Method for extracting immune globulin from fresh pig blood

Abstract

The invention discloses a method for extracting immune globulin in fresh pig blood, and relates to the technical field of fresh pig blood extraction.The method comprises the following steps that firstly, a sample is pretreated, secondly, immune globulin is captured, Protein A / G or an antibody is fixed to the inner wall of a channel in a micro-fluidic chip, and the immune globulin is captured; 3, impurity flushing, wherein the channel is flushed through multi-stage cleaning; 4, immunoglobulin elution and collection, in the method, a micro-fluidic chip mode is adopted, and the immunoglobulin extraction speed can be increased; in the micro-fluidic chip, Protein A / G or an antibody is fixed on the inner wall of a channel to capture the immune globulin, and then the immune globulin is eluted from the channel and collected, so that the extraction speed of the immune globulin is increased; the channel is flushed through multi-stage cleaning, so that the extraction purity of immune globulin can be improved; the immune globulin capturing efficiency is improved through the tuning algorithm, and the concentration of the captured immune globulin is improved.

Description

Technical Field

[0001] The invention relates to the technical field of fresh pig blood extraction, and in particular to a method for extracting immunoglobulin from fresh pig blood. Background Art

[0002] The immune system is composed of immune tissues, organs, immune cells and immune active molecules. Immunoglobulins are a type of immune active molecules, and immune active molecules include immune cell membrane molecules, such as antigen recognition receptors, differentiation antigens, major histocompatibility molecules and some other receptor molecules; they also include molecules synthesized and secreted by immune cells and non-immune cells, such as immunoglobulin molecules, complement molecules and cytokines. Immunoglobulin is a concept in chemical structure. The chemical basis of all antibodies is immunoglobulin, but not all immunoglobulins have antibody activity.

[0003] According to patent number: CN107151268A - A method for extracting immunoglobulins from fresh pig blood, it is recorded that "the crude IgG product is passed through a DEAE52-cellulose chromatography column, eluted with a borate buffer, and the eluate is collected and concentrated to obtain an IgG product; in this step, the chromatography column adopts a DEAE52-cellulose chromatography column, and the immunoglobulin is electrostatically bonded to the chromatography column, and then eluted with a borate buffer to ensure the high purity of the IgG product." From this, a person skilled in the art can know that the reference patent uses a polyphosphate salting-out method for extraction, and although the extraction purity is high, the cost also increases. Not only that, the extraction efficiency is low, which further reduces its scope of application.

[0004] In summary, a method for extracting immunoglobulins from fresh pig blood was designed. Summary of the invention

[0005] In order to overcome the above-mentioned shortcomings, the present invention provides a method for extracting immunoglobulin from fresh pig blood.

