A method and apparatus for improving the accuracy of affinity constant measurements and readable medium

Abstract

The present application relates to the field of life science engineering technology, and particularly relates to a method and device for improving the accuracy of affinity constant measurement and readable medium, the method according to the parameter condition and calculation formula required when obtaining the affinity constant, first divides the initial concentration of the incubate into several concentration combinations, then obtains a large number of random numbers in the limited range of the measured physical concentration value of each incubate concentration combination as the analog value of the measured substance concentration, then substitutes the analog value of the measured substance concentration into the calculation formula to obtain the affinity constant analog value, and finally optimizes the accuracy of the affinity constant by the probability of the affinity constant analog value falling within a certain range of the theoretical value of the affinity constant, so that the affinity constant closer to the theoretical value can be obtained, the accuracy of the affinity constant is improved by the method of the present application.

Description

Technical Field

[0001] This invention relates to the field of life science engineering technology, specifically to the calculation of intermolecular affinity constants, and particularly to a method, apparatus, and readable medium for improving the accuracy of affinity constant measurement. Background Technology

[0002] In the complex systems of life, non-covalent interactions between various molecules are widespread, such as those between proteins and drugs, proteins and proteins, enzymes and substrates, and antigens and antibodies. In vivo, the different forms and degrees of action of these molecules under different environments produce a rich variety of physiological and biochemical effects. The interaction constant (affinity constant) is an important parameter characterizing the strength of intermolecular interactions (affinity). It is typically calculated by obtaining the concentration data of both interacting molecules at equilibrium and inputting them into the corresponding formula. Error reflects how close the calculated result is to the actual situation; therefore, assessing the magnitude of the error and improving the accuracy of the intermolecular affinity constant is crucial.

[0003] In the field of affinity constant analysis, the reported affinity constant values ​​vary considerably across different literatures, often fluctuating within a single order of magnitude. Even when obtained by the same team using the same method at different incubation concentrations, the differences in affinity constant values ​​can exceed 10-fold (Anal ChimActa. 2015; 886:98-106.). To date, few studies have evaluated the accuracy of the determined affinity constants, and there is a lack of systematic methods for assessing the accuracy of affinity constant calculations. Accuracy assessment can provide a theoretical basis for experimental decisions in affinity constant determination and help to unify the reference values ​​for affinity constants. Currently, only a few studies have used the derivative method to assess the error of affinity constants, that is, by observing the variation of the calculation error of affinity constants with experimental conditions through the derivative function, and using the differential to replace the absolute error for quantitative assessment of the error. Even when the derivative method is used to assess the error, an error as high as 100% still occurs (Protein Sci. 2023; 32(4):e4607.). The evaluation methods reported in the literature have significant limitations, as the evaluation results increase with the measurement error. Therefore, developing new affinity constant error evaluation methods that are adapted to actual measurement errors and improving the accuracy of affinity constant calculation is of significant theoretical and practical value. Summary of the Invention

[0004] The purpose of this invention is to establish a novel method, apparatus, and readable medium for improving the accuracy of intermolecular affinity constant determination.

[0005] In a first aspect, the present invention provides a method for improving the accuracy of affinity constant measurement, comprising the following steps:

[0006] S1. Obtain the formula for calculating the affinity constant, the true value range of the affinity constant, the allowable error range of the affinity constant, the instrument measurement error range, and the initial concentration range of different incubators;

[0007] S2. Select at least one point value from different initial concentration ranges of incubators, and divide the different initial concentration ranges of incubators into discontinuous concentration intervals based on the point values; then arrange and combine the selected point values ​​of different initial concentrations of incubators to obtain combinations of different incubator concentrations.

[0008] S3. Under each combination of incubator concentrations, randomly select a point value from the true value range of the affinity constant as the theoretical value of the affinity constant, then calculate the theoretical concentration value of the analyte under the current combination of incubator concentrations, and randomly select a point value from the instrument measurement error range of the theoretical concentration value of the analyte as the simulated value of the concentration of the analyte.

[0009] S4. Using the affinity constant calculation formula, calculate the affinity constant for each combination of analyte concentrations under the simulated values ​​of the analyte concentrations, and obtain the simulated value of the affinity constant; and calculate the relative error between the simulated value of the affinity constant and the theoretical value of the affinity constant.

[0010] S5. Repeat steps S3 and S4 at least 100 times, and calculate the probability that the relative error of the simulated affinity constant for each combination of incubator concentrations is within the allowable error range of the corresponding theoretical affinity constant.

[0011] S6. Evaluate the accuracy of the affinity constant by assessing the probability value and / or optimize the initial concentration of the incubator to optimize the accuracy of the intermolecular affinity constant.

