Method for detecting insoluble immune complex in urine

Abstract

The invention discloses a method for detecting insoluble immune complexes in urine, which comprises the following steps: (1) filtering: filtering a urine sample to be detected by using a microporous filter membrane, and flushing the filter membrane for multiple times by using a PBS (Phosphate Buffer Solution); (2) protein redissolution: putting the filter membrane into a tubular container, then adding a sodium hydroxide solution and a dithiothreitol solution, and performing high-temperature treatment in a water bath for 30-35 minutes; then adding an iodoacetamide solution, standing in a dark place in a room temperature environment, and neutralizing with a formic acid aqueous solution; (3) enzymolysis: carrying out enzymolysis on a product treated in the step (2) by adopting alkaline trypsin; and (4) liquid chromatography-tandem mass spectrometry detection. According to the invention, the insoluble immune complex can be detected, the problem that the detection is influenced by antigenicity reduction caused by redissolution of the insoluble immune complex is avoided, and the accuracy of the detection result is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of medical detection, and particularly relates to a method for detecting insoluble immune complexes in urine. Background Art

[0002] IgA nephropathy (IgAN) is one of the most common primary glomerular diseases worldwide, and its pathological feature is the deposition of immune complexes mainly composed of IgA in the glomerular mesangial region. Its incidence rate among primary glomerular diseases in China is about 50%, and about 30% - 40% of IgAN patients can develop into end-stage kidney disease within 30 years. With the rapid economic growth and lifestyle changes, China is facing an increasing burden of IgA diseases. According to statistics, there are about 5 million potential IgA nephropathy patients in China, and it is estimated that the number of IgA nephropathy patients in China will reach about 2.4 million by 2030. However, the causes of kidney diseases are complex and the progression is slow. The awareness rate of chronic kidney diseases in China is only 12.5%. As a common type among them, the awareness rate of IgA nephropathy is also relatively low. In addition, the early symptoms of IgA nephropathy are not obvious, and many patients already have kidney damage when they are diagnosed, resulting in more than half of the patients missing the best treatment opportunity. Therefore, improving the understanding and early diagnosis of IgAN is particularly crucial for delaying the progression of IgA nephropathy. Previous studies have found that some immune complexes in glomerular deposits can be excreted out of the body through urine, and the content of immune complexes in the urine of IgAN patients is significantly higher than that of the non-IgAN disease control group. In addition, other studies have also shown that in the urine of IgAN patients, IgA mainly appears in the form of insoluble antigen-antibody complexes.

[0003] The characteristic manifestation of IgAN is that deglycosylated IgA1 triggers autoimmunity, forms IgA1-IgG immune complexes, and deposits in the glomerular mesangial region. This process triggers an inflammatory response and glomerular damage. Therefore, the detection of the content of IgA or IgA complexes in patients is an important indicator for the occurrence and development of IgAN. It has been found in the renal biopsy tissues of IgAN patients that in addition to the deposition of IgA immune complexes in the mesangial region, there are also depositions of IgG and / or IgM, complement C3, lectin, and C4d components. These indicate that the expression of serum immunoglobulins is closely related to various immune kidney diseases and is different. The determination of serum IgA level, the evaluation of IgA / C3 ratio, and the determination of serum IgG level are closely related to glomerular damage and the severity of the disease, providing a non-invasive method for the diagnosis and efficacy monitoring of IgAN. However, the specificity and sensitivity of the above detection methods have not yet met the requirements of clinical diagnosis, and they cannot distinguish IgAN from other immune-related diseases, so it is difficult to be used in actual clinical diagnosis.

[0004] Currently, techniques based on the specific reaction of antigen-antibody are commonly used to detect such substances, such as enzyme-linked immunosorbent assay (ELISA), enhanced latex immunoturbidimetry, etc. These detection techniques have been quite mature and are widely used in clinical tests. However, these studies are all premised on the analyte being in a soluble state and are powerless for the detection of insoluble substances. Although there have been various methods attempting to redissolve antibodies by dissociating immune complexes, most of these methods are based on the mechanism of protein denaturation, which may cause irreversible structural changes in some antibodies, thus losing the binding ability to antigens, which will undoubtedly seriously affect the accuracy of the detection results.

