Specific antibody fc fragment glycosylation mrm quantitative analysis method and application thereof

Abstract

The application discloses a specific antibody Fc segment glycosylation MRM quantitative analysis method and application thereof, and comprises the following steps: characterizing isotopic labeling and non-isotopic labeling glycopeptide and polypeptide standard products; adding glycopeptide and polypeptide standard products into a blank matrix to prepare standard solutions with different concentrations; performing pretreatment on a to-be-measured biological sample, including antigen immobilization, immunosorbent assay and protease hydrolysis and cleavage; adopting ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry to perform determination, so as to realize quantitative analysis on specific antibody subtypes and glycosylation types by using characteristic ions of glycopeptide and polypeptide standard products; establishing a standard curve of glycopeptide and polypeptide standard products containing an isotopic internal standard, and calculating antibody subtype and glycosylation type concentrations of the to-be-measured biological sample. The specific antibody subtype and glycosylation type of patient serum are subjected to MRM quantitative analysis, which is rapid, accurate and sensitive, and the fucose deficiency and galactose deficiency levels have good clinical diagnostic values for viral infectious diseases and autoimmune diseases.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a quantitative analysis method for specific antibody Fc fragment glycosylation MRM and its application. Background Technology

[0002] Antibodies are central mediators of the immune system, with IgG being the most prevalent immunoglobulin. When an IgG / antigen complex binds to membrane-bound Fcγ receptors (FcγRs), it triggers a cellular immune response, which is regulated by multiple factors. First, different IgG subclasses have varying affinities for a given FcγR, and different FcγRs exhibit heterogeneity in ligand specificity, expression patterns, and triggering effector functions. Furthermore, post-translational glycosylation modifications of antibodies and Fcγ receptors regulate the affinity of their interactions.

[0003] In existing technologies, multiple studies have shown that the Fc-terminal fucose deficiency of human IgG antibodies plays a key role in enhancing antibody-dependent cytotoxicity (ADCC). Shields et al. revealed the effect of fucoidylated oligosaccharides on antibody effector function, including binding to human FcγR, C1q, FcRn, and ADCC. Fucosylated IgG1s showed a 50-fold increase in binding to FcγRIIIa and enhanced ADCC effects. Toyohide Shinkawa et al. found that using purified human peripheral blood mononuclear cells as effectors, the antibody-dependent cytotoxic ADCC of anti-CD2 chimeric IgG0 generated by anti-human interleukin-5 receptor (hIL-5R), humanized immunoglobulin G1 (IgG1), and rat hybridoma YB20 / 1 cell lines was more than 50-fold higher than that generated by Chinese hamster ovary (CHO) cell lines.

[0004] Under normal circumstances, human plasma IgG is highly fucosylated, while defucosylated antigen-specific IgG responses have played an important role in various pathological processes, including alloimmune responses to blood cells, responses to Plasmodium falciparum antigens expressed on erythrocytes, and immune responses to viruses such as human immunodeficiency virus, dengue virus, and SARS-CoV-2. Regarding the main mechanism of action of Fc core fucosylated IgG1 antibodies, Fc core-fucosylated IgG1 antibodies exhibit a 10-100-fold increase in binding to FcγRIIA and FcγRIIA on indicated immune cells (NK, Mø, and PMN). This leads to increased ADCC on NK cells, enhanced competition with plasma IgG, increased PMN activation, and increased PMN inhibition of FcγRIIA-mediated ADCC. In addition, changes in antibody glycosylation have been observed during active disease in a variety of autoantibody-dependent and autoantibody-independent autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, and inflammatory bowel disease. The most common change is the increase in IgG-GOF glycoform lacking terminal galactose and sialic acid residues during the inflammatory phase.

[0005] In critically ill COVID-19 patients, inhibition of B-cell FUT8 glycosyltransferase leads to a significant increase in non-fucosylated IgG antibody levels. Non-fucosylation elicits a stronger immune response, triggering a cytokine storm and acute respiratory distress syndrome, ultimately resulting in severe COVID-19. Severe COVID-19 is characterized by inflammatory features, including high levels of inflammatory cytokines, alveolar inflammatory infiltration, and vascular microthrombosis. Severe COVID-19 patients exhibit unique serological characteristics, including increased IgG1 with non-fucosylated Fc glycans. This Fc modification of SARS-CoV-2 IgG enhances and activates the interaction of FcγR and FcγRIIIa. When incorporated into immune complexes, fucosylated Fc receptors enhance the production of inflammatory cytokines by monocytes (including IL-6 and TNF).

