Method for Differentiating Activities of Ricin and Abrin Using Heat Pretreatment

Abstract

The present invention relates to an analytical method for distinguishing the activities of two toxins from a sample containing a mixture of lysine and abrine. Specifically, the method comprises: (a) a pretreatment step of heat-treating the sample under predetermined temperature and time conditions; (b) a step of measuring the residual depurination activity of the pretreated sample; The present invention relates to a toxin activity analysis method comprising the step of distinguishing between lysine and abrine by comparing the change in depurination activity before and after heat treatment and distinguishing between an activity pattern that is relatively significantly reduced by heat treatment and an activity pattern that is relatively maintained; a heat treatment pretreatment method for selectively controlling the activity of lysine and abrine, comprising the step of heat treating the sample at a temperature range of 65°C to 85°C for 1 minute to 120 minutes, as a pretreatment method for selectively inhibiting the depurination activity of abrine while maintaining the depurination activity of lysine in a sample containing lysine and abrine; and a kit for distinguishing the activities of the two toxins in a sample containing lysine and abrine, comprising the step of (i) a DNA substrate for inducing a depurination reaction of lysine and abrine; (ii) pretreatment instructions for heat treating the sample under predetermined temperature and time conditions; and (iii) analysis instructions for distinguishing between lysine and abrine by comparing the change in depurination activity before and after heat treatment. According to the present invention, even when lysine and abrine are present together in the same sample, their activities can be distinguished and analyzed by utilizing the difference in the patterns of change in depurination activity of the two toxins through heat pretreatment. Specifically, since the depurination activity of lysine is relatively maintained by heat treatment under predetermined temperature and time conditions, while the depurination activity of abrine is selectively inhibited, the two toxins can be clearly distinguished by comparing the changes in activity before and after heat treatment. Furthermore, the present invention enables the quantitative measurement of activity signals through a depurination reaction using a DNA substrate and mass spectrometry, making it possible to independently evaluate the activity levels of lysine and abrine respectively within a single sample.

Description

Technology Field

[0001] The present invention relates to an analytical method comprising a heat treatment pretreatment for distinguishing the activities of two toxins from a sample containing a mixture of lysine and abrin.

[0002] Specifically, a toxin activity analysis method comprising: (a) a pretreatment step of heat-treating the sample under predetermined temperature and time conditions; (b) a step of measuring the residual depurination activity of the pretreated sample; and (c) a step of distinguishing lysine and abrine by comparing the change in depurination activity before and after heat treatment and distinguishing between an activity pattern that is relatively significantly reduced by heat treatment and an activity pattern that is relatively maintained; a heat treatment pretreatment method for selectively controlling the activity of lysine and abrine, comprising the step of heat-treating the sample for 1 minute to 120 minutes at a temperature range of 65°C to 85°C, as a pretreatment method for selectively inhibiting the depurination activity of abrine while maintaining the depurination activity of lysine in a sample containing lysine and abrine; and a kit for distinguishing the activities of the two toxins in a sample containing lysine and abrine, comprising: (i) a DNA substrate for inducing a depurination reaction of lysine and abrine; The present invention relates to a kit for analyzing toxin activity comprising: (ii) pretreatment instructions for heat-treating a sample under predetermined temperature and time conditions; and (iii) analysis instructions for distinguishing between lysine and abrine by comparing changes in depurination activity before and after heat treatment. Background Technology

[0004] Ricin is a protein toxin derived from the seeds of the castor plant (Ricinus communis) and is known to inhibit protein synthesis and induce strong cytotoxicity by inactivating intracellular ribosomes. Ricin exhibits lethal toxicity even in minute quantities and can have fatal effects on living organisms through various routes, including inhalation, oral ingestion, or injection. Due to these characteristics, ricin is designated as the only protein toxin classified as a chemical agent by the Organization for the Prohibition of Chemical Weapons (OPCW) and is subject to strict international monitoring and regulation.

[0005] Lysine is a double-stranded protein structurally composed of an A chain and a B chain. It is known that after entering the cell via the B chain, the A chain depurates specific adenine bases present in the ribosome's 28S rRNA, thereby causing the ribosome to lose its function. This mechanism of action is a highly efficient toxic mechanism that induces apoptosis by fundamentally blocking protein synthesis.

[0006] Protein toxins that share this mechanism of action are generally classified as Ribosome Inactivating Proteins (RIPs), and among them, abrin is known as the most representative substance along with lysine. Abrin is a protein toxin derived from the seeds of the Indian tropical plant *Avrus precatorius*, and it inhibits protein synthesis through N-glycosidase activity that removes specific adenines from ribosomal rRNA, similar to lysine. According to several studies, abrin has been reported to exhibit toxicity equivalent to or, in some cases, greater than that of lysine (Non-patent Literature 1).

