Micellar mycolate coated carbon electrodes for electrochemical impedance immunoassays

Abstract

A method of forming a solution for a mycolic acid antigen immobilized on a substrate is provided. The method comprises heating a mixture of mycolic acid and a polar organic solvent to a temperature above the melting point of mycolic acid, thereby preparing a solution of a mycolic acid antigen in a polar solvent, where the solution is a micellar solution.

Description

Technical Field

[0001] The present invention is in the field of disease diagnostic devices and methods for using the devices to detect TB biomarker antibodies to antigens in human and animal blood and tissue samples. In particular, the present invention relates to an improved method for preparing a substrate, such as an electrode, coated with immobilized mycolic acid antigens for impedance detection of mycolic acid biomarker antibodies, wherein the mycolic acid antigens serve as capture agents for the biomarker antibodies. Background Art

[0002] Global TB diagnostic needs

[0003] Although curable, tuberculosis (TB) remains the leading cause of death from infectious diseases worldwide. It is caused by Mycobacterium tuberculosis (Mtb). The latest statistics released by the World Health Organization (WHO) in 2021 show that there are more than 1.5 million TB deaths, of which approximately 1.3 million are HIV-negative patients and 200,000 are HIV-positive patients. It is estimated that 1.7 billion people worldwide coexist with latent TB (asymptomatic infection with Mtb), of whom approximately 10% may develop active (symptomatic) TB within two years of initial exposure. The risk of latent TB reactivation is very high in individuals infected with HIV.

[0004] Through its End TB Strategy, WHO aims to reduce TB-related mortality by 95% and incidence by 90% between 2015 and 2035. This will be achieved through more effective vaccines, treatment regimens, and improved diagnostic tests.

[0005] Common diagnostic tools currently in routine clinical use rely primarily on sputum-based tests, which have consistently demonstrated poor sensitivity, particularly in immunocompromised individuals and children who are unable to produce sputum of the required consistency. Diagnostic tests for TB have improved dramatically over the past few years. Despite these advances, no test meets all the necessary regulatory requirements for performance (sensitivity and specificity), ease of use, cost, speed of diagnosis, and the ability to generate same-day results at the point-of-care (POC). The most important contribution to the strategy to end TB will be POC platforms that will make TB diagnosis more accurate, affordable, and widely available to patients and care providers. Despite existing technologies and advances over the past few decades, developing a simple and rapid POC test remains a challenge in the current tuberculosis diagnostic pipeline. To this end, new diagnostic tools and reliable biomarkers in samples other than sputum are needed.

[0006] Advantages of blood-based TB diagnostics

[0007] Blood is an attractive sampling option for TB testing, especially in HIV-infected patients. Blood sampling is easier and more reliable than sputum sampling because the invasiveness of sampling is negligible and only requires a finger prick to obtain blood. Sputum sampling is also insufficient for diagnosing extrapulmonary TB, TB in children, and TB in individuals co-infected with HIV. Blood sampling is better able to overcome these limitations. Many serological tests have been proposed in the past for TB diagnosis. Serological tests include enzyme-linked immunosorbent assays (ELISAs) or lateral flow immunoassays (LFIAs) to detect humoral antibody responses to Mtb antigens by measuring antigen-antibody interactions. Serological tests have the potential to be suitable for and improve diagnosis in resource-limited areas because they are simple and offer the possibility of low-cost, rapid diagnosis with minimal training requirements.

[0008] Antibody biomarkers in TB diagnosis

[0009] Antibodies, or immunoglobulins, are proteins produced by B cells that protect the host from foreign agents such as viruses and bacteria. Antibody-based immunoassays are commonly used for diagnostics. They are based on the affinity and specificity of antibodies to bind and recognize pathogen-associated antigens. Immunoglobulin G (IgG) is the primary blood antibody responsible for secondary immune responses associated with long-term immunity, while immunoglobulin M (IgM) is the primary early blood antibody seen in immune responses to infectious diseases, but generally has a short-lived memory. Immunodiagnostic assays based on the detection of patient IgM and IgG against pathogen-associated antigens in the serum of patients with active TB disease are attractive approaches for rapid point-of-care diagnosis or screening.

[0010] In recent years, research into identifying new biomarkers for TB diagnosis has increased. A good antibody biomarker should be highly specific for the disease, easily detected by standard antibody assays, and able to distinguish between latent and active Mtb infection. Early detection of TB is another important goal of biomarker research, particularly in patients at risk for infection or relapse of active TB. As indicators of disease, antibodies are well-suited for diagnostic purposes due to their renowned sensitivity and specificity. The advantage of detecting antibodies rather than pathogens and their traces is that antibodies are freely available in serum, whereas microorganisms can evade detection by shielding themselves within native cells in various organs of the body.

[0011] Previous work has shown that the production of antibodies to the Mtb antigen mycolic acid (MA), a lipid antigen unique to mycobacteria, could serve as an ideal biomarker for the diagnosis of active TB. This is because these antibodies are produced independently of CD4 helper T cells and are therefore not affected by HIV co-infection. Furthermore, work by Ndlandla (1) using a guinea pig TB infection model has shown that anti-MA antibodies are produced early after infection, are short-lived, and are therefore independent of prior vaccination and diagnostic of active TB even in the presence of HIV co-infection.

[0012] Interference from anti-cholesterol and rheumatoid factor antibodies

[0013] Although MA is an attractive antigen for use in TB diagnostic testing, cholesterol can cause cross-reactivity in TB detection. The stacking structure of cholesterol has been shown to be similar to that of MA, as demonstrated by the indistinguishable binding of amphotericin B (AmB), a cholesterol binder, to both MA and cholesterol. Consequently, the widespread natural anti-cholesterol IgG and IgM antibodies produced by the human body can also recognize MA, leading to cross-reactions that may give false-positive results in TB-negative patients. In addition, some recombinant anti-MA single-chain variable fragments (scFvs) from the Nkuku gene library have been shown to cross-react with cholesterol (2), although the affinity of the recombinant chicken antibodies (gallibodies) for cholesterol is much lower than that for MA.

[0014] Rheumatoid factors (RFs) are antibodies that target the Fc region of IgG. They consist of different isotypes and bind to the fragment crystallizable region (Fc) with varying affinities. Although associated with rheumatoid arthritis, they are also found in over 60% of patients with TB. Through nonspecific cross-linking of IgG, RFs can increase the affinity of low-affinity antibodies to levels that compete with higher-affinity antibodies, thereby interfering with immunoassay accuracy. In active TB, when RFs are also present in the patient's blood, cross-reactivity between MA and cholesterol can be problematic. Therefore, RFs should be controlled for in immunoassays for TB diagnosis using TB biomarker antibody detection.

[0015] MA conformation determined by solvent and surface curvature

[0016] Groenewald et al. (3) demonstrated how MA folding is affected by the solvent in which they are dissolved, allowing different conformations in different solvents. In particular, they determined that the stably folded MA conformation is more hindered in hexane than in water, with the exception of keto-MA, which readily forms the so-called W-conformation in hexane, which Ranchod et al. (2) suggested is likely related to the cholesterol cross-reactive conformer of MA. In addition, the size of the antibody capture particle was shown to be crucial for MA conformation. Baumeister, Shaw, and Verschoor (4) demonstrated that the higher curvature of smaller MA-containing liposomes causes MA to fold into a conformation that better captures anti-MA antibodies, implying that the conformation of MA is also affected by the diameter of the MA-coated antibody capture particle on which MA is immobilized. Therefore, it is important to carefully select the solvent in which MA is dissolved and the properties of the solid-phase sensor surface of the electrode to be coated with MA during the fabrication of MA-coated electrodes.

[0017] MARTI assay for TB diagnosis

[0018] Anti-MA antibodies in human patients were previously considered as biomarkers for the diagnosis of active TB and could potentially be detected by methods suitable for POC TB testing. This idea was first explored by Schleicher et al. (5) who used an ELISA assay and MA as an antigen to detect anti-MA antibodies found in patient sera. The results showed that more anti-MA antibodies were present in TB-positive patients compared to TB-negative patients. As a result of this result, further experiments were conducted to develop diagnostic tests that used MA antigens and anti-MA antibodies to detect active TB (6, 7, 8). The mycolic acid antibody inhibition in real time (MARTI) assay was developed from this concept. MARTI uses immobilized liposomes (carrying MA) on the sensor surface to monitor the binding of anti-MA antibodies. The use of biosensor-based technologies for TB diagnosis has been well demonstrated in waveguide (8) and surface plasmon resonance (SPR) (9) evanescent field biosensors, while proof of concept has also been demonstrated in electrical impedance spectroscopy (EIS) (10, 11). Using this principle, the proposed MARTI test showed positive results in TB diagnosis. However, studies have shown that biosensor technology is difficult and expensive to implement in the laboratory, and the technology remains too advanced to be used to screen large numbers of patient sera in a POC setting.

[0019] The standard ELISA immunoassay is an ineffective tool for TB diagnosis because it inherently records only the highest affinity antibodies binding to antigen in the serum. This is because the washing steps required for the ELISA remove low-affinity antibodies. The MARTI test has higher sensitivity and specificity than the ELISA assay because it does not require a washing step after contacting the sample with the immobilized MA antigen (9, 12). Therefore, the main advantage of the MARTI test is that it can sensitively detect low-affinity antibodies, making it a more accurate diagnostic test.

[0020] Point-of-care version of the MARTI assay

[0021] EIS is more suitable than SPR evanescent field biosensing as a transduction technology for antibody binding detection in MARTI for point-of-care diagnostics because it does not require complex benchtop instrumentation. Signal processing can now be performed using handheld, battery-powered potentiostats. These potentiostats are essentially maintenance-free, unlike SPR, which has moving parts that require periodic maintenance. Point-of-care TB diagnostic devices must be affordable, accurate, simple to use, require minimal biospecimen, be sensitive and specific, be easy to read, enable rapid diagnosis, and produce same-day results. MARTI tests based on EIS, using affordable, disposable electrodes, have the potential to meet these requirements.

[0022] MA antigens have previously been immobilized from hexane solutions onto solvent-resistant screen-printed gold electrodes (where “screen-printed electrodes” may be abbreviated as “SPEs”) coated with octadecylthiol (ODT) to provide a self-assembled monolayer on the gold, a requirement for sensitive detection of bound ligands by EIS. However, this antigen immobilization method on MAs is poorly reproducible and wasteful. Therefore, there is a need to identify a more reproducible and affordable protocol for immobilizing MA antigens on SPEs. Furthermore, nanoparticle antibody capture agents are required in MARTI tests, and to date have been based on MA-containing liposomes and poly(lactic-co-glycolic acid) (PLGA) particles. Both of these particle types have demonstrated extremely limited stability, preventing their incorporation into point-of-care TB diagnostic tools for field use.

[0023] Electrode Progress: Use of Carbon Electrodes

[0024] Previously reported data indicate that the performance of the MARTI assay is highly variable due to inconsistencies in electrode coating. One scientific publication disclosed that the combination of hexacyanoferrate and gold is an inherently unstable system (13). Organic solvents (typically hexane) have been used for MA coating, and many of these solvents are incompatible with carbon electrodes. As disclosed herein, the use of polar organic solvents (e.g., acetone) for MA coating and their compatibility with carbon electrodes and dielectric masks unexpectedly enabled the modification of screen-printed carbon electrodes, as described herein and exemplified in the Examples.

[0025] When using carbon ink as part of the screen printing process for electrode fabrication, variability is inherent due to contamination and variation during the curing process caused by the impurities and organic binders inherent in the ink and improper removal of these impurities and binders. This variability is evident when comparing impedance measurements of cured electrodes. Impedance data typically range from 600Ω to 4000Ω. This variation in inter-electrode impedance values ​​can negatively impact the performance of MA-coated electrodes. The use of carbon electrodes, as implemented in the present invention, requires the investigation of pretreatment methods to minimize inter-electrode variability. Three alternative pretreatment methods were investigated: non-organic treatment at elevated pH; immersion in an ultrasonic bath; and removal by exposure to several milder organic solvents. The data showed that, surprisingly and without any reasonable expectation of success, the third alternative was the most effective. In particular, polar organic solvents (e.g., acetone) successfully pretreated the carbon electrodes prior to MA coating without damaging the electrodes. Acetone exposure time was titrated to determine an effective operating range. This pretreatment step reduced inter-electrode variability to acceptable levels by reducing the presence of organic impurities used in the electrode production process.

