Method of diagnosing tuberculosis

Abstract

Immobilising isolated mycolic acid antigens of tuberculous mycobacterial origin or a synthetic analogue thereof on a screen-printed electrode by binding of the antigens to a self-assembled monolayer comprising a thiolated hydrophobic substance to produce immobilized mycolic acids antigens in the form of a mycolic acid antigen-containing self-assembled monolayer coating on a surface of the electrode.

Claims

1. A method for diagnosing tuberculosis by detecting surrogate marker antibodies indicating active tuberculosis within 24 hours of obtaining a diagnostic sample, the method comprising: taking up isolated mycolic acid antigens of tuberculous mycobacterial origin or a synthetic analogue thereof in a liposome carrier to produce mycolic acid antigen containing liposomes; obtaining a screen-printed electrode containing on its surface immobilised isolated mycolic acid antigens of tuberculous origin or a synthetic analogue thereof; obtaining a diagnostic sample from a human or animal suspected of having active tuberculosis, which sample may contain surrogate marker antibodies to the immobilised antigens; dividing some or all of the diagnostic sample into a first and a second sample; producing a control sample by combining the first sample with a redox probe and exposing the control sample to the mycolic acid antigen containing liposomes; producing a test sample by combining the second sample with a redox probe and exposing the test sample to liposomes not containing mycolic acid antigens; contacting the control sample with the immobilised mycolic acid antigens to allow any antibodies in the control sample to bind to the immobilised mycolic acid antigens; contacting the test sample with the immobilised mycolic acid antigens to allow any antibodies in the test sample to bind to the immobilised mycolic acid antigens; and without a washing step to remove unbound antibody, comparing the degree or extent of the binding of the antibodies to the immobilised mycolic acid antigens in the control sample and the test sample by electrochemical impedance spectroscopy, any observed lesser binding shown by the control sample being an indicator of the presence of antibodies to the mycolic acid antigens in the diagnostic sample indicating active tuberculosis in the human or animal from which the diagnostic sample originated so that active tuberculosis is thereby diagnosed in the human or animal.

2. The method as claimed in claim 1, in which the control sample and the test sample are sequentially contacted with the immobilised antigens of the same screen-printed electrode.

3. The method as claimed in claim 1, in which the test sample substitutes the control sample when sequentially contacted with the immobilised antigens of the same screen-printed electrode.

4. The method as claimed in claim 1, in which the tuberculous mycobacterial antigen source is of the type which causes pulmonary or extra-pulmonary tuberculosis.

5. The method as claimed in claim 1, in which the mycolic acid antigens are extracted from Mycobacterium tuberculosis and the antibodies are antibodies against Mycobacterium tuberculosis, or antibodies against mycolic acid or components thereof.

6. The method as claimed in claim 1, in which the mycolic acid antigens are in a form selected from homogeneous or heterogeneous compound mixtures.

7. The method as claimed in claim 1, in which the diagnostic sample is selected from blood samples, spinal fluid samples and samples that naturally contain antibodies.

8. The method as claimed in claim 1, in which the diagnostic sample is from an HIV-positive human or animal.

9. The method as claimed in claim 1, in which the antibodies are low-affinity antibodies.

10. The method as claimed in claim 1, in which the respective exposure of the control sample and the test sample to the liposomes includes pre-incubating the control sample with the liposomes containing mycolic acid antigens and pre-incubating the test sample with the liposomes not containing mycolic acid antigens.

11. A test kit for use in diagnosing tuberculosis by detecting surrogate marker antibodies indicating active tuberculosis, the kit including one or more screen-printed electrodes containing isolated mycolic acid antigens of tuberculous origin or a synthetic analogue thereof immobilised by hydrophobic interaction with the screen-printed electrodes, isolated mycolic acid antigen of tuberculous mycobacterial origin or a synthetic analogue thereof, buffer forming reagents, redox probe forming reagents and liposome forming reagents.

12. The test kit of claim 11 wherein the one or more screen-printed electrodes are configured to be electrically connected to an electrical impedance measuring circuit that detects surrogate marker antibodies indicating active tuberculosis based on two sequential measurements of electrical impedance of the one or more screen-printed electrodes, and the kit does not include any further component to allow for a wash step between the measurements.

