Multi-parameter condition optimization method based on homogeneous immunodetection and application

Abstract

The invention provides a multi-parameter condition optimization method based on homogeneous immunodetection and application. The optimization method comprises the following steps: defining a multi-parameter combination to be optimized, wherein the multi-parameter combination comprises optimization parameters in a probe preparation stage and optimization parameters in an immune reaction stage; constructing a multi-objective optimization model, and taking at least two of the detection sensitivity, the detection range, the linear correlation, the signal-to-noise ratio and the light-emitting signal as optimization objectives; adopting Box-Behnken design to generate an experimental matrix containing a plurality of parameter combinations, and recording a response value; modeling the experimental matrix based on quadratic polynomial regression, and establishing a nonlinear relation model of parameters and response values; solving the model through a numerical optimization algorithm to obtain a target parameter combination enabling a response value to be optimal; and repeatedly executing homogeneous immunodetection by using the target parameter combination to verify the consistency. When the optimization method provided by the invention is used for screening the optimal condition combination of the marker, the experiment period is short, the trial and error cost is low, and the research and development period is remarkably shortened.

Description

Technical Field

[0001] The present invention relates to the field of detection technologies, and in particular, to a multi-parameter condition optimization method and application based on homogeneous immunoassay. Background Art

[0002] With the innovation of biomedical research paradigms, ultrasensitive quantitative detection technologies for biomarkers have become the core support for precision medicine and disease dynamic monitoring. In this context, Human Chorionic Gonadotropin (HCG), as a key biomarker for pregnancy-related diseases, trophoblastic diseases, and some tumors, the improvement of its detection performance is of great significance for clinical decision-making. HCG is secreted by placental trophoblast cells and is used to assist in the diagnosis of ectopic pregnancy and threatened abortion through the rule of doubling concentration in the early stage of pregnancy. In the field of oncology, it is closely related to the progression of choriocarcinoma, germ cell tumors, and non-reproductive system tumors. However, existing technologies face systematic challenges in balancing detection performance indicators: the improvement of sensitivity is often accompanied by a decrease in detection timeliness, the expansion of the linear range is easily restricted by the stability of the reaction system, and the improvement of a single indicator often leads to an imbalance in the system performance. For example, the traditional ELISA method can improve sensitivity by extending the incubation time, but high-concentration samples are prone to the hook effect, resulting in false negatives; while digital immunoassay technology can achieve single-molecule detection, but the preparation cost of its micro-nano chips is high and the detection range is limited (usually not exceeding 4 orders of magnitude), making it difficult to meet the needs of emergency, point-of-care testing, or resource-constrained scenarios.

[0003] Homogeneous immunoassay technology has been widely used in clinical practice due to its advantage of eliminating cumbersome sample pretreatment steps, but its performance optimization highly depends on the fine regulation of experimental condition parameters. Current research generally faces the collaborative dilemma of parameter optimization: the setting of key parameters such as reaction temperature, antibody conjugation pH value, and probe concentration highly depends on the operator's experience, which is prone to introducing subjective biases. For example, although PBS (pH = 7.4) is a commonly used buffer, its storage stability is poor, and the pH drift between batches may lead to a decrease in precision; during the antibody conjugation process, the non-linear coupling effect between the amount of streptavidin used and the reaction time often traps experimenters in the "local optimum" trap, resulting in reagent waste (the consumption per single optimization reaches 500 μL / condition) and a trial-and-error cycle of up to several months. In addition, the dynamic demand for detection conditions in clinical scenarios further exacerbates the technical challenges - in the early stage of pregnancy, HCG as low as 0.1 - 0.5 mIU / mL needs to be detected to distinguish normal pregnancy from abnormal conditions, while samples from tumor patients or in the second and third trimesters of pregnancy may contain up to 1e 6 mIU / mL of HCG, and a wide detection range is required to avoid missed detection or false positives. Existing methods mostly focus on the improvement of a single indicator, such as enhancing the signal through rare earth elements to improve sensitivity, but ignore the collaborative optimization of the linear range and economy, resulting in detection methods that are difficult to adapt to complex clinical needs. Summary of the Invention

[0004] In view of the above-mentioned defects of the prior art, the present invention provides a multi-parameter condition optimization method and application based on homogeneous immunoassay. The multi-parameter condition optimization method provided by the present invention is a multi-condition trade-off optimization method based on Box-Behnken design to screen the HCG detection conditions in homogeneous long afterglow detection, and verifies the practicability of the model through experiments. Using an algorithm, the optimal condition combination for measuring HCG by homogeneous immunoassay is screened. The specific conditions include antibody screening, optimization of the amount of conjugated antibody, optimization of the amount of conjugated streptavidin, optimization of the conjugation buffer, optimization of the conjugation time, optimization of the pH of the immunoreaction, and immunoreaction time. A condition combination that cannot be simultaneously superior to other solutions in all objectives is found through a multi-objective optimization algorithm, and finally it can be widely applied in the market.

[0005] In the first aspect of the present invention, the multi-parameter condition optimization method based on homogeneous immunoassay includes the following steps:

[0006] Define the multi-parameter combination to be optimized, including the optimization parameters in the probe preparation stage and the optimization parameters in the immunoreaction stage;

[0007] Construct a multi-objective optimization model with at least two of detection sensitivity, detection range, linear correlation, signal-to-noise ratio, and luminescence signal as optimization objectives;

[0008] Use Box-Behnken design to generate an experimental matrix containing multiple parameter combinations, and record the response values of each group of experiments;

[0009] Based on quadratic polynomial regression, model the experimental matrix to establish a non-linear relationship model between parameters and response values;

[0010] Solve the model through a numerical optimization algorithm to obtain the target parameter combination that makes the response value reach the optimum;

[0011] Repeat the homogeneous immunoassay using the target parameter combination to verify the consistency between the response value and the model prediction value.

[0012] In some embodiments, the optimization method further includes constraint conditions that limit the value range of parameters.

[0013] In some embodiments, the optimization parameters in the probe preparation stage include at least one of the amount of conjugated antibody, the amount of conjugated streptavidin, the conjugation buffer, and the conjugation time.

