KIT AND METHOD

Abstract

A method of determining the presence or absence in a sample of a biomarker, the method comprising the steps of (a) providing a porous substrate which carries an antigen; (b) contacting the substrate with the sample; (c) contacting the substrate with a composition comprising a secondary antibody; and (d) observing the substrate. The method may enable a very quick, simple test to be carried out to determine whether or not a particular sample contains a biomarker, for example an antibody indicative of infection with a mycobacterial disease. A kit and a device for determining the presence or absence in a sample of a biomarker are also described.

Claims

1. A method of determining the presence or absence in a sample of a biomarker, the method comprising: (a) providing a porous substrate which carries an antigen; (b) contacting the substrate with the sample; (c) contacting the substrate with a composition comprising a secondary antibody; and (d) observing the substrate.

2. The method of claim 1, wherein step (c) involves contacting the substrate with a composition comprising nanoparticles of a metal or nanoparticles of a polymeric material.

3. The method of claim 2, wherein step (c) involves contacting the substrate with a composition comprising colloidal gold particles wherein the colloidal gold particles carry a secondary antibody.

4. The method of claim 3, the method comprising: (a) providing a porous substrate which carries an antigen; (b) contacting the substrate with the sample; (c) contacting the substrate with a composition comprising colloidal gold particles wherein the colloidal gold particles carry a secondary antibody; and (d) observing the substrate.

5. The method according to claim 3, wherein the composition comprising colloidal gold particles is an aqueous suspension of gold nanoparticles.

6. The method according to claim 1 which is a method of determining the presence or absence in a sample of an antibody indicative of infection with or exposure to mycobacteria.

7. The method according claim 1, wherein the porous substrate is a cellulosic material.

8. The method according to claim 1 wherein the antigen is selected from mycolic acids, derivatives and salts of mycolic acids and wax esters analogous to mycolic acids.

9. The method according to claim 8 wherein the antigen is selected from one or more of the following classes of compounds: (i) mycolic acids obtained from natural sources; (ii) synthetically prepared mycolic acids; (iii) salts of mycolic acids; (iv) esters of mycolic acids (i) and/or (ii); (v) sulfur-containing mycolic acids and/or salts or esters thereof; (vi) simple structural analogues of mycolic acids and/or salts or esters thereof; and (vii) mycolic acid wax esters and/or salts or esters thereof.

10. The method according to claim 1 wherein the substrate carries a synthetic antigen.

11. The method according to claim 1 in which the sample is selected from serum, blood, saliva, urine or sputum.

12. The method according to claim 1 in which step (d) involves visually observing the presence or absence of a colour change.

13. A kit for determining the presence or absence of a biomarker in a sample, the kit comprising: (i) a substrate which carries an antigen; and (ii) a composition comprising a secondary antibody.

14. The kit according to claim 13, wherein (ii) is a composition comprising particles of colloidal gold wherein the colloidal gold particles carry the secondary antibody on their surface.

15. A device comprising a housing and a substrate; wherein the substrate carries an antigen and is located within the housing; and wherein the antigen is selected from mycolic acids, wax esters analogous to mycolic acids and salts and esters thereof.

Description

[0169] The invention will now be further described with reference to FIGS. 1 to 4.

[0170] FIG. 1 shows a cross-sectional view of a device of the third aspect of the present invention;

[0171] FIG. 2 shows a schematic top view of the device of FIG. 1;

[0172] FIG. 3 shows a cross-sectional view of a further device of the present invention; and

[0173] FIG. 4 shows a schematic top view of the device of FIG. 3.

[0174] The device of FIGS. 1 and 2 comprises a plastic housing having an upper portion 1 and a lower portion 2. The upper portion includes an aperture 3. The sides of the aperture are shaped to direct liquid through the aperture. A porous substrate 4 is located within the housing. An antigen is provided on the substrate in the region below the aperture 5.

[0175] The device of FIGS. 3 and 4 includes an array of apertures 6 and an array of spots of antigens 7. This device may be used to test a plurality of samples using the same antigen and/or to test the same sample using a plurality of antigens.

[0176] The invention will now be further described with reference to the following non-limiting examples.

EXAMPLE 1GENERAL PROCEDURE

[0177] An antigen solution (2 mg/ml in hexane) was applied to provide a series of spots on a nitrocellulose membrane.

[0178] The antigen had the following structure:

##STR00007##

[0179] 1 ml of a serum sample (diluted to 50% with a casein/PBS buffer) was applied to each spot and allowed to run through the membrane. The membrane was then washed with distilled water.

[0180] 1 ml of a colloidal gold suspension was applied to the membrane and allowed to flow through. The membrane was again washed. The gold particles were coated with a polymer and had a secondary antibody (IgG/IgM) attached.

[0181] Testing was carried out using two serum samples know to be positive for TB (chips 1 and 3) and two known to be negative for TB (chips 2 and 4). A control sample was also tested.

[0182] On chips 1, 2 and the control chip, amounts of antigen were applied as follows:

##STR00008##

Chips 1, 2 and Control

[0183] On chips 3 and 4 the following amounts of antigen were applied:

##STR00009##

Chips 3 and 4

[0184] The results are shown in FIG. 5.

[0185] Chips 1 and 2 and the control are shown left to right across the top and chips 3 and 4 are left to right on the bottom row.

[0186] These results clearly show that the present invention provides a quick method to distinguish between serum examples which are positive and negative for TB.

[0187] It is also possible to measure the colour quantitatively using digital techniques.

EXAMPLE 2

[0188] A wax template was printed onto a nitrocellulose membrane leaving six blank spots around a central spot.

[0189] 1 g of the antigen of Example 1 (1 mg/ml solution) was applied to five of the blank spots. IgG serum (1 l, 0.05 mg/ml solution) was applied to the central spot. The final spot (bottom right) was left blank.

[0190] A serum sample known to be positive for tuberculosis has passed through a first device and a serum sample known to be negative for tuberculosis has passed through a second device.

[0191] In each case 300 l of each of the following solutions was added to the device and allowed to flow through: [0192] 1. the serum sample diluted in a 1:50 ratio by volume with a composition comprising 0.5% (w/v) casein, 0.1% (v/vs) HMPC (hypromellose), 0.1% (v/v) tween 20 and adjusted to pH 7.4 with phosphate buffered saline (PBS); [0193] 2. distilled water; [0194] 3. a solution of gold nanoparticles which carry Immunoglobulin G (IgG) diluted to an optical density of 1 in a composition comprising 0.5% (w/v) casein, 0.1% (v/vs) HMPC (hypromellose), 0.1% (v/v) tween 20 and adjusted to pH 7.4 with phosphate buffered saline (PBS); [0195] 4. distilled water.

[0196] FIG. 6 shows a photograph of the two devices at the end of the test. Device 1 had the positive serum and it can be seen that the positive control is in the centre and there is one negative control. The other spots have all changed colour.

[0197] For device 2 only the positive control has changed colour.

[0198] A photograph of the device was taken using a mobile phone and an application recorded the average RGB pixel count per spot.

[0199] The number is then subtracted from 255. The test score is then divided by the control spot, and interpreted based on the following criteria: [0200] Positive when t/+c>1 and t/c>1. Negative when t/c<1 and t/+c<1.

[0201] FIG. 7 shows the phone results for each antigen spot graphically.

[0202] ELISA tests were carried out on the same samples using the same antigen as a comparison.

[0203] FIG. 8 shows that the correlation of the mobile phone data of FIG. 7 with the ELISA results for a range of serum samples.