Method for detecting activity of immunoglobulin G

Abstract

The invention relates to the technical field of detection, in particular to a method for detecting the activity of immunoglobulin G. The invention discloses a method for detecting the activity of immunoglobulin G. The method comprises the following specific steps: (1) centrifugally degreasing bovine coloctrum to obtain degreased bovine coloctrum; (2) raising the temperature of the degreased bovine colostrum to 40-50 DEG C, adjusting the pH value of the degreased bovine colostrum to 4.4-4.6, centrifuging, and removing casein to obtain whey; (3) adjusting the pH value of the whey to 3.6-3.8, heating to 80-90 DEG C, heating for 1-5 minutes, cooling to 4 DEG C, and centrifuging to obtain an active immune globulin G solution; (4) adjusting the pH value of the active immune globulin G solution obtained in the step (3), and detecting the activity of IgG in the active immune globulin G solution; according to the method for detecting the activity of the immunoglobulin G, the detection process is simplified, the detection cost is remarkably reduced, and compared with a traditional method, the method is more economical and effective.

Description

Technical Field

[0001] The present application relates to the technical field of detection, and in particular to a method for detecting immunoglobulin G activity. Background Art

[0002] Immunoglobulin G (IgG) is the main antibody in colostrum, accounting for about 80-85% of the total immunoglobulin. IgG is divided into two subclasses, IgG1 (the most abundant immunoglobulin subtype) and IgG2. It is well known that immunoglobulins are beneficial to human health and play an important role in immune response. When the host is exposed to the antigen, the antibody binds and recognizes bacteria, toxins, viruses and other antigens. When exposed to the same antigen again, the body produces the same antibody, effectively eliminating the infection.

[0003] The activity, stability and effectiveness of immunoglobulins are susceptible to significant effects from several factors. Temperature is a key factor, as either too high or too low a temperature can lead to protein denaturation or reduced function. pH is also a key factor, as deviations from the optimal pH can change the structural conformation of immunoglobulins and affect their antigen binding ability. Enzymatic degradation can also threaten IgG activity, as specific enzymes may degrade immunoglobulins, leading to loss of activity. In addition, interactions with other components, such as binding to other proteins or lipids, can enhance or inhibit the activity of immunoglobulins. These interactions may form complexes that alter the effectiveness of immunoglobulins or their ability to bind to pathogens.

[0004] The content and activity detection of bovine colostrum IgG is crucial in various fields, especially in therapeutic development and the formulation of nutritional supplements. Accurate detection of IgG function is important to ensure product effectiveness and safety. However, traditional methods for detecting IgG activity, such as ELISA or turbidimetry, are often complicated, time-consuming and expensive. These techniques usually require specialized laboratory equipment, accurate sample preparation and skilled personnel, which limits their accessibility and scalability. In addition, environmental factors such as temperature and pH can reduce the reproducibility and reliability of the results.

[0005] Therefore, there is a need to develop a simple, more efficient, cost-effective and safe method for rapid and accurate detection of IgG content and activity. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present application provides a method for detecting the activity of immunoglobulin G. The purpose of the present application is to solve the problems of complexity, time consumption and high cost of the current method for detecting the activity of immunoglobulin G (IgG).

[0007] The method for detecting immunoglobulin G activity disclosed in the present application simplifies the detection process, significantly reduces the detection cost, and is more economical and effective than traditional methods. The ultimate goal of the present application is to provide a more practical, efficient and reliable solution for IgG activity detection, thereby improving the effectiveness of products and therapies based on bovine colostrum.

[0008] In a first aspect, the present application provides a method for detecting immunoglobulin G activity, using the following technical solution: A method for detecting immunoglobulin G activity, the specific steps are as follows: (1) centrifugally defatting the bovine colostrum to obtain defatted bovine colostrum; (2) raising the temperature of the defatted bovine colostrum to 40-50° C., adjusting the pH of the defatted bovine colostrum to 4.4-4.6, centrifuging, removing casein, and obtaining whey; (3) adjusting the pH of the whey to 3.6-3.8, heating to 80-90° C., heating for 1-5 min, cooling to 4° C., centrifuging, and obtaining an active immunoglobulin G solution; (4) Adjusting the pH of the active immunoglobulin G solution obtained in step (3), and detecting the activity of IgG in the active immunoglobulin G solution.