[0006] The variable region of immunoglobulin IgG molecules exerts its neutralizing and blocking effects directly by binding to viral envelope proteins. The crystallizable region (Fc) of IgG molecules can bind to complement and also to immune cells expressing Fc receptors, such as monocytes / macrophages and NK cells. After antibodies bind to immune cells via the Fc region, they can regulate the function of these cells. Fc receptors are classified into activating and inhibitory types. The N-glycosylation sites of the Fc region exhibit complex and diverse N-glycan structural modifications, with core fucose being a crucial modification. These N-glycans can regulate the affinity of Fc for Fc receptors. For example, core fucose in the N-glycan regulates the binding of Fc to the activating Fc receptor (FcR IIIa). Modification of the N-glycan with core fucose reduces the affinity for FcR IIIa by tens of times, representing a molecular mechanism to prevent over-activation of FcR IIIa.

[0007] A research group led by Jeffery Ravetch at Rockefeller University discovered that IgG1 lacking core fucose exhibits increased affinity for FcR IIIa, leading to severe dengue fever. Following the outbreak of COVID-19, Wang's lab at Stanford University used biomembrane interferometry (BLI) to detect the affinity of RBD-specific IgG and FcRIIIa in severe infections, finding that RBD-specific IgG bound to recombinant FcRIIIa was significantly enhanced in severe cases compared to mild cases. In experiments activating monocytes, RBD-specific IgG in severe cases induced cytokine release from monocytes, while RBD-specific IgG in mild cases failed to activate cytokine release from monocytes. Simultaneously with Taia Wang's team reporting these findings, Gestur Vidarsson at the University of Amsterdam also reported in Science a similar case of fucose deficiency in anti-spike protein (S protein) IgG in severe infections.

[0008] Studies have shown that abnormal IgG glycosylation (galactose and sialic acid deficiency) is significantly increased in experimental and human autoimmune diseases (such as rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, and infections). A team led by Jörg Köhl at the University of Lübeck discovered that high galactosylation of IgG1 N-glycans promotes synergistic signaling between FcγRIIB and dectin-1, forming an inhibitory pathway that blocks the pro-inflammatory function of C5aR and CXCR2 chemokine receptors. A team led by Jeffery Ravetch revealed that under inflammatory and autoimmune conditions, immune complexes trigger inflammatory macrophage activation through activating FcRs, while hypersialylated Fc induces the secretion of anti-inflammatory mediators by binding to SIGN-R1 lectin on regulatory macrophages in the splenic marginal zone. These mediators act on effector macrophages at the lesion site, upregulating the expression of inhibitory FcgRIIB, thereby increasing the immune complex activation threshold and ultimately reducing autoantibody-mediated inflammatory damage.

[0009] In summary, differences in specific antibody subtypes and glycosylation modifications have a significant impact on the occurrence and development of viral infectious diseases and some autoimmune diseases. Following the global deployment of vaccines against the novel coronavirus, influenza virus, and RSV, the subtypes and fucosylation modifications of virus-specific antibodies remain unclear, especially in the elderly and those with underlying diseases. Deficiencies in Th1 cell-induced antibody subtypes (IgG1 and IgG3 subtypes) and fucosylated antibodies pose potential risks to reduced vaccine efficacy. Several laboratories have conducted rapid detection and analysis studies of specific antibodies against novel coronavirus and influenza vaccines, but standardized analytical methods suitable for widespread use by third-party laboratories are still lacking. Therefore, while optimizing antibody research and treatment, it is crucial to emphasize and strengthen research on the quantitative analysis of antibody Fc fragment glycosylation to achieve timely monitoring and prevention of severe illness. Summary of the Invention

[0010] The main objective of this invention is to provide a quantitative analysis method for Fc glycosylation multiple reaction monitoring (MRM) of specific antibodies. By synthesizing characteristic peptide standards for each IgG subtype and six glycopeptide standards for the Fc segment: G0F, G0NF, G1, G1N, G2, and G2S, an MRM quantitative analysis method suitable for antibody characteristic peptides and glycopeptides is established using ultra-high performance liquid chromatography coupled with a SCIEX 7500 triple quadrupole mass spectrometer. A standard curve containing an internal standard is constructed for quantitative analysis.

[0011] Another objective of this invention is to provide an application of the quantitative analysis method for Fc glycosylation MRM of the specific antibody, including the absolute quantification of the specific antibody content in the serum of patients infected with the novel coronavirus and the absolute quantification of the six glycoforms of the Fc segment, to monitor the fucose and galactose defects of the specific antibody in patients in real time, and to indicate the severity of the disease based on the degree of glycoform defect, thereby achieving a timely preventive monitoring function.

[0012] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for characterizing glycopeptides and characteristic polypeptide standards, the method comprising the following steps: (1) Synthesize isotopically labeled and non-isotopically labeled glycopeptides and characteristic polypeptide standards; (2) The synthesized isotope-labeled glycopeptides and characteristic polypeptide standards were characterized; (3) Characterize the synthesized non-isotope-labeled glycopeptides and characteristic polypeptide standards; (4) A liquid chromatography-mass spectrometry (LC-MS) characterization system was constructed by using an ultra-high performance liquid chromatograph coupled with a triple quadrupole mass spectrometer.

