Method for determining the functional activity of ap50 for the complement alternative pathway of animal sera and use thereof

Abstract

This application discloses a method for determining the functional activity of the alternative complement pathway (AP50) in animal serum and its application. The method includes: preparing a reaction buffer containing magnesium and calcium chelating agents; washing red blood cells with the reaction buffer and adjusting the red blood cell concentration to a preset range by measuring the absorbance value after complete lysis; serially diluting the animal serum to be tested with the reaction buffer, mixing it with a standardized red blood cell suspension, and incubating to induce a hemolytic reaction; after terminating the reaction, measuring the absorbance value of hemoglobin in the supernatant, calculating the hemolysis rate at each dilution, determining the serum dilution factor corresponding to 50% hemolysis, and using its reciprocal as the AP50 value. This application selectively activates the alternative pathway using a disclosed buffer formulation and eliminates batch-to-batch variability in red blood cells through absorbance standardization. It has advantages such as self-controllability, good reproducibility, low cost, and wide species adaptability, and can be widely used in immunological research and drug safety evaluation.

Description

Technical Field

[0001] This application belongs to the field of biotechnology and immunological detection, and in particular relates to a method and application for measuring AP50 functional activity of the complement alternative pathway in animal serum. Background Technology

[0002] The complement system is an important component of the body's innate and adaptive immunity, playing a crucial role in defending against pathogen infection, clearing immune complexes, and maintaining tissue homeostasis. The complement system can be activated through three main pathways: the classical pathway, the lectin pathway, and the alternative pathway. The alternative pathway, in particular, can be activated directly on the surface of pathogens or biological materials without relying on antibodies, making it especially important in early infection defense, regulation of inflammatory responses, and biocompatibility assessment.

[0003] The AP50 assay is an important indicator for evaluating the integrity and functional status of the complement system. The AP50 value is defined as the reciprocal of the serum dilution factor required to induce hemolysis in 50% of target erythrocytes under standard reaction conditions. This value objectively reflects the overall functional activity of the alternative pathway. Currently, AP50 detection is widely used in immunological research, drug development, vaccine evaluation, and biocompatibility studies.

[0004] In existing technologies, AP50 detection primarily relies on classical methods based on erythrocyte hemolysis. This method typically uses rabbit erythrocytes as target cells, incubating them with the test serum in a specific buffer system, and measuring the amount of hemoglobin released to reflect the degree of complement-mediated hemolysis. However, existing technologies generally suffer from the following drawbacks: 1. Existing detection methods largely depend on commercially available kits, whose key reaction components, such as buffer formulations, erythrocyte processing methods, and quality control materials, are provided in a closed system, making it difficult for users to optimize or adjust the reaction system according to their research objectives. This closed approach not only limits the flexibility and scalability of the methodology but also leads to excessive dependence on specific commercial suppliers by laboratories. Once reagent supply is interrupted or batches are changed, it is difficult to independently reconstruct the detection system. 2. The erythrocytes used in the hemolysis method are extremely sensitive to temperature, osmotic pressure, and mechanical vibration. Pre-prepared erythrocytes in commercial kits are prone to membrane damage or spontaneous hemolysis during transportation and storage, resulting in significant differences in the integrity and reaction sensitivity of erythrocytes between different batches. This batch-to-batch instability directly affects the repeatability and reliability of experimental results, increasing the difficulty of quality control. 3. Commercial complement function assay kits are relatively expensive, resulting in high costs per test. This poses a significant economic burden for drug development institutions or long-term animal research units that require large-scale sample testing, limiting the application of this method in large-scale screening, continuous monitoring, or high-throughput studies. 4. Existing kits primarily use human serum, and their reaction conditions and detection sensitivities are designed based on the characteristics of the human complement system. When directly used with serum from non-human primates, rodents, or other experimental animals, problems such as low complement activity response, significant differences in reaction kinetics, or mismatched detection ranges often occur. The concentration and activity regulation mechanisms of complement proteins differ among species, making it difficult for universal kits to meet the detection needs of multiple species, severely restricting the widespread application of complement function assays in preclinical research. Summary of the Invention

[0005] In order to overcome the deficiencies in the prior art, the first objective of this application is to provide a method for measuring the functional activity of AP50 in animal serum complement alternative pathway, the second objective of this application is to provide a kit for performing the above-mentioned measurement method, and the third objective of this application is to provide the application of the above-mentioned kit.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A method for determining the functional activity of AP50 in animal serum complement alternative pathway, comprising the following steps:

[0008] Prepare a reaction buffer solution containing magnesium and calcium ion chelating agents;

[0009] Red blood cells are taken, washed and resuspended with the reaction buffer to obtain a red blood cell suspension. The concentration of the hemoglobin in the red blood cell suspension is adjusted by measuring the absorbance value of the hemoglobin after complete lysis, so that the absorbance value is within a preset range, thus obtaining a standardized red blood cell suspension.

[0010] The serum of the animal to be tested was serially diluted with the reaction buffer to obtain serum samples of a series of dilutions;

[0011] Serum samples of various dilutions were mixed with the standardized red blood cell suspension and incubated to induce a hemolytic reaction. After the hemolytic reaction was terminated, the supernatant was separated, and the hemoglobin absorbance value of the supernatant was measured.

[0012] Calculate the hemolysis rate of each serum sample, determine the serum dilution factor corresponding to a 50% hemolysis rate, and use the reciprocal of this dilution factor as the AP50 value.

