WO1995023969A1

WO1995023969A1 - Rapid immunological test

Info

Publication number: WO1995023969A1
Application number: PCT/GB1995/000467
Authority: WO
Inventors: Michael Stephen Roberts
Original assignee: Aquaculture Diagnostics Limited
Priority date: 1994-03-04
Filing date: 1995-03-03
Publication date: 1995-09-08

Abstract

A rapid antibody test comprising contacting a sample suspected of containing an antigen with means for capturing such antigen, placing said means (with captured antigen) on a support, optionally treating said means with a suitable blocking buffer, and contacting the said supported means with an appropriate binding partner and means for visualisation of any luminescent immunocomplex present by either chemi or bio-luminescence and further providing a sample holder for use in such a test.

Description

RAPID IMMUNOLOGICAL TEST

This invention relates to a rapid antibody assay and in particular to a rapid antibody assay for use in the laboratory, surgery, battlefied, near patient testing and environment.

Conventional testing relies on the traditional enzyme-linked immunosorbent assay "ELISA" method of visualisation which requires sophisticated laboratory equipment. It is a technique used for detecting and quantifying specific serum antibodies. Initially the serum is allowed to react with specific antigens which have been absorbed, e.g. to the surface of a plastic tube. Antibodies which combine with the antigens are then detected by treating the test system with a conjugate, i.e. anti-immunoglobulin which has been linked with a particular enzyme. The test system is washed free of any uncombined conjugate and examined for the presence of bound enzyme. The system is incubated with an appropriate substrate and the products of enzymic cleavage assayed for example by spectrophotometry.

Other test methods of analysing the products from the aforementioned test are by blot or disc immunoassays which are carried out either in petri dishes or on separate absorbent pads . After separation from a complex mixture by electrophoresis in a solid phase such as polyacrylamide or agar gel, antigens can be "blotted" by transverse electrophoresis onto nitrocellulose sheets where they bind non-specifically and can be stained with appropriately labelled antibodies.

Use of either petri-dishes or separate absorbent pads means that there can, for example, be up to five steps involving handling of test discs prior to placing the discs on a flat surface - referred to as a platen. The platen may then be covered with a thin plastic film such as cling film so as to retain the discs thereupon, the platen inverted, placed in a suitable camera such as the Tropix Model ICL-901 camera luminometer system and an instant photograph taken. Since the photograph so produced is a reverse mirror image of the platen, great care needs to be taken in ensuring that the pattern of the discs on the platen is easily read.

There are many problems associated with prior art tests including the fact that they use an antigen to test for an antibody presence in the host, which means that these tests rely on stimulation of the host's immune system. These cannot be used as an environment test, because the environment cannot manufacture antibodies in response to infection.

Another problem is the large number of steps involved in the handling of the test discs when carrying out chemi- or bio-luminescent immunoassay tests. Furthermore the aforementioned methods must be carried out in a reasonably well equipped laboratory, testing and analysis being carried out by skilled scientists and technicians.

The aim of this invention is to provide a rapid antibody test which obviates or mitigates these disadvantages.

According to the present invention there is provided a rapid antibody test comprising contacting a sample suspected of containing an antigen with means for capturing such antigen, placing said means (with captured antigen) on a support, optionally treating said means with a suitable blocking buffer, and contacting the said supported means with an appropriate binding partner and means for visualisation of any luminescent immunocomplex present by either chemi or bio-luminescence. Preferably said means for capturing antigen comprises a semi-permeable membrane, which membrane permits passage of aqueous fluids, but retains macro- molecules and micro-organisms such as viruses, bacteria or parasites.

In one embodiment the test is used as a tracking device for environmental monitoring for example in relation to stack emissions in which a "friendly" organism such as a non pathogenic virus is introduced as a marker at a particular source of pollution. Subsequent detection of the "friendly" organism such as a non pathogenic virus by the test method should confirm the marked source as a pollutant.

According to a further aspect of the present invention there is provided a sample holder comprising a substantially planar member having first and second planar surfaces, characterised in that the planar member has one or more apertures formed therethrough, the/each aperture being capable of receiving a respective membrane therein, the holder further comprising a substantially planar absorbent pad, a first surface of which pad is held in contact with the second surface of the planar member by retaining means when the planar member and pad are arranged such that the pad is uppermost, and an innermost surface of the/each membrane in the/each aperture is in contact with a portion of the first surface of the pad.

