NL2027721B1 - Method of preparing a detection substrate, detection substrate, and uses thereof - Google Patents

Abstract

The present invention relates to a method of preparing‘ a detection. substrate for use in an antigen— antibody interaction determination assay. The invention also relates to a detection substrate, obtainable by said method. The invention also relates to a method for determining antigen—antibody interaction, comprising detection of binding between an antigen and antibody on said detection substrate and a method of determining whether a sample derived from a human or animal contains an antibody indicative of an infection, comprising detection of binding between an antibody in said sample with an antigen coated on said detection substrate, wherein the presence of binding is indicative for the presence of an infection in said human or animal.

Description

METHOD OF PREPARING A DETECTION SUBSTRATE, DETECTION SUB- STRATE, AND USES THEREOF The present invention relates to a method of prepar- ing a detection substrate for use in an antigen-antibody interaction determination assay. The invention also re- lates to a detection substrate, obtainable by said method. The invention also relates to a method for determining an- tigen-antibody interaction, comprising detection of bind- ing between an antigen and antibody on said detection sub- strate. The invention further relates to a method of de- termining whether a sample derived from a human or animal contains an antibody indicative of an infection, compris- ing detection of binding between an antibody in said sam- ple with an antigen coated on said detection substrate, wherein the presence of binding is indicative for the presence of an infection in said human or animal.

Antigen-antibody interaction determination assays of- ten involve coating a detection substrate with an antigen. It is standard procedure to coat the detection substrate by pipetting an amount of antigen dissolved in a solvent on a substrate, wherein a coated detection substrate is obtained after evaporation of the solvent. For instance, in ELISA assays often an amount of 25 to 50 ul dissolved antigen is added into a well of a 96 wells plate using pi- petting. Similar approached are followed in coating gold surface plasmon resonance substrates. Coating of the wells is effected by evaporation of the solvent in which the an- tigen is dissolved. After evaporation the antigen forms a coating on the detection substrate.

The inventor has found that there is room for im- provement with regard to coating detection substrates. Summary of the invention The inventor has observed that application of antigen in the conventional way, i.e. by pipetting often results in a detection substrate that is not coated uniformly and that antigen is locally concentrated at a position where the last solvent has evaporated. Especially in case of sensitive measuring techniques this may have adverse ef- fects on the reliability of determination of antigen- antibody interaction. For instance the inventor observed when antigens were coated onto a substrate by pipetting an amount of dissolved antigen onto the substrate, that after evaporation of the solvent either concentrated on the pe- riphery of wells of a 96 wells plate or on a single spot on a gold surface plasmon resonance substrate. As a result no uniform coating takes place, which is detrimental to, in particular, sensitive detection techniques. It also re- sults in obscuring potential binding partners in antigen- antibody binding. As a result a binding signal may be weak. Moreover, any quantities assessment of binding will also be made unreliable.

The present invention aims to overcome these prob- lems.

The inventor has now surprisingly found that improved performance of a detection substrate can be achieved by spray coating the antigen onto the substrate.

Therefore, in a first aspect the invention relates to a method of preparing a detection substrate for use in an antigen-antibody interaction determination assay, compris- ing providing an antigen in a solvent; spraying said anti- gen onto a substrate; and evaporating said solvent to ob- tain a detection substrate having said antigen coated thereto.

In a second aspect the invention relates to a detec- tion substrate, obtainable by the method according to the first aspect.

In a third aspect the invention relates to a method for determining antigen-antibody interaction, comprising detection of binding between an antigen and antibody on the detection substrate according to the second aspect.

In a fourth aspect the invention relates to a method of determining whether a sample derived from a human or animal contains an antibody indicative of an infection, comprising detection of binding between an antibody in said sample with an antigen coated on the detection sub- strate according to the second aspect, wherein the pres- ence of binding is indicative for the presence of an in- fection in said human or animal.

