Compositions and methods

Abstract

A method of determining whether an individual is infected with a mycobacterial disease, the method comprising: (a) providing a system which comprises an antigen; (b) contacting the system with a sample obtained from the individual; and (c) detecting the presence or absence of binding of a biomarker in the sample with the antigen; wherein the antigen is a mycolic acid wax ester derived antigen.

Claims

1. A method of determining whether an individual is infected with a mycobacterial disease, the method comprising: (a) providing a system which comprises an antigen; (b) contacting the system with a sample obtained from the individual; and (c) detecting the presence or absence of binding of a biomarker for the mycobacterial disease in the sample with the antigen; wherein the antigen is a mycolic acid wax ester derived antigen of formula (I) or an ester or salt thereof: ##STR00020## wherein w is from 2 to 40, x is from 2 to 40, y is from 2 to 40, z is from 4 to 40, and X is a three-carbon fragment including an alkane, alkene, or cyclopropyl moiety.

2. A method according to claim 1 wherein steps (a), (b) and (c) are carried out in the order (a) followed by (b) followed by (c).

3. A method according to claim 1 wherein the antigen is present on a substrate in the system and/or in one or more solutions or suspensions in the system; and/or encapsulated in the system, for example in liposomes.

4. A method according to claim 1 which involves: (a) providing a substrate which carries the mycolic acid wax ester derived antigen; (b) contacting the substrate with a sample obtained from the individual; (c) detecting the presence or absence of binding of a biomarker for the mycobacterial disease in the sample with the mycolic acid wax ester derived antigen.

5. A method according to claim 1 wherein the biomarker is an antibody.

6. A method according to claim 1 wherein the biomarker is a disease antibody indicative of the presence of Mycobacterium avium paratuberculosis.

7. A method according to claim 1 wherein the individual is a ruminant.

8. A method according to claim 4 wherein the substrate carries one or more further antigens 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; (vii) further mycolic acid wax esters or salts thereof.

9. A method according to claim 5 wherein step (c) involves the steps: (i) contacting the system with a composition comprising a secondary antibody that interacts with the antibody in the sample; and (ii) detecting the presence or absence of binding of the antibody in the sample with the secondary antibody.

10. A method according to claim 5 wherein step (c) involves contacting the system with a composition comprising colloidal gold particles wherein the colloidal gold particles carry a secondary antibody that interacts with the antibody in the sample.

11. A kit for determining the presence or absence of a biomarker for a mycobacterial disease in a sample, the kit comprising: (x) a system which comprises a mycolic acid wax ester derived antigen; and (y) a composition comprising a secondary antibody that interacts with the biomarker, wherein the mycolic acid wax ester derived antigen is a compound of formula (I) or an ester or salt thereof: ##STR00021## wherein w is from 2 to 40, x is from 2 to 40, y is from 2 to 40, z is from 4 to 40, and X is a three-carbon fragment including an alkane, alkene, or cyclopropyl moiety.

12. A composition comprising at least 90 wt % of a single compound of formula (I) or an ester or a salt thereof: ##STR00022## wherein w is from 2 to 40, x is from 2 to 40, y is from 2 to 40, z is from 4 to 40 and X is a three carbon fragment including an alkane, alkene or cyclopropyl moiety, which composition comprises at least 90 wt % of a single compound having the formula A, B, C, D, E, F or G: ##STR00023## ##STR00024##

Description

EXAMPLE 1: SYNTHESIS OF WAX ESTER 33

(1) Compound 21 was prepared by the method described in The synthesis of single enantiomers of meromycolic acids from mycobacterial wax esters, Juma'a R. Al Dulayymi, Mark S. Baird, Evan Roberts and David E. Minnikin, Tetrahedron, 2006, 62, 11867-11880.

(2) ##STR00011##

(3) The wax ester 33 was prepared using the following reactions shown in scheme 1. The selection of suitable conditions such as reaction time and temperature are within the competence of the person skilled with the art.

