CN114813900A

CN114813900A - Method for detecting active II type ribosome inactivating protein

Info

Publication number: CN114813900A
Application number: CN202110126805.0A
Authority: CN
Inventors: 郭磊; 杨捷威; 王晨钰; 罗黎; 李治; 谢剑炜
Original assignee: Academy of Military Medical Sciences AMMS of PLA
Current assignee: Academy of Military Medical Sciences AMMS of PLA
Priority date: 2021-01-29
Filing date: 2021-01-29
Publication date: 2022-07-29

Abstract

The application relates to a method for detecting active type II ribosome inactivating protein (RIP-II), a kit for detecting active RIP-II and application of the kit for detecting active RIP-II.

Description

Method for detecting active II type ribosome inactivating protein

Technical Field

The application belongs to the field of analytical chemistry, and particularly relates to a method for detecting active II type ribosome inactivating protein (RIP-II) and a kit for detecting the active RIP-II.

Background

Ricin (ricin) is a toxic protein extracted from seeds of Ricinus communis (Ricinus communis L.) belonging to the family euphorbiaceae, originating from africa (nat. biotechnol.,2010,28:951-953), and is the only biotoxin protein listed simultaneously under the "international banned chemical weapons convention" and the "international banned biological and toxin weapons convention" (forensic. sci. int.,2011,209: 70-79). Ricin is also classified by the united states center for disease control and prevention (CDC) as a class B bioterrorism agent. The half lethal dose of ricin is 3 μ g/kg (mouse, i.v.) (Molecules,2016,21: 556). Ricin has a molecular weight of about 60-66kDa, and is typically RIP-II, which is formed by a disulfide bond linkage of an A chain (RTA) having a catalytic effect and a B chain (RTB) having a binding effect and a lectin function.

Castor seeds also contain trace ricin lectin (RCA120), a heterotetrameric protein with a molecular weight of about 120kDa, consisting of two ricin-like structures linked by disulfide bonds between the a chains (Proteins,1997,28:586 589), which has a high degree of sequence similarity to ricin, with 93% and 84% sequence homology between the a and B chains, respectively (biosens. bioelectron.,2016,78: 111-.

Abrin (abrin) is derived from seed of abrin (Abrus precatorius L.) of abrin of Leguminosae of Rosales, and is RIP-II and mouse LD ₅₀ 0.04 μ g/kg, which is 75 times of ricin (bioorg.Med.chem.Lett.,2007,17: 5690-. Similar to castor seeds, minute quantities of abrin (AAG) are also present in abrin seeds.

Due to the structural similarity and toxicity similarity between ricin, RCA120, abrin, AAG, for toxin proteins in complex matrices such as food, biomedical sample matrices in various toxic and intoxication incidents, firstly, the correct distinction between active and inactive toxin proteins is required, and then the quantitative analysis of the active toxin is required.

At present, various analysis and detection methods are developed aiming at RIP-II such as ricin and the like, and mainly comprise immunoassay methods such as Radioimmunoassay (RIA), colloidal gold test paper, enzyme-linked immunosorbent assay (ELISA), immune Polymerase Chain Reaction (PCR) and the like, and instrument analysis and detection methods such as liquid chromatography, mass spectrometry, liquid chromatography-mass spectrometry and the like. Most of the analysis and detection methods can realize the rapid, sensitive and accurate detection of ricin, but active toxin and inactive toxin cannot be distinguished, and whether the detected sample is threatened or not cannot be judged in practical application. Therefore, there is a need to further develop and establish assays that can distinguish between active and inactive toxins.

Disclosure of Invention

The inventor of the application screens out a hybrid oligonucleotide substrate (such as a nucleotide sequence shown in SEQ ID NO: 5) with high reactivity with the active RIP-II through intensive research, and develops a method for qualitatively and/or quantitatively detecting the active RIP-II (such as ricin) based on a matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) technology, wherein the method has the characteristics of high sensitivity, quick detection, accurate quantification, capability of distinguishing active toxins from inactive toxins and the like. The flow diagram of the method is shown in figure 1, and comprises the steps of desalting pretreatment of a sample to be detected, mixed incubation with an oligonucleotide substrate, MALDI-MS detection and the like.

Thus, in one aspect, the present application provides a method for detecting active RIP-II in a sample, comprising:

providing a sample to be tested;

contacting a sample to be tested with an oligonucleotide substrate, wherein the oligonucleotide substrate is an oligonucleotide containing a stem-loop structure, and the sequence of the stem-loop structure contains 5 '-r (GdAGA) -3';

detecting whether the oligonucleotide substrate is deprived of adenine (a);

judging whether the active RIP-II exists in the sample to be detected;

optionally, the test sample is pretreated prior to contacting the test sample with the oligonucleotide substrate.

In certain embodiments, the method can be combined with mass spectrometry to identify a particular species of active RIP-II if present in the test sample.

In certain embodiments, the oligonucleotide substrate is of the general formula 5' -r ((GC) _n GdAGA(GC) _n ) -3 'or of the general formula 5' -r (C (GC) _n GdAGA(GC) _n G) -3', wherein n ═ 1,2, 3, 4, 5, 6, and,7. 8, 9 or 10, for example n-2 or 3.

In certain embodiments, the oligonucleotide substrate is a nucleic acid sequence selected from SEQ ID NOs: 5-7.

In certain embodiments, the oligonucleotide substrate is SEQ ID NO: 5.

In certain embodiments, the RIP-II is one or more selected from ricin, RCA120, abrin, AAG.

In certain embodiments, the RIP-II is ricin.

In certain embodiments, the methods of detecting active RIP-II in a sample described herein comprise contacting a test sample with an oligonucleotide substrate in a buffer solution comprising: water, capable of providing NH ₄ ⁺ An ammonium salt of (a), a metal ion chelating agent, and a pH adjusting agent.

In certain embodiments, the ammonium salt is an ammonium citrate salt, preferably the ammonium salt is Diammonium Hydrogen Citrate (DHC).

In certain embodiments, the DHC is not less than 98% pure, more preferably at a concentration of not less than 99%.

In certain embodiments, the metal ion chelating agent is ethylenediaminetetraacetic acid (EDTA).

In certain embodiments, the pH adjusting agent is Formic Acid (FA), Citric Acid (CA), or trifluoroacetic acid (TFA), preferably FA.

In certain embodiments, the buffer solution has a final concentration of DHC of about 0.2 to 5g/L, e.g., about 0.5 to 5g/L, about 0.8 to 2g/L, about 0.8 to 1.5g/L, about 1g/L, about 2g/L, about 3 g/L.

In certain embodiments, the final concentration of EDTA in the buffer solution is about 0.05 to about 0.375g/L, such as about 0.05 to about 0.3g/L, about 0.1 to about 0.35g/L, about 0.15 to about 0.3g/L, about 0.25 to about 0.35g/L, about 0.3 g/L.

In certain embodiments, the pH of the buffer solution is from about 4.0 to about 4.6, preferably from about 4.1 to about 4.4, and more preferably from about 4.2 to about 4.4.

In certain embodiments, the buffer solution contains DHC at a final concentration of about 1g/L, EDTA at a final concentration of about 0.3g/L, and FA, and the pH of the buffer solution is about 4.2.

In certain embodiments, the oligonucleotide substrate is present in the buffer solution at a final concentration of about 1-100. mu.M, preferably about 1-60. mu.M or about 1-30. mu.M, more preferably about 20-30. mu.M.

In certain embodiments, the methods of detecting active RIP-II in a sample described herein comprise contacting a test sample with an oligonucleotide substrate in a buffered solution at a temperature of about 4 ℃ to about 55 ℃ (e.g., about 25 ℃ to about 55 ℃, about 37 ℃).

In certain embodiments, the methods of detecting active RIP-II in a sample described herein comprise contacting a test sample with an oligonucleotide substrate in a buffered solution at a temperature of about 4-55 deg.C (e.g., about 25-55 deg.C, e.g., about 35-40 deg.C, e.g., about 37 deg.C) for about 3-60min, preferably about 8-35min, e.g., about 10-30 min.

In certain embodiments, the methods of detecting active RIP-II in a sample described herein comprise contacting a test sample with an oligonucleotide substrate in a buffer solution at a temperature of about 35-40 ℃ (e.g., about 37 ℃) for about 8-35min, e.g., about 10-30min, wherein the oligonucleotide substrate is SEQ ID NO: 5.