[0026] Purpose of the Invention

[0027] Objects of the present invention include providing EIS detection of TB biomarker antibodies to MA antigens in human and animal blood and tissue samples, where SPE can be reproducibly and affordably coated with MA.

[0028] The present invention also seeks independently but also cumulatively to provide a more reliable method to replace the nanoparticle immunoadsorption step of existing methods, avoiding cross-reactivity and interference of RF antibodies.

[0029] To achieve its objectives, the present invention discloses, inter alia, a novel use of a polar organic solvent to prepare a mycolic acid solution, which is used to prepare a substrate, such as an electrode, to immobilize mycolic acid antigens on the electrode for binding to anti-mycolic acid antibodies to detect active tuberculosis infection in patients. MA, a substance present in the Mtb cell wall, is insoluble in polar solvents such as acetone. Unexpectedly, the inventors discovered that MA forms a micellar solution in acetone upon heating and cooling. This micellar solution can then be used to coat biosensor electrodes with MA. Furthermore, acetone is a polar solvent (unlike n-hexane, which is widely used in similar technologies), and it was advantageously and unexpectedly discovered that MA can be coated on biosensor electrodes for sensitive electrical impedance measurements by self-assembling into a capacitive surface with the desired conformation. This was found to be advantageous compared to earlier technologies using n-hexane because it eliminates the step associated with using n-hexane, which requires pre-coating SPE (particularly gold SPE) with long-chain alkane thiols to provide a surface for antigen immobilization.

[0030] More specifically, when an acetone micelle solution was applied to an electrode surface, the MA unexpectedly folded itself into the desired antigenic conformation to detect and bind to TB biomarker antibodies. Therefore, there was no need to form a primed hydrophobic self-assembled monolayer, as required by previous techniques using n-hexane as a solvent for mycolic acids. Summary of the Invention

[0031] According to a first aspect of the present invention, there is provided a method for forming a mycolic acid antigen solution, the method comprising heating a mixture of mycolic acid and a polar organic solvent to a temperature above the melting point of the mycolic acid, thereby preparing a solution of the mycolic acid antigen in the polar solvent.

[0032] The solution of the mycolic acid antigen in the polar solvent may in particular be a micellar solution.

[0033] The mycolic acid antigen solution can be a solution specifically used to immobilize the mycolic acid antigen on a substrate, so as to bind to anti-mycolic acid antibodies during use. Such anti-mycolic acid antibodies can generally be contained in a blood or tissue sample of a human or animal patient suffering from tuberculosis, and the anti-mycolic acid antibodies can be brought into contact with the substrate on which the mycolic acid antigen is immobilized during use.

[0034] Throughout this specification, the singular term "mycolic acid" should also be understood to include the plural form (i.e., "mycolic acids"), since in most, if not all, instances, mycolic acids will include a combination of two or more different mycolic acids. This is a natural consequence of mycolic acid biosynthesis.

[0035] Furthermore, in this specification, unless expressly stated otherwise or the context clearly requires otherwise, the terms "mycolic acid" and "mycolic acid antigen" are used interchangeably, as mycolic acid is an antigen, more specifically, an Mtb antigen. In this sense, the singular "mycolic acid antigen" should also be understood to include the plural (i.e., "mycolic acid antigens") for the same reasons discussed above for the singular "mycolic acid."

[0036] Furthermore, unless expressly stated otherwise or the context clearly requires otherwise, references to mycolic acids should be interpreted as including not only mycolic acids themselves but also mycolate esters comprising mycolic acids. It follows that anti-mycolic acid antibodies may also be anti-mycolate ester antibodies.

[0037] Mycolate esters are a general term for waxy compounds derived from the cell envelope of Mycobacterium tuberculosis species. More specifically, a mycolate ester can be any salt or ester of a mycolic acid. Examples include α-, keto-, and methoxymycolic acids. Examples of mycolate esters include glycolipids—monosaccharide and disaccharide esters of mycolic acids—including glucose monomycolate, trehalose monomycolate, trehalose dimycolate, or arabinogalactan mycolate.

[0038] The mycolic acid may in particular be derived from tuberculous Mycobacterium, such as Mycobacterium tuberculosis, or may be a synthetic analogue of such a mycolic acid.

[0039] The temperature above the melting point of mycolic acids may be a temperature between 60°C and 90°C.

[0040] The method may comprise cooling the solution of the mycolic acid antigen in the polar solvent to a temperature between 25°C and 35°C.

[0041] The polar organic solvent may be acetone.

[0042] The method may comprise a prior step of forming a mixture of mycolic acids and a polar organic solvent.

[0043] The step of forming a mixture of mycolic acids and a polar solvent may comprise adding mycolic acids to the polar solvent. Such addition may provide a mycolic acid concentration of 0.05 to 0.25 mg / ml in the polar organic solvent. It will be appreciated that such addition may require an appropriate amount and volume to achieve the desired mycolic acid concentration.

[0044] As a second aspect of the present invention, the present invention extends to a solution of mycolic acid antigens in a polar organic solvent prepared according to the method of the first aspect of the present invention.

[0045] The solution may be a micellar solution.

[0046] According to a third aspect of the present invention, there is provided a method of immobilizing a mycolic acid antigen on a substrate, the method comprising applying a solution of the mycolic acid antigen according to the second aspect of the present invention in a polar solvent to the substrate on which the mycolic acid antigen is to be immobilized.

[0047] The mycolic acid antigens may be immobilized on the substrate so as to bind to anti-mycolic acid antibodies during use. Such anti-mycolic acid antibodies may typically be contained in a blood or tissue sample of a human or animal patient suffering from tuberculosis and may be brought into contact with the substrate to which the mycolic acid antigens are immobilized during use.

[0048] The method may comprise the prior step of preparing a solution of mycolic acid antigens in a polar solvent according to the method of the first aspect of the invention.

[0049] After heating the mixture of mycolic acid and polar organic solvent according to the method of the first aspect of the invention, a step of applying a solution of mycolic acid antigens in a polar solvent to a substrate may be performed.

[0050] More specifically, the step of applying the solution of mycolic acid antigens in a polar solvent to the substrate may be performed after heating and after cooling the mixture.

[0051] The step of applying the solution of mycolic acid antigens in a polar solvent to the substrate can be performed, for example, within about 4 hours after such heating and preferably cooling.

[0052] Several aliquots of the solution may be applied sequentially to the substrate.

[0053] According to the present invention, application of the solution to the substrate causes the mycolic acid antigens to self-assemble into a capacitive surface on the substrate as the mycolic acids will appropriately fold themselves into the required antigenic conformation to detect and bind anti-mycolic acid antibodies in a sample that is in contact with the substrate in use.

[0054] The method may include leaving or allowing a solution of the mycolic acid antigen in a polar solvent to dry on a substrate. The substrate is dried to remove all volatiles. The substrate may be dried under high vacuum for a period of up to 16 hours.

[0055] The substrate may be free of a surface-modifying monolayer.

[0056] The method may comprise a subsequent step of blocking non-specific binding sites on the substrate according to the method of the seventh aspect of the invention.

[0057] The substrate may be a conductive substrate.

[0058] The substrate may more particularly be an electrode. Typically, the substrate will comprise the solid surface of the electrode.

[0059] The electrodes may in particular be screen-printed carbon electrodes.

[0060] The screen-printed carbon electrode may be, for example, the screen-printed carbon electrode prepared according to the method of the fifth aspect of the present invention, that is, the screen-printed carbon electrode according to the sixth aspect of the present invention.

[0061] The method may comprise the prior step of preparing a screen printed carbon electrode according to the method of the fifth aspect of the invention.

[0062] Applying the solution to the substrate may include depositing the solution onto the substrate. Such deposition may be performed, for example, by spraying, jetting, printing, dispensing, or the like.

[0063] As a fourth aspect, the present invention extends to a screen-printed carbon electrode having mycolic acid antigens immobilized thereon, the screen-printed carbon electrode being prepared according to the method of the third aspect of the present invention.

[0064] According to a fifth aspect of the present invention there is provided a method of preparing a screen printed carbon electrode for immobilising an antigen thereon in the absence of a surface modification monolayer, the method comprising treating the electrode to which the antigen is to be immobilised with acetone.

[0065] Treating the electrode with acetone may include contacting the electrode with acetone.

[0066] Contacting the electrode with acetone may include:

[0067] Immerse the electrode in acetone, spray the electrode with acetone, or allow acetone to flow over the electrode; and / or

[0068] Rinse the electrode with acetone,

[0069] Wherein, if both immersion and rinsing are to be performed, rinsing shall be performed after immersion.

[0070] The electrodes can be immersed in acetone to remove some of the organic binder and impurities. This typically takes 2 to 7 minutes.

[0071] The electrode can be immersed in acetone and / or rinsed with excess acetone.

[0072] In the case of rinsing, for example, a volume of acetone ≤ 0.5 ml may be used.

[0073] The method may include drying the electrode after treating the substrate with acetone.

[0074] The electrodes can be dried in an oven and cooled. The electrodes can be dried, for example, in an oven at approximately 80°C for 10-30 minutes and allowed to cool to room temperature for 3-7 minutes.

[0075] The method can be performed under conditions wherein the method omits the step of applying the surface-modified monolayer to the electrode.

[0076] The method may include subsequently performing the method of the third aspect of the present invention. The method may also include subsequently performing the method of the seventh aspect of the present invention.

[0077] As a sixth aspect of the present invention, the present invention extends to a screen-printed carbon electrode without a surface-modified single layer prepared according to the method of the fifth aspect of the present invention.

[0078] According to a seventh aspect of the present invention, there is provided a method for blocking non-specific binding sites on a substrate, the method comprising treating the substrate with a protein hydrolysate.

[0079] The protein hydrolysate may be provided as a solution thereof.For example, the solution may comprise 0.1 to 3% w / v of the protein hydrolysate.

[0080] The protein may be casein.

[0081] Treating the substrate with the casein hydrolysate may comprise incubating the substrate in the protein hydrolysate or, more particularly, dispersing the protein hydrolysate onto the substrate in a protein hydrolysate solution.

[0082] The incubation period of the mycolic acid coated electrode in casein hydrolysate may range from 1 to 16 hours.

[0083] The substrate may in particular be a substrate on which antigens from a pathogen have been immobilized.

[0084] For example, the substrate may be a substrate on which the mycolic acid antigen has been immobilized according to the method of the third aspect of the invention. Thus, the method may comprise a preceding step comprising the method of the third aspect of the invention.

[0085] The substrate may be an electrode. In one embodiment of the present invention, the electrode may be a screen-printed carbon electrode.

[0086] For example, the substrate or electrode may be according to the fourth aspect of the invention.Thus, the method may comprise a preceding step comprising the method of the third aspect of the invention.

[0087] As an eighth aspect, the present invention extends to a substrate whose non-specific binding sites have been blocked according to the method of the seventh aspect of the invention.

[0088] According to a ninth aspect of the present invention, there is provided a method for detecting tuberculosis biomarker antibodies in a human or animal blood or tissue sample, the method comprising contacting a substrate on which a mycolic acid antigen has been immobilized according to the method of the third aspect of the present invention or a substrate according to the fourth aspect of the present invention with a sample from a patient suspected of having active tuberculosis, so as to allow any biomarker anti-mycolic acid antibodies in the sample to bind to the immobilized mycolic acid antigen.

[0089] The method may include using electrochemical impedance spectroscopy to measure the extent of antibody binding to immobilized mycolic acid antigens in the sample, wherein any measured signal that is above a previously measured baseline signal for the same substrate is a result of binding of biomarker anti-mycolic acid antibodies to the immobilized mycolic acid antigens and indicates that the patient has active tuberculosis.

[0090] Furthermore, the substrate may be subjected to the method of the seventh aspect of the invention before contacting the substrate with the sample.

[0091] Therefore, the substrate may be a substrate or an electrode according to the fourth aspect or the eighth aspect of the present invention.