Preferably the planar member and the pad are substantially rectangular in shape.

Preferably also, the planar member and the pad are substantially the same size in plan section.

Preferably also, the planar member is suitably held in association with or integrally formed with a first open end of a hollow frame member, the frame member having an inner space suitable for receiving the pad therein.

Preferably also, the hollow frame member has a height substantially less than its breadth.

Preferably also, the hollow frame member is substantially rectangular in planar section.

Preferably also, the retaining means comprises a further planar member a first surface of which is adjacent a second surface of the pad, such that when the holder is arranged so that the pad is above the planar member, the further planar member is above and presses down on the pad.

The first surface of the further planar member may be adhered to the second surface of the pad.

The membrane (s) and pad may be made of fibrous materials.

The fibres of the pad may be substantially aligned parallel with opposing planar surfaces of the pad.

Preferably also, there are provided a plurality of apertures, said apertures being spaced from one another in a non-symmetric pattern on the planar member.

According to a yet further aspect of the present invention there is provided a method of performing an antibody test employing a sample holder of this invention as described above.

Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, which are: Fig 1 a sectional view of a first embodiment of a sample holder according to the present invention;

Fig 2 an exploded perspective view from the front of the sample holder of Fig 1;

Fig 3 a view from above of the sample holder of Fig 1 being loaded into a suitable camera;

Fig 4 a perspective view from one side and to the front of the sample holder and camera shown in Fig 3 ; and

Fig 5 a view from above of a part of a second embodiment of a sample holder according to the present invention.

According to one embodiment of the invention a rapid antibody test is provided in which antigens are captured by microfiltration of an aqueous medium or other medium (air) through a predetermined optimum capture means which may take the form of a generally disc shaped membrane or tubular membrane which may be either plain or pre- blocked. Alternatively antigen capture may be carried out by using a suitable, in-line air filter. Following antigen capture the membranes are placed, capture side exposed on a suitable platen.

The non pre-blocked membranes are blocked by a suitable blocking buffer such as 0.5% - 1% Casein in maleic acid buffer or bovine serum albumin etc. Other suitable blocking agents are available commercially under proprietory names Tween 20, Tween 40, Tween 60. The membranes are then washed in 200 microlitres of appropriate buffer to remove endogenous enzymes. Following washing, 50-100 microlitres of an appropriate monoclonal or polyclonal antibody bound to an enzyme capable of illiciting a luminescent reaction is applied to the membrane discs. The conjugated antibody is dissolved in an appropriate buffer at optimum pH and containing a blocking reagent. The chemi- or bio- luminescent compounds utilize alkaline phosphatase, beta galactosidase or another as yet unidentified enzymes.

The membranes are further washed with at least 200 microlitres of appropriate buffer containing blocking buffer at optimum pH to remove any unbound secondary antibody. Finally 50 microlitres of a suitable chemi- luminescent compound is applied to the membranes. The membranes are then incubated for approximately 15 minutes before being placed into a Polaroid camera.

In an alternative embodiment the membranes either alone or on the platen can be placed in a luminometer to measure the level of chemiluminescence.

It should be understood that the membranes although generally disc shaped could be replaced by any other suitable membranes or hollow fibre.

Referring to the drawings there is further described a platen which may have the form of a holder 5 which comprises a substantially rectangular planar member 10 having first and second planar surfaces 15, 20. A plurality of substantially circular spaced apertures 21 are formed through the planar member 10.

The planar member 10 is formed integrally with a first open end of a hollow rectangular frame member 25. The frame member 25 has a hollow inner space 30.

Further, the frame member 25 has a height substantially less than its planar dimensions. The planar member 10 and frame member 25 together may be conveniently referred to as a platen 35.

The holder 5 further comprises a substantially rectangular planar absorbent pad 40 having first and second surfaces 45, 50. The thickness of the pad 40 (between surfaces 45, 50) may be around 7 mm. The pad 40 is dimensioned so as to be a snug fit in the hollow inner space 30 of the frame member 25 so that the first surface 45 of the pad 40 comes into intimate contact with the second surface 20 of the planar member 10.