[0012] This invention provides a method for improving the accuracy of affinity constant measurement. Based on the parameters and calculation formula required to obtain the affinity constant, the initial concentration of the incubator is first divided into several concentration combinations. Then, a large number of random numbers within the theoretical concentration range of the analyte for each incubator concentration combination are obtained as simulated analyte concentration values. These simulated analyte concentration values ​​are then substituted into the calculation formula to obtain simulated affinity constant values. Finally, the accuracy of the affinity constant is optimized by statistically analyzing the probability that the simulated affinity constant values ​​fall within the theoretical range, thus obtaining an affinity constant closer to the theoretical value. This invention transforms the accuracy problem of the affinity constant into a probability problem, thereby optimizing the calculation error of the affinity constant under conditions where measurement errors of any magnitude exist. This method is widely applicable, simple, and easy to implement, greatly improving the accuracy and reliability of the affinity constant calculation results, and has great application potential.

[0013] In step S1, the formula for calculating the affinity constant varies depending on the method used to determine the affinity constant.

[0014] The initial concentration range of the incubator is a point value and / or a range value.

[0015] Wherein, the true value interval of the affinity constant is either a point value or a range value; when the accurate affinity constant of the incubator can be confirmed by other means, the accurate affinity constant is taken as the true value interval of the affinity constant, which is a point value; if the accurate affinity constant cannot be confirmed by other means, it is obtained through preliminary experiments. The specific method includes: obtaining the measured concentration value of the analyte under any initial concentration condition of the incubator through preliminary experiments, then calculating the error range value of the measured concentration of the analyte based on the measurement error, and using the affinity constant calculation formula to calculate the range of the affinity constant within the error range value, which is the true value interval of the affinity constant, which is a range value. Wherein, the analyte is determined according to the affinity constant calculation formula and can be a complex or a free substance.

[0016] Preferably, the preliminary experiment includes at least one of ultrafiltration, equilibrium dialysis, magnetic bead fishing, and capillary electrophoresis.

[0017] In step 2, preferably, the number of point values ​​is not less than 10; the more points there are, the smaller the concentration range is divided, the larger the number of concentration combinations, the more accurate the probability statistics are, and the better the effect on improving the accuracy of the calculation of the intermolecular affinity constant; more preferably, the number of point values ​​is not less than 20.

[0018] Preferably, the point values ​​are discontinuous point values ​​selected in a geometric sequence.

[0019] Preferably, in step S3, the instrument measurement error range of the theoretical concentration value of the analyte is equal to the theoretical concentration value of the analyte multiplied by (1 ± instrument measurement error range).

[0020] Preferably, in step S3, the method for randomly selecting the simulated concentration value of the analyte includes a uniformly distributed random number selection method and / or a normally distributed random number selection method.

[0021] Preferably, in step S5, steps S3 and S4 are repeated at least 200 times; the greater the number of simulated affinity constant values ​​obtained, the more accurate the probability statistics, and the better the effect on improving the accuracy of the calculation of intermolecular affinity constants; more preferably, steps S3 and S4 are repeated at least 500 times.

[0022] Preferably, step S5 further includes: generating tables and / or relationship diagrams based on different combinations of incubator concentrations, corresponding simulated affinity constants, and probability values; through tables and relationship diagrams, the relationship between concentration combinations, simulated affinity constants, and probability values ​​can be displayed more intuitively.

[0023] Preferably, in step S6, the method for evaluating the accuracy of the affinity constant includes: judging the accuracy based on the probability value of the concentration combination corresponding to the concentration of the incubator to be evaluated; the higher the probability value, the higher the accuracy of the affinity constant to be evaluated.

[0024] Preferably, in step S6, the method for optimizing the initial concentration of the incubator includes: (1) determining the accuracy cutoff value; (2) taking the combination of incubator concentrations with a probability value not lower than the accuracy cutoff value as the preferred concentration combination, and confirming the preferred concentration range based on the preferred concentration combination; (3) selecting the incubator concentration within the preferred concentration range as the initial concentration of the incubator for calculating the affinity constant.

[0025] In a second aspect, the present invention also provides an apparatus for improving the accuracy of intermolecular affinity constant measurement, comprising at least one processor and a memory communicatively connected to the at least one processor; the memory stores instructions executable by the at least one processor, the instructions being executed by the at least one processor to enable the at least one processor to perform the aforementioned method for improving the accuracy of affinity constant measurement.

