Patent Application
20180328927
The present invention relates to the detection of biological samples, more in particular to a method and a kit for determining the biological activity of a methionine adenosyltransferase (MAT).
In addition to host-dependent parasites, Methionine Adenosyltransferase (MAT, EC2.5.1.6), also known as S-adenosylmethionine synthetase, is present in the cells of all species of organisms. The MAT sequence is very highly conserved, with a 59% homology in the gene sequence of MAT between human and E. coli. In mammals, MAT contains three isoenzymes encoded by three genes. MAT-I and MAT-III are composed of the catalytic subunit α1 encoded by the same gene (MAT1a). MAT-I is a tetramer and MAT-III is a dimer mainly present in adult liver cells. MAT-II is composed of catalytic subunit α2 encoded by another MAT gene (MAT2a) and is present in cells other than liver, embryonic liver and liver cancer cells. The MAT2 beta gene encodes a regulatory subunit beta that primarily regulates MAT-II. The catalyzed reaction of MAT in vivo is divided into two steps: (1) Catalyzing methionine (Met) and adenosine triphosphate (ATP) to produce S-adenosylmethionine (commonly known as activated or active methionine, S-adenosylmethionine, SAM, AdoMet) and tripolyphosphate (PPPi). SAM and PPPi remain on the MAT surface; and (2) the phosphatase activity of MAT will further decompose PPPi into diphosphoric acid (PPi) and inorganic monophosphoric acid (Pi).
MAT catalyzes the production of S-adenosylmethionine from L-methionine or methionine (L-Met) and adenosine triphosphate (ATP). MAT has a triphosphatase activity that degrades triphosphates into pyrophosphate and phosphoric acid. It's enzymatic activity is dependent on Mg2+ and K+, and the enzymatic reaction is as follows:
ATP and SAM are the most common metabolic intermediates in organisms. In the methionine cycle, methionine is first activated to form SAM. SAM provides methyl groups to important substances in life, such as DNA, RNA, lipoproteins, hormones, neurotransmitters, etc., and becomes homocysteine (HCY). Finally, through the methyl group provided by tetrahydrofolate, HCY becomes methionine. In the liver, the methionine cycle has an additional function, mainly, to quickly reduce the high level of methionine in the blood after a high methionine or high-protein diet, and finally through homocysteine, cysteine, cystathionine, and glutathione, the metabolites are transported to other organs.
SAM is the only sulphur-containing bioactive substance in nature with extremely diversified and important functions. It is the key substance in the methionine cycle and is the only methyl donor in the methylation modification process that is critical to humans. It plays an important role in transmethylation, trans-sulfuration and transamidation reactions. There are direct effects on methylation-related cellular functions, polyamine synthesis, and the ratio of methionine to homocysteine. It is of great importance in various life-related metabolic reactions and cell proliferation and differentiation. The SAM-dependent methyltransferase accounts for 1% of the total number of genes in the human genome. Studies have shown that maintaining a certain concentration of SAM in the liver plays a crucial role in the function of liver function. About 85% of methylation reactions and about 50% of methionine metabolism are performed in the liver, not only suggesting that the liver plays an important role in regulating blood methionine levels (by enhancing MAT-I/III activity), but also that SAM plays important role in the liver cell regeneration, differentiation and sensitivity to hepatocellular damage caused by various factors (alcohol, chemicals, radiation, pathogenic microorganisms, viruses, parasites, etc.). Therefore, it is possible to accurately, sensitively, and conveniently measure the biological activity of MAT and the content of SAM under different conditions, which provides an important insight for deepening the understanding of the methionine cycle and provides critical research tools for studying the regulations between different tissues and organs under different physiological and pathological conditions.
Partial inactivation of the MAT enzyme was observed in liver injury due to hepatic inflammation or oxidative stress. The MAT1a gene is not expressed at all in liver cirrhosis and liver cancer. The level of SAM decreased in MATI/III-deficient mice; in mice lacking glycine N-methyltransferase (GNMT), the level of SAM increased. In both cases, the probability of liver cancer in mice increased significantly.
Many studies have shown that MAT and its catalytically synthesized SAMs play crucial roles in different stages of human life, such as embryogenesis, development, growth, differentiation, health, and disease, because SAM has very important special roles in the methionine cycle and carbon metabolism, and is directly related to the metabolism of the human body. The body's SAM content fluctuates based on age, gender, race, weight, diet, medication status, health, and illness. In view of this characteristic, both the synthesis and decomposition of SAM should be considered at the same time. Therefore, understanding the levels of biological activity of MAT under different situations has great practical significance for us to study many vital metabolic and health problems.
There are two configurations of the MAT enzyme active center, with the first configuration favoring SAM synthesis, and the second configuration having triphosphate hydrolysis activity. The enzyme configuration of the second configuration is significantly slower than the first configuration. The conversion from the first configuration to the second configuration takes some time. The difference between the two configurations lies in the sensitivity to the triphosphatase and nitrosylation.
There are currently two methods for determining MAT activity: In method 1, MAT eventually produces Pi under the conditions of the saturated substrates methionine, adenosine triphosphate and PPPi. The content of inorganic phosphorus Pi was measured by direct staining of malachite green and ammonium molybdate. In method 2, the amount of SAM catalyzed by MAT was determined by high pressure liquid chromatography (HPLC) or mass spectrometry (LC-MS/MS).
Among the above methods, method 1 has a big limitation: (1) PPPi, PPi and Pi coexist in the reaction system, and malachite green and ammonium molybdate also have some combination with PPPi and PPi, and the measured Pi value is inaccurate; (2) Sensitivity is not high; (3) MAT's triphosphatase activity requires configuration transformation, therefore, measurement of the reaction product Pi for the determination of the triphosphatase has a hysteresis effect. The indirect method measures the delayed product Pi, hence the variability of this method is high; (4) The triphosphatase activity of MAT is greatly affected by Met, which impacts the stability of the method; (5) The use of triphosphatase activity of MAT to represent the SAM synthesis ability (or activity) of MAT has a limitation or defect. It is very likely that the two enzyme activities of MAT are disproportionately related. It is highly possible that different MAT enzyme activities (referring to the ability of SAM synthesis and the ability of PPPi to hydrolyze) are differently influenced by different factors, making it difficult to use MAT's phosphatase hydrolysis activity to accurately reflect the SAM synthesis activity of MAT.
