably inserted inside the reaction chamber (8) with said filter element (27) directly contacting said support medium (3) .

5) A device as claimed in claim 4, characterized in that said filter element (27) is pressed to contact said support medium (3) by said clamping means (5) .

6) A device as claimed in any one of claims 2 to

5, characterized in that said clamping means (5)

comprise at least a clip (37) positioned at an end of

said case (2) and comprising an elastically deformable

U-shaped body (38) having opposite teeth (41) for

engaging blocking seats (42) formed in said filtering

unit (4) and in said case (2) respectively.

7) A device as claimed in claim 6, characterized

in that said clamping means (5) comprise two clips (37)

positioned at opposite ends of said case (2) .

8) A device as claimed in any on of claims 2 to

7, characterized in that said filter element (27) is

made of a filter material capable to separate the blood

liquid part, which pass through the filter material,

from the blood corpuscle part, in particular from

erythrocytes .

9) A device as claimed in claim 8, characterized

in that said reaction chamber (8) is provided with an

absorbent layer (20) , preferably made of spongy

material, over which said support medium (3) is

applied; said filter element (27) being pressed against

said absorbent layer (20) with interposition of said

support medium (3) .

10) A device as claimed in any one of the preceding claims, characterized in that said support , medium (3) is defined by a membrane (21) made of nitrocellulose .

11) A device as claimed in any one of the preceding claims, characterized in that said case (2) is provided with a plurality of wells (15) , each well (15) being provided with a reactant agent.

12) A device as claimed in claim 11, characterized in that said filtering unit (4) comprises a plate (23) provided with a plurality of seats (24), said seats

(24) being closed by respective filter elements (27) .

13) A device as claimed in claim 12, characterized in that said seats (24) are removably housed inside respective wells (15) , each seat (24) being separated from the respective well (15) by a filter elements (27) .

14) A device as claimed in any one of the preceding claims and especially designed for detecting HIV-specific antibodies in human blood, characterized in that said support medium (3) carries a sequence of bands (51) coated with respective HIV antigens.

15) A device as claimed in any one of the preceding claims 1 to 13 and especially designed for the qualitative and/or quantitative multiparameter screening of tumour markers, in particular of AFP, CEA,

( HCG, Pepsinogen I, Pepsinogen II, H. pylori, PSA, in human blood, characterized in that said support medium (3) carries a plurality of reactive sites constituted by respective spots (50) ; each spot including a monoclonal antibody specific to a respective tumour marker and being associated to a reference spot (54) .

16) A diagnostic kit for detecting the presence of predetermined substances in a biological fluid, in particular in human blood samples, characterized by comprising the device as claimed in any one of the preceding claims, and at least an enzyme conjugate capable of reacting with the substances to be detected.

17) A kit as claimed in the preceding claim, characterized in that said enzyme conjugate is an enzyme AP-conjugate.