[0026] In a third aspect, the present invention also provides a computer-readable medium having stored thereon instructions executable by a processor, which, when executed by the processor, cause the processor to perform the aforementioned method for improving the accuracy of affinity constant measurement.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] 1. The present invention provides a method for improving the accuracy of affinity constant measurement. Based on the parameter conditions and calculation formula required to obtain the affinity constant, the initial concentration of the incubator is first divided into several concentration combinations. Then, a large number of random numbers within the theoretical concentration range of the analyte for each incubator concentration combination are obtained as simulated values ​​of the analyte concentration. The simulated values ​​of the analyte concentration are then substituted into the calculation formula to obtain the simulated value of the affinity constant. Finally, the accuracy of the affinity constant is optimized by statistically analyzing the probability that the simulated value of the affinity constant falls within the theoretical value range, thereby obtaining an affinity constant that is closer to the theoretical value.

[0029] 2. The method for improving the accuracy of affinity constant measurement in this invention transforms the accuracy problem of affinity constant into a probability problem. It can optimize the calculation error of affinity constant under the condition that any size of measurement error exists, thereby obtaining an affinity constant with higher accuracy.

[0030] 3. The method for improving the accuracy of affinity constant measurement in this invention has wide applicability, is simple and easy to implement, and greatly improves the accuracy and reliability of affinity constant calculation results, thus having great application potential. Attached image description:

[0031] Figure 1 This is a flowchart of the method for improving the accuracy of affinity constant measurement in Embodiment 1 of the present invention;

[0032] Figure 2 This is a graph showing the relationship between the combination of incubator concentrations and the probability value of the affinity constant in Example 1 of the present invention. Detailed Implementation

[0033] The present invention will be further described in detail below with reference to experimental examples and specific embodiments. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0034] Example 1

[0035] This invention provides a method for improving the accuracy of affinity constant measurement (see flowchart). Figure 1 To determine the interaction constant (affinity constant) between acetazolamide and human carbonic anhydrase I, the following steps are included:

[0036] S1. Obtain the formula for calculating the affinity constant and the true value interval of the affinity constant (the accurate affinity constant is confirmed to be 2.7 μM by other means (AnalChimActa. 2015; 886:98-106.), therefore, the true value interval of this affinity constant is a point value, i.e., K). pre =2.7μM), tolerance range of affinity constant, range of instrument measurement error, and range of initial concentrations of different incubators (calculated using the formula: Where P0 represents the initial incubation concentration of human carbonic anhydrase I, L0 represents the initial incubation concentration of acetazolamide, x represents the concentration of the acetazolamide-human carbonic anhydrase I complex when the interaction reaches equilibrium, i.e., the concentration of the analyte; Y represents the affinity constant; the instrument measurement error range RE1 = ±15%, the allowable error range of the affinity constant RE2 = ±20%; the minimum concentration of acetazolamide L... 0min =0.1 μM, maximum concentration of acetazolamide (L) 0max =10μM, minimum concentration P of human carbonic anhydrase I 0min =0.1 μM, maximum concentration P of human carbonic anhydrase I 0max =10μM; accuracy cutoff value is 95%;

[0037] S2. Select 10 discontinuous points from the initial concentration range of different incubators in a proportional sequence (the concentration range of acetazolamide L...). 0min ~L 0max Divide into a geometric sequence of 10 elements: 0.1, 0.17, 0.28, ..., 10; and define the concentration range P of human carbonic anhydrase I.0min ~P 0max Divide the data into a geometric sequence of 10 elements (0.1, 0.17, 0.28, ..., 10), and arrange and combine the discontinuous values ​​of different incubators to obtain different incubator concentration combinations ([0.1 0.1; 0.1 0.17; 0.1 0.28; ..., 10 10]).

[0038] S3. Under each combination of incubator concentrations, the true range of the affinity constant (K) is used. pre =2.7μM) was used as the theoretical value of the affinity constant, and then solved by computer. (AnalChem.2020;92(18):12630-12638.) The theoretical value of free protein concentration P was obtained, and then the theoretical concentration value of the complex PLe under the current incubator concentration combination was calculated. A point value was randomly selected within the instrument measurement error range (PLe±15%) of the theoretical concentration of the complex as the simulated concentration value of the complex; K is the affinity constant;

[0039] S4. Calculate using the affinity constant formula. Calculate the affinity constant for each combination of incubator concentrations under simulated analyte concentrations, and obtain the simulated affinity constant (K). cal ); and calculate the relative error (e) between the simulated value and the theoretical value of the affinity constant;

[0040] S5. Repeat steps S3 and S4 a total of 500 times, and calculate the probability (π) that the relative error of the simulated affinity constant for each incubator concentration combination is within the allowable error range (|e|≤20%) of the corresponding theoretical affinity constant; based on the different incubator concentration combinations and their corresponding simulated affinity constant values ​​and probability values, generate a relationship graph ( Figure 2 );