13. The test kit of claim 11 wherein the one or more screen-printed electrodes are each configured to be electrically connected to an electrical impedance measuring circuit that detects surrogate marker antibodies indicating active tuberculosis based on two sequential measurements of electrical impedance of the one or more screen-printed electrodes, and the kit is structured to disallow a wash step between the two sequential measurements.

14. The test kit of claim 11 wherein the one or more screen-printed electrodes are each configured to be electrically connected to an electrical impedance measuring circuit that detects surrogate marker antibodies indicating active tuberculosis based on plural sequential measurements of electrical impedance of the one or more screen-printed electrodes without any wash step between the plural sequential measurements.

15. A test kit for use in diagnosing tuberculosis by detecting surrogate marker antibodies indicating active tuberculosis, the kit consisting of at least one screen-printed electrode containing isolated mycolic acid antigens of tuberculous origin or a synthetic analogue thereof immobilised by hydrophobic interaction with the screen-printed electrodes, isolated mycolic acid antigen of tuberculous mycobacterial origin or a synthetic analogue thereof, buffer forming reagents, redox probe forming reagents and liposome forming reagents, the at least one screen-printed electrode configured for electrical measurements of plural electro-impedances thereof with no required washing of the at least one screen-printed electrode between the electrical measurements of the plural electro-impedances.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows Dropsens™ [Llanera (Asturias), Spain] screen-printed electrodes without and with insulating layer with a conductive layer (a) and an insulating layer (b);

(2) FIG. 2 shows scanning electron microscope images of “ink” cured at high temperature on screen-printed electrodes (Dropsense™ Screen-Printed Gold Electrodes catalogue);

(3) FIG. 3 shows scanning electron microscope images of “ink” cured at low temperature on screen-printed electrodes (Dropsense™ Screen-Printed Gold Electrodes catalogue);

(4) FIG. 4 shows a typical cyclic voltammogram of potential (E) in millivolts (mV) against current (i) in micro Amperes (μA) for the binding of a complexed SAM onto a gold electrode in 5 mM K.sub.3[Fe(CN).sub.6] solution at a scan rate of 10 mV/s; (a) gold plus thiocitic acid, (b) gold plus thiocitic acid plus antibody, (c) gold plus thiocitic acid plus antibody plus 1-dodecanethiol (Berggren & Johansson 1997);

(5) FIG. 5 shows a Randle's equivalent circuit (a) which describes the response of a single-step charge-transfer process with diffusion of reactants and/or products to the interface and its associated Nyquist plot (b); C.sub.dl is the capacitance of a double layer, R.sub.s is solution resistance, R.sub.ct is charge transfer resistance (speed of electron transfer), and Z.sub.w is Warburg impedance (electrolyte resistance); for the Nyquist plot (b). Z′ is real frequency and Z″ is imaginary frequency. The positive slope at mass transfer control is during the diffusion process (linear or radial) (Dijksma, 2003, Barsoukov & MacDonald, 2005, p70)

(6) FIG. 6 is a Nyquist plot (Z.sub.imaginary against Z.sub.real) showing an increase in R.sub.ct during immobilization steps of (a) electro-deposition of gold layer (b) assembly of 11-MUA monolayer (c) immobilization of anti-human IgG (d) binding of human IgG antigen (e) regeneration (Zhang et al., 2009):

(7) FIG. 7 shows mean values of percentage binding inhibition of TB negative (ASPA004) and TB positive (ASPA019) patient sera using solvent resistant electrodes. Error bars represent the standard deviation, n=4.

DETAILED DESCRIPTION OF EXAMPLE EMBODIMENTS

Example 1

(8) Detection of Low Affinity Anti-Mycolic Acids Antibodies

(9) Materials

(10) Mycolic acids were purchased from Sigma Aldrich, Germany. Assay reagents were purchased from Sigma. Ethanol, acetone, chloroform, hexane and dimethylformamide were purchased from Merck South Africa.