[0013] Traditional methods typically standardize red blood cells using volume-based concentrations (e.g., 2% red blood cell suspension). However, this method cannot eliminate subtle differences in size, membrane stability, and intrinsic state between different batches of red blood cells. These differences directly affect the sensitivity of the hemolytic reaction, leading to poor experimental repeatability. Those skilled in the art would not readily consider using the absorbance value after red blood cell lysis to quantify red blood cells and use it as a basis for batch-to-batch calibration. This application transforms the absorbance measurement after complete lysis into a quality control step for red blood cells. Specifically, by measuring the absorbance of hemoglobin released after complete red blood cell lysis, the total number of red blood cells participating in the reaction and their potential hemoglobin release capacity can be accurately reflected, thus providing a benchmark for fine calibration of red blood cell concentration. This step ensures that regardless of the source, initial state, or batch differences of the red blood cells, the final red blood cells participating in the reaction have the same functional reaction equivalent. Theoretically, even if the red blood cells are in poor condition, stable and reliable detection results can be obtained by adjusting according to the preset absorbance range. This fundamentally eliminates the influence of batch differences in red blood cells on the detection results, significantly improving the repeatability and reliability of the detection.

[0014] This application employs a reaction buffer containing magnesium and calcium chelating agents. By chelating calcium ions, it inhibits the activation of both the classical and lectin pathways, while simultaneously providing magnesium ions to ensure the normal cascade reaction of the alternative pathway, thus achieving specific detection of the complement alternative pathway. The entire reaction system preparation, serum dilution, hemolysis reaction, and data processing steps utilize publicly available materials and well-defined procedures, making this method completely independent of commercial reagent kits and possessing a high degree of technological autonomy and scalability.

[0015] Optionally, the reaction buffer solution containing magnesium and calcium ion chelating agents comprises:

[0016] A mixture is prepared by dissolving a buffer, an osmotic pressure regulator, a magnesium ion donor, a calcium ion chelating agent, and a protein stabilizer in water.

[0017] Adjust the pH of the mixture to 7.2–7.6;

[0018] Adjust the pH of the mixture to the target volume, so that the concentration of the buffer in the resulting reaction buffer is 1-20 mM, the concentration of the osmotic pressure regulator is 120-160 mM, the concentration of the magnesium ion donor is 2-10 mM, the concentration of the calcium ion chelator is 5-15 mM, and the concentration of the protein stabilizer is 0.05%-0.5% by mass / volume percentage.

[0019] The reaction buffer, serving as the fundamental medium of the entire detection system, provides a stable pH environment and suitable ionic strength for complement proteins, maintaining the native conformation and biological activity of complement components. Through ionic composition, it selectively regulates different complement activation pathways, suppressing interference from classical and lectin pathways and allowing only alternative pathways to be activated. Simultaneously, the addition of protein stabilizers reduces non-specific adsorption and inactivation of complement proteins during dilution and incubation, contributing to the stability of the reaction system and the accuracy of the detection results.

[0020] Specifically, the reaction buffer contains a buffer, an osmotic pressure regulator, a magnesium ion donor, a calcium ion chelator, and a protein stabilizer.

[0021] HEPES (4-hydroxyethylpiperazine ethanesulfonic acid) can be used as a buffer, as it has good pH buffering capacity and can effectively resist pH fluctuations caused by complement activation or metabolite accumulation during the reaction within the pH range of 7.2-7.6, providing a relatively stable acid-base environment for the complement cascade reaction. HEPES is a zwitterionic buffer with low permeability to biological membranes and minimal interaction with complement proteins.

[0022] Sodium chloride can be used as an osmotic pressure regulator to maintain the system's osmotic pressure within the isotonic range, approximately 280-320 mOsm / kg. Appropriate osmotic pressure is crucial for maintaining the morphological integrity and membrane stability of red blood cells. Excessively high or low osmotic pressure can lead to damage to the red blood cell membrane structure or abnormal volume, thus affecting the accuracy and repeatability of the hemolytic reaction.

[0023] Magnesium chloride can be used as a magnesium ion donor. Activation of the complement substitution pathway depends on magnesium ions as a cofactor. Magnesium ions participate in the formation and stabilization of C3 invertase, helping to promote the binding of factor B and C3b, and forming an enzymatically active C3 invertase under the action of factor D. Exogenous addition of magnesium ions can support the smooth progress of the substitution pathway cascade reaction.

[0024] EGTA (ethylene glycol bis(2-aminoethyl ether)tetraacetic acid) can be used as a calcium ion chelating agent. EGTA exhibits high selective chelating ability for calcium ions, with its binding constant for calcium ions being significantly higher than that for magnesium ions. By adding EGTA, free calcium ions in the system are effectively chelated, thereby inhibiting the activation of the calcium-dependent classical pathway and lectin pathway; at the same time, sufficient magnesium ions are retained to support the function of the alternative pathway. This ion regulation mechanism is beneficial for achieving specific detection of the complement alternative pathway.

[0025] Gelatin can be used as a protein stabilizer. During serum dilution and incubation, complement proteins may be non-specifically adsorbed onto the reaction vessel walls or gas-liquid interface, leading to a decrease in effective concentration and altered reaction kinetics. Gelatin, as a macromolecular protective agent, can reduce the non-specific loss of complement proteins through competitive adsorption or steric hindrance effects, while also helping to maintain the native conformation of complement components and improving reaction stability at low serum dilutions.

[0026] This application controls the pH of the reaction buffer within the range of 7.2-7.6. Within this pH range, the conformational stability and enzyme activity of serum complement proteins are relatively good; key enzymatic steps in the complement cascade reaction, such as the formation of C3 convertase and the activity of C5 convertase, are sensitive to this pH range, and deviations from this range may lead to a decrease in complement activity; at the same time, the membrane integrity and functional stability of erythrocytes are also ideal within this pH range, which is conducive to the hemolysis reaction accurately reflecting complement activity. Therefore, controlling the pH at 7.2-7.6 is an important aspect of ensuring the accuracy and repeatability of the detection results.