[0005] Renal tissue biopsy is the gold standard for the diagnosis of IgAN. Pathological immunofluorescence examination shows the deposition of IgA or IgA-dominated immune complexes in the glomerular mesangial region. At the same time, observe whether there are other symptoms such as endothelial hyperplasia, crescent bodies, nephritis, etc., and combine the renal injury-related indicators (urinary protein, occult blood, and creatinine) in the urine sample and the immune-related indicators (immunoglobulins, cytokines, complements, etc.) in the blood sample. Using these detection indicators, the progression and severity of the disease can be described according to methods such as Lee grading, Haas classification, and Oxford classification, and then corresponding treatment strategies can be formulated.

[0006] However, renal biopsy is an invasive examination. In addition to having potential risks such as bleeding and infection, the cost of renal biopsy is relatively high, about 6,000 yuan per time, and it cannot be repeatedly detected. More importantly, early-stage nephropathy patients do not have the indication for renal biopsy, so this method is not applicable to the early diagnosis of IgAN patients.

[0007] Searching for early diagnostic markers and establishing corresponding detection methods are of great significance for the diagnosis and treatment of IgAN. Summary of the Invention

[0008] The purpose of the present invention is to provide a method for detecting insoluble immune complexes in urine. After treatment, it can detect insoluble immune complexes, avoid the problem that the antigenicity reduction caused by the redissolution of insoluble immune complexes affects the detection, and thus can improve the accuracy of the detection results.

[0009] The technical solution adopted by the present invention to solve its technical problems is: A method for detecting insoluble immune complexes in urine, comprising the following steps: (1) Filtration: The urine sample to be tested is filtered using a microporous filter membrane, and the filter membrane is rinsed multiple times with PBS buffer to remove residual soluble antibodies; (2) Protein reconstitution: Place the filter membrane in a tubular container, then add sodium hydroxide solution and dithiothreitol solution, and perform high-temperature treatment in a water bath for 30 - 35 min; then add iodoacetamide solution, let it stand in the dark at room temperature, and neutralize with formic acid aqueous solution; (3) Enzymatic digestion: Use alkaline trypsin to digest the product obtained in step (2). (4) Liquid chromatography - tandem mass spectrometry detection: Select the DTLMISR polypeptide sequence as the marker for IgG, and select SAVQGPPER as the marker for IgA. Use liquid chromatography - tandem mass spectrometry for detection. After calculation, obtain the concentration of insoluble immune complexes. During the detection process, add isotope - labeled DTL*MISR and SAV*QGPPER as internal standards. L* represents isotope - labeled leucine, and V* represents isotope - labeled valine. Establish standard curves for IgG and IgA by the internal standard method, and then substitute the detection results of the urine sample to be tested into the standard curves to calculate the concentrations of IgG and IgA in the insoluble immune complexes.

[0010] The binding between immune complexes mainly relies on the formation of hydrogen bonds. Heating can disrupt the hydrogen bonds and dissolve the complexes. However, heating may cause protein denaturation and coagulation. Therefore, in the heating process of the present invention, reducing substances such as dithiothreitol are added to break the disulfide bonds of proteins, making the antibody form smaller heavy - chain and light - chain subunits, and using iodoacetamide to protect the free sulfhydryl groups to reduce the possibility of its coagulation.

[0011] Although the above - mentioned method can reconstitute insoluble immune complexes, it loses antigenicity and cannot be detected by traditional methods such as ELISA. Therefore, the present invention then selects to digest the protein and then use liquid chromatography - tandem mass spectrometry for detection. Select DTLMISR and SAVQGPPER, where IgG and IgA subtypes are both stably present, as markers, so that antibody subunits without antigenicity can be measured.