(11) PBS buffer (20×) consisted of: 160 g NaCl, 4 g KCl, 4 g KH.sub.2PO.sub.4, 4 g anhydrous Na.sub.2HPO.sub.4, dissolved in 1 litre dddH.sub.2O. PBS/AE buffer (1×): 0.25 g NaN.sub.3 and 0.3802 g EDTA were dissolved in 50 ml 20× PBS buffer and 800 ml dddH.sub.2O, adjusted to pH 7.44 with 1 M acetic acid and made up to a final volume of 1 litre with dddH.sub.2O, before filtering through 0.22 μm filter disc using a Solvac® filtration system.

(12) A 1 mM solution of hexacyannoferrate (Ferri/Ferrocyanide) i.e. [Fe(CN).sub.6].sup.4−/[Fe(CN).sub.6].sup.3− (from Bio-zone Chemicals, South Africa) was made up in 1× PBS/AE buffer. Analysis was done using an Autolab PGstat302 (Autolab, the Netherlands) with the GPES and FRA software.

(13) (4) The blood samples collected from HIV positive patients were delivered freshly to the laboratory for serum preparation. Samples were kept at 15° C. before they were processed within eight hours after sampling. After the blood clotted (2-4 hours after sampling), serum was removed from the blood clot using plastic Pasteur pipettes and transferred to 1.5 ml Eppendorf tubes. The serum samples were then centrifuged in a microfuge (360 g, 5 minutes, 4° C.) to remove any cellular matter still present. The serum samples were then aliquoted (500 μL portions) into 1.8 ml cryotubes (NUNC© Brand products, Nunc international, Denmark) and stored at −70° C. When an adequate number of samples were obtained in this way, they were thawed once more and γ-irradiated (30 Gy, 5 minutes on each side of the box, Pretoria Academic Hospital) as a safety precaution (Vermaak, 2005), in order to inactivate any viral activity. From this step, samples were finally frozen at −70° C. until used for MARTI-analysis. Here, a MARTI positive (ASPA019) and a MARTI negative control (ASPA004) were used,

(14) Methods

(15) Electra-analysis: Bare screen-printed electrodes [DRP-220AT, Dropsens, Llanera (Asturias), Spain] were briefly polished, rinsed in hexane and characterized in 0.1 mM Ferri/Ferrocyanide in PBS/AE (the redox probe solution). Cyclic voltammetry was performed between a window potential of −0.336 V to 0.6 V with a step potential of 0.00244 V and a scan rate of 0.025 V/s. This allowed for the determination of the E.sub.1/2 of each specific electrode in the flow cell. By performing a peak search the E.sub.1/2 was found to be about 0.15 V. The analysis was performed using the EIS (frequency response analysis) on FRA software (Metrohm Autolab, the Netherlands) at the E.sub.1/2 of the cell from 2 kHz to 1 Hz.

(16) Electrodes were then coated with a 0.1 M ODT-hexane solution, rinsed in absolute ethanol and then coated in 40 μl of a 0.5 mg/ml MA-hexane solution. This was washed with absolute ethanol again and then analysed to determine the baseline using the EIS technique at the E.sub.1/2 mentioned in (1),

(17) Serum samples were diluted and incubated with liposomes to produce a “control step” solution and a “test step” solution.

(18) Inhibition studies were performed using a patient serum that was left at room temperature to thaw completely. A 20 minute pre-incubation of 1/500 dilutions of serum with solutions of liposomes containing mycolic acids “control step” or liposomes not containing mycolic acids “test step” was allowed. These were then sequentially injected for binding inhibition studies to the electrode surface. Control and test solutions were allowed to bind for 10 min as can be seen in (4) and (5) respectively.

(19) (4) “Control step”. After incubation the inhibited sample (the diluted serum sample with MA containing liposomes and redox probe in PBS/AE buffer) was pumped onto the surface, which displaced the redox probe solution. The “control step” solution was allowed to bind for a brief fixed period and then analysed by EIS.

(20) (5) “Test step”: The “control step” solution was then displaced by the “test step” (non-inhibited sample) i.e. the serum sample that had not been exposed to antigen and in which antibody binding activity remains maximal. The signal was analysed a third time, after binding was allowed to occur over the same time as in (4). The difference between the inhibited and non-inhibited sample was compared.