[0009] By adopting the above technical scheme, the present application discloses a method for detecting the activity of immunoglobulin G, which utilizes the aggregation-dispersion behavior of IgG molecules under specific pH and ionic strength conditions, and accurately infers the activity of IgG, especially its interaction with pathogens or antigens, by monitoring the changes in ionizable amino acids and their effects on the net charge and isoelectric point of IgG molecules.

[0010] Preferably, in step (1), the centrifugal defatting step is as follows: centrifugal defatting of bovine colostrum is performed using a centrifugal device, the rotation speed of the centrifugal device is 8000-12000 rpm, the centrifugal defatting time is 20-30 minutes, and the temperature of the centrifugal device is 4°C-10°C.

[0011] Preferably, in step (2), the solution used to adjust the pH of the defatted bovine colostrum to 4.4-4.6 is an acid solution.

[0012] Preferably, in step (2), the acid solution is a hydrochloric acid solution or a citric acid solution.

[0013] Further preferably, in step (2), the acid solution is a 0.1 M hydrochloric acid solution.

[0014] Preferably, the centrifugation step in step (2) is as follows: the defatted bovine colostrum is centrifuged using a centrifugal device, the rotation speed of the centrifugal device is 8000-12000 rpm; the centrifugation time is 20-30 minutes; the temperature of the centrifugal device is 4°C-10°C.

[0015] Preferably, during the centrifugation in step (2), the temperature of the defatted bovine colostrum is controlled at 40-50°C.

[0016] Preferably, the centrifugation step in step (3) is as follows: the whey is centrifuged using a centrifugal device, the rotation speed of the centrifugal device is 8000-12000 rpm, the centrifugation time is 20-30 minutes, and the temperature of the centrifugal device is 4°C-10°C.

[0017] In the step (3), the solution used to adjust the pH of the whey to 3.6-3.8 is an acid solution.

[0018] Preferably, in step (3), the acid solution is a hydrochloric acid solution or a citric acid solution.

[0019] Further preferably, in step (3), the acid solution is a 0.1 M hydrochloric acid solution.

[0020] Preferably, the method for detecting the activity of IgG in the active immunoglobulin G solution in step (4) is any one of spectrophotometry, ultraviolet absorption spectroscopy, precipitation weight method or colorimetry.

[0021] Preferably, the method for detecting the activity of IgG in the active immunoglobulin G solution in step (4) is: when detecting the activity of IgG in the active immunoglobulin G solution by spectrophotometry, first adjust the pH of the active immunoglobulin G solution to 5.4-6.4, and then detect the activity of IgG by spectrophotometry; When the activity of IgG in the active immunoglobulin G solution is detected by ultraviolet absorption spectroscopy, the pH of the active immunoglobulin G solution is first adjusted to 3-8, and then the activity of IgG is detected by ultraviolet absorption spectroscopy.

[0022] When the activity of IgG in the active immunoglobulin G solution is detected by the precipitation weight method, the pH of the active immunoglobulin G solution is first adjusted to 5.4-6.4, and then the activity of IgG is detected by the precipitation weight method; When the activity of IgG in the active immunoglobulin G solution is detected by the colorimeter method, the pH of the active immunoglobulin G solution is first adjusted to 5.4-6.4, and then the activity of IgG is detected by the colorimeter method.

[0023] By adopting the above technical scheme, in the present application, the pH of the active immunoglobulin G solution is adjusted by adding an alkaline solution or an acid solution to the IgG solution to adjust the pH value so that the ionizable amino acids are deprotonated. This step changes the net charge of IgG; when the pH value is close to the isoelectric point, the color changes to white, indicating that the active IgG is responding to the change in pH value; the positive and negative charges attract each other to obtain a precipitate, or continue to use sodium hydroxide to repel each other.

[0024] The IgG activity detection method disclosed in the present application focuses on the ionizable amino acids (charge analysis) inside IgG, reducing the risks associated with handling antigens or pathogens. This method does not directly interact with dangerous biological agents, but detects IgG activity by analyzing the net charge and isoelectric point of the protein, improving safety and paving the way for the widespread application of advanced technologies for protein activity analysis in immunology and diagnostics.

[0025] In a specific embodiment, the activity of IgG in the active immunoglobulin G solution is detected by spectrophotometry, ultraviolet absorption spectroscopy, precipitation weight method or colorimetry: (1) Spectrophotometry: When the IgG activity is measured by spectrophotometry, the steps are as follows: the pH value of the active immunoglobulin G solution obtained in step (4) is adjusted to 5.4-6.4 using a sodium hydroxide solution, and then spectrophotometric detection is performed at 405 nm.