[0007] Since lysine and abrine share very similar characteristics not only in structural similarity but also in terms of their mechanisms of action, existing toxin activity assays have limitations in clearly distinguishing between the two toxins. Representative activity assays include (i) assays that directly detect adenine depurination reactions, (ii) cytotoxicity assays using cultured cells, and (iii) assays that measure the degree of inhibition of protein synthesis in cellular or cell-free systems. However, since these methods are all based on the same toxicity mechanism of ribosome inactivation, they are limited in independently evaluating the activity of each toxin when lysine and abrine are present simultaneously in a sample (Non-patent Literature 2).

[0008] In particular, existing analytical methods based on depurination reactions are useful for measuring the presence or total amount of a reaction, given that adenine, the same reaction product, is produced by both lysine and abrine; however, they are not suitable for distinguishing which of the two toxins the activity originates from. Cytotoxicity and protein synthesis inhibition analyses also have limitations in the selective analysis of mixed samples, as lysine and abrine induce similar patterns of cell death.

[0009] Consequently, when ricin and abrin are detected simultaneously in samples with complex matrices, such as actual environmental, food, or biological samples, it has been virtually impossible with existing technologies to calculate the quantitative proportion of each toxin's contribution to total toxicity. This has acted as a significant technical limitation in terms of sample risk assessment, pathological analysis, or surveillance of biological threats.

[0010] Therefore, there has been a demand for the development of new analytical methods capable of distinguishing the activities of the two toxins in samples containing a mixture of lysine and abrine, and furthermore, independently evaluating the activity levels of each toxin. In particular, a technology capable of selectively differentiating activities by utilizing differences in reactivity to physical or chemical treatments, despite the two toxins inducing the same biochemical reaction, is attracting attention as an alternative to overcome the limitations of existing activity analysis methods.

[0012] Against this backdrop, the inventors of the present invention have made research efforts to develop a new analytical method capable of distinguishing and analyzing the activities of the two toxins from a sample containing both lysine and abrine, and further evaluating the activity levels of each toxin independently. As a result, they discovered that although both lysine and abrine have generally been recognized as being relatively stable to heat, the patterns of change in the depurination activity of the two toxins appear significantly different when heat treatment pretreatment is applied under specific temperature and time conditions. Specifically, they confirmed that a difference in activity patterns appears in which the depurination activity of lysine is relatively maintained, while the depurination activity of abrine is selectively inhibited. By utilizing this difference, they identified that the activities of lysine and abrine mixed within the same sample can be effectively distinguished and analyzed, thereby completing the present invention. Prior art literature

[0014] Olsnes, S., Toxicon, 2004Stirpe, F. & Battelli, M.G., Cell Mol Life Sci, 2006 The problem to be solved

[0015] The object of the present invention is an analytical method for distinguishing the activity of two toxins from a sample containing a mixture of lysine and abrin, wherein

[0016] (a) a pretreatment step of heat-treating the above sample under predetermined temperature and time conditions;

[0017] (b) a step of measuring the residual depurination activity of the above-mentioned pretreated sample; and

[0018] (c) A step of distinguishing between lysine and abrine by comparing the change in depurination activity before and after heat treatment and distinguishing between an activity pattern that is relatively significantly reduced by heat treatment and an activity pattern that is relatively maintained, comprising

[0019] It is to provide a method for analyzing toxin activity.

[0020] Another objective of the present invention is to provide a heat treatment pretreatment method for selectively controlling the activity of lysine and abrine, comprising the step of heat treating the sample at a temperature range of 65°C to 85°C for 1 minute to 120 minutes, as a pretreatment method for selectively inhibiting the depurination activity of abrine while maintaining the depurination activity of lysine in a sample containing a mixture of lysine and abrine.

[0021] Another objective of the present invention is a kit for distinguishing the activity of two toxins from a sample containing a mixture of lysine and abrin,

[0022] (i) DNA substrate for inducing the depurination reaction of lysine and abrine;

[0023] (ii) pretreatment instructions for heat-treating the sample under specified temperature and time conditions; and

[0024] (iii) Provides a kit for analyzing toxin activity, comprising analytical guidelines for distinguishing between lysine and abrine by comparing changes in depurination activity before and after heat treatment.

[0026] The problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below. means of solving the problem

[0028] One aspect of the present invention for achieving the above objective is an analytical method for distinguishing the activity of two toxins from a sample containing lysine and abrin, wherein

[0029] (a) a pretreatment step of heat-treating the above sample under predetermined temperature and time conditions;

[0030] (b) a step of measuring the residual depurination activity of the above-mentioned pretreated sample; and

[0031] (c) A method for analyzing toxin activity, comprising the step of distinguishing between lysine and abrine by comparing the change in depurination activity before and after heat treatment and distinguishing between an activity pattern that is relatively significantly reduced by heat treatment and an activity pattern that is relatively maintained.