In certain embodiments, the methods of detecting active RIP-II in a sample described herein further comprise, after contacting the test sample with the oligonucleotide substrate in a buffer solution, adding ammonia to the buffer solution to bring the pH of the buffer solution to about 8 to 10, e.g., about 9, to provide a test solution.

In certain embodiments, the concentration of the aqueous ammonia is about 5 to 28 wt%, for example about 12 to 14 wt%.

In certain embodiments, the aqueous ammonia has a concentration of about 12 to 14 wt% and is added in an amount of about 0.8 to 1.2. mu.L (e.g., about 1. mu.L) of aqueous ammonia per 50. mu.L of the buffer solution.

In certain embodiments, the methods of detecting active RIP-II in a sample described herein comprise detecting whether the oligonucleotide substrate is deprived of adenine (a) using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS).

In certain embodiments, the MALDI-MS is matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).

In certain embodiments, the methods described herein for detecting active RIP-II in a sample employ a target plate having a hydrophilic enrichment effect (e.g., an ancorochip target plate) as the sample target plate.

In certain embodiments, the methods of detecting active RIP-II in a sample described herein employ a reflex mode to detect whether the oligonucleotide substrate is adenine (A) depleted.

The method for detecting active RIP-II in a sample adopts a negative ion reflex mode to detect whether the oligonucleotide substrate is deprived of adenine (A).

In certain embodiments, the methods of detecting active RIP-II in a sample described herein, wherein the oligonucleotide is tested for adenine (a) depletion by a method comprising:

mixing the solution to be detected with a MALDI matrix to obtain a mixed sample;

adding the mixed sample into a sample target plate;

collecting the spectrum of the mixed sample by using MALDI-MS;

it is judged whether or not the oligonucleotide substrate in the mixed sample is deprived of adenine (A).

In certain embodiments, the methods of the present application for detecting active RIP-II in a sample, wherein if the obtained profile shows a peak characteristic of the product after the removal of adenine (a) from the oligonucleotide substrate, determining that adenine (a) is removed from the oligonucleotide substrate, and determining the presence of active RIP-II in the sample to be tested.

In certain embodiments, the methods of the present application for detecting active RIP-II in a sample, wherein if the obtained profile shows a characteristic peak of the product after the oligonucleotide is deprived of adenine (a) and the peak area ratio (Prod/sub) of the product to the peak area of the substrate spectrum is not less than 0.003, determining that the oligonucleotide substrate is deprived of adenine (a), and determining that active RIP-II is present in the sample to be detected.

In certain embodiments, the methods of detecting active RIP-II in a sample described herein utilize the magnitude of the Prod/sub ratio to quantify active RIP-II.

In certain embodiments, the methods of detecting active RIP-II in a sample described herein, wherein: the oligonucleotide substrate is SEQ ID NO: 5, the product characteristic peak is located at 3754 ± 1 (mass-to-charge ratio, m/z); the oligonucleotide substrate is SEQ ID NO: 6, and the characteristic peak of the product is located at 4404 +/-1 (mass-to-charge ratio, m/z); the oligonucleotide substrate is SEQ ID NO: 7, and the characteristic peak of the product is located at 5055 +/-1 (mass-to-charge ratio, m/z).

In certain embodiments, the MALDI matrix comprises 3-hydroxy-2-pyridinecarboxylic acid (3-HPA), DHC, Acetonitrile (ACN), and water.

In certain embodiments, the concentration of 3-HPA in the MALDI matrix is about 1-80g/L, preferably about 14-70g/L, e.g., about 30-60g/L, e.g., about 50 g/L.

In certain embodiments, the concentration of DHC in the MALDI matrix is about 0-2g/L, preferably about 0.5-2g/L, e.g., about 0.8-1.2g/L, e.g., about 1 g/L.

In certain embodiments, the concentration of ACN in the MALDI matrix is about 0-75%, preferably about 10-50%, for example about 20-30%, for example about 25%.

In certain embodiments, the mixing ratio of the test solution to the MALDI matrix is about 1:1 to 10:1, preferably about 1:1 to 6: 1.

In certain embodiments, the purity of 3-HPA in the MALDI matrix is not less than 98%, more preferably the concentration is not less than 99%.

In certain embodiments, the purity of DHC in the MALDI matrix is not less than 98%, more preferably the concentration is not less than 99%.

In certain embodiments, the methods of detecting active RIP-II in a sample described herein, wherein the pretreatment is one or more selected from the group consisting of dilution, concentration, and desalting.

In certain embodiments, the methods of detecting active RIP-II in a sample described herein comprise desalting the sample to be tested by extraction and/or chromatography.

In certain embodiments, the methods of detecting active RIP-II in a sample described herein comprise desalting the sample to be tested using size exclusion chromatography.

In certain embodiments, the methods of detecting active RIP-II in a sample described herein comprise desalting the sample to be tested with a Zeba spin desalting spin column.

In another aspect, the present application also provides a kit comprising: oligonucleotide substrate capable of providing NH ₄ ⁺ An ammonium salt of (a), a metal ion chelating agent, and a pH adjusting agent.

In certain embodiments, in the kits described herein, the oligonucleotide substrate is an oligonucleotide comprising a stem-loop structure, and the sequence of the stem-loop structure comprises 5 '-r (GdAGA) -3'.

In certain embodiments, in the kits described herein, the oligonucleotide substrate is of the general formula 5' -r ((GC) _n GdAGA(GC) _n ) -3 'or of the general formula 5' -r (C (GC) _n GdAGA(GC) _n G) -3', wherein n is1, 2, 3, 4, 5, 6,7, 8, 9 or 10, for example n is 2 or 3.

In certain embodiments, in the kits described herein, the oligonucleotide substrate is a nucleic acid sequence selected from SEQ ID NOs: 5-7.

In certain embodiments, in the kits described herein, the oligonucleotide substrate is SEQ ID NO: 5 under the condition of high-efficiency DNA polymerase chain reaction.

In certain embodiments, in the kits described herein, the ammonium salt is an ammonium citrate salt, preferably the ammonium salt is Diammonium Hydrogen Citrate (DHC).

In certain embodiments, the DHC is not less than 98% pure, and more preferably is not less than 99% pure in the kits described herein.

In certain embodiments, in the kits described herein, the metal ion chelating agent is ethylenediaminetetraacetic acid (EDTA).

In certain embodiments, in the kits described herein, the pH adjusting agent is Formic Acid (FA), Citric Acid (CA), or trifluoroacetic acid (TFA), preferably FA.

In certain embodiments, the kits described herein further comprise aqueous ammonia.

In certain embodiments, the kit described herein, the concentration of ammonia is about 5 to 28 wt%, e.g., about 12 to 14 wt%.

In certain embodiments, the kits described herein further comprise 3-hydroxy-2-pyridinecarboxylic acid (3-HPA).

In certain embodiments, the kit described herein provides 3-HPA in a purity of no less than 98%, and more preferably at a concentration of no less than 99%.

In certain embodiments, the kits described herein further comprise Acetonitrile (ACN).

In certain embodiments, the kits described herein further comprise a desalting chromatography column, such as a size exclusion chromatography column, for example a Zeba spin desalting centrifugation column.

In certain embodiments, the kits described herein further comprise instructions.

In certain embodiments, the specification recites at least the detection method described in any one of the embodiments of the present application.

In another aspect, the application also provides the use of the kit for detecting (including qualitatively and/or quantitatively detecting) active RIP-II, or

Use in the detection (including qualitative and/or quantitative detection) of active RIP-II using MALDI-MS.

In another aspect, the present application also provides the use of an oligonucleotide substrate as described herein for detecting (including qualitatively and/or quantitatively detecting) active RIP-II or for detecting (including qualitatively and/or quantitatively detecting) active RIP-II using MALDI-MS.

Description and explanation of related terms in this application

In the present application, unless otherwise indicated, scientific and technical terms used herein have the meanings that are commonly understood by those of skill in the art.

When the terms "for example," "such as," "including," "containing," or variants thereof are used herein, these terms are not to be considered limiting terms, but rather are to be construed to mean "without limitation" or "without limitation".

The terms "a" and "an" and "the" and similar referents in the context of describing the application (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

As used herein, the term "RIP-II" refers to type II Ribosome-Inactivating Protein (RIP), which is formed by the linkage of two chains (A-and B-chains) via a disulfide bond. RIP-II is RNA N-glycosidase, has N-glycosidase activity, and can hydrolyze adenine at specific site on stem-loop structure of biological ribosome large subunit rRNA, so that ribosome can lose protein synthesis function. Non-limiting examples of RIP-II include, but are not limited to, ricin, RCA120, abrin, AAG, and the like. It can be derived from a biological sample, e.g., a plant (castor, acacia, cinnamomum camphora, balanophora, elderberry, mistletoe, etc.), an animal, a fungus, a prokaryote (e.g., escherichia coli expressing RIP-II), a virus, etc.; but also from non-biological samples such as protein libraries, synthetic products, etc.

