Lectin-based assay for detecting microbial contamination

Abstract

An analytical method for detecting microbial contaminants in oily liquids, comprising: a) preparing an aqueous liquid from the oily liquid to obtain polysaccharide-containing biomicrobial material derived from any microbial contaminants contained in the oily liquid; b) determining the presence of polysaccharides in the aqueous liquid using a lectin-based lateral flow chromatography assay (LFA), wherein the LFA uses mannose-binding protein (MBP) as a trapping agent in the detection zone of the lateral flow chromatography membrane and as a trapping agent binding to flowable, identifiable reporter nanoparticles, using a capture reaction buffer (CRB) containing 100–500 mM free calcium ions, detergent, buffer, and a pH range of 6.5 to 9.0, thereby allowing the identification of MBP sandwich trap products at the detection zone, the MBP sandwich trap products indicating microbial contamination; and a detection kit used in the method, the detection kit comprising: a) A lectin-based lateral flow chromatography (LFA) apparatus that uses mannose-binding protein (MBP) as a trapping agent, which binds to flowable, identifiable reporter nanoparticles in the reaction zone of the lateral flow chromatography membrane; and b) a running buffer containing detergent, buffer, and a pH range of 6.5 to 9.0, wherein the running buffer sets the amount of free calcium ions in the reaction zone to 100–500 mM.

Description

Technical Field

[0001] This invention relates to a method for determining microbial contamination in oily liquids, such as oil, particularly crude oil, petroleum, or their fractions. The method employs a lateral-flow assay (LFA) that uses mannose-binding protein (MBP) as a trapping agent to react with the glycans of contaminating microorganisms, particularly mannose or mannan. By using a specific reaction buffer, the sensitivity of LFA can be improved even in the presence of hydrophobic substances in the sample to be tested using LFA (substances that can interfere with the MBP-glycan reaction or the MBP-mannose reaction). Background Technology

[0002] Detecting microbial contamination in non-aqueous fluids, such as diesel and kerosene, is notoriously difficult because many different organisms can grow in these liquids or at the interface between the hydrophobic and aqueous phases. Detecting such microbial growth can serve as an indication of problems during the storage or use of these liquids.

[0003] There are many different methods available for detecting microbial contamination, such as growth assays on solid or liquid matrices, assays using ATP as a substitute biomarker for metabolic activity, nucleic acid-based assays, and assays that detect microbial antigens of the target microorganism using specific antibodies. Among these assays, the simplest and fastest form is lateral flow chromatography because it can be performed on-site and requires relatively little equipment and expertise.

[0004] Lateral flow immunoassay (LFIA) has been used to detect microbial contamination in fuel tanks, utilizing antibodies that target microbial structural antigens. ASTM International has adopted the standard test method ASTM D8070 for screening fuel and fuel-related aqueous samples for microbial contamination via LFIA. This semi-quantitative test method can be used to determine whether contaminants in samples collected from fuel tanks and fuel systems are within specific limits. The test method detects the presence of microbial antigens and metabolite antigens in the sample, which are produced by metabolites generated by living cells and some fungi and bacteria during their growth on fuel. The presence of antigens is an indication of microbial contamination in the fuel system. This test method aims to provide a tool for assessing the presence of microbial growth in fuel storage and distribution equipment or end-user fuel tanks, and to alert fuel suppliers or users to potential problems with fuel quality or operation, or to suggest the need for preventative or remedial actions, or both.

[0005] A drawback of ASTM D8070 is its limited sensitivity: the ASTM D8070 test method may be less sensitive than other methods, potentially leading to false negative results. Furthermore, this LFIA is an antibody-based assay used to detect some microorganisms or microbial material commonly found in fuel tanks. Therefore, while the antibodies used in such assays may achieve acceptablely high levels of sensitivity for some target molecules, their limited specificity means that the assay may not detect all microorganisms and microbial material. This issue of relatively concentrated specificity can be addressed by selecting detection antibodies that target universal microbial targets (broad-spectrum common antigens) or by increasing the number of targets (and thus correspondingly increasing the number of specific antibody species in the assay).

[0006] It has been proposed that such assays utilize lectins and Toll-like receptors as ligands to target common antigens, enabling the detection of microbial material with broad specificity (e.g., WO2021252902A1, EP3081937B1, and US9593160B2), thus exhibiting high practical detection sensitivity. This method is known to involve reactions in aqueous matrices, as lectins and TLRs have been selected in nature to function effectively in such matrices.

[0007] However, the specificity and affinity of lectins can be affected by trace amounts of hydrophobic substances in the assay buffer. The presence of hydrophobic substances in the assay buffer may affect the conformation of glycoproteins or the accessibility of specific glycan structures, thereby affecting the binding affinity of lectins. Furthermore, hydrophobic substances may potentially interfere with the binding sites of lectins, leading to changes in their specificity and affinity for glycan motifs.

[0008] To detect most bacterial and fungal substances (live or dead) in fuels such as kerosene or diesel with high sensitivity, the oil or grease is typically extracted with an aqueous extraction buffer before initiating the LFA step. However, the extract may still contain hydrophobic residues. The signal attenuation over time in lateral flow immunochromatography can be challenging, particularly affecting sensitivity, robustness, accuracy, and the reliability of the results. Standard and well-known measures to combat signal attenuation in LFA include: using suitable nanoparticles, using covalently linked chemistry, using optimized membranes, removing interfering substances, controlling flow rate, and appropriate readout time.

[0009] A crucial universal target on microorganisms and their products is the mannose motif, which can be part of high-mannose glycoprotein structures, polymannose, or the cell wall. As broad-spectrum, specific lectins, mannose-binding proteins (MBPs) (also known as mannan-binding proteins) are therefore candidate binding molecules for detecting microbial structures in samples. MBPs are known to recognize mannose-containing structures, such as those present on the surfaces of bacteria, fungi, and viruses. Some MBPs can also recognize N-acetylglucosamine, a sugar molecule present on the surfaces of some bacteria and fungi; they can also bind to other sugar molecules (such as fucose and glucose) and lipopolysaccharide (LPS, a component of the outer membrane of Gram-negative bacteria).

[0010] MBP, or C-type lectin, is a calcium-dependent carbohydrate-binding protein that typically forms oligomeric structures to exert its effective function. The native MBP polypeptide chain can consist of several distinct regions, such as collagen-like regions, α-helical coils, and the C-type lectin domain (CTLD). Collagen-like and coil regions promote polymerization, while the C-type lectin domain confers specificity to carbohydrates. The C-type lectin fold structure is known to be a compact domain of 110–130 amino acid residues with a bicyclic two-stranded antiparallel β-sheet structure formed by amino-terminal and carboxyl-terminal residues linked by two α-helices and a triple antiparallel β-sheet. Calcium binding in C-type lectins is mediated by specific amino acid residues that coordinate calcium and bind to the hydroxyl groups of sugars. The CTLD has two highly conserved disulfide bonds and at most four Ca2+ bonds. ++ Binding sites, the occupancy of which depends on the lectin. Amino acid residues with carbonyl side chains typically bind with Ca in the carbohydrate recognition domain (CRD). 2+ Coordination, and when combined with Ca ++ Then, these residues can bind directly to sugars. This can happen with sugars and calcium compounds in glycans. 2+ Ions form ternary complexes with amino acids within the CRD. Changes in amino acids within the CRD may alter sugar specificity. This includes the binding of mannose and Ca... ++ Key conserved residues typically include the “EPN” and “WND” amino acid motifs in the CRD of C-type lectins.

[0011] The presence of cofactors, such as divalent cations other than calcium, can affect the binding activity of MBL. Typically, calcium concentrations in the range of 1–10 mM are used to promote MBL recognition of mannose-containing structures. US10696733B2 discloses microparticles for diagnosing infection and their reaction with mannose structures in samples, said microparticles being coated with mannose-binding lectins. After adding a processing buffer to remove detergent, DNase, plasmin, and salts, the sample typically contains 5 mM of calcium.

[0012] WO2013012924A2 discloses engineered microbial-targeting or microbial-binding molecules for capturing microorganisms. The microbial-targeting molecule may contain a CRD of a type C lectin (e.g., human mannose-binding lectin). After adding a treatment buffer, in addition to removing contaminants, DNase, plasmin, and salts, the sample also contains 5 mM of calcium.

[0013] WO2021252902 discloses the use of LFA targeting microbial molecules, which includes, for example, human mannose-binding lectin. It is described that a suitable running buffer, in addition to removing detergents, glucose, and salts, contains 10 mM CaCl2.

[0014] WO02 / 068959A2 discloses a detection method for analyzing the presence of microorganisms in hydrocarbon fuels using antibodies that react with microorganisms. WO2010102285A1 discloses a side-flow chromatography glycan detection device that uses a conjugate containing a first lectin and a label, as well as a fixed second lectin, to detect specific types of glycans in body fluids.

[0015] US2019077850A1 discloses a microbial binding molecule comprising a microbial surface binding domain connected to a portion of its Fc region, such as mannose-binding lectin (FcMBL) that binds to the Fc region of an antibody. Microorganisms expressing protein A and protein G can bind to the microbial binding molecule through two independent (but additive) mechanisms: Fc-mediated binding and the microbial surface binding domain. Free calcium ions can be removed using chelating agents to bind microorganisms expressing protein A or protein G through the Fc region of the molecule.

[0016] WO2019064463A1 discloses a method for concentrating target viruses or bacteria, which captures the target viruses or bacteria by means of metal ion-dependent ligand-binding molecules and separates them from the ligand-binding molecules by treatment with a chelating agent.

[0017] US2021364491A1 discloses a method for detecting biocontamination in fuel samples using specific biometric elements.

[0018] DE102014116204B3 discloses a method for detecting microbial contamination in organic liquids using qPCR.

[0019] Li Zezhen et al. (American Journal of Biochemistry and Biotechnology 2018, 14(2):117-123) described the detection of sulfate-reducing bacteria in aviation kerosene by loop-mediated isothermal amplification combined with lateral flow chromatography test strips.

[0020] Climent Estela et al. (ACS Sensors 2020, 6(1): 2379-3694) described the detection of microbial contamination in fuel by identifying microbial genomic DNA in fuel extracts.

[0021] There is a need for a highly sensitive and robust assay method to identify broad-spectrum microbial contamination in hydrophobic fluids, such as oily liquids, and especially fuels. Summary of the Invention

[0022] This summary is provided to introduce, in a simplified form, some concepts that will be further described in the detailed description below. This summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to limit the scope of protection of the claimed subject matter. Other features, details, utility, and advantages of the claimed subject matter will become apparent from the following detailed written description, including those shown in the accompanying drawings and defined in the appended claims.

[0023] The object of this invention is to provide novel strategies or methods for identifying, detecting, and / or controlling microbial contamination in hydrophobic fluids (e.g., oily liquids, particularly fuels). A specific object is to ensure the quality of any such hydrophobic fluid by determining the presence or absence of microbial contaminants using sensitive assay methods. A specific object is to provide an improved LFA using MBP as a trapping agent.

[0024] This objective is addressed by the claimed subject matter, which is further described herein.

[0025] This invention provides an analytical method for detecting microbial contaminants in oily liquids (especially oily liquid samples), comprising: a) Prepare an aqueous liquid from an oily liquid to obtain polysaccharide-containing microbial material derived from any microbial contaminants contained in the oily liquid; b) The presence of glycans in aqueous liquids was determined using a lectin-based lateral flow chromatography assay (LFA) that used mannose-binding protein (MBP) as a trapping agent in the detection zone of the lateral flow chromatography membrane and as a trapping agent that binds to flowable, identifiable reporter nanoparticles. A capture reaction buffer (CRB) containing 100–500 mM free calcium ions, detergent, buffer, and a pH range of 6.5 to 9 was used to allow the identification of MBP sandwich trap products at the detection zone, which indicate microbial contamination.

[0026] This invention provides an LFA with unexpected sensitivity and robustness under specific assay conditions by coupling MBP with an aqueous extract or fraction of an oily liquid. It has been found that hydrophobic or oily residues in such aqueous extracts or fractions do not interfere with the high sensitivity and robustness of the assay.

[0027] It has been found, as described in this paper, that MBP capturing its target structure in the presence of CRB can greatly improve LFA.

[0028] The target structure of the MBP (also referred to as the target analyte in this document) should be understood as a structure or compound that is recognized and bound by the MBP to detect analytes containing such structures. These are typically polysaccharide structures, particularly those containing mannan or mannose.

[0029] Unexpectedly, the presence of high concentrations of free calcium ions increased the binding of MBP to its target structure, both in terms of sensitivity and binding stability. Specifically, robust and sensitive LFAs containing 100–500 mM of free calcium ions were provided to support the binding of MBP to the target structure.

