Measurement method based on antibody binding rate

Abstract

The invention discloses a measurement method based on an antibody binding rate, and particularly relates to the field of antibody drug development and immunodetection in biopharmacy, and the method comprises the steps of antigen fixation, antibody dilution, antibody binding rate measurement and specificity index calculation. According to the scheme, the cross reactivity of the antibody is measured based on the antibody binding rate, quantification of the binding strength of the antibody and different antigens is achieved, and compared with a traditional static method, dynamic quantitative analysis of the binding rate more comprehensively evaluates the specificity and the cross reactivity of the antibody, and the detection accuracy is improved. And a more reliable analysis result is provided for antibody drug development and immunodetection.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody drug development and immune detection in biopharmaceuticals, and more specifically, to a measurement method based on antibody binding rate. Background Art

[0002] The antibody binding rate refers to the ratio of the antibody to the antigen to form a complex, which reflects the strength and efficiency of the antibody-antigen binding. It is usually expressed as the ratio of the amount of antibody binding to the total amount of antigen. The higher the binding rate, the stronger the affinity between the antibody and the antigen, and the higher the binding efficiency. It is an important parameter to measure the function of the antibody and affects the application effect of the antibody in experiments and diagnosis. Antibody cross-reactivity refers to the phenomenon that an antibody can not only bind to its target antigen, but also to other non-target antigens. This situation is usually due to the similar epitope structure between different antigens, which makes it impossible for the antibody to completely distinguish between the target antigen and the non-target antigen, thereby inducing non-specific binding and affecting the accuracy of the experimental results.

[0003] There is an important problem in the quantification of antibody cross-reactivity in the existing technology, that is, the antibody binding rate is not fully utilized to calculate the specificity of the antibody. Most traditional cross-reactivity evaluation methods rely only on static experiments, such as ELISA, Western blot, etc., and infer cross-reactivity by comparing the binding of antibodies to target antigens and non-target antigens, without using the antibody binding rate as the core parameter for specificity evaluation. This results in the inability to accurately quantify the binding dynamics of antibodies to different antigens under complex experimental conditions, thereby limiting the comprehensive analysis and measurement of antibody specificity. Summary of the invention

[0004] In order to overcome the above-mentioned defects of the prior art, an embodiment of the present invention provides a measurement method based on antibody binding rate. By measuring antibody cross-reactivity based on antibody binding rate, the binding strength of antibodies to different antigens is quantified to solve the problems raised in the above-mentioned background technology.

[0005] To achieve the above object, the present invention provides the following technical solution: a measurement method based on antibody binding rate, comprising:

[0006] Antigen fixation: Select target antigens and potential non-target antigens and fix them to the experimental platform. The target antigens and non-target antigens are used for cross-reactivity detection;

[0007] Antibody dilution: dilute the antibody sample to be tested and generate antibody solutions with different concentration gradients;

[0008] Antibody binding rate measurement: Add the diluted antibody solution to the wells where different antigens have been fixed for binding reaction and measure the strength of antibody-antigen binding; propose the antibody binding rate R b It is used to quantify the degree of complex formation between antibodies and antigens. The calculation formula is as follows:

[0009]

[0010] Where R b It is used to indicate the ratio of the number of complexes formed by the binding of antibodies and antigens to the total number of antigens;

[0011] Specificity index calculation: by R b Calculate the specificity index to assess the selectivity of the antibody:

[0012]

[0013] Among them, R b (target antigen) represents the binding rate between the antibody and the target antigen, R b (Non-target antigen) indicates the binding rate of the antibody to the non-target antigen.

[0014] In a preferred embodiment, by introducing the affinity constant K D To correct the specificity index, affinity constant K D Indicates the strength of antibody-antigen binding, affinity constant K D Including the dissociation rate v diss , combined frequency f bind Determined by the relationship between temperature and T;

[0015] The modified formula for the affinity constant is:

[0016]

[0017] Substitute the specificity index to form the modified affinity specificity index formula:

[0018]

[0019] where v diss is the dissociation rate, which indicates the rate at which the antibody dissociates from the antigen, v diss The unit is the reciprocal of seconds; f bind is the binding frequency, which indicates the frequency of antibody-antigen binding per unit time, f bind The unit is the reciprocal of seconds; T is temperature, which affects the molecular movement rate of antibodies and antigens; T 目 and T 非 represent the experimental temperatures for target antigen and non-target antigen, respectively.

[0020] In a preferred embodiment, by introducing the dissociation rate k off To correct the specificity index, the dissociation rate k off The dissociation rate k represents the rate at which the antibody dissociates from the antigen; off Including the dissociation activation energy E diss and the ionic strength of the solution I sol The modified formula of dissociation rate is:

[0021] k off =E diss I sol

[0022] Substituting the corrected specificity index, we obtain:

[0023]

[0024] Where E diss E is the dissociation activation energy, which represents the energy that the antibody and antigen need to overcome when they dissociate. diss The unit is joule; I sol is the ionic strength of the solution, which indicates the concentration of ions in the solution.

[0025] In a preferred embodiment, the specificity index is modified by introducing an electrostatic interaction factor, which is used to evaluate the charge attraction or repulsion between the antibody and the antigen. The electrostatic interaction factor includes the charge distribution difference ΔQ between the antibody and the antigen, the charge center distance d charge The mutual relationship between

[0026] The correction formula for electrostatic interaction is:

[0027]

[0028] Substituting it into the correction formula:

[0029]

[0030] Where ΔQ is the charge distribution difference, which represents the difference in charge between the antibody and the antigen; d charge is the charge center distance, which represents the distance between the surface charge centers of the antibody and antigen.