(4) ##STR00012##

(5) In order to generate the full wax ester, the -dicarboxylic acid mono-ester 27 needed to be coupled to (S)-2-eicosanol 31.

(6) The -dicarboxylic acid mono-ester 27 was esterified with S-2-eicosanol 31 through a Steglisch esterfication reaction (Scheme 2):

(7) ##STR00013##

(8) Removal of the silyl ether and methyl ester protection was achieved in two steps to produce the free wax ester mycolic acid 33 (Scheme 3):

(9) ##STR00014##

(10) The -dicarboxylic acid 34 has been previously isolated from cells as a component of a complex mixture. The compound 34 was also synthesized starting from the -dicarboxylic acid mono-ester 27 after TBDMS deprotection and hydrolysis. (Scheme 4):

(11) ##STR00015##

EXAMPLE 2: SYNTHESIS OF TREHALOSE MONOMYCOLATE AND TREHALOSE DIMYCOLATE OF WAX ESTER 33

(12) The protected wax ester mycolic 35 was prepared from the corresponding free hydroxy wax ester 33 by reaction with an excess of tert-butyldimethylsilyl chloride and imidazole in the presence of 4-dimethylaminopyridine for 24 h at 70 C., followed by hydrolysis of the TBDMS ester on the acid group by stirring in THF for 15 minutes in the presence of (4%) of aqueous solution of tetrabutyl ammonium hydroxide.

(13) The compound 35 was coupled to hexatrimethylsilyl trehalose 36 using 1-(3-dimethylaminopropyl-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine and 4 molecular sieves in dichloromethane for six days at ambient temperature. This gave the protected TDM (trehalose dimycolate) 37 (52%) and the protected TMM (trehalose monomycolate) 38 (32%). Both of the compounds 37 and 38 were deprotected in two steps, including sugar deprotection to give 39 and 40 and finally TBDMS deprotection to give the free TDM 41 and the free TMM 42 (Scheme 5).

(14) ##STR00016## ##STR00017##

EXAMPLE 3

(15) The following additional wax esters were synthesised by the above or analogous methods:

(16) ##STR00018## ##STR00019##

EXAMPLE 4

(17) The binding of antibodies in serum of cattle infected with M. tuberculosis or M. avium with the antigens detailed in example 3 was measured in an ELISA assay.

(18) ELISA assays were carried out as known to those practiced in the art using 96 well plates. In the first procedure, the ELISA assay was carried out on 96-well microplates and the purified antigens were dissolved in n-hexane at a concentration of 62.5 g/ml. The antigen solutions were diluted and 50 l and placed in each well. The plates were left to dry at room temperature overnight. Blocking was done with 0.5% casein/PBS (400 l/well) and the plates are left to incubate at 25 C. for 1 h. The casein was aspirated using the LT-3500 plate washer and the plates flicked dry. Then the serum (1:20 dilution in 0.5% casein/PBS, pH 7.4) was added to the plate (50 l/well) and left to incubate at 25 C. for 1 h. The serum was aspirated and washed three times with (casein/PBS, 400 l/well) using the same plate washer and the plates were flicked dry again. After that, the secondary antibody, in the example given IgG Fc (1:1000 dilution in 0.5% casein/PBS) was added to the plate (50 l/well) and the plates were left to incubate at 25 C. for 30 min. The plates were washed three times again with (casein/PBS, 400 l/well) and dried. Then, the OPD substrate was added to the plates, which were left to incubate at 25 C. for 30 min. Finally, 2.5 M H.sub.2SO.sub.4 (50 l/well) was added and the absorbances of each well were read at 492, 450 and 630 nm using an LT-4000 plate reader.

(19) Serum used in this work was obtained from cattle infected with MAP, diagnosed as naturally infected with bovine TB, or known not to be infected using standard assays.

(20) All measurements were taken as four replicates unless otherwise stated. These were averaged to provide the data in the table which are optical density measurements at 492 nm.