[0092] More specifically, according to a ninth aspect of the present invention, there is provided a method for detecting TB biomarker antibodies in human and animal blood and tissue samples, the method comprising:

[0093] incubating a sample from a patient suspected of having active tuberculosis with a rheumatoid factor blocking solution to produce a rheumatoid factor-blocked sample;

[0094] The rheumatoid factor-blocked sample was diluted in redox buffer to produce a dilution of the rheumatoid factor-blocked serum sample;

[0095] Alternatively, if the electrode has not previously been subjected to the method according to the third aspect of the invention to achieve such chemical blocking, non-specific antibody binding sites on the mycolic acid antigen coated electrode prepared according to the third aspect of the invention are preferably chemically blocked according to the seventh aspect of the invention;

[0096] The electrical impedance baseline signal of the chemically blocked mycolic acid-coated electrode in redox buffer was measured using a potentiostat;

[0097] contacting dilutions of rheumatoid factor-blocked serum samples with chemically blocked screen-printed electrodes containing immobilized mycolic acid antigens to allow any biomarker anti-mycolic acid antibodies in each sample to bind to the immobilized mycolic acid antigens; and

[0098] Electrochemical impedance spectroscopy is used to measure the extent of binding of anti-mycolic acid antibodies to immobilized mycolic acid antigens in the sample, wherein any measured signal that is above a previously measured baseline signal at the same electrode is a result of binding of the biomarker anti-mycolic acid antibodies to the immobilized mycolic acid antigens and indicates that the patient has active tuberculosis.

[0099] Anti-cholesterol IgG and IgM antibodies cross-react with MA and may produce false positive results (5). Rheumatoid factor affects the accuracy of immunoassays because it binds non-specifically to IgG and increases the binding of low-affinity antibodies. By treating human blood-derived samples with a rheumatoid factor blocker prior to electrochemical detection of TB biomarker antibodies, the specificity of the assay is improved because the rheumatoid factor blocker inhibits the presence of rheumatoid factor in the sample. The use of a rheumatoid factor blocker eliminates the need for an antibody binding inhibition step using MA-coated antibody capture particles prior to electrochemical endpoint detection of the antibodies. This simplifies the test and results in a direct binding assay that successfully distinguishes TB-positive from TB-negative patient sera, which is not achieved in standard ELISA immunoassays.

[0100] The sample can be incubated with the rheumatoid factor blocking reagent at room temperature for up to 15 minutes.

[0101] Chemical blocking of mycolic acid-coated electrode surfaces can involve incubating the electrodes in an aqueous solution of up to 1% casein hydrolysate for more than 16 hours at room temperature.

[0102] The redox buffer may be 0.05 to 10 mM hexacyanoferrate.

[0103] The redox buffer may comprise the same protein hydrolysate, preferably in the same concentration, as characterized according to the seventh aspect of the invention.

[0104] The potentiostat can be any potentiostat with EIS capability, preferably a handheld potentiostat for convenient use in point-of-care testing.

[0105] The diluted rheumatoid factor blocking sample can be 0.05 to 10 mM [Fe(CN)6] in phosphate buffered saline. 4+ / [Fe(CN)6] 3- Dilutions of serum between 1:100 and 1:1600 in 5% paraformaldehyde (hexacyanoferrate redox probe).

[0106] The screen-printed electrodes can be any carbon electrodes used for impedance analysis.

[0107] Screen-printed electrodes can be disposable electrodes.

[0108] The screen-printed electrodes can be exposed to the mycolic acid solution for a period of time to allow the heated solution to cool, but not so long that the micelles precipitate from the solution.

[0109] According to a tenth aspect of the present invention, there is provided a diagnostic kit for diagnosing tuberculosis in a human or animal subject by electrical impedance spectroscopy, the kit comprising a screen-printed carbon electrode according to the fourth or eighth aspect of the present invention.

[0110] More specifically, according to the tenth aspect of the present invention, there is provided a point-of-care tuberculosis diagnostic kit comprising: an individually packaged, mycolic acid antigen-coated screen-printed carbon electrode according to the fourth or eighth aspect of the present invention; a rheumatoid factor blocking reagent to be added to a sample; a dried protein hydrolysate optionally used to block nonspecific antibody binding sites on the mycolic acid-coated electrode; a redox buffer solution; and standard equipment for measuring electrical impedance spectroscopy.

[0111] The mycolic acid antigen-coated screen-printed carbon electrode may be the electrode prepared according to the third aspect of the present invention, ie, the fourth aspect of the present invention.

[0112] Protein hydrolysates can be used to produce a chemical blocking solution, which can be characterized with reference to the seventh aspect of the invention, preferably comprising 0.1 to 3% w / v protein, such as casein hydrolysate. If the substrate has already been subjected to non-specific antibody binding site blocking according to the seventh aspect of the invention, the protein hydrolysate can be omitted. Otherwise, it will typically be included.

[0113] The redox buffer solution can be 0.05 to 10 mM hexacyanoferrate redox probe to optimize electrical signal strength while minimizing reagent usage.

[0114] Standard equipment may be known to those skilled in the art of electrochemistry and may include a portable or benchtop potentiostat equipped with software for integrating the electrochemical signal.

[0115] As an eleventh aspect of the present invention, the present invention also extends to the use of a polar organic solvent in preparing a mycolic acid antigen solution for immobilizing the mycolic acid antigen on a substrate to bind the antigen to an anti-mycolic acid antibody.

[0116] As a twelfth aspect of the present invention, the present invention also extends to the use of acetone in the preparation of a screen-printed carbon electrode without a surface-modified monolayer to immobilize antigens on the electrode without a surface-modified monolayer.

[0117] As a thirteenth aspect, the present invention also extends to the use of a protein hydrolyzate in blocking non-specific binding sites on a substrate comprising an antigen or antibody immobilized thereon.

[0118] The protein hydrolysate may be casein hydrolysate.

[0119] Such uses of the eleventh, twelfth and thirteenth aspects of the present invention may be according to the corresponding method aspects of the present invention.

[0120] The use of polar solvents such as acetone is advantageously milder than n-hexane typically used for mycolic acid solubilization and surprisingly eliminates the need for a hydrophobic, primed ODT self-assembled monolayer according to the present invention.

[0121] Previously, custom fabrication of gold electrodes was required because they were solvent-resistant and provided strong sulfur-gold bonding via ODT to form a self-assembled monolayer. Since this development eliminates the need for this, carbon electrodes can effectively serve as a more economical and environmentally friendly alternative to gold electrodes.

[0122] Another benefit of carbon electrodes compared to gold electrodes is that they do not require any mechanical polishing, only a pretreatment step to remove organic impurities, which the present invention unexpectedly successfully achieved using acetone. These factors provide the desired electrode reproducibility for the assay. BRIEF DESCRIPTION OF THE DRAWINGS

[0123] The invention will now be described by way of example with reference to the accompanying drawings and the accompanying non-limiting examples.

[0124] In the attached figure:

[0125] Figure 1 Shown are electrical impedance spectroscopy (EIS) readings on carbon electrodes in the Examples: an unmodified carbon electrode from the manufacturer, a carbon electrode treated with sodium carbonate, and a carbon electrode treated with acetone for 5 minutes, with error bars indicating standard deviation (n=3).

[0126] Figure 2 The figure shows thin layer chromatograms of the upper and lower phase fractions of selected tubes in the countercurrent distribution tube train during the purification of mycolic acids from a crude extract of Mycobacterium tuberculosis in the Examples. A is a compound with a specific migration value (Rf) of 1, B is a compound with an Rf of 0.6, and C is a compound with an Rf of 0.1. Lanes indicate tube numbers / CCD upper (U) or lower (L) phase solvents.

[0127] Figure 3The present invention shows how to determine the solubility of MA in acetone by immunoassay (indirect ELISA) using a monoclonal antibody (gallibody) in the examples to evaluate the residual amount of MA in the acetone solution 1 hour and 4 hours after cooling from 90°C to room temperature compared with pure MA of the same initial concentration. The error bars represent the standard deviation, and n=3 (biological replicates);

[0128] Figure 4 The feasibility of spotting MA in acetone (M / A) compared to MA in hexane (M / H) on three substrate types (nitrocellulose, filter paper, and TLC silica gel plates) is shown in the Examples, as determined by immunoblotting using a monoclonal antibody against MA. Spots of MA in hexane are indicated by arrows marked with an "*," and spots of MA in acetone are indicated by arrows marked with an "X." (A) The darker spots on nitrocellulose indicate indentations.

[0129] Figure 5 The figures show a comparison of the antigenicity of MA in acetone (M / A) and MA in hexane (M / H) spotted on a silica gel TLC plate and developed with an anti-MA monoclonal antibody in an immunoblotting assay in the Examples. The spots of MA in hexane are indicated by arrows marked with "*", and the spots of MA in acetone are indicated by arrows marked with "X".

[0130] Figure 6 The absorbance change at 420 nm of 0.25 mg / ml MA in acetone when cooled from 85° C. in the Examples is shown to indicate the dynamic micelle properties of MA in acetone solution;

[0131] Figure 7 The absorbance change at 420 nm of 0.125 mg / ml MA in acetone when cooled from 85° C. in the Examples is shown to indicate the dynamic micelle properties of MA in acetone solution;

[0132] Figure 8 The absorbance change at 420 nm of 0.05 mg / ml MA in acetone when cooled from 85° C. in the Examples is shown to indicate the dynamic micelle properties of MA in acetone solution;

[0133] Figure 9 Figure 2 shows dynamic light scattering of the examples, showing the change in micelle size (nm) over time (minutes) for a 0.1 mg / ml solution of MA in acetone heated at 85°C and then cooled at 25°C. The first data point at t=0 is a blank acetone sample, and the second data point at t=0 is the measurement of MA in acetone immediately after removal from the 85°C heat source.

[0134] Figure 10The figures show the size (in nm) of micelles formed when a 0.1 mg / ml acetone solution of MA in the example was removed from an 85°C heat source and the temperature was lowered to an end point of 37°C to 20°C within 3 minutes, as measured by dynamic light scattering. The error bars indicate the standard deviation, and n=10.

[0135] Figure 11 The electrode pretreated with acetone, coated with a 0.1 mg / ml acetone solution of MA, and blocked with 1% casein hydrolyzate (CasH) for nonspecific antibody binding sites in the embodiment is shown. Error bars represent standard deviations. n=12 before and after acetone pretreatment (acetone washing), n=11 for casein hydrolyzate treatment of MA.

[0136] Figure 12 The comparison of TB-positive and TB-negative sera at dilutions of 1:1000 and 1:200 in the Examples is shown. The fold difference of MVT112 / 116 at a dilution of 1:1000 is 1.3-fold, and the fold difference at a dilution of 1:200 is 1.7-fold. The fold difference of MVT034 / MVT225 at a dilution of 1:1000 is 1.9-fold, and the fold difference at a dilution of 1:200 is 2.2-fold. TB-positive sera n=6, TB-negative sera n=5);

[0137] Figure 13 The advantages between the previous electrode coating process in the examples and the improved system using screen-printed carbon electrodes are shown;

[0138] Figure 14 The advantages of using casein hydrolysate as a chemical blocking agent for MA-coated electrodes (used as a baseline) compared to PVP in the Examples are shown, and impedance values ​​of TB- patient serum and TB+ patient serum on carbon electrodes coated with 0.1 mg / ml of MA in acetone and blocked with 0.01% PVP or 0.01% casein hydrolysate are shown. These values ​​are baseline-corrected, and error bars represent standard deviations. For MA containing 0.01% PVP and MA containing 0.01% casein hydrolysate, n=4, and error bars represent the range between replicate values ​​for TB-positive serum on electrodes blocked with PVP and casein hydrolysate, and replicate values ​​for TB-negative serum on electrodes blocked with PVP and casein hydrolysate; and

[0139] Figure 15 The advantages of mixing a rheumatoid factor blocker solution into a serum sample in the examples to improve the resolution between TB+ and TB- patient sera in EIS analysis are shown, and the impedance values ​​of TB- serum and TB+ serum with and without a rheumatoid factor blocker on a carbon electrode coated with a 0.1 mg / ml acetone solution of MA and blocked with 0.1% casein hydrolysate are compared, with n=2 for all impedance measurements. DETAILED DESCRIPTION

[0140] A method for detecting TB biomarker antibodies is described through selected examples. These examples demonstrate how to overcome limitations of previously published inventions, particularly with respect to fabricating functional MA-coated electrodes for use in assays where MA antigens are presented as capture agents for antibody biomarkers.