[0031] In a preferred embodiment, the specificity index is modified by introducing a multivalency factor of the antibody, which represents the ability of the antibody to bind to the antigen through multiple sites. The multivalency factor of the antibody includes the number of binding sites n 位点 and binding site cooperativity σ 协同 The multivalency factor correction formula is:

[0032] Multivalency factor = n 位点 ·σ 协同

[0033] Substituting this into the specificity index, the final multivalency factor correction formula is formed:

[0034]

[0035] Where n 位点 is the number of binding sites, indicating the number of binding sites between antibody and antigen; σ 协同 is the binding site cooperativity, which indicates the synergistic effect between multiple binding sites of the antibody;

[0036] Taking all the above factors into consideration, the final revised specificity index formula SI is formed 最终 :

[0037]

[0038] The specificity and cross-reactivity of antibodies on target antigens and non-target antigens were evaluated by introducing the antibody's binding rate, affinity constant, dissociation rate, electrostatic interaction factor, and antibody multivalency factor.

[0039] In a preferred embodiment, the antibody binding rate R is optimized by a gradient adjustment algorithm. b , the gradient regulation algorithm includes a dynamic binding efficiency regulation model;

[0040]

[0041] Among them, v bind is the binding rate, which indicates the binding speed between antibody and antigen; f bind A is the binding frequency, which indicates the number of times the antibody binds to the antigen per unit time; 位点 and A 总 Represent the number of binding sites and the total number of binding sites, respectively; F 静电 and F 流水 represent electrostatic force and hydrophobic force respectively; To evaluate electrostatic and hydrophobic forces by combining driving force threshold as a benchmark value; 1-r 占用 is the proportion of unoccupied binding sites; O 占用 is the site occupancy factor, which indicates the utilization efficiency of the binding site; E 抗原 is the antigen exposure; T is the experimental temperature, the temperature factor that affects the binding rate;

[0042] When the binding rate v bind Above its threshold And combined with the driving force F 结合 Exceeding its threshold Entering the high binding driving state, in the high binding driving state, the electrostatic force and hydrophobic force are reduced, and the site occupancy efficiency is increased; the specific adjustment formula is:

[0043]

[0044] in represents the adjusted electrostatic force; Indicates the electrostatic force before adjustment; ΔF 静电 Indicates the amplitude of electrostatic force adjustment; represents the adjusted hydrophobicity; Indicates the hydrophobic force before adjustment; ΔF 疏水 Indicates the amplitude of hydrophobic force adjustment; represents the adjusted occupancy factor; represents the occupancy factor before adjustment; ΔO 占用 Represents the increment of the occupancy factor.

[0045] In a preferred embodiment, the gradient adjustment algorithm includes a dilution equilibrium correction model;

[0046]

[0047] in C is the antibody concentration corrected after dilution; 初 is the initial concentration of the antibody; C 目标 is the target concentration of the antibody; is the flow rate actually measured during the antibody dilution process; V is the standard flow rate set during the dilution process; 修正 Indicates the actual dilution volume after correction during the dilution process; V 稀释 Indicates the standard dilution volume set during the dilution process; R 分布 Represents the actual distribution ratio of the antibody in the solution; R 理想 It indicates the ideal distribution ratio of the antibody in the solution;

[0048] When the dilution concentration Above target concentration C 目标 And flow rate Then it enters the high dilution concentration state. In the high dilution concentration state, the flow rate is reduced and the dilution volume is increased. The specific adjustment formula is:

[0049]

[0050] in Indicates the actual flow rate after adjustment; Indicates the actual flow rate before adjustment; ΔF 流速 Indicates the amount by which the flow rate is reduced;

[0051] in represents the corrected dilution volume; is the dilution volume before correction; ΔV 修正 Indicates the amount of volume added.

[0052] Technical effects and advantages of the present invention:

[0053] 1. This scheme measures antibody cross-reactivity based on antibody binding rate, thereby quantifying the binding strength of antibodies to different antigens. Compared with traditional static methods, dynamic quantitative analysis of binding rate more comprehensively evaluates the specificity and cross-reactivity of antibodies, providing more reliable analysis results for antibody drug development and immune detection;

[0054] 2. By introducing affinity constants, combined with the binding frequency and dissociation rate of antibodies and antigens, the binding performance of antibodies in different environments is corrected, the stability of the binding rate is improved, and it is suitable for antibody measurement under complex experimental conditions;

[0055] 3. The solution solves the influence of different solution environments on the measurement of antibody binding rate by dynamically adjusting the dissociation rate. It combines the dissociation activation energy and the solution ionic strength to ensure the stable binding of antibodies under different physiological conditions, reduce the data deviation caused by environmental changes, and improve the reliability of measurement.