[0014] In some embodiments, the optimization parameters in the immunoreaction stage include at least one of the pH of the immunoreaction, the immunoreaction time, the incubation temperature, and the surfactant.

[0015] In some embodiments, the amount of the antibody ranges from 10 μg to 300 μg.

[0016] In some embodiments, the amount of the conjugated streptavidin ranges from 10 μg to 500 μg.

[0017] In some embodiments, the types of the conjugation buffer include one of morpholine ethanesulfonic acid buffer, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, phosphate buffer, and tris(hydroxymethyl)aminomethane buffer.

[0018] In some embodiments, the pH range of the conjugation buffer is 6 - 8, and the conjugation time range is 0.5 - 4 h.

[0019] In some embodiments, the pH range of the immunoreaction is 7.0 - 8.5, and the immunoreaction time range is 1 - 20 min.

[0020] In the second aspect of the present invention, there is provided an application of the foregoing optimization method in the condition screening of a chorionic gonadotropin kit.

[0021] When the multi-parameter condition optimization method based on homogeneous immunoassay provided by the present invention is used to screen the optimal condition combination of markers, the experimental period is short and the trial-and-error cost is low. By simulating with an algorithm to replace a large number of experiments, the R & D cycle is significantly shortened, and the solutions that do not meet the experimental feasibility (such as the antibody concentration being lower than the lowest effective threshold) are automatically excluded. For example, an invalid combination that causes signal weakening due to too low an amount of streptavidin is avoided. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Shows a schematic diagram of the detection principle of the homogeneous immunoluminescence method in the prior art;

[0023] Figure 2 Shows a schematic diagram of the detection principle of the homogeneous immunoluminescence method in the embodiments of the present invention;

[0024] Figure 3 Shows a flowchart of the multi-parameter condition optimization method based on homogeneous immunoassay in the embodiments of the present invention;

[0025] Figure 4 Shows the results of testing different standards with different HCG monoclonal antibodies;

[0026] Figure 5 Shows the graph of the amount of HCG antibody used in the conjugation reaction and the change in the long afterglow luminescence signal;

[0027] Figure 6 Shows the graph of the amount of streptavidin used in the conjugation reaction and the change in the long afterglow luminescence signal;

[0028] Figure 7Shows the change diagram of the long afterglow luminescence signal with the buffer pH in the coupling reaction;

[0029] Figure 8 Shows the change diagram of the long afterglow luminescence signal with the coupling time;

[0030] Figure 9 Shows the test results of the pH optimization experiment of the homogeneous immunoassay reaction solution;

[0031] Figure 10 Shows the test results of the incubation time optimization experiment before homogeneous immunoassay;

[0032] Figure 11 Shows the standard curve diagram of the HCG long afterglow homogeneous detection;

[0033] Figure 12 Shows the correlation between the HCG detection of long afterglow homogeneous immunoassay and Abbott chemiluminescence method. Detailed implementation manners

[0034] The following will further elaborate in more detail on the multi-parameter condition optimization method and application based on homogeneous immunoassay of the present invention in combination with specific embodiments. It should be understood that the following embodiments are only for illustrative explanation of the present invention and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0035] It should be understood that the various steps recorded in the method embodiments of the present disclosure can be executed sequentially and / or in parallel. In addition, the method embodiments may include additional steps and / or omit the steps shown. The scope of the present disclosure is not limited in this regard.

[0036] The term "including" and its variations used herein are open-ended, that is, "including but not limited to". The term "based on" means "at least partially based on". The term "one embodiment" means "at least one embodiment"; the term "another embodiment" means "at least one additional embodiment"; the term "some embodiments" means "at least some embodiments". The relevant definitions of other terms will be given in the following description.

[0037] The human chorionic gonadotropin (HCG) mentioned in the context of the present invention is a glycoprotein hormone secreted by placental trophoblast cells and has important clinical application value in the field of in vitro diagnosis. HCG detection is not only widely used for the diagnosis and monitoring of pregnancy-related diseases, but also plays a key role in the diagnosis and treatment monitoring of trophoblastic diseases, germ cell tumors, and certain non-reproductive system tumors. During pregnancy, changes in HCG levels are important indicators for diagnosing early pregnancy, ectopic pregnancy, threatened abortion, and trophoblastic diseases. For example, in normal pregnancy, HCG levels increase significantly with the increase of gestational weeks, while in ectopic pregnancy and threatened abortion, the changes in HCG levels are significantly different from those in normal pregnancy. In addition, abnormal elevation of HCG levels may also indicate trophoblastic diseases and tumor diseases. For example, in some germ cell tumors and non-reproductive system tumors, the increase in HCG levels is closely related to the presence and progression of tumors. Therefore, the change in HCG expression level provides a decision-making basis for the diagnosis and management of related diseases, and accurate detection of HCG is crucial for early diagnosis and timely intervention. With the continuous progress of detection technology, the sensitivity and specificity of HCG detection have been significantly improved. However, in order to meet clinical needs, HCG detection requires a wide detection range. On the one hand, the HCG levels in the middle and late pregnancy or some disease tumor patients may be extremely high, and a wide detection range is needed to accurately reflect the condition. On the other hand, the HCG levels in early pregnancy and some tumor patients may be at a relatively low level, and sensitive detection methods are needed to meet the early diagnosis requirements. Therefore, the development of HCG detection methods with a wide detection range helps to improve the accuracy of diagnosis, monitor the progression of diseases, and evaluate the treatment effect. At the technical level, in recent years, the detection sensitivity of biomarkers has been significantly improved, but there are still relatively few reported homogeneous wash-free detection methods that can simultaneously achieve sensitivity, rapidity, and wide range. Digital immunoassay technology can even achieve immunoassay at the single molecule or single particle level through the action of hundreds of thousands of tiny reaction chambers and mathematical operations in the chip. However, the detection range is often limited and difficult to break through four orders of magnitude. Moreover, these detection technologies have very high requirements in terms of time and cost, and cannot meet the application requirements of emergency, point-of-care testing, or resource-poor scenarios. Therefore, the development of optimal combination screening for HCG is particularly important when developing HCG test kits.