[0006] The present invention achieves the above-mentioned purpose through the following technical solutions: A method for extracting immunoglobulin from fresh pig blood comprises the following steps: Step 1: Sample pretreatment: blood cells in the sample are directly intercepted by a microfluidic chip, plasma enters the reaction area, and immunoglobulins are released; Step 2: Immunoglobulin capture: In the microfluidic chip, Protein A / G or antibodies are fixed on the inner wall of the channel to capture immunoglobulins; Step 3: Flushing impurities, flushing the channel through multi-stage cleaning; Step 4: eluting and collecting immunoglobulins, eluting the immunoglobulins from the channel and collecting them; The step 2 comprises the following steps: S21, channel design and surface treatment, a polydimethylsiloxane channel is designed on polydimethylsiloxane, and the polydimethylsiloxane channel passes through Plasma treatment to generate -SiOH groups, and then immersed in 2% ethanol solution at 60°C for 2 hours to form an amino surface on the surface of the polydimethylsiloxane channel; S22, ligand immobilization and functionalization, MES buffer containing 1mM EDC and 0.2mM NHS was perfused into the polydimethylsiloxane channel, the pH of the MES buffer was 5.5, and the carboxyl group was activated for 30 minutes. The activated solid phase carrier was rinsed with PBS buffer, the pH of the PBS buffer was 7.4, to remove unreacted EDC and NHS, and then 0.1mg / mL recombinant Protein A solution was injected into the treated polydimethylsiloxane channel and reacted at room temperature for about 2 hours. At this time, the amino groups on the Protein A molecules reacted with the activated carboxyl groups to form covalent bonds, thereby coupling Protein A to the surface of the solid phase polydimethylsiloxane channel; S23, blocking treatment. After the coupling reaction is completed, in order to prevent nonspecific adsorption, a blocking agent, such as 1% BSA solution, is used to block the surface of the polydimethylsiloxane channel. The solution is incubated at room temperature for 1 hour. The blocking agent can occupy the unreacted active sites on the surface of the carrier, reduce the nonspecific binding of other substances in subsequent experiments, and improve the specificity of detection or purification. S24, wash and store, rinse the blocked polydimethylsiloxane channel with PBS buffer to remove excess blocking agent and unbound Protein A; S25, capturing immunoglobulins, passing the sample through a polydimethylsiloxane channel to capture immunoglobulins in the sample; S26. Real-time monitoring and optimization: the flow rate of the sample flowing through the polydimethylsiloxane channel is monitored in real time, and real-time calculation is performed according to the optimization algorithm, and the optimal data is fed back to the terminal to control the flow rate of the sample flowing through the polydimethylsiloxane channel.

[0007] Preferably, the tuning algorithm comprises the following steps: S261, system modeling, assuming that the state variables of the chromatography process are , the output variable is , which is the flow rate of the sample flowing through the polydimethylsiloxane channel, and the output variable is , set as the concentration of immunoglobulin, the system can be described by a discrete state space model: + , where A, B, C, and D are system matrices; S262, prediction model, predict the future based on the system model Output at each moment: ,i= , ,i= , in, is the output at time k+i predicted at time k, is the predicted state, is the input of the prediction; S263, Objective Function, defines an objective function J to measure the error between the predicted output and the expected output and the change in the control input: ; in, and is the weight coefficient, is the expected output, - is the change in control input; S264, optimization solution, at each sampling time k, solve the minimum value of the objective function J to obtain the optimal control output sequence , , , and the first control output As the actual control input at the current moment ; S265, particle swarm optimization, each particle represents a possible control input sequence , the objective function J is used as the fitness function of the particle. The smaller the fitness value, the better the particle. S266, particle update, each particle is updated according to its own historical optimal position and the global optimal position of the group Update your position and velocity: + ; in, is the velocity of particle i in the jth dimension, is the position of particle i in the jth dimension, is the inertia weight, and is the learning factor, and is a random number between 0 and 1.

[0008] Preferably, the step 1 comprises the following steps: S11, direct whole blood sampling: no centrifugation is required, a 3μm filter is integrated at the chip entrance of the microfluidic chip to intercept blood cells, and plasma enters the reaction area; S12. Online lysis: The lysis buffer is mixed with plasma to release immunoglobulins. The lysis buffer includes TritonX-100 at a concentration of 0.1%, and the lysis takes less than 2 minutes.

[0009] Preferably, in step S24, the carrier coupled with Protein A is filled with a storage solution containing 0.05% NaN3 and stored in a sealed container at 4°C away from light to maintain its activity and stability. The shelf life is up to 6 months.