Fernández-Irigoyen reported that the MAT-I and MAT-III enzyme activities of different mutant strains were different from those of wild-type MAT-I and MAT-III in that their triphosphate hydrolase activity and SAM synthesis activity were affected differently. The activity of triphosphatase was unchanged, but the SAM synthesis activity was significantly reduced in some mutants; other mutant strains had enhanced SAM synthesis ability but triphosphoric acid hydrolase activity decreased; and yet some others had two types of enzyme activities that were both reduced to various degrees. In short, all kinds of possibilities exist.
Sanxhez del Pino et al. found that MAT-III introduces an NO group at 121 cysteine residue (nitrosation), which results in a marked decrease in SAM synthesis activity but no inhibition of triphosphatase activity of MAT. Therefore, determining MAT activity by measuring Pi can only reflect the overall results of SAM synthetase activity and triphosphatase activity, but cannot reflect its SAM synthesis ability. The result is inaccurate and therefore has great limitations.
In the Fernández-Irigoyen's report, measurement of SAM synthesis activity is based on the use of method 2 mentioned above, that is, HPLC method to measure the amount of SAM generated. In fact, no matter which method is used, either HPLC or LC-MS/MS to measure the concentration of SAM, and hence evaluate MAT activity, it is very inaccurate. We know that SAM is bound to MAT for a period of time after its synthesis, whereas HPLC and LC-MS/MS methods (1) only measure free SAM molecules; (2) samples used for HPLC and LC-MS/MS platforms have to undergo special treatment, after a long time and many steps, the content of SAM must results in some loses; (3) the amount of SAM cannot be determined in time (SAM molecule itself is very unstable, therefore SAM needs to be measured timely without delay). All these factors will cause the measured SAM value rather inaccurate, let alone trying to achieve the purpose of reflecting the enzyme kinetic reaction.
Therefore, it is necessary to develop a detection method that can accurately, rapidly and directly determine the biological activity of methionine adenosyltransferase.
The object of the present invention is to provide a method for accurately, rapidly and directly measuring the biological activity of methionine adenosyltransferase and a kit developed by the method. The one-step method or the simultaneous method to directly determine the concentration of the synthesized SAM product involved in the present invention is an innovation in the field of MAT activity measurement. MAT is the only enzyme synthesizing SAM. The present invention can measure the concentration of SAM most rapidly and directly for the first time to determine the level of MAT activity and SAM concentration at the same time ex vivo or in vitro. In the present invention, the MAT catalyzed biochemical reaction and the determination of the SAM immunoreaction are performed simultaneously and the two processes are seamlessly combined, which is particularly significant for accurately measuring the SAM molecule that is unstable by nature. The novelty of the present invention is the determination of the biological activity of methionine adenosyltransferase using anti-S-adenosylmethionine-specific antibodies. The method for determining the biological activity of the methionine adenosyltransferase comprises the following steps: (1) In a buffer system that ensures the biological activity of methionine adenosyltransferase, the sample containing the enzyme and the substrate react to produce certain amount of S-adenosylmethionine product; (2) The use of immunological methods to measure the level of S-adenosylmethionine, in order to determine whether the reaction system possesses methionine adenosyltransferase activity, as well as the level of its activity. Among them, the substrate contains methionine, adenosine triphosphate, magnesium ion and potassium ion in a suitable acid-alkaline buffer system; the sample can be derived from genetic engineering products, purified samples from cells of biological tissues, methionine adenosyltransferases in the tissue cells, biological fluids or tissue and cell cultured fluids, biological fluids including blood, plasma, serum, saliva, urine, cerebrospinal fluid, abdominal or thoracic exudates and tissue fluids. Immunoassays can be made up of anti-S-adenosyl-methionine-specific antibody, various tracer-coupled conjugates to such antibody, S-adenosyl-methionine or its analogues, and conjugated-S-adenosylmethionine or its conjugated analogues. Catalytic reaction of methionine adenosyltransferase and its resulting product S-adenosine methionine competes with the coated S-adenosylmethionine antigen or tracer-labeled S-adenosylmethionine antigen in binding tracer-labeled anti-S-adenosylmethionine antibody or coated anti-S-adenosine antibody. The immune response is based on the one-step principle that enzymatic reaction and immunoassay are performed simultaneously.
In order to solve the problems existing in the prior art (measurement of inorganic phosphorus Pi content by direct staining of malachite green and ammonium molybdate; HPLC or LC-MS/MS method), the present invention provides a method for determining the biological activity of methionine adenosyltransferase in a sample. One of the formats is as follows:
(1) Preparation of a standard with different concentrations: Take the S-adenosyl-methionine standard in the kit and prepare it in the proper buffer at different concentrations used for a standard curve;
(2) In a microtiter plate coated with a protein (or polymer)-SAM antigen conjugates or protein (or polymer)-SAM analogue antigen conjugates, add the standards of different concentrations to the standard wells and samples to be tested to sample wells;
(3) Add horseradish peroxidase or alkaline phosphatase labeled anti-S-adenosyl-methionine monoclonal antibody, methionine, adenosine triphosphate, and methionine adenosine transferase positive controls, and mix them for 60 min at 37° C. The plate is washed after the reaction;
(4) Add horseradish peroxidase or alkaline phosphatase substrate, allow color development at 37° C. for 15 min, stop solution was added to terminate the reaction, and read optical absorbance at 450 nm wavelength per well;
(5) In the same enzyme-linked immunoassay plate, a series of SAM with known gradients are used as standards to plot a standard curve. The curve equation is obtained based on the values of OD450 and the known standard concentrations, and then the sample OD450 is substituted into the equation to obtain the corresponding SAM concentration;
(6) The MAT enzyme activity is calculated based on the concentration of catalyzed product SAM per unit time of a certain mass of MAT sample.