The inventor has surprisingly found that when anti- gens are coated on a detection substrate by means of spray coating a homogenous and uniform coating takes place. This leads to optimal distribution of the antigens over the coated surface. As a result exposure of the antigens to their respective antibodies is improved, leading to im- proved sensitivity of detection. In line with this, the present invention allows reliable detection results while requiring less antigens immobilized on a substrate than conventional methods that apply other means of applica- tion. In view of the large costs of antigens, in particu- lar in view of synthesis and purification, this is an im- portant advantage. Further, because of the improved dis- tribution of the antigens the invention allows a more quantitive approach with regard to the detection of inter- action between antibodies and antigens.

Short description of the drawings Fig.l shows a comparison between binding signals ob- tained from a gold detection substrate coated in a conven- tional prior art manner and coated by spray coating in ac- cordance with the invention.

Fig.2 shows a surface plasmon analysis using a spray coated gold chip.

Fig.3 shows a comparison between binding signals ob- tained from ELISA wells coated in a conventional prior art manner and coated by spray coating in accordance with the invention. Detailed description of the invention The present invention is based on the finding that when antigens are coated on a detection substrate by means of spray coating a homogenous and uniform coating takes place leading to improved sensitivity and reliability of detection as well quantitively as qualitively.

With the term spraying is meant that the dissolved antigens are blown or driven through the air in the form of tiny drops, preferably in the form of a nebula, fog or mist. It is preferred that the liquid is applied on the substrate in the form of atomized mist. Droplets prefera- bly have an average diameter size of 0,7 mm or less such as 0,5 mm or less, such as 0,3 mm or less, such as 0,2 mm or less. The droplets preferably have a diameter in the micrometer range, i.e. between 1 and 999 um, such as be- tween 1 and 700 um, between 1 and 500 um, between 1 and 250 um or between 1 and 100 um, for instance.

Spraying the dissolved antigens onto the substrate can be performed with any suitable spray equipment. Given the often small volumes that are used to coat a substrate it is preferred that the equipment is configured to spray the liquid locally on specific areas in the millimetre range or less.

In order to obtain the tiny droplets of the above specified sizes is preferred that spraying is carried out of a system configured to spray the dissolved antigens us- ing air pressure. In a preferred embodiment use is made of an airbrush system. Such systems are equipped to spray liquid using air pressure.

Suitable nozzles for spraying can be applied with di- ameters of the above specified diameter sizes, such as nozzles with a size in the micrometer range, for instance between 0,01 and 0,7 mm, for instance nozzles with a 0,5 5 mm, 0,3 mm, 0,2 mm or 0,1 mm diameter size or even small- er. Standard nebulizers as known in the art can be used for these purposes. Such nebulizers are capable to produce the droplets of the above specified sizes. Pressures may be applied between 1 and 3,5 bar, although for the method of the invention pressures in the lower range of this, such as pressures between 1 and 1,5 bar will be most suit- able.

Preferably the spray system is a multichannel system. In a preferred embodiment the air brush system is a multi- channel airbrush system configured to coat multiple detec- tion substrates simultaneously. This is in particular ad- vantageous in case detection arrays are used. Such arrays include for instance ELISA plates with arrays of ELISA wells, such as the commonly used 96-wells plates. Spray coating can for instance be automated using computer nu- merical control (CNC).

The inventor has found that advantageous effects of the present invention are in particular well expressed when lipid based antigens are used. The inventor has ob- served that application of lipid antigens in the conven- tional way, i.e. by pipetting, results in a detection sub- strate that is not coated uniformly and that the lipids are locally concentrated at a position where the last sol- vent has evaporated. Without wishing to be bound by theory it is thought that the hydrophilic tails of lipid antigens may be a contributing factor in these problems. As men- tioned above, this may have adverse effects on the relia- bility of determination of antigen-antibody interaction. The present invention solves these problems in particular in case of lipid based antigens. It is therefore preferred that the antigen coated on the substrate is a lipid based antigen.

The inventor has observed excellent results in re- spect of coating detection substrates with lipid based my- cobacterial antigens, in particular antigens of Mycobacte- rium tuberculosis, the causative agent of tuberculosis.

This is in particular advantageous because this opens the possibility to achieve more sensitive and reliable diagno- sis of mycobacterium borne diseases, such as tuberculosis. This will be explained below in more detail.