[0027] When preparing the reaction buffer, after adjusting the pH, the volume must be brought to the target volume. This step helps ensure the accuracy of the concentration of each component in the buffer and the consistency between batches. Since the detection method of this application does not rely on commercial kits but uses a self-prepared reaction system, the precise control of the concentration of each batch of buffer components is of positive significance for ensuring the reproducibility of the methodology across different times and different operators. The target volume can be set according to the actual preparation scale, for example, it can be set to 100 ml, 500 ml, 1 L or larger to meet the needs of different detection throughputs. After this step, the resulting reaction buffer can be filtered through a 0.22 micrometer filter membrane for sterilization and stored at 2℃-8℃ for later use.

[0028] Optionally, the concentration range of each component in the reaction buffer is as follows: buffer 1-20mM, osmotic pressure regulator 120-160mM, magnesium ion donor 2-10mM, calcium ion chelator 5-15mM, and protein stabilizer 0.05%-0.5% (mass-volume ratio).

[0029] Specifically, for example: buffer concentrations can be any value between 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, or 1-20 mM; osmotic pressure regulator concentrations can be 120 mM, 130 mM, 140 mM, 150 mM, or 160 mM; magnesium ion donor concentrations can be any value between 2 mM, 4 mM, 6 mM, 8 mM, 10 mM, or 2-10 mM; calcium ion chelating agent concentrations can be any value between 5 mM, 7.5 mM, 10 mM, 12.5 mM, 15 mM, or 5-15 mM; and protein stabilizer concentrations can be any value between 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, or 0.05%-0.5% (mass-volume ratio).

[0030] Within this concentration range, the buffer provides sufficient buffering capacity to maintain pH stability in the reaction system. An osmolarity regulator in the range of 120-160 mM maintains an osmotic pressure close to that of physiological saline, preventing non-specific hemolysis of erythrocytes due to osmotic pressure changes. A magnesium ion concentration in the range of 2-10 mM adequately supports the activity of C3 convertase in the alternative pathway while avoiding non-specific activation that may be caused by excessively high concentrations. A calcium ion chelator concentration in the range of 5-15 mM is sufficient to effectively chelate free calcium ions in the reaction system, ensuring complete inhibition of both the classical and lectin pathways. A protein stabilizer in the range of 0.05%-0.5% (mass-volume ratio) effectively prevents protein adsorption and denaturation without interfering with the reaction due to excessively high concentrations. The combination of the above concentration ranges provides an optimal ionic environment and protein stability conditions for the activation of the alternative pathway.

[0031] Optionally, adjusting the concentration of hemoglobin by measuring the absorbance value of the hemoglobin after complete lysis of the red blood cell suspension, so that the absorbance value is within a preset range, includes:

[0032] Rabbit red blood cells were taken, washed with the reaction buffer, centrifuged and the supernatant was discarded, the precipitated cells were retained, and then resuspended in the reaction buffer to obtain a red blood cell suspension.

[0033] Take a portion of the red blood cell suspension, add lysis buffer for complete lysis, and measure the absorbance of the lysis buffer at a wavelength of 405 nm to 415 nm.

[0034] The concentration of the red blood cell suspension is adjusted based on the measured absorbance value to control the absorbance value of the completely lysed red blood cell suspension within the range of 0.35 to 0.60, preferably 0.40 to 0.50. Specifically, the preset absorbance value can be any value between 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, or 0.35 to 0.60.

[0035] Rabbit erythrocytes are highly sensitive to hemolysis in the complement alternative pathway and are commonly used target cells in complement activity assays. Through the standardization process described above, rabbit erythrocytes from different batches and sources can be calibrated to a uniform functional response level, effectively eliminating the influence of erythrocyte differences on the test results. The preset absorbance range of 0.35-0.60 is an optimized result; within this range, the hemolysis reaction exhibits good sensitivity and linear response characteristics. If the absorbance is too low, insufficient erythrocyte count may lead to incomplete complement reaction, resulting in a weak hemolysis signal and affecting detection sensitivity. If the absorbance is too high, excessive erythrocyte concentration may conversely inhibit complement activity or cause excessively rapid complement consumption and reaction system saturation, affecting the accuracy of the hemolysis curve. Therefore, controlling the absorbance of complete lysis within the range of 0.35–0.60 is crucial for ensuring the accuracy and reliability of the test results.

[0036] Preferably, the lysis solution is deionized water or an aqueous solution containing a surfactant.

[0037] Optionally, the step of serially diluting the serum of the animal to be tested with the reaction buffer to obtain serum samples of a series of dilutions includes:

[0038] Take serum from the animal to be tested and perform serial dilutions of 2-fold, 4-fold, or logarithmic gradients (e.g., logarithmic gradients to base 10) using the reaction buffer to obtain multiple serum samples with different dilutions.

[0039] The choice of dilution method should consider the complement activity range of the sample. For samples with strong complement activity, a wider dilution range or a higher dilution factor can be used to ensure that the hemolysis curve covers the entire range from low to high hemolysis rate, thereby accurately locating the 50% hemolysis point. Serial dilutions of 2-fold are commonly used in complement activity testing, providing sufficient data points for curve fitting without increasing the workload.

[0040] Optionally, the step of mixing serum samples of various dilutions with the standardized red blood cell suspension, incubating to induce a hemolytic reaction, terminating the hemolytic reaction, separating the supernatant, and measuring the hemoglobin absorbance value of the supernatant includes:

[0041] Mix serum samples of various dilutions with the standardized red blood cell suspension in equal volumes and incubate at 35-39°C for 20-60 minutes; specifically, the incubation temperature can be 35°C, 36°C, 37°C, 38°C or 39°C; the incubation time can be 20 minutes, 30 minutes, 40 minutes, 50 minutes or 60 minutes.

[0042] The hemolysis reaction was terminated by adding a reaction terminator, and the supernatant was collected after centrifugation; the reaction terminator included EDTA or EGTA.

[0043] The absorbance of the supernatant is measured at the characteristic absorption wavelength of hemoglobin; the characteristic absorption wavelength of hemoglobin is 405-415 nm. Specifically, for example, the detection wavelength can be 405 nm, 412 nm, or 415 nm.