The sample to be detected in step (2) above may be derived from a genetically engineered sample, a purified sample from a biological tissue cell, a biological fluid or a tissue culture fluid. The reaction time at 37° C. in step (3) above is 20 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, and 90 min.
Compared with the prior art, the beneficial advantages of the present invention are:
1. Make direct determination of the concentration of SAM possible instead of indirect determination of Pi, which directly reflects the MAT's capability of SAM synthesis, and the process is not affected by MAT configuration changes;
2. The SAM production and measurement in the present invention are done in one step in the same system. There is no lag, therefore the result is accurate and timely;
3. Immunoassays for SAM are not only specific but also very sensitive, and are not affected by the presentation form of SAM, which can be in both bound and dissociated forms;
4. Compared with the prior art, the present invention does not require special instruments. It only needs a conventional microplate reader.
Therefore, the use of the kit from the present invention makes the measurement of MAT activity more accurate, reliable, direct, simple and rapid, and can simultaneously obtain MAT activity and SAM concentration in a sample.
The great significance of the present invention lies in providing new MAT assay methods and products that allow researchers to conveniently and accurately determine MAT enzyme activity at any time. It plays important roles in understanding the expression profiles of MAT1A, MAT1B, MAT2 genes in different populations; any other in-depth understanding of the research and development work relating to methionine cycle; answering questions relevant to methionine metabolism; in vivo methylation status (methylation process); for the discussion of methylation mechanisms in various situations; in-depth studies of epigenetics; research and monitoring of metabolic diseases, oncogenesis, development, prognosis, nutrition, disease, and health. In addition, many metabolic processes such as homocysteine-cysteine-glutathione production (trans-sulfuration process) and polyamine (aminopropylation process) are also related to the production of SAM.
Another object of the present invention is to provide a methionine adenosyltransferase biological activity assay kit comprising the following components: a microplate coated with an anti-S-adenosylmethionine antibody or S-adenosylmethionine antigen or its analogues; tracer-labeled anti-S-adenosylmethionine antibody or tracer-labeled S-adenosylmethionine antigen or labeled S-adenosylmethionine analogues; S-adenosylmethionine standard; enzyme substrate solution consisting of methionine, adenosine triphosphate and a buffer of magnesium ions and potassium ions; a positive control (methionine adenosyltransferase); a tracer detection system; and an appropriate buffer system. Among them: microplates coated with S-adenosylmethionine antigen that can be in the form of polylysine (PLL), bovine serum albumin (BSA) or other carrier protein coupled S-adenosylmethionine or S-adenosylmethionine analogues, or directly coated onto the microplates with anti-S-adenosylmethionine antibodies (monoclonal and polyclonal) or indirectly coated through goat or rabbit anti-mouse IgG and then S-adenosylmethionine monoclonal antibody to the microplate; tracers may include enzymes, fluorescein, colloidal gold, chemiluminescent substances, biotin, digoxin (or digoxigenin), radioactive substances, various types of latex microspheres including fluorescent microspheres and colored Latex microspheres, etc.; display system corresponding to the tracer used; microplates are microplates consisting of removable ELISA strips; the substrate fluid and buffer system are determined after repeated tests to determine the optimal concentration of each component, pH and reaction time and so on.
The examples provided by the present invention are based on an enzyme-linked immunosorbent assay (ELISA), i.e. coated with S-adenosylmethionine antigen, or S-adenosylmethionine analogues; horseradish peroxidase or other tracer-labeled anti-SAM-specific antibody; S-adenosylmethionine or S-adenosylmethionine analogues as the standard; MAT positive control; substrate solution consisting of methionine, adenosine triphosphate, etc.; and proper buffer system; HRP substrate TMB (3,3′,5,5′-tetramethylbenzidine) or other tracer display or detection system and ELISA stop solution. The amounts of methionine, adenosine triphosphate and methionine adenosyltransferase reported in the examples of the present invention are summarized here. The concentration of methionine and the concentration of adenosine triphosphate vary from micromolar to millimolar depending on the level of MAT activity tested. Because the substrate cross-reaction should be excluded and control is set for each experiment, in most cases excess substrate should bear no adverse effects. The pH of the enzyme reaction is 7.42-8.50. The concentration of MAT also varies depending on the source. The purified product of E. coli MAT gene expression used in this laboratory is employed at a concentration of 0.3-1.0 mg/ml. Suitable buffer systems include 50-100 mM magnesium ion, 50-400 mM potassium ion, 50-200 mM Tris-HCl buffer system.
Compared with the prior art, the key points of the present invention are:
1. One-step or simultaneous method, that is, the MAT enzymatic reaction and the quantification of the key product produced are performed together at the same time, or the MAT catalyzed chemical reaction is first performed for a period of time, about 20 min, which is very useful for the case when MAT activity is very low, and then the detection of product by immunological reaction is introduced. Since antigen-antibody binding takes a relatively longer time (it is generally assumed that 37° C. for 1 h is needed), during the course of the immunological reaction, the MAT-catalyzed chemical reaction is still going on, i.e., the SAM is continuously generated. Therefore, regardless of whether the chemical reaction is first performed for a period of time alone or not, it is not contradictory to the key point in the present invention, i.e., the design in which the catalytic reaction and the immunological reaction are combined together. It is simply used in order to achieve the best detection sensitivity. For this purpose, (1) reflects the dynamic changes of MAT activity most precisely and without any delay; (2) Since the product SAM is very unstable, any SAM quantification methods, even though accurate, if they cannot measure the unstable SAM upon being produced, they are deemed to fail to calculate MAT enzyme activity efficiently, reliably and accurately.
2. Simultaneously measure the MAT activity and SAM concentration in the sample in the shortest possible time.
3. The combination of immunological methods and biochemical reactions enables substances in biochemical reactions to be detected efficiently, rapidly, specifically and sensitively.
4. The liquid formulation of the one-step method or the simultaneous method (that can sensitively reflect the reaction that changes the MAT activity) not only ensures that the enzymatically catalyzed biochemical reaction can proceed normally, but also enables the immunoassay step to be effectively implemented.