[0032] The above step (a) heat treatment is characterized by being performed in a temperature range of 75°C to 85°C.

[0033] The heat treatment in step (a) above is characterized by being performed at 80°C for 5 minutes or more and less than 10 minutes.

[0034] In step (b) above, the depurination activity is characterized by being measured through a reaction with a DNA substrate.

[0035] The above DNA substrate is characterized as single-stranded DNA containing the base sequence 5'-CGCGCGAGAGCGCG-3'.

[0036] Step (b) above may be performed by mass spectrometry including LC-MS / MS.

[0037] The above LC-MS / MS analysis is characterized by being performed in an SRM manner that monitors the generated ions for the precursor ions of adenine at m / z 136 in ESI cation mode.

[0038] The change in depurinization activity in step (c) above is characterized by being compared based on the relative activity reduction rate after heat treatment compared to before heat treatment.

[0039] The above relative activity reduction rate is characterized by being calculated based on the value obtained by subtracting the reduction rate of lysine from the reduction rate of abrin.

[0040] In step (c) above, abrin is characterized by exhibiting a pattern in which its depurinization activity is reduced relatively more significantly than that of lysine by heat treatment.

[0041] In step (c) above, lysine is characterized by exhibiting a pattern in which its depurinization activity is maintained relatively more than that of abrine by heat treatment.

[0042] The above step (c) is characterized by including a step of quantitatively calculating the contribution of lysine and abrin using the activity signal of the sample before heat treatment and the activity signal of the sample after heat treatment.

[0043] The above sample is characterized as being an environmental sample, a food sample, or a biologically derived sample.

[0044] The above method is characterized by being used for the detection or monitoring of biological hazardous substances.

[0045] In addition, another aspect of the present invention for achieving the above objective provides a heat treatment pretreatment method for selectively controlling the activity of lysine and abrine, comprising the step of heat treating the sample at a temperature range of 65°C to 85°C for 1 minute to 120 minutes, as a pretreatment method for selectively inhibiting the depurinization activity of abrine while maintaining the depurinization activity of lysine in a sample containing a mixture of lysine and abrine.

[0046] The above heat treatment is characterized by being set to conditions that maintain the depurinization activity of lysine while significantly reducing the depurinization activity of abrine.

[0047] The above heat treatment is characterized by being performed at 80°C for 5 minutes or more and less than 10 minutes.

[0048] In addition, another aspect of the present invention for achieving the above objective is a kit for distinguishing the activity of two toxins from a sample containing lysine and abrin, wherein

[0049] (i) DNA substrate for inducing the depurination reaction of lysine and abrine;

[0050] (ii) pretreatment instructions for heat-treating the sample under specified temperature and time conditions; and

[0051] (iii) Provides a kit for analyzing toxin activity, comprising analytical guidelines for distinguishing between lysine and abrine by comparing changes in depurination activity before and after heat treatment.

[0052] The above DNA substrate is characterized by containing a 5'-CGCGCGAGAGCGCG-3' base sequence designed to release adenine by the depurination reaction of lysine and abrine.

[0053] The above kit is characterized by being used to distinguish the activity of lysine and abrin from environmental samples, food samples, or biologically derived samples. Effects of the invention

[0055] According to the present invention, even when lysine and abrine are present together in the same sample, their activities can be distinguished and analyzed by utilizing the difference in the patterns of change in depurination activity of the two toxins through heat pretreatment. Specifically, since the depurination activity of lysine is relatively maintained by heat treatment under predetermined temperature and time conditions, while the depurination activity of abrine is selectively inhibited, the two toxins can be clearly distinguished by comparing the changes in activity before and after heat treatment. Furthermore, the present invention enables the quantitative measurement of activity signals through a depurination reaction using a DNA substrate and mass spectrometry, making it possible to independently evaluate the activity levels of lysine and abrine respectively within a single sample. Brief explanation of the drawing

[0057] Figure 1 shows the results of analyzing adenine, a product of the depurination reaction, by LC-MS / MS. Figure 2 shows the relative depurination activity of lysine and abrine according to heat treatment conditions (temperature and time) ((a): change in relative activity of lysine, (b): change in relative activity of abrine). Figure 3 shows the results of comparing the differences in depurination activity of lysine and abrine according to heat treatment conditions ((a): bar graph showing the relative activity difference values, (b): heat map showing the activity difference according to heat treatment temperature and time). Figure 4 shows the standard activity curves for the depurination activity of lysine and abrine before and after heat treatment ((a): standard activity curve of lysine before and after heat treatment, (b): standard activity curve of abrine before and after heat treatment). Specific details for implementing the invention

[0058] Preferred embodiments according to the present invention will be described in detail below with reference to the attached drawings.