[0044] An incubation temperature of 35-39℃, close to mammalian body temperature, is beneficial for maintaining optimal complement protein enzyme activity. An incubation time of 20-60 minutes ensures sufficient hemolysis while avoiding excessively long incubation periods that could lead to increased nonspecific hemolysis or complete complement depletion. A reaction terminator rapidly blocks the complement cascade reaction by chelating metal ions, ensuring that the degree of hemolysis accurately reflects the state at the end of incubation. After centrifugation, the amount of hemoglobin released in the supernatant is directly proportional to the degree of hemolysis; quantitative analysis can be performed by measuring absorbance at a characteristic wavelength.

[0045] Optionally, the calculation of the hemolysis rate of each serum sample, determining the serum dilution factor corresponding to a 50% hemolysis rate, and using the reciprocal of this dilution factor as the AP50 value, includes:

[0046] A negative control and a complete hemolysis control were set up. The negative control was a reaction system without serum or with EDTA added to inhibit complement activity. The complete hemolysis control was a reaction system with lysis buffer added to completely lyse red blood cells.

[0047] The hemolysis rate of serum samples at each dilution was calculated using the following formula:

[0048] Hemolysis rate = (sample absorbance - negative control absorbance) / (absorbance of complete hemolysis control - absorbance of negative control) × 100%;

[0049] Plot a hemolysis curve with the logarithm of serum dilution on the x-axis and hemolysis rate on the y-axis;

[0050] The serum dilution factor corresponding to a 50% hemolysis rate is determined by linear interpolation or four-parameter logistic regression fitting; the AP50 value is the reciprocal of this dilution factor.

[0051] Negative controls are used to subtract background hemolysis and instrument background, while complete hemolysis controls are used to calibrate the absorbance value at 100% hemolysis. Together, they form the benchmark for calculating the hemolysis rate. Hemolysis curves typically exhibit a typical S-shaped distribution, with an approximately linear relationship between the hemolysis rate and the logarithm of serum dilution within the 20%-80% hemolysis rate range. Linear interpolation is suitable for situations where data points are evenly distributed and the hemolysis curve is well-linear; four-parameter logistic regression fitting is suitable for various curve shapes and can more accurately fit the entire hemolysis curve, especially when there are few data points or the curve is asymmetrical. The AP50 value, as the reciprocal of the dilution factor, indicates that a higher serum dilution factor is required to achieve 50% hemolysis, i.e., stronger complement alternative pathway activity; conversely, a lower AP50 value indicates weaker complement activity.

[0052] This application also provides a kit for performing the above-described assay method, comprising the following components, each individually packaged:

[0053] A reaction buffer containing magnesium and calcium ion chelators to provide an ionic environment for selective activation of the complement alternative pathway;

[0054] Rabbit erythrocyte suspension, used as target cells for hemolysis reaction, wherein the rabbit erythrocyte suspension is standardized so that the absorbance value after complete lysis is in the range of 0.35 to 0.60;

[0055] Complete hemolysis control reagent, used to provide a 100% hemolysis reference;

[0056] Negative control reagent, used to provide a reference for background hemolysis;

[0057] Reaction terminator, used to terminate complement-mediated hemolysis.

[0058] All components of the above-described kit are prepared using publicly available formulations, and users can prepare them themselves or use them directly according to the instructions. Unlike existing commercial kits, the kit described in this application adopts an open design, with all components being traceable and adjustable. Users can optimize reaction conditions according to specific experimental needs, providing greater flexibility and scalability.

[0059] In addition, this application also provides the application of the above-mentioned kit in the preparation of products for evaluating the functional activity of the alternative complement pathway in mammalian serum, wherein the mammal is a human, non-human primate, rodent, dog or pig, and the evaluation includes complement function evaluation, immunotoxicity evaluation, drug safety studies, disease model evaluation or quality control of biological products.

[0060] Compared with the prior art, this application has the following beneficial effects:

[0061] 1. Unlike existing technologies that rely on physical standardization through cell counting or volumetric concentration, this application calibrates the hemolytic functional equivalent of red blood cells by measuring the absorbance of hemoglobin after complete lysis, ensuring uniform functional equivalents for red blood cells from different batches and sources before they enter the reaction system. This output calibration method effectively eliminates reaction fluctuations caused by differences in red blood cell size, membrane stability, and endogenous state, controlling batch-to-batch deviations to a very small range and solving the long-standing problem of repeatability in complement hemolysis detection.

[0062] 2. The alternative pathway activation buffer, red blood cell reaction system, hemolysis control, and reaction termination system involved in this application all use clearly disclosed formulations. Laboratories can independently prepare all reagents without being limited by the closed formulations and supply stability of commercial kits. This open system not only significantly reduces the cost per test, making it suitable for large-scale screening and long-term monitoring, but also allows researchers to flexibly optimize key parameters such as ion concentration and reaction time according to their research objectives, facilitating mechanistic studies and personalized experiments.

[0063] 3. This application employs a buffer system containing magnesium ions and chelating calcium ions, which effectively blocks the activation of the classical and lectin pathways while ensuring the necessary cofactors for the alternative pathway. This ion regulation strategy ensures that the hemolytic reaction is driven solely by the complement alternative pathway, avoiding cross-interference from multiple pathways. This allows the AP50 measurement to specifically reflect the true functional activity of the alternative pathway, improving the accuracy of the interpretation of the test results.

[0064] 4. This application does not rely on pre-made reagents customized for specific species. By adjusting the serum dilution range and reaction conditions, it can be adapted to sera from various mammals, including humans, non-human primates, rodents, dogs, and pigs, thus solving the problem of insufficient species compatibility in existing reagent kits. Furthermore, the system has a clear composition and a high degree of modularity, facilitating further development into standardized reagent kit products that can widely serve multiple fields such as basic immunological research, drug safety evaluation, disease model assessment, and biopharmaceutical quality control.