[0010] Preferably, the step three comprises the following steps: S31. Preliminary buffer flushing: Use phosphate buffer to flush the functionalized channel bound to immunoglobulins at a flow rate of 10-20μL / min for 3-5 minutes. Phosphate buffer is a commonly used physiological buffer, and its composition and pH value are similar to the in vivo environment. By flushing, a large amount of impurities in the channel that are not bound to the functionalized surface can be removed, such as albumin in serum and other non-specifically adsorbed proteins. Since these impurities do not specifically bind to the ligands (such as Protein A) fixed on the channel surface, they are flushed away under the flow of the buffer; S32, low concentration of chaotropic agent flushing, using a buffer containing 0.5M NaCl, continue to flush the channel at a flow rate of 8-15μL / min, and the flushing time is 5-8 minutes. Chaotropic agents (such as NaCl) can destroy weak interactions between proteins and channel surfaces, such as ionic bonds, hydrogen bonds, and van der Waals forces. Some impurities may non-specifically adsorb to the channel surface or bound immunoglobulins through these weaker interactions. Low concentration NaCl solution can elute these weakly bound impurities without affecting the specific binding of immunoglobulins to ligands, further improving purity; S33, rinse with deionized water, rinse the channel with deionized water at a flow rate of 20-30μL / min for 2-3 minutes. After the previous rinsing steps, buffer and salt ions may remain in the channel. The role of deionized water is to remove these residual salts to avoid their impact on the subsequent elution steps and the properties of immunoglobulins. Because too high salt concentration may interfere with the elution process of immunoglobulins, or interfere with the results in subsequent analytical tests.

[0011] Preferably, the step three also includes a step of checking the flushing effect, and the flushing effect is judged by detecting the absorbance, conductivity and other parameters of the effluent, using a UV-visible spectrophotometer to detect the absorbance of the effluent at a wavelength of 280 nm. When the absorbance drops to a low and stable value, it indicates that most of the impurities have been flushed away; or using a conductivity meter to detect the conductivity of the effluent. When the conductivity is close to the conductivity of deionized water, it indicates that the salt has been basically removed. Protein has a characteristic absorption peak at a wavelength of 280nm. By detecting the absorbance, the content of protein impurities in the effluent can be roughly determined. The conductivity mainly reflects the concentration of ions in the solution, and detecting the conductivity can understand the residual salt in the channel.

[0012] Preferably, the step 4 comprises the following steps: S41. Prepare the elution buffer, using glycine buffer with pH=3.0 as the elution buffer. The preparation of this buffer requires precise control of its composition and concentration to ensure that it can effectively destroy the binding force between Protein A and immunoglobulin without causing irreversible damage to the immunoglobulin. S42, perform elution, inject the eluent into the functionalized channel at a flow rate of 5-10μL / min for 5-20 minutes. The flow rate of the eluent needs to be optimized according to factors such as the size of the channel, the amount of bound protein, and the nature of the eluent to ensure that the elution process is both efficient and does not cause the immunoglobulin to be washed away without being fully eluted due to too fast a flow rate. The elution time of the eluent is to ensure that most of the bound target immune proteins can be eluted. During the elution process, the progress of the elution can be judged by monitoring certain indicators of the effluent (such as absorbance, conductivity, etc.); S43, in-situ neutralization, after the eluent flows out of the channel, the eluent is immediately mixed with the neutralizing solution, and the pH value of the eluent is quickly adjusted to near neutrality to prevent the immunoglobulin from being denatured due to long-term exposure to acidic conditions; S44. Collect immunoglobulins. Use a microcentrifuge tube to collect the neutralized effluent, which contains the eluted immunoglobulins.

[0013] The beneficial effects of the present invention are as follows: in the method for extracting immunoglobulin from fresh pig blood: 1. The microfluidic chip method is used to increase the speed of immunoglobulin extraction; 2. In the microfluidic chip, Protein A / G or antibodies are fixed on the inner wall of the channel to capture immunoglobulins, and then the immunoglobulins are eluted from the channel and collected, thereby increasing the extraction speed of immunoglobulins; 3. Flushing the channel through multi-stage cleaning can improve the purity of immunoglobulin extraction; 4. By tuning the algorithm, the efficiency of immunoglobulin capture can be improved, thereby increasing the concentration of captured immunoglobulins. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The present invention will now be described by way of example with reference to the accompanying drawings, in which: Figure 1 It is a method step diagram of the present invention; Figure 2 is a diagram of the steps of sample pretreatment of the present invention; Figure 3 is a step diagram of immunoglobulin capture of the present invention; Figure 4 is a step diagram of impurity flushing of the present invention; Figure 5 is a step diagram of immunoglobulin elution and collection of the present invention; Figure 6 It is a step diagram of the tuning algorithm of the present invention. DETAILED DESCRIPTION

[0015] The present invention will now be described in further detail with reference to the accompanying drawings. These drawings are simplified schematic diagrams, which only illustrate the basic structure of the present invention in a schematic manner, and therefore only show the components related to the present invention.