[0038] It should be understood that in the embodiments of the present invention, only a biomarker such as HCG (human chorionic gonadotropin) is used as an example to demonstrate the advantages and steps of the multi-parameter condition optimization method. This method lies in establishing an extensible parameter optimization framework, which can precisely control the detection performance of different biomarkers by dynamically adjusting the combination of key variables in the detection system. This modular design concept enables the rapid establishment of optimization schemes even in the face of newly discovered biomarkers, significantly shortening the development cycle of detection methods. Therefore, the multi-parameter condition optimization method provided in the embodiments of the present invention can also be applied to the condition screening of other biomarkers using homogeneous immunoassays, such as infection and inflammation biomarkers, including C-reactive protein (CRP), procalcitonin (PCT), interleukin-6 (IL-6), etc., and can also be nephropathy biomarkers such as serum creatinine (Scr), blood urea nitrogen (BUN), urinary β2-microglobulin (β2-MG), urinary microalbumin / creatinine ratio (ACR), or cancer biomarkers. It can be understood that the constraint conditions, optimization parameters, and optimization objectives of these biomarkers may not be exactly the same, but based on the principle of this multi-parameter condition optimization method, it can be applied to these biomarkers and should all be within the scope of protection of this patent.

[0039] Those skilled in the art can know that generally, in the process of optimizing the conditions of a homogeneous immunoassay system, the method of controlling variables, as a classic research paradigm, has irreplaceable advantages in clarifying the causal relationship between a single parameter and the reaction result. By fixing other variables and adjusting the target parameter one by one, this method can efficiently establish a quantitative relationship between a single factor and the response value, and is particularly suitable for systems with clear action mechanisms and strong independence between variables. However, when facing actual biomedical detection scenarios, the limitations of this method gradually emerge. Since the immune reaction involves the complex coupling of multi-dimensional parameters such as antibody affinity, antigen concentration, buffer pH value, ionic strength, and reaction time, there are often non-linear interaction effects between variables. For example, a change in antibody concentration may change the colloidal stability of the solution system, thereby affecting the antigen-antibody binding kinetics; while fluctuations in pH value not only directly affect protein conformation but may also indirectly regulate electrostatic interactions by changing ionic strength. At this time, the strategy of optimizing single factors one by one cannot capture the synergistic effects between variables, resulting in the optimization result deviating from the optimal parameter combination. As the number of parameter dimensions increases, the number of experiments grows exponentially, and the cost consumed is relatively high.

[0040] Based on the above deficiencies existing in the prior art, the embodiments of the present invention provide a multi-parameter condition optimization method and application based on homogeneous immunoassay, and particularly illustrate it by taking the condition screening of human chorionic gonadotropin as an example.

[0041] Homogeneous immunoassay is a homogeneous assay method that can be used for immunoassay of analytes. With its unique energy transfer mechanism and chemiluminescence principle, it realizes homogeneous non-washing, rapid, highly sensitive and high-throughput detection, and can be applied to basic medical research, DNA molecule detection, food safety, new drug development and other fields. The detection principle diagram of homogeneous immunoassay based on photochemical long afterglow refers to Figure 1 , that is, mainly based on the distance effect of energy transfer between donor microspheres and acceptor microspheres, and the concentration information of analytes is indicated by the intensity of long afterglow luminescence signal. Generally speaking, the donor microspheres are coated with sensitizers, and the acceptor microspheres are coated with buffers and luminophores. The acceptor microspheres approach the donor microspheres through immune reaction binding and other means. The light source selectively excites the donor microspheres, and then activates the acceptor microspheres through the energy transfer process, and finally emits a luminescence signal, which is detected by an optical detector and used for result analysis. In other words, that is, the sensitized microspheres and the luminescent microspheres coexist in the solution, and the two microspheres are respectively coupled with antibody pairs for immunoassay by using surface carboxyl groups. When the corresponding analyte antigen exists, the sensitized microspheres and the luminescent microspheres are closely connected together through immune reaction, and the photochemical energy storage and transfer process can be carried out completely to emit photon signals. Therefore, the sensitized microspheres and the luminescent microspheres modified with HCG antibodies form a long afterglow luminescence probe with HCG detection function, and long afterglow luminescence occurs only when HCG antigen exists in the solution, and effective long afterglow homogeneous immunoassay can be realized without washing.

[0042] The homogeneous immunoassay principle in the embodiments of the present invention refers to Figure 2 From Figure 2It can be seen that there are some differences between the homogeneous immunoassay in the embodiments of the present invention and the prior art. This is mainly reflected in that in order to amplify the signal, streptavidin (abbreviated as SA) is usually conjugated. In the prior art, streptavidin is usually conjugated to the donor microspheres, while in the embodiments of the present invention, streptavidin is conjugated to the receptor microspheres. When streptavidin is conjugated to the surface of the donor microspheres, the fluorescently labeled antibody / antigen is mainly anchored through the biotin-streptavidin system. When the donor is irradiated with excitation light, its emission spectrum needs to overlap with the absorption spectrum of the receptor microspheres, so as to transfer energy to the receptor through FRET (fluorescence resonance energy transfer). At this time, on the one hand, the tetrameric structure of SA is utilized, and each microsphere can conjugate more biotinylated fluorescent molecules. On the other hand, by controlling the conjugation density of SA, the distance between the donor and the receptor microspheres (the ideal distance is 1-10 nm) is adjusted to maximize the FRET efficiency. When SA is conjugated to the receptor microspheres, a quencher (such as Dabcyl) or a long-wavelength fluorescent molecule (such as Cy5) is usually anchored. The quencher absorbs the energy of the donor through FRET to inhibit non-specific fluorescence interference. In competitive immunoassay, the SA on the receptor microspheres can bind to the biotinylated antibody not occupied by the target, and the analyte is quantitatively detected by the change in signal intensity (such as the release of quenching).