Preferred mycobacterial antigens for this application may be selected from one or more of a) a mycolic acid derived antigen; b) a tuberculosinyl adenosine antigen; c) a diacyl glycolipid antigen; and d) a mannosyl phosphoketide antigen.

Such antigens and examples are described in detail in pa- tent applications WO 2020/119669 Al, WO 2020/030034 Al, WO 2017/211314 Al and WO 2017/211316 Al of the present appli- cants. The antigens disclosed in these documents can be used for purposes of the present invention and are incor- porated in the present patent application by reference.

The solvent in which the antigen is dissolved may be any suitable solvent chosen on the basis of the antigen. In other words, they should dissolve well in the solvent and, if present, their secondary or tertiary structure should be stable in the solvent.

Solvents may be adueous solvents or organic solvents and are preferably volatile such that evaporation can take place, optionally by heating. In particular in view of their hydrophobic tails it is preferred that the above specified lipid antigens are dissolved in an organic sol- vent. Such organic solvent may include without limitation pentane, hexane, heptane, petroleum ethers, diethyl ether, dichloromethane, chloroform polar organic solvents such as alcohols, esters, ketones and aldehydes, mixtures thereof, and mixtures thereof containing water. Further exemplary solvents include methanol, ethanol, propanol, isopropanol,

and acetone or mixtures of any of the solvents mentioned herein.

For the purpose of spray coating lipid antigens such as the above mentioned mycobacterial antigens it is pre- ferred that the organic solvent is or comprises hexane.

With the combination lipid antigens and hexane excellent results are obtained in terms of the advantageous effects specified above.

Very good results, as evidenced in the examples section of this application have been obtained when the antigen is a mycolic acid dissolved in hexane.

It is therefore preferred that the solvent is hexane and the antigen is a mycolic acid or a derivative thereof.

The invention is in principle applicable to any de- tection surface that can be used in an antigen-antibody interaction determination assay, because the advantageous effects are obtained regardless of the surface character- istics of the substrate.

What matters is that the antigens are applied in the form of tiny drops such as in the form of atomized mist, so that a high evaporation surface is obtained, leading to evaporation and immobilisation of the antigens before they get the chance to concentrate local- ly.

Such substrates include without limitation a sensing surface of a surface plasmon resonance device, electrochem- ical impedance spectroscopy device, isothermal titration calorimetry device, bio-layer interferometry device, opti- cal gratings device, photonic crystal device, acoustic res- onant profiling device, or quartz crystal microbalance de- vice.

The substrate may further be a detection surface in an enzyme-linked immunosorbent assay (ELISA), a Western blotting assay, radioactive labelling assay, photospectro- metric assay, immunofluorescence, imunoprecipitation as- say, immunocytochemistry assay, immunchistochemistry assay, amperometric detection assay, voltametric detection assay, or electrochemical impedance spectroscopy assay.

Excellent results have been obtained with ELISA plates as detection substrates.

These plates are in general made of polystyrene. In one embodiment the substrate therefore may be a polystyrene substrate such as an ELISA plate.

Excellent results have also been obtained with gold detection substrates. In another embodiment the substrate therefore may be a gold substrate. Such gold substrates are suitably used in detection techniques such as surface plas- mon resondance. Therefore, the gold substrate may suitably be a surface plasmon resonance detection substrate.

The detection substrate obtained by the method of preparing a detection substrate according to the invention, i.e. the detection substrate according to the invention, can be used in a method for determining antigen-antibody interaction, comprising detection of binding between an antigen and antibody on the detection substrate.

As can be understood from the above such a method for determining antigen-antibody interaction may suitably comprise one of the techniques selected from the group consisting of surface plasmon resonance, electrochemical impedance spectroscopy, isothermal titration calorimetry, bio-layer interferometry, optical gratings, photonic crystal assay, acoustic resonant profiling, quartz crystal microbalance assay, enzyme-linked immunosorbent assay (ELISA), a Western blotting assay, radioactive labelling assay, photospectrometric assay, imunofluorescence, immunoprecipitation assay, immunocytochemistry assay, immunohistochemistry assay, amperometric detection assay, voltametric detection assay, or electrochemical impedance spectroscopy assay.