[0016] like Figure 1-Figure 5 As shown, a method for extracting immunoglobulin from fresh pig blood comprises the following steps: Step 1: Sample pretreatment: blood cells in the sample are directly intercepted by a microfluidic chip, plasma enters the reaction area, and immunoglobulins are released; Step 2: Immunoglobulin capture: In the microfluidic chip, Protein A / G or antibodies are fixed on the inner wall of the channel to capture immunoglobulins; Step 3: Flushing impurities, flushing the channel through multi-stage cleaning; Step 4: eluting and collecting immunoglobulins, eluting the immunoglobulins from the channel and collecting them; The step 2 comprises the following steps: S21, channel design and surface treatment, a polydimethylsiloxane channel is designed on polydimethylsiloxane, and the polydimethylsiloxane channel passes through Plasma treatment to generate -SiOH groups, and then immersed in 2% ethanol solution at 60°C for 2 hours to form an amino surface on the surface of the polydimethylsiloxane channel; S22, ligand immobilization and functionalization, MES buffer containing 1mM EDC and 0.2mM NHS was perfused into the polydimethylsiloxane channel, the pH of the MES buffer was 5.5, and the carboxyl group was activated for 30 minutes. The activated solid phase carrier was rinsed with PBS buffer, the pH of the PBS buffer was 7.4, to remove unreacted EDC and NHS, and then 0.1mg / mL recombinant Protein A solution was injected into the treated polydimethylsiloxane channel and reacted at room temperature for about 2 hours. At this time, the amino groups on the Protein A molecules reacted with the activated carboxyl groups to form covalent bonds, thereby coupling Protein A to the surface of the solid phase polydimethylsiloxane channel; S23, blocking treatment. After the coupling reaction is completed, in order to prevent nonspecific adsorption, a blocking agent, such as 1% BSA solution, is used to block the surface of the polydimethylsiloxane channel. The solution is incubated at room temperature for 1 hour. The blocking agent can occupy the unreacted active sites on the surface of the carrier, reduce the nonspecific binding of other substances in subsequent experiments, and improve the specificity of detection or purification. S24, wash and store, rinse the blocked polydimethylsiloxane channel with PBS buffer to remove excess blocking agent and unbound Protein A; S25, capturing immunoglobulins, passing the sample through a polydimethylsiloxane channel to capture immunoglobulins in the sample; S26. Real-time monitoring and optimization: the flow rate of the sample flowing through the polydimethylsiloxane channel is monitored in real time, and real-time calculation is performed according to the optimization algorithm, and the optimal data is fed back to the terminal to control the flow rate of the sample flowing through the polydimethylsiloxane channel.

[0017] Specifically, the step 1 includes the following steps: S11, direct whole blood sampling: no centrifugation is required, a 3μm filter is integrated at the chip entrance of the microfluidic chip to intercept blood cells, and plasma enters the reaction area; S12. Online lysis: The lysis buffer is mixed with plasma to release immunoglobulins. The lysis buffer includes TritonX-100 at a concentration of 0.1%, and the lysis takes less than 2 minutes.

[0018] Specifically, in the step S24, the carrier coupled with Protein A is filled with a storage solution containing 0.05% NaN3 and stored in a sealed container at 4°C away from light to maintain its activity and stability. The shelf life is up to 6 months.