[0059] The advantages and features of the present invention and the method for achieving them will become clear by referring to the embodiments described in detail below together with the accompanying drawings.

[0060] However, the present invention is not limited by the embodiments disclosed below but may be implemented in various different forms, and these embodiments are provided merely to make the disclosure of the present invention complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined only by the scope of the claims.

[0061] In addition, in describing the present invention, if it is determined that related known technologies, etc., may obscure the essence of the present invention, a detailed explanation thereof will be omitted.

[0062] The present invention will be described in detail below.

[0064] Analysis method for lysine and abrin activity

[0065] One aspect of the present invention for achieving the above objective provides an analytical method comprising a heat treatment pretreatment to distinguish the activities of two toxins from a sample containing a mixture of lysine and abrin.

[0066] Specifically,

[0067] (a) A pretreatment step of heat-treating a sample containing a mixture of ricin and abrin under predetermined temperature and time conditions;

[0068] (b) a step of measuring the residual depurination activity of the above-mentioned pretreated sample; and

[0069] (c) A step of distinguishing between lysine and abrine by comparing the change in depurination activity before and after heat treatment and distinguishing between an activity pattern that is relatively significantly reduced by heat treatment and an activity pattern that is relatively maintained, comprising

[0070] Provides a method for analyzing toxin activity.

[0071] The term “ricin” in this invention refers to a ribosome inactivating protein (RIP) derived from the seeds of the castor plant (Ricinus communis), which inhibits protein synthesis through N-glycosidase activity that removes specific adenine bases present in the 28S rRNA of ribosomes. Ricin is generally known as a double-stranded protein composed of an A chain and a B chain.

[0072] In the present invention, the lysine may refer to a toxin having the characteristic of exhibiting depurinization activity and maintaining such activity relatively under specific heat treatment pretreatment conditions. That is, the lysine in the present invention includes a protein toxin that exhibits an activity pattern distinguishable from abrine based on the change in depurinization activity before and after heat treatment. In the present invention, the lysine is interpreted as a concept that includes not only natural lysine but also recombinant lysine, modified lysine, lysine derivatives, lysine fragments, or variants exhibiting substantially the same depurinization activity as lysine. Furthermore, regardless of source, manufacturing method, degree of purification, or binding type, it is included in the lysine of the present invention as long as it exhibits ribosomal inactivation activity.

[0073] In one embodiment of the present invention, in-house purified ricin was used as an analytical standard material, but is not limited thereto (Example 1).

[0074] The term “abrin” in this invention refers to a ribosomal inactivation protein derived from the seeds of Abrus precatorius, which inhibits protein synthesis through depurinization activity that removes specific adenines from ribosomal rRNA similar to lysine.

[0075] In the present invention, the abrin may refer to a toxin having the characteristic of exhibiting depurination activity, wherein such activity is relatively significantly reduced or inhibited compared to lysine under specific heat treatment pretreatment conditions. That is, in the present invention, abrin is defined as a toxin that exhibits a pattern of reduced activity distinct from lysine based on the pattern of change in depurination activity before and after heat treatment. Furthermore, in the present invention, the abrin may include not only natural abrin but also recombinant abrin, modified abrin, abrin derivatives, abrin fragments, or variants exhibiting substantially the same depurination activity as abrin, and may be interpreted as a concept that includes homologues, isoforms, or RIP-family proteins having similar heat treatment reactivity of abrin. Additionally, regardless of source, manufacturing method, degree of purification, or binding type, it may be included in the abrin of the present invention as long as it exhibits ribosome inactivation activity.

[0076] The term “heat treatment” in this invention refers to a process of inducing physical or chemical changes by applying thermal energy to a sample. According to previous studies, ribosome inactivating proteins (RIPs), such as lysine and abrine, have been reported to possess relatively high thermal stability. For example, lysine and abrine are known to retain a significant portion of their depurinization activity associated with ribosome inactivation even at temperatures of approximately 70°C to 80°C; accordingly, heat treatment has been recognized as a process that partially reduces, rather than completely eliminates, the activity of these toxins. For this reason, in the prior art, heat treatment was not typically considered as a means to distinguish between lysine and abrine.

[0077] However, in the present invention, the heat treatment refers to a pretreatment process that induces differential changes in the depurination activity of the two toxins by applying predetermined temperature and time conditions to a sample containing a mixture of lysine and abrine. In particular, the present invention is based on the observation that, although lysine and abrine have both been recognized as relatively stable against heat in previous studies, a difference in activity patterns appears under specific heat treatment conditions, in which the depurination activity of lysine is relatively maintained while the depurination activity of abrine is selectively inhibited.