[0065] To make the above and other objects, features and advantages of this application more apparent and understandable, preferred embodiments are described below in detail with reference to the accompanying drawings. Attached Figure Description

[0066] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0067] Figure 1The hemolysis curve is the natural logarithm of the serum dilution factor versus the hemolysis rate in the blank control group 1 in Example 4;

[0068] Figure 2 The hemolysis curve is the natural logarithm of the serum dilution factor versus the hemolysis rate in the blank control group 2 in Example 4;

[0069] Figure 3 The hemolysis curve is the natural logarithm of the serum dilution factor versus the hemolysis rate in the blank control group 3 in Example 4;

[0070] Figure 4 The hemolysis curve is the natural logarithm of serum dilution factor versus hemolysis rate in complement inhibitor administration group 1 in Example 4.

[0071] Figure 5 The hemolysis curve is the natural logarithm of serum dilution and hemolysis rate in the complement inhibitor administration group 2 in Example 4.

[0072] Figure 6 The hemolysis curve is the natural logarithm of serum dilution and hemolysis rate in the complement inhibitor administration group 3 in Example 4. Detailed Implementation

[0073] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0074] Example 1: A method for determining AP50, the functional activity of the complement alternative pathway in animal serum, comprising the following steps:

[0075] S1, Preparation of reaction buffer

[0076] A buffer, osmotic pressure regulator, magnesium ion donor, calcium ion chelator, and protein stabilizer are dissolved in deionized water to obtain a mixture. The concentration of the buffer is 1–20 mM, the concentration of the osmotic pressure regulator is 120–160 mM, the concentration of the magnesium ion donor is 2–10 mM, the concentration of the calcium ion chelator is 5–15 mM, and the concentration of the protein stabilizer is 0.05%–0.5% (mass-volume ratio).

[0077] Adjust the pH of the mixture to 7.2-7.6, bring the pH-adjusted mixture to the target volume, filter it through a 0.22-micron filter membrane for sterilization, and store it at 2℃-8℃ for later use.

[0078] S2, Standardized processing of red blood cells

[0079] Rabbit red blood cells were taken, washed twice with the reaction buffer prepared above, and centrifuged at 2000 rpm for 5 minutes each time. After centrifugation, the supernatant was discarded and the cells were resuspended to the target volume to obtain a red blood cell suspension.

[0080] A portion of the red blood cell suspension was added to a lysis buffer (deionized water or surfactant) for complete lysis, and the absorbance of the lysis buffer at the characteristic absorption wavelength of hemoglobin (405–415 nm) was measured.

[0081] The red blood cell concentration is adjusted according to the measured absorbance value so that the absorbance value of the red blood cell suspension after complete lysis is controlled at 0.35-0.60, preferably 0.40-0.50.

[0082] S3, Serial dilution of serum samples

[0083] Take the serum of the animal to be tested and perform serial dilutions with the reaction buffer. The dilution method can be any one of 2-fold serial dilution, 4-fold serial dilution or logarithmic serial dilution. The preferred dilution gradients are 1:5, 1:10, 1:20, 1:40, 1:80, 1:160 and 1:320. Each dilution is set with 3 replicate wells to obtain multiple serum samples with different dilutions.

[0084] S4. Hemolytic reaction and termination

[0085] Mix serum samples of various dilutions with an equal volume of standardized red blood cell suspension and incubate at 35–39°C for 20–60 minutes, gently shaking every 10 minutes to prevent red blood cell sedimentation.

[0086] After incubation, add 0.2M EDTA or EGTA at pH 7.4 to terminate the hemolysis reaction, centrifuge at 3000 rpm for 5 minutes, and collect the supernatant.

[0087] S5. Absorbance Measurement and Hemolysis Rate Calculation

[0088] The absorbance of the supernatant was measured at the characteristic absorption wavelength of hemoglobin (405-415 nm). A negative control and a complete hemolysis control were set up. The negative control was a mixture of equal volume of reaction buffer and red blood cell suspension, and the complete hemolysis control was a mixture of equal volume of deionized water and red blood cell suspension that was completely lysed.

[0089] The hemolysis rate of serum samples at each dilution was calculated using the following formula, with the average absorbance of three replicate wells at the same dilution used for the calculation to reduce experimental error:

[0090]

[0091] S6 and AP50 value calculation

[0092] A hemolysis curve was plotted with the natural logarithm of the serum dilution factor on the x-axis and the hemolysis rate on the y-axis. The serum dilution factor corresponding to a 50% hemolysis rate was determined by linear interpolation or a four-parameter logistic regression fitting method. The AP50 value was the reciprocal of the dilution factor. The criterion was that the smaller the AP50 serum fraction (i.e., the AP50 value), the stronger the function of the complement alternative pathway.

[0093] Example 2: A method for determining AP50, the functional activity of the complement alternative pathway in animal serum, comprising the following steps:

[0094] S1, Preparation of reaction buffer

[0095] A buffer, osmotic pressure regulator, magnesium ion donor, calcium ion chelator, and protein stabilizer were dissolved in deionized water to obtain a mixture. The buffer concentration was 1 mM, the osmotic pressure regulator concentration was 120 mM, the magnesium ion donor concentration was 2 mM, the calcium ion chelator concentration was 5 mM, and the protein stabilizer concentration was 0.05% (mass-volume ratio).

[0096] Adjust the pH of the mixture to 7.2, bring the pH-adjusted mixture to the target volume, filter it through a 0.22-micron filter membrane for sterilization, and store it at 2℃~8℃ for later use.

[0097] S2, Standardized processing of red blood cells

[0098] Rabbit red blood cells were taken, washed twice with the reaction buffer prepared above, and centrifuged at 2000 rpm for 5 minutes each time. After centrifugation, the supernatant was discarded and the cells were resuspended to the target volume to obtain a red blood cell suspension.