[0043] Specifically, as Figure 3 shown, the steps of a multi-parameter condition optimization method based on homogeneous immunoassay in the embodiments of the present invention are as follows:

[0044] First, referring to Figure 3 the S1 step in, define the multi-parameter combination to be optimized, including the optimization parameters in the probe preparation stage and the optimization parameters in the immunoassay stage.

[0045] The "multi-parameter combination to be optimized" here generally refers to that in a specific reaction process, there are often multiple factors that affect the reaction result, and these factors usually exist in the form of parameters. The "multi-parameter combination to be optimized" means that a set of optimal parameter values need to be found through experiments, simulations or other methods so that the reaction can achieve the expected optimal effect. It can be understood that the "parameters" in the embodiments of the present invention can be parameters in the form of numerical ranges or type-selection parameters. For example, in many actual reaction processes, the values of each influencing factor usually change within a continuous interval, and this interval is the specific numerical selection range. Researchers need to explore the influence of different parameter combinations on the reaction result within this range to find the optimal solution. In addition to specific numerical ranges, some parameters are selected in different types or categories. These different types of parameters may represent different substances, methods, conditions, etc. Selecting the appropriate type is equally crucial for achieving the expected effect of the reaction. These parameters may interact, cross-interact or synergistically affect the result of homogeneous immunoassay.

[0046] In some embodiments, the optimized parameters in the probe preparation stage include at least one of the amount of conjugated antibody, the amount of conjugated streptavidin, the conjugation buffer, and the conjugation time.

[0047] More specifically, the amount of antibody ranges from 10 μg to 300 μg, for example, it can be 10 μg, 50 μg, 100 μg, 200 μg, 300 μg.

[0048] More specifically, the amount of conjugated streptavidin ranges from 10 μg to 500 μg, for example, it can be 10 μg, 50 μg, 100 μg, 200 μg, 500 μg.

[0049] More specifically, the types of conjugation buffers include one of morpholineethanesulfonic acid buffer (MES buffer), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (HEPES buffer), phosphate buffer (PBS buffer), and tris(hydroxymethyl)aminomethane buffer (Tris buffer);

[0050] More specifically, the pH range of the conjugation buffer is 6 - 8, for example, it can be 6.0, 6.5, 7.0, 7.5, 8.0.

[0051] For conjugation buffers with different pH values, for example, it can be MES buffer with pH 6.0, MES buffer with pH 6.5, HEPES buffer with pH 7.0, PBS buffer with pH 7.5, and Tris buffer with pH 8.0.

[0052] More specifically, the coupling time ranges from 0.5 to 4 h. For example, the coupling reaction time can be controlled to be 0.5 h, 1 h, 2 h, 3 h, and 4 h respectively.

[0053] In some embodiments, the optimized parameters in the immune reaction stage include at least one of the immune reaction pH, immune reaction time, incubation temperature, and surfactant.

[0054] More specifically, the immune reaction pH ranges from 7.0 to 8.5. For example, it can be HEPES buffer with pH 7.0, HEPES buffer with pH 7.4, Tris buffer with pH 8.0, and Tris buffer with pH 8.5.

[0055] More specifically, the immune reaction time ranges from 1 to 20 min. For example, the incubation time of the immune reaction can be controlled to be 1 min, 5 min, 10 min, 15 min, and 20 min respectively.

[0056] It can be understood that the range selection of the optimized parameters in both the probe preparation stage and the immune reaction stage is based on the modeling and algorithm simulation with HCG as an example. Therefore, when applied to other markers, these ranges and parameter selections can be adjusted adaptively to match the conditions of the target marker to screen out the optimal condition combination.

[0057] Secondly, referring to Figure 3 Step S2 in, construct a multi-objective optimization model with at least two of detection sensitivity, detection range, linear correlation, signal-to-noise ratio, and luminescence signal as the optimization objectives.

[0058] For example, when optimizing the objective, this method is generally superior to the optimization applicable to at least two objectives, such as the combination of detection sensitivity and detection range, or the combination of detection range and linear correlation, or the combination of linear correlation and signal-to-noise ratio, or the combination of signal-to-noise ratio and luminescence signal, or the combination of detection sensitivity and linear correlation, or the combination of detection sensitivity and signal-to-noise ratio, or the combination of detection sensitivity and luminescence signal, or the combination of detection range and signal-to-noise ratio, or the combination of detection range and luminescence signal, or the combination of linear correlation and luminescence signal, or the combination of detection sensitivity, detection range, and linear correlation, or the combination of detection sensitivity, linear correlation, and signal-to-noise ratio, or the combination of detection sensitivity, signal-to-noise ratio, and luminescence signal, or the combination of detection range, linear correlation, and signal-to-noise ratio, or the combination of linear correlation, signal-to-noise ratio, and luminescence signal, or the combination of detection sensitivity, detection range, linear correlation, and signal-to-noise ratio, or the combination of detection range, linear correlation, signal-to-noise ratio, and luminescence signal, or the combination of detection sensitivity, detection range, linear correlation, and luminescence signal, or the combined combination of detection sensitivity, detection range, linear correlation, signal-to-noise ratio, and luminescence signal.

[0059] Secondly, referring to Figure 3 the S3 step in

[0060] generate an experimental matrix containing multiple parameter combinations using the Box-Behnken design, and record the response values of each group of experiments. For example, the objective is to simultaneously improve detection sensitivity, detection range, linear correlation, signal-to-noise ratio, and luminescence signal, and the parameter factors include the amount of conjugated antibody, the amount of conjugated streptavidin, the conjugation buffer, the conjugation time, the pH of the immunoreaction, and the immunoreaction time. Then, in the Box-Behnken design, it can be defined as follows:

[0061] The amount of antibody is defined as X1, and the coding levels are 10 μg, 50 μg, 100 μg, 200 μg, and 300 μg;

[0062] The amount of conjugated streptavidin is defined as X2, and the coding levels are 10 μg, 50 μg, 100 μg, 200 μg, and 500 μg;

[0063] The pH of the conjugation buffer is defined as X3, and the coding levels are 6.0, 6.5, 7.0, 7.5, and 8.0;

[0064] The conjugation time is defined as X4, and the coding levels are 0.5 h, 1 h, 2 h, 3 h, and 4 h;

[0065] The pH of the immunoreaction is defined as X5, and the coding levels are 7.0, 7.4, 8.0, and 8.5;

[0066] The immune reaction time is defined as X6, and the coding levels are 1 min, 5 min, 10 min, 15 min, and 20 min.