[0141] In the Examples, and generally in the specification, the following abbreviations are occasionally used:

[0142] -MA = mycolic acid

[0143] -EIS = Electrical Impedance Spectroscopy

[0144] -TB = Tuberculosis

[0145] - Mtb = Mycobacterium tuberculosis

[0146] -POC = Point-of-care testing

[0147] -ELISA = Enzyme-linked immunosorbent assay

[0148] - LFIA = Lateral Flow Immunoassay

[0149] -IgG = Immunoglobulin G

[0150] -IgM = Immunoglobulin M

[0151] -AmB = Amphotericin B

[0152] -RF = rheumatoid factor

[0153] -RFB = Rheumatoid factor blocker

[0154] -Fc = fragment crystallizable region

[0155] -MARTI = Mycolic acid antibody real-time inhibition

[0156] -SPR = Surface Plasmon Resonance

[0157] -SPEs = Screen Printed Electrodes

[0158] -ODT = Octadecanethiol

[0159] -PLGA = poly(lactic-co-glycolic acid)

[0160] -Rf = ratio shift value

[0161] -U=up

[0162] -L = Down

[0163] -M / A = MA in acetone

[0164] -M / H = MA in hexane

[0165] -CasH = casein hydrolysate

[0166] -Ac wash = acetone pretreatment

[0167] -PBS = Phosphate buffered saline

[0168] -OD = Optical density

[0169] -CCD=Countercurrent Distribution

[0170] -DLS = Dynamic Light Scattering

[0171] -PVP = Polyvinylpyrrolidone

[0172] Example 1

[0173] Pretreatment of carbon electrodes

[0174] To minimize inter-electrode variation and improve the reproducibility of the EIS signal, three groups of carbon electrodes were tested: an unmodified electrode, an electrode treated with Na2CO3, and a carbon electrode treated with acetone. The electrodes were exposed to a saturated aqueous Na2CO3 solution and subjected to three electrochemical cycles between -0.4 and 0.4 V at 23°C, or immersed in acetone at 23°C for 2 to 7 minutes, in this example 5 minutes, followed by a rinse with 500 μl of acetone. Impedance data were generated using a carbon electrode that was exposed to a redox buffer hexacyanoferrate (0.05 mM potassium ferrocyanide (II) and potassium ferricyanide [Fe(CN)6]) in phosphate-buffered saline (PBS) containing 3.8 mM sodium azide and 1 mM EDTA, as described in Example 4, without the addition of a stabilizer. 4- / [Fe(CN)6] 3- )middle.

[0175] result

[0176] data( Figure 1 ) showed that treatment with acetone successfully reduced inter-electrode variation, as indicated by the reduction in error bar size when the electrodes were pretreated with acetone. As expected, effectiveness showed a distribution over time, but the acetone exposure time was titrated to an optimal level of approximately 5 minutes, although the effect could be achieved in a range of 2 to 7 minutes.

[0177] in conclusion

[0178] Pretreatment of screen-printed carbon electrodes with acetone improved the electrode-to-electrode reproducibility in preparing MA coatings.

[0179] Example 2

[0180] Solubility and Antigenicity of Mycolic Acids in Acetone

[0181] 1. Materials and Methods

[0182] 1.1MA purification

[0183] 1.1.1. Saponification of Crude Bacterial Extracts

[0184] To release MA from the mycobacterial cell wall, a homogenized bacterial cell pellet cultured according to the conditions established by Ndlandla et al. (6) was resuspended in 300 ml of saline. The bacterial resuspension was allowed to settle to the bottom of a Schott flask, and 250 ml of saline was removed. The bacterial cells were resuspended in the remaining saline and divided into equal parts, which were then centrifuged at 1500×g for 20 minutes. The remaining saline was removed by decantation, ensuring that the remaining bacterial pellet was not disturbed. The pellet was resuspended in reagent A to obtain an optical density (OD) of approximately McFarland standard 4, i.e., an OD of 0.837 measured using a spectrophotometer at 486 nm.

[0185] Saponification was performed using autoclavable 2.5 L tightly closed Schott bottles equipped with red heat stable caps and rings. The Schott bottles were wrapped in foil and incubated overnight in a 70°C water bath covered with foil.

[0186] 1.1.2. Crude extracts of mycolic acids

[0187] The saponified cell suspension in Reagent A was funnel-extracted to obtain a crude MA extract. After cooling to room temperature, 1.5 ml of Reagent B (HCl (32% (v / v)) dissolved in double-distilled deionized water (dddH2O) at a volume ratio of 1:1) was added for every 2 ml of Reagent A (KOH (25% (m / v)) dissolved in a 1:1 volume ratio of methanol-water) used. The cell suspension was carefully shaken in a 2.5 L reagent bottle until no fumes were present, then shaken vigorously to ensure uniform mixing. The pH of the suspension was adjusted to 1 using Reagent B to protonate all MA and dissolve it in chloroform. . Add chloroform (600 ml) and shake the suspension vigorously in a tightly covered reagent bottle for 10 minutes. Transfer the crude MA extract to a separatory funnel and allow it to separate into two phases at room temperature overnight. Collect the lower phase containing the crude MA extract and evaporate it at 60°C in a pre-weighed round-bottom flask using a rotary evaporator. Add acetone (5-10 ml) to completely dry the crude MA extract. Repeat this process until the crude MA extract is completely dry. Store the dried crude MA at 4°C for further use.

[0188] 1.1.3. Crude extraction of MA in a phase solvent without saline

[0189] Chloroform, methanol, and water were mixed in a 2.5 L reagent bottle in a ratio of 42:39:19. The solvent was shaken vigorously to form two phases in a separatory funnel. The two phases were collected and stored separately. The crude MA extract was reconstituted with equal volumes of the lower phase solvent and the upper phase solvent. The two phases were mixed together and transferred to a separatory funnel, shaken vigorously, and allowed to separate completely at room temperature. The lower phase extract was collected and evaporated at 60°C in a pre-weighed round-bottom flask on a rotary evaporator. Acetone (5-10 ml) was added to completely dry the crude MA extract. This process was repeated until the crude MA extract was completely dry. The crude MA was stored in a desiccator with indicating silica gel as a desiccant at room temperature overnight to ensure that all moisture was removed before weighing. The dried crude MA was stored at 4°C for further use.

[0190] 1.1.4. Purification of MA by countercurrent distribution

[0191] Prepare the phase solvent for countercurrent distribution (CCD) by mixing chloroform, methanol, and 0.2 M sodium chloride in a ratio of 42:39:12. Allow the solvent to form two phases in a separatory funnel at room temperature. Extract and save each phase solution separately.

[0192] The crude MA extract was purified using the countercurrent distribution (CCD) method previously described by Goodrum et al. (14). The CCD instrument was constructed using an array of positions representing a test tube chain and consisted of four interconnected glass test tube racks.

[0193] The principle is as follows: equal proportions of each phase (upper phase solution and lower phase solution) are loaded into each test tube. The lower phase of the two-phase solvent system is the "stationary phase" and the upper phase is the "mobile phase". The process starts with test tube No. 0, which contains the mixture of substances to be separated in the lower phase solvent. All other test tubes are loaded with the same volume of the same solvent. The upper phase solvent is added to test tube No. 0, where it is equilibrated and the phases are allowed to separate. The upper phase of test tube No. 0 is then transferred to test tube 1, and new solvent is added to test tube No. 0, after which the two phases are allowed to equilibrate again. After each separation cycle, the clear upper phase is moved from the first test tube to the adjacent test tube along the test tube rack. In each transfer cycle, a clean upper phase solution is added to the first test tube, which means that after 60 cycles, the initial volume of the upper phase added to test tube No. 0 will end up in test tube 60.

[0194] Before purifying crude MA, the tubes of the CCD were cleaned by running the device in wash mode, followed by 1 L of 10% Contrad TMSolution, 2L of dddH2O to rinse out the Contrad solution and 1L of 96% ethanol to rinse the test tubes. Invert the device to drain all liquids and ensure it is completely dry before use. First load the lower phase in each test tube. Load the crude MA extract into the first test tube of the CCD device. Rinse the crude MA sample bottle with the lower phase containing saline to ensure quantitative transfer of MA to the CCD device. Fill the first two test tubes of the CCD with the crude MA sample solution and adjust each volume to exactly 10ml using clean CCD lower phase solvent. Add the lower phase to the first test tube to balance to the third test tube, then readjust to 10ml and balance to the fourth test tube. In this way, the volume of each of the first four test tubes is 10ml, and the fifth test tube has approximately 5ml remaining after balancing.

[0195] The CCD upper phase solvent (650ml) that is enough for 60 circulations is loaded into the large upper phase tank of machine. Manually the upper phase CCD solvent containing normal saline of 10ml volume is transferred to No. 0 test tube. Then, after the 5th test tube, every other test tube is loaded with 10ml CCD lower phase solvent. Manually mix content 20 times by shaking machine, and make content be separated into two phases in static position. This is first circulation. Clean upper phase is transferred to first test tube to start second circulation, repeats this process, until 60 circulations are completed. In initial first test tube, upper phase has absorbed some lower phases, by in each cycle, adding the clean lower phase CCD solvent containing normal saline to No. 0 test tube and correcting absorption, until no longer needed. By regulating the rod that controls the volume of the upper phase that enters test tube, the volume of upper phase is adjusted to just in time 10ml.

[0196] 1.1.5 Analysis of CCD-purified MA using TLC

[0197] To analyze the purity and distribution of MA, 10 ml of the lower phase sample and 10 ml of the upper phase sample from every other identical tube among 60 test tubes were spotted on a TLC plate.

[0198] TLC was performed as follows. The MA sample (10 ml) was loaded onto an aluminum backed TLC silica gel plate by spotting on a pencil line 1 cm from the bottom of the silica gel plate using a Hamilton syringe. The spot was allowed to dry at room temperature. After the spot was dried, the silica gel plate was placed in a TLC developing tank, which contained a mobile solution of ether (30% (v / v)) dissolved in hexane, and the mobile solution was pulled up by capillary action until it reached approximately 2-3 cm from the top of the TLC plate. Subsequently, the plate was immersed in a visualization reagent container for 5 seconds, which in this case was the developing solution. The plate was removed from the developing solution using tweezers, placed on laboratory tissue paper, and immediately scorched with a heat gun. The plate was analyzed and the retention factor value was calculated. Thus, the MA in test tubes 0-15 was identified. The lower and upper phases of test tubes 0-15 were removed from the CCD test tubes, combined, and evaporated at 80°C until substantially dry.

[0199] 1.1.6. Acetone precipitation of CCD-purified MA

[0200] To achieve the correct yield of pure MA, the salts in the saline solution must be removed. Mix the semi-dried MA with 2 ml of chloroform and 1 ml of water and shake vigorously. Transfer the mixture to a separatory funnel and allow the mixture to separate into two phases at room temperature. Discard the upper phase containing the saline solution and evaporate the lower phase using a rotary evaporator at 80°C. Repeat the saline removal process three times. Add 5-10 ml of acetone to completely dry the MA. Repeat this process until the MA is completely dry.

[0201] To remove contaminants attached to or associated with the CCD-purified MA, dissolve the dried MA from the previous step in 4 ml of chloroform and concentrate to 1 ml on a rotary evaporator at 85°C. Do this for 5 minutes to ensure that all the MA is dissolved. Add acetone to precipitate the MA from the solution. Add 20 ml of acetone to the MA extract and heat at 85°C in a pre-weighed round-bottom flask. Allow the MA to precipitate from the acetone solution at 4°C overnight.

[0202] Filtration through filter paper previously rinsed with chloroform and acetone was used to separate the MA precipitate from the contaminants. The MA precipitate on the filter paper was rinsed with cold acetone (10 ml). The precipitate was retained on the filter paper and the acetone filtrate was filtered into a round-bottom flask labeled "Acetone filtrate / MA supernatant". This process was repeated three times until the flask containing the MA precipitate was completely clean. The precipitate on the filter paper was then rinsed three times with 10 ml of chloroform, placed in a pre-weighed round-bottom flask labeled "MA precipitate", and then evaporated using a rotary evaporator at 60°C until completely dry. The precipitation of MA from the contaminants was repeated a second time. Acetone (100 ml) and chloroform (4 ml) were added to the MA precipitate recovered from the filter paper and heated at 85°C for 5 minutes. As before, MA was precipitated and recovered from the acetone solution at 4°C overnight.

[0203] The MA precipitate and supernatant collected in the round-bottom flask were stored in a desiccator overnight with indicating silica gel as a desiccant to ensure that all moisture was removed before weighing the flask using an analytical balance.

[0204] It is important to note that the use of acetone described in Sections 1.1.2 to 1.1.5 is not the use of acetone characterized in accordance with the present invention, but rather the more conventional use of acetone in the art for precipitation.