(21) (6) If a substantial signal difference between the control and test samples was detected the patient was considered TB positive.

(22) Results and Discussion

(23) Patients with active TB will exhibit increased levels of antibodies to mycobacterial cell wall mycotic acids. The antibodies have previously been detected by various techniques including ELISA, waveguide and resonant mirror biosensors. Results with ELISA published by Schleicher et al. (2002) provided 57% accuracy. With the resonant mirror biosensor 82% accuracy was achieved for detection biomarker anti-mycotic acids antibodies (Thanyani et al., 2008). The Applicant has now shown that the principles of an inhibition assay in the MARTI assay as described by Verschoor et al. (2005) can be applied to detect a response by means of an amperometric technique, namely electrochemical impedance spectroscopy (EIS). The ability to distinguish between TB-positive and TB-negative sera occurs as a result of a difference in bound antibody to a surface.

(24) To investigate if the principles of MARTI could be incorporated into the EIS system the Applicant investigated the addition of the redox probe to the serum dilution, together with either the inhibiting or non-inhibiting liposomes. In theory this would be expected to eliminate the need for a washing step after antibody exposure, thereby retaining the ability to detect the binding of low affinity biomarker antibodies. The liposomes were made as described (Thanyani et al., 2008), but a redox probe containing PBS/AE (0.1 mM Ferri/Ferrocyanide in PBS/AE) was used as the suspension liquid. The serum dilutions and liposomes were prepared in the presence of the redox probe. The PBS/AE buffer containing the redox probe was also used during the inhibition step and during measurements after immobilisation onto the electrode surface. A second measurement was performed after exposure of non-inhibited serum sample to the sensor surface. This sample comprised of serum, redox probe, PBS/AE buffer and liposomes not containing MA. Without being limited thereto, the Applicant believes that this could take place by a mechanism in which unbound antibodies and liposomes from the “control step” are removed from the electrode simultaneous with injection of the “test step” solution. The Applicant also believes that the method of the embodiments herein can be automated.

(25) The difference between TB-negative and TB-positive serum was observed by a difference in percentage inhibition of antibody binding to sensor surface immobilized MA calculated from R.sub.ct values. In this instance, an increase in charge transfer resistance was an indicator of antibody binding to mycolic acids on the surface. This occurred due to the formation of an immuno-complex of immobilized antigen (mycolic acids) and antibody from the serum sample. Because the mycolic acids were immobilized in the SAM across the electrode surface, the immune-complex lowered the capacitance by increasing the distance between the outer layer and the electrode, and thereby increased the resistance to charge transfer (R.sub.ct). This hindered the interfacial electron transfer kinetics at the electrode-electrolyte interface (Hays et al., 2006).

(26) The data from FIG. 7 indicated a near 50% difference in signal between TB-positive and TB-negative sera that was statistically significant. This represented the degree to which anti-mycolic acid antibodies bound to the sensor surface in TB-infected serum. The results suggested that for the purpose of TB diagnostics, antibody binding to mycolic acids using SPEs can only be used for MARTI TB testing purposes if the solvent resistant SPEs are used that were designed specifically for the purpose of the technology herein.

(27) In conclusion, Mathebula et al. (2009) and Ozoemena et al. (2010) paved the way for the use of EIS in TB serodiagnosis, providing the groundwork and principles. The Applicant has now shown that a high sample throughput diagnosis can be achieved by alleviating tedious electrode polishing, costly electrodes and complex carbodiimide surface chemistry. Previously an EIS TB test based on anti-MA antibody detection could take up to one week. An example method of the present technology allows this time to be cut back to one day using screen-printed electrodes, with the potential to decrease this to under an hour. Screen-printed electrodes have been used previously in an attempt to diagnose TB (Diaz-Gonzalez et al., 2005). Their system made use of carbon screen-printed electrodes, an enzyme-dependent sandwich assay, highly diluted serum sample (1:10 000) and 2×90 minute incubation steps. Their potential time to diagnosis is longer and limited to detecting high affinity antibodies because of the necessity to separate free from bound antibodies by means of a wash step. Limited published information pertains to solvent resistant electrodes for immunosensors. In 1997, Kröger & Turner investigated the use of solvent resistant carbon screen-printed electrodes for the detection of hydrogen peroxide. Potyrailo et al., (2004) used solvent resistant electrodes to assess polymer-solvent interactions. The Applicant is, however, not aware of the application of solvent resistant screen-printed electrodes for the diagnosis of TB.