[0067] Define the above X1-X4 as the coupling stage, and define the above X5-X6 as the immune reaction stage. Perform Box-Behnken design separately. In the first stage, optimize the coupling parameters (4 factors, 27 groups of experiments are required). In the second stage, optimize the immune parameters (2 factors, 13 groups of experiments are required). If some parameters have been determined through preliminary experiments (such as the ionic strength of the buffer), they can be fixed at the intermediate level (such as X3 = 100 mM), reducing it to 5 factors.

[0068] Normalize the obtained response values to eliminate the dimension difference. For example:

[0069]

[0070] Again, refer to Figure 3 In step S4, based on quadratic polynomial regression, model the experimental matrix to establish a non-linear relationship model between the parameters and the response values.

[0071]

[0072] Again, refer to Figure 3 In step S5, solve the model through a numerical optimization algorithm to obtain the target parameter combination that makes the response value reach the optimal.

[0073] In the embodiment of the present invention, for the biomarker HCG, the optimal target parameter combination is:

[0074] The amount of conjugated antibody is 200 μg, the amount of conjugated streptavidin is 200 μg, the conjugation buffer is HEPES buffer with pH 7.0, the conjugation time is 3 h, the pH of the immune reaction is Tris buffer with pH 8.0, and the immune reaction time is 15 min.

[0075] In the embodiment of the present invention, the optimization method further includes constraint conditions, which limit the value range of the parameters. In the multi-parameter optimization of probe preparation, the constraint conditions are used to ensure that the experimental parameters meet physical feasibility, process stability, or cost limitations. The constraint conditions here limit the physically feasible range of a single parameter, avoid failure or dangerous conditions caused by parameter combinations, and ensure that key performance indicators are met. Therefore, after performing the Box-Behnken design, delete the combinations that violate the interaction constraints, and measure the response values of the remaining matrix combinations.

[0076] Finally, refer to Figure 3 In step S6, repeat the homogeneous immunoassay using the target parameter combination to verify the consistency between the response value and the model prediction value.

[0077] In this step, experimental verification is carried out according to the selected optimal combination to confirm the effectiveness of the model.

[0078] In the optimization method provided by the embodiments of the present invention, during the probe preparation and detection process, the detection sensitivity and detection range can be effectively adjusted through the optimization of components and processes. The analysis of HCG in blood samples shows that the long afterglow homogeneous detection range is 0.5 - 10000 U / L, and the detection interval spans up to 4 - 5 orders of magnitude, which can effectively cover the HCG concentration changes in important application scenarios. Therefore, the present invention establishes a wide-range photochemical long afterglow homogeneous immunoassay method. The ultra-wide long afterglow homogeneous detection range can meet the diverse needs of clinical detection. By quickly and accurately evaluating the HCG level and status of patients, it is expected to provide more powerful support for the early diagnosis, treatment monitoring, and prognosis evaluation of related diseases. Utilizing the characteristic of collecting signals after the excitation light is turned off in long afterglow luminescence, it is possible to avoid excitation light and biological background interference and establish an application advantage in the immunoassay of blood samples. The long afterglow homogeneous detection method not only has the advantage of convenient operation but also has the characteristic that the detection performance indicators are easy to regulate.

[0079] The following is the experimental verification part of the optimal combination given based on this optimization method to elaborate and verify the consistency of this optimization method.

[0080] The reagents and their abbreviations involved in the embodiments of the present invention include: sodium chloride (NaCl), bovine serum albumin (BSA), polyethylene glycol (PEG6000), 2-morpholinoethanesulfonic acid (MES), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), N-hydroxysuccinimide (NHS), tris(hydroxymethyl)aminomethane (Tris), sodium dihydrogen phosphate (Na2HPO4), disodium hydrogen phosphate (NaH2PO4), tetrahydrofuran (THF), octylphenoxypolyethoxyethanol (Triton X-100), human chorionic gonadotropin monoclonal antibody, antigen (HCG), biotinylated human chorionic gonadotropin monoclonal antibody (Bio-HCG).

[0081] Example 1 Antibody Optimization and Probe Microsphere Preparation

[0082] Step 1: Three pairs of HCG monoclonal antibodies involved in the HCG project screening and optimization experiment are provided by Taihui Biotech Co., Ltd. Different pairs of the three paired antibodies are used to label luminescent microspheres and biotin respectively. Samples with different concentrations of HCG are tested, and the incubation time is 15 minutes. The photochemical long afterglow luminescence signals are tested before and after the addition of HCG antigen. By analyzing the test linear correlation and signal-to-noise ratio of different antibody pairs, the most suitable antibody pair is selected for further research.

[0083] Step 2. Optimization of probe microsphere preparation conditions

[0084] Optimization of the amount of conjugated antibody: During the conjugation of antibody and luminescent microspheres, different concentrations of antibody were added for the conjugation reaction, and the amount of antibody used in the preparation ranged from 10 μg to 300 μg. Then, the immunodetection system composed of the obtained luminescent microsphere probes and sensitized microsphere probes was tested. HCG antigen samples with a concentration of 500 U / L were measured respectively, the incubation time was 15 minutes, and the photochemical long afterglow luminescence signals were tested respectively. By analyzing the intensity and change trend of the photochemical long afterglow luminescence signals, the most suitable amount of antibody was screened out.

[0085] Optimization of the amount of conjugated streptavidin: During the conjugation of streptavidin and luminescent microspheres, different concentrations of streptavidin were added for the conjugation reaction, and the amount of streptavidin used in the preparation ranged from 10 μg to 500 μg. Then, the immunodetection system composed of the obtained luminescent microsphere probes and sensitized microsphere probes was tested. HCG antigen samples with a concentration of 500 U / L were measured respectively, the incubation time was 15 minutes, and the photochemical long afterglow luminescence signals were tested respectively. By analyzing the intensity and change trend of the photochemical long afterglow luminescence signals, the most suitable amount of antibody was screened out.