[0056] 4. Electrostatic interaction correction ensures the accurate correspondence between antibody binding rate and specificity by regulating the difference in charge distribution and the distance between charge centers. This correction reduces nonspecific binding on non-target antigens, makes the antibody binding rate measurement more accurate, and improves the accuracy of specificity assessment;

[0057] 5. Multivalency correction makes the evaluation of antibody binding rate more comprehensive, and the multi-site binding of multivalent antibodies to antigens can be quantitatively analyzed. By adjusting the number and cooperativity of binding sites, the scheme improves the binding effect of antibodies to target antigens, while reducing the nonspecific binding rate of antibodies to non-target antigens. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 It is a flow chart of the present invention. DETAILED DESCRIPTION

[0059] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0060] Refer to the instruction manual Figure 1, a method for measuring antibody binding rate according to an embodiment of the present invention comprises:

[0061] Antigen fixation: Select target antigens and potential non-target antigens and fix them to the experimental platform. The target antigens and non-target antigens are used for cross-reactivity detection;

[0062] Antibody dilution: dilute the antibody sample to be tested and generate antibody solutions with different concentration gradients; these dilutions will be used to evaluate the antibody's ability to bind to the antigen at different concentrations; through the diluted antibody concentration, multiple sets of binding rate data can be generated to avoid non-specific binding that may occur under high concentration conditions;

[0063] Antibody binding rate measurement: Add the diluted antibody solution to the wells where different antigens have been fixed for binding reaction and measure the strength of antibody-antigen binding; propose the antibody binding rate R b It is used to quantify the degree of complex formation between antibodies and antigens. The calculation formula is as follows:

[0064]

[0065] Where R b It is used to indicate the ratio of the number of complexes formed by the binding of antibodies and antigens to the total number of antigens. The accurate measurement of the binding rate can provide data on the binding strength of antibodies on different antigens, providing a basis for subsequent specificity analysis.

[0066] Specificity index calculation: by R b Calculate the specificity index to assess the selectivity of the antibody:

[0067]

[0068] Among them, R b (target antigen) represents the binding rate between the antibody and the target antigen, R b (Non-target antigen) represents the binding rate of antibody to non-target antigen; specificity index SI is used to quantify the difference in antibody binding to target antigen and non-target antigen, reflecting the selectivity of the antibody; by calculating the specificity index, the specificity of the antibody to the target antigen can be clearly determined; the larger the value, the higher the selectivity of the antibody to the target antigen and the lower the cross-reactivity; the closer the value is to 1, the closer the antibody's binding ability to the target antigen and non-target antigen is, and there is a higher cross-reactivity.

[0069] By introducing the affinity constant K D To correct the specificity index, affinity constant K D Indicates the strength of antibody-antigen binding, affinity constant K D Including the dissociation rate v diss , combined frequency f bindThe relationship between K and temperature T determines that the lower the D Values ​​represent higher affinity;

[0070] The modified formula for the affinity constant is:

[0071]

[0072] Substitute the specificity index to form the modified affinity specificity index formula:

[0073]

[0074] where v diss is the dissociation rate, which indicates the rate at which the antibody dissociates from the antigen, v diss The unit is the reciprocal of seconds; f bind is the binding frequency, which indicates the frequency of antibody-antigen binding per unit time, f bind The unit is the reciprocal of seconds; T is temperature, which affects the molecular movement rate of antibodies and antigens; T 目 and T 非 Represent the experimental temperature of target antigen and non-target antigen respectively; the target and non in the formula represent the target antigen and non-target antigen respectively; the affinity of the antibody is further corrected by the binding frequency and dissociation rate to ensure that the binding rate reflects the difference in binding strength of the antibody at different temperatures.

[0075] By introducing the dissociation rate k off To correct the specificity index, the dissociation rate k off The dissociation rate k represents the rate at which the antibody dissociates from the antigen; off Including the dissociation activation energy E diss and the ionic strength of the solution I sol The modified formula of dissociation rate is:

[0076] k off =E diss I sol

[0077] Substituting the corrected specificity index, we obtain:

[0078]

[0079] Where E diss E is the dissociation activation energy, which represents the energy that the antibody and antigen need to overcome when they dissociate. diss The unit is joule or electron volt; I solis the ionic strength of the solution, indicating the concentration of ions in the solution; the target and non-target antigens in the formula represent the target antigen and the non-target antigen, respectively; the ionic strength affects the stability of the binding between the antibody and the antigen. The higher the ion concentration, the stronger the charge shielding effect and the slower the dissociation rate; by considering the dissociation activation energy of the target antigen and the non-target antigen and the ionic strength of the solution, the difference in the dissociation rate of the antibody on the two antigens is corrected, making the specificity index more accurate.

[0080] The specificity index is modified by introducing an electrostatic interaction factor, which is used to evaluate the charge attraction or repulsion between the antibody and the antigen. The electrostatic interaction factor includes the charge distribution difference ΔQ between the antibody and the antigen, the charge center distance d charge The mutual relationship between

[0081] The correction formula for electrostatic interaction is:

[0082]

[0083] Substituting it into the correction formula:

[0084]

[0085] Where ΔQ is the charge distribution difference, which indicates the difference in charge between the antibody and the antigen. The greater the charge difference, the stronger the attraction and the tighter the binding; d charge The charge center distance represents the distance between the surface charge centers of the antibody and antigen. The shorter the distance, the stronger the electrostatic interaction. By considering the charge difference and charge distance between the target antigen and the non-target antigen, the electrostatic interaction of the antibody is corrected and the specificity index is further adjusted. In the formula, SI 静电 It is through SI 解离速率 Multiply it by a correction factor, which is Among them, 1 represents the electrostatic interaction of the target antigen, which is used as the baseline or reference value because the target antigen is considered to be the ideal binding partner of the antibody and therefore no additional correction is required, while the electrostatic interaction of the non-target antigen is calculated by ΔQ 非 and d charge非 Make corrections; ΔQ 非 represents the charge difference between the antibody and the non-target antigen, d charge非 It represents the distance between the charge center of the antibody and the non-target antigen. This correction factor reflects the difference in electrostatic interaction between the antibody and the non-target antigen, helping to more accurately evaluate the cross-reactivity of the antibody on non-target antigens.