[0030] A certain amount of free calcium ions can exist in the buffer system, which provides 100-500 mM of free calcium ions in CRB, thereby enabling the capture reaction to proceed with high sensitivity and without interference from oily liquid residues in aqueous liquids prepared from oily liquids.

[0031] In addition to the amount of free calcium ions mentioned above, CRB advantageously also contains detergents and buffers, and has a pH range of 6.5 to 9.0, preferably about (i.e. ±0.5 or ±0.25) 7 or 8.

[0032] Specifically, a buffer system comprising one or more liquids or buffer solutions can be conveniently used in LFA. For example, an extraction buffer or buffer solution can be used to obtain an extract of an oily liquid. This extract can be used with or without a running buffer. If the extract is applied directly to the LFA device, particularly to the sample region of the side-flow chromatography membrane, it can serve as the extraction medium for side-flow chromatography within the LFA device, thereby enabling LFA without the need for a separate running buffer. This extraction medium can be, for example, an extraction buffer that is used as a running buffer. In other words, the running buffer can also be used as an extraction buffer. However, a separate running buffer can also be used in addition to the extraction buffer. Furthermore, a chase buffer can also be used.

[0033] Typically, buffer solutions may be contained in specific areas of the LFA device, particularly at or on the side-flow chromatography membrane, preferably in a dry form, such as dried on the specific area. The reaction zone of the LFA device may contain a medium, typically a buffer solution, contained in specific areas within the reaction zone. Specifically, a conjugate buffer is contained in the conjugate zone, and / or a detection buffer is contained in the test zone and / or control zone. Additionally, a sample buffer may be used, typically contained in the sample zone of the LFA device, such as a sample pad contained at or on the side-flow chromatography membrane.

[0034] Specifically, the accumulation of free calcium ions originates from the buffer system, resulting in a total amount of 100-500 mM of free calcium ions during the reaction in which MBP binds to the MBP target structure.

[0035] Specifically, a buffer system is used to set the composition of CRB in the reaction zone of the LFA device, particularly in the side-flow chromatography membrane reaction zone of the LFA device, thereby providing the required amount of 100-500 mM of free calcium ions in the region where the MBP binds to its target structure (i.e., is captured).

[0036] The reaction zone of the LFA unit, especially the reaction zone of the side-flow chromatography membrane in the LFA unit, specifically includes: (i) A binding region (also known as a binding pad) comprising an MBP (also known as an MBP reporter nanoparticle) bound to a reporter nanoparticle; and (ii) A detection area, which includes a test area and an optional control area.

[0037] A test area typically includes test lines, test points, or a test area region, or is composed of test lines, test points, or a test area region, with an MBP fixed in the test area.

[0038] A control area typically includes a control line, a control point, or a control region, or is composed of a control line, a control point, or a control region, and a control object is fixed in the control area.

[0039] Specifically, the methods described herein provide the composition of the CRB as described herein: a) At the junction region of the side-flow chromatography membrane; b) At the reaction zone of the side-flow chromatography membrane, particularly at the test zone of the side-flow chromatography membrane, especially at the test zone within the detection zone, and optionally at the control zone within the detection zone.

[0040] Specifically, a buffer system is used to set a total amount of 100-500 mM of free calcium ions in the CRB, thereby providing a high amount of free calcium ions when the MBP captures the target structure.

[0041] Specifically, a running buffer containing 100-500 mM of free calcium ions, or supplementing the amount of free calcium ions in an aqueous liquid (e.g., an aqueous extract), and / or the device used for LFA, is used to set the total amount of free calcium ions in the CRB to 100-500 mM, thereby providing a higher amount of free calcium ions when the MBP captures the target structure.

[0042] In addition to, or as an alternative to, the run buffer, an extraction buffer or catch-up buffer may be used, which contains 100-500 mM of free calcium ions, or the amount of free calcium ions provided by the device used for LFA (e.g., the binding buffer) and optionally the run buffer, to set the total amount of free calcium ions in the CRB to 100-500 mM, thereby providing a higher amount of free calcium ions when the MBP captures the target structure.

[0043] Typical buffer systems include any one or more of an extraction medium (particularly an extraction solution, such as an extraction buffer), a running buffer, an optional catch-up buffer, and / or an additional buffer at the binding pad, to obtain or set up a CRB with the desired composition in the reaction zone of the LFA device, thereby supporting the capture reaction in a medium containing or composed of CRB.

[0044] Specifically, when MBP binds to its target structure, CRB is first present in the binding region or binding pad, where the MBP reporter nanoparticle captures the target structure; secondly, it is present in the test region, where the immobilized MBP captures the target structure bound to the MBP reporter nanoparticle, thus forming an MBP sandwich capture product. If the target structure of the MBP (e.g., mannan) is used as a control (also called the control target structure), and the MBP reporter nanoparticle captures the control target structure in this region, a third reaction may optionally occur in the control region.

[0045] Specifically, if present, the MBP capture product comprises or consists of MBP, which binds to (“captures”) the target structure. As described herein, the formation of the MBP capture product is specifically carried out in the presence of capture reaction buffer (CRB).

[0046] MBP capture products are typically found in the region of the corresponding LFA device where the MBP reporter nanoparticle (sometimes referred to as the "binding") first contacts the target structure, such as in the reaction region including a "binding region" (also known as a "binding-release region"). This region may contain the MBP reporter nanoparticle, which binds to the target structure (if present) and migrates along the LFA membrane. Such MBP capture products are also referred to herein as "first MBP capture products".

[0047] Typically, the first MBP capture product is allowed to flow along the membrane of the LFA device. When a target structure or analyte is present, the first MBP capture product accumulates along the test region, such as the test line (which is also located within the reaction region, downstream of the binding region).

[0048] The MBP capture product can be an MBP sandwich capture product, which includes or is composed of a target structure sandwiched between an MBP reporter nanoparticle and an MBP at the test region or test line. The MBP sandwich capture product is typically present in the region of contact between the first MBP capture product and another MBP in the corresponding LFA device, such as at the test region or test line. The other MBP is typically an MBP adsorbed to or otherwise bound (“immobilized”) to the test region or test line of the chromatography membrane.

[0049] Control lines are typically used to verify that the test is functioning correctly, and / or to assist in quantifying the test lines.

[0050] MBP capture products can also be found in the region of the corresponding LFA device where the MBP reporter nanoparticles contact the control target structure (e.g., mannan on the control line), where the MBP reporter nanoparticles aggregate after binding to the control target structure. Such MBP capture products are also referred to as "control MBP capture products" in this paper.

[0051] Alternatively, microbial structure-independent ligands and ligand-specific targets can be used as controls (i.e., test controls that are non-mannose-specific), where the ligands are provided as nanoparticle conjugates at the binding site and the ligand-specific targets are immobilized on the side-flow chromatography membrane.

[0052] Specifically, CRB is used during all MBP capture reactions, especially during the following reactions: (i) Formation of a first MBP capture product; specifically, if present, such a first MBP capture product consists of MBP reporter nanoparticles, wherein the MBP reacts with the target structure (particularly by binding to or being bound to the target structure). (ii) Forming an MBP sandwich capture product; specifically, such an MBP sandwich capture product comprises a first MBP capture product, wherein the first MBP capture product is further bound to a test region or test line, the test region or test line containing a fixed MBP that reacts with (particularly bound to or bound by) the target structure of the first MBP capture product; and (iii) Optionally, a control MBP capture product is formed; specifically, such control MBP capture product consists of MBP reporter nanoparticles that react with (particularly bind to or be bound to) a control target structure.

[0053] Using this LFA and the corresponding LFA settings or configurations, most microorganisms in the fuel tank can be detected, with 1 CFU extracted per 100 mL of oily liquid (i.e., a detection limit of 1 CFU / 100 mL). It has been found that the detection sensitivity for a broad spectrum of microorganisms has been greatly improved, covering at least indicator microorganisms, which are any one or more (or all) of species from the genera *Methylobacterium*, *Burkholderia*, *Rhodococcus erythropolis*, *Hormoconis resinae*, *Bacillus pumilus*, *Yarrowia lipolytica*, or *Candida tropicalis*.

[0054] Specifically, recombinant cell lines are used to express MBP. Such recombinant MBPs are preferably used in the LFA described herein.

[0055] Specifically, the formulation uses MBP containing less than 10% monomeric MBP. Specifically, the formulation uses MBP containing less than 10% of MBP polymers (including, for example, dimers, trimers, or oligomers) of 140 kDa or higher. Preferably, the vast majority of the MBP in the formulation is contained in the formulation as polymers of less than 140 kDa (preferably MBP dimers or trimers). Specifically, the polymers of less than 140 kDa are MBP dimers or MBP trimers.

[0056] The polymerization of MBP is associated with increased affinity for multivalent ligands, thereby enhancing binding to mannose-rich substances from microorganisms.

[0057] Depending on the specific aspect, the MBP formulation contains MBP, which contains MBP polymers, wherein 10%-90% of the MBP is contained in the MBP formulation in a polymeric form of less than 140 kDa (preferably MBP dimer and / or MBP trimer).

[0058] Specifically, MBP is mannose-binding lectin (MBL), such as human MBP or non-human mammalian MBP. As described herein, MBP is preferably MBP.

[0059] Preferably, MBP or MBL: a) Containing at least one mannose-binding C-type lectin domain (CTLD), preferably, wherein the MBP has at least 90% sequence identity with any one of SEQ ID NO: 1 to SEQ ID NO: 6, or with the CTLD of any one of SEQ ID NO: 1 to SEQ ID NO: 6; or b) The MBP contains or is composed of a CTLD, wherein the CTLD contains SEQ ID NO: 15.

[0060] Specifically, an MBP or MBL formulation is used, comprising less than 10% of monomeric MBL. Specifically, an MBL formulation is used, comprising less than 10% of MBL polymers (including, for example, dimers, trimers, or oligomers) of 140 kDa or higher. Preferably, the vast majority of the MBL in the formulation is contained in a polymeric form of less than 140 kDa (preferably MBL dimers or MBL trimers). MBL polymerization is associated with increased affinity for multivalent ligands, enhancing binding to mannose-rich substances derived from microorganisms.

[0061] Depending on the specific aspect, the MBP (or MBL) formulation contains MBL, and the MBP (or MBL) formulation contains MBL in the form of MBL polymers, wherein 10%-90% of the MBL is contained in the MBP (or MBL) formulation in the form of polymers less than 140 kDa (preferably MBL dimers and / or MBL trimers).

[0062] Specifically, the enhanced binding of MBP (or MBL) to mannose-rich substances from microorganisms provides specific improvements to the LFA described herein, such as avoiding or reducing test line decay for at least 30 minutes after test line formation.

[0063] Depending on the specific aspect, the oily liquid is oil, an oily aqueous condensate, or an emulsion; preferably, the oily liquid is derived from crude oil, petroleum, mineral oil, or a fraction of any of the foregoing, or is a fuel such as kerosene, gasoline, diesel, or biodiesel. Specifically, the oily liquid may be derived from plants (e.g., biodiesel), microorganisms, or animals, or from oils or fats derived from plants (e.g., biodiesel), microorganisms, or animals.

[0064] Depending on the specific aspect, the aqueous liquid is prepared by extracting an oily liquid using an extraction solution, particularly an extraction buffer. The aqueous liquid may contain an extraction solution or an extraction buffer. Specifically, an aqueous liquid containing an extraction solution or an extraction buffer can be used directly in an LFA, for example, by applying the aqueous liquid directly to the sample area of ​​the LFA device (e.g., to the sample pad).

[0065] According to specific implementation methods, the extraction buffer contains 100-500 mM of free calcium ions, detergent, and buffer, and has a pH range of 6.5 to 9, preferably a pH of about (i.e. ±0.5 or ±0.25) 7 or 8.

[0066] Specifically, CRB does not contain calcium chelating agents such as EDTA.

[0067] Specifically, the buffer system does not contain calcium chelating agents, such as EDTA.

[0068] Specifically, CRB may contain one or more different buffering substances, such as phosphates, borates, MES, HEPES, or Tris.

[0069] Specifically, CRB may contain one or more different detergents, such as NP-40, Triton-X100, Tween 20, Tween 80, CHAPS, CA-630, Brij-35, Triton-X114, GDN, GLC and Pluronic-F68.

[0070] Additional substances included in the buffer system or CRB can be sugars (such as sucrose, trehalose), PVP (polyvinylpyrrolidone) or PVA (polyvinyl acetate).

[0071] Depending on the specific aspect, CRB is used as a run buffer for LFA.