The principle of the direct in vitro determination of MAT activity of the present invention is summarized as follows: An enzyme-linked plate coated with SAM antigen is added with an appropriate amount of HRP-labeled anti-SAM antibody. Then, a SAM standard or a MAT activity assay system (a suitable buffer containing methionine and adenosine triphosphate, plus a MAT positive control or a sample having MAT activity to be measured) was added as a SAM test sample. Incubate at 37° C. for a certain period of time, then add HRP substrate TMB to develop color, and read the optical density at 450 nm. Based on the standard curve of SAM, the concentration of SAM can be calculated. Based to the amount of SAM, the ability of MAT to produce SAM can be calculated. MAT samples can be derived from genetically engineered MAT, purified tissue samples from biological tissue cells, MAT within tissues and cells, biological or tissue cell culture fluids. Examples of biological fluids are blood, plasma, serum, saliva, urine, cerebrospinal fluid, abdominal or thoracic exudate, tissue fluid, etc. The detailed technical principle and method of this invention is as follows:
1. MAT catalytic reaction principle:
2. Principle and method of direct competitive ELISA measurement:
(A) Antigen-coating method: A PLL or BSA-conjugated SAM antigen is pre-coated on a solid-phase ELISA micro-titer plate that is then blocked with BSA solution. The antigen to be detected (SAM sample), then HRP-conjugated anti-SAM antibody are added and incubated at the same time. The antigen to be tested and the coating antigen compete for the enzyme-labeled antibody. After incubation and washing, the enzyme-labeled antibody bound to the coating antigen is retained, and the enzyme-labeled antibody that binds to the antigen to be detected (free antigen) is washed away. Finally, adding TMB substrate to the reaction wells. The final coloration result is inversely proportional to the amount of the antigen to be detected, that is, the higher the OD450 value, the lower the SAM concentration in the sample to be measured. In the course of the experiment, a series of SAM solutions with a known concentration were used as standards in the same ELISA micro-titer plate for the standard curve of the assay. Based on the standard curve, an equation showing how OD450 readings and values of the known standard concentration are related can be obtained. Put the sample OD450 into the equation to get the corresponding concentration value of the sample. The principle of this direct competitive ELISA is shown in
(B) Antibody-coating method: First coat goat or rabbit anti-mouse IgG onto micro-titer plate at 4° C. overnight, add the proper amount of anti-SAM antibody to the wells and incubate for 30-60 min, or directly coat the optimal amount of anti-SAM antibody to the micro-titer plate. Wash plate, add HRP conjugated SAM antigen or its analogues, then add SAM standard and samples. The standards and samples compete with HRP conjugated SAM antigen or its analogues to bind anti-SAM specific antibody on the plate. Incubate for 30 min and then wash plate. Add TMB substrate and incubate for 15 min, then add stop solution. Record OD450. According to the standard curve, the concentration of SAM from the samples can be calculated.
3. Combine the catalytic reaction of the MAT and the direct competitive ELISA to investigate the feasibility of a one-step method or a simultaneous method. In order to make it feasible and workable, the present invention is divided into the following parts to elaborate the technical details of the present invention:
Part 1: Exclude the existence of cross-reaction between anti-SAM antibody and methionine adenosyltransferase substrate ATP and L-Met in order to avoid interference of cross-reaction to the competitive ELISA.
Part 2: Explore the optimal reaction ratio between methionine adenosyltransferase and its substrate ATP and L-Met under different pH values.
Part 3: Determine the optimal reaction time of MAT and set the appropriate time to measure the amount of SAM synthesis reaction. Plot a curve showing SAM concentration over time. The reaction of various anti-SAM monoclonal antibodies 118-6, 84-3 and rabbit anti-SAM polyclonal antibodies with enzymatically synthesized SAM are confirmed.
Part 4: Determine the enzymatic activity of MAT purified from mouse liver cells under optimal reaction conditions.
Part 5: Under optimal reaction conditions, measure MAT enzymatic activity in normal liver cell line L02 and hepatoma cell line HepG2 under different MAT regulatory factors, and observe the changes.
Part 6: Experiment with different coating antigens and SAM standards to determine their impacts on MAT activity under the determined optimal reaction conditions.
4. Repeated tests are done to optimize the optimum concentration of each component, reaction time, buffer formulation and pH value, standard curve and the range of standard concentrations.
The above technical route and methods have been fully confirmed by the following examples of the invention. It is apparent from the above, that the advantages of the method of the present invention include, but are not limited to the following: (1) Direct determination of the SAM, the key product of the MAT catalyzed reaction, but not of the secondary product Pi, and thus the most direct reflection of the MAT's ability to synthesize SAM; (2) SAM production and measurement are performed in one step at the same time in the same reaction and incubation system, with which the changes of MAT activity under different conditions can be captured timely and accurately and reaction time of the enzyme can be controlled better. Therefore, the results will be very accurate and timely; (3) Using anti-SAM antibodies with very high specificity, sensitivity and affinity and measure it timely, the results are not only specific, but also very sensitive and are not influenced by the different forms of SAM (binding and dissociative); (4) Being convenient, direct, fast, and therefore very useful in studying the effects of various factors on the activities of MAT from samples. Even if the influence was subtle, it could be identified and captured.
The present invention also studied BSA and hemocyanin (KLH) conjugated SAM or SAM antigen analogues and used them to coat micro-titer plates as in the 2 (A) Antigen-coating method above; and a direct method competitive ELISA for the measurement of SAM as in the 2 (B) Antibody-coating method, the results were consistent with those described in the Examples.
In order to clearly describe the technical solutions in the embodiments of the present invention or in the prior art, the drawings used in the description of the embodiments or the prior art are briefly described below.
The following further describes the present invention in detail with reference to the experimental results and data. Raw materials not mentioned are conventional commercial reagents and are commercially available.