(28) The technology herein thus provides a real-time antibody detection method. A real time detection method means the actual time during which an antibody binds to antigen and the event is recorded. For the MARTI diagnostic this terminology refers to the detection of antibody binding to immobilized antigen at any time during the process without the need to wash away any excess of unbound antibody. In the present embodiment as opposed to surface plasmon resonance biosensors that allow the continuous recording of antibody binding to immobilized antigen over time, EIS measures antibody binding at particular time stamps after exposure to antigen, but still does not require a washing step to remove unbound antibody to enable recording of the events.

(29) The method of the technology herein accordingly provides a novel approach for tuberculosis diagnostics and the Applicant is of the view that the method may lead to point-of-care devices for TB diagnostics.

BIBLIOGRAPHY

(30) Barsoukov, E., Macdonald, J. R., 2005. Impedance spectroscopy theory, experiment and applications (Second Edition), Chapter 3. Measuring techniques and data analysis, McKckubre M. C. H, Macdonald, D. D., (Eds.) John Wiley & Sons Inc. (USA). Benadie, Y., Deysel, M., Siko, D. G. R., Roberts, V. V., Van Wyngaardt, S., Thanyani, S. T., Sekanka, G., Ten Bokum, A, M. C., Collett, L. A., Grooten, J., Baird, M. S., Verschoor, J. A., 2008. Cholesteroid nature of free mycolic acids from M. tuberculosis. Chemistry and Physics of Lipids 152, 95-103. Berggren, C., Johansson, G., 1997. Capacitance Measurements of Antibody-Antigen Interactions in a Flow System. Analytical Chemistry 69, 3651-3657. Campuzano, S., Pedrero, M., Montemayor, C., Fatás, E., Pingarrón, J. M., 2006. Characterization of alkanethiol-self-assembled monolayers-modified gold electrodes by electrochemical impedance spectroscopy. Journal of Electroanalytical Chemistry 586, 112-121. Das, M., Sumana, G., Nagarajan, R., Malhotra, B. D., 2010. Zirconia based nucleic acid sensor for M. tuberculosis detection, Applied Physics Letters 96, 133703-133706 Diaz-González, M., González-Garcia, M. B., Costa-Garcia, A., 2005. Immunosensor for M. tuberculosis on screen-printed carbon electrodes. Biosensors and Bioelectronics 20, 2035-2043. Dijksma, M., 2003. Development of electrochemical immunosensors based on self-assembled monolayers, PhD Thesis, Farmacie. Universiteit Utrecht, Utrecht. Duman, M., Erhan, P., 2010. Detection of Mycobacterium tuberculosis complex and Mycobacterium gordonae on the same portable surface plasmon resonance sensor. Biosensors and Bioelectronics 26, 2, 908-912. Garcia-González, R., Fernández-Abedul, M. T., Pernia, A., Costa-Garcia, A., 2008. Electrochemical characterization of different screen-printed gold electrodes. Electrochimica Acta 53, 3242-3249. Gebbert, A., Alvarez-Icaza, M., Stoecklein, W., Schmid, R. D., 1992. Real-time monitoring of immunochemical interactions with a tantalum capacitance flow-through cell. Analytical Chemistry 64, 997-1003. Hays, H. C. W., Millner, P. A., Prodromidis, M. I., 2006. Development of capacitance based immunosensors on mixed self-assembled monolayers. Sensors and Actuators B: Chemical 114, 1064-1070. He, F., Zhao, J., Zhang, L., Su, X., 2003. A rapid method for determining M. tuberculosis based on a bulk acoustic wave impedance biosensor. Talanta 59, 935-941. He, F., Zhang, L., Zhao, J., Hu, B., Lei, J., 2002. A thickness shear mode immunosensor for detection of M. tuberculosis with a new membrane material. Sensors and Actuators