Excellent results have been obtained with surface plasmon resonance and ELISA. The method for determining antigen-antibody interaction may therefore suitably comprise one of surface plasmon resonance and ELISA.

In a preferred embodiment of the method for determining antigen-antibody interaction said detection substrate has a mycobacterial antigen, in particular a lipid based mycobacterial antigen, coated thereto, and the method comprises detection of binding between said antigen and an antibody indicative of tuberculosis in a human or animal.

As follows form the above the method for determining antigen-antibody interaction according to the invention can be applied to detect antibodies that are associated with an infection, Therefore the invention also relates to a method of determining whether a sample derived from a human or animal contains an antibody indicative of an infection. This method comprises detection of binding between an antibody in said sample with an antigen containing an epitope that can be recognized by said antibody, said antigen being coated on the detection substrate of the invention, wherein the presence of binding is indicative for the presence of an infection in said human or animal. In this method the presence of antibodies specific for an infectious agent in a sample is determined using a substrate spray coated with antigens that specifically bind to the antibodies in the sample.

The sample may be any human or animal sample, including sputum or blood samples. In case the sample is a whole blood sample, the sample may depending on the way of detection of binding of antibodies to the antigen be filtered or separated to plasma or serum. The sample is preferably a blood derived sample. The sample may be a whole blood sample, a plasma sample or a serum sample. Blood serum is blood plasma without clotting factors and is preferred as plasma. The word plasma in this application may therefore as well refer to (blood) serum. About 55% of whole blood consists of plasma/serum. If a whole blood sample is not filtered perfectly or if the patient’s physical situation necessitates it, it may be desired to dilute the whole blood sample or plasma or serum. The words plasma or serum in this application may therefore also refer to diluted plasma or serum. A dilution of the blood or plasma may therefore be implemented in the real time method of the invention, such as a 250 to 5000 x dilution, a 750 to 1250 x dilution, such as for instance a 4000, 2000 or 1000 x dilution.

Dilution may be performed with any suitable diluent, for example a PBS based buffer.

Such buffer may for example be a PBS/AE buffer comprising NaCl, KCl, KH:P04, Na HPO; and EDTA in water at physiological pH.

Such buffer may be a PBS based buffer consisting of 8.0 g NaCl, 0.2 g KCl, 0.2 g KH;PO,;, and 1.05 g Na:2HPO,4 per liter of double distilled, deionized water containing 1 mM EDTA and 0.025% (m/v) sodium azide which is adjusted to pH 7.4. The whole blood sample or plasma or serum may be further diluted with agents that prevent blood clotting, such as EDTA, heparine or citrate.

Optionally a detergent may be added in low concentration to the blood/plasma/serum to avoid sticking of components to walls of tubings, vessels or containers.

In principle the method is applicable to any infection wherein antigens elicit an immunogenic response accompanied with the generation of antibodies.

The method can be applied to diagnose a disease in humans or animals, preferably humans.

The inventor has found a particular applicability in diagnosis of tuberculosis, which is caused by Mycobacterium tuberculosis.

The infection in the context of the invention may therefore suitably be a tuberculosis infection.

Suitable methods and approaches for determining whether a sample derived from a human or animal contains an antigen indicative of tuberculosis infection are described in WO 2017/143985 Al, WO 2016/111619 Al, WO 2020/11%669 Al, WO 2020/030034 Al, WO 2017/211314 Al and WO 2017/211316 Al of the present applicants, the content of which is incorporated in the present patent application by reference.

Examples The following examples and explanation are meant to illustrate the invention and not to limit the scope of the invention.

Example 1 - gold substrates In an exemplary setup the principle of the invention was tested by spray coating a lipid antigen on a gold sub- strate. For this purpose gold chips were used. These chips were each placed on the bottom of a well of a polystyrene 6 wells plate. The gold chips are visualised in Fig.l. The dark halos in panels A, B and C represent the peripheral edges of the disc shaped chips.

An antigen solution was prepared as follows. A 50 pg/mL mycolic acid solution in hexane was prepared.