Adhered to the second surface 50 of the pad 40 is a first surface 55 of a further substantially rectangular planar member 60, which further planar member 60 is also dimensioned so as to be a snug fit in the hollow inner space 30 of the frame member 25.

As can best be seen from Fig 1, each aperture 21 is capable of receiving a respective membrane 65 therein. Each membrane 65 is substantially disc-shaped having first and second planar surface 70, 75. The membranes 65 are typically of the order of 50-60 microns thick. When a membrane 65 is loaded into an aperture 21 the second surface 75 of the membrane 65 comes into intimate contact with a respective portion 80 of the first surface 45 of the pad 40.

In use of the present invention the antigen is captured by one of the aforementioned methods and each membrane 65 is then placed, capture side exposed, in an aperture 21 of the holder 5. If the membranes 65 are plain they would now be blocked using a suitable blocking buffer. Following blocking, or insertion into the holder 5 without blocking, the membranes 65 are washed with the appropriate buffer to remove any endogenous enzymes. Following washing, the membranes have applied thereto an appropriate antibody, bound to an enzyme to form a conjugate capable of producing a chemi-luminescent reaction with a target antigen, in buffer of optimum pH and containing blocking reagent.

Each membrane 65 contained within the holder 5 is washed with buffer at optimum pH and containing the blocking agent and finally the chemi-luminescent substrate is applied. After a minimum time lapse of 15 minutes at ambient temperature the platen 35 is placed into a suitable camera 85, as shown in Figs 3 and 4 with the first surface 15 of the planar member 10 lowermost. A suitable camera is the ICL-901 camera luminometer system manufactured by Tropix Inc which uses Polaroid® instant developing film. Whilst it is expected that the above test uses photographic film such as Polaroid® instant film, the test can also be carried out using x- ray film to detect photoemission. Alternatively after a suitable incubation time a luminometer can be used to obtain a reading from the chemi- or bio-luminescence on the discs.

It is apparent that when the membrane 65 are placed in the apertures 21 they come into contact with and are held in place by the pad 40. Thus when the holder 5 is inverted, as shown in Figs 1-4, the membranes 65 are retained within the apertures 21.

Once the holder 5 has been placed in the camera 85, a cover 90 of the camera 85 is closed so as to close the holder 5 within the camera 85. An exposure may then be made.

If using a 3,000 ASA black and white film the exposure should be approximately 3 minutes; if using a 20,000 ASA black and white film the exposure should be approximately 30 seconds; and if using a 100 ASA colour film the exposure should be approximately 3 hours.

Time from capture of antigen to visualisation of result can be decreased if the test is carried out under temperature control at, for example, 37°C.

After exposure, the holder 5 may be removed from the camera 85 and the pad 40 removed and disposed of.

It can be seen from the foregoing description that the holder according to the present invention removes a number of handling steps from the "blot" immuno-assay test and seeks to ensure intimate contact between test substance and reagents.

Further, the pad 40 assists in adhering the membranes 65 within the apertures 21 and also filters excess liquids away from the membrane 65 thereby seeking to ensure optimum test conditions.

Referring now to Fig 5, there is illustrated a second embodiment of a platen 35' . The platen 35' is substantially identical to the platen 35 hereinbefore described excepting that the apertures 21' are formed in a non-symmetric pattern. This formation assists in ease of analysis of the photograph produced by the test.

The main advantages of the present invention are that the test can be carried out outwith laboratory conditions and also the test from antibody to substrate is now carried out directly on the discs. Therefore there is no need for further handling, and at the end of the test the platen is simply inverted, placed in the camera and a photograph taken. The test can be carried out at speed and at a significantly lower cost. The test also offers better sensitivity. Whereas the invention has been particularly described herein by way of an illustrative example it will be apparent to those of appropriate skills and knowledge that variants in the types of reagents and quantities thereof are possible without departing from the essential concept of the invention.

Claims

1. A rapid antibody test comprising contacting a sample suspected of containing an antigen with means for capturing such antigen, placing said means (with captured antigen) on a support, optionally treating said means with a suitable blocking buffer, and contacting the said supported means with an appropriate binding partner and means for visualisation of any luminescent immunocomplex present by either chemi or bio-luminescence.