[0026] Spectrophotometric determination: IgG activity was measured at 405 nm using a spectrophotometer, and the calculation formula was: IgG activity (U / mL) = C*OD*V / W; Where: C---IgG concentration after casein removal (mg / mL); OD---absorbance at 405nm; V---NaOH titration volume (mL); W---lipid weight (g); (2) Sedimentation weight method: When the IgG activity is determined by the precipitation weight method, the steps are as follows: the pH value of the active immunoglobulin G solution obtained in step (4) is adjusted to 5.4-6.4 using a sodium hydroxide solution, centrifuged at 8000-12000 rpm for 20-30 minutes at 4°C, and after removing the supernatant, the solid mass is weighed to detect its activity.

[0027] Amount of precipitation: IgG has no activity when its net charge is zero, so it will precipitate; the precipitate can be collected and the IgG activity and content can be calculated using a formula; IgG precipitation ratio = IgG precipitation amount (mg) / IgG concentration (mg / mL) * IgG solution volume (mL) * 100%; (3) Ultraviolet absorption spectroscopy: When the IgG activity is measured by ultraviolet absorption spectroscopy, the steps are as follows: the pH value of the active immunoglobulin G solution obtained in step (4) is adjusted to 3-8 using hydrochloric acid solution or sodium hydroxide solution, and the sample is subjected to ultraviolet absorption spectroscopy detection.

[0028] UV absorption spectrum: The activity of IgG is characterized by analyzing the absorbance spectra at different wavelengths; changes in the spectral curve indicate changes in the structure and activity of IgG; Option 1: Compare the absorbance difference between pH 3 and pH 6, or between pH 6 and pH 7 within the wavelength range of 250-280nm, 281-309nm, or above 310nm.

[0029] Option 2: The absorbance differences between pH 3, pH 6 and pH 7 can be compared within the wavelength ranges of 250-280nm, 281-309nm and above 310nm.

[0030] (4) Using the colorimeter method: When the IgG activity is measured by the colorimeter method, the steps are as follows: the pH value of the active immunoglobulin G solution obtained in step (4) is adjusted to 5.4-6.4 using a sodium hydroxide solution, and the solution is detected using a colorimeter.

[0031] Colorimeter: Calculates the total color change (ΔE) using a formula to quantify the change in color intensity.

[0032] Where: ΔL * Represents brightness changes; Δa * and Δb * Represents the change in the anti-color dimension.

[0033] The present application discloses a method for detecting the activity of IgG in bovine colostrum, the principle of which is that IgG molecules are affected by the pH and ionic strength of the solution to aggregate and disperse. The activity of IgG molecules can be inferred by measuring ionizable amino acids; the present application provides a simple, fast and economical method to replace traditional technologies.

[0034] The present application discloses a method for detecting IgG activity in bovine colostrum, comprising the following steps: (1) Sample: bovine colostrum liquid or bovine colostrum freeze-dried powder (dissolved before use); (2) Centrifugal degreasing: Place the colostrum in a centrifuge for degreasing; (3) Removing casein: Raise the temperature of the defatted colostrum to 40-50°C, use an acid solution to adjust the pH of the colostrum to precipitate casein, and centrifuge to remove casein, inactive IgG and some proteins to obtain whey (containing highly active IgG); (4) pH adjustment and heat treatment: The pH of the whey is adjusted using an acid solution, and then heat treated to precipitate the impurities in the immunoglobulin G and obtain active immunoglobulin G; This step is to denature and remove unwanted proteins by centrifugation, effectively removing impurities; (5) Titration: By adding alkaline or acid solution to the active immunoglobulin G solution, the pH is adjusted to deprotonate the ionizable amino acids. This step changes the net charge of IgG. When the pH is close to the isoelectric point, the color changes to white, indicating that the active IgG is responding to the pH change. The positive and negative charges attract each other and precipitate, or continue to repel each other with sodium hydroxide.

[0035] (6) Activity detection: Use spectrophotometry, ultraviolet absorption spectroscopy, precipitation weight method or colorimetry to reflect the color change of IgG activity reaction.

[0036] The method provided in this application can be used to detect the activity of immunoglobulin G, showing its structural stability and antibody activity. The method of this application is applicable to foods, health products, beverages or other related products containing IgG to ensure the activity and function of IgG.