As used herein, the term "N-glycosidase" refers to an enzyme that cleaves an N-glycosidic bond to a specific base site in a DNA or RNA species, forming an apurinic or apyrimidinic site.

As used herein, the term "N-glycosidic linkage" refers to a chemical linkage that links the non-sugar moiety (i.e., aglycon) of the glycoside molecule to the sugar moiety, or sugar moiety to a sugar moiety, by nitrogen atom, and the linkage of a substance containing glycosidic linkages is referred to as a glycoside. For example, a chemical bond is formed between a base and a ribose in an oligonucleotide.

As used herein, the term "active RIP-II" refers to a RIP-II with an intact active structure that can cause toxicity in an organism. For example, the term "active ricin", the forbidden chemical weapon tissue (OPCW) is given the following definition: all forms of ricin "derived from ricin (Ricinus communis L.), including any possible variation of the molecular structure, whether naturally occurring or artificially engineered, should be considered ricin, so long as they conform to the basic 'native' double-stranded molecular structure of ricin (a-S-B), as well as the toxic structure for mammals. Once the disulfide linkage (-S-S-) between the A and B chains is broken or the protein is denatured, it is no longer ricin. "(OPCW Scientific advertisement Board. Twenty-First Session Ricin fat Sheet [ EB/OL ])

As used herein, the term "oligonucleotide" generally refers to a polynucleotide fragment in which 2 to 30 nucleotide residues are ester-linked, and which can exist stably in a linear state or stably in a form of a specific secondary structure (e.g., hairpin structure, stem-loop structure). One or more modifications may be present in the nucleobase, sugar moiety in a single nucleotide at one or more positions in the oligonucleotide. The internucleoside linkages of the oligonucleotides of the present application may be phosphodiester linkages, or alternatively, one or more of the internucleoside linkages may comprise modified linkages, such as, but not limited to: phosphorothioate, phosphorodithioate, phosphoroselenoate (phosphoroselenate), or phosphorodiselenoate (phosphorodiselelenate) linkages, which are resistant to some nucleases.

As used herein, the term "hybridize" refers to a non-single type of ribose sugar that is the ribose moiety of the nucleotide units that make up the oligonucleotide substrate. For example, "r (NNNN)dNNN) "means a hybrid oligonucleotide formed by 7 nucleotide units linked by an ester bond, in which the nucleotide at position 5 is a deoxyribonucleotide and the nucleotides at the remaining positions are ribonucleotides.

As used herein, the term "analytical test drill" refers to the evaluation activity of OPCW in its ability to analytically test one of the biotoxin analytical test techniques conducted in the designated laboratory.

As used herein, the term "about," when used to modify a value or range of values, means that the value or range of values and the range of errors acceptable to one of skill in the art for that value or range of values, for example, the range of errors is ± 10%, ± 5%, ± 4%, ± 3%, ± 2%, ± 1%, ± 0.5%, etc. Unless otherwise apparent from the context, all numbers provided herein are modified by the term "about".

As used herein, the term "ultrapure water" refers to water having a resistivity of up to 18M Ω cm (25 ℃).

Advantageous effects of the present application

The method for detecting the active RIP-II has the following beneficial technical effects:

(1) the sensitivity is high, the LOD is about 1ng/mL, and the lowest LOD can reach about 0.01 ng/mL;

(2) the detection is rapid, and the deadenination (A) reaction of the oligonucleotide substrate can be completed within about 3-60min (for example, about 8-35min, about 10-30 min);

(3) the quantification is accurate, and the linear range of the detection is about 1-5000 ng/mL;

(4) the detection conditions are mild, and detection can be carried out under temperature conditions of about 4-55 ℃ (e.g., about 25-55 ℃, e.g., about 35-40 ℃, e.g., about 37 ℃);

(5) the method can quickly detect the active toxin and can be applied to the detection of the active RIP-II in the actual biological sample;

the hybrid oligonucleotide substrate obtained by screening of the present application, for example, S5 (nucleotide sequence shown in SEQ ID NO: 5), has high reactivity with ricin, and can be applied to the detection of various active RIP-II.

The buffer solution provided by the application can rapidly exert the N-glycosidase activity of ricin.

The MALDI matrix provided by the application can realize high-sensitivity and accurate detection of oligonucleotide substrates. After the MALDI matrix is mixed with a solution to be detected, a white uniform crystal with darker color can be formed, and the peak intensity of a MALDI-MS spectrum is obviously improved.

Embodiments of the present application will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only for illustrating the present application and do not limit the scope of the present application. Various objects and advantageous aspects of the present application will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiments.

Drawings

FIG. 1 is a schematic flow chart of the method for detecting active RIP-II, which comprises the steps of desalting pretreatment of a sample to be detected, mixed incubation with an oligonucleotide substrate, MALDI-MS detection and the like.

FIG. 2 shows the spectrum peak morphology and intensity of MALDI-MS detection after reacting three substrates S1, S2 and S5 with ricin for different times. The results show that S1 requires more than 240min to react with ricin to hydrolyze less than 30% of the substrate, S2 requires 30min to almost completely convert to depurination product, S5 is more susceptible to hydrolysis by ricin, and only 3min, about 50% of S5 has been converted to its hydrolysis product.

Figure 3 shows two products resulting from the reaction of S5 with ricin: m-136[ M-adenine-H] ^- And M-118[ M-adenine + H ₂ O-H] ^- With unreacted substrate M-1[ M-H ] under corresponding conditions] ^- Peak area ratio of (a). The results show that the main product molecular ion of the reaction of S5 and ricin is [ M-118 ]] ^- The product molecular ion is [ M-136 ] with time and temperature increase] ^- The ratio rises.

FIG. 4 shows the peak area ratios (Prod/sub values) of the product to unreacted substrate for different times of reaction of seven substrates S1, S2, S3, S4, S5, S6 and S7 with ricin. The results show that the reactivity of S5, S6 and S7 is superior to that of S1, S2, S3 and S4 in the enzymatic reaction system in which ricin exerts N-glycosidase activity. In the substrate, the order of the reactivity was S5> S6> S7 in descending order, and the fourth name was S3. Among all substrates, S5 showed the highest sensitivity, the fastest reaction rate, and had good stability without spontaneous depurination.

FIG. 5 shows the Prod/sub values (A) and the peak morphologies and intensities (B) of the MALDI matrices prepared with reagents of different purities, after MALDI-MS detection. The result shows that the high-purity matrix 3-HPA is beneficial to improving the ionization efficiency of the substrate detected by MALDI-MS and reducing the interference degree of the substrate by alkali metal ions in the MALDI-MS; the high-purity eutectic reagent DHC is beneficial to improving Prod/sub and promoting ricin to exert N-glycosidase activity.

FIG. 6 shows the effect of adding ammonia water on the crystallization of a MALDI matrix at the time of termination of the reaction, wherein A is the crystal morphology formed by spotting the sample directly on the MALDI target plate without adding ammonia water at the time of termination of the reaction, and B is the matrix crystal morphology formed by spotting the sample on the MALDI target plate with adding ammonia water at the time of termination of the reaction. The results show that after the ammonia water sample and the MALDI matrix are mixed in the same volume, the pH value of the mixed solution is between 4 and 5, and crystals with smoother and more smooth surfaces can be formed.

FIG. 7 shows the optimization results of the reaction buffer system composition and mixture ratio of ricin and substrate, wherein A shows the detected Prod/sub value of MALDI-MS after reaction with different regulation systems (formic acid, FA or trifluoroacetic acid, TFA); b1 and B2 and C1 and C2 respectively show the Prod/sub value and the product peak intensity detected by MALDI-MS after the reaction is carried out under different Diammonium Hydrogen Citrate (DHC) contents (the concentration of EDTA is 0.3g/L) and different ethylenediaminetetraacetic acid (EDTA) contents (the concentration of DHC is1 g/L).

Fig. 8 shows the results of a linear fit of the ratio of the peak area of S5 to the peak area of the internal standard IS10b to the initial concentration of substrate S5. The results show that S5 is linear well at a final concentration of 1-60. mu.M in the reaction system, R ² 0.9926; the linearity is better when the final concentration is 1-30 mu M, R ² Is 0.9946.