B: Chemical 85, 284-290. Kröger, S., Turner, A. P. F., 1997. Solvent-resistant carbon electrodes screen printed onto plastic for use in biosensors, Analytica Chimica Acta 347, 9-18. Lemmer, Y., Thanyani, S. T., Vrey, P. J., Driver, C. H. S., Venter, L., van Wyngaardt, S., ten Bokum, A. M. C., Ozoemena, K. I, Pilcher, L. A., Fernig, D. G., Stoltz, A. C., Swai, H. S., Verschoor, J. A., 2009. Detection of antimycolic acid antibodies by liposomal biosensors, Methods in Enzymology 464, 79-104. Mathebula, N. S., Pillay, J., Toschi, G., Verschoor, J. A., Ozoemena, K. I., 2009. Recognition of anti-mycolic acid antibody at self-assembled mycolic acid antigens on a gold electrode: A potential impedimetric immunosensing platform for active tuberculosis. Chemical Communications 23, 3345-3347. Ozoemena, K., Mathebula, N. S., Jeseelan, P., Toschi, G., Verschoor, J. A., 2010. Electron transfer dynamics across self-assembled N-(2-mercaptoethyl)octadecanamide/mycolic acid layers: impedimetric insights into the structural integrity and interaction with anti-mycolic acid antibodies. Physical Chemistry Chemical Physics 12, 345-357. Pang, P., Cai, Q., Yao, S., Grimes, C. A., 2008. The detection of M. tuberculosis in sputum sample based on a wireless magnetoelastic-sensing device. Talanta 76, 360-364. Potyrailo, R. A., Morris, W. G., Wroczynski, R. J., McCloskey, P. J., 2004. Resonant multisensor system for high-throughput determinations of solvent/polymer interactions. Journal of Combinatorial Chemistry 6, 869-873. Ren, J., He, F., Yi, S., Cui, X., 2008. A new multi-channel series piezoelectric quartz crystal for rapid growth and detection of M. tuberculosis. Biosensors and Bioelectronics 24, 403-409. Schleicher, G. K., Feldman, C., Vermaak, Y., Verschoor, J. A., 2002. Prevalence of anti-mycolic acid antibodies in patients with pulmonary tuberculosis co-infected with HIV, Clinical Chemistry and Laboratory Medicine 40, 882-887. Thanyani, S. T., Roberts, V., Siko, D. G. R., Vrey, P., Verschoor, J. A., 2008. A novel application of affinity biosensor technology to detect antibodies to mycolic acid in tuberculosis patients. Journal of Immunological Methods 332, 61-72. Tudorache, M., Bala, C., 2007. Biosensors based on screen-printing technology, and their applications in environmental and food analysis. Analytical and Bioanalytical Chemistry 388, 565-578. Vermaak Y. 2005. Properties of anti-mycolic acids antibodies in human Tuberculosis patients. MSc dissertation, Faculty of Natural and Agricultural Sciences, University of Pretoria, Pretoria. Verschoor, J. A., Lenaerts, A., Johannsen, E. (1998) A composition comprising a carrier and a purified mycobacterial lipid cell-wall component and its use in the prevention, treatment and diagnosis of disease. Patent Application no. PCT/GB98/00681; U.S. Pat. No. 7,122,577. Verschoor, J. A., Siko, D. G. R. & Van Wyngaardt, S. (2005). A serodiagnostic method to detect antibodies to mycolic acid in tuberculosis patients as surrogate markers for infection. International patent application no. PCT/IB2005/051548, U.S. Pat. No. 7,851,830 B1; ZA 2006/09113. Weyer, K., Mirzayev, F., van Gemert, W., Gilpin, C., 2011. Commercial serodiagnostic tests for diagnosis of tuberculosis: policy statement, World Health Organisation, WHO/HTM/TB/2011.5. Zhang, Y., Wang, H., Nie, J., Zhang, Y., Shen, G., Yu, R., 2009. Individually addressable microelectrode arrays fabricated with gold-coated pencil graphite particles for multiplexed and high sensitive impedance immunoassays. Biosensors and Bioelectronics 25, 34-40.