[0041] S6. Determine the accuracy cutoff value (95%); select the incubator concentration combination with a probability value not lower than the accuracy cutoff value (≥95%) as the preferred concentration combination (based on the statistical results of the probability values: when P0 is 0.1~0.17μM, the selectable L0 is 0.1~0.77μM; when P0 is 0.28~0.46μM, the selectable L0 is 0.1~0.28μM; when P0 is 0.77μM, the selectable L0 is 0.1~0.28μM; there is no recommended value for P0≥1.29μM); randomly select 5 groups of initial incubator concentrations from the preferred concentration combinations, then obtain the corresponding complex concentrations by ultrafiltration, and finally calculate the optimized intermolecular affinity constant using the affinity constant calculation formula (see Table 1 for the results).

[0042] Table 1. Optimized incubator concentrations and corresponding intermolecular affinity constants

[0043]

[0044] As shown in Table 1, by selecting the optimized concentration combination of the present invention as the initial concentration of the incubator, the relative error between the obtained intermolecular affinity constant and the theoretical value can be less than 15%, with a minimum error of only 2.9%. This demonstrates that the accuracy of the intermolecular affinity constant obtained by the method of the present invention has been significantly improved.

[0045] Example 2

[0046] This invention evaluates and further optimizes the accuracy of the obtained warfarin-human serum albumin interaction constant by using a method to improve the accuracy of affinity constant measurement;

[0047] (1) Evaluate the accuracy of the experimental affinity constant, including the following steps:

[0048] S1. Obtain the affinity constant calculation formula, the true value range of the affinity constant (obtain the measured concentration of the analyte (PLec = 1.62 μM) at the initial concentration of the incubator (5 μM) using dialysis, then calculate the error range of the measured concentration of the complex (PLec / 1.15~PLec / 0.85) based on the measurement error, and use the affinity constant calculation formula to calculate the range of the affinity constant within this error range, which is the true value range of the affinity constant (5.28 μM~9.53 μM)), the allowable error range of the affinity constant, the instrument measurement error range, and the range of different initial concentrations of the incubator (calculation formula: Where P0 represents the initial incubation concentration of human serum albumin, L0 represents the initial incubation concentration of warfarin, x represents the concentration of the warfarin-human serum albumin complex when the interaction reaches equilibrium, i.e., the required experimental measurement value; Y represents the affinity constant; the instrument measurement error range RE1 = ±15%, the allowable error range of the affinity constant RE2 = ±20%; the minimum concentration of warfarin L... 0min =5μM, maximum concentration of warfarin (L) 0max =5μM, minimum concentration P of human serum albumin 0min =5μM, maximum concentration of human serum albumin P 0max =5μM;

[0049] S2. Arrange and combine the point values ​​of warfarin and human serum albumin to obtain the incubator concentration combination (

[55] );

[0050] S3. Under this combination of incubator concentrations, within the true range of the affinity constant (K... pre A point value is randomly selected from the range of 5.28 μM to 9.53 μM as the theoretical value of the affinity constant, and then the value is solved by computer. (AnalChem.2020;92(18):12630-12638.) The theoretical value P of the free protein concentration was obtained, and then the theoretical concentration value of the complex under the current incubator concentration combination was calculated. A point value was randomly selected within the instrument measurement error range (PLe±15%) of the theoretical concentration of the complex as the simulated concentration value of the complex; K is the affinity constant;

[0051] S4. Calculate using the affinity constant formula. Calculate the affinity constant for each combination of incubator concentrations under simulated analyte concentrations, and obtain the simulated affinity constant (K). cal ); and calculate the relative error (e) between the simulated value and the theoretical value of the affinity constant;

[0052] S5. Repeat steps S3 and S4 a total of 500 times, and calculate the probability (π) that the relative error of the simulated affinity constant for each combination of incubator concentrations is within the allowable error range of the corresponding theoretical affinity constant (|e|≤20%).

[0053] S6. According to statistics, under the initial concentration combination of incubator

[55] , the probability value π = 66.6% of the affinity constant is within the allowable error range of the theoretical value of the affinity constant; that is, the accuracy of the experimental affinity constant is only 66.6%, and the incubation concentration needs to be adjusted to obtain a more accurate affinity constant.