Chip A of Fig. 1 was not coated. Only hexane was placed on the chip and left to evaporate.

Chip B of Fig. 1 was coated by placing 200 pl of hex- ane dissolved mycolic acid on the chip by pipetting. The place of application is indicated with the black arrow.

Chip C of Fig. 1 was coated by spray coating hexane dissolved mycolic acid on the chip, using an air brush de- vice. Spraying was performed until the full chip was cov- ered. It is estimated that a total of 20 ul was required for this.

After coating the gold chips were allowed to dry and antibody antigen interaction was performed in accordance with the following steps (indicated per well of the 6 wells plate): e Blocking in 3 ML PBS 1% carbohydrate free BSA for 30 minutes; e Incubation for 30 minutes with 3 ml 1 1 :1000 diluted anti-mycolic acid in PBS; e Washing 3x with 3 ml PBS; e Incubation for 30 minutes with 3 ml 1 :1000 diluted anti-human IgG-biotin in PBS; e Washing 3x with 3 ml PBS e Incubation for 30 minutes with 3 ml 1 :1000 diluted streptavidin HRP in PBS e Washing with 3 ml PBS. Chips were put in a grip bag with a pen marker strip as a positive control. To this 0,5 mL of a Biorad substrate liquid was added for 6 minutes to develop a black stain- ing. The uncoated gold chip shown in panel A of Fig.l shows no staining. The droplet coated gold chip shown in panel B of Fig.l shows intense staining at the point of application as indicated with the arrow. The intensity of staining rapidly decreases with the distance from the point of application as can be seen by the staining pat- tern around the intense black spot. A large part of this chip (i.e. the top and right portions) shows no signifi- cant staining. The spray coated gold chip shown in panel C of Fig.l shows a uniform staining all over the gold chip. Apart from some minor local variations the intensity of staining is the same all over the chip.

Example 2 -surface plasmon resonance analysis A non-coated chip and a chip spray coated with mycol- ic acid as described for the gold chips of Fig. 1, panels A and C were subjected to surface plasmon resonance (SPR) analysis. The graph shown in Fig.2 shows in graph A the SPR signal of the non-coated chip and in graph B the SPR signal of the spray coated chip is shown. During the meas- urement washing steps take place until point I. At point I a so-called sealer is added, a thiol compound which fills the empty spaces of gold not covered with antigen. At point II a human monoclonal anti-mycolic acid antigen is added in a 1:200 dilution factor, followed by washing steps just before point III. At point III anti-human IgG is added. As shown in graph B, this results in a high in- crease in signal, indicating that the gold chips uniformly spray coated with antigen are well applicable in SPR anal- ysis. Example 3 — ELISA substrates In another exemplary setup the principle of the in- vention was tested by spray coating a lipid antigen on an ELISA substrate. For this purpose polystyrene 6 wells plates were used.

The bottoms of the wells are visualised in Fig.3. An antigen solution was prepared as follows. 1 ml of a 200 uM mycolic acid in DCM methanol was evaporated and subsequently dissolved in hexane with sonification to ob- tain a 200 uM mycolic acid solution.

Well A of Fig. 3 was not coated.

Only hexane was placed on the chip and left to evaporate.

Well B of Fig. 3 was coated by placing 200 ul of hex- ane dissolved mycolic acid in the well by pipetting.

The place of application is indicated with the black arrow.

Well C of Fig. 3 was coated by spray coating hexane dissolved mycolic acid on the bottom of a well using an air brush device.

In this case the antigen was spray coat- ed in a straight line extending between opposite sides of the bottom of the well as indicated by the arrow in panel C of Fg.3. It is estimated that a total of 20 ul was sprayed.

After coating the wells were allowed to dry and anti- body antigen interaction was performed in accordance with the following steps (indicated per well of the 6 wells plate): e blocking for 30 minutes with 3000 ul PBS 5% car- bohydrate free BSA; e incubation for 30 minutes with 3 mL 1 :1000 di- luted anti-MA in PBS-BSA; e 3x wash with 3000 ul PBS e Incubation for 30 minutes with 3 mL ul 1 :2000 diluted anti-human IgG-biotin in PBS BSA e 3x wash with 3 ml PBS e Incubation for 30 minutes with 3 ml 1 :1000 di- luted streptavidin HRP in PBS-BSA and e wash with 3 ml PBS ImL Clarity Western ECL substrate liquid was added to each wells to develop staining.