[0072] Depending on the specific aspect, mannan was used as a positive control.

[0073] Specifically, mannan is used in the control area (e.g., on the control line) of a side-flow chromatography membrane, particularly by fixing mannan onto the control line or control area of ​​an LFA device.

[0074] Specifically, the mannan used is a highly branched complex carbohydrate with an α-1,6 backbone and mannose side chains linked by α-1,2 and α-1,3, such as yeast mannan.

[0075] Alternatively, other mannose-containing compounds can be used as positive controls and therefore can be used in the control area or on the control line.

[0076] Alternatively, mannose-independent or microbial structure-independent ligands and corresponding ligand-specific targets can be used as controls (i.e., test controls), wherein the ligands are provided as nanoparticle conjugates in the binding region and the ligand-specific targets are immobilized on the side-flow chromatography membrane.

[0077] Depending on the specific aspect, the compound bound to the reporter nanoparticle can be used in combination with the MBP reporter nanoparticle in the LFA, which is used to react with a control that specifically binds to such a compound. Such a compound can be, for example, an antibody or antigen that does not interfere with the MBP reaction, and the corresponding positive control is another reactant (e.g., another antibody) that specifically binds to the corresponding antibody or antigen on the control line.

[0078] Depending on the specific aspect, MBP is a protein containing a lectin domain (e.g., the lectin domain of C-type lectins), which depends on Ca2+. 2+ It identifies mannose-containing structures present on glycoproteins, such as ICAM-2 and ICAM-3, in a way that allows it to recognize these structures.

[0079] Specifically, MBP is selected from mannose-binding lectin (MBL), dendritic cell-specific ICAM-3-binding non-integrin (DC-SIGN, e.g., R&D Systems, catalog number 9136-DC), Dectin-2 (e.g., R&D Systems, catalog number 3114-DC), MMR (e.g., R&D Systems, catalog number 2534-MR), or Langerin (e.g., R&D Systems, catalog number 2088-LN).

[0080] Preferably, the MBP contains or is composed of C-type lectins.

[0081] Depending on the specific aspect, MBP comprises (or consists of): at least one naturally occurring mannose-binding C-lectin or a corresponding C-lectin domain; or a recombinant protein comprising, or consisting of, an amino acid sequence of such a lectin or lectin domain.

[0082] Preferably, the MBP is a mannose-binding lectin (MBL), for example, containing mannose-binding C-type lectin or a corresponding C-type lectin domain, or composed of mannose-binding C-type lectin or a corresponding C-type lectin domain; preferably human MBP or non-human mammalian MBP.

[0083] Exemplary lectins are human MBPs or MBPs derived from other eukaryotes, particularly MBPs comprising any one of SEQ ID NO: 1 to SEQ ID NO: 6 or any one of SEQ ID NO: 6 CTLDs, or MBPs composed of any one of SEQ ID NO: 1 to SEQ ID NO: 6 CTLDs. Specifically, the MBP is derived from humans or non-human animals, such as mammalian species (e.g., human, mouse, or bovine species), preferably human MBP or MBL.

[0084] Specific examples are as follows: a) Human MBP containing SEQ ID NO: 1 (Uniprot database number PRO_0000017401, 21-248, mannose-binding protein C), SEQ ID NO: 6 or SEQ ID NO: 10; b) Mouse MBP, comprising SEQ ID NO: 2 (Uniprot database number PRO_0000017413), SEQ ID NO: 3 (Uniprot database number PRO_0000017405), SEQ ID NO: 11 or SEQ ID NO: 12; c) A bovine MBP containing either SEQ ID NO: 4 (Uniprot database number PRO_0000017397) or SEQ ID NO: 13; d) Human CD209, which contains SEQ ID NO: 5 (Uniprot database number PRO_0000046595, human DC-SIGN) or SEQ ID NO: 14; or e) A functional variant of any of the above, which is capable of binding to a target structure, optionally characterized by having a certain sequence identity with any corresponding sequence or with at least its corresponding CTLD.

[0085] Specifically, MBP includes (or consists of) at least one of the sequences having 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one of SEQ ID NO: 1 to SEQ ID NO: 6; or at least one of the sequences having 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one of SEQ ID NO: 1 to SEQ ID NO: 6 CTLD.

[0086] Specifically, the MBP includes (or consists of): a C-type lectin domain of any one of SEQ ID NO: 10 to SEQ ID NO: 14; and at least one of the C-type lectin domains having 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one of SEQ ID NO: 10 to SEQ ID NO: 14; preferably, the MBP includes at least one of the sequences having 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one of SEQ ID NO: 1 to SEQ ID NO: 6.

[0087] Depending on the specific aspect, MBP includes: any one of SEQ ID NO: 10 to SEQ ID NO: 14; and at least one of the sequences having 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one of SEQ ID NO: 1 to SEQ ID NO: 6.

[0088] SEQ ID NO: 15 is the common sequence for mannose-binding CTLD.

[0089] Depending on the specific aspects, a MBP may include or consist of the following: a) CTLD, which includes or consists of SEQ ID NO: 15; or b) CTLD, comprising (or consisting of): SEQ ID NO: 15; and at least one of the sequences having 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one of SEQ ID NO: 10 to SEQ ID NO: 14; or b) CTLD, which includes (or consists of): SEQ ID NO: 15; and at least one of the sequences having 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with any one of SEQ ID NO: 1 to SEQ ID NO: 6.

[0090] Preferably, the MBP includes: SEQ ID NO: 15; and at least one of the sequences having 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one of SEQ ID NO: 1 to SEQ ID NO: 6.

[0091] Specifically, MBP is a recombinant protein expressed in eukaryotic or prokaryotic expression systems.

[0092] An exemplary expression system is a mammalian host cell, preferably a host cell derived from a human or a non-human animal. Preferably, the host cell is selected from Chinese hamster ovary (CHO) cell line, mouse myeloma (NS0) cell line, HEK-293, HT1080, H9, HepG2, MCF7, MDBK, Jurkat, MDCK, NIH3T3, PC12, BHK (juvenile hamster kidney cells), VERO, SP2 / 0, YB2 / 0, Y0, C127, L cells, COS (e.g., COS1 and COS7), QC1-3, VERO, PER.C6, HeLA, EB1, EB2, EB3, or hybridoma cell line.

[0093] Alternative expression systems include insects, plants, yeast, or bacteria.

[0094] Specifically, MBP is a recombinant protein expressed in Escherichia coli or other non-glycosylated expression systems.

[0095] Depending on the specific aspect, the MBP formulation comprises the MBP, the MBP formulation comprises MBP polymers, wherein the MBP is 10%-90% (w / w), preferably, the MBP is any one of at least 10% (w / w), at least 20% (w / w), at least 30% (w / w), at least 40% (w / w), at least 50% (w / w), at least 60% (w / w), at least 70% (w / w), or at least 80% (w / w); the MBP is contained in the MBP formulation in the form of polymers, including, for example, dimers, trimers, and / or oligomers.

[0096] Specifically, the MBP polymer is less than 140 kDa; preferably, the MBP is contained in the MBP formulation in the form of a dimer and / or a trimer.

[0097] Specifically, the vast majority of MBP molecules contained in the MBP formulation (e.g., any one of at least 50% (w / w), at least 60% (w / w), at least 70% (w / w) or at least 80% (w / w)) are MBP dimers or MBP trimers.

[0098] Specifically, the amounts of MBP monomers and MBP polymers (especially dimers and trimers) can be determined by non-reducing SDS-PAGE.

[0099] In specific examples, human MBP formulations are used, wherein the molecular weights of the MBP dimer or MBP trimer are approximately 55 kDa and 85 kDa, respectively.

[0100] Depending on the specific aspect, the same MBP formulation is used both at the test area and on the MBP reporter nanoparticles. Specifically, the MBP formulation immobilized at the test area and the MBP formulation bound to the reporter nanoparticles have the same quality or origin.

[0101] Depending on the specific aspect, the reporting nanoparticles include or consist of tracers. Specifically, the tracers possess unique optical properties. Specifically, the reporting nanoparticles include tracers, such as visually identifiable tracers, which allow for visual identification of MBP capture products. Specifically, by using such tracers, MBP sandwich capture products are visually identifiable. If a control area or control line is used, the MBP capture products at the control area or control line are also visually identifiable.

[0102] Specifically, under natural light, the presence of tracers at the test line and optional control line of the LFA device can be detected visually, particularly the presence of colored or fluorescent reporter nanoparticles containing MBP. For fluorescent tracers or other tracers that cannot be visually identified, side-flow chromatography measurements may not be visually readable, thus a suitable reading device can be conveniently used.

[0103] Specifically, the tracer includes gold, silver, platinum, carbon, fluorescent, colored emulsion or magnetic tracer, or is composed of gold, silver, platinum, carbon, fluorescent, colored emulsion or magnetic tracer, preferably colloidal gold.

[0104] Specifically, the reported nanoparticles are dyed latex. In one embodiment, the size of the dyed latex nanoparticles is about 100 nm to about 200 nm. In another embodiment, the size of the dyed latex nanoparticles is about 150 nm.

[0105] Specifically, the reported nanoparticles are colloidal gold, such as gold nanoparticles or gold nanoshells. In one embodiment, the size of the gold nanoparticles or gold nanoshells is from about 10 nm to about 80 nm. In another embodiment, the size of the gold nanoparticles or gold nanoshells is about 40 nm.

[0106] Depending on the specific aspect, the microbial contaminant is any one or more microorganisms, such as bacteria, yeast, fungi, or spores. Specifically, the microorganism contains glycan structures, particularly target structures, that can be recognized and bound by the MBP, preferably mannan or mannose-containing structures. Some MBPs may also additionally recognize other glycan structures, such as N-acetylglucosamine, fucose, glucose, and lipopolysaccharide (LPS).

[0107] Specifically, the methods described herein are used to identify microbial contaminants from mannose-containing microorganisms, particularly microorganisms that can grow in oily liquids, such as fuels (e.g., diesel, kerosene, or biodiesel), thereby contaminating such oily liquids.

[0108] Contaminants can originate from target microorganisms, such as those that contaminate diesel, kerosene, or biodiesel. Exemplary microorganisms are selected from the following phyla: Trichocomaceae, Yeastaceae, Acetobacteraceae, Thermomycetes, Spirochetes, Firmicutes, Bacteroidetes, Clostridium, Lentisphaerae, Thermoprotei, Methanocytoceti, Methanosaeta, Methanobacteria, Alpha-Proteobacteria, Beta-Proteobacteria, and Gamma-Proteobacteria; particularly Methylobacteria. Acteria, Pseudomonas, Coxella, Amorpotheka, Aspergillus, Fusarium, Penicillium, Methylobacterium, Bacillus, Bacillus subtilis, Micrococcus, Breobrachiobacterium, Balloonella, Zimmermannella, Serratia, Sphingosomalids, Gnatifida, Flavobacterium, Arthrobacter, Amorphotheca, and Alcaligenes.

[0109] Specifically, the target microorganism includes any one or more of the following: species of the genus *Methylobacterium*, species of the genus *Burkholderia*, *Rhodococcus rubrum*, *Cladosporium spp.*, *Bacillus pumilus*, *Yersinia lipolytica*, or *Candida tropicalis*.

[0110] Specifically, the indicator microorganisms used to determine the sensitivity and robustness of LFA include any one or more of the following: species of Methylobacterium, species of Burkholderia, Rhodococcus rubrum, Cladosporium styracifolium, Bacillus pumilus, Yersinia lipolytica, or Candida tropicalis.

[0111] Depending on the specific aspect, this method detects microbial contaminants with a sensitivity of 1 cfu or higher per 100 mL of oily liquid.

[0112] Specifically, the detection limit can be about (±1) 10 cfu / 100 mL, preferably about 9 cfu / 100 mL, 8 cfu / 100 mL, 7 cfu / 100 mL, 6 cfu / 100 mL, 5 cfu / 100 mL, 4 cfu / 100 mL, 3 cfu / 100 mL or 2 cfu / 100 mL, or it can be 1 cfu / 100 mL.

[0113] Depending on the specific example, LFA is performed as follows: a) Treating an oily fluid to prepare an aqueous liquid, for example by preparing an aqueous extract or by liquid-liquid separation to separate an aqueous condensate. To prepare the aqueous liquid, an extract may be used, which may contain all or part of the free calcium ions required for the CRB described herein.

[0114] b) Apply the aqueous liquid to the LFA device, for example, to the sample area of ​​the device, particularly to the sample pad.