[0054] (2) Optimize the incubator concentration to obtain a more accurate affinity constant, including the following steps:

[0055] S1. Obtain the affinity constant calculation formula, the true value range of the affinity constant (obtain the measured concentration of the analyte (PLec = 1.62 μM) at the initial concentration of the incubator (5 μM) using dialysis, then calculate the error range of the measured concentration of the analyte (PLec / 1.15~PLec / 0.85) based on the measurement error, and use the affinity constant calculation formula to calculate the range of the affinity constant within this error range, which is the true value range of the affinity constant (5.28 μM~9.53 μM)), the allowable error range of the affinity constant, the instrument measurement error range, and the range of different initial concentrations of the incubator; (instrument measurement error range RE1 = ±15%, allowable error range of affinity constant RE2 = ±20%; minimum concentration of warfarin L 0min =0.1μM, maximum warfarin concentration (L) 0max =5μM, minimum concentration P of human serum albumin 0min =5μM, maximum concentration of human serum albumin P 0max =5μM; accuracy cutoff value is 75%)

[0056] S2. Arrange the concentration range of warfarin in a geometric sequence L0min ~L 0max The values ​​were divided into a geometric sequence of 10 elements: 0.1, 0.15, 0.24, ..., 5; and the discontinuous values ​​of warfarin and human serum albumin were arranged and combined to obtain different combinations of incubator concentrations ([0.15; 0.155; 0.245; ..., 55]).

[0057] S3. Under each combination of incubator concentrations, within the true range of the affinity constant (K... pre A point value is randomly selected from the range of 5.28 μM to 9.53 μM as the theoretical value of the affinity constant, and then the value is solved by computer. (Anal Chem. 2020; 92(18):12630-12638.) The theoretical value P of the free protein concentration was obtained, and then the theoretical concentration of the complex under the current combination of incubator concentrations was calculated. A point value was randomly selected within the instrument measurement error range (PLe±15%) of the theoretical concentration of the complex as the simulated concentration value of the complex; K is the affinity constant;

[0058] S4. Calculate using the affinity constant formula. Calculate the affinity constant for each combination of incubator concentrations under simulated analyte concentrations, and obtain the simulated affinity constant (K). cal ); and calculate the relative error (e) between the simulated value and the theoretical value of the affinity constant;

[0059] S5. Repeat steps S3 and S4 a total of 500 times, and calculate the probability (π) that the relative error of the simulated affinity constant for each combination of incubator concentrations is within the allowable error range of the corresponding theoretical affinity constant (|e|≤20%).

[0060] S6. Based on the accuracy cutoff value (75%), select the incubator concentration combinations with probability values ​​not lower than the accuracy cutoff value (≥75%) as the preferred concentration combinations (based on the statistical results of probability values: when P0 is 5 μM, L0 can be selected in the range of 0.1 to 2.1 μM); randomly select 5 initial incubator concentrations from the preferred concentration combinations, then obtain the corresponding complex concentrations by ultrafiltration, and finally calculate the optimized intermolecular affinity constants using the affinity constant calculation formula (see Table 2 for the results).

[0061] Table 2. Optimized incubator concentrations and corresponding intermolecular affinity constants

[0062]

[0063] As shown in Table 2, by selecting the optimized concentration combination of the present invention as the initial concentration of the incubator, the relative error between the obtained intermolecular affinity constant and the theoretical value can be less than 25%, with a minimum error of only 3.06%. Compared with the original experimental affinity constant, its accuracy has been significantly improved.

[0064] Example 3

[0065] This invention provides a method to improve the accuracy of affinity constant measurement for determining the interaction constant (affinity constant) in a mixed system of a single target and multiple ligands (target A, ligands B, C, and D). The method includes the following steps:

[0066] S1. Obtain the affinity constant calculation formula and the true value interval of the affinity constant (based on other methods, the accurate affinity constants are confirmed to be 2.7μM, 0.27μM, and 2.2μM. Therefore, the true value interval of this affinity constant is a point value, i.e., K). pre =2.7μM, 0.27μM, 2.2μM), tolerance range of affinity constant, range of instrument measurement error, and range of initial concentrations of different incubators (calculation formula: Where P0 represents the initial incubation concentration of target A, L0 represents the initial incubation concentration of each ligand, x represents the concentration of the ligand-target complex (analyte) when the interaction reaches equilibrium; Y represents the affinity constant; the instrument measurement error range RE1 = ±15%, the affinity constant allowable error range RE2 = ±100%; and the minimum concentration of each ligand L... 0min =1μM, maximum concentration of each ligand L 0max =100μM, minimum concentration P at target site A 0min =1μM, maximum concentration P at target site A 0max =100 μM; known affinity constants K between the three ligands (B, C, D) and target A. pre The effective values ​​were 2.7 μM, 0.27 μM, and 2.2 μM, respectively; the accuracy cutoff was 95%.