[0012] In the present invention, the urine sample to be tested is filtered through a 0.22 - μm aqueous filter membrane to retain insoluble immune complexes on the filter membrane. Use PBS buffer to repeatedly rinse the filter membrane to remove residual soluble antibodies. After placing the filter membrane in a test tube, add sodium hydroxide and dithiothreitol solution, and perform high - temperature treatment to break the hydrogen bonds and disulfide bonds between immune complexes and between antibody subunits, enabling them to redissolve. After treatment, add iodoacetamide to block free sulfhydryl groups. Subsequently, perform enzymatic digestion with alkaline trypsin. Select the DTLMISR polypeptide sequence as the marker for IgG, and select SAVQGPPER as the marker for IgA. Use liquid chromatography - tandem chromatography for detection. During the detection process, add isotope - labeled DTL*MISR and SAV*QGPPER as internal standards to calibrate the matrix effect during mass spectrometry ionization. Among them, L* is13 C 6 , 15 N]-Leucine, and V is 13 C 5 , 15 N]-Valine.

[0013] In step (4), the chromatographic conditions are as follows: Chromatographic column: Prochrom300 C18 reversed-phase chromatographic column, specification: 2.1x100mm, particle size 3μm; Column temperature: 40°C; Mobile phase A: Ultra-pure water containing 0.1% formic acid (by volume); Mobile phase B: Acetonitrile; Gradient elution; Injection volume: 3 μL Flow rate: 0.4 ml / min.

[0014] The gradient elution parameter settings are as follows: At the start, the proportion of mobile phase B is 5%; At 5 min, the proportion of mobile phase B is 100%; At 5.4 min, the proportion of mobile phase B is 100%; At 5.5 min, the proportion of mobile phase B is 5%; At 7.5 min, the proportion of mobile phase B is 5%.

[0015] Mass spectrometry parameters: Ionization mode: ESI+ Ion source temperature: 150°C Capillary voltage: 0.5 kV Desolvation gas flow rate: 800 L / hr Desolvation temperature: 500°C Cone hole back purge gas flow rate: 150 L / hr The monitoring mode adopts the multiple reaction monitoring mode MRM.

[0016] When detecting the biomarker of IgG, the parent ion is 418.2, and the daughter ions are 175.1 and 217.1; when detecting the biomarker of IgA, the parent ion is 470.7, and the daughter ions are 159.1 and 555.3.

[0017] Specifically, the protein reconstitution in step (2) is as follows: Place the filter membrane in a tubular container, then add 200 μL of sodium hydroxide solution and 100 μL of dithiothreitol solution, and perform high-temperature treatment in a water bath for 30 - 35 min; then add 100 μL of iodoacetamide solution, let it stand in the dark at room temperature, and then neutralize it with 200 μL of formic acid aqueous solution; Among them, the concentration of the sodium hydroxide solution is 1 mol / L, the concentration of the dithiothreitol solution is 100 mmol / L, the concentration of the iodoacetamide solution is 300 mmol / L, and the concentration of the formic acid aqueous solution is 1 mol / L.

[0018] The high temperature for the high-temperature treatment is 80 - 90 °C.

[0019] The specific enzymatic hydrolysis in step (3) is as follows: Add 200 μL of ammonium bicarbonate buffer and 10 μL of trypsin solution to the product obtained after the treatment in step (2), incubate overnight in a water bath at 37 °C, and then add 10 μL of the termination solution; The concentration of the ammonium bicarbonate buffer is 200 mmol / L, the concentration of the trypsin solution is 1 mg / mL, and the termination solution is a 30% (by volume) formic acid aqueous solution.

[0020] The beneficial effects of the present invention are as follows: A simple heating method can be used to redissolve the insoluble immune complexes, and protein coagulation and denaturation can be avoided. Further combined with enzymatic hydrolysis and liquid chromatography-tandem mass spectrometry detection, precise detection is carried out using specific polypeptide markers, avoiding interference factors brought by the redissolution operation. Description of the Drawings

[0021] Figure 1 It is the standard curve graph of IgG; Figure 2 It is the standard curve graph of IgA; Figure 3 It is the relationship graph between the dosage of sodium hydroxide and the recovery rate, blue: IgG specific peptide; orange: IgA specific peptide; Figure 4 It is the relationship graph between the incubation temperature and the recovery rate, blue: IgG specific peptide; orange: IgA specific peptide; Figure 5 It is the comparison graph of the tolerance of different polypeptides in a strong base and high-temperature environment. Detailed Embodiments

[0022] The following are specific examples to further illustrate the technical solutions of the present invention.