[0115] c) The sample flows through the reaction zone of the LFA device, where, if a polysaccharide-containing microbial contaminant is present, it can first be captured by identifiable (e.g., using identifiable tracers) MBP-containing reporter nanoparticles (MBP adsorbed onto colloidal gold), thereby forming a first MBP capture product. For this first capture reaction, CRB as described herein is used as the reaction buffer.

[0116] d) The first MBP capture product is moved to the detection zone within the reaction zone of the LFA device, where the MBP is immobilized (e.g., adsorbed) onto the test zone (e.g., the test line). The immobilized MBP captures the first MBP capture product, particularly by binding to microbial contaminants present in the first captured MBP product, thus forming an MBP sandwich capture product. Such MBP sandwich capture products, bound by immobilized MBP to the test zone or test line, can be identified visually or by an identification reader, depending on the tracer used for identification. For this second capture reaction, CRB is also used as the reaction buffer.

[0117] The LFA device is conveniently configured to allow capillary flow within the LFA device using the buffer system described herein.

[0118] Optionally, the MBP-containing reporter nanoparticles continue to flow through the LFA device and reach a control area or control line, which includes a fixed (e.g., by adsorption) positive control, such as a target structure (e.g., mannan), thereby forming another MBP capture product, particularly by binding the MBP reporter particles to the fixed target structure, and this MBP capture product can be identified visually or by an identification reader, depending on the tracer used for identification.

[0119] Optionally, the buffer solution terminates at a wick or absorbent pad after flowing through the LFA device to absorb residual liquid.

[0120] The present invention further provides an analytical detection kit for detecting microbial contamination in oily liquids (especially oily liquid samples), comprising: a) A lectin-based lateral flow chromatography (LFA) apparatus that uses mannose-binding protein (MBP) as a trapping agent, which binds to flowable, identifiable reporter nanoparticles at the reaction zone of the lateral flow chromatography membrane; and b) A running buffer containing detergent, buffer and pH range of 6.5 to 9.0, wherein the running buffer sets the amount of free calcium ions in the reaction zone to 100-500 mM.

[0121] Specifically, the reaction region includes a first reaction region (also known as a “binding region”) in which, for example in the presence of a binding buffer, MBP reporter nanoparticles are placed.

[0122] Specifically, the reaction zone includes at least a first reaction zone and a second reaction zone (also known as a "detection zone"), wherein the MBP is fixed at or within the test zone, or fixed on the test line, and serves as a capture agent at the test zone or test line.

[0123] Specifically, the reaction zone contains the compound in dry form and a buffer solution.

[0124] Depending on the specific aspects, the run buffer includes: a) 100-500 mM of free calcium ions; or b) The amount of free calcium ions, which replenishes the amount of free calcium ions provided within the LFA device (e.g., any one or more zones or regions of the LFA device); or c) The amount of free calcium ions, which supplements the amount of free calcium ions provided in the sample (or an aqueous liquid prepared from an oily liquid) applied to the LFA device; and / or supplements the amount of free calcium ions provided in the sample applied to the LFA device; and / or supplements the amount of free calcium ions provided in the LFA device (e.g., any one or more regions of the LFA device (e.g., the reaction zone)).

[0125] In addition to the running buffer, the kit may further include an extraction buffer. Specifically, an extraction solution (e.g., extraction buffer) can be used to prepare an aqueous extract from an oily liquid.

[0126] Specifically, the running buffer can also be used as an extraction buffer for extracting oily liquids.

[0127] Specifically, the running buffer is the extraction buffer.

[0128] Specifically, the kit may include an extraction buffer, which is also used as a run buffer. Preferably, the extraction buffer has the same characteristics as the run buffer described herein.

[0129] Depending on the specific implementation, the test kit includes an extraction buffer rather than a running buffer.

[0130] Specifically, the test kit provides a buffer system for LFA, which includes any one or more of a running buffer, an extraction buffer (e.g., an extraction buffer), a reaction buffer (CRB), a binding buffer, or a catch-up buffer, or is composed of any one or more of a running buffer, an extraction buffer (e.g., an extraction buffer), a reaction buffer (CRB), a binding buffer, or a catch-up buffer.

[0131] Specifically, the test kit provides a buffer system to set the amount of free calcium ions in the capture reaction buffer (CRB), particularly the amount of free calcium ions in the CRB present in the reaction zone of the LFA device.

[0132] Specifically, the test kit provides a buffer system to set 100-500 mM of free calcium ions, detergent, and buffer in CRB, particularly in the reaction zone of the LFA device, with a pH range of 6.5 to 9.0.

[0133] Specifically, the run buffer (or the extraction buffer used as the run buffer) contains 100-500 mM of free calcium ions, or contains the amount of free calcium ions supplied by the buffer system to replenish the LFA and / or the amount of free calcium ions provided in the LFA device, so as to set a total amount of 100-500 mM of free calcium ions when the MBP captures the target structure.

[0134] Specifically, a running buffer is used, which contains 100-500 mM of free calcium ions, or contains a amount of free calcium ions to replenish the amount of free calcium ions provided in the extraction buffer and / or LFA device, so as to set a total amount of 100-500 mM of free calcium ions when the MBP captures the target structure.

[0135] Depending on the specific aspect, the reaction zone is configured to receive a sample, for example, when fluid flows through the sample zone and moves forward to the reaction zone. Specifically, the reaction zone includes: an MBP bound to the reporter nanoparticle at the binding zone; and a detection zone, for example, a detection zone located downstream of the binding zone, wherein the detection zone includes a test zone, preferably, the test zone includes a test line.

[0136] Specifically, the reaction zone includes: an MBP bound to the reporter nanoparticle at the binding zone; a test zone including a test line, wherein the immobilized MBP is used as a trapping agent at the test zone; and optionally a control zone or control line, wherein a control, such as a positive control or test control, is immobilized at the control zone or control line.

[0137] Depending on the specific aspect, the test kit is characterized by having one or more features of LFA or analytical methods as described herein.

[0138] Depending on the specific aspect, the kit is characterized by having one or more features as described herein regarding analytical methods and LFA components, such as the features of MBP.

[0139] Preferably, the MBP formulation contains MBP, the MBP formulation contains MBP polymers, wherein 10%-90% of the MBP is contained in the MBP formulation in the form of polymers less than 140 kDa, preferably, wherein the polymers less than 140 kDa are dimers and / or trimers.

[0140] Preferably, MBP is mannose-binding lectin (MBL).

[0141] Specifically, the MBP or MBL: a) Containing at least one mannose-binding C-type lectin domain (CTLD), preferably, wherein the MBP has at least 90% sequence identity with any one of SEQ ID NO: 1 to SEQ ID NO: 6 or with any one of the CTLDs of SEQ ID NO: 1 to SEQ ID NO: 6; or b) The MBP contains or is composed of a CTLD, the CTLD including SEQ ID NO: 15.

[0142] Specifically, the LFA device includes one or more of the following features: a) The reported nanoparticles include or consist of tracers, preferably gold, silver, platinum, carbon, fluorescent, colored latex or magnetic tracers, preferably colloidal gold; b) The side-flow chromatography membrane is a cellulose-based or synthetic fiber-based side-flow chromatography membrane, preferably a nitrocellulose membrane or other cellulose-based paper; c) Using the same MBP formulation as a trapping agent, which is bound to the reporter nanoparticles and the test line; d) The MBP located in the test area or on the test line is fixed inside or on the side-flow chromatography membrane and is located in a fixed position on the membrane; e) Use mannan as a positive control, or use microbial structure-independent ligands and ligand-specific targets as test controls, wherein the ligands are provided as nanoparticle conjugates at the binding region, and the ligand-specific targets are immobilized on the side-flow chromatography membrane.

[0143] Specifically, the binders described herein (e.g., MBP or control substances) are immobilized within or on the side-flow chromatography membrane via physical adsorption, chemical binding, or affinity binding, for example, through: i) Dispersing the binder in a specific area of ​​the membrane and fixing it onto the membrane by direct physical adsorption, for example, by drying; or ii) Covalently attaching the binder to an epoxy-functionalized membrane, such as a nitrocellulose membrane; or iii) Biotinylated binders are immobilized by binding to membrane-bound streptavidin-anchored proteins, such as proteins anchored to nitrocellulose membranes.

[0144] This invention further provides the application of the detection kit described herein in the analytical methods described herein.

[0145] The present invention further provides the method described herein, wherein the detection kit described herein is used.

[0146] Specifically, the assay kit is used in combination with a buffer system containing 100-500 mM of free calcium ions, detergent, and buffer in the CRB, with a pH range of 6.5 to 9.0. Specifically, the CRB is used during one or more reactions in which the trapping agent (i.e., MBP) binds to the target structure.

[0147] The present invention further provides a method for reducing or avoiding test signal intensity attenuation in the reaction zone of a lateral flow chromatography membrane in a lectin-based lateral flow chromatography assay (LFA) device. The assay determines the presence of glycans in an aqueous liquid prepared from an oily liquid sample by reacting mannose-binding protein (MBP) as a trapping agent with glycans in the reaction zone. The assay uses a capture reaction buffer (CRB) containing 100-500 mM of free calcium ions, detergent, buffer, and a pH range of 6.5 to 9.0.

[0148] Specifically, at least within any one of the following time periods, 30, 40, 50, or 60 minutes after the formation of the test signal, the attenuation or degree of attenuation of the detection signal is reduced to less than any one of 50%, 40%, 30%, 20%, or 10% of the signal strength loss.

[0149] When the attenuation is reduced to less than 10% of the signal strength, or when the attenuation cannot be determined visually (i.e., the person implementing LFA cannot observe the attenuation with the naked eye), it is considered that attenuation has been avoided.

[0150] Specifically, attenuation is reduced or avoided for at least 30 minutes after the detection signal is generated.

[0151] The attenuation or degree of attenuation can be determined at room temperature, especially in the environment of a room or laboratory where LFA is implemented.

[0152] Specifically, methods to reduce or avoid attenuation include assays for detecting microbial contaminants in oily liquids according to the methods described herein, or the use of assay kits described herein.

[0153] All features disclosed in the analytical methods described herein are also applicable, where appropriate, to the methods described herein for reducing or avoiding attenuation, and vice versa.

[0154] All features disclosed regarding the methods (in particular any one or both of the analytical methods and methods for reducing or avoiding attenuation described herein) are applicable, where appropriate, to the test kits described herein, and vice versa. Attached Figure Description

[0155] Figure 1 A schematic diagram of a typical side-flow chromatography assay described herein. A) shows a side view of the LFA test strip according to the invention; B) shows a top view of the test strip; C) shows the flow of the target molecule, a mannose-containing structure, which, under the conditions described herein, is captured in the reaction zone to form a first capture product and a sandwich product that generates a signal at the test line.

[0156] Figure 2 The sequences provided in this article. Detailed Implementation

[0157] Unless otherwise stated or defined, all terms used herein have their ordinary meaning in the art, which will be clear to those skilled in the art. See, for example, standard manuals such as Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY); Lewin, "Genes IV", Oxford University Press, New York, (1990), and Janeway et al., "Immunobiology" (5th Ed., or more recent editions), GarlandScience, New York, 2001, Ausubel et al., Current Protocols in MolecularBiology, John Wiley and Sons, Baltimore, Md. (1989), Vega et al., GeneTargeting, CRC Press, Ann Arbor Mich. (1995); Vectors: A Survey of MolecularCloning Vectors and Their Uses, Butterworths, Boston Mass. (1988); "TheImmunoassay Handbook": Wild, D. (2013), ISBN: 978-0-08-097037-0; and "LateralFlow Immunoassay" by Raphael Wong and Harley Tse (2009), Humana Press, ISBN:978-1-58829-908-6.

[0158] The terms “comprise,” “contain,” “have,” and “include” used herein are used interchangeably and should be understood as open-ended definitions, allowing for the existence of further components, parts, or elements. “Comprising of…” is considered a closed-ended definition, excluding elements other than those defined in the compositional definition. Therefore, “comprise” has a broader scope and includes the definition of “comprising of…”.

[0159] Unless otherwise stated herein, the terms “about” or “around” as used herein refer to the value itself, or to a value that differs from a given value by ±10% or ±5%.

[0160] The specific terms used throughout this instruction manual have the following meanings.

[0161] As used herein, the term "aqueous liquid" refers to a water-based liquid, such as an aqueous solution or suspension of a compound in water. For the LFA described herein, typically, an aqueous liquid containing the target analyte is prepared from an oily liquid, and this aqueous liquid is applied to the LFA to determine the analyte.