FIG. 9 shows the results of curve fitting of the reaction rate and input substrate concentration by the Michaelis-Menten equation.

FIG. 10 shows Log ₁₀ [ ricin concentration]And Log ₁₀ [Prod/sub]The linear fitting result of (2). The results show that the ricin concentration is good in linearity within the range of 1-5000ng/mL, and R is obtained in 10min of reaction ² 0.9780; r when the reaction time is 30min ² 0.9800, and a limit of detection (LOD) of 1 ng/mL.

FIG. 11 shows the spectral peak morphology and intensity of MALDI-MS detection after ricin has reacted with different concentrations of substrate for 10min (A) and 30min (B). The results show that ricin is not linear below LOD due to the catalytic nature of the enzyme in the reaction system, but is still detectable, with a minimum detectable level of 0.01 ng/mL. For samples with lower active toxin content, the detection rate can be improved by prolonging the reaction time.

FIG. 12 shows the Prod/sub values (A) and the peak morphologies and intensities (B) of MALDI-MS when different RIP-II (ricin, abrin, AAG, RCA120) activities were measured.

FIG. 13 shows the peak morphology and intensity of MALDI-MS when detecting ricin activity before and after acid-base washout.

FIG. 14 shows the Prod/sub values (A) and the peak morphologies and intensities (B) of MALDI-MS measured for ricin activity before and after treatment with different solvents, different temperatures (55 ℃, 80 ℃, 100 ℃) or sterilization conditions.

FIG. 15 shows the spectrum peak morphology and intensity of MALDI-MS when the sample to be tested is subjected to activity detection after being diluted by different times.

Sequence information

Information on the sequences to which the present application relates is provided in table 1 below.

TABLE 1

SEQ ID NO:	Name (R)	Nucleotide sequence
			1	S1	d(GCGCGAGAGCGC)
2	S2	r(GCGCGAGAGCGC)
			3	S3	r(CGCGCGAGAGCGCG)
4	S4	r(GCGCGCGAGAGCGCGC)
			5	S5	r(GCGCGdAGAGCGC)
6	S6	r(CGCGCGdAGAGCGCG)
			7	S7	r(GCGCGCGdAGAGCGCGC)

Detailed Description

The present application will now be described with reference to the following examples, which are intended to illustrate, but not limit, the present application.

The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are conventional products which are commercially available, and are not indicated by manufacturers. The examples are described by way of example and are not intended to limit the scope of the claims to this application, as those skilled in the art will recognize.

Instruments and reagents

The apparatus and reagents used in the exemplary embodiments of the present application include:

ricin, purity > 95% (electrophoretically pure); abrin, AAG, purity > 95% (electrophoresis pure), preparation methods can be found in: preparation of high-grade ricin and abrin standard substances and quantitative analysis and rapid detection technology thereof [ D ] military medical science institute of liberty military, 2014, national people.

RCA120 was purchased from Sigma-Aldrich, USA;

the oligonucleotides listed in Table 1, all synthesized by Olympic Biotech, Beijing, and purified by HPLC;

3-HPA (98% and 99% pure), DHC (99% pure), EDTA (99.995% pure) was purchased from Sigma-Aldrich, USA;

TFA (purity 98%), ammonia (concentration 25-28 wt%, diluted as required) and FA (LC-MS grade) were purchased from siemer heschel technologies (shanghai);

acetonitrile (ACN) was chromatographically pure and purchased from DUKSAN corporation (korea);

a multiparameter tester (SevenExcellence) available from mettler-toledo instruments (shanghai) ltd;

an Autoflex III Smartbeam MALDI TOF-TOF mass spectrometer (containing FlexControl acquisition software, Flexanalysis data processing software, anchichip 400/384 target plate) was purchased from Bruker Daltonik (germany);

MALDI matrix, 3-HPA 50mg, ultrapure water 730 μ L, ACN 250 μ L, 20 μ L DHC water solution with concentration of 50g/L (DHC dissolved in ultrapure water), and mixing well to obtain the final product;

ultrapure water (18.2M Ω. cm), Milli-Q A10 purification system from Millipore corporation (MA, USA);

zeba spin desalting column (cut-off molecular weight 7000Da) from Saimer Feishel scientific Co., USA;

hydrochloric acid and sodium hydroxide, both available from Beijing national pharmaceutical Chemicals, Inc.

Both AS1901 and BT1904 were samples provided by OPCW in the international analytical testing drill, with AS1901 being ricin positive and BT1904 being unknown.

In the following examples, the oligonucleotides, EDTA, DHC and biotoxin listed in Table 1 were dissolved in ultrapure water to the desired concentrations.

Example 1: comparison of reactivity of three 12nt oligonucleotides with ricin

In an in vitro reaction system, the reaction activities of ricin and three 12nt substrates of S1, S2 and S5 are compared by selecting a higher reaction temperature (for example, 55 ℃). The substrate sequence and molecular weight information are shown in Table 2.

S1, S2 and S5 are respectively dissolved to 100 mu M by ultrapure water; EDTA and DHC are weighed and dissolved in ultrapure water respectively to obtain 0.5g/L EDTA and 50g/L DHC.

mu.L of 100. mu.M substrate (S1, S2 or S5), 30. mu.L of EDTA 0.5g/L, 1. mu.L of DHC 50g/L, were mixed well, the pH was adjusted to about 4.2 with FA, 1. mu.L of 25. mu.g/mL ricin aqueous solution was added, and finally ultrapure water was added to a final volume of 50. mu.L. Reacting S1 at 55 ℃ for 1, 60, 180 and 240min respectively; s2, reacting for 1, 10, 30 and 60min respectively; s5 is reacted for 1, 3, 5 and 10min respectively. After the reaction, the temperature was lowered and 1. mu.L of 12 to 14 wt% aqueous ammonia was added to terminate the reaction. Taking 5 mu L of the terminated sample, taking 5 mu L of MALDI matrix, mixing uniformly, taking 1 mu L of sample to be spotted on an AnchorChip400/384 target plate, naturally drying and then carrying out MALDI-MS detection.

TABLE 2 substrate sequences and molecular weights

This embodiment contrasts the influence of the positive and negative ion reflection mode on the detection effect. The detection mode is set to be a positive ion reflection mode or a negative ion reflection mode, the total laser intensity is 95%, and the cumulative irradiation times are 1000 times. The difference of the detection effect of S1 in the positive and negative ion reflection mode is shown in Table 3, and the result shows that the MALDI-MS detection effect in the negative ion reflection mode is better. Therefore, the present application selects the negative ion reflection mode when MALDI-MS detection is performed.

TABLE 3 Positive and negative ion reflectance mode comparison

Under the condition that the total laser intensity is 95% in the negative ion reflection mode and the cumulative irradiation frequency is 1000 times, the spectrum peak shape and intensity detected by MALDI-MS of each reaction system are shown in figure 2, and the result shows that S1 needs to react with ricin for more than 240min to hydrolyze less than 30% of the substrate, and S2 needs 30min to almost completely convert into depurination products. In contrast, S5 was more susceptible to hydrolysis by ricin, only 3min, with about 50% of S5 having been converted to its hydrolysate. The product substrate ratios of the three substrates at the same time and concentration are in the relation of S5> S2> S1, which shows that S5 has the strongest reactivity, S2 is the next and S1 is the weakest.

Example 2: comparison of two different products after reaction of oligonucleotide substrate with Ricin

mu.L of 100. mu.M substrate S515. mu.L, 0.5g/L EDTA 30. mu.L, 50g/L DHC 1. mu.L were mixed well, adjusted to pH 4.2 with FA, 1. mu.L of 25. mu.g/mL ricin was added, and finally ultrapure water was added to a final volume of 50. mu.L. The reaction is carried out at 55 ℃ for 10, 20, 25, 30, 40, 50 and 60min respectively. After the reaction, the temperature was lowered and 1. mu.L of 12 to 14 wt% aqueous ammonia was added to terminate the reaction. Taking 5 mu L of the terminated sample, taking 5 mu L of MALDI matrix, mixing uniformly, taking 1 mu L of sample to be spotted on an AnchorChip400/384 target plate, naturally drying, and then carrying out MALDI-MS detection, wherein the detection mode is a negative ion reflection mode, the total laser intensity is 95%, and the cumulative irradiation frequency is 2000 times.