[0099] Take 100 μL of the red blood cell suspension and add 900 μL of deionized water for complete lysis. After thorough mixing, measure the absorbance value at a wavelength of 405 nm.

[0100] The red blood cell concentration is adjusted based on the measured absorbance value so that the absorbance value of the red blood cell suspension after complete lysis is precisely controlled at 0.35.

[0101] S3, Serial dilution of serum samples

[0102] Take the serum from the cynomolgus monkeys to be tested and perform serial 2-fold dilutions with the reaction buffer. Set the dilution gradients to 1:5, 1:10, 1:20, 1:40, 1:80, 1:160, and 1:320, and set 3 replicate wells for each dilution.

[0103] S4. Hemolytic reaction and termination

[0104] In a 96-well V-bottom reaction plate, 50 μL of serum sample of various dilutions was added to each well, followed by 50 μL of standardized red blood cell suspension. At the same time, negative control wells (50 μL reaction buffer + 50 μL red blood cell suspension) and complete hemolysis control wells (50 μL deionized water + 50 μL red blood cell suspension) were set up.

[0105] Incubate the reaction plate in a 35°C incubator for 20 minutes, gently shaking it every 10 minutes to prevent erythrocyte sedimentation.

[0106] After incubation, 100 μL of 0.2 M EDTA solution (pH 7.4) was added to each well to terminate the hemolysis reaction. The reaction plate was centrifuged at 3000 rpm for 5 minutes, and 100 μL of the supernatant from each well was carefully transferred to a new 96-well plate.

[0107] S5. Absorbance Measurement and Hemolysis Rate Calculation

[0108] The absorbance of the supernatant in each well was measured at a wavelength of 405 nm using an ELISA reader. The absorbance of the sample well (OD_sample), the negative control well (OD_negative), and the complete hemolysis control well (OD_total) was recorded. The average absorbance of the three replicate wells at the same dilution was used for calculation.

[0109] The hemolysis rate of serum samples at each dilution was calculated using the following formula:

[0110]

[0111] S6 and AP50 value calculation

[0112] A hemolysis curve was plotted with the natural logarithm of the serum dilution factor on the x-axis and the hemolysis rate on the y-axis. The serum dilution factor corresponding to a 50% hemolysis rate was determined by linear interpolation, and the AP50 value was the reciprocal of this dilution factor.

[0113] Example 3: A method for determining AP50, the functional activity of the complement alternative pathway in animal serum, comprising the following steps:

[0114] S1, Preparation of reaction buffer

[0115] A buffer, osmotic pressure regulator, magnesium ion donor, calcium ion chelator, and protein stabilizer were dissolved in deionized water to obtain a mixture. The buffer concentration was 20 mM, the osmotic pressure regulator concentration was 160 mM, the magnesium ion donor concentration was 10 mM, the calcium ion chelator concentration was 15 mM, and the protein stabilizer concentration was 0.5% (mass-volume ratio).

[0116] Adjust the pH of the mixture to 7.6, bring the pH-adjusted mixture to the target volume, filter it through a 0.22-micron filter membrane for sterilization, and store it at 2℃-8℃ for later use.

[0117] S2, Standardized processing of red blood cells

[0118] Rabbit red blood cells were taken, washed twice with the reaction buffer prepared above, and centrifuged at 2000 rpm for 5 minutes each time. After centrifugation, the supernatant was discarded and the cells were resuspended to the target volume to obtain a red blood cell suspension.

[0119] Take 100 μL of the erythrocyte suspension and add 900 μL of deionized water for complete lysis. After thorough mixing, measure the absorbance value at a wavelength of 415 nm.

[0120] The red blood cell concentration is adjusted based on the measured absorbance value so that the absorbance value of the red blood cell suspension after complete lysis is precisely controlled at 0.60.

[0121] S3, Serial dilution of serum samples

[0122] Take the serum from the cynomolgus monkeys to be tested and perform a 4-fold serial dilution with the reaction buffer. Set the dilution gradients to 1:4, 1:16, 1:64, 1:256, and 1:1024, and set 3 replicate wells for each dilution.

[0123] S4. Hemolytic reaction and termination

[0124] In a 96-well V-bottom reaction plate, 50 μL of serum sample of various dilutions was added to each well, followed by 50 μL of standardized red blood cell suspension. At the same time, negative control wells (50 μL reaction buffer + 50 μL red blood cell suspension) and complete hemolysis control wells (50 μL deionized water + 50 μL red blood cell suspension) were set up.

[0125] Incubate the reaction plate in a 39°C incubator for 60 minutes, gently shaking it every 15 minutes to prevent erythrocyte sedimentation.

[0126] After incubation, 100 μL of 0.2 MEGTA solution (pH 7.4) was added to each well to terminate the hemolysis reaction. The reaction plate was centrifuged at 3000 rpm for 5 minutes, and 100 μL of the supernatant from each well was carefully transferred to a new 96-well plate.

[0127] S5. Absorbance Measurement and Hemolysis Rate Calculation

[0128] The absorbance of the supernatant in each well was measured at a wavelength of 415 nm using an ELISA reader. The absorbance of the sample well (OD_sample), the negative control well (OD_negative), and the complete hemolysis control well (OD_total) was recorded. The average absorbance of the three replicate wells at the same dilution was used for calculation.

[0129] The hemolysis rate of serum samples at each dilution was calculated using the following formula:

[0130]

[0131] S6 and AP50 value calculation

[0132] A hemolysis curve was plotted with the natural logarithm of the serum dilution factor on the x-axis and the hemolysis rate on the y-axis. The serum dilution factor corresponding to a 50% hemolysis rate was determined by a four-parameter logistic regression fitting method, and the AP50 value was the reciprocal of the dilution factor.