[0205] 1.2 Solubility of MA in acetone

[0206] To determine the extent to which MA can be dissolved in acetone, acetone was added to an excess of MA, dissolved by heating, and cooled to room temperature to provide a saturated solution of MA in the vial at room temperature, including insoluble MA that adhered to the walls during cooling. Soluble and insoluble MA were quantified by immunoassay using a recombinant monoclonal antibody developed by Ranchod et al. (2).

[0207] 1.2.1 Purification of recombinant chicken antibodies

[0208] According to the study by Ranchod et al. in 2018 (2), recombinant chicken antibody clone 12CH1-4 was purified using nickel affinity chromatography. 5 ml of cell culture medium containing the recombinant chicken antibody was diluted with 45 ml of 1× lysis buffer and passed through the column. The eluate was passed through the column a second time, and the final flow-through was discarded. The column was washed twice with 1× lysis buffer, and the protein retained in the column was eluted into 1 ml fractions using elution buffers 1, 2, 3, and 4 in sequence. Borate buffer was added to the purified recombinant chicken antibody in a 1:1 ratio, and then concentrated by ultrafiltration centrifugation at 3500×g for 20 minutes using a Vivaspin 10000MW PES centrifuge (VivaScience, Satorius Group, UK). The protein concentration was determined by Bradford assay.

[0209] 1.2.2 Determination of the solubility of MA in acetone

[0210] To characterize the solubility of MA in acetone, two vials (i and ii), each containing 1 mg of MA and 2 ml of acetone, were heated at 90°C for 5 minutes on a Reacti-Therm III heating block. After mixing, each vial was allowed to cool at room temperature. After 1 hour, the MA / acetone solution in the first vial (i) was transferred to a clean amber glass vial (iii) by pipetting. After 4 hours, the MA / acetone solution in the second glass vial (ii) was transferred to a clean amber glass vial (iv). The four vials were placed on a heating block at 80°C and the contents of each vial were evaporated and dried under a stream of nitrogen. All vials were stored in a desiccator at room temperature overnight to allow complete drying before weighing.

[0211] Nunc-Maxisorp ELISA plates were coated with 50 μl of purified MA and residual MA from vials i-iv in the previous step. The residual MA concentration was calculated assuming each vial still contained the original 1 mg of MA. The residual MA in the vials was first dissolved in freshly distilled hexane, with decreasing dilutions ranging from 0.25 to 0.1 mg / ml. All MA samples were coated three times. Nonspecific binding sites in each well were blocked with 300 μl / well of 4% (m / v) casein hydrolysate / PBS, pH 7.4, for 2 hours at room temperature, followed by three washes with 300 μl / well of PBS / 0.1% Tween 20. Each well was then incubated with 50 μl / well of recombinant chicken antibody 12CH1-4, diluted to a concentration of 0.031 mg / ml in 4% (m / v) casein hydrolysate / 1×PBS, pH 7.4, 0.1% Tween 20, for 1 hour at room temperature. Unbound antibody was removed by washing the wells with PBS / 0.1% Tween 20 as described above. A secondary antibody conjugate (goat anti-chicken Fc:HRP) (AbD Serotic, Kidlington, UK) was diluted 1:1000 in 4% casein hydrolysate / 1×PBS-0.1% Tween 20, pH 7.4, and 50 ml was added to each well. The plates were incubated at room temperature for 1 hour. The conjugate solution was discarded and the plates were washed as described above. The signal was visualized by adding 50 μl of TMB single solution developer for ELISA to each well and then incubating at room temperature for 5 minutes. To stop the reaction, 50 μl of 1 M H2SO4 was added to each well. The plates were read at 450 nm.

[0212] 1.3 Antigenicity of MA in different solvents: comparison between acetone and hexane

[0213] Immunoblotting of acetone-soluble MA

[0214] A 4 ml volume of acetone was added to a 1 mg aliquot of MA, heated at 90°C on a heating block for 5 minutes, and shaken by hand to mix. After the MA / acetone solution was allowed to cool for 1 hour, 10 μl was spotted on a silica gel plate at a concentration of 0.25 mg / ml using a Hamilton syringe. Another 1 mg / ml aliquot of MA was dissolved in hexane (4 ml), spotted as above, and allowed to dry for 30 minutes. The silica gel plate spotted with MA was placed in a covered petri dish and immersed in 4% (m / v) casein hydrolysate / pH 7.4 PBS so that the entire plate was covered with the solution. It was then incubated at room temperature for 2 hours. After 2 hours, the buffer solution was poured out of the covered petri dish, making sure not to pour out the silica gel plate, and then the silica gel plate was washed three times with PBS / 0.1% Tween 20 by gently tilting the buffer solution in the covered petri dish from side to side without moving the silica gel plate. The buffer solution was poured out and the excess buffer solution was removed with a disposable plastic dropper. The silica gel plate was then immersed in a solution of recombinant chicken antibody 12CH1-4 diluted to a concentration of 0.031 mg / ml in 4% (m / v) casein hydrolysate / 1× PBS-0.1% Tween 20, pH 7.4, and incubated at room temperature for 1 hour. After washing as described above, a secondary antibody solution (goat anti-chicken Fc:HRP) (AbD Serotic, Kidlington, UK) diluted 1:1000 in 4% casein hydrolysate / 1× PBS-0.1% Tween 20, pH 7.4, was added to the plate in a culture dish and incubated at room temperature for 1 hour. The plate was washed three times with PBS / 0.1% Tween 20 to remove unbound secondary antibody, and then 5 ml of TMB colorimetric solution was added for 3 minutes. The color signal on the plate was then visually analyzed and photographed with a mobile phone camera. Because TMB oxidizes rapidly, color development must be recorded within 5 minutes, after which the color stain precipitates randomly, reducing resolution.

[0215] 2. result

[0216] 2.1. Countercurrent purification of mycolic acids

[0217] To analyze the distribution of MA after CCD purification, TLC was performed on tubes 0-60, using the upper and lower phases of every other tube from tube 1 to tube 15. The TLC results are shown in Figure 2. Figure 2 shown.

[0218] from Figure 2It can be seen that the MA spot with an Rf of 0.1 is visible in test tubes 1 to 13, reaching a peak at test tube 5L. The compound with an Rf of 1 also reaches a peak at test tube 5L and begins to fade as the MA spot begins to fade from test tube 7L, indicating an association with MA and the lower phase solvent. The compound with an Rf of 0.6 reaches a peak in test tube 5L and fades after test tube 13L. Since similar spots were found at the same Rf of 0.6 in the channel containing only the lower phase solvent, it is speculated that they come from the lower phase solvent. The black spot at the origin of the peak in test tube 5L is speculated to be the ionized (more polar) form of MA. Nothing was seen in the upper phase of each test tube, so this makes chloroform most likely the culprit for the Rf 0.6 contaminant in the fractions collected from the CCD. The absence of material in the upper phase indicates that most of the contaminants from the crude MA extract are moving. No spots of MA or contaminants were seen in tubes 15-60.

[0219] MAs show low mobility on TLC because they are not far from the origin. Figure 2 As shown, MA also did not move in the countercurrent column, but remained in the first tube, indicating that they were extremely hydrophobic. More hydrophilic substances moved further along the column. Figure 2 Only three compounds / pollutants are shown, excluding MA, but in Figure 2 Most of the contaminants seen in the TLC chromatogram of the crude MA extract will migrate along the remaining tubes in the tube train. Smaller-scale CCD purifications load the crude extract only in tube 1, yielding MA-enriched samples in tubes 1-10. With increased sample loading of the crude extract, MA-containing fractions are pooled from tubes 1-15, as the sample is loaded across five tubes instead of one. Therefore, separation efficiency is unaffected by increasing the scale of the CCD purification, while benefiting from improved MA detection in the CCD tubes by TLC to aid in identifying MA within the tube train after separation.

[0220] 2.2. Determination of the solubility limit of MA in acetone

[0221] To determine the acetone solubility of MA in acetone, a recombinant chicken-derived antibody was applied in a quantitative ELISA immunoassay to avoid the inaccuracies inherent in gravimetric determination of small amounts of MA, which can be transferred from a vial where MA is dissolved in acetone at elevated temperature and cooled for a defined time before being transferred to a new vial.

[0222] It is reasonable to assume that the functional groups of MA have the physicochemical property of folding the conformational freedom of the long methylene units in the MA backbone in solution at high temperatures, because it is well known that mycolic acids have relatively low melting points despite their high molecular weight and are highly soluble in nonpolar organic solvents such as chloroform, hexane, heptane, dichloromethane, toluene, and xylene (15). Therefore, it is reasonable to infer that MA can be dissolved in almost any nonpolar solvent at high temperatures.

[0223] Two vials of MA (1 mg per vial) were prepared. MA was dissolved in acetone (2 ml), heated at 90°C for 5 minutes, shaken by hand, and then cooled for 1 hour (vial 1) or 4 hours (vial 2). After cooling, the contents of the vials were transferred to separate clean vials. All vials were placed in a heating block at 80°C and the contents were evaporated to dryness in a stream of nitrogen. The dried vial contents were then dissolved in hexane at a dilution range of 0.25, 0.05, and 0.01 mg / ml for ELISA analysis using recombinant chicken antibody 12-1. The results are shown in Figure 2. Figure 3 shown.

[0224] Heating the MA solution in acetone at 90°C revealed that the MA remained in solution after 1 hour, as a clear solution was observed. Four hours after cooling, the solution gradually became opaque, but the amount of MA that could be transferred remained around 50% of the original content. This increase in opacity over time is likely due to the micellar MA dynamically enlarging and eventually settling at the bottom of the vial. This suggests that MA solutions in acetone are unstable at saturated concentrations and should be used within 4 hours of cooling or at lower concentrations.

[0225] from Figure 3 As can be seen from the results, the amount of MA that can be transferred and the amount of MA left behind (residue) are roughly the same after 1 hour and 4 hours of cooling. This is also true for antigenicity, as the ELISA signal is comparable and almost identical to that of pure MA at the same concentration.

[0226] Surprisingly, it was concluded that MA is soluble in acetone at room temperature to a certain limit, with a first indication of an upper limit of approximately 0.25 mg / ml. However, the micellar solubility of MA in acetone was unstable for at least 4 hours after cooling.

[0227] 2.3. Immunoblotting of acetone-soluble MA

[0228] A 10 μl aliquot of MA in acetone at 0.25 mg / ml was applied twice to nitrocellulose to demonstrate the incompatibility effect with acetone. Similarly, the same concentration of MA in n-hexane was applied twice to the same substrate. This was compared to two different substrate types, whatman No. 1 paper and silica gel TLC plates, both of which were coated with MA in the same manner as the nitrocellulose substrate. Hexane and acetone were spotted separately as negative controls. The coated and dried substrates were incubated in recombinant chicken antibody type 12-CH1-4 at a concentration of 0.031 mg / ml. The results of immunoblotting with secondary antibody HRP conjugate are shown in Figure 2. Figure 4 The blue spots indicate that the recombinant chicken antibody positively binds to MA.

[0229] The results of immunoblotting test ( Figure 4 ) shows that when MA in acetone was spotted on nitrocellulose, the nitrocellulose dissolved at the spots, confirming the incompatibility, compared with when MA was spotted from a hexane solution ( Figure 4 A in Figure 1). Whatman filter paper substrate No. 1 could not be used because there were no visible spots ( Figure 4 B in the Figure). The results of the blotting test on the silica gel TLC plate showed that the spots of MA in acetone and MA in hexane ( Figure 4 C), but only one spot of MA in hexane is visible, while both spots of MA in acetone are visible. The positive visible signal of MA in acetone is much stronger than that of MA in hexane. The negative control (hexane and acetone only) does not show detectable signal. Figure 4 The MA signal in C indicates that MA is antigenic when spotted on silica gel, and MA in acetone maintains antigenicity and is even more antigenic than when spotted from a hexane solution. To further investigate the antigenicity of MA in acetone compared to MA in hexane on silica gel, a dilution range was completed at spotting concentrations of 0.25, 0.05, and 0.01 mg / ml. The results of the blot tests with MA in acetone and MA in hexane spotted at decreasing concentrations are shown in Figure 2. Figure 5 shown.

[0230] exist Figure 5In the figure, MA concentration-dependent positive antibody binding spots are visible for MA spotted in acetone and hexane at concentrations of 0.25 mg / ml and 0.05 mg / ml, but the spots for MA in hexane are significantly weaker, indicated by the arrows marked with "*". Repeated spots of MA in acetone, indicated by the arrows marked with "X", show clear repeated spots, and the intensity of the repeated spots decreases with decreasing concentration, becoming more distinct than those spotted with MA in hexane at a concentration of 0.05 mg / ml. The repeatability of MA spotted in acetone on silica gel is also better than that of MA spotted in hexane. Negative controls (hexane and acetone as spotting reagents only) showed no detectable signal, demonstrating that background was successfully washed out during the immunoblotting process.