[0086] The specificity index is modified by introducing the antibody multivalency factor, which indicates the ability of the antibody to bind to the antigen through multiple sites. The antibody multivalency factor includes the number of binding sites n 位点and binding site cooperativity σ 协同 The multivalency factor correction formula is:

[0087] Multivalency factor = n 位点 ·σ 协同

[0088] Substituting this into the specificity index, the final multivalency factor correction formula is formed:

[0089]

[0090] where n 位点 is the number of binding sites, indicating the number of binding sites between the antibody and the antigen. The more binding sites there are, the more stable the binding is; σ 协同 The binding site synergy indicates the synergistic effect between multiple binding sites of an antibody. The stronger the synergistic effect, the more stable the binding. The specificity index is corrected by the number and synergy of binding sites so that the multivalent binding behavior of the antibody can be accurately reflected.

[0091] Taking all the above factors into consideration, the final revised specificity index formula SI is formed 最终 :

[0092]

[0093] By introducing the antibody binding rate, affinity constant, dissociation rate, electrostatic interaction factor and antibody multivalency factor, the specificity and cross-reactivity of the antibody on the target antigen and non-target antigen are evaluated, which makes up for the neglect of complex interactions in the existing technology. These correction factors ensure that the specificity index is not only based on the binding rate, but also reflects the complex interactions between antibodies and antigens during the binding process, making the specificity index more accurate and valuable for application.

[0094] The method for correcting the specificity index in this scheme introduces four key parameters. The introduction of each parameter has a clear physical or chemical meaning. The purpose is to more accurately evaluate the cross-reactivity and specificity of antibodies in different environments. Each correction parameter is based on specific molecular behavior and environmental conditions, which solves the defect that the existing technology only focuses on the simple mode of antibody-antigen surface binding and cannot explain complex interactions. First, the correction of the affinity constant accurately evaluates the binding strength between the antibody and the target antigen and the non-target antigen by introducing the two parameters of binding frequency and dissociation rate. The binding frequency f bind It reflects the efficiency of antibody binding to antigen within a certain period of time. A higher frequency indicates that the antibody can quickly find and bind to the antigen. The dissociation rate v dissIt indicates the speed at which the antibody detaches from the antigen. The slower the speed, the more stable the binding between the antibody and the antigen. The introduction of these two parameters significantly enhances the explanation of antibody-antigen binding behavior, especially at different temperatures, the binding frequency and dissociation rate will be affected by the ambient temperature. The existing technology usually only focuses on whether the antibody is successfully bound, but ignores these dynamic changes. This solution can more accurately distinguish the binding differences between the antibody and the target antigen and the non-target antigen by quantifying these dynamic processes, ensuring that the specificity evaluation of the antibody is more scientific.

[0095] The correction of the dissociation rate solves the defect of ignoring the performance of antibodies in different environments in the prior art by introducing the dissociation activation energy and the ionic strength of the solution. diss It represents the energy required for the antibody to dissociate from the antigen. The higher the energy, the more difficult it is for the antibody to separate from the antigen, indicating that the binding is more stable. The ionic strength of the solution is I sol Directly affects the charge interaction between antibodies and antigens. Especially in electrolyte solutions, the ionic strength determines the electrostatic shielding effect between antibodies and antigens, which means that in a high ionic strength environment, the binding stability of antibodies and antigens may decrease. By adding these parameters, the binding and dissociation behavior of antibodies under physiological conditions can be better simulated, so that the evaluation results can be closer to actual application scenarios, which relatively overcomes the limitation of existing technologies that only focus on ideal experimental conditions;

[0096] The correction of electrostatic interaction ensures that the surface charge difference between the antibody and the antigen is fully considered by introducing the charge distribution difference and the charge center distance. The charge distribution ΔQ of the antibody and the antigen determines the electrostatic attraction or repulsion between them. The greater the charge difference, the stronger the electrostatic attraction and the more stable the binding. If the charge distribution difference of the antibody to the non-target antigen is small, it may lead to nonspecific binding, thereby triggering cross-reactions. The charge center distance d charge It reflects the charge position between the antibody and the antigen. The shorter the distance, the stronger the electrostatic interaction. Traditional specificity assessment methods usually ignore the details of this electrostatic interaction. Through this correction, this scheme can identify non-specific binding caused by charge differences, further improve the accuracy of the specificity index, and ensure that the electrostatic effect is taken into account.