[0013] Secondly, this invention provides a method for quantitative analysis of specific antibody Fc fragment glycosylation MRM, the method comprising the following steps: (i) MRM parameters were set by coupling ultra-high performance liquid chromatography with triple quadrupole mass spectrometry; (ii) Construct a standard curve of IgG1-GPSVFPLAPSSK containing an isotopic internal standard based on the MRM parameter settings; (iii) Based on the MRM parameter settings, construct a mixed standard curve of six glycopeptides including G0F, G0NF, G1, G1N, G2, and G2S with isotopic internal standards.

[0014] Preferably, in step (ii), the concentration gradient of the IgG1-GPSVFPLAPSSK standard curve is set to 5 ng / μL, 1 ng / μL, 500 pg / μL, 100 pg / μL, and 20 pg / μL, and the internal standard concentration is set to 1 ng / μL.

[0015] Preferably, in step (iii), the concentration gradient of the six glycopeptide mixed standard curves is set to 500 pg / μL, 200 pg / μL, 100 pg / μL, 50 pg / μL, 10 pg / μL, and 2 pg / μL, and the internal standard concentration is set to 100 pg / μL.

[0016] Thirdly, the present invention provides a pretreatment method for patient serum samples, the method comprising: (a) Immobilization of disease antigens in patients to be tested, immunoadsorption assay, and protein hydrolysis; (b) Based on the characterization method of the glycopeptide and characteristic polypeptide standards, the synthesized isotopically labeled glycopeptide and characteristic polypeptide standards, and the standard curve containing the isotopic internal standard obtained by the quantitative analysis method of the specific antibody Fc fragment glycosylation MRM, the specific antibody subtype and glycopeptide MRM of the patient's serum sample are absolutely quantified.

[0017] Preferably, the diseases include novel coronavirus, influenza virus, and RSV virus.

[0018] This invention uses synthesized isotope-labeled characteristic peptides of various IgG subtypes and six glycopeptides of the Fc segment: G0F, G0NF, G1, G1N, G2, and G2S. The synthesized isotope standards are used as internal standards for standard curves to correct for instrument response fluctuations and matrix effects, thereby improving quantitative accuracy and enhancing detection sensitivity and specificity, thus providing a powerful tool for clinical diagnosis.

[0019] Compared with the prior art, the beneficial effects of the present invention are at least as follows: 1. High sensitivity and accuracy: By combining an ultra-high performance liquid chromatograph (Exion LC AD) with a SCIEX 7500 triple quadrupole mass spectrometer and the MRM method, a highly sensitive and accurate quantitative analysis technique is proposed, which helps to accurately detect and quantify target peptides and glycopeptides in complex biological samples.

[0020] 2. Use of isotope internal standards: This invention uses isotope internal standards to establish standard curves for characteristic peptides and glycopeptides, which helps to correct matrix effects, instrument fluctuations and losses during sample preparation in the analysis process, and further improves the accuracy and repeatability of quantitative analysis.

[0021] 3. Highly targeted: This invention performs quantitative analysis on specific antibody subtypes and glycotype deficiencies in the serum of patients infected with the novel coronavirus, especially in the detection of fucose and galactose deficiency levels, providing new biomarkers for the diagnosis of viral infectious diseases and autoimmune diseases.

[0022] 4. Clinical diagnostic value: This invention provides valuable information for the clinical diagnosis of viral infectious diseases and autoimmune diseases by detecting fucose and galactose deficiency levels, which helps in the early detection, treatment monitoring and prognosis assessment of diseases.

[0023] 5. Rapid, accurate, and sensitive analysis: This invention combines the MRM quantitative method of liquid chromatography-mass spectrometry with the specificity of immunoadsorption, enabling rapid, accurate, and sensitive analysis of disease-related specific antibody levels and glycoform defect levels, which is of great significance for clinical diagnosis and patient management.

[0024] 6. Methodological Innovation: This invention provides a new methodology that applies liquid chromatography coupled with mass spectrometry to the precise quantitative analysis of serum-specific antibody subtypes and glycoform deficiencies, filling a gap in existing technologies to some extent.

[0025] 7. Broad application prospects: This invention is not only applicable to the analysis of serum from patients infected with the novel coronavirus, but can also be extended to the research and diagnosis of other diseases, and has broad application prospects and potential. Attached Figure Description

[0026] Figure 1 This is a schematic diagram illustrating the quantitative analysis method and application of antibody Fc fragment glycosylation MRM in the examples.

[0027] Figure 2 This is a schematic diagram illustrating the characterization of six isotopically labeled glycopeptide standards in the examples.