[0231] These results demonstrate that aluminum-backed silica gel plates serve well as substrates for immunochemical detection of MA spotted onto silica gel. Clear antibody-bound spots were obtained when MA was spotted from an acetone solution, while the unspotted silica gel background could be washed cleanly with aqueous buffer, indicating that there was no nonspecific binding of the antibody to the silica gel. Furthermore, the silica gel plates remained intact after extensive washing with aqueous buffer.

[0232] discuss

[0233] The use of MA to detect anti-mycolic acid antibody biomarkers in TB patients was investigated and used as a handheld diagnostic device in a POC setting, which will meet a huge need in the management and control of TB epidemics.

[0234] The availability of recombinant monoclonal anti-MA recombinant chicken antibodies provides an opportunity to quantify small amounts of MA and characterize it in terms of its antigenic properties. Previous work in this research area has shown that the use of recombinant chicken antibodies in immunoassays faces several challenges, mainly due to the nature of the large lipid MA antigen. MA can be successfully immobilized in an antigenic conformation from hexane onto nitrocellulose (15). Although this was successful, hexane is incompatible with the bioprinting machinery used for electrode coating. Surprisingly, according to the present invention, solubilization of MA in polar solvents such as acetone is possible, which will solve this problem. Due to the incompatibility of acetone, lipid immobilization on nitrocellulose is not suitable (16), and the typical low affinity of serum anti-lipid antibodies presents an almost insurmountable labeling problem (15).

[0235] MA was dissolved in acetone at high temperature and then cooled. The amount of MA remaining in the solution was studied. Figure 3Quantitative results are shown, in which MA was titrated down to where a reduced signal was obtained, indicating that the amount of MA in the transferred vial was comparable to the amount remaining as insoluble material in the original vial. It is assumed that MA dissolves in acetone as dynamic micelles at elevated temperatures. After cooling, it remains in solution for approximately 4 hours. Under these conditions, MA was found to have a solubility of approximately 0.25 mg / ml or less, likely as a micellar suspension that condenses into a precipitate after several hours at saturation concentrations.

[0236] The significant contribution of the solubility of the MA of the present invention in polar solvents such as acetone finds application in overcoming challenges in the development of TB immunodiagnostics due to the chemical compatibility of acetone with antigen printing instruments, on electrodes and other substrates for MA immobilization (16). However, acetone is incompatible with printing on nitrocellulose, as Figure 4 In contrast, TLC silica gel plates were found to be a suitable substrate for spotting MA antigens from acetone solutions, as shown in Figure A, because the structural integrity of nitrocellulose failed due to dissolution in the spotted area. Figure 4 As shown in Figure C, MA antigenicity was also confirmed after dot-blotting and immunoblotting. Figure 5 The superiority of acetone over hexane as a solvent for MA was demonstrated in

[17] , where the spots produced from MA-acetone were darker than those from MA-hexane. It appears that the antigenicity presented by MA spotted from acetone on silica gel is superior to that achieved using MA-hexane solutions. A possible explanation for this is the micellar nature of the acetone solutions of MA as observed by the homogeneous opacity of the solutions, which gradually increased within 4 hours after heating. The hexane solutions of MA remained stable and clear. It is thought that the micellar packing of MA helps them fold into an antigenic conformation before and during adsorption onto silica gel. The importance of MA folding into a variable antigenic structure was demonstrated by the study of Beukes et al. in 2010 (17).

[0237] Example 3

[0238] Micellar properties of MA dissolved in acetone

[0239] According to the present invention, the solubility of the newly disclosed MA in acetone is exemplified in Example 2 above. After removal from heating at 90°C, the MA appears to remain soluble in acetone for a limited time at room temperature. This data suggests that the more polar nature of acetone, when used as a solvent for MA, may better direct the folding of MA into an antigenic state for immobilization on solid substrates such as silica gel, compared to hexane.

[0240] There is no information in the literature on the solubility of MA in acetone, which requires characterization of the behavior of MA in acetone solution. Therefore, the following work was carried out:

[0241] Determine the "solubility" limit of MA in acetone, and

[0242] Characterize the behavior of MA in acetone at different temperatures.

[0243] 1. method

[0244] 1.1 Determine the solubility limit of MA in acetone

[0245] The solubility limit of MA in freshly distilled acetone was determined spectrophotometrically using quartz cuvettes at concentrations of 0.25, 0.125, and 0.05 mg / ml ( Figure 6 、 Figure 7 and Figure 8 ). MA samples were weighed and dissolved in acetone by heating to 85°C for 5 minutes in a capped amber glass vial.

[0246] Observation: The MA solution in the vial became clear immediately after the vial was removed from the heat source. Within one minute of removing the vial from the heat source, the solution became cloudy.

[0247] The MA solution was transferred from the glass vial to a quartz cuvette, the opening of the cuvette was covered with a PTFE lid, heated on a heat block at 85 °C for one minute, and then immediately transferred to a spectrophotometer, where absorbance measurements were recorded at 420 nm at intervals of 2, 15, 30, and / or 60 minutes at room temperature after removal from the heat source.

[0248] Observation: When the MA-acetone in the quartz cuvette is transferred from the hot block to the spectrophotometer, the solution appears clear.

[0249] 1.2 Characterization of the behavior of MA in acetone at different temperatures

[0250] The obvious change in absorbance over time at a concentration of 0.125 mg / ml indicated that the micelles might be in a dynamic state. Therefore, a concentration of 0.1 mg / ml MA in acetone was chosen as the concentration to characterize the behavior of MA in acetone in the temperature-dependent study.

[0251] A 0.1 mg / ml MA solution in acetone was heated at 85°C on a hot plate for 5 minutes. The vial was removed from the hot plate and allowed to cool on the laboratory bench for 5 minutes. 2 ml of the MA-acetone solution was transferred to a clean quartz cuvette with a rectangular, non-airtight Teflon cap. The vial was heated at 85°C on a hot plate for 1 minute and immediately placed in the Zetasizer container. The Zetasizer equilibrated the cuvette temperature for 180 seconds using a temperature-controlled air flow over the cuvette walls.

[0252] Dynamic light scattering (DLS) is used as a subsequent characterization method due to its high resolution in detecting both scattered and transmitted light. The Zetasizer Nano zs uses dynamic light scattering to provide data on the size (diameter) and polydispersity index (size distribution) of the nanoparticles.

[0253] To investigate the effect of temperature changes on the dynamic light scattering of MA in acetone, a 0.1 mg / ml solution of MA in acetone was heated to 85°C in a sealed glass vial with a screw cap. The hot solution was then transferred to a clean quartz cuvette with a cap, heated at 85°C for an additional minute, and allowed to equilibrate in the Zetasizer vessel over a range of decreasing temperatures for three minutes. Size measurements were taken at 50°C, 40°C, 37°C, 25°C, and 20°C.

[0254] 2. result

[0255] Neither the acetone nor the MA solution has a distinct yellow color, so it is expected that the MA solution in acetone will not exhibit significant absorption of light at a wavelength of 420 nm. If the MA solution in acetone exhibits any absorption at 420 nm, it can be assumed that this is due to light scattering, indicating the particulate nature of the MA in solution, possibly micelles. Figures 6 to 8 In the acetone, a rapid increase in light scattering was measured within the first 10 minutes of cooling from 85°C, indicating the rapid formation of MA micelles in the solution. Between 10 minutes and 1 hour, the rate of increase in absorption leveled off and entered a plateau phase, with the slope of the absorption increase proportional to the MA concentration. Therefore, during cooling from 85°C, the growth of MA micelles was proportional to the initial MA concentration in acetone. At the highest MA concentration of 0.25 mg / ml, the solution also developed visible turbidity most rapidly, likely due to the tendency of aggregated MA micelles to precipitate out of solution after cooling for more than 4 hours.

[0256] Figure 6 It was shown that 0.25 mg / ml MA in acetone solution rapidly aggregated into growing micelles, and the solution might be considered too unstable for reliable EIS electrode coating. Figure 7 It was shown that 0.125 mg / ml MA in acetone solution showed a relatively stable light scattering platform with only a 0.1 absorbance unit increase between 10 min and 4 h, indicating a relatively stable micellar solution that can satisfy MA coating of the electrode. Figure 8 The data at a MA concentration of 0.05 mg / ml showed the most stable light scattering plateau after 10 min, but the plateau height was significantly lower, which may require a larger volume of solution to coat the electrode.

[0257] Figures 6 to 8The data in indicate that 0.1 mg / ml MA in acetone is a practical concentration for coating electrodes with MA within 4 hours of cooling. Therefore, this concentration was chosen to study the behavior of MA in acetone at different temperatures. Dynamic light scattering allows estimation of the micelle size of MA solutions over time. Figure 9 In a study published in Nature Communications, a significant logarithmic growth of micelles was observed in a 0.1 mg / ml solution of MA after cooling from 85°C for 3 hours, reaching 400 nm within this time, while remaining in an apparently transparent solution. The data showed that the micelle diameter increased over time from 225 nm (t = 0 minutes) to 400 nm (t = 180 minutes). To the inventors' knowledge, this is the first description of spontaneous micelle formation by mycolic acids in acetone.

[0258] The next task was to investigate whether temperature had an effect on the size of micelles in MA solutions. A 0.1 mg / ml MA solution in acetone was heated to 85°C, transferred to a clean quartz cuvette, heated at 85°C for another minute, and allowed to equilibrate within the instrument over a range of decreasing temperatures for 180 seconds.

[0259] At 50°C and 45°C, no dynamic light scattering was detected. At 37°C, the instrument detected a signal indicating that the micelles had an average size of 270 nm. When the temperature was lowered to 30°C, nanoparticles with a significantly narrow polydispersity index were obtained, which had an average diameter of 280 nm. The size of the micelles increased with decreasing temperature. Figure 10 An increase in micelle size can be observed from the data, from approximately 270 nm at 37 °C to nearly 400 nm at 20 °C.

[0260] 3. discuss

[0261] Immediately after removing the MA-acetone solution from the heat source, a rapid increase in light absorption and diffraction is observed, which can be attributed to micelle formation. The gradual increase in absorption and diffraction after 10 minutes is interpreted as a gradual increase in micelle size, and as the solution cools, a state is reached after several hours in which the aggregated micelles tend to precipitate out of solution. Visual observation noted that precipitation occurred more rapidly at the highest MA concentration in acetone.

[0262] Previous work by Baumeister, Shaw, and Verschoor (4) demonstrated that smaller sized MA-presenting liposomes were better able to present MA in an antigenic form that enabled detection of active TB biomarker antibodies.

[0263] 4. in conclusion

[0264] There are several advantages to using acetone as a solvent for MA for its application as a coating electrode for the purpose of detecting anti-MA antibodies, including improving the antigen orientation of the immobilized MA (Example 2). In addition, acetone is compatible with antigen deposition or printing equipment, which is suitable for the scale-up required for automatic antigen coating of screen-printed electrodes. For coating of biosensor electrode surfaces, the behavior of MA in micellar acetone solution points to a concentration of about 0.1 mg / ml, which needs to be heated to 85°C or higher to allow complete chemical dissolution, and then needs to be cooled to between 25°C and 35°C and applied to the electrode within about 4 hours after heating to ensure reliable MA antigen coating in the desired conformation from a relatively stable MA micellar solution.

[0265] Example 4

[0266] Detection of TB biomarker anti-MA antibodies in human serum by electrical impedance spectroscopy (EIS) on MA-coated carbon SPE

[0267] Patients with active TB will show elevated levels of antibodies to mycolic acid, a substance found on the mycobacterial cell wall. Antibodies have previously been detected using various techniques, including ELISA, waveguides, and resonant mirror biosensors. The ELISA results published by Schleicher et al. (5) provided an accuracy of 57%. Using a resonant mirror biosensor, the biomarker anti-mycolic acid antibodies were detected with an accuracy of 82% (8). The latter approach employed an inhibition assay format of the so-called MARTI assay described by Lemmer et al. (9). To meet the requirements of affordability and high-throughput sample analysis, the MARTI assay was adapted to detect anti-MA antibodies on screen-printed MA-coated gold electrodes using amperometric detection (i.e., EIS). The ability to distinguish between TB-positive and TB-negative sera was achieved by measuring the difference in bound antibodies on the MA-coated surface. Previous approaches were plagued by poor reproducibility due to the need to polish the gold electrodes before coating, the need to create a self-assembled resistive lipid monolayer, the challenges of using the unstable dimethylformamide solvent for MA coating, and the instability of the PLGA nanoparticles used as immunosorbents in the pretreatment of serum samples.