[0078] The heat treatment in the present invention includes physical heat treatment performed without chemical or enzymatic treatment, and may include all forms of thermal treatment that increase the temperature of a sample and affect the activity of the toxin, regardless of the type of heating method, heating medium, or heating device. For example, water baths, dry heat, moist heat, microwave heating, heating using an incubator or a heat block may all be included.

[0079] In the present invention, the heat treatment specifically means to be performed for 1 minute to 120 minutes at a temperature range of 65°C to 85°C, and more specifically means to be performed for 5 minutes or more and less than 10 minutes at 80°C, but is not limited thereto.

[0080] In one embodiment of the present invention, lysine and abrine samples were heat-treated at temperatures of 65°C, 70°C, 75°C, and 80°C for 0, 5, 10, and 15 minutes, respectively, and then reacted with a DNA substrate under the same conditions. The reaction products generated were then quantitatively analyzed using LC-MS / MS. As a result, it was confirmed that lysine maintained relatively high activity regardless of whether it was before or after heat treatment (Fig. 2a). On the other hand, in the case of abrine, the relative activity decreased significantly as the treatment time increased under the 80°C condition. Thus, it was confirmed that the patterns of decrease in depurination activity differed between lysine and abrine under the same heat treatment conditions (Fig. 2b).

[0081] In addition, in one embodiment, the difference in relative activity between lysine and abrin according to heat treatment conditions was calculated, and the difference in relative activity between lysine and abrin was greatest when treated at 80°C for 5 minutes, confirming that this condition can maximize the difference in activity between the two toxins (Fig. 3).

[0082] The term “pretreatment” in this invention refers to a process performed prior to analysis or reaction to control the state of a sample or to improve analytical efficiency. In conventional studies on the analysis of lysine and abrine, pretreatment has primarily been used to eliminate analytical interference or improve detection sensitivity, such as through immunological concentration, chemical denaturation, or enzymatic degradation. However, these pretreatments focused on confirming the presence or structural characteristics of toxins rather than inducing differences in the activity of lysine and abrine. In particular, as mentioned above, since both lysine and abrine have been recognized as relatively stable to heat, no attempt to selectively distinguish the activities of the two toxins through heat treatment has been proposed in the prior art.

[0083] The pretreatment in the present invention refers to a process that induces differential changes in the depurination activity of lysine and abrine by applying heat treatment to a sample, unlike conventional immunological, chemical, or enzymatic pretreatments.

[0084] Specifically, this may refer to a process of performing heat treatment on a sample containing a mixture of lysine and abrine so that the difference in activity between the two toxins becomes clearly apparent in a subsequent analysis of depurination activity. The pretreatment may be performed in a single step or multiple steps, and as long as heat treatment is included, it is interpreted regardless of the order, repetition, or parallelity thereof. Additionally, the pretreatment may be applied to the entire sample or a part of the sample to be analyzed.

[0085] The term “depurination” in the present invention refers to a reaction in which a purine base (adenine or guanine) is removed from nucleic acid.

[0086] In the present invention, the depurination may refer to a reaction in which adenine is removed from a DNA or RNA substrate by the N-glycosidase activity of lysine or abrine, and includes a reaction in which the activity of a toxin is evaluated through the amount or signal of adenine produced as a result. In the present invention, the depurination may include a purine base removal reaction occurring in any nucleic acid substrate, including single-stranded or double-stranded nucleic acids, synthetic nucleic acids, or natural nucleic acids, and it is interpreted that not only the direct products of the depurination reaction but also signals or indicators indirectly reflecting it are included in the depurination activity.

[0087] Specifically, the above depurinization activity is measured through a reaction between a sample containing heat-pretreated lysine and labrin and a DNA substrate containing the base sequence 5'-CGCGCGAGAGCGCG-3', and can be performed by mass spectrometry including LC-MS / MS.

[0088] Here, the above LC-MS / MS analysis can be performed in an SRM manner that monitors the generated ions for the precursor ions of adenine at m / z 136 in ESI cation mode, and the change in depurination activity may mean that the relative activity reduction rate after heat treatment is compared to before heat treatment.

[0089] More specifically, in the present invention, the change in depurification activity is characterized in that abrine exhibits a pattern in which the depurification activity of abrine is reduced relatively more significantly than that of lysine upon heat treatment, and lysine exhibits a pattern in which the depurification activity of lysine is maintained relatively more than that of abrine upon heat treatment, and the relative activity reduction rate is calculated based on the value obtained by subtracting the reduction rate of lysine from the reduction rate of abrine.