[0097] Finally, the multivalency correction accurately defines the binding capacity of antibodies and antigens by introducing the number of binding sites and the cooperativity of binding sites. 位点 It directly determines how many positions the antibody can bind to the antigen. The more sites, the stronger the binding and the stronger the affinity of the antibody. 协同It represents the synergistic effect between multiple binding sites. The stronger the synergy, the stronger the stability of the antibody when it binds to multiple sites at the same time. In addition, most of the existing technologies only evaluate the situation of a single binding site, ignoring the special properties of multivalent antibodies. The multivalent correction can evaluate antibodies bound to multiple sites, ensuring that the specificity index is not only applicable to simple monovalent antibodies, but also to complex multivalent antibodies, thereby expanding the scope of application of the method; through these corrections, the present method can provide a more accurate and comprehensive antibody specificity evaluation than the existing technology. The existing technology often only focuses on whether the antibody can bind to the target antigen, ignoring complex factors such as dynamic processes, solution environment, electrostatic effects and multivalent binding, which are the key points that cannot be ignored in actual biological systems. By introducing these parameters, this scheme can better simulate and reflect the actual performance of antibodies under physiological conditions, ensure the practical application value of the evaluation results, not only improve the accuracy of antibody screening, but also provide a more reliable scientific basis for antibody drug development.

[0098] Optimizing antibody binding rate R by gradient adjustment algorithm b , the gradient adjustment algorithm includes a dynamic binding efficiency regulation model; this model optimizes the binding rate measurement by adjusting the dynamic binding efficiency during the binding process between the antibody and the antigen to ensure that the binding process is not affected by too high or too low binding driving force. The binding efficiency model comprehensively considers the binding frequency, binding rate, and the physical and chemical interactions between the antibody and the antigen. The formula formation process is based on the comprehensive regulation of the binding driving force and the site occupancy factor. The binding driving force includes electrostatic and hydrophobic effects, which determine the strength and rate of antibody binding, while the site occupancy factor represents the availability of the antigen epitope and the occupancy of the binding site. These factors jointly affect the actual binding rate of the antibody, thereby forming a binding efficiency regulation formula;

[0099]

[0100] Among them, v bind is the binding rate, which indicates the binding speed between antibody and antigen; f bind A is the binding frequency, which indicates the number of times the antibody binds to the antigen per unit time; 位点 and A 总 Represent the number of available binding sites and the total number of binding sites, respectively; F 静电 and F 流水 They represent electrostatic force and hydrophobic force, which represent the two main physical and chemical effects driving the binding of antibodies and antigens. The electrostatic force originates from the mutual attraction or repulsion of the surface charges of antibodies and antigens, affecting the strength of the binding. The hydrophobic force originates from the interaction of hydrophobic regions, which will be brought closer by reducing contact with water, thereby enhancing the binding force between antibodies and antigens. In order to evaluate the strength of electrostatic force and hydrophobic force by combining the driving force threshold as a benchmark value; 1-r 占用 is the ratio of unoccupied binding sites, indicating the availability of antibody binding sites; 占用 E is the site occupancy factor, which indicates the utilization efficiency of the binding site. The more binding sites there are, the more stable the binding between the antibody and the antigen. 抗原 is the antigen exposure, which is used to reflect the accessibility of the antigen epitope; T is the experimental temperature, which is the temperature factor that affects the binding rate;

[0101] When the binding rate v bind Above its threshold And combined with the driving force F 结合 Exceeding its threshold Entering the high binding driving state, in the high binding driving state, by reducing the electrostatic force and hydrophobic force, increasing the site occupancy efficiency, ensuring the balance between the binding rate and the physical and chemical effects; the specific adjustment formula is:

[0102]

[0103] in represents the adjusted electrostatic force; Indicates the electrostatic force before adjustment; ΔF 静电 It represents the magnitude of electrostatic force adjustment, which is used to weaken or enhance the electrostatic interaction between antibody and antigen; represents the adjusted hydrophobicity; Indicates the hydrophobic force before adjustment; ΔF 疏水 Indicates the amplitude of hydrophobic force adjustment, which is used to increase or decrease the effect of hydrophobic interaction on antibody binding; represents the adjusted occupancy factor, where an increased value indicates that the antibody occupies more binding sites; represents the occupancy factor before adjustment; ΔO 占用 It represents the increment of occupancy factor, indicating how much the binding sites of antibody and antigen have increased.

[0104] The gradient adjustment algorithm includes a dilution equilibrium correction model. The dilution equilibrium correction model ensures the uniformity of antibody concentration distribution and avoids concentration deviation during the dilution process by dynamically regulating the antibody dilution process. The dilution equilibrium correction model introduces two major factors: concentration control factor and distribution dynamic adjustment. The concentration control factor takes into account the difference between the initial concentration and the target concentration of the antibody, and is corrected in combination with the dilution volume and flow rate. The distribution dynamic adjustment considers whether the antibody is evenly distributed in the solution, and specifically controls the uniformity of antibody concentration during the dilution process through flow rate distribution and volume correction factor.