The uncoated gold chip shown in panel A of Fig.3 shows no staining.

The droplet coated gold chip shown in panel B of Fig.l shows intense staining at the periphery of the well.

No significant staining rapidly was observed in the rest of the well bottom.

The spray coated well shown in panel C of Fig.l shows a uniform staining all over the application line indicated with the arrow.

Apart from some minor local variations the intensity of staining is the same all over the application line.

Claims (1)

CONCLUSIONS A method for preparing a detection substrate for use in an antigen-antibody interaction assay, comprising providing an antigen in a solvent; spraying the antigen onto a substrate; and evaporating the solvent to obtain a detection substrate with the antigen coated thereon. A method according to claim 1, wherein the spraying is performed with a system adapted to spray the dissolved antigen using air pressure. Method according to claim 2, wherein the spraying is carried out by means of a nozzle with a diameter size in the micrometer range with an air pressure between 1 and 3.5 bar. The method of claim 2 or 3, wherein the system is a multi-channel system. A method according to any one of the preceding claims, wherein the antigen is a lipid-based antigen. The method of claim 5, wherein the antigen is a mycobacterial antigen. The method of claim 6, wherein the mycobacterial antigen is selected from one or more of: a) a mycolic acid-derived antigen; b) a tuberculosinyladenosine antigen; c) a diacylglycolipid antigen; d) a mannosylphosphoketide antigen. A method according to any one of the preceding claims, wherein the solvent is an organic solvent. The method of claim 8, wherein the organic solvent is hexane. The method of claim 9, wherein the organic solvent is hexane and the antigen is a mycolic acid or a derivative thereof. A method according to any preceding claim, wherein the substrate is a sensing surface of a surface plasmon resonance device, electrochemical impedance spectroscopy device, isothermal titration calorimetry device, biolayer interferometry device, optical grating device, photonic crystal device , acoustic resonance profiling device, or quartz crystal microbalance device; or wherein the substrate has a detection surface in an enzyme-linked immunosorbent assay (ELISA), a western blot assay, radioactive labeling assay, photospectrometric assay, immunofluorescence assay, immunoprecipitation assay, immunocytochemistry assay, immunohistochemistry assay, amperometric detection assay, voltametric detection assay, or electrochemical impedance spectroscopy test. A method according to any one of the preceding claims, wherein the detection substrate is a gold substrate or a polystyrene substrate. The method of claim 12, wherein the gold substrate is a surface plasmon resonance detection substrate. A detection substrate obtainable by the method according to any one of the preceding claims. A method for determining antigen-antibody interaction comprising detecting binding between an antigen and antibody on the detection substrate of claim 14. The method of claim 15, wherein the method comprises one of the techniques selected from the group consisting of surface plasmon resonance, electrochemical impedance spectroscopy, isothermal titration calorimetry, biolayer interferometry, optical grid test, photonic crystal test, acoustic resonance profiling, quartz crystal microbalance test, enzyme-linked immunosorbent test (ELISA), a Western blot test, radiolabelling test, photospectrometric test, immunofluorescence test, immunoprecipitation test, immunocytochemistry test, immunohistochemistry test, amperometric detection test, voltametric detection test, or electrochemical impedance spectroscopy test. The method of claim 16, wherein the method comprises surface plasmon resonance or ELISA. The method of any one of claims 15 to 17, wherein the detection substrate has a mycobacterial antigen coated thereon, and wherein the method comprises detecting binding between the antigen and an antibody indicative of tuberculosis in a human or animal. A method for determining whether a sample derived from a human or animal contains an antibody indicative of an infection, comprising detecting binding between an antibody in the sample with an antigen coated on the detection substrate of claim 14 wherein the presence of binding is indicative of the presence of an infection in the human or animal. The method of claim 19, wherein the infection is a tuberculosis infection. -0-0-0-