[0037] In summary, the present application includes at least one of the following beneficial technical effects: 1. This application discloses a method for detecting immunoglobulin G activity, which focuses on the ionizable amino acids inside IgG (charge analysis), reducing the risks associated with handling antigens or pathogens; 2. The detection method disclosed in this application does not directly interact with dangerous biological agents, but detects IgG activity by analyzing the net charge and isoelectric point of the protein, thereby improving safety; 3. The present application discloses a method for detecting immunoglobulin G activity, which is simple and straightforward, does not require complex and specialized equipment, is suitable for various laboratory settings, and can obtain results faster and reduce overall costs compared to traditional techniques; 4. This application discloses a method for detecting the activity of immunoglobulin G, which is less affected by external factors such as temperature and pH, thereby ensuring the stability and repeatability of the results. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1Immunoglobulin G activity was measured for net charge and isoelectric point; Figure 2 The effect of heating time on IgG activity; Figure 3 For UV absorption spectroscopy analysis, IgG was analyzed after being heated at 85°C for different time periods and adjusted to different pH values; Figure 4 pH-adjusted IgG activity for UV absorption spectroscopy analysis; Figure 5 Rocket immunoelectrophoresis was used to analyze the kinetic stability of IgG heated at 85°C for different time periods; Figure 6 The stability of IgG after heating was analyzed by SDS-PAGE; Figure 7 The effect of heating time and precipitation amount on IgG activity; Figure 8 IgG activity was detected by colorimeter. DETAILED DESCRIPTION

[0039] The technical solution of the present application is further illustrated by specific embodiments below. The specific embodiments do not limit the protection scope of the present application; some non-essential modifications and adjustments made by others based on the concept of the present application still fall within the protection scope of the present application.

[0040] The raw materials involved in this application are all commercially available products, among which: The standard sample used in this example is bovine colostrum liquid, bovine colostrum liquid batch ZD202241001 (IgG content 61.2 mg / mL), purchased from Nanxiaoniu Food (Nanjing) Co., Ltd.

[0041] The present application is further described in detail below in conjunction with embodiments and comparative examples.

[0042] Example 1: Detection of IgG activity in bovine colostrum using spectrophotometry A method for detecting IgG activity in bovine colostrum by spectrophotometry, the specific steps are as follows: 1. Standard sample: bovine colostrum liquid (IgG content 61.2 mg / mL); 2. Centrifugal degreasing: Put the bovine colostrum standard sample into the centrifuge for degreasing to obtain defatted bovine colostrum; The speed of the centrifuge degreasing is 8000 rpm, the time of centrifugal degreasing is 20 minutes, and the temperature of the centrifuge is 4°C; 3. Remove casein: The temperature of the defatted bovine colostrum was raised to 45°C, the pH value of the defatted bovine colostrum was adjusted to 4.6 using a 0.1M hydrochloric acid solution, and the defatted bovine colostrum was centrifuged to remove casein to obtain whey; The centrifuge was used for centrifugation. The speed of the centrifuge was 8000 rpm. The centrifuge time was 20 minutes. The temperature of the centrifuge was 4° C. During the centrifugation process, the temperature of the bovine colostrum was controlled at 45° C.

[0043] 4. pH adjustment and heat treatment: The pH of the whey was adjusted to 3.8 using 0.1 M hydrochloric acid solution, the temperature was raised to 85°C, heated for 5 min, cooled to 4°C, and centrifuged to obtain an active immunoglobulin G solution; The centrifuge was centrifuged at a speed of 10,000 rpm, a centrifugal time of 30 minutes, and a temperature of 4°C.

[0044] 5. Detect the activity of IgG in the active immunoglobulin G solution: 5.1 Titration: Take the active immunoglobulin G solution obtained in step 4 and carefully titrate it with 0.1M NaOH solution to check the pH value until the pH is between 5.4-6.4. In this pH range, the positive and negative charges are balanced (the net charge is equal to zero), and the solution is white, indicating the presence of active IgG antibodies. The results are shown in Figure 1 .