[0162] The term "free calcium ion" or "Ca" as used in this article ++ "Free calcium ions" refers to calcium ions that are not complexed with any molecule or compound, such as a chelating agent, which prevents calcium ions from reacting with other molecules or ions, thereby mediating the binding between carbohydrate patterns on the surface of microbial cells and microbial surface binding domains (e.g., MBLs) of engineered microbial binding molecules. Specifically, free calcium ions can be present in the absence of a chelating agent. In some embodiments, free calcium ions can be present in a solution containing a chelating agent and calcium ions, wherein the amount of calcium ions present in the solution is at least about 30%, 40%, or 50% more than the amount of calcium ions required for substantially all chelating agent molecules present in the solution to interact to form a chelate complex.

[0163] Specifically, free calcium ions can be obtained from water-soluble calcium salts, which can be understood as calcium salts that are significantly soluble in water at room temperature, for example, at least 1 gram per 100 mL of water, at least 10 grams per 100 mL of water, or at least 25 grams or more per 100 mL of water. Examples of calcium salts include, but are not limited to, calcium chloride, calcium fluoride, calcium bromide, calcium iodide, calcium nitrate, calcium citrate, calcium formate, calcium acetate, calcium gluconate, calcium ascorbate, calcium lactate, calcium glycinate, and mixtures thereof. Specifically, calcium chloride can be used as a source of calcium ions.

[0164] As used herein, the term "oily liquid" refers to a liquid that is an oil, an oil-based liquid, or an oil phase in an emulsion. Additionally, aqueous condensates derived from oil or emulsions formed by an oil phase and an aqueous phase may contain hydrophobic substances derived from oil and are therefore also considered oily liquids. Oily liquids may consist of, or be derived from, fractions of crude oil, petroleum, mineral oil, or any of the aforementioned, preferably fuels such as kerosene, gasoline, or diesel, or may be derived from plants (e.g., biodiesel) or from microorganisms or algae.

[0165] The term "lateral flow chromatography assay," abbreviated as LFA, is understood in this context as a method for determining analytes or performing corresponding tests using a lateral flow chromatography apparatus (also known as an LFA apparatus). LFA based on lectins can be understood as LFA using lectins as the trapping agent.

[0166] The term “LFA device” is understood herein to refer to a test strip, housing, or cartridge that includes one or more LFA test strips for detecting target structures (i.e. analytes) in a test sample, such as glycan structures (e.g., mannose or mannan) that can be recognized by lectins (e.g., MBP).

[0167] The typical LFA device or test strip described herein includes a side-flow chromatography membrane. Specifically, it includes: a sample zone for receiving an aqueous liquid; a reaction zone comprising a sample zone and a detection zone, and optionally a control zone or control line, the reaction zone overlapping with or downstream of the sample zone, the reaction zone containing MBP-containing reporter nanoparticles at a binding region, the MBP-containing reporter nanoparticles specifically binding to the target analyte; the detection zone downstream of the binding region, the detection zone including a test zone or test line containing immobilized MBP as a capture agent, the immobilized MBP specifically binding to the target analyte; the control zone or control line containing immobilized control compound bound by the MBP-containing reporter nanoparticles (or, if used in a mixture of MBP-containing reporter nanoparticles, bound by other reporter nanoparticles), wherein the control line region is located within the detection zone, typically downstream of the test zone or test line; and an absorption zone downstream of the detection zone for capturing excess fluid, also referred to as a "wicking pad," "wick pad," or "absorbent pad." "Downstream" refers to a location further downstream than one or more other locations in the fluid flow path.

[0168] As used herein, the term "sample area" or "sample receiving area" refers to the region in an LFA device used to deposit a test sample (e.g., an aqueous liquid as used herein) for flow along the LFA test strip. The sample receiving area can be of any size or shape. In the fluid flow path, the sample receiving area is located upstream of the detection and absorption zones. The sample receiving area may overlap with or be upstream of the reaction zone. Specifically, the sample receiving area is located upstream of the reaction zone. "Upstream" means a position further forward than one or more other locations in the fluid flow path. The sample receiving area can be made of any suitable material to receive the test sample and allow fluid to flow along the side-flow chromatography membrane or test strip, for example, through capillary action. In other words, "upstream" refers to a direction opposite to the direction of liquid flow, while "downstream" refers to a direction in the same direction as the flow.

[0169] The sample area may include or be composed of a sample pad, which is the first part of the LFA test strip to come into contact with the sample. The sample pad may act as a filter, or may include a filter, which prevents unwanted substances in the sample from passing through, or traps unwanted particulate matter in the sample, while allowing sample fluid containing the analyte to flow through the test strip.

[0170] As used herein, the term "reaction zone" refers to the region in an LFA device where the test sample (e.g., an aqueous liquid as used herein) can contact the MBP-containing reporter nanoparticles to form an MBP capture product. The reaction zone may be located on a side-flow chromatography membrane or test strip in the same area as the sample zone, or may partially overlap with the sample zone, or may be located entirely downstream of the sample zone in the fluid flow path. The reaction zone typically includes a binding zone, which may comprise or consist of a binding-release pad. Such binding-release pads are commonly used in LFAs to retain and preserve the capture agent, particularly MBP bound to or conjugated with the reporter nanoparticles, which acts as the capture agent in the binding zone. As the sample flows through the binding zone, the MBP reporter nanoparticles can be released into the sample fluid and, if present, can bind to the target analyte. The sample and the MBP reporter nanoparticles can then co-flow into the detection zone of the reaction zone.

[0171] As used herein, the “detection zone” refers to a region within the LFA device in which the first MBP-captured product moves via fluid flow to the test zone or test line (i.e., the test area) and interacts with the MBPs fixed to the test zone or test line (i.e., the test area) to form an MBP sandwich-captured product. Optionally, the detection zone further includes a control zone or control line. Within the detection zone, the control zone or control line is typically located downstream of the test zone or test line to control the proper functioning of the LFA by confirming fluid flow from the reaction zone to the area containing the control line on the test strip.

[0172] As used herein, the term "control" refers to any compound, molecule, or substance fixed on a control line or control area that is recognized and bound by ligands contained in the medium flowing through the LFA device to verify that the test is functioning correctly.

[0173] Based on specific examples, the control is the target structure, especially mannan, and the MBP in the reporter nanoparticles containing MBP is the ligand that recognizes and binds to the target structure.

[0174] Alternatively, the compound bound to the reporter nanoparticle can be used in combination with the MBP reporter nanoparticle in the LFA, which is used to react with a positive control that is specifically bound to such a compound.

[0175] Alternatively, the control can be completely independent of the MBL / microbial target system. For example, a control nanoparticle-ligand conjugate mixed with the MBL nanoparticle conjugate is released simultaneously with the MBL nanoparticle conjugate and binds to a corresponding ligand binding partner, which can be immobilized on the control area after the liquid front crosses the test line.

[0176] Depending on the specific example, microbial structure-independent ligands and ligand-specific targets can be used as controls (i.e., test controls that are non-mannose-specific), wherein the ligands are provided at the binding site in the form of nanoparticle conjugates and the ligand-specific targets are immobilized on the side-flow chromatography membrane.

[0177] Specifically, the LFA device, test strip, or lateral flow chromatography membrane includes only a single test line or test area in the detection zone, and optionally includes a control line or control area downstream thereof. If the device, test strip, or lateral flow chromatography membrane described herein includes additional identifiable reporter nanoparticles containing lectins or binders other than MBP that are specific to different target analytes, the device or test strip may also include two or more test lines, each immobilized with one type of MBP or another lectin or binder. Test lines or test areas may be used that include not only MBP but also combinations of MBP with other lectins or binders, thereby allowing the identification of combinations of target analytes identified by any one or more of the MBP or other lectins or binders.

[0178] Specifically, the detection area of ​​the LFA device includes one, two, or more test lines or test zones, and the detection trapping agent fixed in each test line or test zone specifically binds to one of the different analytes. In an embodiment, the detection area includes one, two, or more test lines or test zones, and the detection trapping agent fixed in each test line or test zone is the same.

[0179] As used herein, "extraction solution" refers to an aqueous liquid, such as an aqueous solution (e.g., buffer solution) or water, used to extract microbial material from an oily fluid into an aqueous solution to prepare an aqueous extract. It may also contain components capable of lysing whole cells (e.g., lysins, detergents). After thorough mixing with the extraction solution, the aqueous extract can be separated from the hydrophobic liquid by centrifugation or gravity. The aqueous extract can be diluted with a concentrated sample buffer to ensure all necessary substances are incorporated into the sample before applying a portion of the sample to the sample area of ​​the LFA device.

[0180] Run buffer can be added to keep side-flow chromatography running. A catch-up buffer can be used to increase or stabilize the flow rate; this catch-up buffer increases the liquid flow along the side-flow chromatography membrane.

[0181] One option is to incorporate the buffer component (e.g., run buffer or CRB component) within the LFA membrane, for example, by using the buffer component dried on the sample pad, and / or by using a binding buffer containing the buffer component at the binding region. This eliminates or reduces the need to apply the buffer component along with the analyte-containing medium (e.g., sample buffer or extraction buffer) to the side-flow chromatography membrane. The binding buffer should be understood herein as the buffer used when applying the MBP reporter nanoparticle (also referred to herein as the “binding”) to the binding region, such as the binding pad.

[0182] The terms "run buffer" and "sample buffer" are not synonymous, although both play important roles in lateral flow chromatography assays. The run buffer is used to adjust the sample pH, minimize nonspecific binding, neutralize interfering substances, and control the flow rate during the assay. The sample buffer, on the other hand, is used to prepare the sample for the assay, helping to solubilize the analyte, maintain its stability, and promote sample flow through the test strip or along the lateral flow chromatography membrane. While both buffers contribute to the overall performance of the assay, their purposes differ and they are not interchangeable.

[0183] The term “mannose-binding protein (MBP)” as used herein refers to a monomeric or polymerized polypeptide, whether natural or recombinant, comprising at least one C-type lectin domain capable of binding to a mannose-containing structure. The C-type lectin-like folded protein superfamily is classified as SCOP ID: 3001261 ​​(Structural Classification of Proteins; Andreeva A, Kulesha E, Gough J, Murzin AG. The SCOP database in 2020: expanded classification of representative family and superfamily domains of known protein structures. Nucleic Acids Res. 2020;48(D1):D376-D382.doi:10.1093 / nar / gkz1064). The characteristic sequence and map of the C-type lectin domain are described in PROSITE entry PS50041 (PROSITE database of protein families and domains, SIB SwissInstitute of Bioinformatics, Hulo N, Bairoch A, Bulliard V, et al. ThePROSITE database. Nucleic Acids Res. 2006;34(Database issue):D227-D230. doi:10.1093 / nar / gkj063). The common sequence of the appropriate C-type lectin domain is provided in this paper as SEQ ID NO: 15. The corresponding common pattern is as follows: Figure 2 As shown.

[0184] Depending on the molecular complexity of the identified carbohydrate, two closely related types of carbohydrate binding specificity can occur at the carbohydrate binding site of the MBP of the present invention: 1. Mannose monosaccharide binding specificity, which allows MBP to specifically recognize mannose (Man) and its derivatives, such as methylmannosides. This recognition of mannose monosaccharides by MBP corresponds to the "broad-spectrum sugar-binding specificity" of lectins, which depends on the presence of a monosaccharide binding pocket within the carbohydrate binding site.

[0185] 2. Oligosaccharide binding specificity, which consists of multiple sugar units that simultaneously accommodate complex N-glycans (such as high-mannose glycans), is also known as the "fine sugar binding specificity" of lectins. This oligosaccharide recognition method involves most of the surface of carbohydrate binding sites, including monosaccharide binding sites.

[0186] Specifically, MBP has the ability to bind to target structures, particularly to specific target structures.

[0187] Specifically, the MBP used in this paper is characterized by its ability to bind mannose structures (e.g., mannose structures contained in mannans), which can be identified by standard ELISA using mannans (e.g., yeast mannans) that include, for example, a main chain with α(1-6) linkages and side chains with α(1-2) linkages and α(1-3) linkages.

[0188] The term "mannan" as used herein refers to a biopolymer of mannose, which is a linear polymer of pyranomannose residues linked by β-(1,4) bonds, or fungal cell wall mannan having an α(1,6)-linked backbone modified by α(1,2) and α(1,3)-linked side chains. The term "mannan" includes glycomannans or mannan derivatives in reduced or oxidized forms. Generally, mannan is a collective term for the polysaccharide portion of glycoproteins or mannose proteins. Mannans typically include mannose structures that can be targeted by MBPs. Different types of mannans include galactomannans, glucomannans, galactoglucomannans, arabinomannans, mannooligosaccharides, linear mannans, or branched mannans. Galactomannans typically contain mannose and galactose linked by β(1-4) bonds, and occasionally have α(1-6) galactose branches. Glucomannans are found in yeast and have different structures, possessing an α(1-6) linked backbone and α(1-2) and α(1-3) linked glucose side chains. Galactoglucomannans have a β(1-4) backbone composed of a mixture of mannose and glucose. Mannooligosaccharides (MOS) are smaller molecules obtained by hydrolyzing long-chain mannans into shorter chains. They can be produced from insoluble galactomannans or soluble glucomannans. Linear mannans are linear polymers of pyranomannose residues.