2. A test according to claim 1 wherein said means for capturing antigen comprises a semi-permeable membrane, which membrane permits passage of aqueous fluids, but retains micro-organisms such as viruses, bacteria or parasites.

3. A test according to claim 1 or 2 in which antigens are captured by microfiltration through a generally disc shaped membrane.

4. A test according to claim 1 or 2 in which the antigens are captured on an affinity column.

5. A test according to claim 1 in which antigens are captured by an in-line air filter.

6. A test according to claim 2 or 3 wherein the membrane is pre-blocked.

7. A test according to claim 6 wherein the blocking buffer is 0.5% Casein maleic acid in appropriate buffer optimum pH and molarity.

8. A test according to claim 6 wherein the blocking agent 0.05% Tween 20, or Tween 40 or Tween 60 in appropriate buffer at optimum pH and molarity.

9. A test according to claim 6 wherein the blocking buffer is bovine serum albumin in appropriate buffer at optimum pH and molarity.

10. A test according to any of the preceding claims wherein the membranes are washed to remove any endogenous enzymes and an appropriate monoclonal or polyclonal antibody bound to an enzyme capable of illiciting a luminescent reaction is applied to the membrane discs and the conjugated antibody is dissolved in an appropriate buffer at optimum pH and containing a blocking reagent and subsequently the membranes are further washed with an appropriate buffer, treated with a suitable chemi- luminescent compound and incubated after which the results are scrutinised.

11. A test according to any one of the preceding claims wherein a non-pathogenic organism (e.g virus) is introduced as a marker antigen at a particular source of pollution to act as a tracer and the test is carried out on samples taken at a site remote from the source.

12. A sample holder for use in the test of claim 1 comprising a substantially planar member (10) having first and second planar surfaces (15,20) , characterised in that the planar member has one or more apertures (21) formed therethrough, the/each aperture being capable of receiving a respective membrane (65) therein, the holder further comprising a substantially planar absorbent pad (40) , a first surface (45) of which pad is held in contact with the second surface of the planar member (20) by retaining means when the planar member and pad are arranged such that the pad is uppermost, and an innermost surface of the/each membrane (65) in the/each aperture (21) is in contact with a portion of the first surface of the pad (45) .

13. A sample holder according to claim 12 wherein the planar member (10) and the pad (45) are substantially rectangular in shape.

14. A sample holder according to claim 12 or claim 13 wherein the planar member (10) and the pad (45) are substantially the same size in plan section.

15. A sample holder according to claim 12, 13 or 14 wherein the planar member (10) is suitably held in association with or integrally formed with a first open end of a hollow frame member (25) , the frame member having an inner space (30) suitable for receiving the pad (45) therein.

16. A sample holder according to claim 15 wherein the hollow frame member (25) has a height substantially less than its breadth.

17. A sample holder according to claim 15 or claim 16 wherein the hollow frame member (25) is substantially rectangular in planar section.

18. A sample holder according to claim 12 or claim 13 wherein the retaining means comprises a further planar member (60) a first surface (55) of which is adjacent a second surface of the pad (50) , such that when the holder is arranged so that the pad is above the planar member (10) , the further planar member (60) is above and presses down on the pad (40) .

19. A sample holder according to claims 12, 13 or 14 wherein the first surface (55) of the further planar member (60) is adhered to the second surface of the pad (50) .

20. A sample holder according to claim 12 wherein the membrane(s) (65) and pad (50) is made of fibrous materials.

21. A sample holder according to claim 20 wherein the fibres of the pad (50) is substantially aligned parallel with opposing planar surfaces of the pad.

22. A sample holder according to Claim 12, 13 or 14 wherein the planar member (10) is provided with a plurality of apertures (21) , said apertures being spaced from one another in a non-symmetric pattern on the planar member (10) .

22. A test kit for performing a rapid antibody test including,

(i) means for capturing an antigen (ii) support means for containing said antigen (iii) a suitable blocking buffer (iv) an appropriate binding partner for said antigen (v) a bioluminescent or chemiluminescent reagent capable of illiciting a luminescent reaction (vi) means of visualisation of any luminescent immunocomplex present by either chemi- or bio-luminescence.