[0086] Optimization of conjugation buffer: Antibody conjugation buffers with different pH values were prepared, including MES buffer with pH 6.0, MES buffer with pH 6.5, HEPES buffer with pH 7.0, PBS buffer with pH 7.5, and Tris buffer with pH 8.0. These buffers were used for the conjugation reactions of antibody with sensitized microspheres and streptavidin with luminescent microspheres respectively. Then, the immunodetection system composed of the obtained luminescent microsphere probes and sensitized microsphere probes was tested, in which the added concentration of antigen HCG was 500 U / L for all, the incubation time was 10 minutes, and the photochemical long afterglow luminescence signals were tested respectively. By analyzing the intensity of the photochemical long afterglow luminescence signals, the most suitable conjugation buffer was screened out.

[0087] Optimization of conjugation time: During the preparation of probe microspheres conjugated with antibody, the conjugation incubation times were controlled to be 0.5 hour, 1 hour, 2 hours, 3 hours, and 4 hours respectively. Then, the immunodetection system composed of the obtained luminescent microsphere probes and sensitized microsphere probes was tested, in which the added concentration of antigen HCG was 500 U / L for all, the incubation time was 10 minutes, and the photochemical long afterglow luminescence signals were tested respectively. By analyzing the intensity of the photochemical long afterglow luminescence signals, the most suitable conjugation time was screened out for further research.

[0088] Example 2 Optimization of photochemical long afterglow homogeneous immunoassay conditions

[0089] (1) Immunoreaction pH optimization experiment

[0090] During the long afterglow homogeneous detection process, for the mixed system of streptavidin-luminescent microsphere probe, antibody-sensitized microsphere probe, biotin-antibody probe and the sample to be tested, the pH values of the immune reaction are controlled to be 7.0, 7.4, 8.0, and 8.5 respectively. The immune detection system composed of sensitized microsphere probe and luminescent microsphere probe is tested. The added concentration of antigen HCG is 1000 U / L, and the incubation time is 15 minutes. The chemiluminescent long afterglow luminescence signals are tested before and after the addition of antigen HCG. By analyzing the signal enhancement ratio before and after the addition of the antigen, the most suitable pH value of the dilution buffer is screened for further research.

[0091] (2) Immune reaction time optimization experiment

[0092] During the long afterglow homogeneous detection process, for the mixed system of streptavidin-luminescent microsphere probe, antibody-sensitized microsphere probe, biotin-antibody probe and the sample to be tested, the incubation times of the immune reaction are controlled to be 1 minute, 5 minutes, 10 minutes, 15 minutes and 20 minutes respectively. The concentration of antigen HCG after the addition of the sample to be tested is 500 U / L. The immune detection system composed of sensitized microsphere probe and luminescent microsphere probe is tested. The chemiluminescent long afterglow luminescence signals are tested after a certain incubation time after the addition of the sample. By analyzing the intensity and change trend of the long afterglow luminescence signal, the most suitable immune reaction time is screened for further research.

[0093] Example 3 Performance evaluation of chemiluminescent long afterglow homogeneous immunoassay

[0094] (1) Antibody cross-interference experiment

[0095] Samples with low calibration values of HCG with final concentrations of 10 U / L and 2000 U / L are taken, and TSH, FSH, and LH are added respectively to make the TSH concentration in the sample 20 mIU / L, the FSH concentration 100 IU / L, and the LH concentration 200 IU / L. The long afterglow homogeneous immunoassay method is used for testing, the content of HCG in the sample is determined, and according to the measured long afterglow luminescence signal value, the measured concentration value is obtained by substituting it into the standard curve equation, and the relative deviation is calculated.

[0096] (2) Recovery experiment

[0097] Calibrators with final HCG concentrations of 10 U / L and 2000 U / L are taken and tested using the long afterglow homogeneous immunoassay method. According to the measured long afterglow luminescence signal value, the measured concentration value is obtained by substituting it into the standard curve equation. The recovery rate is calculated by the following formula: Recovery rate = (measured concentration / theoretical concentration). The closer the recovery rate is to 100%, the more accurate the detection method is.

[0098] First, different monoclonal antibody pairs were screened. Three different antibody pairs were used to label luminescent microspheres and sensitized microspheres respectively to construct long afterglow luminescence probes. Then, antigen samples of HCG at different concentrations were tested respectively, with an incubation time of 10 minutes, and the luminescence signals of antigen samples at different concentrations were measured. As Figure 4 shown, Figure 4 Figure Figure 4 shows the results of testing different standards with different HCG monoclonal antibody pairs. The signal-to-noise ratio and linear range of testing antigen samples of HCG at different concentrations with the antibody pair H209-H201 were optimized compared with the other two antibody pairs, and the correlation coefficient r > 0.95%. In addition, in terms of the signal-to-noise ratio at the clinical cutoff value, as shown in Table 1, the signal-to-noise ratio of the antibody pair H209-H201 near the clinical reference value reached 1.78. Through comprehensive analysis, this antibody pair was preliminarily determined for subsequent experiments. Among them, the H209 antibody was used to label the sensitized microspheres, and the H201 antibody was biotinylated and then linked to the streptavidin-labeled luminescent microspheres.

[0099] Table 1 Linear correlation and signal-to-noise ratio of different HCG monoclonal antibody pairs testing different standards

[0100]

[0101] Since the required detection range of HCG is relatively wide, based on the highly sensitive detection project in the previous chapter, it is necessary to re-optimize the dosage of HCG antibody, the dosage of streptavidin, the coupling buffer, and the coupling time in the coupling process to obtain a wider detection range.