[0090] This comparison of changes in depurinization activity before and after heat treatment includes a step of quantitatively calculating the contribution of lysine and abrine using the activity signal of the sample before heat treatment and the activity signal of the sample after heat treatment, thereby enabling the distinction between lysine and abrine.

[0091] In the present invention, a sample containing a mixture of lysine and abrine may include, but is not limited to, environmental samples, food samples, or biologically derived samples, and encompasses all forms of samples in which lysine and abrine may coexist. Specifically, it may refer to any sample used for the detection or monitoring of biological hazardous substances.

[0093] Heat treatment pretreatment method for selective activity control of lysine and abrin

[0094] Another aspect of the present invention for achieving the above objective provides a heat treatment pretreatment method for selectively controlling the activity of lysine and abrine, comprising the step of heat treating the sample at a temperature range of 65°C to 85°C for 1 minute to 120 minutes, for maintaining the depurination activity of lysine while selectively inhibiting the depurination activity of abrine from a sample containing a mixture of lysine and abrine.

[0095] The above terms, lysine, abrine, depurination, pretreatment, and heat treatment are as described above.

[0096] Here, the above heat treatment may mean that the condition is set to significantly reduce the depurination activity of abrine while maintaining the depurination activity of lysine.

[0097] In addition, the above heat treatment may mean being performed at 80°C for 5 minutes or more and less than 10 minutes, but is not limited thereto.

[0099] Analytical kit to distinguish between lysine and abrine activity

[0100] Another aspect of the present invention for achieving the above objective provides a kit for distinguishing the activity of two toxins from a sample containing a mixture of lysine and abrin.

[0101] Specifically,

[0102] (i) DNA substrate for inducing the depurination reaction of lysine and abrine;

[0103] (ii) pretreatment instructions for heat-treating the sample under specified temperature and time conditions; and

[0104] (iii) Provides a kit for analyzing toxin activity that includes analytical guidelines for distinguishing between lysine and abrine by comparing changes in depurination activity before and after heat treatment.

[0105] The above terms, lysine, abrine, depurination, pretreatment, and heat treatment are as described above.

[0106] The term “kit” in the present invention means a set of articles that provide one or more components in combination necessary to perform a specific analysis, measurement, or reaction, and each component may be provided in an individual container, package, or assembled form.

[0107] In the present invention, the kit may refer to a set of components used to analyze and distinguish the depurination activities of the two toxins from a sample containing a mixture of lysine and abrine. For example, it may include at least one of a nucleic acid substrate for inducing the depurination reaction of lysine and abrine, information or instructions regarding heat treatment pretreatment conditions, and analysis instructions for comparing and interpreting changes in depurination activity before and after heat treatment, and the components may be provided together within a single kit or provided in a functionally related form.

[0108] In the present invention, the kit is not limited to cases where the components are provided in physically identical packaging units; rather, even if each is provided in a separate container, packaging, medium, or form, it is interpreted as a concept that includes all of these if they are designed to be used together according to the purpose of the present invention.

[0109] In addition, regardless of the specific quantity, combination, form of provision, or name of the components included in the kit, they are interpreted as being included in the kit of the present invention insofar as they perform the function of enabling the analysis and distinction of the depurination activity of lysine and abrine.

[0110] Furthermore, the kit can be implemented in various forms depending on the purpose of use, such as for laboratory use, field analysis, diagnosis, or research.

[0112] Example 1. Preparation of Lysine and Abrine Samples

[0113] To analyze the depurination activity of lysine and abrine, samples to be used for analysis were prepared, and the activity was compared according to heat treatment conditions.

[0114] First, the lysine sample was immunoprecipitated using magnetic beads conjugated with an anti-lysine monoclonal antibody, and the immunoprecipitated beads were washed three times with reaction buffer (50 mM ammonium formate) and resuspended in 70 μL of deionized water for use in the depurination reaction. In-house purified ricin was used as the analytical standard.

[0115] The abrin sample was also prepared in a form suitable for the analysis of depurination activity and used for heat treatment and subsequent experiments under the same conditions as the lysine sample.

[0117] Example 2. Establishment of an LC-MS / MS-based activity analysis method

[0118] In order to quantitatively analyze the depurination activity of lysine and abrine after the heat treatment performed in Example 1 above, a method for analyzing the DNA substrate to be degraded and the depurination reaction product was established as follows.

[0119] First, to mimic the depurination reaction induced by ribosomal inactivation protein (RIP), the following base sequence (Sequence No. 1), which is a single-stranded DNA (ssDNA) sequence, was used as the DNA substrate to be degraded.

[0120]

[0121] The above DNA substrate is a sequence designed to allow specific adenine bases to be removed by the N-glycosidase activity of lysine and abrine, and is a substrate suitable for quantitative analysis of adenine released by the depurination reaction.