[0023] In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the following examples are conventional methods in the art unless otherwise specified.

[0024] Example 1: Preparation of Reagents PBS buffer: Weigh 8.0 g of sodium chloride (NaCl), 0.2 g of sodium dihydrogen phosphate (NaH 2 PO4·H2O), and sodium hydrogen phosphate (Na 21.15 g of HPO4 was added to 900 mL of ultrapure water and dissolved. The pH value was adjusted to 7.4, and then made up to 1000 mL with ultrapure water; Sodium hydroxide solution (1 mol / L): 4 g of sodium hydroxide was weighed and dissolved in 100 mL of ultrapure water; Formic acid solution (1 mol / L): 5 mL of pure formic acid was measured and added to 95 mL of ultrapure water; Dithiothreitol solution (100 mmol / L): 0.15 g of dithiothreitol was accurately weighed and dissolved in 10 mL of ultrapure water; Iodoacetamide solution (300 mmol / L): 0.555 mg of iodoacetamide was accurately weighed and dissolved in 10 mL of ultrapure water; Ammonium bicarbonate buffer solution (200 mmol / L): 15.8 mg of ammonium bicarbonate was accurately weighed and dissolved in 1000 mL of ultrapure water; Trypsin (1 mg / mL): 10 mg of trypsin was accurately weighed and dissolved in 10 mL of aqueous solution containing 0.1% formic acid; Termination solution: 3 mL of formic acid and 7 mL of water were accurately measured and mixed evenly.

[0025] 5 mg of isotope-labeled DTL*MISR and SAV*QGPPER were accurately weighed and dissolved in 1 mL of 30% aqueous acetonitrile solution, and further diluted to obtain an internal standard solution with the target concentration.

[0026] Example 2: Preparation of spiked samples for standard curve and quality control Human immunoglobulin (Hualan Biological, 2.5 g (5% 50 ml)) was diluted with PBS buffer according to requirements, and 5 times the mass of goat anti-human Ig polyclonal antibody (Quick Science) was added to artificially prepare insoluble immune complexes. The above sample was centrifuged at 8000 g for 10 min, and the supernatant was discarded. After washing the precipitate with PBS buffer three times repeatedly, 500 mL of healthy human urine without antibodies was added, and it was shaken overnight on a shaker to form a suspension again.

[0027] Example 3: Pretreatment method Filtration A microporous filter membrane with a diameter of 13 mm (pore size 0.22 μm) was taken, washed and wetted with ultrapure water, and then loaded into the corresponding filter membrane holder. One end of it was connected to a 50 mL syringe. After adding 50 mL of urine sample (spiked sample or clinical sample) to the syringe, the syringe barrel was pushed to make the sample slowly flow through the filter membrane, avoiding too fast flow rate resulting in the filter membrane rupture. After all the urine sample passed through the filter membrane, 50 mL of PBS buffer was added to the syringe barrel again, and the syringe barrel was pushed in the same way to wash the filter membrane. After repeating the washing 3 times, the filter membrane was taken off and put into a 2 mL EP tube.

[0028] Protein reconstitution Add 200 μL of sodium hydroxide and 100 μL of dithiothreitol solution to an EP tube, heat it in a water bath at 90 °C for 30 min, add 100 μL of iodoacetamide solution, and let it stand in the dark at room temperature for 30 min. Finally, neutralize the solution with 200 μL of formic acid.