[0268] In this example, these technical challenges were overcome by replacing screen-printed gold electrodes with commercially available screen-printed carbon electrodes, preparing these electrodes for MA coating by a simple, short wash in acetone, and using MA dissolved in acetone at a concentration that resulted in its own self-assembled resistive lipid structure, which exhibited better antigenic properties than when coated from a hexane solution. Furthermore, this method eliminated the need to pretreat serum samples with MA-coated nanoparticles by adding a commercially available rheumatoid factor blocking agent solution to inhibit any rheumatoid factor activity in the serum samples.

[0269] 1. Material

[0270] Blood samples were provided by the Department of Internal Medicine (Infectious Diseases), Faculty of Health Sciences, University of Pretoria. Patient consent was obtained before blood sample collection, in accordance with ethical approval provided by the University's Ethics Committee. Blood samples were allowed to clot for 4 h, and serum was aspirated into clean 1.5 ml Eppendorf tubes. The tubes were centrifuged at 4°C to pellet red blood cells, and serum (100 μl) was aliquoted and stored at −80°C until use.

[0271] Unless otherwise stated, all reagents had a purity of at least 99.5%.Distilled deionized water (dddH2O) was used for the preparation of reagents and for rinsing of screen-printed electrodes.

[0272] Materials: acetone (minimum purity 99.5%), deionized water, disodium edetate dihydrate, disodium phosphate, anhydrous ethanol (99.8%), mycolic acid, potassium chloride, potassium dihydrogen phosphate, rheumatoid factor blocking agent (final concentration 1 mg / ml), pre-dried silica gel, sodium azide, sodium chloride, and disodium hydrogen phosphate.

[0273] 2. method

[0274] 2.1. Preparation of hexacyanoferrate redox buffer

[0275] Hexacyanoferrate (1 mM potassium ferrocyanide (II) and potassium ferrocyanide [Fe(CN)6]) was prepared in phosphate-buffered saline (PBS) containing 3.8 mM sodium azide and 1 mM EDTA. 4- / [Fe(CN)6] 3- If necessary, the pH was adjusted to 7.44 with 1 M HCl or 1 M NaOH. The solution was filtered using a Pall filtration system (Washington, D.C., USA) and a 0.2 μm cellulose acetate filter. Casein hydrolyzate was added as a stabilizer at 1% (m / v).

[0276] 2.2. Pretreatment of Carbon SPE with Acetone

[0277] Cyclic voltammetry (CV) and EIS data were generated using a potentiostat, instrument software, and disposable well cells. The carbon electrode was characterized using cyclic voltammetry and EIS. The CV scan cycle was from -0.2 V to +0.4 V (E vertex 1 to E vertex 2) with a scan rate of 50 mV / s and a voltage step of 2.44 mV. The EIS scan occurred at a DC voltage of 0.135 V, an AC amplitude of 0.01 V, and 50 frequencies between 2000 and 0.1 Hz. The electrode was rinsed with dddH2O and air-dried. The sensor surface was immersed in acetone for 2 to 7 minutes, followed by a rinse with approximately 500 μl of acetone. The electrode was dried in an EcoTherm oven (Labotec, ZA) at 80°C for 20 minutes and then cooled in air for 5 minutes. CV and EIS data were then generated.

[0278] 2.3. Use 0.1 mg / ml MA-acetone to coat carbon SPE

[0279] Using a glass micropipette (Blaubrand, Germany) mounted on the robotic arm of a surface plasmon resonance (SPR) biosensor (Metrohm Autolab, The Netherlands), 3 μl of a 0.1 mg / ml MA solution in acetone was dispensed onto the working electrode of each SPE for 10 repetitive depositions at a temperature between 25 and 37° C., in this case 30° C. The acetone was allowed to evaporate for 2 minutes, after which the electrodes were incubated under high vacuum conditions of ≤50 mTorr using a Virtis freeze dryer (SP Scientific, USA) for at least 16 hours to remove traces of acetone.

[0280] 2.4. Blocking of carbon SPE with 1% (m / v) casein hydrolysate

[0281] After incubation in high vacuum for 16 hours, the electrode was transferred to a desiccator for 1 hour. The electrode surface was immersed in 1% (m / v) casein hydrolysate blocking buffer, pH 7.00, for 16 hours. The electrode was removed, rinsed by immersion in deionized water, dried, and stored in a desiccator for at least 1 hour before use.

[0282] 2.5. Preparation of serum dilutions using rheumatoid factor blocker (RfB)

[0283] Serum is diluted to minimize the prozone effect or hook effect in the antibody sample. Multiple serum concentrations can be used to control the prozone effect. In the present embodiment, a concentration of 1:1000 and 1:200 is used. For example, a disposable orifice plate (Metrohm Dropsens, Spain) is attached to the blocking electrode of the coating, and a volume of 150 μl of 1% casein hydrolysate in 1mM hexacyanoferrate buffer is pipetted into the hole, and EIS is used to characterize as described above. Subsequently, serum is thawed and mixed with rheumatoid factor blocker (RfB) to a final concentration of 1mg / ml, and 1% (w / v) casein hydrolysate is added, incubated at 30°C for 15 minutes. Further serum is diluted to 1:500 and 1:100, while keeping the casein hydrolysate final concentration of 1%. A 75 μl volume was removed from the electrode and replaced with 75 μl of a 1:500 dilution (to achieve a 1:1000 effective concentration) of serum / RfB sample solution, 1% casein hydrolysate in 1 mM hexacyanoferrate buffer, triturated, mixed, and incubated for 10 minutes. The electrode was then characterized a second time using EIS as previously described. A 75 μl volume of the 1:1000 sample dilution was removed from the electrode and replaced with 75 μl of a 1:100 sample dilution (1:200 effective concentration), triturated, mixed, and incubated for 10 minutes. The electrode was then characterized a third and final time using EIS as previously described.

[0284] 3. result

[0285] The lowest and highest serum concentrations should be represented by a 1:200 dilution series that includes four unblinded sera. It is important to note that at a dilution of 1:1600, these four serum samples had no EIS signal above background. This allows the 1:1600 serum dilution to be classified as a lower limit concentration, at which not all TB-positive patient sera produce an EIS signal higher than that obtained with TB-negative patient sera.

[0286] Figure 11 Impedance data are shown before and after acetone washing and after antigen immobilization and blocking with casein hydrolysate.

[0287] By using acetone-pretreated carbon electrodes, a micellar MA coating process, and surface blocking with casein hydrolysate, we show here that the MARTI-EIS technique as a direct antibody binding assay can differentiate between two TB-positive and two TB-negative patient sera at dilutions of 1:1000 and 1:200. However, MVT112 and MVT116 were indistinguishable at a dilution of 1:1000. Figure 12 Impedance data comparing TB+ and TB- sera at 1:1000 and 1:200 dilutions are shown.

[0288] 4. discuss

[0289] It is assumed that the demonstrated ability of the method to distinguish between two TB-positive and two TB-negative patients by analyzing their serum samples will be applicable to larger collections of patient samples, e.g. Figure 13 As shown, the present invention has the following advantages over the prior art (EP2997371A1):

[0290] - The carbon screen-printed electrodes used in the present invention are of consistent quality.

[0291] Gold electrodes, which previously required tedious polishing, are now replaced by carbon electrodes, which require a simple, brief chemical treatment with acetone. This is achieved by immersing the carbon electrodes in acetone for 2 to 7 minutes, for example, approximately 5 minutes. Previously, the use of gold electrodes required pre-cleaning with argon plasma and mechanical polishing with aluminum oxide slurry.

[0292] - It is no longer necessary to form an octadecanethiol (ODT) self-assembled monolayer before coating with MA antigen, thus shortening the process.

[0293] - Using gold electrodes and ODT requires repeated washing in ethanol and drying in a nitrogen atmosphere overnight. In the current example, the carbon electrode only needs to be dried in an oven at 80°C for 10-30 minutes, for example about 20 minutes.

[0294] - Immobilized MA from micellar acetone solution is presented in a highly antigenic conformation required for disease-specific antibody detection.

[0295] - Upgraded automated MA antigen coating was achieved by a robotic droplet deposition device. Acetone is generally compatible with this device, but is incompatible with known solvents for MA molecules such as hexane and chloroform.

[0296] After coating, traces of acetone were removed from the carbon electrodes by placing the electrodes under high vacuum. In contrast, the gold electrodes were previously washed three times in n-hexane and dried in a nitrogen atmosphere.

[0297] - Unlike the previous method which did not use blocking, the carbon electrode of this example was blocked with an aqueous solution of casein hydrolysate for 16 hours.

[0298] - The prepared carbon electrodes need to be simply stored in a dry environment, such as in a desiccator at 23°C. Using previous methods, the electrodes need to be individually packaged in dry nitrogen at 23°C.

[0299] - Using previous methods, the serum sample had to be split into two samples, which required more complex liquid handling. In the present invention, three consecutive impedance measurements are required to obtain the test result, thereby simplifying the design of the microfluidic circuit.

[0300] -Previous state-of-the-art techniques required two types of custom-coated nanoparticles. The present invention eliminates the need for immunoadsorbed nanoparticle processing of the sample by simply adding a rheumatoid factor blocking solution instead.

[0301] Table 2 below summarizes the advantages of the carbon electrode approach of the present invention compared to the previous gold electrode approach.

[0302] Table 2: Advantages of the new carbon electrode coating method

[0303]

[0304] Example 5

[0305] Research on chemical blockers

[0306] Most immunoassays require the use of surface blocking agents to prevent the adsorption of nonspecific proteins and antibodies, which can reduce assay accuracy. During the early development of MARTI EIS technology, typical blocking agents proved detrimental, causing impedance data to fall outside the detectable range. Due to differences in the manufacturing process, the change in electrode material from gold to carbon resulted in an increase in surface area, which in turn increased the adsorption of nonspecific proteins and antibodies. Nonspecific adsorption reduced diagnostic test accuracy.

[0307] Approximately 40% of patient samples contain non-analyzed proteins or antibodies. These interfering proteins can have low affinity but be present in high concentrations or high affinity but be present in low concentrations. These interfering proteins also vary from patient to patient (18).

[0308] Inhibiting nonspecific binding of serum improves the signal-to-noise ratio of antibody binding. An ideal chemical blocker must be able to fill all remaining sites after antigen adsorption onto the surface (18). Typically, whole proteins are used as chemical blockers, such as bovine serum albumin (BSA) and casein. In this example, the efficacy of polyvinylpyrrolidone (PVP), a water-soluble polymer, and casein hydrolysate, a bacterial culture medium supplement, as blockers were compared.

[0309] 1. method

[0310] 1.1. Coat the carbon SPE using 0.1 mg / ml MA-acetone under accelerated airflow.

[0311] Using a disposable pipette tip, 4 μl of a 0.1 mg / ml MA solution in acetone was carefully dispensed onto each SPE working electrode at temperatures between 25°C and 35°C (30°C in this example) and 10 repeated depositions were performed under an accelerating gas flow of 15 km / h. The electrodes were incubated under a high vacuum of ≤50 mTorr for 16 hours.

[0312] 1.2. Blocked carbon SPE using 0.01% PVP and casein hydrolysate

[0313] The electrodes were transferred to a desiccator and equilibrated for 1 hour. Some electrode surfaces were immersed in 0.01% (m / v) casein hydrolysate blocking buffer, pH 7.00, for 16 hours, and some were immersed in 0.01% (m / v) PVP blocking buffer, pH 7.00, for 16 hours. The electrodes were removed, rinsed by immersion in deionized water, dried, and stored in a desiccator for at least 1 hour before use.

[0314] 1.3. Comparison of 0.01% (m / v) PVP and casein hydrolysate blocking agents

[0315] A volume of 150 μl of 1 mM hexacyanoferrate buffer was pipetted onto the electrode, and the electrode was characterized using CV and EIS. Cyclic CV scans were performed from -0.2 V to +0.4 V at a scan rate of 50 mV / s in 2.44 mV steps. EIS scans were performed at a DC voltage of 0.135 V, an AC amplitude of 0.01 V, and 50 frequencies ranging from 2000 to 0.1 Hz. Subsequently, serum was thawed and diluted to a concentration of 1:600 ​​with 1 mM hexacyanoferrate buffer. The 1 mM hexacyanoferrate buffer was aspirated and replaced with 150 μl of a 1:1600 serum sample in 1 mM hexacyanoferrate buffer and incubated for 10 minutes. The electrode was then characterized again using EIS as described above.