[0189] The specific mannan composition is mannan derived from yeast. Yeast cell walls are primarily composed of β-1,3-glucan, 1,6-glucan, mannan, and chitin. Yeast mannan contains an α(1-6)-linked backbone and α(1-2)-linked and α(1-3)-linked branches. The mannan found in the cell walls of *Saccharomyces cerevisiae* consists of linear chains of α-(1,6)-D-mannan, modified with side chains and linked to L-asparagine residues of the protein via linker units consisting of two β-(1,4)-N-acetylglucosamine residues. The composition of the side chains varies, but they are primarily composed of α-(1,3)-mannose-α-(1,2)-mannose-α-(1,2)-mannose chains. These side chains can be longer or shorter, and can also be branched. Pure mannans are almost nonexistent in fungi; most contain α(1,6)-linked galactopyranosyl side chains. In some filamentous fungi, furanylgalactosyl rather than galactopyranosyl is present in galactomannans. In Aspergillus fumigatus, the galactomannan linked to the N-glycan consists of a linear mannan core containing α-(1-2)-linked mannotetrasaccharide repeating units interconnected by α-(1-6) bonds, modified by side chains composed of β-(1-5)-galactopyranosyl units linked to the C2 position of the non-reducing mannose residue in each mannotetrasaccharide unit.

[0190] Polysaccharides are also components of bacterial capsules, and may include arabinomannan and mannan.

[0191] Mannan or mannose can be used as a positive control in MBP-based LFA. Specific control mannan formulations are mannan or high-mannose glycoproteins derived from yeast.

[0192] As used herein, the terms "target structure" or "target analyte" refer to any compound, molecule, or substance that is intended to be detected in a test sample. The target structures described herein are the glycan structures of microorganisms, such as bacteria, yeast, fungi, or spores, which can be recognized and bound by the MBP.

[0193] Specifically, the target structures used herein are mannose-containing structures, which should be understood herein as any chemical structure containing at least one mannose residue. Specific glycan structures recognized by MBP are target mannose structures, such as structures contained in mannose, mannose-containing oligosaccharides, or mannans. Manose-containing structures are, for example, contained in mannans, glycoproteins, or glycolipids, and can be contained in glycan-containing microbial materials, such as whole cells, spores, cell debris, release products, or secretion products.

[0194] As used herein, the term "attenuation" refers to a decrease in line intensity over time, such as during LFA and when reading the control line and optional test line (also referred to herein as the signal line). Attenuation is particularly likely to occur in lateral flow chromatography assays after the initial formation of the signal line. Attenuation can be observed in any line in a lateral flow chromatography assay, whether it is the signal line or the control line. Depending on the timescale and magnitude of the signal loss, it can be difficult to standardize or quantify the test results. Reduced signal stability can complicate the interpretation of results. For example, a fainter line might be misinterpreted as a negative result when it may actually indicate a low level of the target analyte. Quantitative assessments based on line intensity can lead to misleading conclusions, especially when signal attenuation is not taken into account. This type of attenuation depends on the dynamic flow of reagents, such as in LFA, but is generally not observed in assays such as ELISA, magnetic bead assays, or classic dipstick assays.

[0195] Therefore, the present invention provides an improved test for determining microbial contamination in oily liquids, such as oils or fuels (including, for example, crude oil), fats, or greases. In particular, the present invention provides an improved LFA method and test kit that reduces or avoids test line decay for at least 30 minutes after test line formation.

[0196] Based on specific examples, it has been found that when using Ca... ++ In the lateral flow chromatography assay of C-type lectins, when used to detect mannose-containing structures in samples from oily fluids, an abnormally high concentration of Ca in the reaction buffer was observed. ++ It can prevent the rapid decay of positive signals.

[0197] It has been confirmed that in various assays using different mannose-binding proteins, oily fluid samples induce signal attenuation in side-flow chromatography. This rapid signal attenuation can be mitigated by utilizing samples with abnormally high Ca2+ concentrations. ++ A specific assay buffer is used to prevent this. A stable assay signal allows for highly sensitive detection of microbial contamination indicators in oily fluids.

[0198] It can be concluded that adding a sufficient amount of Ca to at least one of the extraction buffer, sample buffer, binding buffer, or sample pad buffer will help. ++ It can obtain a stable detection signal and sensitively detect microorganisms with mannose structures from aqueous extracts of oily substances.

[0199] The present invention is further described by one or more of the following items.

[0200] 1. Analytical methods for detecting microbial contaminants in oily liquids, including: a) Prepare an aqueous liquid from an oily liquid to obtain polysaccharide-containing microbial material derived from any microbial contaminants contained in the oily liquid; b) The presence of glycans in aqueous liquids was determined using a lectin-based lateral flow chromatography assay (LFA) that used mannose-binding protein (MBP) as a trapping agent in the test zone of the lateral flow chromatography membrane and as a trapping agent that binds to flowable, identifiable reporter nanoparticles. A capture reaction buffer (CRB) containing 100–500 mM free calcium ions, detergent, buffer, and a pH range of 6.5 to 9.0 was used to allow the identification of MBP sandwich trapping products at the test zone, which indicate microbial contamination.

[0201] 2. The method according to Project 1, wherein the oily liquid is oil, an oily aqueous condensate or an emulsion, preferably wherein the oily liquid is derived from crude oil, petroleum, mineral oil or a fraction of any one of crude oil, petroleum, or mineral oil, or is a fuel such as kerosene, gasoline, diesel or biodiesel.

[0202] 3. The method according to Project 1 or 2, wherein the aqueous liquid is prepared by extracting the oily liquid using an extraction buffer, the extraction buffer containing 100-500 mM free calcium ions, detergent, buffer and having a pH range of 6.5 to 9.0.

[0203] 4. The method according to any one of items 1-3, wherein the CRB is used as a run buffer for the LFA.

[0204] 5. The method according to any one of items 1-4, wherein mannan is used in the control region of the side-flow chromatography membrane.

[0205] 6. The method according to any one of items 1-5, wherein the MBP comprises at least one mannose-binding C-type lectin domain (CTLD), preferably wherein the MBP has at least 90% sequence identity with any one of SEQ ID NO: 1 to SEQ ID NO: 6 or with any one of the CTLDs of SEQ ID NO: 1 to SEQ ID NO: 6.

[0206] 7. The method according to any one of items 1-6, wherein the MBP comprises or is composed of a CTLD, the CTLD including SEQ ID NO: 15.

[0207] 8. The method according to any one of items 1-7, wherein the MBP formulation comprises MBP, the MBP formulation comprises MBP polymers, wherein 10%-90% of the MBP is contained in the MBP formulation in a polymeric form of less than 140 kDa, the polymeric form preferably being an MBP dimer and / or an MBP trimer.

[0208] 9. The method according to any one of items 1-8, wherein the same MBP formulation is used both at the test area and on the reporting nanoparticles.

[0209] 10. The method according to any one of items 1-9, wherein the microbial contaminant is any one or more of bacteria, yeast, fungi or spores, preferably derived from species of the genus *Methylobacterium*, species of the genus *Burkholderia*, *Rhodococcus rubrum*, *Cladosporium spp.*, *Bacillus pumilus*, *Yersinia lipolytica* or *Candida tropicalis*.

[0210] 11. An analytical kit for detecting microbial contamination in oily liquids, comprising: a) A lectin-based lateral flow chromatography (LFA) apparatus that uses mannose-binding protein (MBP) as a trapping agent, which binds to flowable, identifiable reporter nanoparticles at the reaction zone of the lateral flow chromatography membrane; and b) A running buffer containing detergent, buffer and pH range of 6.5 to 9.0, wherein the running buffer sets the amount of free calcium ions in the reaction zone to 100-500 mM.

[0211] 12. The test kit according to item 11, wherein the run buffer comprises: a) 100-500 mM free calcium ions; or b) The amount of free calcium ions, which replenishes the amount of free calcium ions provided within the LFA device; or c) The amount of free calcium ions, which supplements the amount of free calcium ions provided in the sample applied to the LFA device, and / or the amount of free calcium ions provided in the LFA device.

[0212] 13. The detection kit according to item 11 or 12, wherein the reaction zone is configured to receive a sample, the reaction zone comprising: MBP bound to a reporter nanoparticle at a binding zone; and a detection zone comprising a test zone and a control zone, wherein immobilized MBP in the test zone serves as a capture agent, and a positive control is immobilized in the control zone.

[0213] 14. The test kit according to item 13, wherein the LFA device includes one or more of the following features: a) The reported nanoparticles include tracers composed of tracers, preferably gold, silver, platinum, carbon, fluorescent, colored emulsion or magnetic tracers, preferably colloidal gold; b) The side-flow chromatography membrane is a cellulose-based or synthetic fiber-based side-flow chromatography membrane, preferably a nitrocellulose membrane or other cellulose-based paper; c) Using the same MBP formulation as a trapping agent, which is bound to the reporter nanoparticles and the test line; d) The MBP located in the test area is fixed inside or on the side-flow chromatography membrane and is located in a fixed position on the membrane; e) Use mannan as a positive control, or use microbial structure-independent ligands and ligand-specific targets as test controls, wherein the ligands are provided at the binding region in the form of nanoparticle conjugates and the ligand-specific targets are immobilized on a side-flow chromatography membrane.

[0214] 15. The use of the test kit according to any one of items 11-14 in the method described in any one of items 1-10.

[0215] The embodiments listed below are intended to aid in understanding the invention and are not intended, nor should they be construed, as limiting the scope of the invention in any way. The embodiments do not include a detailed description of conventional methods and apparatus, which are well known to those skilled in the art.

[0216] In particular, the present invention is further described by one or more of the following items.

[0217] 1. Analytical methods for detecting microbial contaminants in oily liquids, including: a) Prepare an aqueous liquid from an oily liquid to obtain polysaccharide-containing microbial material derived from any microbial contaminants contained in the oily liquid; b) The presence of polysaccharides in aqueous liquids is determined using a lectin-based lateral flow chromatography assay (LFA), which uses mannose-binding protein (MBP) as a trapping agent in the test zone of the lateral flow chromatography membrane and as a trapping agent that binds to flowable, identifiable reporter nanoparticles. A capture reaction buffer (CRB) containing 100-500 mM free calcium ions, detergent, buffer, and a pH range of 6.5 to 9.0 is used to allow the identification of MBP sandwich trap products at the test zone, which indicate microbial contamination. The MBP formulation contains MBP polymers, wherein 10%-90% of the MBP is contained in the MBP formulation as polymers less than 140 kDa.

[0218] 2. The method according to Project 1, wherein the polymer less than 140 kDa is an MBP dimer and / or an MBP trimer.

[0219] 3. The method according to Project 1 or 2, wherein the MBP is mannose-binding lectin (MBL).

[0220] 4. The method according to any one of items 1-3, wherein the MBP: a) Containing at least one mannose-binding C-type lectin domain (CTLD), preferably, wherein the MBP has at least 90% sequence identity with any one of SEQ ID NO: 1 to SEQ ID NO: 6 or with any one of the CTLDs of SEQ ID NO: 1 to SEQ ID NO: 6; or b) The MBP includes or is composed of CTLDs, the CTLDs including SEQ ID NO: 15.

[0221] 5. The method according to any one of items 1-4, wherein the same MBP formulation is used both at the test area and on the reporting nanoparticles.

[0222] 6. The method according to any one of items 1-5, wherein the oily liquid is oil, an oily aqueous condensate or an emulsion, preferably, wherein the oily liquid is derived from crude oil, petroleum, mineral oil or a fraction of any one of crude oil, petroleum, or mineral oil, or is a fuel such as kerosene, gasoline, diesel or biodiesel.

[0223] 7. The method according to Project 6, wherein the oily liquid is a fuel.

[0224] 8. The method according to any one of items 1-7, wherein the aqueous liquid is prepared by extracting the oily liquid using an extraction buffer, the extraction buffer comprising 100-500 mM free calcium ions, detergent, buffer and having a pH range of 6.5 to 9.0.

[0225] 9. The method according to any one of items 1-8, wherein CRB is used as the run buffer for the LFA.

[0226] 10. The method according to any one of items 1-9, wherein mannan is used in the control region of the side-flow chromatography membrane.