[0102] During the coupling process of the antibody and the sensitized microspheres, different masses of HCG antibody were added respectively for the coupling reaction. The obtained sensitized microsphere probes and luminescent microspheres were combined to form an immunoassay system to measure the long afterglow luminescence signals. As Figure 5 shown, Figure 5 Figure Figure 5 shows the graph of the dosage of HCG antibody in the coupling reaction and the change of long afterglow luminescence signal. The obtained luminescent microsphere probes and sensitized microsphere probes were combined with biotin antibody to form an immunoassay system for testing. The added concentration of the antigen HCG was 500 U / L, and the incubation time before testing was 15 minutes. When the amount of antibody increased from 10 μg to 200 μg, the long afterglow luminescence signal gradually increased. Further increasing the antibody dosage to 300 μg, the long afterglow luminescence signal tended to be balanced. By analyzing the intensity and change trend of the photochemical long afterglow luminescence signal, 200 μg was determined as the subsequent antibody coupling dosage. For the sensitized microsphere probes and luminescent microsphere probes, a high antibody dosage is beneficial to obtaining a larger upper limit of HCG detection and delaying the adverse effects brought by the HOOK effect.

[0103] Furthermore, the amount of streptavidin coupling was optimized. Different amounts of streptavidin were added for the coupling reaction to obtain luminescent microsphere probes with different concentrations. The immunodetection system composed of the sensitized microsphere probes and the luminescent microsphere probes was used to test the long afterglow luminescence signals respectively. As Figure 6 shown, Figure 6 Figure Figure 6 shows the relationship between the amount of streptavidin used in the coupling reaction and the change of long afterglow luminescence signal. The immunodetection system composed of the sensitized microsphere probes and the luminescent microsphere probes obtained by coupling was used for testing. The added concentration of antigen HCG was 500 U / L, and the incubation time before testing was 15 minutes. When the amount of streptavidin increased from 50 μg to 200 μg, the long afterglow luminescence signal gradually increased, but it was already close to the maximum value at 200 μg. By analyzing the intensity and change trend of the photochemical long afterglow luminescence signal, 200 μg was determined as the subsequent amount of streptavidin coupling.

[0104] When optimizing the coupling buffer, coupling buffers with different pH values were prepared, including MES buffer with pH 6.0, MES buffer with pH 6.5, HEPES buffer with pH 7.0, PBS buffer with pH 7.5, and Tris buffer with pH 8.0. 200 μg of antibody was added, and these buffers were used for the coupling reactions of antibody with sensitized microspheres and streptavidin with luminescent microspheres respectively. The immunodetection system composed of the obtained sensitized microsphere probes and luminescent microsphere probes was used to test the long afterglow luminescence signals respectively. As Figure 7 shown, Figure 7 Figure Figure 7 shows the relationship between the pH of the buffer and the change of long afterglow luminescence signal in the coupling reaction. The immunodetection system composed of the sensitized microsphere probes and the luminescent microsphere probes obtained by coupling was used for testing. The added concentration of HCG antigen was 500 U / L, and the incubation time before testing was 15 minutes. Under the condition of HEPES buffer with pH = 7.0, the strongest long afterglow luminescence signal was obtained. By analyzing the intensity of the photochemical long afterglow luminescence signal, HEPES with pH = 7.0 was determined as the preferred coupling buffer.

[0105] When optimizing the microsphere coupling time, the coupling reaction times were controlled to be 0.5 h, 1 h, 2 h, 3 h, and 4 h respectively. During the microsphere coupling process, the amounts of antibody and streptavidin added were both 200 μg / mL, and the coupling reaction was carried out in HEPES buffer with pH 7.0. After the coupling reaction was carried out for a certain time, the immunodetection system composed of the obtained sensitized microsphere probes and luminescent microsphere probes was used to test the long afterglow luminescence signal. As Figure 8 shown, Figure 8It is a graph showing the change of the coupling time and the long afterglow luminescence signal. The sensitized microsphere probe, luminescent microsphere probe and biotin antibody probe obtained by coupling are used to form an immunoassay system for testing. The added concentration of the antigen HCG is 500 U / L, and the incubation time before testing is 15 minutes. The long afterglow luminescence signal is obtained under the condition that the coupling reaction time is 3 hours. By analyzing the intensity of the photochemical long afterglow luminescence signal, 3 hours is determined as the most suitable coupling reaction time and is used for subsequent research.

[0106] Meanwhile, in order to further broaden the homogeneous detection range, the composition of the test buffer during the incubation process of the immunoreaction was adjusted.

[0107] (1) Optimization of the pH of the homogeneous immunoassay reaction solution

[0108] During the long afterglow homogeneous detection process, for the mixed system of the sensitized microsphere probe, luminescent microsphere probe, antibody-biotin probe and the sample to be tested, the pH of the immunoreaction was controlled to be 7.0 (HEPES), 7.4 (HEPES), 8.0 (Tris), and 8.5 (Tris) respectively. For the immunoassay system composed of the luminescent microsphere probe and the sensitized microsphere probe in proportion, the photochemical long afterglow luminescence signals were tested before and after the addition of the antigen HCG under different pH conditions of the test buffer. Among them, the added concentration of the HCG antigen is 500 U / L, and the incubation time before testing is 15 minutes. As Figure 9 shown, Figure 9 It is the test result of the pH optimization experiment of the homogeneous immunoassay reaction solution. The dosage ratio of the luminescent microsphere probe to the sensitized microsphere probe is 1:1. Under different pH conditions of the dilution buffer, the ratio change of the long afterglow luminescence signal before and after the addition of HCG. The added concentration of the antigen HCG is 500 U / L, and the incubation time before testing is 15 minutes. By analyzing the signal enhancement ratio before and after the addition of the antigen, Tris (pH = 8.0) is determined as the preferred acid-base buffer component of the homogeneous immunoassay dilution solution. After the pH of the buffer is adjusted to weakly alkaline, although the sensitivity of the immunoreaction decreases, since the HCG item has low requirements for sensitivity, the overall detectability can be improved.

[0109] During the long afterglow homogeneous detection process, for the mixed system of the sensitized microsphere probe, luminescent microsphere probe and the sample to be tested, the incubation time of the immunoreaction was controlled to be 1 minute, 5 minutes, 10 minutes, 15 minutes and 20 minutes respectively. The sample to be tested with the added concentration of the HCG antigen of 500 U / L was incubated for a certain time, and then the photochemical long afterglow luminescence signals were tested respectively. As Figure 10 shown, Figure 10 It is the test result of the incubation time optimization experiment before the homogeneous immunoassay. By analyzing the strength and change trend of the long afterglow luminescence signal, 15 minutes is determined as the most suitable incubation time for subsequent research.