[0028] Figure 3 The chromatograms of total MRM for the standard and the sample to be tested are shown in the examples.

[0029] Figure 4 The diagram below shows the basic structure of the glycopeptide in the example; a) Oligosaccharide composition: blue squares represent n-acetylglucosamine, green circles represent mannose, red triangles represent fucose, yellow circles represent galactose, and purple quadrilaterals represent sialic acid; b) Peptide segment is EEQYNSTYR.

[0030] Figure 5 This is a summary curve of the standard curves for the six glycopeptide standards containing isotopic internal standards in the examples.

[0031] Figure 6 The standard curve for the isotopic internal standard IgG1 characteristic polypeptide standard is shown in the examples.

[0032] Figure 7 The concentrations of virus-specific antibodies against the novel coronavirus and influenza virus in patients are shown in the examples.

[0033] Figure 8 The percentage of Fc-terminal glycoform loss of virus-specific antibodies against novel coronavirus and influenza virus in the examples (S-309 is a standard antibody expressed in 293T cells).

[0034] Specific substantive examination methods

[0035] To more fully understand and demonstrate the technical solutions, objectives, and advantages of the present invention, the technical effects produced by the present invention will be further described in detail and completely below with reference to specific embodiments and accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. It should be noted that other embodiments obtained by those skilled in the art without departing from the concept of the present invention are all within the protection scope of the present invention.

[0036] The following examples used an ultra-high performance liquid chromatograph Exion LC AD (AB SCIEX LLC, USA) and a SCIEX 7500 triple quadrupole mass spectrometer (AB SCIEX LLC, USA).

[0037] The following examples present a method for quantitative analysis of specific antibody Fc fragment glycosylation MRM, comprising the following steps: (1) Synthesize isotopically labeled and non-isotopically labeled glycopeptide and polypeptide standards; (2) Glycopeptide and polypeptide standards characterizing isotope-labeled and non-isotope-labeled standards; (3) Prepare standard solutions of different concentrations by adding glycopeptide and polypeptide standards to the blank matrix; (4) Pretreatment of biological samples to be tested, including: antigen immobilization, immunosorbent assay, and protease hydrolysis; (5) Ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry was used to determine the specific antibody subtypes and glycoforms by using the characteristic ions of glycopeptides and polypeptides; (6) Establish standard curves for glycopeptides and polypeptides containing isotopic internal standards, and calculate the antibody subtype and glycoform concentration of the biological sample to be tested.

[0038] In some embodiments, the biological sample to be tested is plasma, serum, or a test solution containing a biological matrix.

[0039] In some embodiments, when the biological sample to be tested is plasma or serum, the pretreatment in step (4) is as follows: the corresponding antigen protein of the sample to be tested is fixed with an antigen-coated plate; after fixing the antigen protein, the diluted serum sample to be tested is added for immunoadsorption; after adsorption, the eluent is concentrated and dried; trypsin solution is added to the dried sample for reaction, and vacuum drying is performed after the reaction is completed.

[0040] In some embodiments, internal standard or external standard methods are used for quantitative determination.

[0041] In some embodiments, in step (5), the quantitative analysis includes the analysis of serum specific antibody subtypes and glycotypes in patients with viral infectious diseases and / or autoimmune diseases, including novel coronavirus, influenza virus, RSV virus. The proportion of fucose deficiency is calculated by normalizing the sum of signal intensities of fucose-deficient glycotypes (G1, G1N, G2, G2S) to the IgG1-GPSVFPLAPSSK peptide intensity, and the proportion of galactose deficiency is calculated by normalizing the sum of signal intensities of galactose-deficient glycotypes (G0F, G0NF) to the IgG1-GPSVFPLAPSSK peptide intensity.

[0042] In some embodiments, the liquid chromatography conditions in step (5) include: an ultra-high performance liquid chromatography system; a reversed-phase column; a water / acetonitrile mobile phase; and gradient elution.

[0043] In some embodiments, in step (5), the liquid chromatography conditions include: an Acquity PREMIER Peptide BEHC18 column; column temperature: 40 °C; mobile phases including: phase A: 0.1% formic acid-water solution and phase B: 0.1% formic acid-acetonitrile solution; flow rate: 0.3 mL / min; gradient elution is performed as follows: the gradient starts with 2% mobile phase B, holds for 0.5 min, then at 9.0 min, the percentage of phase B increases to 38%, then at 10.5 min, it increases to 95%, holds for 12.5 min; finally at 12.6 min, the percentage of phase B decreases to 2%, holds for 2.4 min, and the total run time is 15 min.

[0044] In some embodiments, the triple quadrupole mass spectrometry conditions in step (5) include: ESI ion source, positive ion mode, and MRM detection mode.