[0045] 5.2. IgG activity detection: Spectrophotometric determination: IgG activity was measured at 405 nm using a spectrophotometer (spectrophotometer), and the calculation formula was: IgG activity (U / mL) = C*OD*V / W; Where: C---IgG concentration after casein removal (mg / mL); OD---absorbance at 405nm; V---NaOH titration volume (mL); W---lipid weight (g); 6. Test results: The activity of immunoglobulin G (IgG) was detected by spectrophotometry. IgG was heated at 85°C for different times (0, 5, 15, 30 and 60 minutes). The results are shown in Figure 2. The activity of IgG was not affected by heating at 85°C for 0 and 5 minutes (19.4 U / mL), indicating that IgG remained stable and active at this stage. However, when the heating time reached 15 minutes, the activity of IgG decreased significantly, indicating that IgG began to lose its functional properties due to heat. As the heating time was extended to 30 and 60 minutes, the activity of IgG further decreased (0.2 U / mL), and the activity was almost completely lost at 60 minutes. This loss of activity may be due to the denaturation of IgG molecules. As shown in Table 1, the effect of heating on the amino acid composition may be related to the change in IgG activity, indicating that certain amino acids were affected during the heating process. These results demonstrate that spectrophotometry can be effectively used to detect the activity of immunoglobulin G.

[0046] Table 1 Effect of heating time on the amino acid composition of immunoglobulin G Example 2: Detection of IgG activity in bovine colostrum using ultraviolet absorption spectroscopy A method for detecting immunoglobulin G activity using ultraviolet absorption spectroscopy, the specific steps are as follows: 1. Standard sample: bovine colostrum liquid (IgG content 61.2 mg / mL); 2. Centrifugal degreasing: Put the bovine colostrum standard sample into the centrifuge for degreasing to obtain defatted bovine colostrum; The speed of the centrifuge degreasing is 8000 rpm, the time of centrifugal degreasing is 20 minutes, and the temperature of the centrifuge is 4°C.

[0047] 1. Removal of casein: Raise the temperature of skimmed bovine colostrum to 45°C, adjust the pH value of skimmed bovine colostrum to 4.6 using 0.1M hydrochloric acid solution, centrifuge, remove casein, and obtain whey; The centrifuge was used for centrifugation. The speed of the centrifuge was 8000 rpm. The centrifuge time was 20 minutes. The temperature of the centrifuge was 4° C. During the centrifugation process, the temperature of the bovine colostrum was controlled at 45° C.

[0048] 4. pH adjustment and heat treatment: Use 0.1M hydrochloric acid solution to adjust the pH of whey to 3.8, heat to 85°C, heat for 5 minutes, cool to 4°C, centrifuge to obtain active immunoglobulin G solution; The centrifuge was centrifuged at a speed of 10,000 rpm, a centrifugal time of 30 minutes, and a temperature of 4°C.

[0049] 5. Detect the activity of IgG in the active immunoglobulin G solution: 5.1 Titration: Take the active immunoglobulin G solution obtained in step 4, and adjust the pH to 3, 6, and 7 respectively using 0.1 M hydrochloric acid solution or 0.1 M NaOH solution; 5.2. IgG activity detection: Dilute the solutions of different pH values ​​by the same multiple, use the UV spectrophotometer wavelength scanning mode, set the starting wavelength to 190nm and the ending wavelength to 450nm. First, use purified water to establish the system baseline, then use zero / full scale to calibrate the wavelength measurement range, and finally perform wavelength scanning tests on the diluted solutions of different pH values. Save the file after the scan is completed.

[0050] Method 1: Compare the absorbance difference between pH 3 and pH 6, or between pH 6 and pH 7 within the wavelength range of 250-280nm, 281-309nm or above 310nm. The calculation formula is (1): Method 2: Compare the absorbance differences between pH 3, pH 6 and pH 7 in the wavelength ranges of 250-280nm, 281-309nm and above 310nm. The calculation formula is (2): 6. Test results: The activity of immunoglobulin G (IgG) was detected using ultraviolet absorption spectroscopy. Figure 3 The charge and activity of IgG were analyzed after heating at 85°C for different times (0, 5, 15, 30, and 60 minutes) and then adjusted to different pH values ​​(2-10). In the initial stage (0 minutes), the absorbance was higher at lower pH values, indicating that IgG is more likely to aggregate or be unstable in an acidic environment, which is associated with increased activity of positive charges. As the pH value increases, the absorbance decreases, indicating that IgG maintains higher activity and stability under neutral to alkaline pH conditions, where the charge distribution is more balanced or slightly negative, thereby reducing aggregation.

[0051] like Figure 3 As shown in the figure, as the heating time increases (especially over 15 minutes) and after pH adjustment, the absorbance gradually decreases, indicating that the activity of IgG is reduced due to partial denaturation. At 30 and 60 minutes, the absorbance under all pH conditions is very low or has no significant difference, indicating that IgG has basically lost its activity and structural integrity after prolonged heating. This loss of activity may be caused by the protein denaturation process, in which the protein structure is destroyed and key amino acids and functional charge sites are destroyed. These changes result in a decrease in the ability of IgG to maintain its native conformation and biological function, and the charged areas required for its activity are damaged.