S5 reacts with ricin to produce two products: the molecular ion is [ M-136 ]] ^- (i.e., [ M-adenine-H ]] ^- ) And [ M-118 ]] ^- (i.e., [ M-adenine + H) ₂ O-H] ^- ) Respectively comparing the peak areas of the two products with the unreacted substrate M-1[ M-H ] under corresponding conditions] ^- The peak area ratios are shown in FIG. 3. The molecular ion of the main product of the reaction of S5 and ricin is shown as [ M-118 ]] ^- The product molecular ion is [ M-136 ] with time and temperature increase] ^- The ratio rises.

Example 3: comparison of reactivity of seven oligonucleotides with Ricin

Taking 15 mu L of seven substrates (shown in a sequence table 2) of S1, S2, S5, S3, S6, S4 and S7, wherein the concentrations of the substrates are all 100 mu M, taking 0.5g/L EDTA 30 mu L and 50g/L DHC 1 mu L, uniformly mixing the substrates, adjusting the pH to 4.2 by using FA, adding 1 mu L of 25 mu g/mL ricin, and finally adding ultrapure water to the final volume of 50 mu L. Setting reaction time according to the speed of enzymolysis, wherein the reaction system with the substrates S1, S2, S3, S6, S4 and S7 is reacted at 55 ℃ for 10, 30, 60 and 120min, and the reaction system with the substrate S5 is reacted at 55 ℃ for 1, 3, 5 and 10 min. In addition, the enzymolysis speed of the S5 substrate is too high under the temperature condition of 55 ℃, and the reaction system with the substrate of S5 is used for reactions at 37 ℃ (the physiological temperature of in vivo enzyme digestion) for 10, 30, 60, 100 and 120 min. After the reaction, the temperature was lowered and 1. mu.L of 12 to 14 wt% aqueous ammonia was added to terminate the reaction. Taking 5 mu L of sample after the reaction is ended, taking 5 mu L of MALDI matrix, uniformly mixing the two, taking 1 mu L of sample to be spotted on an AnchorChip400/384 target plate, naturally drying, and then carrying out MALDI-MS detection, wherein the detection mode is a negative ion reflection mode, the total laser intensity is 95%, and the cumulative irradiation frequency is 2000 times.

The peak area ratios (Prod/sub) of the product of the reaction of seven substrates S1, S2, S5, S3, S6, S4 and S7 with ricin over different periods of time to the unreacted substrate are shown in FIG. 4. As can be seen from FIG. 4, in the enzymatic reaction system in which ricin exerts N-glycosidase activity, S5, S6, and S7 all had better reactivity than S1, S2, S3, and S4. In the substrate, the order of the reactivity was S5> S6> S7 in descending order, and the fourth name was S3. Among all substrates, S5 showed the highest sensitivity, the fastest reaction rate, and had good stability without spontaneous depurination. Compared with S3, the reactivity of S5 at 37 ℃ is still better than that of S3 at 55 ℃. Since the reaction of S5 is violent at 55 ℃, the reaction process is not easy to control in subsequent experiments, and the higher temperature can cause the M-136 product proportion to slightly rise, which interferes with the monitoring of the M-118 product, the subsequent reaction temperature is preferably 37 ℃.

Example 4: effect of reagent purity on oligonucleotide detection in MALDI-MS

100 mu M S515 mu L, 0.5g/L EDTA 30 mu L, 50g/L DHC 1 mu L are mixed well, the pH is adjusted to 4.2 with FA, 1 mu L ricin of 25 mu g/mL is added, and finally ultrapure water is added to a final volume of 50 mu L. The reaction was carried out at 37 ℃ for 30 min. After the reaction, the temperature was lowered and 1. mu.L of 12 to 14 wt% aqueous ammonia was added to terminate the reaction. Taking 5 mu L of a sample after termination, taking 5 mu L of four MALDI matrixes prepared by reagents with different purities, respectively uniformly mixing the four MALDI matrixes with the sample after termination of the reaction, then taking 1 mu L of the mixture to be spotted on an AnchorChip400/384 target plate, naturally drying the mixture, and then carrying out MALDI-MS detection, wherein the detection mode is a negative ion reflection mode, the total laser intensity is 95%, and the cumulative irradiation frequency is 2000 times.

The four MALDI matrixes prepared by different purity reagents are prepared by combining two matrixes, namely 3-HPA with the purity of 98% or 99% and DHC with the purity of 98% or 99%, respectively, according to the method.

As a result, as shown in FIG. 5, FIG. 5 shows the Prod/sub values (A in FIG. 5) and the peak morphologies and intensities (B in FIG. 5) of MALDI matrices prepared with reagents of different purities, which were subjected to MALDI-MS detection. The result shows that the high-purity matrix 3-HPA is beneficial to improving the ionization efficiency of the MALDI-MS for detecting the substrate and reducing the interference degree of the substrate in the MALDI-MS by alkali metal ions; the high-purity eutectic reagent DHC is beneficial to improving Prod/sub and promoting ricin to exert N-glycosidase activity.

Example 5: effect of Ammonia on the Crystal morphology of MALDI matrix

100 μ M S515 μ L, 0.5g/L EDTA 30 μ L, 50g/L DHC 1 μ L, mixed well, adjusted pH to 4.2 with FA, added 25 μ g/mL ricin 1 μ L, and finally added ultrapure water to a final volume of 50 μ L. The reaction was carried out at 37 ℃ for 30 min. After the reaction, the temperature is directly reduced without adding ammonia water to terminate the reaction or reduced, and 12 to 14 weight percent of ammonia water is added for 1 mu L. After the reaction without or with ammonia water was terminated, 5. mu.L of the sample was mixed with 5. mu.L of MALDI matrix. Taking 1 mu L of sample to be spotted on an AnchorChip400/384 target plate, naturally drying, and then carrying out MALDI-MS detection, wherein the detection mode is a negative ion reflection mode, the total laser intensity is 95%, and the cumulative irradiation times are 2000 times.

The MALDI matrix crystal form is shown in FIG. 6, and the results show that the matrix crystal form formed by spotting the mixture on the MALDI target plate without adding ammonia water at the time of termination reaction (A in FIG. 6) is greatly different from the matrix crystal form formed by spotting the mixture on the MALDI target plate with adding ammonia water at the time of termination reaction (B in FIG. 6). The pH value of the sample after adding the ammonia water is about 9, the pH value is between 4 and 5 after being mixed with the MALDI matrix in the same volume, the pH value of the sample without adding the ammonia water is about 4.2, and the pH value is about 3 after being mixed with the MALDI matrix in the same volume, which shows that the pH value has important influence on the crystal form of the MALDI-MS matrix. After the sample added with ammonia water and the MALDI matrix are mixed in equal volume, the pH value of the mixed solution is between 4 and 5, and crystals with smoother and more smooth surfaces can be formed.

Example 6: reaction buffer system composition and proportioning optimization of ricin and oligonucleotide substrate

pH is a key factor in extracellular enzymatic reactions because the deadenination of ricin with oligonucleotide substrates can only be accomplished under acidic conditions when oligonucleotides are used in vitro instead of ribosomes, and is ineffective at neutral physiological pH. In addition, the kind of acid directly affects the reaction progress. Based on this, in this example, the composition and ratio of the reaction buffer system were optimized, and the detection effects under different DHC and EDTA contents, different pH values, and different adjustment systems (FA or TFA) were investigated. The method comprises the following steps:

taking 100 mu M S510 mu L, respectively taking 0.5g/

L EDTA

5, 10, 20, 30, 37.5 mu L, respectively taking 50g/L DHC 0.2, 0.5, 1,2, 5 mu L, mixing uniformly, adjusting pH to 3.8, 4.0, 4.2, 4.3, 4.4, 4.7, 4.9 with FA or TFA, adding 25 mu g/mL ricin 1 mu L, and finally adding ultrapure water to a final volume of 50 mu L. The reaction was carried out at 37 ℃ for 30 min. After the reaction, the temperature was lowered and 1. mu.L of 12 to 14 wt% aqueous ammonia was added to terminate the reaction. Taking 5 mu L of the terminated sample, taking 5 mu L of MALDI matrix, mixing uniformly, taking 1 mu L of sample to be spotted on an AnchorChip400/384 target plate, naturally drying, and then carrying out MALDI-MS detection, wherein the detection mode is a negative ion reflection mode, the total laser intensity is 95%, and the cumulative irradiation frequency is 2000 times.