[0105]

[0106] in C is the antibody concentration corrected after dilution; 初 is the initial concentration of the antibody; C 目标 is the target concentration of the antibody; The flow rate is actually measured during the antibody dilution process, reflecting the solution flow during the dilution process. In addition, the flow rate needs to be controlled during the antibody dilution process because the flow rate determines the diffusion rate and uniformity of the antibody in the diluent. Too fast or too slow a flow rate will lead to uneven distribution of antibody concentration, thus affecting the concentration accuracy after dilution and the accuracy of binding rate measurement. The ideal standard flow rate set during the dilution process is used as a benchmark for measuring the actual flow rate; V 修正 Indicates the actual dilution volume after correction during the dilution process, which is used to reflect the possible error correction in the experiment; V 稀释 Indicates the standard dilution volume set during the dilution process, which serves as a reference value for the ideal dilution volume; R 分布 It indicates the actual distribution ratio of the antibody in the solution, reflecting the actual distribution of the antibody in the solution during the dilution process; R 理想 It indicates the ideal distribution ratio of the antibody in the solution, and serves as a reference value for uniform distribution of the antibody during dilution;

[0107] When the dilution concentration Above target concentration C 目标 And flow rate Greater than Then it enters the high dilution concentration state. In the high dilution concentration state, the flow rate is reduced and the dilution volume is increased to ensure balanced dilution. The specific adjustment formula is:

[0108]

[0109] in Indicates the actual flow rate after adjustment; Indicates the actual flow rate before adjustment; ΔF 流速 Indicates the amount of flow rate reduction; when the flow rate is too high, by reducing ΔF 流速 To adjust the flow rate during the dilution process to ensure the accuracy of the antibody concentration distribution;

[0110] in represents the corrected dilution volume; is the dilution volume before correction; ΔV 修正 Indicates the amount of volume added; when the dilution volume is insufficient, by increasing ΔV 修正 To correct the dilution volume and ensure uniform distribution of the antibody in the solution;

[0111] The working principle of the dynamic binding efficiency regulation model is based on the regulation of binding efficiency during the binding process of antibodies and antigens. The binding efficiency of antibodies and antigens depends not only on the natural affinity between antibodies and antigens, but also on a variety of physical and chemical factors, including binding frequency, electrostatic interaction, hydrophobic interaction and the availability of binding sites. In this model, the binding rate is achieved by regulating these interaction forces and the occupancy state of binding sites. When the binding rate of antibodies and antigens is too fast or too slow, or the binding sites are too saturated or insufficient, the algorithm will dynamically adjust the binding efficiency. For example, when the binding rate is higher than the set threshold, the system will reduce the electrostatic force or hydrophobic interaction between antibodies and antigens, thereby reducing the binding driving force, while increasing the availability of unoccupied binding sites, thereby ensuring that the binding efficiency is within a reasonable range and optimizing the antibody binding rate.

[0112] The dilution equilibrium correction model ensures the concentration stability and uniformity of the antibody by dynamically regulating the antibody dilution process. During the antibody dilution process, if the concentration is too high or too low, it may cause measurement deviation of the antibody binding rate. The model dynamically adjusts the concentration distribution of the antibody according to parameters such as the initial concentration of the antibody, the target concentration, the solution flow rate, and the dilution volume. If the antibody concentration distribution is uneven or the flow rate is not appropriate during the dilution process, the system will automatically adjust the flow rate or dilution volume to make the distribution of the antibody in the solution more balanced, thereby ensuring that the antibody can accurately express its actual binding ability during the binding rate measurement process. Through refined adjustment, experimental errors caused by uneven dilution are avoided;

[0113] Based on the joint action of the two models in the gradient adjustment algorithm, the precise regulation of antibody binding rate is ensured; the dynamic binding efficiency control model is responsible for real-time monitoring and regulation of the binding rate and interaction force between antibodies and antigens to prevent measurement distortion caused by excessive or insufficient binding, while the dilution equilibrium correction model ensures the uniformity and stability of antibody concentration during the experiment, avoiding the influence of concentration fluctuations on binding rate measurement. The two ensure accurate measurement of antibody binding rate through coordinated control of the binding process and dilution process, improving the stability of the experiment and the reliability of the results;

[0114] It should be noted that the antibody binding rate and cross-reactivity are closely related. Cross-reactivity is essentially the non-specific binding of antibodies to non-target antigens, while the binding rate directly reflects the binding performance of antibodies on different antigens. By measuring the binding rate of antibodies to target antigens and non-target antigens, it can be clearly shown whether the antibody is specific or whether it also has a significant binding reaction with non-target antigens. The binding rate provides quantitative, real-time dynamic data. By comparing the binding rates of different antigens, the non-specific binding of antibodies can be accurately identified, thereby revealing cross-reactivity. If the binding rate is not used for evaluation and only static single binding experiments are relied upon, the actual binding of antibodies to multiple antigens may not be fully captured. Therefore, the binding rate can not only reveal the affinity of antibodies to target antigens, but also quantify the cross-reactivity of antibodies through the binding of non-target antigens, directly providing a quantitative basis for cross-reactivity.

[0115] In this scheme, a measurement method based on antibody binding rate is used to measure antibody cross-reactivity based on antibody binding rate. In the process of measuring antibody cross-reactivity based on antibody binding rate, the binding strength of antibodies to different antigens is accurately quantified to evaluate the cross-reactivity of antibodies. Antibody binding rate represents the ratio of the number of complexes formed by antibodies and antigens to the total number of antigens. This indicator can directly measure the binding ability of antibodies to antigens. By comparing the binding rates of antibodies to target antigens and non-target antigens, the specificity and cross-reactivity of antibodies can be quantified. If the binding rate of antibodies to target antigens is 80% and the binding rate to non-target antigens is 20%, this indicates that the antibodies have strong specificity to target antigens and weak cross-reactivity. This comparison of binding rates can clearly reflect the performance of antibodies in practical applications and provide reliable data support, which is particularly critical in the fields of antibody drug development and immunoassay.