[0067] S2. Select 10 discontinuous points from the initial concentration ranges of different incubators in a proportional sequence (the concentration ranges of ligands B, C, and D are L...). 0min ~L 0max The values ​​are divided into geometric sequences of 10 elements: 1, 1.67, 2.78, ..., 100; the concentration range P of target A is defined. 0min ~P 0max Divide the data into a geometric sequence of 10 elements (1, 1.67, 2.78, ..., 100), and arrange and combine the discontinuous point values ​​of different incubators to obtain different incubator concentration combinations ([1111; 1111.67; 1112.78; ..., 100100100100]).

[0068] S3. Under each combination of incubator concentrations, the true range of the affinity constant (K) is used. pre =2.7μM, 0.27μM, 2.2μM) were used as theoretical values ​​for the affinity constant, and then the values ​​were solved by computer. (AnalChem.2020;92(18):12630-12638.) The theoretical value P of the free protein concentration was obtained, and then the theoretical concentration value of each complex under the current incubator concentration combination was calculated. Within the instrument measurement error range (PLe±15%) of the theoretical concentration of the complex, a point value is randomly selected as the simulated concentration of the analyte; K is the affinity constant;

[0069] S4. Calculate using the affinity constant formula. Calculate the affinity constant for each combination of incubator concentrations under simulated analyte concentrations, and obtain the simulated affinity constant (K). cal ); and calculate the relative error (e) between the simulated value and the theoretical value of the affinity constant;

[0070] S5. Repeat steps S3 and S4 a total of 100 times, and calculate the probability (π) that the relative error of the simulated affinity constant for each combination of incubator concentrations is within the allowable error range of the corresponding theoretical affinity constant (|e|≤100%).

[0071] S6. Determine the accuracy cutoff value (95%); select the incubation concentration combination with a probability value not lower than the accuracy cutoff value (≥95%) as the preferred concentration combination (when the ligand and target concentrations are both 1 μM, the π value of the three ligands reaches 100%; therefore, select 1 μM as the initial incubation concentration for each component, and obtain the concentrations of the analyte as 0.103 μM, 0.554 μM, and 0.123 μM through experiments, and substitute them into the interaction constant calculation formula to obtain the interaction constants as 1.93 μM, 0.178 μM, and 1.57 μM, with relative errors of -28.59%, -34.36%, and -28.73% compared with the theoretical values.

[0072] Example 4

[0073] This invention provides a method to improve the accuracy of affinity constant measurement for determining the interaction constant (affinity constant) between target E and ligand F, comprising the following steps:

[0074] S1. Obtain the affinity constant calculation formula and the true value interval of the affinity constant (based on other methods, the accurate affinity constant is confirmed to be 400 μM; therefore, the true value interval of this affinity constant is a point value, i.e., K). pre =400μM), tolerance range of affinity constant, instrument measurement error range, and initial concentration range of different incubators; (formula for calculating affinity constant) Where P0 represents the initial incubation concentration of target E, L represents the free concentration of ligand F at equilibrium, Y represents the concentration of the ligand F-target E complex (analyte) at equilibrium of interaction, K represents the affinity constant, the instrument measurement error range RE1 = ±15%, the allowable error range of affinity constant RE2 = ±20%, ligand F concentration L0 = 10 nM, 20 nM, 40 nM, 80 nM, 160 nM, 320 nM, and the minimum concentration of target E P 0min =100μM, maximum concentration P at target site E 0max =100μM; accuracy cutoff value is 75%)

[0075] S2. Arrange and combine the concentrations of ligand F and target E to obtain different combinations of incubator concentrations ([10100; 20100; 40100; ..., 320100]);

[0076] S3. Under each combination of incubator concentrations, the true range of the affinity constant (K) is used. pre =400μM) was used as the theoretical value of the affinity constant, and then solved by computer. (AnalChem.2020;92(18):12630-12638.) The theoretical value P of the free protein concentration was obtained, and then the theoretical concentration value of each complex under the current incubator concentration combination was calculated. Within the instrument measurement error range (PLe±15%) of the theoretical concentration of the complex, a point value was randomly selected as the simulated concentration of the analyte; K is the affinity constant;

[0077] S4. Substitute all different combinations of incubator concentrations and the corresponding simulated values ​​of analyte concentrations into the affinity constant calculation formula. Calculate (Method 1: Use L0 instead of L for curve fitting, substitute PLes and L0 into formula Y to obtain the simulated affinity constant value; or Method 2: Use L for curve fitting, and the value of L is obtained through (L0 - PLes), substitute PLes and L into formula Y to obtain the simulated affinity constant value) to obtain the simulated affinity constant value for all different combinations of incubator concentrations (K). cal ); and calculate the relative error (e) between the simulated value and the theoretical value of the affinity constant;

[0078] S5. Repeat steps S3 and S4 a total of 100 times, and calculate the probability (π) that the relative error of the simulated affinity constant for each combination of incubator concentrations is within the allowable error range of the corresponding theoretical affinity constant (|e|≤20%).