[0029] Enzymatic digestion: Add 200 μL of ammonium bicarbonate buffer and 10 μL of trypsin solution to an EP tube, incubate it overnight in a water bath at 37 °C, add 10 μL of termination solution and 10 μL of internal standard solution (containing 1000 ng / mL of DTL*MISR and 100 ng / mL of SAV*QGPPER), and wait for injection.

[0030] Example 4: LC-MS Conditions Instrument: Waters Xevo TQ-S (Waters Corporation, USA) Chromatographic conditions: Chromatographic column: Prochrom300 C18 reversed-phase chromatographic column (2.1 x 100 mm, 3 μm) Mobile phase A: Ultra-pure water containing 0.1% formic acid Mobile phase B: Acetonitrile Mobile phase ratio: The elution gradient is shown in Table 1 Flow rate: 0.4 ml / min Injection volume: 3 μL Column temperature: 40 °C Table 1 Time Proportion of Mobile Phase B Start 5 5 min 100 5.4 min 100 5.5 min 5 7.5 min 5

[0031] Mass spectrometry conditions: Instrument: Waters Xevo TQ-XS Ionization mode: ESI+ Ion source temperature: 150 °C Capillary voltage: 0.5 kV Desolvation gas flow rate: 800 L / hr Desolvation temperature: 500 °C Cone hole backflush gas flow rate: 150 L / hr The monitoring mode adopts the multiple reaction monitoring mode MRM.

[0032] Multiple reaction ion monitoring (MRM) parameters L* is 13 C 6 , 15 N]-leucine, and V is 13 C 5 , 15 N]-valine.

[0033] Example 5: Linear Range When preparing the spiked samples of the standard curve solution described in "Example 2", by adding human immunoglobulin solutions with different concentrations, the concentrations of IgG and IgA in the samples reach 1 - 100 μg / mL and 0.1 - 10 μg / mL respectively.

[0034] After subjecting the above spiked samples to pretreatment according to the method of Example 3 of the present invention, they are injected for detection. A standard curve is established with the peak area ratio of the standard substance to the internal standard as the ordinate and the concentration as the abscissa. The results show that the method has good linearity in the corresponding ranges of IgG and IgA (correlation coefficient R 2 > 0.99)( Figure 1-2 ).

[0035] Example 6: Inter - batch Precision Test Prepare spiked samples of IgG and IgA with three different concentrations for detection, and divide them into 3 batches for detection, with 7 parallels each time. The spiked concentrations and the measured results are shown in Table 2 - Table 3. The calculated coefficient of variation of the detection results is less than 15%, meeting the requirements of clinical tests.

[0036] Table 2: Precision Results of IgG

[0037] Table 3: Precision Results of IgA

[0038] Example 7: Recovery Test In this study, spiked samples of IgG and IgA with three different concentrations are prepared for detection, divided into 3 batches for detection, with 7 parallels each time. The recoveries are shown in Table 4 - 5, all between 85 - 1115%, meeting the requirements of clinical tests.

[0039] Table 4: IgG Recovery

[0040] Table 5: IgA Recovery

[0041] Comparative Example 1: Anti - interference Ability (Soluble Antibody) Prepare an insoluble immune complex suspension of urine matrix (prepared according to the method of Example 2), in which the concentrations of IgG and IgA reach 25 μg / mL and 2.5 μg / mL respectively.

[0042] In addition, soluble immunoglobulins were added to blank urine to bring the IgG and IgA concentrations to 25 μg / mL and 2.5 μg / mL, respectively. The present method and a commercial immunoturbidimetry kit were used for detection. The results are shown in Table 6.

[0043] From the detection results, it can be seen that the traditional immunoturbidimetry can only detect part of the soluble immunoglobulins, and has problems such as high background signal and large fluctuations in results. In contrast, the method developed in the present invention can selectively detect insoluble immune complexes, exclude the interference of soluble immunoglobulins in the sample, and has high precision and strong specificity in results.