[0045] The following examples also propose that the above-mentioned quantitative analysis method for Fc fragment glycosylation MRM of specific antibodies be applied to the analysis of serum specific antibody subtypes and glycotypes in patients with viral infectious diseases and / or autoimmune diseases, including novel coronavirus, influenza virus, RSV virus. Example

[0046] 1. Characterization of standard products 1.1 Information on synthesized isotopically labeled and non-isotopically labeled glycopeptides (G0F, G0NF, G1, G1N, G2, G2S) and peptides Isotope-labeled glycopeptides and polypeptides were characterized using a high-resolution ZenoTOF 7600 system. The isotope labeling positions of the glycopeptides were […]. 13 C6, 15 [N4]-Arginine, the polypeptide isotope labeling position is [ 13 C6,15 N4]-Arginine and [ 13 C6, 15 [N2]-Lysine, as shown in Table 1 and Figure 3 As shown.

[0047] Table 1: Information on Glycopeptides and Polypeptides Standards 1 Glycopeptides G0F-EEQYNSTYR 2 Glycopeptides G1-EEQYNSTYR 3 Glycopeptides G2-EEQYNSTYR 4 Glycopeptides G0NF-EEQYNSTYR 5 Glycopeptides G1N-EEQYNSTYR 6 Glycopeptides G2S-EEQYNSTYR 7 Isotope-labeled glycopeptides G0F-EEQYNSTYR* 8 Isotope-labeled glycopeptides G1-EEQYNSTYR* 9 Isotope-labeled glycopeptides G2-EEQYNSTYR* 10 Isotope-labeled glycopeptides G0NF-EEQYNSTYR* 11 Isotope-labeled glycopeptides G1N-EEQYNSTYR* 12 Isotope-labeled glycopeptides G2S-EEQYNSTYR* 13 IgG1 characteristic polypeptide GPSVFPLAPSSK 14 IgG2 characteristic polypeptide GLPAPIEK 15 IgG3 characteristic polypeptide WYVDGVEVHNAK 16 IgG4 characteristic polypeptide TTPPVLDSDGSFFLYSR 17 IgG characteristic polypeptide DTLMISR 18 Isotope-labeled peptides GPSVFPLAPSSK* 19 Isotope-labeled peptides GLPAPIEK* 20 Isotope-labeled peptides WYVDGVEVHNAK* 21 Isotope-labeled peptides TTPPVLDSDGSFFLYSR* 22 Isotope-labeled peptides DTLMISR* 1.2 Characterization of synthesized isotopically labeled and non-isotopically labeled glycopeptides and polypeptides The liquid chromatography and mass spectrometry methods used for characterization are as follows: a. Liquid phase method The SCIEX ExionLC™ AC liquid chromatography system was used, with a Kinetex 2.6 μm C18 100 Å column and an LC column size of 100 × 2.6 mm. Mobile phase A consisted of 2% acetonitrile / water and 0.1% formic acid; mobile phase B consisted of 98% acetonitrile / water and 0.1% formic acid. The flow rate was 0.3 mL / min, the column temperature was 45 °C, the injection volume was 1 μL, and the separation gradient was shown in Table 2.

[0048] Table 2: Liquid Phase Gradient 0.00 0.3 2 0 0.50 0.3 2 0 9.00 0.3 38 0 9.50 0.3 95 0 11.50 0.3 95 0 11.60 0.3 2 0 b. Mass spectrometry Data acquisition was performed using a SCIEX Zeno TOF 7600 mass spectrometer system. Source gas parameters were as follows: spray gas: 55 psi, auxiliary heating gas: 55 psi, curtain gas: 35 psi, ion source temperature: 500℃, ion source voltage: 5500 V, collision gas: 7 psi, declustering voltage: 60 V. Acquisition mode: Information-dependent acquisition (IDA), primary scan range 400-2500 nm, primary accumulation time 0.25 s, Time bins to sum set to 8; secondary scan range 100-3000 nm, secondary accumulation time 0.1 s, Time bins to sum set to 8; collision energy: dynamic collision energy.

[0049] c. Based on the above liquid chromatography-mass spectrometry method, the characterization results are shown in Table 3.