[0122] The adenine produced after the depurination reaction was analyzed using LC-MS / MS. Specifically, liquid chromatography analysis was performed using a Thermo Scientific Ultimate 3000 UPLC, a Waters CORTECS C18+ column (100 mm × 2.1 mm × 1.6 μm) was used, and the column temperature was maintained at 30℃. The injection volume was 10 μL.

[0123] The mobile phase used was (A) 0.1% formic acid in deionized water and (B) 0.1% formic acid in acetonitrile, and the elution conditions were set as shown in Table 1 below, with a flow rate of 200 μL / min.

[0124] Time (min) 0 2 3 4 5 6 %B 0 0 50 50 0 0

[0126] Mass spectrometry was performed using a Thermo Scientific TSQ Quantiva, and the ionization method was applied using the ESI cation mode. The analysis was conducted under SRM conditions monitoring generated ions at m / z 92.000, 94.000, and 119.000 relative to precursor ions at m / z 136. The collision energies were set to 25 eV, 18 eV, and 25 eV, respectively, and argon was used as the collision gas.

[0127] As a result, as shown in Figure 1, adenine, the product of the depurination reaction, was detected as a single peak at a retention time of about 1.4 to 1.5 minutes, and the peak showed a sufficiently high intensity compared to the background signal.

[0128] In addition, MS / MS analysis was performed on the same peak, and generated ions of m / z 92, 94, and 119 were detected from the precursor ion m / z 136, and in particular, the generated ion of m / z 119 showed the highest relative intensity.

[0130] From this, it was confirmed that the peak detected under the above analysis conditions corresponds to adenine, a product of the depurination reaction, and it was confirmed that the above LC-MS / MS conditions can quantify adenine rapidly and reproducibly.

[0132] Example 3. Analysis of changes in lysine and abrin activity according to heat treatment conditions

[0133] Subsequently, the depurination activity of lysine and abrine was analyzed according to heat treatment conditions of time and temperature.

[0134] Specifically, the lysine and abrin samples prepared above were heat-treated at temperatures of 65°C, 70°C, 75°C, and 80°C for 0 minutes, 5 minutes, 10 minutes, and 15 minutes, respectively, and then reacted with a DNA substrate under the same conditions to induce a depurination reaction.

[0135] The reaction products generated after the depurination reaction were quantitatively analyzed using LC-MS / MS under the activity analysis conditions described above.

[0136] As a result, as shown in Fig. 2, in the case of lysine, the relative activity was maintained without significant change in the range of 65°C to 75°C, and it was confirmed that relatively high activity was maintained up to 5 minutes even under 80°C conditions (Fig. 2a). On the other hand, in the case of abrin, the relative activity decreased significantly as the treatment time increased above 75°C, especially under 80°C conditions, confirming that the pattern of decrease in depurination activity differed between lysine and abrin under the same heat treatment conditions (Fig. 2b).

[0138] Example 4. Derivation of conditions to maximize the difference in lysine and abrin activity according to heat treatment conditions

[0139] Based on the data obtained in Example 3 above, the difference in relative activity between lysine and abrin according to heat treatment conditions was calculated.

[0140] The reduction rate of abrin activity and the reduction rate of lysine activity calculated under each condition were compared, and the value of (reduction rate of abrin activity) - (reduction rate of lysine activity) was calculated.

[0141] As a result, as shown in Figure 3, the difference in relative activity between lysine and abrin was greatest when treated at 80°C for 5 minutes, and it was confirmed that this condition can maximize the difference in activity between the two toxins.

[0143] Example 5. Establishment of a quantitative model for lysine and abrin based on standard activity curves before and after heat treatment

[0144] A model was established to quantitatively separate and calculate the contributions of lysine and abrine in mixed samples using standard activity curves of lysine and abrine before and after heat treatment.

[0145] Specifically, lysine and adenine samples were prepared under conditions of no heat treatment and heat treatment at 80°C for 5 minutes, respectively. Then, each sample was serially diluted to different concentration levels, and a depurination reaction was performed using the LC-MS / MS-based analysis method established in Example 2. The signal intensity of the adenine released after the reaction was measured, and standard activity curves were constructed for the period before and after heat treatment.

[0146] As a result, as shown in Figure 4, in the case of lysine, an excellent linear relationship between the adenine signal and the activity concentration of the standard activity curve was maintained both before and after heat treatment, and it was confirmed that the slope and linearity of the standard activity curve did not significantly decrease even after heat treatment (Figure 4a). On the other hand, in the case of abrine, linearity between the adenine signal and the activity concentration was observed before heat treatment, but after heat treatment at 80°C for 5 minutes, the increase in the adenine signal within the same concentration range was significantly reduced, and it was confirmed that the slope of the standard activity curve decreased significantly.