Cross-Reactivity of SAM and Methionine Adenosyltransferase Substrates ATP and L-Met Experimental Materials
Anti-SAM and HRP-conjugated anti-SAM antibody: Arthus Biosystems, MA00201, MA00202, PA00201, MAH00201; polylysine (PLL) or bovine serum albumin (BSA) conjugated SAM antigens (for coating ELISA plates): Arthus Biosystems, ACT00201, ACT00204; S-adenosylmethionine (aza-SAM) standard: Arthus Biosystems, AST00201; S-adenosylmethionine (adenosine methionine disulfide methionine, SAMe) standards: Sigma, A2408, and the same named purchased from Shaanxi Pioneer Biotechnology Co., Ltd.; Methionine adenosyltransferase (MAT): Beijing Aibixin Biotech Co., Ltd. Abt-P-005; BSA, Tris, also known as tris (hydroxymethyl) aminomethane, NaCl, NaH2PO4, Na2HPO4: Beijing Huameijiacheng Biotechnology Co., Ltd.; KCl: Tianjin Damao Chemical Reagents Co. Ltd.; MgSO4: Hunan Honghao Gene Technology Co., Ltd.; Adenosine triphosphate (ATP): Shanghai Jingchun Chemical Reagents Co., Ltd.; L-Met, ProClin 300: Sigma; TMB: Huzhou Yingchuang Biotechnology Co., Ltd.; Sulfuric acid: Hunan Kangdu Pharmaceutical Co., Ltd.; 96-well enzyme-linked immunoassay micro-titer plate: US Corning high adsorption enzyme-linked immunoassay plate.
Enzyme Reaction Buffer: 100 mM Tris, 100 mM KCl, 20 mM MgSO4, ProClin 1%, adjusted pH values pH 7.42, pH 8.0, pH 8.5 respectively; SAM analogue standard (aza-SAM): 960 nM, 480 nM, 240 nM, 120 nM, 60 nM, 30 nM, 15 nM, 0 nM, in enzyme reaction buffer pH7.42; SAM analogous quality control: 200 nM aza-SAM in enzyme reaction buffer pH 7.42; SAM standard (SAMe): 1-40 μM in enzyme reaction buffer pH7.42; SAM quality control in: 8 μM SAMe in enzyme reaction buffer pH7.42; the enzyme reaction buffer: 0.5% or 0.2% IB (Incubation Buffer): 10 nM PB (Phosphate Buffer), 150 mM NaCl, 0.5% or 0.2% BSA, 0.1% ProClin.
The ELISA plates were coated with PLL SAM antigen. The SAM standard curve was created using 0, 15, 30, 60, 120, 240, and 480 nM standards IB containing 0.5% BSA. The concentrations of the cross-reacting substances ATP and L-Met were 0, 120, 1200, 3960 and 12000 nM. The HRP-anti-SAM antibody was diluted at 1:25000 with HRP antibody diluent. Added 30 μl standards and cross-reacting substances ATP and Met per well, and 70 μl diluted HRP-anti-SAM antibody per well and incubated at 37° C. for 1 h. Washed the plate, added 100 μl TMB at 37° C. for 15 min, added 50 μl stop solution and read OD450. The results are shown in Table 1.
A/A0; reaction rate (A0 is the OD450 reading at 0 nM, A represents any OD450 reading of wells that have different amount of free small molecule antigen competing.)
Cross-reaction rate=(Concentration of free antigen when 50% inhibition is achieved/Concentration of cross-reaction substances when 50% inhibition is achieved)*100%. The results of repeated experiments showed that the cross-reaction rates of ATP, ADP and Met with anti-SAM monoclonal antibodies 118-6, 84-3 and rabbit anti-SAM polyclonal antibody. R3 were much less than 1%. The cross-reaction of anti-SAM antibody with methionine, adenosine, S-adenosylhomocysteine and methylthioadenosine are all far less than 1% (see antibody product data from Arthus Biosystems). Therefore, this experiment does not have any cross-reaction with other components in the reaction system.
Using ELISA strips that were pre-coated with PLL-SAM antigen, and different concentrations of ATP, Met, and MAT enzyme were formulated with the enzyme reaction buffer at pH 7.42, pH 8.0, and pH 8.5 respectively. As shown below, codes A\B\C\D represents four Met concentrations, adenosine triphosphate components, and code 1\2\3 represents three MAT concentrations. The combination of the two codes was used to prepare the chessboard design. A total of 12 different combinations of methionine adenosyltransferase, methionine, adenosine triphosphate solutions have been obtained.
The reaction was performed at 37° C. for 1 hour. After the plate was washed, 100 μL of TMB was added to each well. After incubation at 37° C. for 15 minutes, 50 μl of the stop solution was added before reading OD450. The results are shown in Table 2 below.
The synthetic yield of SAM is expressed as A/A0, i.e. the ratio of OD450 of sample well to control well. The lower the A/A0, the greater the ability of the SAM synthesized to compete with the coating antigen for HRP-labeled antibodies. The higher the SAM production, the greater is the MAT activity. The results are shown in Table 3 below.
The resulting reaction rate, i.e. the A/A0 and methionine concentrations were plotted to obtain the results shown in
Based on the results from three different conditions above, the strongest factor influencing the SAM formation reaction is the amount of MAT, followed by the increase in substrate Met, and the smallest factor is the buffer pH value. In addition, because the MAT used in this experiment is a product of E. coli MAT gene expression, it's catalytic activity might not be ideal, therefore the required substrate concentration was relatively higher, but the experiment shows that even in the milli-molar level of Met and ATP, there was no cross-reaction. Since the aim of this experiment was not to screen for highly active MAT, the MAT selected here is able to meet the needs of establishing a MAT activity determination methods. Due to the relatively high activity of MAT under alkaline conditions such as pH 8.5, the antigen-antibody binding is reduced at pH 8.5 compared to pH 7.42, thus A/A0 from
Take PLL-SAM-coated antigen ELSIA strips, and add SAM standards at concentration of 0, 15, 30, 60, 120, 240, 480, 960 nM, and quality control 200 nM that were prepared in an enzyme reaction buffer at pH 7.42. Add ATP and Met at 2.4 mM, plus MAT enzyme at 0.6 mg/ml, or ATP and Met at 2.4 mM, plus MAT at 1.0 mg/ml (all final concentrations) as SAM substrate and enzyme mixture. Add 30 μl per standard well and enzyme samples, and 70 μl per well of HRP-anti-SAM antibody. Then incubated at 37° C. for 30 min, 60 min, 90 min, after washing the plate, 100 μl TMB was added and left at 37° C. for 15 min, added 50 μl stop solution and read OD450. Results are shown in Table 4.