[0050] Table 3: Characterization results of glycopeptides and polypeptide standards G0F 2634.55 2634.3 G1 2650.54 2650.4 G2 2812.69 2812.5 G0NF 2837.74 2837.1 G1N 2853.74 2853.1 G2S 3103.94 3103.2 IS_G0F 2643.91 2644.2 IS_G1 2659.90 2660.2 IS_G2 2821.96 2822.3 IS_G0NF 2846.90 2847.3 IS_G1N 2862.98 2863.3 IS_G2S 3113.81 3113.5 IgG1_GPSVFPLAPSSK 1186.34 1186.5 IgG2_GLPAPIEK 823.98 824.0 IgG3_WYVDGVEVHNAK 1416.55 1416.7 IgG4_TTPPVLDSDGSFFLYSR 1902.04 1902.0 IgG_DTLMISR 834.97 834.9 IS_IgG1_GPSVFPLAPSSK 1194.34 1194.4 IS_IgG2_GLPAPIEK 831.98 831.9 IS_IgG3_WYVDGVEVHNAK 1424.55 1424.5 IS_IgG4_TTPPVLDSDGSFFLYSR 1912.04 1912.0 IS_IgG_DTLMISR 844.97 844.8 2. Construct a standard curve containing isotopic internal standards. 2.1 Constructing a mixed standard curve containing isotopic internal standard IgG1-GPSVFPLAPSSK and 6 glycopeptides: a. Prepare the loading buffer: 2% CAN (0.1% HA), the standard curve is a 100 μL system, the concentration of the isotope internal standard IgG1-GPSVFPLAPSSK is set to 1 μg / mL, the concentration of the isotope internal standard glycopeptide is set to 100 ng / mL, and 2 μL is injected.

[0051] b. Preparation of internal standard mixture (IS-MIX): 10 μL 10 μg / mL glycopeptides (G0F, G0NF, G1, G1N, G2, G2S) + 10 μL 100 μg / mL IgG1-GPSVFPLAPSSK.

[0052] c. Prepare the concentration points for each standard curve, as shown in Table 4: Table 4: Preparation of Standard Curve

[0053] 2.2 MRM Liquid Chromatography and Mass Spectrometry Methods Ultra-high performance liquid chromatography (UHPLC) using an Exion LC-AD system (AB SCIEX LLC, USA) coupled with a SCIEX 7500 triple quadrupole mass spectrometer (AB SCIEX LLC, USA) was employed for chromatographic separation using an Acquity PREMIER Peptide CSH C18 column (2.1 x 100 mm, 1.7 μm, Waters). Mobile phase A consisted of water containing 0.1% formic acid, and mobile phase B consisted of acetonitrile containing 0.1% formic acid. For glycopeptide analysis, the gradient started with 2% mobile phase B, held for 0.5 min, then increased to 38% at 9.0 min; subsequently increased to 95% at 10.5 min and held for 12.5 min; finally, decreased to 2% at 12.6 min and held for 2.4 min, for a total run time of 15 min. The column temperature was 40 °C, and the flow rate was 0.3 mL / min.

[0054] The SCIEX Triple Quad™ 7500 mass spectrometer was equipped with an OptiFlow Pro electrospray ionization source for positive multiple reaction monitoring (MRM) quantification. MS analysis conditions were as follows: turbine temperature set to 450°C; gas values ​​set as follows: curtain gas 40 psi, GS1 gas 50 psi, GS2 gas 50 psi; collision-activated dissociation (CAD) gas pressure set to 10 psi. Other parameters included ion ejection voltage (1.7 kV), pause time (5 ms), and analyte-specific MRM parameters and m / z ratios, as shown in Table 5.

[0055] Table 5: MRM parameters used for monitoring glycopeptides (HexNAc)4(Hex)3(Fuc)1 G0F 878.69 204.1 15 10 19 11.5 (HexNAc)4(Hex)4 G1 884.021 204.1 15 10 17 11.5 (HexNAc)4(Hex)5 G2 938.039 366.1 15 10 17 11.5 (HexNAc)5(Hex)3(Fuc)1 G0NF 946.384 204.1 15 10 23 11.5 (HexNAc)5(Hex)4 G1N 951.715 204.1 15 10 30 11.5 (HexNAc)4(Hex)5(NeuAc)1 G2S 1035.071 366.1 15 10 39 11.5 (HexNAc)4(Hex)3(Fuc)1 IS_G0F 882.0 204.1 15 10 19 11.5 (HexNAc)4(Hex)4 IS_G1 887.4 204.1 15 10 17 11.5 (HexNAc)4(Hex)5 IS_G2 941.4 366.1 15 10 17 11.5 (HexNAc)5(Hex)3(Fuc)1 IS_G0NF 949.7 204.1 15 10 23 11.5 (HexNAc)5(Hex)4 IS_G1N 955.1 204.1 15 10 30 11.5 (HexNAc)4(Hex)5(NeuAc)1 IS_G2S 1038.4 366.1 15 10 39 11.5 IgG1_GPSVFPLAPSSK GPSVFPLAPSSK 593.8 699.4 15 10 28.1 11.5 IgG2_GLPAPIEK GLPAPIEK 412.8 327.7 15 10 19.2 11.5 IgG3_WYVDGVEVHNAK WYVDGVEVHNAK 708.9 469.3 15 10 33.9 11.5 IgG4_TTPPVLDSDGSFFLYSR TTPPVLDSDGSFFLYSR 951.5 850.4 15 10 45.9 11.5 IgG_DTLMISR DTLMISR 418.2 506.3 15 10 19.5 11.5 IgG1_GPSVFPLAPSSK IS_GPSVFPLAPSSK 597.8 707.4 15 10 28.3 11.5 IgG2_GLPAPIEK IS_GLPAPIEK 416.8 331.7 15 10 19.4 11.5 IgG3_WYVDGVEVHNAK IS_WYVDGVEVHNAK 712.9 477.3 15 10 33.9 11.5 IgG4_TTPPVLDSDGSFFLYSR IS_TTPPVLDSDGSFFLYSR 956.5 855.4 15 10 46.9 11.5 IgG_DTLMISR IS_DTLMISR 423.2 516.3 15 10 19.7 11.5 BSA LGEYGFQNALIVR 740.4 813.49 15 10 15 11.5 EP: entry potential; CE: collision energy; CXP: collision exit potential.