(21) The results are given in Table 1 below:

(22) TABLE-US-00001 TABLE 1 ELISA assays to detect antibodies in serum of cattle infected with bovine M. tuberculosis (B tb) or M. avium paratuberculosis (MAP). Shaded boxes represent absorbances above a cut-off set for positive response for the particular antigen. The first two samples are infected with M. avium paratuberculosis (MAP). Antigen B shows the ability to identify MAP infection and gives no signal with B tb infected serum. Other wax esters identify the MAP infected serum, but also give selective responses with some of the B tb+ samples and with B tb samples, either due to co-infection with MAP or infection with other mycobacteria. Antigens A B C D E F G MAP 3.70 0.76 3.07 3.29 2.97 1.44 4.38 (experimentally infected) MAP (Naturally 4.38 0.72 4.06 4.00 3.88 4.00 4.38 infected) VLA (Naturally 1 1.49 0.17 2.74 3.12 0.38 1.07 3.68 infected) bovis 2 2.59 0.15 1.81 3.20 0.54 0.67 2.78 3 1.85 0.13 2.94 3.08 1.24 2.47 4.06 4 2.51 0.16 2.15 1.69 1.82 0.67 0.63 5 0.68 0.20 0.61 3.05 1.56 0.29 1.04 6 1.70 0.21 0.26 1.43 2.03 0.18 0.88 7 1.20 0.16 0.61 3.07 2.77 0.51 2.34 8 3.02 0.21 1.86 1.51 3.29 2.23 1.39 9 0.91 0.14 0.20 0.73 2.62 0.18 0.40 10 0.57 0.19 0.22 0.82 1.02 0.24 0.52 11 0.52 0.19 0.36 1.64 0.98 0.31 1.63 12 3.94 0.27 0.64 0.95 4.05 1.08 1.54 13 0.14 0.05 0.37 0.28 0.17 0.66 0.26 14 0.19 0.05 0.99 0.40 0.13 0.23 0.22 15 1.85 0.06 0.29 0.31 0.98 0.12 0.15 16 1.30 0.07 0.12 0.18 1.25 0.62 0.15 17 3.05 0.10 1.80 0.78 1.20 0.47 0.11 18 0.87 0.08 0.70 0.19 0.39 0.52 0.27 19 0.69 0.07 0.68 0.28 1.45 0.69 0.11 20 0.59 0.10 0.36 0.18 0.73 1.08 1.54 VLA (Non- 21 1.34 0.12 0.24 0.47 0.33 0.25 0.37 vaccinates 22 0.47 0.10 0.51 0.27 0.21 0.25 0.14 23 0.65 0.11 0.14 0.45 0.47 0.17 0.24 24 0.47 0.10 0.34 1.17 0.32 0.21 0.51 25 0.60 0.12 0.18 0.50 0.87 0.15 0.27 26 0.43 0.12 4.03 1.13 1.03 3.66 0.82 27 0.50 0.15 3.79 1.18 0.62 1.96 0.55 28 0.79 0.17 0.71 0.91 0.96 0.36 0.46 29 0.33 0.16 0.64 2.86 0.45 0.29 1.58 30 0.76 0.19 0.26 0.30 0.74 0.26 0.16 31 0.35 0.22 0.70 1.58 0.67 0.45 1.99 32 0.14 0.06 0.21 0.11 0.16 0.06 0.06 33 0.14 0.05 0.07 0.08 0.14 0.08 0.10 34 0.14 0.05 0.08 0.08 0.15 0.11 0.07 35 0.14 0.06 0.22 0.11 0.20 0.31 0.07 36 0.13 0.06 0.50 0.10 0.16 0.12 0.07 Cut-off for 0.85 0.30 0.85 0.85 0.85 0.85 0.85 conditional formating Cut-off for >.85 >0.30 >.85 >.85 >.85 >.85 >.85 sensitivity and specificity In Table 1, VLA means Veterinary Laboratories Agency, the source of the samples.