The results above indicated that the amount of SAM synthesized at 60 minutes was significantly higher than that at 30 minutes, but was almost the same at 90 minutes and 60 minutes. Measured SAM synthesis from the 20-90 minutes time period, the relationship of SAM production change over time could be obtained.
Take PLL-SAM-coated antigen ELSIA strips, and add SAM standards and quality control standards were prepared in an enzyme reaction buffer at pH 7.42, added ATP and Met at 2.4 mM, plus MAT enzyme at 0.6 mg/ml, or ATP and Met at 2.4 mM, plus MAT at 1.0 mg/ml (all final concentrations) as SAM substrate and enzyme mixture. Added 30 μl per standard well and enzyme samples, and 70 μl per well of HRP-anti-SAM antibody. Incubation time at 37° C. is shown in Table 5. After washing the plate, 100 μl TMB was added and left at 37° C. for 15 min, added 50 μl stop solution and read at OD450. Data processing is the same as done previously. The concentrations measured are as follows.
The concentration of synthesized SAM and reaction time were plotted and the results are shown in
Using a system similar to the example above, the coating PLL-aza-SAM was used at 0.05 μg/ml. The HRP-anti-SAM antibody 118-6 was diluted at 1:30000 and was incubated for 1 hour. The pH value of the prepared solution was 7.40±0.05, and the ATP was at 5 mM, Met at 4 mM, MAT at 1 mg/ml. When titrating the concentration of Mg2+, the buffer contained 250 mM KCl and 100 mM Tris. When titrating the concentration of K+, the buffer contained 20 mM MgSO4 and 100 mM Tris. After the optimal concentrations of Mg2+ and K+ were determined, Tris was titrated again. The results are shown in Table 6. The A/A0 was gradually decreased by 0.475 when the concentration of MgSO4 was from 4 mM to 100 mM. However, A/A0 decreased rapidly when the concentration of MgSO4 was from 4 to 50 mM, and decreased slowly when the concentration of MgSO4 was from 50 to 100 mM. Thus, the suitable MgSO4 concentration was selected between 50 mM and 100 mM. The A/A0 has small changes when the concentration of KCl was from 50-400 mM, There was no significant decrease and increase trend, and chose about 150 mM. The concentration of Tris was 100 mM and remained optimal.
Sterile PBS, 0.25% trypsin (Aladdin), 1640 medium (Gibco), 10% FBS (Yuanheng St. Ma), serum-free MEM medium (Gibco), 10 g/ml Met aseptic concentrate, GSNO, 15 cm2 square bottle, 75 cm2 square bottle (Corning), FITC-goat anti-mouse IgG (abcam), DAPI (Life Technologies); Triphenylene blue powder (Aladdin).
Phosphate buffer (PBS): NaCl 8 g, Na2HPO4.12H2O 2.885 g, KCl 0.2 g, KH2PO4 0.2 g, ultrapure water 1000 ml; trypsin (0.25% trypsin): trypsin 0.1 g, 4% EDTA solution 200 μl 39.8 ml of 1×PBS solution; Trypsin is fully dissolved and filtered with a 0.2 μm sterile filter (Pall). Trypan blue solution (4% Triphenyl blue solution): Triphenyl blue 1.6 g, ultrapure water 40 ml, filtered with filter paper.
The experimental design is shown in Table 7.
1. When the cells in the flask are at about 80% confluence, remove the 1640 medium (with 10% fetal bovine serum) and wash with PBS once;
2. Take the two groups containing GSNO, treat the cells with serum-free MEM medium containing 1 mM GSNO and leave in the incubator for 30 min.
3. Remove the serum-free medium containing GSNO and wash it with PBS once;
4. Add Met-containing serum-free MEM medium (containing 5% fetal bovine serum and 2 mM glutamine) into the square flask according to the parameters in the table above. Add the Met-free MEM as the control group and put into the incubator for 24 h;
5. Remove the culture medium from the flask and add an appropriate volume of trypsin to ensure that the pancreatin covers the bottom of the entire square flask;
6. Place the flask in a 37° C., 5% CO2 incubator for 2-3 minutes. When cells start to fall out, add 5 ml of 1640 medium containing 10% FBS to terminate the action of pancreatin.
7. Resuspend with about 1 ml of PBS after centrifugation, and count with trypan blue staining.
8. Take 1 ml of each cell suspension, place on an ice bath and sonicate it (see Example 5). Centrifuge at 15000 g and then collect the supernatant.
9. Reaction is performed as described in Example 3 at 37° C. for 60 min to determine the MAT activity in the cell supernatant.
Since the amount of cells in each experimental group used for the ELISA assay was not exactly the same, L02, HepG2 cells were normalized by cell count (adjusted to 2×107). The amounts of SAM synthesized by MAT under different conditions are shown in Table 8 and
Human normal liver cell line L02 expresses the dimer MAT-III or tetramer MAT-I consisting of the catalytic subunit α1 encoded by the MAT1a gene, whereas HepG2 hepatoma cells express tetramer MAT-II consisting of the catalytic subunit α2 and regulatory subunit β encoded by the MAT2a/2b gene. The responses to Met stimulation, cell types of expression and effects of the two MAT enzymes encoded by these two genes are not the same. The presence of MAT-III or MAT-I in adult hepatocytes is sensitive to Met stimulation, and its function is to rapidly reduce Met levels in the blood during high Met diets. Therefore, MAT-III or MAT-I activity is increased by Met stimulation [5]. Our results show that 500 μM Met stimulated the activity of MAT-III or MAT-I in vitro for 24 hours, and the inhibition of MAT-III or MAT-I after nitrosation of GSNO was obvious. No stimulatory effects of Met at 2 mM dosage on MAT-III or MAT-I activity was observed, and MAT activity on the contrary was slightly decreased. The reason was unclear. However, GSNO nitrosylation-inhibitory effects on MAT activity in the presence of 2 mM Met remained obvious. In contrast, the dose-response relationship of Met inhibits MAT-II activity in hepatoma cell lines is consistent with the literature [12], i.e. the higher the Met concentration, the lower the activity of MAT-II. In addition, our results also showed that nitrosylation has a minor effect on MAT-II, which is in agreement with the reported elsewhere that NO in GSNO inhibits MAT activity by binding to the cysteine at position 121 of MAT-II [5]. GSNO mainly inhibits the activity of MAT-III/I post Met stimulation.