[0116] In order to improve the accuracy of antibody binding rate, this scheme introduces affinity constant correction. The affinity constant measures the strength of antibody-antigen binding, which is mainly determined by the frequency of antibody-antigen binding and the rate of dissociation. Assuming that the affinity constant of the antibody and the target antigen is 1.2 micromoles per liter, the binding rate will be significantly improved, while for non-target antigens with an affinity constant of only 0.8 micromoles per liter, the binding rate will drop significantly. This correction ensures that the binding rate not only reflects the strength of the initial binding, but also can more accurately reflect the affinity of the antibody for different antigens through the correction process; through affinity correction, the binding rate of the antibody and the target antigen can be further improved, while reducing the non-specific binding of non-target antigens and enhancing the specificity of the antibody;

[0117] Dynamic regulation of the dissociation rate is the key to further improving the accuracy of the binding rate. The dissociation rate reflects the speed at which the antibody dissociates from the antigen epitope and is affected by the dissociation activation energy and the ionic strength of the solution. For example, when the ionic strength of the antibody is 50 mmol / l, the dissociation rate is low and the binding is more stable. When the ionic strength is increased to 100 mmol / l, the dissociation rate is accelerated and the binding is weakened. Therefore, the dissociation rate is adjusted to ensure the stability and accuracy of the antibody binding rate under different experimental conditions. If the dissociation rate of the antibody to the target antigen is 0.2s -1 (inverse per second), while the dissociation rate for non-target antigens is 1.0s -1 (inverse per second), this rate difference can effectively improve specificity and reduce cross-reactivity through dynamic regulation;

[0118] Electrostatic interaction is also one of the important factors affecting the antibody binding rate. The electrostatic interaction between the antibody and the antigen is regulated by the difference in charge distribution and the distance between the charge centers. For example, when the charge difference between the antibody and the target antigen is 15 charge units, the electrostatic attraction is strong and the binding is relatively stable; when the charge difference between the antibody and the non-target antigen is only 5 charge units, the binding is relatively loose. In order to avoid nonspecific binding, the antibody binding rate is dynamically adjusted by correcting the electrostatic interaction. If the electrostatic attraction of the antibody to the non-target antigen is weak, the binding rate will be reduced accordingly, thereby reducing the possibility of nonspecific binding. Through electrostatic correction, the antibody binding rate to the target antigen can be further improved, while the binding rate to the non-target antigen will be reduced.

[0119] Multivalency refers to the ability of an antibody to bind to an antigen through multiple binding sites, which has an important impact on the antibody binding rate. The more binding sites there are, the greater the chance of the antibody binding to the antigen. For example, the number of binding sites for an antibody to bind to a target antigen is 3, while the number of binding sites for an antibody to bind to a non-target antigen is only 1, which indicates that the binding rate of the antibody to the target antigen is significantly higher than that of the non-target antigen. Synergy is the synergistic effect between binding sites. The higher the synergy, the higher the binding rate. Through multivalency correction, the measurement of antibody binding rate is further optimized. When the number of binding sites for an antibody to bind to a target antigen is 4 and the synergy is high, the binding rate can reach 85%; when the number of binding sites for an antibody to bind to a non-target antigen is small and the synergy is weak, the binding rate is only 30%. Through this multivalency correction, the scheme can more accurately distinguish the binding of the antibody to the target antigen and the non-target antigen, and improve the accuracy of the binding rate measurement.

[0120] Dilution balance is another important factor in the measurement of antibody binding rate. If the initial concentration of the antibody is high or low, the dilution process will cause the binding rate to deviate from the true value. Assuming that the initial concentration of the antibody is 100μg / mL and the target concentration is 10μg / mL, the uniformity and stability of the antibody concentration can be ensured through reasonable dilution steps and balanced control. The scheme uses the dilution balance correction model to ensure that the antibody concentration is evenly distributed during the dilution process. When the diluted antibody concentration is stabilized at 10μg / mL, the binding rate can accurately reflect the binding ability of the antibody; if the dilution process is uneven, resulting in the antibody concentration in some areas reaching 50μg / mL, the binding rate measurement will be biased. Through dilution balance correction, the binding rate can remain stable, thereby providing accurate experimental results.

[0121] 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, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A measurement method based on antibody binding rate, characterized in that: include: Antigen fixation: Select target antigens and potential non-target antigens and fix them to the experimental platform. The target antigens and non-target antigens are used for cross-reactivity detection; Antibody dilution: dilute the antibody sample to be tested and generate antibody solutions with different concentration gradients; Antibody binding rate measurement: Add the diluted antibody solution to the wells where different antigens have been fixed for binding reaction and measure the strength of antibody-antigen binding; b It is used to quantify the degree of complex formation between antibodies and antigens. The calculation formula is as follows: Where R b It is used to indicate the ratio of the number of complexes formed by the binding of antibodies and antigens to the total number of antigens; Specificity index calculation: by R b Calculate the specificity index to assess the selectivity of the antibody: Among them, R b (target antigen) represents the binding rate between the antibody and the target antigen, R b (Non-target antigen) indicates the binding rate of the antibody to the non-target antigen.