[0316] 2. result

[0317] In the case of PVP, the impedance data were out of range, even with titrations as low as 0.01%. No differentiation between TB-positive and TB-negative sera was obtained using PVP as a surface blocker, providing evidence of PVP's incompatibility with this system. However, 0.1% casein hydrolysate successfully differentiated TB-positive and TB-negative sera.

[0318] Figure 14 The results showed that PVP blocking significantly increased the impedance signal of MA coating and reduced the detection window, resulting in no statistically significant difference between TB-positive and TB-negative serum samples. In contrast, casein hydrolysate blocking improved antibody detection and obtained a difference in impedance values ​​between TB-positive and TB-negative serum samples.

[0319] Example 6

[0320] Rheumatoid factor blockers as serum additives to improve accuracy

[0321] Elkayam et al. (19) demonstrated that most TB patients with active disease have high levels of rheumatoid factor. Rheumatoid factor binds to the nonspecific, fragment crystallizable (Fc) region of antibodies, forming complexes that effectively minimize the amount of TB antigen-specific antibody available for detection. Immunoassays can be affected by interfering antibodies (e.g., autoantibodies) that alter the measurable concentration of specific antibodies against the antigen. Consequently, data can be misinterpreted due to erroneous concentrations of specific antibody binding (20).

[0322] In this example, a rheumatoid factor (RF) interference inhibitor (RFI blocker) consisting of a proprietary, highly concentrated anti-RF antibody cocktail was used to reduce interference and improve antibody binding accuracy.

[0323] 1. method

[0324] 1.1. Coat the carbon SPE using 0.1 mg / ml MA-acetone under accelerated airflow.

[0325] Using a plastic pipette, carefully dispense 4 μl of a 0.1 mg / ml MA solution in acetone onto each SPE working electrode at temperatures between 25°C and 35°C (30°C in this case). Repeat the deposition process 10 times under an accelerating airflow of 15 km / h. The electrodes were incubated under a high vacuum of ≤50 mTorr for 16 hours.

[0326] 1.2. Carbon SPE was blocked using 1% (m / v) casein hydrolysate

[0327] The electrode was transferred to a desiccator and equilibrated for 1 hour. The electrode surface was immersed in 1% (m / v) casein hydrolysate blocking buffer at pH 7.00 for 16 hours. The electrode was removed, rinsed by immersion in deionized water, dried, and stored in a desiccator for at least 1 hour before use.

[0328] 1.3. Use of rheumatoid factor blockers (RfB) to reduce interference

[0329] A volume of 150 μl of 1 mM hexacyanoferrate buffer was pipetted onto the electrode and the electrode was characterized using CV and EIS. CV scans were performed cyclically from -0.2 V to +0.4 V at a scan rate of 50 mV / s and voltage steps of 2.44 mV. EIS scans were performed at a DC voltage of 0.135 V, an AC amplitude of 0.01 V, and 50 frequencies from 2000 to 0.1 Hz. The serum was then thawed and diluted to a concentration of 1:600 ​​with either 1 mM hexacyanoferrate buffer alone or with 1 mM hexacyanoferrate buffer and RfB (1 mg / ml). The 1 mM hexacyanoferrate buffer on the electrodes was aspirated and replaced with 150 μl of a 1:1600 serum sample in 1 mM hexacyanoferrate buffer on one electrode and 150 μl of a 1:1600 serum sample and 1 mM hexacyanoferrate containing 1 mg / ml RfB on the second electrode. Both electrodes were then incubated for 10 minutes. Impedance signals were then generated using EIS techniques as described previously.

[0330] 2. result

[0331] Using the same serum sample set as used in Example 5, each sample was incubated with or without RfB to generate an impedance signal to detect the binding of anti-mycolic acid antibodies. Figure 15 It was shown that RfB enhanced the impedance signal of antibody binding, thereby improving the discrimination quality of sera from TB-positive patients and TB-negative controls.

[0332] 3. in conclusion

[0333] By blocking the activity of rheumatoid factor, nonspecific antibody binding is impaired and specific anti-mycolic acid antibodies are enhanced. Since more than half of TB patients have rheumatoid factor circulating in their blood (19), it is important to demonstrate that the use of rheumatoid factor blockers in serum samples can improve the results of tests that detect antibodies to mycolic acids, a surrogate marker of active TB.

[0334] In earlier versions of the MARTI assay, nanoparticles were shown to be an essential immunoadsorbent, minimizing the effects of cross-reactive anti-cholesterol antibodies and amplifying the signal difference between suppressed and non-suppressed TB patient samples. Our data (Example 4, Table 1) demonstrate that PLGA nanoparticle immunoadsorbents are no longer required to ensure the accuracy of the MARTI diagnostic test. Rheumatoid factor blockers effectively eliminate the need for nanoparticles by minimizing the effects of antibody cross-linking of rheumatoid factor.

[0335] This discovery simplifies the diagnostic procedure by eliminating the inhibition step, which requires splitting the sample into two parts, each of which needs to be subjected to EIS measurement on a sample-by-sample basis. Direct antibody binding within a single sample exposed to the electrode is now sufficient, providing an ample signal window to distinguish TB-positive from TB-negative patient sera.

[0336] References

[0337] 1. Ndlandla FL (2017). Diagnostic antibody biomarkers for tuberculosis characterized by natural and chemically synthesized mycolic acid antigens. PhD thesis, Faculty of Natural and Agricultural Sciences, University of Pretoria.

[0338] 2. Ranchod H, Ndlandla F, Lemmer Y, Beukes M, Niebuhr J, Al Dulayymi JR, Wemmer S, Fehrsen J, Baird MS, Verschoor JA (2018). Antigenicity and cholesterol-like properties of mycolic acids determined by recombinant chicken antibodies. PLoS One, vol. 13(no. 8): e02002984.

[0339] 3.Groenewald W, Parra-Cruz RA, CM, Croft AK (2019), Solvent-dependent folding behavior of mycolic acids from Mycobacterium tuberculosis revealed by advanced simulation analysis, Journal of Molecular Modeling, Vol. 25, No. 68, https: / / doi.org / 10.1007 / s00894-019-3943-5.

[0340] 4. Baumeister CR, Shaw WA, and Verschoor JA (2019). Liposomal compositions for TB diagnostics. US 10,228,371.

[0341] 5. Schleicher GK, Feldman C, Vermaak Y, Verschoor JA (2002). Prevalence of antimycolic acid antibodies in patients with pulmonary tuberculosis and HIV infection. Clinical Chemistry and Laboratory Medicine, 40(9): 882-887.

[0342] 6. Ndlandla F, Ejoh V, Stoltz A, Naicker B, Cromarty A, van Wyngaardt S, Khti M, Rotherham L, Lemmer Y, Niebuhr J, Baumeister C, Al Dulayymi JR, Swai H, Baird MS, Verschoor JA (2016). Standardization of the composition and production of natural mycolic acid antigens for biomarker antibody assays to diagnose active tuberculosis. Journal of Immunological Methods, vol. 435: pp. 50–59.

[0343] 7. Thanyani ST (2003). Novel application of affinity biosensor technology for the detection of mycolic acid antibodies in tuberculosis patients. Master of Science thesis, Faculty of Natural and Agricultural Sciences, University of Pretoria.

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Claims

1. A method for forming a mycolic acid antigen solution for immobilization on a substrate, the method comprising heating a mixture of mycolic acid and a polar organic solvent to a temperature above the melting point of the mycolic acid, thereby preparing a solution of the mycolic acid antigen in the polar solvent, wherein the solution is a micellar solution.

2. The method according to claim 1, wherein the temperature above the melting point of the mycolic acid is a temperature between 60°C and 90°C.

3. A method according to claim 1 or 2, comprising cooling the micellar solution of mycolic acid antigens in the polar solvent to a temperature between 25°C and 35°C.

4. The method according to any one of claims 1 to 3, wherein the polar organic solvent is acetone.

5. The method according to any one of claims 1 to 4, comprising a preceding step of forming said mixture of mycolic acids and said polar organic solvent at a concentration between 0.05 and 0.25 mg / ml by adding said mycolic acids to said polar organic solvent. 6 . A micellar solution of mycolic acid antigen in a polar solvent prepared according to the method of claim 1 .

7. A method for immobilizing a mycolic acid antigen on a substrate, the method comprising applying a micellar solution of the mycolic acid antigen in a polar solvent according to claim 6 to a substrate on which the mycolic acid antigen is to be immobilized.

8. A method according to claim 7, comprising the preceding step of preparing a micellar solution of mycolic acid antigens in a polar solvent according to the method of any one of claims 1 to 5.

9. The method according to claim 8, wherein the step of applying the micellar solution of the mycolic acid antigen in the polar solvent to the substrate is carried out within four hours after heating the mixture of mycolic acids and polar organic solvent according to the method of any one of claims 1 to 5.

10. A method according to any one of claims 7 to 9, comprising leaving or allowing the micellar solution of the mycolic acid antigen in the polar solvent to dry on the substrate.

11. The method of any one of claims 7 to 10, wherein the substrate is free of a surface-modified monolayer.

12. The method according to any one of claims 7 to 11, comprising the subsequent step of blocking non-specific binding sites on the solid surface according to any one of claims 24 to 26.

13. The method according to any one of claims 7 to 12, wherein the substrate is an electrode.

14. The method of claim 13, wherein the electrode is a screen-printed carbon electrode.

15. The method of claim 14, wherein the screen-printed carbon electrode is a screen-printed carbon electrode according to claim 23.

16. A method according to claim 15, comprising the preceding step of preparing the screen-printed carbon electrode according to the method of any one of claims 18 to 22.

17. A screen-printed carbon electrode comprising mycolic acid antigens immobilized thereon, prepared according to the method of any one of claims 14 to 16.

18. A method for preparing a screen-printed carbon electrode for immobilizing an antigen thereon without a surface modification monolayer, thereby removing organic binders and impurities from the electrode, the method comprising treating the electrode to which the antigen is to be immobilized with acetone.

19. The method of claim 18, wherein treating the substrate with acetone comprises contacting the substrate with acetone.

20. The method of claim 19, wherein contacting the substrate with acetone comprises: immersing the electrode in acetone, spraying the electrode with acetone, or allowing acetone to flow through the electrode; and / or Rinse the solid surface with acetone, Wherein, if immersion and rinsing are performed simultaneously, rinsing is performed after immersion.

21. A method according to any one of claims 18 to 20, comprising drying the substrate after treating the substrate with acetone.

22. A method according to any one of claims 18 to 21, provided that the method omits the step of applying a surface-modifying monolayer to the electrode.

23. A screen-printed carbon electrode without a surface-modified single layer prepared according to the method of any one of claims 18 to 22.

24. A method for blocking non-specific binding sites on a substrate comprising an antigen immobilized thereon, the method comprising treating the substrate with a protein hydrolysate.

25. The method of claim 24, wherein the protein hydrolysate is provided as a solution of casein hydrolysate.

26. The method of claim 24 or 25, wherein treating the substrate with a casein hydrolysate comprises incubating the substrate in the casein hydrolysate.

27. A diagnostic kit for diagnosing tuberculosis in a human or animal subject by electrical impedance spectroscopy, the kit comprising the screen-printed carbon electrode according to claim 17.

28. A method for detecting tuberculosis biomarker antibodies in a human or animal blood or tissue sample, the method comprising contacting a substrate onto which a mycolic acid antigen has been immobilized according to the method of any one of claims 7 to 16 or an electrode according to claim 17 with a sample from a patient suspected of having active tuberculosis, such that any biomarker anti-mycolic acid antibodies in the sample bind to the immobilized mycolic acid antigen.

29. Use of a polar organic solvent in preparing a mycolic acid antigen solution, for immobilizing the mycolic acid antigen on a substrate, so as to bind anti-mycolic acid antibodies via the antigen.

30. Use of acetone in preparing a screen-printed carbon electrode without a surface-modified monolayer for immobilizing an antigen thereon without a surface-modified monolayer.

31. Use of a casein hydrolysate for blocking non-specific binding sites on a substrate comprising an antigen or antibody immobilized thereon.

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