[0019] Specifically, the step three includes the following steps: S31. Preliminary buffer flushing: Use phosphate buffer to flush the functionalized channel bound to immunoglobulins at a flow rate of 10-20μL / min for 3-5 minutes. Phosphate buffer is a commonly used physiological buffer, and its composition and pH value are similar to the in vivo environment. By flushing, a large amount of impurities in the channel that are not bound to the functionalized surface can be removed, such as albumin in serum and other non-specifically adsorbed proteins. Since these impurities do not specifically bind to the ligands (such as Protein A) fixed on the channel surface, they are flushed away under the flow of the buffer; S32, low concentration of chaotropic agent flushing, using a buffer containing 0.5M NaCl, continue to flush the channel at a flow rate of 8-15μL / min, and the flushing time is 5-8 minutes. Chaotropic agents (such as NaCl) can destroy weak interactions between proteins and channel surfaces, such as ionic bonds, hydrogen bonds, and van der Waals forces. Some impurities may non-specifically adsorb to the channel surface or bound immunoglobulins through these weaker interactions. Low concentration NaCl solution can elute these weakly bound impurities without affecting the specific binding of immunoglobulins to ligands, further improving purity; S33, rinse with deionized water, rinse the channel with deionized water at a flow rate of 20-30μL / min for 2-3 minutes. After the previous rinsing steps, buffer and salt ions may remain in the channel. The role of deionized water is to remove these residual salts to avoid their impact on the subsequent elution steps and the properties of immunoglobulins. Because too high salt concentration may interfere with the elution process of immunoglobulins, or interfere with the results in subsequent analytical tests.

[0020] Specifically, the step three also includes a step of checking the flushing effect, and the flushing effect is judged by detecting the absorbance, conductivity and other parameters of the effluent. The absorbance of the effluent is detected at a wavelength of 280 nm using a UV-visible spectrophotometer. When the absorbance drops to a low and stable value, it indicates that most of the impurities have been washed away; or the conductivity of the effluent is detected using a conductivity meter. When the conductivity is close to the conductivity of deionized water, it indicates that the salt has been basically removed. Protein has a characteristic absorption peak at a wavelength of 280 nm. By detecting the absorbance, the content of protein impurities in the effluent can be roughly determined. The conductivity mainly reflects the concentration of ions in the solution, and the detection of the conductivity can understand the residual salt in the channel.

[0021] Specifically, the step 4 includes the following steps: S41. Prepare the elution buffer, using glycine buffer with pH=3.0 as the elution buffer. The preparation of this buffer requires precise control of its composition and concentration to ensure that it can effectively destroy the binding force between Protein A and immunoglobulin without causing irreversible damage to the immunoglobulin. S42, perform elution, inject the eluent into the functionalized channel at a flow rate of 5-10μL / min for 5-20 minutes. The flow rate of the eluent needs to be optimized according to factors such as the size of the channel, the amount of bound protein, and the nature of the eluent to ensure that the elution process is both efficient and does not cause the immunoglobulin to be washed away without being fully eluted due to too fast a flow rate. The elution time of the eluent is to ensure that most of the bound target immune proteins can be eluted. During the elution process, the progress of the elution can be judged by monitoring certain indicators of the effluent (such as absorbance, conductivity, etc.); S43, in-situ neutralization, after the eluent flows out of the channel, the eluent is immediately mixed with the neutralizing solution, and the pH value of the eluent is quickly adjusted to near neutrality to prevent the immunoglobulin from being denatured due to long-term exposure to acidic conditions; S44. Collect immunoglobulins. Use a microcentrifuge tube to collect the neutralized effluent, which contains the eluted immunoglobulins.

[0022] like Figure 6 As shown, the tuning algorithm includes the following steps: S261, system modeling, assuming that the state variables of the chromatography process are , the output variable is , which is the flow rate of the sample flowing through the polydimethylsiloxane channel, and the output variable is , set as the concentration of immunoglobulin, the system can be described by a discrete state space model: + , where A, B, C, and D are system matrices; S262, prediction model, predict the future based on the system model Output at each moment: ,i= , ,i= , in, is the output at time k+i predicted at time k, is the predicted state, is the input of the prediction; S263, Objective Function, defines an objective function J to measure the error between the predicted output and the expected output and the change in the control input: ; in, and is the weight coefficient, is the expected output, - is the change in control input; S264, optimization solution, at each sampling time k, solve the minimum value of the objective function J to obtain the optimal control output sequence , , , and the first control output As the actual control input at the current moment ; S265, particle swarm optimization, each particle represents a possible control input sequence , the objective function J is used as the fitness function of the particle. The smaller the fitness value, the better the particle. S266, particle update, each particle is updated according to its own historical optimal position and the global optimal position of the group Update your position and velocity: + ; in, is the velocity of particle i in the jth dimension, is the position of particle i in the jth dimension, is the inertia weight, and is the learning factor, and is a random number between 0 and 1.