The results are shown in FIG. 7, where: a shows the Prod/sub values detected by MALDI-MS after the reaction with different adjusting systems (FA or TFA) when the final concentration of EDTA is 0.3g/L and the final concentration of DHC is1 g/L; b1 and B2 show the Prod/sub value detected by MALDI-MS after reaction under different DHC content (the final concentration of EDTA is 0.3g/L) and the product after substrate deadenination (M-118[ M-adenine + H) ₂ O-H] ^- ) The signal strength of (a); c1 and C2 respectively show the Prod/sub value detected by MALDI-MS after reaction under different EDTA content (the final concentration of DHC is 1g/L) and the product after substrate deadenination (M-118[ M-adenine + H) ₂ O-H] ^- ) The signal strength of (c).

As can be seen from A in FIG. 7, using the most acid-TFA to adjust the pH of the oligonucleotide substrate buffer for polypeptide ionization in MALDI-MS, FA provided better response intensity than FA with reduced activity, with the optimal pH between 4.2-4.4, and most preferably pH 4.2.

As can be seen from B1 and B2 in FIG. 7, the reactivity of the substrate is low at the final concentrations of DHC of 0.2g/L and 0.5g/L, and the reactivity of the substrate is high at the final concentrations of 1,2 and 5 g/L; the final concentration of DHC is 5g/L, the signal intensity of the product after the adenine-removing reaction is strongest, and the final concentration of DHC is 0.2, 0.5, 1 and 2g/L, the signal intensity of the product is not greatly different. Thus, the final concentration of DHC in the reaction system can be from 0.2 to 5g/L, e.g., from 0.5 to 5g/L, from 0.8 to 2g/L, from 0.8 to 1.5g/L, 1g/L, 2g/L, 3 g/L.

As can be seen from C1 and C2 in FIG. 7, the reactivity of the substrate was relatively high at the final concentration of EDTA of 0.2, 0.3, 0.375g/L in the reaction system, while the signal intensity of the product was low at the final concentration of 0.375 g/L. Thus, the final concentration of EDTA in the reaction system may be 0.05 to 0.375g/L, for example, 0.05 to 0.3g/L, 0.1 to 0.35g/L, 0.15 to 0.3g/L, 0.25 to 0.35g/L, 0.3 g/L.

Example 7: linear investigation of Signal response in MALDI-MS of different concentrations of oligonucleotides

200. mu.M of S50, 1.25, 2.5, 5, 7.5, 10, 12.5 and 15. mu.L were taken, 250. mu.M of an internal standard substance IS10b (r (GCGCGCGGGCGC)) 1. mu.L was taken, 0.5g/L of EDTA 30. mu.L and 50g/L of DHC 1. mu.L were taken, the mixture was mixed well, the pH was adjusted to 4.2 with FA, ultrapure water was added to a final volume of 50. mu.L, and then 1. mu.L of 12-14 wt% aqueous ammonia was added to obtain a sample. Taking 5 mu L of the terminated sample and 5 mu L of MALDI matrix, mixing the two uniformly, then taking 1 mu L of sample to be spotted on an AnchorChip400/384 target plate, naturally drying the sample, and then carrying out MALDI-MS detection, wherein the detection mode is a negative ion reflection mode, the total laser intensity is 95%, and the cumulative irradiation frequency is 2000 times.

The linearity was examined by plotting the ratio of the peak area of S5 to the peak area of the internal standard IS10b on the ordinate and the initial concentration of S5 on the abscissa.

The results are shown in FIG. 8, and indicate that the final concentration of S5 in the reaction system is1 to 60. mu.LGood linearity at M, R ² 0.9926; the linearity is better when the final concentration is 1-30 mu M, R ² Is 0.9946.

Example 8: determination of enzyme kinetic parameters of hybrid oligonucleotide substrate (S5\ S6\ S7) and ricin

Enzyme kinetic studies of ricin with hybrid oligonucleotide substrates (S5, S6, S7, sequences shown in table 2) set ricin concentration to 0.16% of the lowest concentration of hybrid oligonucleotide substrate. Taking 30 mu L of EDTA (0.5g/L), 1 mu L of DHC (50g/L), 0.5 mu g/mL of ricin and 1.25-17 mu L of hybrid oligonucleotide substrate (200 mu M), adding ultrapure water to make up to 50 mu L, and uniformly mixing. The reaction times of S5, S6 and S7 are respectively 10, 20 and 90 min. The reaction temperature was 37 ℃. After the reaction, the temperature is reduced, 1 mu L of 12-14 wt% ammonia water is added to terminate the reaction, the terminated sample is mixed with an equal volume MALDI matrix, 1 mu L of the mixture is spotted on an Anchorchip400/384 target plate, MALDI-MS detection is carried out in a negative ion reflection mode, the total laser intensity is 95%, and the cumulative irradiation is carried out for 2000 times. According to the formula I, the average reaction rate V of the substrate with each concentration can be calculated according to Prod/sub, the amount of the substrate put into reaction and the reaction time:

taking V as the y axis and the input substrate concentration as the x axis, curve fitting can be carried out by the Michaelis-Menten equation, and then relevant parameters of enzyme kinetics, including the maximum reaction rate (V) _max ) Mie constant (K) _m ) Catalytic constant (k) _cat ) And catalytic efficiency (k) _cat /K _m ). The fitted curve is shown in FIG. 9, and the enzyme kinetics-related parameters are shown in Table 4.

TABLE 4 enzyme kinetics-related parameters

From the results in table 4 and fig. 9, it can be seen that:

i) k of S5, S6, S7 _m The value is 5.5-7.5μ M, indicating a similar affinity between the hybrid oligonucleotide substrate and ricin;

ii) V of S5 _max About 2 times S6 and 7 times S7;

iii) k of S5 _cat /K _m Very high, 1.61X 10 ⁵ M ^-1 .s ^-1 Approximately 1.5-fold over S6 and 6-fold over S7, indicating that the catalytic efficiency of ricin towards the hybrid oligonucleotide substrate decreases progressively with increasing substrate length.

Example 9: linear range and LOD of the detection method of the present application

Taking 100 mu M S515 mu L, taking 0.5g/L EDTA 30 mu L, taking 50g/L DHC 1 mu L, mixing uniformly, adjusting pH to 4.2 with FA, adding 1 mu L ricin to make the

final concentration

1,2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10000ng/mL, and finally adding ultrapure water to make the final volume 50 mu L. The reaction was carried out at 37 ℃ for 10min or 30min, respectively. After the reaction, the temperature was lowered and 1. mu.L of 12 to 14 wt% aqueous ammonia was added to terminate the reaction. Taking 5 mu L of terminated sample, taking 5 mu L of MALDI matrix, mixing uniformly, taking 1 mu L of sample to be spotted on an AnchorChip400/384 target plate, naturally drying, and carrying out MALDI-MS detection, wherein the detection mode is a negative ion reflection mode, the total laser intensity is 95%, and the cumulative irradiation times is 2000 times.

By Log ₁₀ [ ricin concentration]As abscissa, in Log ₁₀ [Prod/sub]And (4) taking a scatter diagram for the abscissa, performing linear fitting on the diagram, and judging the detection linear range. The fitting results are shown in FIG. 10, and the results show that ricin concentrations in the range of 1-5000ng/mL are linear, and R is good ² 0.9780 (10 min reaction); r ² 0.9800 (at 30min reaction), and an LOD of 1ng/mL was determined. The slope of the curve is similar, the linear range and the LOD are also the same for different reaction times. Indicating that the reaction rate is proportional to the reaction time over the selected time range.

FIG. 11 shows the spectral peak morphology and intensity of MALDI-MS detection after ricin has reacted with substrate for 10min (A) and 30min (B). The results in FIG. 11 show that ricin is detectable at a minimum of 0.01ng/mL, although not linear below LOD, due to the catalytic nature of the enzyme in the reaction system.

Example 10: selectivity of the detection method in detection of different RIP-II activities

Abrin, AAG, and RCA120 were dissolved in ultrapure water, respectively.

100 mu M S515 mu L, 0.5g/L EDTA 30 mu L, 50g/L DHC 1 mu L are taken, mixed evenly, pH is adjusted to 4.2 by FA, 25 mu g/mL ricin, abrin, RCA120 and AAG 1 mu L are respectively added, and finally ultrapure water is added until the final volume is 50 mu L. The reaction was carried out at 37 ℃ for 30 min. After the reaction, the temperature was lowered and 1. mu.L of 12 to 14 wt% aqueous ammonia was added to terminate the reaction. Taking 5 mu L of the terminated sample, taking 5 mu L of MALDI matrix, mixing uniformly, taking 1 mu L of sample to be spotted on an AnchorChip400/384 target plate, naturally drying, and then carrying out MALDI-MS detection, wherein the detection mode is a negative ion reflection mode, the total laser intensity is 95%, and the cumulative irradiation frequency is 2000 times.