Immunofluorescence staining of these two cell lines was performed simultaneously with the competitive ELISA described above for quantification of SAM, the procedure is as follows:
(1) Digest the HepG2 and L02 cells from the bottom of the square flask;
(2) Centrifuge at 1050 rpm for 5 min and re-suspend the cells in 1640 medium containing 10% FBS in (Gibco);
(3) After counting with trypan blue, cells were seeded onto 24 wells at a density of 7.5×104 cells/well, and 8 wells were seeded each cell line;
(4) Add the medium to 24-well culture plate to 1 ml/well and place it in the incubator for 24 h;
(5) Remove the original culture medium from the wells. Add 1 ml/well MEM medium containing 5% FBS and 500 μM Met according to Table 1, and put the culture plate into the incubator for 24 h.
(6) After 24 h. discard the medium and wash it twice with 37° C. pre-warmed PBS. Add 500 μl 80% ice acetone per well at −20° C. for 20 min. Wash 3 times with PBS.
(7) Add primary antibody: Anti-SAM monoclonal antibody was diluted at 1:400 with PBS containing 0.5% skimmed milk, 50 μL per well, 37° C. wet-box for 1 h, and PBS washed 3 times.
(8) Add secondary antibody: FITC-labeled goat anti-mouse IgG diluted at 1:500 with PBS containing 0.5% non-fat dry milk, 50 μL per well, 37° C. wet-box for 45 min, PBS washed 5 times (to avoid light); observed by ordinary fluorescence microscope and took pictures.
(9) 100 μL of 0.5 mg/ml DAPI (diamidino-2-phenylindole) was added and stained the cell nucleus for 20 min; after washing with PBS for 5 times, observed the cells under laser scan confocal microscope directly and photographed as needed.
PPS protein purification system (Institute of Engineering, Chinese Academy of Sciences); Ultrasonic crusher (Ningbo Xinzhi); High-speed refrigerated centrifuge (Xiangli Centrifuge)
Tissue homogenates: sucrose 0.25 M, Tris 10 mM, EGTA 0.1 M, beta-mercaptoethanol 0.1%. pH 7.5; Buffer A: MgSO4 10 mM, EDTA 1 mM, Tris 10 mM, pH 7.5; Buffer B: Buffer A+600 mM KCl; Dialysate: Buffer A+75 mM KCl; pH7.42 Enzyme Reaction Buffer: 100 mM Tris, 100 mM KCl, 20 mM MgSO4, ProClin 1% adjusted to pH 7.42; 50% DMSO solution: Buffer A+equal volume of DMSO.
1. Dissect mouse, remove connective tissue from liver, place it in a de-contracted 1.5 ml tube, and weigh the liver. The net weight was 0.6 g.
2. Wash the liver 3 to 5 times with 1 ml normal saline until the solution is clear and transparent.
3. The liver was cut into pieces and placed into a clean tissue homogenizer. About 3-fold more ice-cold tissue homogenate solution was added to the liver tissue pieces. Rotated and grinded in an ice bath for 6-8 min and grinded it thoroughly. The homogenate was removed, and the homogenizer was filled with 1 volume of hepatic tissue homogenate to wash the wall and aspirated into the homogenate.
4. The homogenate was placed in the liquid ice bath in an ultrasonic crusher for comminution, with φ6 probe, power 80%, working time was 3 seconds, pause time was 3 seconds, sonication was 30 minutes.
5. Homogenate was centrifuged under refrigerated condition at 15,000×g high speed for 20 min, the supernatant was filtered with qualitative filter paper. A total of 150 ml homogenate was obtained.
6. Slowly added 31.3 g of ammonium sulfate per 100 ml of the homogenate with stir, and added 47 g of ammonium sulfate to make the saturation to be 50%. Stirred for 30 min and let it stand for 60 min.
7. Supernatant from high-speed refrigerated centrifuge was discarded. The precipitate was dissolved with 150 ml of pre-cooled dialysate, dialyzed in 2 L dialysate, and exchanged once.
Purify with DEAE Column:
1. Dialysis sample processing: The dialysis sample was taken out and centrifuged at high speed to discard precipitate. The supernatant is filtered with filter paper. About 230 ml of sample was obtained.
2. Took 60 ml of DEAE packing, packed the column (3×20 cm), and applied to the PPS chromatography system, rinsed with deionized water at a flow rate of 10 ml/min for 30 min, and equilibrated with dialysis buffer.
3. Mixed the sample and DEAE packing in a 4° C. refrigerator using a low speed shaker for 90 minutes.
4. Packed the column, rinsed on the chromatographic system, and dialysis buffer was set to 6 ml/min for 30 min, set UV to zero.
5. Elution: 100% buffer B salt eluted for 20 min with a linear gradient, flow-rate was 6 ml/min. When the UV monitoring peak appeared, started to collect 5 ml per tube till the end of the peak, A total of 38 tubes were collected.
Used the PLL-SAM antigen coated ELSIA plate. Prepared MAT reaction system with reaction buffer with pH 7.42, ATP and Met at 2 mM, and elution sample accounted for 50% of the reaction mixture. Control group had not ATP and Met substrates. The SAM quantification kit was used to measure the amount of SAM produced by each eluent, which indirectly reflected MAT activity and elution range. The activity unit was defined as the amount of SAM in nM produced per minute per milligram of MAT enzyme.