[0227] 11. The method according to any one of items 1-10, wherein the microbial contaminant is any one or more of bacteria, yeast, fungi or spores, preferably derived from species of the genus *Methylobacterium*, species of the genus *Burkholderia*, *Rhodococcus rubrum*, *Cladosporium spp.*, *Bacillus pumilus*, *Yersinia lipolytica* or *Candida tropicalis*.

[0228] 12. An analytical kit for detecting microbial contamination in oily liquids, comprising: a) A lectin-based lateral flow chromatography (LFA) apparatus that uses mannose-binding protein (MBP) as a trapping agent, which binds to flowable, identifiable reporter nanoparticles at the reaction zone of the lateral flow chromatography membrane; and b) A running buffer containing detergent, buffer and pH range of 6.5 to 9.0, wherein the running buffer sets the amount of free calcium ions in the reaction zone to 100-500 mM.

[0229] The MBP formulation contains the MBP, and the MBP formulation contains MBP polymers, wherein 10%-90% of the MBP is contained in the MBP formulation in the form of polymers with a mass less than 140 kDa.

[0230] 13. The detection kit according to Item 12, wherein the polymer less than 140 kDa is an MBP dimer and / or an MBP trimer.

[0231] 14. The test kit according to item 12 or 13, wherein the MBP is mannose-binding lectin (MBL).

[0232] 15. The detection kit according to any one of items 12-14, wherein the MBP: a) Containing at least one mannose-binding C-type lectin domain (CTLD), preferably, wherein the MBP has at least 90% sequence identity with any one of SEQ ID NO: 1 to SEQ ID NO: 6 or with any one of the CTLDs of SEQ ID NO: 1 to SEQ ID NO: 6; or b) The MBP includes or is composed of CTLDs, the CTLDs including SEQ ID NO: 15.

[0233] 16. The detection kit according to any one of items 12-15, wherein the run buffer comprises: a) 100-500 mM free calcium ions; or b) The amount of free calcium ions, which replenishes the amount of free calcium ions provided within the LFA device; or c) The amount of free calcium ions, which supplements the amount of free calcium ions provided in the sample applied to the LFA device and / or the amount of free calcium ions provided within the LFA device.

[0234] 17. The detection kit according to any one of items 12-16, wherein the reaction region is configured to receive a sample, the reaction region comprising: MBP bound to a reporter nanoparticle at a binding region; and a detection region comprising a test region and a control region, wherein the MBP immobilized in the test region serves as a capture agent, and a positive control is immobilized in the control region.

[0235] 18. The test kit according to any one of items 12-17, wherein the LFA device comprises one or more of the following features: a) The reported nanoparticles include or consist of tracers, preferably gold, silver, platinum, carbon, fluorescent, colored emulsion or magnetic tracers, preferably colloidal gold; b) The side-flow chromatography membrane is a cellulose-based or synthetic fiber-based side-flow chromatography membrane, preferably a nitrocellulose membrane or other cellulose-based paper; c) Using the same MBP formulation as a trapping agent, which is bound to the reporter nanoparticles and the test line; d) The MBP located in the test area is fixed inside or on the side-flow chromatography membrane and is located in a fixed position on the membrane; e) Use mannan as a positive control, or use microbial structure-independent ligands and ligand-specific targets as test controls, wherein the ligands are provided at the binding region in the form of nanoparticle conjugates and the ligand-specific targets are immobilized on a side-flow chromatography membrane.

[0236] 19. The use of the test kit according to any one of items 12-18 in the method described in any one of items 1-11.

[0237] Example Example 1: The effect of high calcium concentration on the stability of test signals 1.1 Labeling of mannose-binding proteins To prepare various gold nanoparticle-mannose-binding protein conjugates, BioReady was used. TM 40 nm Bare Gold Nanoparticles (nanoComposix, USA; Product No. AUCR40). Follow the manufacturer's recommendations. In short, perform pH titration according to the manufacturer's protocol to bind mannose-binding protein to 250 μL of BioReady OD20. TM On 40 nm bare gold nanoparticles (colloidal gold concentration is usually expressed as optical density or absorbance at 530 nm in a 1 cm path length cuvette).

[0238] The pH range of 7, 8, and 9 was evaluated to determine the optimal conditions for passive binding. Proteins can be purified to a suitable buffer using spin columns or dialysis tubes with appropriate molecular weight cutoffs. The protein concentration used to prepare the conjugate is 1 mg / mL. For passive adsorption of 40 nm gold nanoparticles, 100 μg of protein can be loaded per mL of OD20 gold nanoparticles. To obtain the optimal adsorption pH, pH titration is required according to the manufacturer's protocol. 1. Prepare a set of 3 EP tubes. Label each tube as 7A, 8A, and 9A to correspond to each pH point.

[0239] 2. Add 12.5 μL of the corresponding 100 mM buffer to the respective labeled tube. The volume of buffer added should be approximately 1 / 20 of the total working volume of the gold nanoparticles.

[0240] 3. Add 20 μg of mannose-binding protein to each tube containing buffer from step 2.

[0241] 4. Add 250 μL of OD20 gold nanoparticles to each tube from step 3. Vortex mix and incubate on a rotary mixer for 10 minutes.

[0242] 5. Aliquot 50 μL of 10% NaCl into three separate EP tubes. Label each tube as 7B, 8B, and 9B.

[0243] 6. After incubating tube "A" (buffer solution + MBP + gold nanoparticles) for approximately 10 minutes: a. Transfer 50 μL of the conjugate from each “A” tube and add it to the corresponding “B” tube containing NaCl solution; b. Incubate the remaining volume in tube "A" for an additional 20 minutes.

[0244] 7. Vortex the NaCl+ conjugate mixture in tube "B". Incubate on a rotary mixer for approximately 10 minutes and observe for any color change: a. Stable conjugate: Gold nanoparticles remain red; b. Unstable conjugates: Gold nanoparticles turn purple, gray, transparent, or precipitate.

[0245] Choose the pH conditions that produce a stable conjugate and proceed with subsequent operations.

[0246] 8. After incubating the “A” tubes from step 6b for a total of 30 minutes, select the tubes corresponding to the stable “B” tubes. Add 1 / 10 volume of the conjugate blocking buffer to bring the final BSA concentration in each “A” tube to approximately 1%.

[0247] 9. Incubate on a rotary mixer for 30 minutes.

[0248] 10. Centrifuge the conjugate at 3600 RCF for 3 minutes.

[0249] 11. Carefully remove the supernatant and resuspend the conjugate in conjugate dilution buffer to the target OD value (e.g., 200 μL for OD20). If complete resuspension of the conjugate is required, vortex and sonicate in a water bath.

[0250] 12. Perform UV-Vis analysis and check the final OD value.

[0251] 13. Store the conjugate at 4°C for later use. Do not freeze.

[0252] Table 1

[0253] 1.2 Establishment of lateral flow chromatography determination: The Lateral Flow Material Starter Kit (nanoComposix, USA, product number: MSKR) can be used to screen for assay components. This kit contains various components, such as membranes, aspiration pads, binding pads, and sample pads. The following materials are tested according to the manufacturer's protocol: Membrane: UniSart® CN 95 from Sartorius, Vivid from Pall TM120 samples were selected from MDI 70, MDI 90, and MDI 150; a Wickpad Grad 222 from Ahlstrom; a cotton sample pad 1281 from Ahlstrom; and binding pads: all from Ahlstrom, made of 8950 glass fiber, 8951 glass fiber, 6613 polyester fiber, and 6614 polyester fiber. Optimization was performed according to the manufacturer's instructions. The signal-to-noise ratio was selected using yeast mannan (Sigma M7504) as a positive sample.

[0254] 1.3 Sideflow chromatography test system using recombinant human MBP 1.3.1 Preparation of gold conjugates In a 2 mL Eppendorf tube, dilute MBL (R&D Systems, USA; catalog number 9086-MB) to 4 μg / mL in 1.5 mL binding buffer (25 mM borate, pH 7.0). Add 150 μL of gold nanoparticles (40 nm gold nanoparticles, OD10, from Abcam, UK; catalog number ab269930), and immediately invert the Eppendorf tube several times to ensure uniform dispersion. Incubate the mixture at room temperature on a rotary mixer for 15 minutes. Then, add 75 μL of blocking buffer (2 mM borate, 10% BSA, 0.1% Tween 20, 0.1% sodium azide, pH 9.0), and incubate the tube again at room temperature on a rotary mixer for 15 minutes. The mixture was then centrifuged at 2500×g for 10 minutes. The supernatant was carefully removed, and the precipitate was resuspended in buffer (2 mM borate, 1% BSA, 0.1% Tween 20, 0.1% sodium azide, pH 9.0) by gently tapping the tube wall.

[0255] 1.3.2 Quality Control and Quantification The absorbance of the resuspended conjugates at 530 nm, 550 nm, and 600 nm was measured using a Tecan SPARK 10 spectrophotometer (1 cm path length for the cuvette). The OD value of the pure resuspension buffer was subtracted from the corresponding sample measurements. The measurement at 530 nm was defined as GU / μL. The aggregation factor was determined by the ratio of OD550 / OD600. For quality control of the MBP-gold nanoparticle conjugates, this aggregation factor should be below 3.5.

[0256] The test was conducted using the half-strip spotting method, in which MBP was spotted onto the LFA test strip in the form of droplets.

[0257] Spot 5 μL of mannose-binding protein (0.1 mg / mL in 5 mM borate; 150 mM NaCl; 0.0625% Tween 20; pH 8) onto the test area of ​​a nitrocellulose membrane test strip (CN95; Sartorius). Dry the test strip at 37°C for 30 minutes. Load the gold-mannose-binding protein conjugate onto the conjugation pad at an OD15 concentration per test strip (binding resuspension buffer: 2 mM borate, 0.1% Tween 20, 0.1% azide, 1% BSA, pH 9). Dry the test strip at 37°C for 1 hour. Use immediately after drying, or store at room temperature in a desiccator. Prepare 500 μL of sample diluted in capture reaction buffer. The sample consisted of mannan (50 ng / mL; sample buffer was 12.5 mM borate buffer, pH 8.0) diluted 1:5 in capture reaction buffer, or mannan from the aqueous extract of Jet A1 fuel (i.e., fuel spiked with 100 ng mannan (Sigma M7504) / mL). The spiked fuel was extracted using sample buffer at a 1:150 aqueous buffer / fuel ratio, with vigorous shaking for 30 seconds. After separation of the two phases by gravity (and standing on the test bench for approximately 5 minutes), 100 μL of the aqueous phase was added to the test system. The capture reaction buffer was 15 mM Tris, pH 8, containing 0.3% Tween 20. Variations in the capture reaction buffer conditions included 12.5 mM EDTA, 12.5 mM CaCl2, 125 mM CaCl2, and 625 mM CaCl2. Apply 100 μL of each sample to the sample pad of the LFA test strip and observe the color change at the sampling area. Visually observe the reaction and rate it as 0 ( The color intensity is indicated by a light red (+), a light red (++), or a strong, deep red (+++). The signal in the test area develops after approximately 2 minutes. The signal is rechecked and recorded periodically for 30 minutes. Table 2 summarizes the results at 4 minutes and 30 minutes after the start of the assay using recombinant human MBL protein (R&D Systems, catalog number #: 9086-MB) coated in the test area and used as a gold binder.

[0258] Table 2

[0259] As shown in Table 2, the addition of abnormally high concentrations of calcium stabilized the signal derived from the fuel extraction sample. Replacing the mannose-binding protein in the coating and / or conjugate with other mannose-binding proteins and adjusting the pH of the capture reaction buffer between 6.5 and 9 did not significantly alter the results in Table 2.

[0260] MBL is an exemplary MBP described herein. Other mannose-binding proteins also exist, such as DC-SIGN (e.g., R&D Systems, USA, catalog number 9136-DC), Dectin-2 (e.g., R&D Systems, USA, catalog number 3114-DC), MMR (e.g., R&D Systems, USA, catalog number 2534-MR), or Langerin (e.g., R&D Systems, USA, catalog number 2088-LN), which, in the lateral flow chromatography assay system described herein, determine the same Ca for signal stability. ++ Ion concentration dependence.

[0261] In LFA assays using MBP (or MBL) as a capture agent, a signal attenuation effect has been established at low calcium ion concentrations, but this phenomenon is generally not observed in similar sandwich ELISA assays.