The results are shown in fig. 12, and fig. 12 shows the Prod/sub value (a) and the peak morphology and intensity (B) of MALDI-MS when different RIP-II activities are detected, and the results show that the method has the highest activity detection sensitivity for ricin and has significant selectivity for ricin, RCA120, abrin and AAG, and the second is that the detection sensitivity for RCA120, abrin and AAG is low.

Example 11: evaluation of ricinoleic acid alkali decontamination effect

The U.S. CDC reports that ricin can still maintain certain stability in a high-temperature environment of 80 ℃, and RIP-II represented by ricin has a serious practical threat due to high toxicity. Therefore, how to quickly and effectively inactivate and decontaminate the toxin infected by the environment and eliminate potential harm has important significance.

Selecting 1g/L ricin, treating with 0.1M hydrochloric acid solution or 0.1M sodium hydroxide solution (both final concentrations) at room temperature for 30min or 60min, respectively, and diluting the treated ricin with ultrapure water to 25 μ g/mL.

100 mu M S515 mu L, 0.5g/L EDTA 30 mu L, 50g/L DHC 1 mu L are mixed evenly, the pH is adjusted to 4.2 by FA, 25 mu g/mL ricin (after decontamination) 1 mu L is added respectively, and finally ultrapure water is added until the final volume is 50 mu L. The reaction was carried out at 37 ℃ for 30 min. After the reaction, the temperature was lowered and 1. mu.L of 12 to 14 wt% aqueous ammonia was added to terminate the reaction. Taking 5 mu L of the terminated sample, taking 5 mu L of MALDI matrix, mixing uniformly, taking 1 mu L of sample to be spotted on an AnchorChip400/384 target plate, naturally drying, and then carrying out MALDI-MS detection, wherein the detection mode is a negative ion reflection mode, the total laser intensity is 95%, and the cumulative irradiation frequency is 2000 times.

The results are shown in FIG. 13, and FIG. 13 shows the spectrum peak shape and intensity of MALDI-MS when ricin is active before and after acid and alkali decontamination, and the results show that ricin can maintain a certain activity after being treated in 0.1M hydrochloric acid solution for 30min, and is almost completely inactivated after being treated in 0.1M sodium hydroxide for 30 min. Therefore, the ricin has better washing effect in alkaline environment.

Example 12: evaluation of decontamination effect of ricin under high temperature and sterilization conditions

Ricin at a concentration of 1g/L was subjected to thermal stability studies in aqueous solution and substrate buffer of the present application (including 30. mu.M substrate, 0.3g/L EDTA, 1g/L DHC, FA adjusted to pH 4.2) at 55 deg.C, 80 deg.C, 100 deg.C for 10min, and maintained at 121 deg.C for 10min under high pressure (0.2MPa) sterilization conditions, respectively. After the reaction, the reaction mixture was cooled on ice for 5 min. mu.L EDTA (0.5g/L), 1. mu.L DHC (50g/L), 15. mu.L S5 (100. mu.M) were added with 2. mu.L ultrapure water and mixed well. Diluting the treated sample by 40 times, taking 1 μ L to the mixed solution, reacting at 37 ℃ for 30min, reducing the temperature, adding 12-14 wt% ammonia water to 1 μ L to terminate the reaction, mixing the terminated sample with an equal volume of MALDI matrix (3-HPA 50g/L, DHC 1g/L, ACN 25%), taking 1 μ L to spot on an Anchorchip400/384 target plate, performing MALDI-MS detection in a negative ion reflection mode, and irradiating cumulatively for 2000 times.

As shown in FIG. 14, A of FIG. 14 shows the Prod/sub values after treatment of active ricin with different solvents, different temperatures (55 ℃, 80 ℃, 100 ℃) or autoclaving conditions, and B shows the peak morphology and intensity after treatment of active ricin with different temperature autoclaving conditions in water. As can be seen from fig. 14, the weakly acidic substrate buffer did not maintain the stability of active ricin at high temperature better than the aqueous solution, and there was little difference between the two solvents; active ricin can maintain good activity at 55 deg.C, and is almost completely inactivated after 10min of high temperature or high pressure sterilization at 80 deg.C or above.

Example 13: actual sample Activity detection

A real sample of biotoxin mixed with pesticide microemulsion from an international biotoxin assay test run (BT1904) was desalted using a Zeba spin desalting cartridge to remove salts and polyethylene/propylene glycol from the sample. The sample desalting step was as follows:

a sample desalting step: removing the bottom seal of the desalting column and placing into a 1.5mL centrifuge tube, loosening the cap, and centrifuging (1000g, 1min) to remove the stock solution; adding ultrapure water, and centrifuging (1000g for 1min) twice; the column was transferred to a new 1.5mL centrifuge tube, 100. mu.L of sample was added, and the flow-through from this step was collected after centrifugation (1000g, 2 min).

mu.L of EDTA (0.5g/L), 1. mu.L of DHC (50g/L), 1. mu.L of FA (0.4%), 15. mu.L of S5 (100. mu.M), and 2. mu.L of ultrapure water were taken and mixed to obtain a substrate buffer. Diluting the above samples with ultrapure water by 2 times and 4 times, adding 1 μ L into substrate buffer solution, reacting at 37 deg.C for 10min, reducing temperature and adding 12-14 wt% ammonia water 1 μ L to terminate the reaction, mixing the terminated sample with equal volume MALDI matrix, spotting 1 μ L on Anchorchip target plate, performing MALDI-MS detection in negative ion reflection mode, and irradiating cumulatively for 2000 times.

The results are shown in FIG. 15, and FIG. 15 shows the spectrum peak morphology and intensity of MALDI-MS when the sample is subjected to activity detection after dilution by different times. The sample is shown to have extremely significant N-glycosidase activity, Prod/sub is substituted into a linear regression equation (see figure 10), and the activity of the sample is calculated to be equivalent to ricin of about 26.01 +/-0.57 mu g/mL. And determining the ricin as the result of mass spectrum structure identification.

While specific embodiments of the present application have been described in detail, those skilled in the art will understand that: various modifications and changes in detail can be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present application. The full scope of the application is given by the appended claims and any equivalents thereof.

SEQUENCE LISTING

<110> military medical research institute of military science institute of people's liberation force of China

<120> method for detecting active II type ribosome inactivating protein

<130> IDC200497

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 12

<212> DNA

<213> Artificial Sequence

<220>

<223> S1 nucleotide sequence

<400> 1

gcgcgagagc gc 12

<210> 2

<211> 12

<212> RNA

<213> Artificial Sequence

<220>

<223> S2 nucleotide sequence

<400> 2

gcgcgagagc gc 12

<210> 3

<211> 14

<212> RNA

<213> Artificial Sequence

<220>

<223> S3 nucleotide sequence

<400> 3

cgcgcgagag cgcg 14

<210> 4

<211> 16

<212> RNA

<213> Artificial Sequence

<220>

<223> S4 nucleotide sequence

<400> 4

gcgcgcgaga gcgcgc 16

<210> 5

<211> 12

<212> RNA

<213> Artificial Sequence

<220>

<223> S5 nucleotide sequence

<220>

<221> misc_feature

<222> (6)..(6)

<223> n represents adenine deoxynucleotide

<400> 5

gcgcgngagc gc 12

<210> 6

<211> 14

<212> RNA

<213> Artificial Sequence

<220>

<223> S6 nucleotide sequence

<220>

<221> misc_feature

<222> (7)..(7)

<223> n represents adenine deoxynucleotide

<400> 6

cgcgcgngag cgcg 14

<210> 7

<211> 16

<212> RNA

<213> Artificial Sequence

<220>

<223> S7 nucleotide sequence

<220>

<221> misc_feature

<222> (8)..(8)

<220>

<221> misc_feature

<222> (8)..(8)

<223> n represents adenine deoxynucleotide

<400> 7

gcgcgcgnga gcgcgc 16

Claims

1. A method of detecting active RIP-II in a sample, comprising:

providing a sample to be tested;

detecting whether the oligonucleotide substrate is adenine (a) depleted;

judging whether the active RIP-II exists in the sample to be detected or not;

optionally, pre-treating the test sample prior to contacting the test sample with the oligonucleotide substrate;

optionally, if the active RIP-II exists in the sample to be detected, identifying the specific type of the active RIP-II by combining mass spectrometry;

preferably, the oligonucleotide substrate is of the general formula 5' -r ((GC) _n GdAGA(GC) _n ) -3 'or of the general formula 5' -r (C (GC) _n GdAGA(GC) _n G) -3', wherein n ═ 1,2, 3, 4, 5, 6,7, 8, 9, or 10;

preferably, the oligonucleotide substrate is a nucleotide sequence selected from SEQ ID NO: 5-7;

preferably, the oligonucleotide substrate is SEQ ID NO: 5;

preferably, the RIP-II is one or more selected from ricin, RCA120, abrin, AAG;

preferably, the RIP-II is ricin.