[0056] 3. Preparation of patient serum samples by pretreatment 3.1. Antigen immobilization: a. Dilute Recombinant 2019-nCoV S-trimer Protein (novoprotein, Cat. No.:DRA49) to 2 μg / mL in phosphate-buffered saline (PBS; pH 7.4), and mix tetravalent influenza protein antigens (H1N1, BY, H3N2 and BV subtypes; Chengdu Oulin Biotechnology) in equimolar proportions, and dilute with phosphate-buffered saline (PBS; pH 7.4) to a total protein concentration of 2 µg / mL.

[0057] b. Transfer the diluted antigen to the wells of a 96-well plate, 200 μL / well, seal the plate and incubate at 37°C for 3 hours to ensure that the antigen is coated on the plate.

[0058] c. Washing: Remove the protein solution and wash three times with 250 μL PBS-T, 2 min each time.

[0059] 3.2. Immunosorbent assay: a. 20 μL serum + 180 μL PBS-T, 10-fold dilution, 200 μL / well, incubate overnight at 4°C to capture IgG.

[0060] b. Washing: Wash each well 3 times with 250 μL PBS-T, then wash twice with 250 μL PBS, and then twice with 250 μL 50 mM ABC, each time for 2 min.

[0061] c. Add 200 μL of 100 mM formic acid to each well using a multi-channel pipette, and elute the IgG by shaking and incubating at room temperature for 5 minutes (300 rpm). Transfer the eluent to a 0.5 mL EP tube (Eppendorf Protein LoBind® Tubes (0030108094)).

[0062] d. Dry the eluent in a centrifugal vacuum dryer (speed vac) at 50-60 °C for 4-6 hours.

[0063] 3.3 Protease hydrolysis and cleavage a. Redissolve the dried IgG in 20 μL of 50 mM ABC for 5 minutes, and shake at 450 rpm at room temperature.

[0064] b. Dilute the acidic trypsin solution in ice-cold water at a concentration of 1:20 (v / v) to 50 ng / μL to obtain a trypsin lysis mixture. Add 20 μL of the trypsin lysis mixture to each well and shake at 450 rpm and room temperature for 5 minutes.

[0065] c. Seal with an adhesive sealing plate and incubate overnight at 37°C (recommended incubation time: 18-22 h).

[0066] d. Dry the eluent in a centrifugal vacuum dryer (speed vac) at 37°C for 4-6 hours, and store at -80°C after drying.

[0067] 3.4 LC-MS Measurement Before loading, the dried enzyme-digested sample was redissolved in 10 μL loading buffer (2% ACN (0.1% HA), containing 6 glycopeptides and isotopic internal standards of characteristic peptides) for 5 minutes, shaken at 450 rpm at room temperature, centrifuged at 780 g for 10 minutes at room temperature, and stored in the thermostatic autosampler of the LC-MS instrument at 4℃ until injection.

[0068] 4. Results Analysis Absolute quantification of specific antibody subtypes and MRMs of various glycopeptides in patient serum samples was performed using a constructed standard curve. The percentages of fucose and galactose defects were analyzed, as shown in Table 6. Figure 7 and Figure 8 As shown in the results, the mean Spike-specific IgG1 concentration in SARS-CoV2 infected individuals (2 months post-infection) was 7.65 μg / mL (51.00 nM), while the mean hemagglutinin-specific IgG1 concentration in influenza virus infected individuals was 5.37 μg / mL (35.80 nM). Analysis of influenza and COVID-19 infected individuals using a calibration curve-based detection method revealed significant differences in glycotypes: core fucosylation levels were significantly reduced in influenza infected individuals (22%), while galactosylation levels were increased in both disease groups.

[0069] In addition, the standard curve concentration was set at the second and third points, and the test was repeated three times. Three samples were randomly selected from the patient samples and tested three times. As shown in Table 7, the coefficient of variation of the repeated injection samples was less than 10%, indicating that the detection system has high precision, high consistency in sample processing and preparation, and strong data reliability.