[0023] As a specific example, the goal is to maximize the extraction rate of immunoglobulins while ensuring that the purity of the immunoglobulins is not less than 90%.

[0024] 1. System modeling: the state space model of the ion exchange chromatography process is obtained by fitting the experimental data: A= ,B= , C , D = 0, state variable Indicates the protein concentration and impurity concentration in the column, input variable Indicates flow rate, output variable Indicates the purity of immunoglobulin.

[0025] 2. Parameter setting, prediction time domain =10, control time domain =5, weight system =1, =0.1, number of particles N=20, inertia weight =0.7, learning factor = =1.4, maximum number of iterations T=50.

[0026] 3. Algorithm implementation steps: A. Initialization: Initialize the position and velocity of the particle and set the initial state x(0).

[0027] B. Based on the current state x(k), use the prediction model to calculate the output at Np moments in the future.

[0028] C. Solve the minimum value of the objective function J through particle swarm optimization to obtain the optimal control input sequence ; D. Control execution, the first element of the optimal control input sequence As the actual control input at the current moment, update the system status .

[0029] E. Repeat steps BD until the preset termination condition is reached, that is, the maximum number of iterations is reached or the purity of the immunoglobulin meets the requirements.

[0030] The above is based on the present invention as an inspiration. Through the above description, relevant staff can make various changes and modifications without departing from the technical idea of ​​this invention. The technical scope of this invention is not limited to the content in the specification, and its technical scope must be determined according to the scope of the claims.

Claims

1. A method for extracting immunoglobulin from fresh pig blood, characterized in that: The following steps are involved: Step 1: Sample pretreatment: blood cells in the sample are directly intercepted by a microfluidic chip, plasma enters the reaction area, and immunoglobulins are released; Step 2: Immunoglobulin capture: In the microfluidic chip, Protein A / G or antibodies are fixed on the inner wall of the channel to capture immunoglobulins; Step 3: Flushing impurities, flushing the channel through multi-stage cleaning; Step 4: eluting and collecting immunoglobulins, eluting the immunoglobulins from the channel and collecting them; The step 2 comprises the following steps: S21, channel design and surface treatment, a polydimethylsiloxane channel is designed on polydimethylsiloxane, and the polydimethylsiloxane channel passes through Plasma treatment to generate -SiOH groups, and then immersed in 2% ethanol solution at 60°C for 2 hours to form an amino surface on the surface of the polydimethylsiloxane channel; S22, ligand immobilization and functionalization, MES buffer containing 1mM EDC and 0.2mM NHS was perfused into the polydimethylsiloxane channel, the pH of the MES buffer was 5.5, and the carboxyl group was activated for 30 minutes. The activated solid phase carrier was rinsed with PBS buffer, the pH of the PBS buffer was 7.4, to remove unreacted EDC and NHS, and then 0.1mg / mL recombinant Protein A solution was injected into the treated polydimethylsiloxane channel and reacted at room temperature for about 2 hours. At this time, the amino groups on the Protein A molecules reacted with the activated carboxyl groups to form covalent bonds, thereby coupling Protein A to the surface of the solid phase polydimethylsiloxane channel; S23, blocking treatment, using a blocking agent to block the surface of the polydimethylsiloxane channel and incubating at room temperature for 1 hour; S24, wash and store, rinse the blocked polydimethylsiloxane channel with PBS buffer to remove excess blocking agent and unbound Protein A; S25, capturing immunoglobulins, passing the sample through a polydimethylsiloxane channel to capture immunoglobulins in the sample; S26. Real-time monitoring and optimization: the flow rate of the sample flowing through the polydimethylsiloxane channel is monitored in real time, and real-time calculation is performed according to the optimization algorithm, and the optimal data is fed back to the terminal to control the flow rate of the sample flowing through the polydimethylsiloxane channel.