[0133] Example 4: A method for determining AP50, the functional activity of the complement alternative pathway in animal serum, comprising the following steps:

[0134] S1, Preparation of reaction buffer

[0135] Prepare the reaction buffer solution according to the following formula:

[0136] HEPES: 5mM; NaCl: 145mM; MgCl2: 7mM; EGTA: 10mM; Gelatin: 0.1% (by mass / volume).

[0137] Dissolve all the above components in deionized water, adjust the pH to 7.4, bring the volume to 1 liter, filter through a 0.22-micron filter membrane for sterilization, and store at 4°C for later use.

[0138] S2, Standardized processing of red blood cells

[0139] Take commercially available rabbit red blood cell suspension (2% suspension), wash twice with the reaction buffer prepared above, centrifuge at 2000 rpm for 5 minutes each time, discard the supernatant and resuspend to the original volume to obtain red blood cell suspension.

[0140] Take 100 μL of the red blood cell suspension and add 900 μL of deionized water for complete lysis. After thorough mixing, measure the absorbance value at a wavelength of 405 nm.

[0141] Adjust the red blood cell concentration according to the measured absorbance value: if the absorbance value is higher than 0.50, dilute the red blood cell suspension appropriately with reaction buffer; if the absorbance value is lower than 0.40, concentrate the red blood cell suspension by centrifugation; finally, control the absorbance value of the red blood cell suspension after complete lysis within the range of 0.45±0.05.

[0142] The standardized red blood cell suspension was stored at 4°C and used on the same day.

[0143] S3, Serial dilution of serum samples

[0144] Serum from cynomolgus monkeys was collected and divided into three parallel samples for the blank control group and three parallel samples for the complement activity inhibitor administration group. The serum was serially diluted with the reaction buffer solution. The serum dilution factors were set to 2, 6, 18, 54, 162, and 486, and three replicate wells were set for each dilution.

[0145] S4. Hemolytic reaction and termination

[0146] In a 96-well V-bottom reaction plate, 50 μL of serum sample at various dilutions was added to each well, followed by 50 μL of standardized red blood cell suspension. At the same time, negative control wells (50 μL reaction buffer + 50 μL red blood cell suspension) and complete hemolysis control wells (50 μL deionized water + 50 μL red blood cell suspension) were set up, with 3 replicate wells for each.

[0147] Incubate the reaction plate in a 37°C incubator for 30 minutes, gently shaking it every 10 minutes to prevent erythrocyte sedimentation.

[0148] After incubation, 100 μL of 0.2 M EDTA solution (pH 7.4) was added to each well to terminate the hemolysis reaction.

[0149] Centrifuge the reaction plate at 3000 rpm for 5 minutes, and carefully aspirate 100 μL of the supernatant from each well to a new 96-well flat plate.

[0150] S5. Absorbance Measurement and Hemolysis Rate Calculation

[0151] The absorbance of the supernatant in each well was measured at a wavelength of 405 nm using an ELISA reader. The absorbance of the sample well (OD_sample), the negative control well (OD_negative), and the complete hemolysis control well (OD_total) was recorded. The average absorbance of the three replicate wells at the same dilution / control well was used for subsequent calculations.

[0152] The hemolysis rate of serum samples at each dilution was calculated using the following formula:

[0153]

[0154] S6 and AP50 value calculation

[0155] A hemolysis curve was plotted with the natural logarithm of serum dilution factor on the x-axis and hemolysis rate on the y-axis. A four-parameter logistic regression model was used to fit the hemolysis curve, and the fitting equation is as follows:

[0156]

[0157] in, The percentage is the rate of hemolysis (%). This refers to the serum dilution factor. The lower asymptote indicates hemolysis close to 0%. The upper asymptote indicates near 100% hemolysis. This represents the serum dilution factor corresponding to 50% hemolysis. This is the slope factor.

[0158] Based on the fitting results, the serum dilution factor C corresponding to a 50% hemolysis rate was determined, and the AP50 value was the reciprocal of this dilution factor (i.e., the AP50 serum fraction).

[0159] The hemolysis rate data of blank control group 1 are shown in Table 1, and the corresponding hemolysis curves are shown in Table 1. Figure 1 .

[0160]

[0161] Table 1

[0162] The hemolysis rate data of the blank control group 2 are shown in Table 2, and the corresponding hemolysis curves are shown in Table 2. Figure 2 .

[0163]

[0164] Table 2

[0165] The hemolysis rate data of the blank control group 3 are shown in Table 3, and the corresponding hemolysis curves are shown in Table 3. Figure 3 .

[0166]

[0167] Table 3

[0168] The hemolysis rate data of complement inhibitor group 1 are shown in Table 4, and the corresponding hemolysis curves are shown in [Figure 4]. Figure 4 .

[0169]

[0170] Table 4

[0171] The hemolysis rate data of complement inhibitor group 2 are shown in Table 5, and the corresponding hemolysis curves are shown in [Table 5]. Figure 5 .

[0172]

[0173] Table 5

[0174] The hemolysis rate data of complement inhibitor group 3 are shown in Table 6, and the corresponding hemolysis curves are shown in [Table 6]. Figure 6 .

[0175]

[0176] Table 6

[0177] The results above show that the serum AP50 scores of the three parallel samples in the blank control group ranged from 0.004647 to 0.006858, with very small differences, indicating that the method of the present invention has good repeatability and stability and can effectively control experimental errors.

[0178] The serum AP50 scores of the three parallel samples in the complement activity inhibitor administration group ranged from 0.128134 to 0.170066, which was 20 to 36 times that of the blank control group. This indicates that the method of the present invention can sensitively and accurately detect changes in the functional activity of the complement alternative pathway and can effectively evaluate the effect of complement inhibitors.

[0179] The hemolysis rate of all samples decreased in a gradient with increasing serum dilution factor, which is consistent with the concentration-dependent law of complement activity, proving that the detection results of the method of the present invention are reasonable and reliable.