[0070] Table 6: Serum-specific antibodies, molar amounts of various glycopeptides, and glycoform defect levels in influenza and novel coronavirus infected patient samples calculated based on standard curves. Note: Fucose deficiency (%): [(G1+G1N+G2+G2S) / GPS]; Galactose deficiency (%): [(G0F+G0NF) / GPS]; GPS: GPSVFPLAPSSK; jyc, gl, ywh, wn, jj, hy are samples from influenza patients; A11, A10, A13, A8, D1, D13, D3, D11, D6, D2 are samples from patients infected with the novel coronavirus; s309 is a standard antibody with complete core fucose expressed in 293T cells.

[0071] Table 7: Statistical analysis of coefficient of variation for sample and standard curve jyc 2.54% 2.54% 3.25% 4.00% 0.61% 6.37% 0.94% A11 4.32% 4.32% 7.66% 1.81% 4.95% 9.81% 4.40% D1 2.10% 2.10% 2.35% 9.88% 6.15% 3.00% 2.77% gs1 - 6.3% 8.8% 3.2% 5.9% 8.5% 8.3% gs2 - 5.2% 2.2% 2.6% 5.5% 1.3% 9.3% ps1 5.2% - - - - - - ps2 2.8% - - - - - - Note: gs1: glycopeptide-standard1 (100 ng / mL); gs2: glycopeptide-standard1 (50 ng / mL); ps1: peptide standard1 (1 μg / mL); ps2: peptide standard1 (0.5 μg / mL).

[0072] The conditions for use of this invention are: the antibody concentration against a specific antigen reaches 1 μg / mL.

[0073] The above are merely preferred embodiments of the present invention and are not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for quantitative analysis of specific antibody Fc fragment glycosylation MRM, characterized in that, Includes the following steps: (1) Synthesize isotopically labeled and non-isotopically labeled glycopeptide and polypeptide standards; (2) Glycopeptide and polypeptide standards characterizing isotope-labeled and non-isotope-labeled standards; (3) Prepare standard solutions of different concentrations by adding glycopeptide and polypeptide standards to the blank matrix; (4) Pretreatment of biological samples to be tested, including: antigen immobilization, immunosorbent assay, and protease hydrolysis; (5) Ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry was used to determine the specific antibody subtypes and glycoforms by using the characteristic ions of glycopeptides and polypeptides; (6) Establish standard curves for glycopeptide and polypeptide standards containing isotopic internal standards, and calculate the antibody subtype and glycoform concentration of the biological sample to be tested; The biological samples to be tested are serum from patients with viral infectious diseases such as novel coronavirus and influenza virus; In step (5), the liquid chromatography conditions include: an Acquity PREMIER Peptide BEH C18 column; column temperature: 40 ℃; mobile phases including phase A: 0.1% formic acid-water solution and phase B: 0.1% formic acid-acetonitrile solution; flow rate: 0.3 mL / min; gradient elution is performed as follows: the gradient starts with 2% mobile phase B, holds for 0.5 min, then at 9.0 min, the percentage of phase B increases to 38%, then at 10.5 min, it increases to 95%, holds for 12.5 min; finally at 12.6 min, the percentage of phase B decreases to 2%, holds for 2.4 min, and the total run time is 15 min. The triple quadrupole mass spectrometry conditions include: ESI ion source, positive ion mode, and MRM detection mode; the MS analysis conditions are as follows: turbine temperature is 450℃, gas values ​​are as follows: curtain gas 40 psi, GS1 gas 50 psi, GS2 gas 50 psi, collision-activated dissociation gas pressure is 10 psi, and ion injection voltage is 1.7 kV. The proportion of fucose defects was calculated by normalizing the sum of the signal intensities of fucose-deficient glycoforms G1, G1N, G2, and G2S to the IgG1-GPSVFPLAPSSK peptide intensity, and the proportion of galactose defects was calculated by normalizing the sum of the signal intensities of galactose-deficient glycoforms G0F and G0NF to the IgG1-GPSVFPLAPSSK peptide intensity.

2. The method for quantitative analysis of specific antibody Fc fragment glycosylation MRM according to claim 1, characterized in that, In step (4), the pretreatment is as follows: the corresponding antigen protein of the sample to be tested is fixed with an antigen-coated plate; after fixing the antigen protein, the diluted serum sample to be tested is added for immunoadsorption; after adsorption, the eluent is concentrated and dried; trypsin solution is added to the dried sample for reaction, and after the reaction is completed, it is vacuum dried.

3. The method for quantitative analysis of specific antibody Fc fragment glycosylation MRM according to claim 1, characterized in that, Quantitative determination was performed using either the internal standard method or the external standard method.

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