2. The method of claim 1, comprising contacting the sample to be tested with an oligonucleotide substrate in a buffer solution comprising: water, capable of providing NH ₄ ⁺ The ammonium salt of (a), a metal ion chelating agent, and a pH adjusting agent;

preferably, the ammonium salt is an ammonium citrate salt, preferably, the ammonium salt is Diammonium Hydrogen Citrate (DHC);

preferably; DHC purity is not less than 98%, more preferably concentration is not less than 99%;

preferably, the metal ion chelating agent is ethylenediaminetetraacetic acid (EDTA);

preferably, the pH adjusting agent is Formic Acid (FA), Citric Acid (CA) or trifluoroacetic acid (TFA), preferably FA;

preferably, the buffer solution has a final concentration of DHC of 0.2-5g/L, e.g., 0.5-5g/L, 0.8-5g/L, 0.8-2g/L, 0.8-1.5g/L, about 1g/L, about 2g/L, about 3 g/L;

preferably, the final concentration of EDTA in the buffer solution is 0.05-0.375g/L, such as 0.05-0.3g/L, 0.1-0.35g/L, 0.15-0.3g/L, 0.25-0.35g/L, about 0.3 g/L;

preferably, the pH value of the buffer solution is 4.0-4.6, preferably 4.1-4.4, and more preferably 4.2-4.4;

preferably, the buffer solution contains DHC at a final concentration of about 1g/L, EDTA and FA at a final concentration of about 0.3g/L, and the pH of the buffer solution is about 4.2;

the oligonucleotide substrate is present in the buffer solution at a final concentration of 1-100. mu.M, preferably 1-60. mu.M or 1-30. mu.M, more preferably 20-30. mu.M.

3. The method of claim 1 or 2, comprising contacting the test sample with an oligonucleotide substrate in a buffered solution at a temperature of 4-55 ℃ (e.g., 25-55 ℃, about 37 ℃);

preferably, the sample to be tested is contacted with the oligonucleotide substrate at a temperature of 4-55 deg.C (e.g., 25-55 deg.C, e.g., 35-40 deg.C, e.g., about 37 deg.C) for 3-60min, preferably 8-35min, e.g., 10-30 min;

preferably, the oligonucleotide substrate is SEQ ID NO: 5, the sample to be tested is contacted with the oligonucleotide substrate under the condition of 35-40 ℃ (for example, 37 ℃) for 8-35min, for example, 10-30 min;

preferably, the method further comprises, after contacting the sample to be tested with the oligonucleotide substrate in the buffer solution, adding ammonia water to the buffer solution to adjust the pH of the buffer solution to 8 to 10, for example, about 9, to obtain a test solution;

preferably, the concentration of the aqueous ammonia is from 5 to 28 wt%, for example from 12 to 14 wt%;

preferably, the concentration of the ammonia water is 12-14 wt%, and the amount is 0.8-1.2. mu.L (e.g., about 1. mu.L) of ammonia water per 50. mu.L of the buffer solution.

4. The method of any one of claims 1-3, wherein the oligonucleotide substrate is tested for adenine (A) depletion using matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS);

preferably, the MALDI-MS is matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS);

preferably, the method employs a target plate having a hydrophilic enrichment effect (e.g., an Anchorchip target plate) as the sample target plate;

preferably, the method is performed using a reflex mode to detect whether the oligonucleotide substrate is adenine (a) depleted;

preferably, the method is performed using negative ion reflex mode to detect whether the oligonucleotide substrate is adenine (A) depleted.

5. The method of any one of claims 1-5, wherein the oligonucleotide is tested for adenine (A) depletion by a method comprising:

adding the mixed sample into a sample target plate;

collecting the spectrum of the mixed sample by using MALDI-MS;

judging whether the oligonucleotide substrate in the mixed sample is subjected to adenine (A) removal;

preferably, if the obtained map has a product characteristic peak after the removal of adenine (A) from the oligonucleotide substrate, judging that the adenine (A) is removed from the oligonucleotide substrate, and further determining that the activity RIP-II exists in the sample to be detected;

preferably, if the obtained map has a product characteristic peak after the removal of adenine (A) by the oligonucleotide and the peak area ratio (Prod/sub) of the product to the substrate peak is not less than 0.003, determining that the adenine (A) is removed by the oligonucleotide substrate, and further determining that the activity RIP-II exists in the sample to be detected;

preferably, the activity RIP-II is quantified by using the size of the Prod/sub ratio;

preferably, the oligonucleotide substrate is SEQ ID NO: 5, the product characteristic peak is located at 3754 ± 1 (mass-to-charge ratio, m/z); the oligonucleotide substrate is SEQ ID NO: 6, and the characteristic peak of the product is located at 4404 +/-1 (mass-to-charge ratio, m/z); the oligonucleotide substrate is SEQ ID NO: 7, the product characteristic peak is located at 5055 +/-1 (mass-to-charge ratio, m/z);

preferably, the MALDI matrix comprises 3-hydroxy-2-pyridinecarboxylic acid (3-HPA), DHC, Acetonitrile (ACN), and water;

preferably, the concentration of 3-HPA in the MALDI matrix is 1-80g/L, preferably 14-70g/L, such as 30-60g/L, for example about 50 g/L;

preferably, the concentration of DHC in the MALDI matrix is 0-2g/L, preferably 0.5-2g/L, such as 0.8-1.2g/L, e.g., about 1 g/L;

preferably, the concentration of CAN in the MALDI matrix is 0-75%, preferably 10-50%, e.g., 20-30%, e.g., about 25%;

preferably, the mixing ratio of the solution to be measured to the MALDI matrix is 1:1-10:1, preferably 1:1-6: 1;

preferably, the purity of 3-HPA is not less than 98%, more preferably the concentration is not less than 99%;

preferably, the DHC is not less than 98% pure, more preferably not less than 99% concentrated.

6. The method of any one of claims 1-5, wherein the pretreatment is one or more selected from the group consisting of dilution, concentration, desalting;

preferably, the sample to be tested is desalted by extraction and/or chromatography;

preferably, desalting the sample to be detected by using a molecular exclusion chromatography desalting method;

preferably, the sample to be tested is desalted using a Zeba spin desalting spin column.

7. KitWhich comprises the following steps: oligonucleotide substrate capable of providing NH ₄ ⁺ The ammonium salt of (a), a metal ion chelating agent, and a pH adjusting agent;

preferably, the oligonucleotide substrate is an oligonucleotide comprising a stem-loop structure, and the sequence of the stem-loop structure comprises 5 '-r (GdAGA) -3';

preferably, the oligonucleotide substrate is SEQ ID NO: 5;

preferably, the pH adjusting agent is Formic Acid (FA), Citric Acid (CA) or trifluoroacetic acid (TFA), preferably FA.

8. The kit of claim 7, further comprising ammonia,

preferably, the kit further comprises 3-hydroxy-2-pyridinecarboxylic acid (3-HPA);

preferably, the kit further comprises Acetonitrile (ACN);

preferably, the kit further comprises a desalting chromatography column, such as a size exclusion chromatography column, for example a Zeba spin desalting centrifugation column;

preferably, the kit further comprises instructions;

preferably, the specification at least describes the detection method according to any one of claims 1 to 6.

9. Use of a kit according to claim 7 or 8 for the detection (including qualitative and/or quantitative detection) of active RIP-II, or

Use in the detection (including qualitative and/or quantitative detection) of active RIP-II using MALDI-MS;

preferably, the RIP-II is one or more selected from ricin, RCA120, abrin, AAG.

10. Use of an oligonucleotide substrate for detecting (including qualitatively and/or quantitatively detecting) active RIP-II or for detecting (including qualitatively and/or quantitatively detecting) active RIP-II using MALDI-MS, wherein the oligonucleotide substrate is an oligonucleotide comprising a stem-loop structure and the sequence of the stem-loop structure comprises 5 '-r (gdaga) -3';

preferably, the oligonucleotide substrate is SEQ ID NO: 5.