[0044] Table 6: Recovery rates of different spiked samples

[0045] Example 8: Comparison of protein resolubilization protocols The precipitate formed by immune complexes is difficult to resolubilize. Some common methods in proteomics, such as heating and breaking disulfide bonds, although can partially increase the protein concentration, still cannot achieve ideal results for the already formed precipitate of immune complexes. Therefore, more intense reaction conditions need to be added during the resolubilization process, such as adding strong base sodium hydroxide and using a relatively high temperature.

[0046] On the one hand, the above intense reaction conditions can destroy the protein spatial structure. However, on the other hand, they can also destroy the chemical structure of the target polypeptide, resulting in the inability to measure the enzymatic hydrolysis products. Therefore, a comparative study was conducted on the addition amount of sodium hydroxide and the heating temperature. The optimized range of the addition amount of sodium hydroxide solution (1 mol / L) was 10, 50, 100, 150, 200, 250, 300 μL, and the corresponding formic acid solution (1 mol / L) was also kept consistent with sodium hydroxide to neutralize the reaction system. The optimized range of the heating temperature was room temperature, 40 °C, 50 °C, 60 °C, 70 °C, 80 °C, 90 °C, boiling water bath. The results are shown in Figure X and Figure X.

[0047] The results of IgG showed that the recovery rate was low when the addition amount of sodium hydroxide was low, indicating that most proteins were still not resolubilized and thus difficult to measure. When the addition amount of sodium hydroxide was increased to 150 - 200 μL, the recovery rate increased significantly, and the optimal addition volume was 200 μL, at which time the recovery rate was as high as 96.1%. As the addition amount of sodium hydroxide further increased, the recovery rate of proteins decreased, probably due to the instability of the product polypeptides in the strong base and high temperature environment. The results shown by IgA were similar to those of IgG, but its optimal addition volume was between 200 - 250 μL ( Figure 3 ). Considering the need to detect both proteins simultaneously, the addition amount of sodium hydroxide in the present invention was set at 200 μL.

[0048] Based on the optimized results of the volume of sodium hydroxide added above, the present invention further optimizes the incubation temperature. As the temperature rises, the recovery rates of IgG and IgA also gradually increase. When the incubation temperature reaches 80 - 90 °C, the recovery rate reaches the highest ( Figure 4 ). Considering that the IgG recovery rate is slightly lower at 80 °C and the coefficient of variation of IgA is relatively large, 90 °C is selected as the optimal temperature.

[0049] Example 9: Comparison of the tolerance of polypeptides in a strong base and high temperature environment Polypeptides are prone to destruction in a strong base and high temperature environment. Therefore, it is important to select polypeptides with relatively high chemical stability.

[0050] Through comparison, it is found that there are 3 general polypeptides in IgG, namely DTLMISR (SEQ ID No.1), NQVSTCLVK (SEQ ID No.2), and LTVDK (SEQ ID No.3). After synthesizing the above three polypeptides respectively, the present invention places them in a strong base and high temperature environment to verify their stability. There are 5 specific polypeptides suitable for testing in IgA, namely SVTCHVK (SEQ ID No.4), SAVQGPPER (SEQ ID No.5), WLQGSQELPR (SEQ ID No.6), YLTWASR (SEQ ID No.7), VAAEDWK (SEQ ID No.8). The present invention also uses the same method to verify their stability.

[0051] Prepare a polypeptide solution with a concentration of 1000 ng / mL. Take 10 μL of the polypeptide solution, 200 μL of sodium hydroxide solution, and 100 μL of dithiothreitol solution, heat them in a water bath at 90 °C for 5 - 60 min, add 100 μL of iodoacetamide solution, and let it stand in the dark at room temperature for 30 min. Finally, neutralize the solution with 200 uL of formic acid. The subsequent enzymatic digestion operation is the same as that in Example 3. Calculate the recovery rate of the polypeptide after detection. The results show that there are significant differences in the stability of different polypeptides. Among them, the IgG general peptide DTLMISR and the IgA general peptide SAVQGPPER selected by the present invention have the best stability, which can reach more than 40 minutes ( Figure 5 ). Therefore, the above two polypeptides are suitable for a reaction system with a strong base and high temperature. And combined with the results of protein resolubilization, the optimal reaction time is set at 30 min.