2. The method for extracting immunoglobulin from fresh pig blood according to claim 1, characterized in that: The tuning algorithm includes the following steps: S261, system modeling, assuming that the state variables of the chromatography process are , the output variable is , which is the flow rate of the sample flowing through the polydimethylsiloxane channel, and the output variable is , set as the concentration of immunoglobulin, the system can be described by a discrete state space model: + , where A, B, C, and D are system matrices; S262, prediction model, predict the future based on the system model Output at each moment: ,i= , ,i= , in, is the output at time k+i predicted at time k, is the predicted state, is the input of the prediction; S263, Objective Function, defines an objective function J to measure the error between the predicted output and the expected output and the change in the control input: ; in, and is the weight coefficient, is the expected output, - is the change in control input; S264, optimization solution, at each sampling time k, solve the minimum value of the objective function J to obtain the optimal control output sequence , , , and the first control output As the actual control input at the current moment ; S265, particle swarm optimization, each particle represents a possible control input sequence , the objective function J is used as the fitness function of the particle. The smaller the fitness value, the better the particle. S266, particle update, each particle is updated according to its own historical optimal position and the global optimal position of the group Update your position and velocity: + ; in, is the velocity of particle i in the jth dimension, is the position of particle i in the jth dimension, is the inertia weight, and is the learning factor, and is a random number between 0 and 1.

3. The method for extracting immunoglobulin from fresh pig blood according to claim 1, characterized in that: The step 1 comprises the following steps: S11, direct whole blood sampling: no centrifugation is required, a 3μm filter is integrated at the chip entrance of the microfluidic chip to intercept blood cells, and plasma enters the reaction area; S12. Online lysis: The lysis buffer is mixed with plasma to release immunoglobulins. The lysis buffer includes TritonX-100 at a concentration of 0.1%, and the lysis takes less than 2 minutes.

4. The method for extracting immunoglobulin from fresh pig blood according to claim 1, characterized in that: In the step S24, the carrier coupled with Protein A is filled with a storage solution containing 0.05% NaN3 and stored in a sealed container at 4°C away from light.

5. The method for extracting immunoglobulin from fresh pig blood according to claim 1, characterized in that: The step three comprises the following steps: S31, preliminary buffer flushing, using phosphate buffer to flush the functionalized channel bound to the immunoglobulin at a flow rate of 10-20 μL / min, and the flushing time lasts for 3-5 minutes; S32, low concentration chaotropic agent flushing, using a buffer containing 0.5 M NaCl, continue to flush the channel at a flow rate of 8-15 μL / min, and the flushing time is 5-8 minutes; S33. Rinse with deionized water. Rinse the channel with deionized water at a flow rate of 20-30 μL / min for 2-3 minutes.

6. The method for extracting immunoglobulin from fresh pig blood according to claim 4, characterized in that: The step three also includes a step of checking the flushing effect, and judging the flushing effect by detecting the absorbance and conductivity parameters of the effluent.

7. The method for extracting immunoglobulin from fresh pig blood according to claim 6, characterized in that: The step 4 comprises the following steps: S41, prepare a elution solution, using a glycine buffer with a pH of 3.0 as an elution solution; S42, performing elution, injecting the eluent into the functionalized channel at a flow rate of 5-10 μL / min for 5-20 minutes; S43, in-situ neutralization, immediately mixing the eluent with a neutralizing solution after the eluent flows out of the channel, and quickly adjusting the pH value of the eluent to near neutrality; S44. Collect immunoglobulins. Use a microcentrifuge tube to collect the neutralized effluent, which contains the eluted immunoglobulins.

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