2. The method for measuring antibody binding rate according to claim 1, characterized in that: By introducing the affinity constant K D To correct the specificity index, affinity constant K D Indicates the strength of antibody-antigen binding, affinity constant K D Including the dissociation rate v diss , combined frequency f bind Determined by the relationship between temperature and T; The modified formula for the affinity constant is: Substitute the specificity index to form the modified affinity specificity index formula: where v diss is the dissociation rate, which indicates the rate at which the antibody dissociates from the antigen, v diss The unit is the reciprocal of seconds; f bind is the binding frequency, which indicates the frequency of antibody-antigen binding per unit time, f bind The unit is the reciprocal of seconds; T is the temperature, which affects the molecular movement rate of antibodies and antigens; T 目 and T 非 represent the experimental temperatures for target antigen and non-target antigen, respectively.

3. The method for measuring antibody binding rate according to claim 2, characterized in that: By introducing the dissociation rate k off To correct the specificity index, the dissociation rate k off The dissociation rate k represents the rate at which the antibody dissociates from the antigen; off Including the dissociation activation energy E diss and the ionic strength of the solution I sol The modified formula of dissociation rate is: k off =E diss ·I sol Substituting the corrected specificity index, we obtain: Where E diss E is the dissociation activation energy, which represents the energy that the antibody and antigen need to overcome when they dissociate. diss The unit is joule; I sol is the ionic strength of the solution, which indicates the concentration of ions in the solution.

4. The method for measuring antibody binding rate according to claim 3, characterized in that: The specificity index is modified by introducing an electrostatic interaction factor, which is used to evaluate the charge attraction or repulsion between the antibody and the antigen. The electrostatic interaction factor includes the charge distribution difference ΔQ between the antibody and the antigen, the charge center distance d charge The mutual relationship between The correction formula for electrostatic interaction is: Substituting it into the correction formula: Where ΔQ is the charge distribution difference, which represents the difference in charge between the antibody and the antigen; d charge is the charge center distance, which represents the distance between the surface charge centers of the antibody and antigen.

5. The method for measuring antibody binding rate according to claim 4, characterized in that: The specificity index is modified by introducing the antibody multivalency factor, which indicates the ability of the antibody to bind to the antigen through multiple sites. The antibody multivalency factor includes the number of binding sites n 位点 and binding site cooperativity σ 协同 The multivalency factor correction formula is: Multivalency factor = n 位点 ·σ 协同 Substituting this into the specificity index, the final multivalency factor correction formula is formed: Where n 位点 is the number of binding sites, indicating the number of binding sites between antibody and antigen; σ 协同 is the binding site cooperativity, which indicates the synergistic effect between multiple binding sites of the antibody; Taking all the above factors into consideration, the final revised specificity index formula SI is formed 最终 : The specificity and cross-reactivity of antibodies on target antigens and non-target antigens were evaluated by introducing the antibody's binding rate, affinity constant, dissociation rate, electrostatic interaction factor, and antibody multivalency factor.

6. A method for measuring antibody binding rate according to claim 5 or 1, characterized in that: Includes gradient adjustment algorithm; Optimizing antibody binding rate R by gradient adjustment algorithm b , the gradient regulation algorithm includes a dynamic binding efficiency regulation model; Among them, v bind is the binding rate, which indicates the binding speed between antibody and antigen; f bind A is the binding frequency, which indicates the number of times the antibody binds to the antigen per unit time; 位点 and A 总 Represent the number of binding sites and the total number of binding sites, respectively; F 静电 and F 流水 represent electrostatic force and hydrophobic force respectively; To evaluate electrostatic and hydrophobic forces by combining driving force threshold as a benchmark value; 1-r 占用 is the proportion of unoccupied binding sites; O 占用 is the site occupancy factor, which indicates the utilization efficiency of the binding site; E 抗原 is the antigen exposure; T is the experimental temperature, the temperature factor that affects the binding rate; When the binding rate v bind Above its threshold And combined with the driving force F 结合 Exceeding its threshold Entering the high binding driving state, in the high binding driving state, the electrostatic force and hydrophobic force are reduced, and the site occupancy efficiency is increased; the specific adjustment formula is: in represents the adjusted electrostatic force; Indicates the electrostatic force before adjustment; ΔF 静电 Indicates the amplitude of electrostatic force adjustment; represents the adjusted hydrophobicity; Indicates the hydrophobic force before adjustment; ΔF 疏水 Indicates the amplitude of hydrophobic force adjustment; represents the adjusted occupancy factor; represents the occupancy factor before adjustment; ΔO 占用 Represents the increment of the occupancy factor.

7. The method for measuring antibody binding rate according to claim 6, characterized in that: The gradient adjustment algorithm includes a dilution equilibrium correction model; in C is the antibody concentration corrected after dilution; 初 is the initial concentration of the antibody; C 目标 is the target concentration of the antibody; is the flow rate actually measured during the antibody dilution process; V is the standard flow rate set during the dilution process; 修正 Indicates the actual dilution volume after correction during the dilution process; V 稀释 Indicates the standard dilution volume set during the dilution process; R 分布 Represents the actual distribution ratio of the antibody in the solution; R 理想 It indicates the ideal distribution ratio of the antibody in the solution; When the dilution concentration Above target concentration C 目标 And flow rate Then it enters the high dilution concentration state. In the high dilution concentration state, the flow rate is reduced and the dilution volume is increased. The specific adjustment formula is: in Indicates the actual flow rate after adjustment; Indicates the actual flow rate before adjustment; ΔF 流速 Indicates the amount by which the flow rate is reduced; in represents the corrected dilution volume; is the dilution volume before correction; ΔV 修正 Indicates the amount of volume added.

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