[0148] Let P be the total depurinization activity signal of the sample before heat treatment and Q be the total depurinization activity signal of the sample after heat treatment, let x be the concentration of lysine in the sample and y be the concentration of abrine, let k₁ and k₂ be the slopes of the standard activity curves of lysine before and after heat treatment, respectively, let k₁ and k₂ be the slopes of the standard activity curves of abrine, respectively, and let b be the y-intercept of each standard activity curve. Then, P and Q can be expressed by the following relationship.

[0149]

[0150] From the two equations above, the values ​​of lysine (x) and abrin (y) in the mixed sample can be calculated as follows.

[0151]

[0153] From this, it was confirmed that by using the measured values ​​of depurinization activity before and after heat treatment according to the present invention, the contributions of lysine and abrine mixed within a single sample can be separated and quantitatively calculated.

[0155] From the above results, it was confirmed that the heat treatment pretreatment of the present invention can be effective for the selective differentiation of the activities of lysine and abrine and for reliable quantitative analysis, as it significantly reduces the activity reactivity of abrine without affecting the quantification of the depurination activity of lysine.

[0157] Although specific embodiments regarding the distinction and quantitative analysis of two toxins using the difference in depurination activity resulting from heat pretreatment for lysine and abrine according to one embodiment of the present invention have been described so far, it is obvious that various modifications are possible within the scope of the present invention.

[0158] Therefore, the scope of the present invention should not be limited to the described embodiments, but should be defined by the claims set forth below as well as equivalents thereof.

[0159] That is, the aforementioned embodiments should be understood as exemplary in all respects and not limiting, and the scope of the invention is defined by the claims set forth below rather than by the detailed description, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts thereof should be interpreted as being included within the scope of the invention.

Claims

Claim 1 A method for analyzing the activity of two toxins in a sample containing a mixture of lysine and abrine, comprising: (a) a pretreatment step of heat-treating the sample under predetermined temperature and time conditions; (b) a step of measuring the residual depurination activity of the pretreated sample; and (c) a step of distinguishing lysine and abrine by comparing the change in depurination activity before and after heat treatment, and distinguishing an activity pattern in which the decrease rate of abrine is greater than the decrease rate of lysine based on the decrease rate of depurination activity after heat treatment compared to before heat treatment. Claim 2 A method according to claim 1, characterized in that the heat treatment in step (a) is performed in a temperature range of 75°C to 85°C. Claim 3 A method according to claim 1, characterized in that the heat treatment in step (a) is performed at 80°C for 5 minutes or more and less than 10 minutes. Claim 4 delete Claim 5 delete Claim 6 delete Claim 7 A method according to claim 1, characterized in that the change in depurination activity of step (c) is compared based on the rate of decrease in depurination activity after heat treatment compared to before heat treatment. Claim 8 A method according to claim 7, characterized in that the reduction rate of depurination activity is calculated based on the value obtained by subtracting the reduction rate of lysine from the reduction rate of abrine. Claim 9 A method according to claim 1, characterized in that, based on the rate of reduction in depurination activity after heat treatment compared to before heat treatment in step (c), the rate of reduction in depurination activity of abrine is greater than the rate of reduction in depurination activity of lysine. Claim 10 A method according to claim 1, wherein step (c) comprises a step of quantitatively calculating the contribution of lysine and abrine based on the difference in depurinization activity before and after heat treatment. Claim 11 A method according to claim 1, characterized in that the sample is an environmental sample, a food sample, or a biologically derived sample. Claim 12 A method according to claim 1, characterized in that the method is used for the detection or monitoring of biological hazardous substances. Claim 13 A heat treatment pretreatment method for selectively controlling the activity of lysine and abrine, comprising the step of heat-treating the sample at a temperature range of 65°C to 85°C for 1 minute to 120 minutes, as a pretreatment method for selectively inhibiting the depurination activity of abrine while maintaining the depurination activity of lysine in a sample containing a mixture of lysine and abrine. Claim 14 delete Claim 15 A method according to claim 13, characterized in that the heat treatment is performed at 80°C for 5 minutes or more and less than 10 minutes. Claim 16 A kit for distinguishing the activities of two toxins from a sample containing a mixture of lysine and abrine, comprising: (i) a DNA substrate for inducing a depurination reaction of lysine and abrine; (ii) pretreatment instructions for heat-treating the sample under predetermined temperature and time conditions; and (iii) analysis instructions for distinguishing lysine and abrine by comparing the change in depurination activity before and after heat treatment. Claim 17 delete Claim 18 In claim 16, the kit is characterized by being used for distinguishing the activity of lysine and abrine from environmental samples, food samples, or biological samples.

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