[0180] This application uses specific embodiments to illustrate the principles and implementation methods of this application. The description of the above embodiments is only for the purpose of helping to understand the method and core ideas of this application. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of this application. Therefore, the content of this specification should not be construed as a limitation of this application.

Claims

1. A method for determining AP50, the functional activity of the complement alternative pathway in animal serum, characterized in that, Includes the following steps: Prepare a reaction buffer solution containing magnesium and calcium ion chelating agents; Red blood cells are taken, washed and resuspended with the reaction buffer to obtain a red blood cell suspension. The concentration of the hemoglobin in the red blood cell suspension is adjusted by measuring the absorbance value of the hemoglobin after complete lysis, so that the absorbance value is within a preset range, thus obtaining a standardized red blood cell suspension. The serum of the animal to be tested was serially diluted with the reaction buffer to obtain serum samples of a series of dilutions; Serum samples of various dilutions were mixed with the standardized red blood cell suspension and incubated to induce a hemolytic reaction. After the hemolytic reaction was terminated, the supernatant was separated, and the hemoglobin absorbance value of the supernatant was measured. Calculate the hemolysis rate of each serum sample, determine the serum dilution factor corresponding to a 50% hemolysis rate, and use the reciprocal of this dilution factor as the AP50 value.

2. The method according to claim 1, characterized in that, The reaction buffer solution containing magnesium and calcium ion chelating agents comprises: A mixture is prepared by dissolving a buffer, an osmotic pressure regulator, a magnesium ion donor, a calcium ion chelating agent, and a protein stabilizer in water. Adjust the pH of the mixture to 7.2–7.6; Adjust the pH of the mixture to the target volume, so that the concentration of the buffer in the resulting reaction buffer is 1-20 mM, the concentration of the osmotic pressure regulator is 120-160 mM, the concentration of the magnesium ion donor is 2-10 mM, the concentration of the calcium ion chelator is 5-15 mM, and the concentration of the protein stabilizer is 0.05%-0.5% by mass / volume percentage.

3. The method according to claim 2, characterized in that, The calcium ion chelating agent is EGTA, the buffer is HEPES, the osmotic pressure regulator is NaCl, the magnesium ion donor is MgCl2, and the protein stabilizer is gelatin.

4. The method according to claim 1, characterized in that, The step of adjusting the concentration of hemoglobin by measuring the absorbance value of the completely lysed red blood cell suspension to ensure that the absorbance value is within a preset range includes: Rabbit red blood cells were taken, washed with the reaction buffer, centrifuged and the supernatant was discarded, the precipitated cells were retained, and then resuspended in the reaction buffer to obtain a red blood cell suspension. Take a portion of the red blood cell suspension, add lysis buffer for complete lysis, and measure the absorbance of the lysis buffer at a wavelength of 405 nm to 415 nm. The concentration of the red blood cell suspension is adjusted according to the measured absorbance value so that the absorbance value of the red blood cell suspension after complete lysis is controlled within the range of 0.35 to 0.

60.

5. The method according to claim 4, characterized in that, The lysis solution is deionized water or an aqueous solution containing a surfactant.

6. The method according to claim 1, characterized in that, The step of serially diluting the serum of the animal to be tested with the reaction buffer to obtain serum samples of a series of dilutions includes: Serum from the animal to be tested was taken and serially diluted 2-fold, 4-fold, or logarithmically using the reaction buffer solution to obtain multiple serum samples with different dilutions.

7. The method according to claim 1, characterized in that, The process of mixing serum samples of various dilutions with the standardized red blood cell suspension, incubating to induce a hemolytic reaction, terminating the hemolytic reaction, separating the supernatant, and measuring the hemoglobin absorbance value of the supernatant includes: Mix serum samples of each dilution with an equal volume of the standardized red blood cell suspension and incubate at 35-39°C for 20-60 minutes. The hemolysis reaction was terminated by adding a reaction terminator, and the supernatant was collected after centrifugation; the reaction terminator included EDTA or EGTA. The absorbance of the supernatant was measured at the characteristic absorption wavelength of hemoglobin, which is 405-415 nm.

8. The method according to claim 1, characterized in that, The calculation of the hemolysis rate of each serum sample, determining the serum dilution factor corresponding to a 50% hemolysis rate, and using the reciprocal of this dilution factor as the AP50 value, includes: A negative control and a complete hemolysis control were set up. The negative control was a reaction system without serum or with EDTA added to inhibit complement activity. The complete hemolysis control was a reaction system with lysis buffer added to completely lyse red blood cells. The hemolysis rate of serum samples at each dilution was calculated using the following formula: Hemolysis rate = (sample absorbance - negative control absorbance) / (absorbance of complete hemolysis control - absorbance of negative control) × 100%; Plot a hemolysis curve with the logarithm of serum dilution on the x-axis and hemolysis rate on the y-axis; The serum dilution factor corresponding to a 50% hemolysis rate is determined by linear interpolation or four-parameter logistic regression fitting; the AP50 value is the reciprocal of this dilution factor.

9. A kit for implementing the method according to any one of claims 1 to 8, characterized in that, Includes the following components, each packaged separately: A reaction buffer containing magnesium and calcium ion chelators to provide an ionic environment for selective activation of the complement alternative pathway; Rabbit erythrocyte suspension, used as target cells for hemolysis reaction, wherein the rabbit erythrocyte suspension is standardized so that the absorbance value after complete lysis is in the range of 0.35 to 0.60; Complete hemolysis control reagent, used to provide a 100% hemolysis reference; Negative control reagent, used to provide a reference for background hemolysis; Reaction terminator, used to terminate complement-mediated hemolysis.

10. The use of the kit according to claim 9 in evaluating the functional activity of the alternative complement pathway in mammalian serum, characterized in that, The mammals mentioned are humans, non-human primates, rodents, dogs, or pigs, and the evaluations include complement function evaluation, immunotoxicity evaluation, drug safety studies, disease model evaluation, or quality control of biological products.

Images