The elution portion with the reaction rate A/A0<90% in Table 9 was significantly competing. The eluent from tubes 10-23 were pooled and dialyzed overnight to prepare for applying to a Phenyl Sepharose Fast Flow column. The protein concentration after dialysis was 5.98 mg/ml in a total volume of 70 ml. Activity of this component: 28 nM/60/0.015 ml×5.95 mg/ml=5.3 U (nM/min/mg).
1. Took 20 ml of Phenyl Sepharose Fast Flow packing, packed a column, rinse with deionized water at a flow rate of 10 ml/min for 10 min, and equilibrated with buffer A+220 mM KCl.
2. Mixed the sample and the packing in a 4° C. refrigerator using a low speed shaker for 90 minutes.
3. Rinsed on the chromatographic system, set UV to zero.
4. Eluted with pre-cooled 50% DMSO solution at 6 ml/min. Collected elution peaks and dialyzed the eluent.
Three tubes of the eluent samples were pooled and assayed for MAT activity in the same manner as above with a load of 15 μl. SAM production was tested as 15 nM. The specific activity of this fraction was 15 nM/60/0.015 ml×2.93 mg/ml=5.7 U (nM/min/mg).
Due to the limited amount of sample left, no further purification was done. However, MAT activity has been measured after the purification with two chromatography columns. It is suggested that the method of determining MAT activity of the present invention is also applicable to the activity assay of MAT enzyme purified from mouse or rat liver.
To clarify the effect of different steps or methods on the amount of SAM production, the MAT enzyme and the substrate were first applied to the PLL-aza-SAM coated strips using the optimal buffer and substrate concentrations described above. The reaction was carried out at 37° C. for 20 min and reacted with HRP-anti-SAM antibody for 40 min. At the same time, MAT was directly reacted with the substrate and HRP-anti-SAM antibody for 1 h. The results showed that the amount of SAM synthesized by the step-by-step or two-step reaction was higher than that produced by the one-step method because the total volume of the system was only 30 μl during the first 20 min reaction. The concentrations of MAT and the substrates were both 2.33-fold higher than those of the one-step method. Table 10 shows the compared effects of these two different operating procedures on the amount of SAM synthesized. When the MAT concentration was lower at 0.3 mg/ml, the SAM synthesized by the two-step method was 2.81 times higher than that of the one-step method. When the MAT concentration was 0.6 mg/ml, the SAM synthesized by the two-step method was 1.76 times higher than that of the one-step method. When the MAT concentration was increased to 1 mg/ml, the SAM synthesized by the two-step method was 1.42 times higher than that of the one-step method. If MAT concentration continues to increase, it can be foreseen that the stepwise or two-step approach and one-step approach will be similar to each other in the SAM synthesis. The increases in the amount of SAM production (2.81, 1.76, 1.42 times) were not higher than the increases of the substrate and the MAT concentration (3.33 times). The stepwise method was helpful for the improvement of the SAM yield. Especially, in the test when MAT concentration or activity was low, the effects and advantages of the stepwise method would be more pronounced. If the MAT activity of the sample to be tested was high, and it was meant to compare the differences between groups of samples under different conditions, the one-step method is more convenient and easy, and the operating procedures have little influence on results.
Numerous examples from the present invention demonstrate that the system we have established is very sensitive and can accurately and reliably determine the biological activity of MAT. Although the step-by-step method was used in this example, it was only used to selectively allow the MAT-catalyzed chemical reaction to proceed more efficiently in an independently optimal environment first, and immediately followed by performing quantitative immunological assay. In the second step 40-minute immunoassay, the MAT catalyzed chemical reaction continues to progress. The newly synthesized SAM immediately participates in the competitive immunological reaction. With the optimal system, all synthesized SAM molecules participate in the competition of tracer-labeled anti-SAM antibody. Due to the high specificity of the anti-SAM antibody, the results are very accurate There is no issue of inaccurate measurement caused by SAM loss due to various reasons. Therefore, the present invention proposes that both operating procedures are all very useful and can be used properly based on specific circumstances.
In addition to 1 μg/ml BSA-SAMe that was used for coating the ELISA plate, the same standard SAMe as the coating antigen was used as the standard antigen. HRP-anti-SAM 84-3 (Arthus Biosystems, MAH00202) was used as the anti-SAM antibody. Table 11 shows the result of such an experiment. The linearity of SAMe as a standard product was very good. When MAT was at 0.3 mg/ml, the SAM synthesized was about 3 μM. The result indicated that the use of SAM analogue aza-SAM plates and standard analogue as well as the SAMe plates and standard can both nicely reflect the changes of MAT activity. Due to differences in binding affinity or other factors, for the same densitometric readings, different amounts of antigens might be required or presented, resulting in differences in the values measured and the linear ranges of the two systems. However, the standard curves are linear and assays are sensitive enough to reflect the changes of MAT activity in both systems described in the present invention. Therefore, both of them may be used.
Several tests have proved that the results of using the SAMe coated plated and the standard in the present example were generally associated with poor repeatability and unstable linear range compared to those used in Examples 1-7 (coating the SAM analog antigen and using a stable analog as a standard). However, if the source of the raw material SAMe are well controlled and improved, and some related processes and methods are further improved and optimized, the solution in this example is also feasible.
The above embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the foregoing embodiment, and any other changes, modifications, substitutions, combinations, and other modifications made without departing from the spirit and principle of the present invention which are also possible and included in the present invention. Simplifications should all be equivalent to the replacement methods, and all are included in the protection scope of the claims of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201510755262.3 | Nov 2015 | CN | national |
This application is a 371 application of International Application No. PCT/IB2016/056666 filed Nov. 6, 2016; (published on May 11, 2017 as WO/2017/077509) titled “Methionine Adenosyltransferase (MAT) Biological Activity Assay Method and Reagent Kit”; the entire contents of which are hereby incorporated by reference herewith. This application also claims priority to CN Application No. 201510755262.3 filed Nov. 7, 2015.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2016/056666 | 11/6/2016 | WO | 00 |