[0052] The activity of immunoglobulin G (IgG) was detected using ultraviolet absorption spectroscopy. Figure 4 The charge and activity of IgG were analyzed by adjusting it to different pH values ​​(3, 6, 7). The results showed that there were significant differences in absorbance between pH 3, 6 and 7. From pH 3 to pH 6, the absorbance decreased by 77%, 84% and 95% in the wavelength ranges of 250-280nm, 280-310nm and above 310nm, respectively. Similar trends of change were observed between pH 6 and pH 7. This shows that this method can be effectively used to evaluate the activity of the sample. If the IgG is inactivated, the percentage change in absorbance will be low or almost unchanged, indicating that there is no significant difference. Therefore, the activity of IgG may be affected by temperature, storage time, microbial contamination of colostrum or other factors. The net charge of amino acids is a criterion for detecting protein activity, and it also affects the ability of IgG to adsorb pathogens. This method can be reliably applied to monitor the activity and stability of IgG, especially in evaluating the effectiveness of its immune function.

[0053] Figure 5 and Figure 6 The visual changes of IgG samples after different heating times (0, 5, 15, 30 and 60 minutes) are shown. It can be observed that the band intensity of the IgG sample gradually weakens with increasing heating time, indicating that the activity and structural integrity of IgG are reduced during long-term heating. Prolonged heating leads to a significant decrease in IgG activity, which is related to the degradation of amino acids and the loss of functional charge sites. These visual observations confirm the effectiveness of the method for evaluating IgG activity and denaturation state by adjusting pH and observing changes in ionizable amino acids.

[0054] Example 3: Detection of IgG activity in bovine colostrum using the precipitation weight method The method of detecting the activity of IgG in bovine colostrum by sedimentation weight method is as follows: 1. Standard sample: bovine colostrum liquid (IgG content 61.2 mg / mL); 2. Centrifugal degreasing: Put the bovine colostrum standard sample into the centrifuge for degreasing to obtain defatted bovine colostrum; The speed of the centrifuge degreasing is 8000 rpm, the time of centrifugal degreasing is 20 minutes, and the temperature of the centrifuge is 4°C; 3. Removing casein: Raise the temperature of the skimmed bovine colostrum to 45°C, adjust the pH value of the skimmed bovine colostrum to 4.6 using 0.1M hydrochloric acid solution, centrifuge, remove casein, and obtain whey; The centrifuge was used for centrifugation. The speed of the centrifuge was 8000 rpm. The centrifuge time was 20 minutes. The temperature of the centrifuge was 4° C. During the centrifugation process, the temperature of the bovine colostrum was controlled at 45° C.

[0055] 4. pH adjustment and heat treatment: Use 0.1M hydrochloric acid solution to adjust the pH of whey to 3.8, heat to 85°C, heat for 5 minutes, cool to 4°C, centrifuge to obtain active immunoglobulin G solution; The centrifuge was centrifuged at a speed of 10,000 rpm, a centrifugal time of 30 minutes, and a temperature of 4°C.

[0056] 5. Detect the activity of IgG in the active immunoglobulin G solution: 5.1. Take the active immunoglobulin G solution obtained in step 4, carefully titrate with 0.1 M NaOH solution, check the pH value until the pH is between 5.4-6.4, centrifuge at 4°C (12000 rpm, 20 minutes), remove the supernatant, and obtain immunoglobulin G solid.

[0057] 5.2. Collect the precipitate (i.e., immunoglobulin G solid) and use the formula to calculate the IgG activity and content. The calculation formula is: IgG precipitation ratio = IgG precipitation amount (mg) / IgG concentration (mg / mL) * IgG solution volume (mL) * 100%; 6. Test results: The activity of immunoglobulin G (IgG) was detected using the amount of immunoglobulin G precipitation. Figure 7 The effect of different heating times on the activity of immunoglobulin G (IgG) is shown. At 0 minutes of heating, the IgG activity was about 40%, indicating that the protein remained fully active without heating, and IgG maintained its structural integrity and function without thermal stress. When heated continuously for 30 and 60 minutes, the IgG activity dropped to almost zero. This sharp drop indicates complete denaturation and loss of function of IgG. Prolonged high-temperature heating can cause irreversible melting and aggregation of protein molecules, thereby losing antibody activity. The loss of IgG activity indicates that the protein is heat-sensitive. This aggregation affects the tertiary structure of IgG, changes its antigen binding site and reduces its activity. Monitoring changes in the state of ionizable amino acids can provide insight into the activity and function of IgG.