[0079] S6. The probability values ​​show that, under the current incubation concentration settings, Method 1 has a 63% chance of obtaining an accurate affinity constant, while Method 2 has a 97% chance. Therefore, these conditions can be used to determine the affinity constants of target E and ligand F, but Method 2 should be preferred for obtaining the affinity constant.

[0080] Therefore, the concentrations of the analyte at six different concentrations were obtained using the saturation binding method: 2.20 nM, 3.35 nM, 8.31 nM, 15.66 nM, 26.78 nM, and 38.22 nM. Then, curve fitting was performed using L, yielding an affinity constant of 405.6 nM, with an error of 1.40% compared to the theoretical value of 400 nM (using L0 instead of L for curve fitting yielded an affinity constant of 475.9 nM, with an error of 18.97% compared to the theoretical value of 400 nM).

[0081] Example 5:

[0082] (1) The method of the present invention for improving the accuracy of affinity constant measurement is used to determine the accuracy of evaluating the interaction constant (affinity constant) between target G and ligand H, comprising the following steps:

[0083] S1. Obtain the affinity constant calculation formula, the true value range of the affinity constant (obtain the measured concentration of the analyte (PLec = 3.18 μM) at the initial concentration of the incubator (5 μM) using dialysis, then calculate the error range of the measured concentration of the complex (PLec / 1.15~PLec / 0.85) based on the measurement error, and use the affinity constant calculation formula to calculate the range of the affinity constant within this error range, which is the true value range of the affinity constant (0.49 μM~1.95 μM)), the allowable error range of the affinity constant, the instrument measurement error range, and the range of different initial concentrations of the incubator; Calculation formula: Where P0 represents the initial incubation concentration of target G, L0 represents the initial incubation concentration of ligand H, and x represents the concentration of the ligand-target complex (analyte) when the interaction reaches equilibrium, i.e., the required experimental measurement value; the instrument measurement error range RE1 = ±15%, the affinity constant allowable error range RE2 = ±20%; the minimum concentration of ligand H L... 0min =5μM, maximum concentration of ligand H L 0max =5μM, minimum concentration of target G P 0min =5μM, target G maximum concentration P 0max =5μM;

[0084] S2. Arrange and combine the point values ​​of target G and ligand H to obtain the incubator concentration combination (

[55] );

[0085] S3. Under this combination of incubator concentrations, within the true range of the affinity constant (K... pre A point value is randomly selected from the range of 0.49 μM to 1.95 μM as the theoretical value of the affinity constant, and then the solution is obtained by computer. (AnalChem.2020;92(18):12630-12638.) The theoretical value P of the free protein concentration was obtained, and then the theoretical concentration value of each complex under the current incubator concentration combination was calculated. A point value was randomly selected within the instrument measurement error range (PLe±15%) of the theoretical concentration of the complex as the simulated concentration value of the complex; K is the affinity constant;

[0086] S4. Calculate using the affinity constant formula. Calculate the affinity constant for each combination of incubator concentrations under simulated analyte concentrations, and obtain the simulated affinity constant (K). cal ); and calculate the relative error (e) between the simulated value and the theoretical value of the affinity constant;

[0087] S5. Repeat steps S3 and S4 a total of 100 times, and calculate the probability (π) that the relative error of the simulated affinity constant for each combination of incubator concentrations is within the allowable error range of the corresponding theoretical affinity constant (|e|≤20%).

[0088] S6. According to statistics, under the initial concentration combination of incubator

[55] , the probability value π = 17% of the affinity constant is within the allowable error range of the theoretical value of the affinity constant; that is, the accuracy of the experimental affinity constant is only 17%, and this incubation concentration is not suitable for the determination of the interaction constant between target G and ligand H.

[0089] (2) The accuracy of evaluating the interaction constant (affinity constant) between target G and ligand H using the differentiation method: The formula for calculating the affinity constant is as follows: Where P0 represents the initial incubation concentration of target G, L0 represents the initial incubation concentration of ligand H, x represents the ligand-target analyte concentration at which the interaction reaches equilibrium, i.e., the required experimental measurement value, and the function value y is the affinity constant. Substituting P0 = 5 μM and L0 = 5 μM into the equation and differentiating with respect to y, we obtain the derivative function as follows: Based on the truth values ​​of P0, L0, and affinity constant K pre =0.5μM, yielding a theoretical analyte concentration PLe = 3.65μM. Substituting PLe as x into y', we get y' = -0.88μM. With an error of ±15%, the affinity constant error ΔK = |y' × 0.15 × PLe| = 0.066μM, ΔK / K pre =13.2%. The derivative results show that the current incubation concentration has good accuracy and low calculation error.