[0262] Example 2: Detection of microbial contamination in fuel samples Microorganisms or microbial material in fuel were detected using the assay method described in Example 1. The sample used for LFA was an aqueous sample from a fuel tank or kerosene. The sample was processed according to the above example: the sample was diluted and mixed with sample buffer (1:2), extracted by vortexing, and then centrifuged at room temperature to separate the phases; subsequently, 10 μL of the aqueous phase was transferred to 40 μL of capture reaction buffer containing 10 mM and 500 mM CaCl2 respectively (“LFA 10 mM Ca” and “LFA 500 mM Ca”), mixed, and applied to the sample pad of the side-flow chromatography test strip. The same samples were tested using standard methods for determining the viable aerobic microbial content of Fuels and Fuel Components Boiling Below 390 °C - Filtration and Culture Method (for bacteria and fungi, respectively, i.e., "IP385 bacteria" and "IP385 fungi"). (For the IP385 standard method, see ASTM manual "IP385-99 Determination of the Viable Aerobic Microbial Content of Fuels and Fuel Components Boiling Below 390 °C - Filtration and Culture Method" (ASTM International, USA, June 2003).

[0263] The results in Table 3 show that, under conditions where the reaction buffer contains a high concentration of calcium, the mannose-binding protein lateral flow chromatography assay can detect low concentrations of CFU / mL equivalents in the sample.

[0264] Table 3

[0265] The unit of IP385 value is cfu / mL; bdl... is below the detection limit.

[0266] Example 3: Detection of indicator strains The following indicator strains of bacteria and fungi can be grown on IP385 medium. Colony samples of each strain were suspended in sample buffer and then mixed with capture reaction buffer containing 500 mM CaCl2 (1:5). After centrifugation, 50 μL of the clear supernatant was applied to the lateral flow chromatography test strip as described in Example 1. The results showed positive reactions for Rhodococcus rubrum, Burkholderia species, Methylobacterium species, Bacillus pumilus, Yersinia lipolytica, Candida tropicalis, and Cladosporium resinosum.

[0267] Example 4: Expression of recombinant human mannose-binding protein in CHO 4.1. Cloning: Protein sequence source: Uniprot P11226 Expression vector: VB230228 Sleeping Beauty gene expression vector system (Vector Builder) Host cell line: CHO-K1 (The Antibody Lab, Austria) The expressed recombinant human MBP protein sequence includes the N-terminal albumin signal peptide ( italics (removed during secretion), C-terminal 6 x His tag (bold), and 2 additional amino acids ( underline (Clonality specific; 27 kDa), SEQ ID NO:16: .

[0268] A gene cassette containing optimized hMBP DNA from the Chinese hamster (Cricetulus griseus) and an N-terminal albumin leader sequence, along with a neomycin resistance gene cassette controlled by the PGK promoter, was cloned into the Sleeping Beauty cloning vector. CHO-K1 cells were transfected with this cloning vector and a Sleeping Beauty helper vector encoding a transposase, which cleaves the IR / DR sequence and inserts the gene cassette into the cell genome. Cells underwent rigorous antibiotic selection, and surviving cells were amplified using limiting dilution. Twenty-eight cell clones were obtained through limiting dilution and single-clone amplification, and their genotypes were identified to determine the presence of the recombinant MBP gene. Two positive clones were amplified for cell bank storage, and their expression yield during fed-batch production was tested.

[0269] 4.2. Protein Production: Cultivation conditions: Cells were cultured in CD CHO medium (Gibco, 10743-029) / L-glutamine / anti-cell clumping agent (Gibco, 01-0057D6) at 37°C and 5% CO2, with shaking at 160 rpm and a 45° tilt angle on a shaker. Injection was performed every 3-4 days at 1×10⁻⁶ mg / L. 5 Passaged at a density of / mL.

[0270] Replenishment and batch production: Cells used for protein production were 3 × 10 5 Cells were seeded at a density of [number] cells / mL in shake flasks containing HyClone ActiPro medium (GE, SH31039.02) / superglutamine / anti-clustering agent and cultured at 37°C, 5% CO2 with shaking at 160 rpm for 10 days. On days 3, 6, 7, and 8, 4% HyClone Cell Boost7A (GE, 15805261) was added to the culture. The culture was harvested by centrifugation at 4000 rpm for 20 minutes and aseptically filtered. MBP expression in the production supernatant was analyzed by SDS-PAGE.

[0271] 4.3. Purification Binding / washing buffer: 10 mM Tris (pH 7.5) / 150 mM NaCl / 5 mM imidazole.

[0272] Elution buffer: 10 mM Tris (pH 7.5) / 0.5 M NaCl / 0.5 M imidazole.

[0273] Dilute 20 mL of cell culture supernatant with 10 mM Tris / 150 mM NaCl / 5 mM imidazole (pH 7.5) buffer at a ratio of 1:1.

[0274] Add 1 mL of Ni-NTA agarose gel beads (Qiagen, 30210) to a new 15 mL centrifuge tube (adjust the volume of the gel beads if the supernatant volume exceeds 4 mL).

[0275] Centrifuge at 2000 rcf for 2 minutes, resuspend the gel beads in 2 mL binding buffer, and centrifuge again.

[0276] The gel beads were resuspended in 20 mL of supernatant and incubated end-to-end by rotation at 4 °C for 1 h.

[0277] The suspension was loaded into a gravity flow column, and the flow through was collected as a control.

[0278] Wash twice with 2 mL of binding buffer (retain the flow solution as a control).

[0279] Add 4 × 0.5 mL of elution buffer and collect the elution fractions E1-E4.

[0280] Store the sample at 4°C.

[0281] The quality of the purified sample was determined by PAGE.

[0282] The elution fractions containing visible protein test strips of the expected size were combined, concentrated using a Vivaspin centrifuge with a molecular weight cutoff (MW) of 10 K, and the buffer was replaced with PBS.

Claims

1. Analytical methods for detecting microbial contaminants in oily liquids, including: a) Prepare an aqueous liquid from an oily liquid to obtain polysaccharide-containing microbial material derived from any microbial contaminants contained in the oily liquid; b) The presence of glycans in aqueous liquids was determined using a lectin-based lateral flow chromatography assay (LFA) that used mannose-binding protein (MBP) as a trapping agent in the test zone of the lateral flow chromatography membrane and as a trapping agent that binds to flowable, identifiable reporter nanoparticles. A trapping reaction buffer (CRB) containing 100–500 mM free calcium ions, detergent, buffer, and a pH range of 6.5 to 9.0 was used to allow the identification of MBP sandwich trapping products at the test zone, which indicate microbial contamination.

2. The method according to claim 1, wherein, The MBP contains at least one mannose-binding C-type lectin domain (CTLD), preferably wherein the MBP has at least 90% sequence identity with any one of SEQ ID NO: 1 to SEQ ID NO: 6 or with any one of the CTLDs of SEQ ID NO: 1 to SEQ ID NO:

6.

3. The method according to claim 1 or 2, wherein, The MBP includes or is composed of CTLDs, the CTLDs including SEQ ID NO:

15.

4. The method according to any one of claims 1-3, wherein, The MBP formulation contains the MBP, and the MBP formulation contains MBP polymers, wherein 10%-90% of the MBP is contained in the MBP formulation in the form of polymers less than 140 kDa. Preferably, the polymers less than 140 kDa are MBP dimers and / or MBP trimers.

5. The method according to any one of claims 1-4, wherein, The MBP is mannose-binding lectin (MBL).

6. The method according to any one of claims 1-5, wherein, The MBP: a) Containing at least one mannose-binding C-type lectin domain (CTLD), preferably, wherein the MBP has at least 90% sequence identity with any one of SEQ ID NO: 1 to SEQ ID NO: 6 or with any one of the CTLDs of SEQ ID NO: 1 to SEQ ID NO: 6; or b) The MBP includes or is composed of CTLDs, the CTLDs including SEQ ID NO:

15.

7. The method according to any one of claims 1-6, wherein, The same MBP formulation was used both in the test area and on the report nanoparticles.

8. The method according to any one of claims 1-7, wherein, The oily liquid is oil, an oily aqueous condensate, or an emulsion. Preferably, the oily liquid is derived from crude oil, petroleum, mineral oil, or a fraction of any of these, or is a fuel such as kerosene, gasoline, diesel, or biodiesel.

9. The method according to any one of claims 1-8, wherein, The aqueous liquid is prepared by extracting the oily liquid using an extraction buffer solution containing 100-500 mM free calcium ions, detergent, buffer, and having a pH range of 6.5 to 9.

0.

10. The method according to any one of claims 1-9, wherein, CRB is used as the running buffer for the LFA.

11. The method according to any one of claims 1-10, wherein, Mannan was used in the control region of the side-flow chromatography membrane.

12. The method according to any one of claims 1-11, wherein, The microbial contaminant is any one or more of bacteria, yeast, fungi, or spores, preferably derived from species of the genus *Methylobacterium*, species of the genus *Burkholderia*, *Rhodococcus rubrum*, *Cladosporium spp.*, *Bacillus pumilus*, *Yersinia lipolytica*, or *Candida tropicalis*.

13. An analytical kit for detecting microbial contamination in oily liquids, comprising: a) A lectin-based lateral flow chromatography (LFA) apparatus that uses mannose-binding protein (MBP) as a trapping agent, which binds to flowable, identifiable reporter nanoparticles at the reaction zone of the lateral flow chromatography membrane. as well as b) A running buffer containing detergent, buffer and pH range of 6.5 to 9.0, wherein the running buffer sets the amount of free calcium ions in the reaction zone to 100-500 mM.

14. The test kit according to claim 13, wherein, The MBP formulation contains the MBP, and the MBP formulation contains MBP polymers, wherein 10%-90% of the MBP is contained in the MBP formulation in the form of polymers less than 140 kDa. Preferably, the polymers less than 140 kDa are dimers and / or trimers.

15. The detection kit according to claim 12 or 13, wherein, The MBP is mannose-binding lectin (MBL).

16. The test kit according to any one of claims 13-15, wherein, The MBP: a) Containing at least one mannose-binding C-type lectin domain (CTLD), preferably, wherein the MBP has at least 90% sequence identity with any one of SEQ ID NO: 1 to SEQ ID NO: 6 or with any one of the CTLDs of SEQ ID NO: 1 to SEQ ID NO: 6; or b) The MBP contains or is composed of a CTLD, the CTLD including SEQ ID NO:

15.

17. The test kit according to any one of claims 13-16, wherein, The running buffer contains: a) 100-500 mM free calcium ions; or b) The amount of free calcium ions, which replenishes the amount of free calcium ions provided within the LFA device; or c) The amount of free calcium ions, which supplements the amount of free calcium ions provided in the sample applied to the LFA device, and / or the amount of free calcium ions provided in the LFA device.

18. The detection kit according to any one of claims 13-17, wherein, The reaction zone is configured to receive a sample and includes: an MBP bound to a reporter nanoparticle at a binding region; and a detection region including a test region and a control region, wherein the immobilized MBP in the test region serves as a capture agent, and the control region contains an immobilized positive control.

19. The detection kit according to any one of claims 13-18, wherein, The LFA device includes one or more of the following features: a) The reported nanoparticles include or consist of tracers, preferably gold, silver, platinum, carbon, fluorescent, colored emulsion or magnetic tracers, preferably colloidal gold; b) The side-flow chromatography membrane is a cellulose-based or synthetic fiber-based side-flow chromatography membrane, preferably a nitrocellulose membrane or other cellulose-based paper; c) Using the same MBP formulation as a trapping agent, said trapping agent is bound to the reporter nanoparticles and the test line; d) The MBP located in the test area is fixed inside or on the side-flow chromatography membrane and is located at a fixed position on the membrane; e) Use mannan as a positive control, or use microbial structure-independent ligands and ligand-specific targets as test controls, wherein the ligands are provided at the binding region in the form of nanoparticle conjugates and the ligand-specific targets are immobilized on a side-flow chromatography membrane.

20. The use of the test kit according to any one of claims 13-19 in the method according to any one of claims 1-12.

21. The method according to any one of claims 1-12, wherein, Use the test kit according to any one of claims 13-19.

22. A method for reducing the attenuation of test signal intensity in the reaction zone of a lateral flow chromatography membrane in a lectin-based lateral flow chromatography assay (LFA) device, the assay determining the presence of glycans in an aqueous liquid prepared from an oily liquid sample by reacting mannose-binding protein (MBP) as a capture agent with glycans in the reaction zone, the assay using a capture reaction buffer (CRB) containing 100-500 mM of free calcium ions, detergent, buffer, and a pH range of 6.5 to 9.

0.

23. The method according to claim 22, wherein, The determination is performed using the method according to any one of claims 1-12 or using the detection kit according to any one of claims 13-19 to detect microbial contaminants in oily liquids.

24. The method according to claim 22 or 23, wherein, Attenuation is reduced or avoided for at least 30 minutes after the test signal is generated.

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