[0058] When IgG is at its isoelectric point (pI), its net charge is zero, solubility is minimal, and precipitation increases. Precipitation typically occurs in a pH range close to the isoelectric point, suggesting a correlation between solubility and the functional activity of the protein. In addition, factors such as protein charge, ionizable amino acids, and isoelectric point play a key role in the stability and activity of IgG. Solubility decreases near the isoelectric point, promoting aggregation, which affects the structural integrity and antigen binding ability of IgG. Therefore, controlling pH, temperature, and ionic strength is essential to maintain the active form of IgG, ensuring its use in immune responses and efficacy in the formulation of IgG-containing products such as colostrum supplements and functional foods.

[0059] Example 4: Detection of IgG activity in bovine colostrum using a colorimeter A method for detecting IgG activity in bovine colostrum using a colorimeter method, the specific steps are as follows: 1. Standard sample: bovine colostrum liquid (IgG content 61.2 mg / mL); 2. Centrifugal degreasing: Put the bovine colostrum standard sample into the centrifuge for degreasing to obtain defatted bovine colostrum; The speed of the centrifuge degreasing is 8000 rpm, the time of centrifugal degreasing is 20 minutes, and the temperature of the centrifuge is 4°C; 3. Removing casein: Raise the temperature of the skimmed bovine colostrum to 45°C, adjust the pH value of the skimmed bovine colostrum to 4.6 using 0.1M hydrochloric acid solution, centrifuge, remove casein, and obtain whey; The centrifuge was used for centrifugation. The speed of the centrifuge was 8000 rpm. The centrifuge time was 20 minutes. The temperature of the centrifuge was 4° C. During the centrifugation process, the temperature of the bovine colostrum was controlled at 45° C.

[0060] 4. pH adjustment and heat treatment: The pH of the whey was adjusted to 3.8 using 0.1 M hydrochloric acid solution, the temperature was raised to 85°C, heated for 5 min, cooled to 4°C, and centrifuged to obtain an active immunoglobulin G solution; The centrifuge was centrifuged at a speed of 10,000 rpm, a centrifugal time of 30 minutes, and a temperature of 4°C.

[0061] 5. Detect the activity of IgG in the active immunoglobulin G solution: 5.1 Titration: Take the active IgG solution obtained in step 4 and carefully titrate it with 0.1M NaOH solution to check the pH value until the pH is between 5.4-6.4 and the color changes to white; 5.2. IgG activity detection: The color of the immunoglobulin G solution was measured using a colorimeter (CR-40, Japan Minolta Camera Co., Ltd.) with illuminant D65, a viewing angle of 0 degrees, and an observation area of ​​0.13 mm in diameter to determine the color of the precipitated immunoglobulin G, and the calculation formula was: Colorimeter: The total color change (ΔE) was calculated using the formula to quantify the change in color intensity.

[0062] Where: ΔL * Represents brightness changes; Δa * and Δb * Represents the change of anti-color dimension; 6. Test results: The activity of immunoglobulin G (IgG) was tested using a colorimeter. This was achieved by measuring the color difference (ΔE) change during the precipitation process before and after heating at 85°C. The experimental results showed that as the heating time increased, the total color difference value (ΔE) of IgG gradually decreased, such as Figure 8 As shown. At 0 minutes, the color difference is the largest, but it decreases significantly after 15 minutes and almost drops to the minimum at 30 minutes and 60 minutes, indicating that the IgG activity is reduced by heating. At the same time, the IgG precipitation content decreases with the increase of heating time. There is significant IgG precipitation at the beginning (0 minutes), but as the heating time increases, the amount of precipitation gradually decreases and almost disappears at 60 minutes. These changes indicate that IgG denatures during heating at 85°C, resulting in reduced activity, decreased solubility, and ultimately reduced precipitation. These data show that the activity of IgG is not only affected by heating, but also related to pH changes. When heating and pH adjustment are used in combination, significant changes in IgG activity can be observed. The ΔE value measured by the colorimeter is directly related to the changes in the physical and functional state of IgG. The use of a colorimeter can clearly monitor the changes in IgG activity with heating and pH changes, proving that it is an effective tool for detecting changes in IgG activity and its precipitation content.