[0090] However, according to other methods, the true value of the affinity constant between target G and ligand H is 0.5 μM. That is, the experimentally obtained affinity constant (1.04 μM) has an error of 108% compared with the theoretical affinity constant. Obviously, the error is very large, which is consistent with the evaluation results of the method of the present invention. Therefore, the method of the present invention is more accurate in evaluating the accuracy of the experimental affinity constant, while the evaluation results of the existing derivative method have limitations.

[0091] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for improving the accuracy of affinity constant measurement, characterized in that, Includes the following steps: S1. Obtain the formula for calculating the affinity constant, the true value range of the affinity constant, the allowable error range of the affinity constant, the instrument measurement error range, and the initial concentration range of different incubators; S2. Select at least one point value from different initial concentration ranges of incubators, and divide the different initial concentration ranges of incubators into discontinuous concentration intervals based on the point values; then arrange and combine the selected point values ​​of different initial concentrations of incubators to obtain combinations of different incubator concentrations. S3. Under each combination of incubator concentrations, randomly select a point value from the true value range of the affinity constant as the theoretical value of the affinity constant, then calculate the theoretical concentration value of the analyte under the current combination of incubator concentrations, and randomly select a point value from the instrument measurement error range of the theoretical concentration value of the analyte as the simulated value of the concentration of the analyte. S4. Using the affinity constant calculation formula, calculate the affinity constant for each combination of analyte concentrations under the simulated values ​​of the analyte concentrations, and obtain the simulated value of the affinity constant; and calculate the relative error between the simulated value of the affinity constant and the theoretical value of the affinity constant. S5. Repeat steps S3 and S4 at least 100 times, and calculate the probability that the relative error of the simulated affinity constant for each combination of incubator concentrations is within the allowable error range of the corresponding theoretical affinity constant. S6. Evaluate the accuracy of the affinity constant by assessing the probability value and / or optimize the initial concentration of the incubator to optimize the accuracy of the intermolecular affinity constant. The accuracy assessment of affinity constants includes: determining the accuracy based on the probability value of the concentration combination corresponding to the incubator concentration of the affinity constant to be assessed; Optimization of the initial concentration of the incubator includes: (1) Determine the accuracy cutoff value; (2) Select the combination of incubator concentrations with a probability value not lower than the accuracy cutoff value as the preferred concentration combination, and confirm the preferred concentration range based on the preferred concentration combination; (3) Select the incubator concentration within the preferred concentration range as the initial incubator concentration for calculating the affinity constant.

2. The method for improving the accuracy of affinity constant measurement according to claim 1, characterized in that, In step S1, the true value interval of the affinity constant is a point value or a range value; the specific method for obtaining the range value includes: obtaining the measured concentration value of the analyte under any initial concentration condition of the incubator through a preliminary experiment, then calculating the error range value of the measured concentration of the analyte based on the measurement error, and calculating the affinity constant range corresponding to the error range value using the affinity constant calculation formula, which is the true value interval of the affinity constant; the preliminary experiment includes at least one of ultrafiltration, equilibrium dialysis, magnetic bead fishing, and capillary electrophoresis.

3. The method for improving the accuracy of affinity constant measurement according to claim 1, characterized in that, In step S2, the number of point values ​​is not less than 10; the point values ​​are discontinuous point values ​​selected in a geometric sequence.

4. The method for improving the accuracy of affinity constant measurement according to claim 1, characterized in that, In step S3, the method for randomly selecting the simulated concentration value of the analyte includes the method of selecting uniformly distributed random numbers and / or the method of selecting normally distributed random numbers.

5. The method for improving the accuracy of affinity constant measurement according to claim 1, characterized in that, In step S5, steps S3 and S4 are repeated at least 200 times.

6. The method for improving the accuracy of affinity constant measurement according to claim 1, characterized in that, Step S5 further includes generating tables and / or relationship diagrams based on different combinations of incubator concentrations, corresponding simulated affinity constants, and probability values.

7. A device for improving the accuracy of intermolecular affinity constant measurement, characterized in that, The device includes at least one processor and a memory communicatively connected to the at least one processor; the memory stores instructions executable by the at least one processor to enable the at least one processor to perform a method for improving the accuracy of affinity constant measurement according to any one of claims 1-6.

8. A computer-readable medium having stored thereon instructions executable by a processor, said instructions, when executed by the processor, causing the processor to perform a method for improving the accuracy of affinity constant measurement as claimed in any one of claims 1-6.

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