[0052] The above-described embodiments are only a preferred solution of the present invention, and do not impose any form of limitation on the present invention. There are other variations and modifications without exceeding the technical solutions recorded in the claims.

Claims

1. A method for detecting insoluble immune complexes in urine, characterized in that: The steps include: (1) Filtration: The urine sample to be tested is filtered through a microporous membrane and the membrane is rinsed multiple times with PBS buffer to remove residual soluble antibodies; (2) Protein resolubilization: Place the filter membrane in a tubular container, then add sodium hydroxide solution and dithiothreitol solution, and treat at high temperature in a water bath for 30-35 minutes; then add iodoacetamide solution, let stand in the dark at room temperature, and then neutralize with formic acid aqueous solution; (3) Enzymatic hydrolysis: using alkaline trypsin to enzymatically hydrolyze the product treated in step (2); (4) Liquid chromatography tandem mass spectrometry detection: The DTLMISR polypeptide sequence was selected as a marker for IgG, and SAVQGPPER was selected as a marker for IgA. Liquid chromatography tandem mass spectrometry was used for detection. The concentration of the insoluble immune complex was calculated. During the detection process, isotope-labeled DTL*MISR and SAV*QGPPER were added as internal standards. L* represents isotope-labeled leucine, and V* represents isotope-labeled valine.

2. The method according to claim 1, characterized in that In step (4), the chromatographic conditions are as follows: chromatographic column: Prochrom 300C18 reverse phase chromatographic column, specification: 2.1 x 100 mm, particle size 3 μm; Column temperature: 40°C; Mobile phase A: ultrapure water containing 0.1% formic acid; Mobile phase B: acetonitrile; Gradient elution; Injection volume: 3 μL Flow rate: 0.4ml / min.

3. The method according to claim 2, characterized in that The gradient elution parameters were set as: Initially, mobile phase B accounted for 5%; 5 min, mobile phase B 100%; 5.4min, mobile phase B 100%; 5.5min, mobile phase B 5%; 7.5min, mobile phase B accounted for 5%.

4. The method according to claim 1, characterized in that Mass spectrometry parameters: Ionization mode: ESI+ Ion source temperature: 150°C Capillary voltage: 0.5kV Desolvation gas flow rate: 800L / hr Desolventization temperature: 500℃ Cone hole backflush gas flow rate: 150L / hr The monitoring mode adopted was multiple reaction monitoring mode MRM.

5. The method according to claim 1, characterized in that When the marker mass spectrometry of IgG was detected, the parent ion was 418.2, and the daughter ions were 175.1 and 217.1; when the marker mass spectrometry of IgA was detected, the parent ion was 470.7, and the daughter ions were 159.1 and 555.

3.

6. The method according to claim 1, characterized in that Step (2) protein redissolution is specifically as follows: place the filter membrane in a tubular container, then add 200 μL of sodium hydroxide solution and 100 μL of dithiothreitol solution, and treat at high temperature in a water bath for 30-35 minutes; then add 100 μL of iodoacetamide solution, let stand in a dark place at room temperature, and then neutralize with 200 uL of formic acid aqueous solution; Among them, the concentration of sodium hydroxide solution is 1 mol / L, the concentration of dithiothreitol solution is 100 mmol / L, the concentration of iodoacetamide solution is 300 mmol / L, and the concentration of formic acid aqueous solution is 1 mol / L.

7. The method according to claim 1 or 6, characterized in that: The high temperature for high temperature treatment is 80-90°C.

8. The method according to claim 1, characterized in that The enzymatic hydrolysis in step (3) is specifically as follows: adding 200 μL of ammonium bicarbonate buffer and 10 μL of trypsin solution to the product treated in step (2), incubating in a 37° C. water bath overnight, and adding 10 μL of stop solution; The concentration of ammonium bicarbonate buffer was 200 mmol / L, the concentration of trypsin solution was 1 mg / mL, and the stop solution was a formic acid aqueous solution with a volume concentration of 30%.

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