[0063] The present application discloses a method for detecting the activity of immunoglobulin G, which focuses on the ionizable amino acids (charge analysis) inside IgG, reducing the risks associated with handling antigens or pathogens; the detection method disclosed in the present application does not directly interact with dangerous biological agents, but detects IgG activity by analyzing the net charge and isoelectric point of the protein, thereby improving safety and paving the way for the widespread application of advanced technologies for protein activity analysis in immunology and diagnostics; the method is simple and clear, does not require complex and special equipment, and is suitable for various laboratory settings; the present application discloses a method for detecting the activity of immunoglobulin G, which obtains results faster than traditional technologies, which is crucial for applications that require quick decision-making; the method is more economical and affordable than traditional methods such as ELISA and turbidimetry, reducing overall costs; the method focuses on ionizable amino acids, net charge and isoelectric point, and the results are consistent and reliable under different environmental conditions; the method disclosed in the present application is less affected by external factors such as temperature and pH, ensuring the stability and repeatability of the results.

Claims

1. A method for detecting immunoglobulin G activity, characterized in that: The specific steps are as follows: (1) Centrifugally defatting the bovine colostrum to obtain defatted bovine colostrum; (2) raising the temperature of the defatted bovine colostrum to 40-50° C., adjusting the pH of the defatted bovine colostrum to 4.4-4.6, centrifuging, removing casein, and obtaining whey; (3) adjusting the pH of the whey to 3.6-3.8, heating to 80-90°C for 1-5 min, cooling to 4°C, centrifuging, and obtaining an active immunoglobulin G solution; (4) Adjusting the pH of the active immunoglobulin G solution obtained in step (3), and detecting the activity of IgG in the active immunoglobulin G solution.

2. A method for detecting immunoglobulin G activity according to claim 1, characterized in that: In step (1), the centrifugal degreasing step is as follows: centrifugal degreasing is performed on bovine colostrum using a centrifugal device, the speed of the centrifugal device is 8000-12000 rpm, the time of centrifugal degreasing is 20-30 minutes, and the temperature of the centrifugal device is 4°C-10°C.

3. The method for detecting immunoglobulin G activity according to claim 1, characterized in that: In the step (2), the solution used to adjust the pH of the defatted bovine colostrum to 4.4-4.6 is an acid solution; wherein the acid solution is a hydrochloric acid solution or a citric acid solution.

4. The method for detecting immunoglobulin G activity according to claim 1, characterized in that: The centrifugation step in step (2) is as follows: centrifuge the defatted bovine colostrum with a centrifuge device, the speed of the centrifuge device is 8000-12000 rpm; the centrifugation time is 20-30 minutes; the temperature of the centrifuge device is 4°C-10°C.

5. The method for detecting immunoglobulin G activity according to claim 1, characterized in that: During the centrifugation in step (2), the temperature of the defatted bovine colostrum is controlled at 40-50°C.

6. The method for detecting immunoglobulin G activity according to claim 1, characterized in that: The centrifugation step in step (3) is as follows: centrifuge the whey with a centrifugal device, the rotation speed of the centrifugal device is 8000-12000 rpm, the centrifugation time is 20-30 minutes, and the temperature of the centrifugal device is 4°C-10°C.

7. The method for detecting immunoglobulin G activity according to claim 1, characterized in that: In the step (3), the solution used to adjust the pH of the whey to 3.6-3.8 is an acid solution; wherein the acid solution is a hydrochloric acid solution or a citric acid solution.

8. The method for detecting immunoglobulin G activity according to claim 1, characterized in that: The method for detecting the activity of IgG in the active immunoglobulin G solution in step (4) is any one of spectrophotometry, ultraviolet absorption spectroscopy, precipitation weight method or colorimetry.

9. The method for detecting immunoglobulin G activity according to claim 8, characterized in that: When the activity of IgG in the active immunoglobulin G solution is detected by spectrophotometry, the pH of the active immunoglobulin G solution is adjusted to 5.4-6.4; When the activity of IgG in the active immunoglobulin G solution is detected by ultraviolet absorption spectroscopy, the pH of the active immunoglobulin G solution is adjusted to 3-8; When the activity of IgG in the active immunoglobulin G solution is detected by the precipitation weight method, the pH of the active immunoglobulin G solution is adjusted to 5.4-6.4; When the activity of IgG in the active immunoglobulin G solution is detected by the colorimeter method, the pH of the active immunoglobulin G solution is adjusted to 5.4-6.4.

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