[0110] Based on the above optimization tests, the long afterglow homogeneous detection standard curve of HCG was plotted. For HCG antigen calibrators with different concentrations, they were prepared in a dilution buffer of 25 mM HEPES (pH = 7.0, 1% NaCl, 0.1% BSA, 1 mg / mL Dextran-500, 0.5% Triton X-100). HCG antibody 1-biotin probe, HCG antibody 2-sensitized microspheres, and streptavidin-luminescent microsphere probe were mixed according to the dosage ratio, and HCG antigen samples with different concentrations were added. After incubating for 15 minutes, the long afterglow luminescence signal was measured.

[0111] As Figure 11 shown, the obtained HCG detection standard curve has a very wide detection range (nonlinear fitting R 2 > 0.995). By analyzing the HCG biomarker in blood samples, the long afterglow homogeneous detection limit is 0.5 U / L, and the detection range is 0.5 - 10,000 U / L, which can effectively meet the clinical needs.

[0112] As shown in Table 2, the relative deviations of the measured values of the interference samples are all less than 10%, indicating that the three interference substances of TSH, FSH, and LH commonly found in HCG clinical samples have no effect on the test of HCG.

[0113] Table 2 Recovery rates of long afterglow homogeneous detection of PCT spiked samples

[0114]

[0115] The recovery rates of long afterglow homogeneous detection of PCT spiked samples were tested. As shown in Table 3, when the concentrations of HCG spiked samples are 10 U / L and 2000 U / L, the recovery rates are 102% and 97% respectively, and the recovery rates are close to 100%, indicating that the detection results of this method are accurate.

[0116] Table 3 Recovery rates of long afterglow homogeneous detection of HCG spiked samples

[0117]

[0118] Through clinical comparison experiments, the correlation between the long afterglow homogeneous immunoassay and the Abbott chemiluminescence method for detecting HCG was preliminarily evaluated (as Figure 12 shown, Figure 12Correlation between long afterglow homogeneous immunoassay and Abbott chemiluminescence method for HCG detection. Fifty clinically-sampled specimens with determined values by chemiluminescence method were selected, and the same specimens were then analyzed by long afterglow homogeneous immunoassay. A scatter plot was drawn with the results of long afterglow homogeneous reagent detection as the abscissa and the results of chemiluminescence detection as the ordinate to compare the methodologies of long afterglow homogeneous detection and chemiluminescence detection. The results showed that there was no significant difference between the measured values of the two methods, and the Pearson correlation coefficient r>0.99, indicating that the homogeneous immunoassay based on photochemical long afterglow and chemiluminescence method had good correlation in detecting HCG.

[0119] The sensitized microspheres and luminescent microspheres modified with HCG antibody form a long afterglow luminescence probe with HCG detection function. Only when there is a specific antigen-antibody immune reaction in the solution can the photochemical energy storage and transfer process proceed completely to emit long afterglow signals, and effective long afterglow homogeneous immunoassay can be achieved without washing. The analysis of HCG in blood samples showed that the long afterglow homogeneous detection range was 0.5-10000 U / L, and the span of the detection interval was as high as 4-5 orders of magnitude, which could effectively cover the HCG concentration changes in important application scenarios. The ultra-wide long afterglow homogeneous detection range could meet the diverse needs of clinical detection. Therefore, a wide-range photochemical long afterglow homogeneous immunoassay method was established, which was expected to provide a wash-free and rapid immunoassay tool for markers such as HCG with large concentration changes in biological samples.

Claims

1. A multi-parameter condition optimization method based on homogeneous immunoassay, the optimization method is used for condition screening of chorionic gonadotropin kit, characterized in that: The steps include: Define the multi-parameter combination to be optimized, including the optimization parameters in the probe preparation stage and the optimization parameters in the immune response stage; Constructing a multi-objective optimization model, with at least two of the detection sensitivity, detection range, linear correlation, signal-to-noise ratio, and luminescence signal as optimization targets; The Box-Behnken design was used to generate an experimental matrix containing multiple parameter combinations, and the response values ​​of each group of experiments were recorded; The experimental matrix was modeled based on quadratic polynomial regression to establish a nonlinear relationship model between parameters and response values; Solving the model by numerical optimization algorithm to obtain the target parameter combination that makes the response value reach the optimal value; The homogeneous immunoassay was repeated using the target parameter combination to verify the consistency of the response value with the model prediction value.

2. The optimization method according to claim 1, characterized in that: The optimization method further includes constraints, which limit the range of parameter values.

3. The optimization method according to claim 1, characterized in that: The optimization parameters in the probe preparation stage include at least one of the amount of coupled antibody, the amount of coupled streptavidin, the coupling buffer, and the coupling time.

4. The optimization method according to claim 1, characterized in that: The optimization parameters in the immune response stage include at least one of immune response pH, immune response time, incubation temperature, and surfactant.

5. The optimization method according to claim 3, characterized in that: The amount of antibody ranges from 10 μg to 300 μg.

6. The optimization method according to claim 3, characterized in that: The dosage of the coupled streptavidin ranges from 10 μg to 500 μg.

7. The optimization method according to claim 3, characterized in that: The coupling buffer type includes one of morpholineethanesulfonic acid buffer, hydroxyethylpiperazineethanesulfonic acid buffer, phosphate buffer, and tris(hydroxymethyl)aminomethane buffer.

8. The optimization method according to claim 3, characterized in that: The pH range of the coupling buffer is 6-8, and the coupling time range is 0.5-4h.

9. The optimization method according to claim 3, characterized in that: The immune reaction pH range is 7.0-8.5, and the immune reaction time range is 1-20 minutes.

10. Use of the optimization method according to any one of claims 1 to 9 in screening conditions of a chorionic gonadotropin kit.

Images