A hybrid LC-MS / MS method for detecting immunoglobulin E in monkey serum / plasma

Abstract

This invention discloses a method for detecting immunoglobulin E in monkey serum / plasma based on hybrid LC-MS / MS. The method includes immunoaffinity capture of the target protein (IgE) using a biotin-labeled antibody and magnetic beads, protein denaturation and reduction, enzymatic digestion with trypsin to generate stable characteristic peptides, correction of pretreatment and instrument fluctuations using an internal standard, and specific detection of target ion pairs in triple quadrupole mass spectrometry using multiple reaction monitoring (MRM) mode. It can accurately and linearly quantify IgE in the concentration range of 0.1-50 ug / mL, which has higher specificity and accuracy than enzyme-linked immunosorbent assay (ELISA) and can meet the requirements of practical applications.

Description

Technical Field

[0001] This invention relates to the field of biological detection, specifically to a method for detecting immunoglobulin E in monkey serum / plasma and its application. Background Technology

[0002] IgE (Immunoglobulin E) is one of the immune antibodies in the human body. IgE is mainly produced by mucosal lymphoid tissues, tonsils, and adenoids, and its concentration in the blood is very low. When the body comes into contact with allergens, IgE binds to Fc receptors on mast cells and basophils, triggering the release of inflammatory mediators and causing allergic reactions such as asthma, allergic rhinitis, and urticaria. Clinically, detecting serum total IgE levels can help diagnose type I hypersensitivity diseases, assess allergic constitution, and evaluate treatment effectiveness.

[0003] Monkey serum / plasma IgE detection is primarily used in three major areas: research on allergic / immune-related diseases, preclinical evaluation of anti-IgE drugs, and evaluation of vaccine immunotoxicity. It mainly relies on immunological methods such as enzyme-linked immunosorbent assay (ELISA). ELISA, based on the principle of antigen-antibody specific binding, uses an enzyme-catalyzed colorimetric reaction to qualitatively or quantitatively detect total or specific IgE in serum. It is widely used as an adjunct to the diagnosis of diseases such as allergic asthma, atopic dermatitis, and parasitic infections, as well as for allergen screening (e.g., identifying sensitizing substances by detecting IgE). In research, ELISA is also frequently used to evaluate the pharmacodynamic effects of anti-IgE drugs (such as omalizumab) in mouse, rat, and guinea pig animal models.

[0004] However, when using ELISA kits to detect IgE in monkey serum / plasma, the results are subject to significant fluctuations due to several factors: (1) monkey serum / plasma contains approximately 16 mg / mL of IgG, making ELISA detection susceptible to interference from IgG in the sample matrix; (2) the capture / detection antibodies in the kit have different recognition efficiencies for endogenous IgE and the standards in the kit, and the antibodies used in kits from different manufacturers may differ, or even if it is the same antibody, the affinity of different batches of antibodies may vary. For example, using Thermoinvitrogen's Catalog Number EEL235 (96 tests) kit, the measured endogenous IgE in monkeys was 5.3-43.7 ng / ml, but according to literature reports, the range of endogenous monkey IgE is 4-23 ug / ml, indicating a significant deviation. Furthermore, ELISA often struggles to accurately distinguish between free IgE (a direct biomarker of drug efficacy) and drug-bound IgE (a pharmacokinetic and safety indicator), limiting the precise assessment of the relationship between pharmacokinetic (PK) and pharmacodynamic (PD) during drug development. Summary of the Invention

[0005] To address the aforementioned problems in the existing technology, this invention provides a method for detecting immunoglobulin E (IgE) in monkey serum / plasma based on hybrid LC-MS / MS. The method uses immunoaffinity to capture IgE in monkey serum / plasma, and after denaturation, reduction, and enzymatic digestion, stable characteristic peptides are generated. The target ion pairs are then specifically detected by LC-MS / MS to achieve absolute quantification of IgE.

[0006] In some embodiments, the detection method specifically includes the following steps: (1) Capture IgE in monkey serum / plasma using IgE-specific antibodies; (2) After adding the IgE captured in step (1) of the Rapi Gest denaturation process, further denature it at high temperature to obtain denatured IgE; (3) Add the denatured IgE from step (2) of DTT reduction; (4) Add the reduced IgE from the IAA alkylation step (3) to obtain alkylated IgE; (5) Alkylate IgE in enzymatic hydrolysis step (4) to obtain IgE hydrolysis product; (6) After pretreatment of IgE enzymatic hydrolysis products, LC-MS / MS analysis was performed to obtain the IgE concentration in monkey serum / plasma based on the analysis results; Preferably, the test is for non-diagnostic purposes.

[0007] In some embodiments, step (1) uses magnetic beads coated with IgE-specific antibodies to capture IgE in monkey serum / plasma. Preferably, the IgE-specific antibody is a monoclonal antibody or a polyclonal antibody; Preferably, the IgE-specific antibody is a biotin-labeled goat anti-human IgE antibody; Preferably, the IgE-specific antibody is an antibody that specifically binds to free IgE or specifically binds to drug-bound IgE; Preferably, the capture conditions are as follows: add the sample to be tested and PBS containing 0.1% Tween 20 to magnetic beads coated with IgE specific antibody, vortex mix, and then shake at room temperature.

[0008] Preferably, the magnetic beads are washed with PBS containing 0.1% Tween 20 after oscillation; Preferably, the washing is followed by elution with 0.1 M glycine and then neutralization.

[0009] In some embodiments, the high-temperature denaturation condition in step (2) is incubation at 95°C for 1 hour.

[0010] In some embodiments, the reduction conditions in step (3) are 100 mM DTT incubation at 95°C for 30 minutes.

[0011] In some embodiments, the alkylation conditions in step (4) are 100 mM IAA at 30°C and 1000 rpm for 30 minutes.

[0012] In some embodiments, step (5) involves enzymatic hydrolysis of alkylated IgE using trypsin; Preferably, the enzymatic hydrolysis conditions are 37°C and 1000 rpm overnight.

[0013] In some embodiments, the pretreatment in step (6) involves adding formic acid, shaking, and then terminating the reaction with acetonitrile.

[0014] Preferably, the formic acid is a 20% formic acid solution, and the shaking conditions are shaking at 800 rpm for 10 minutes at room temperature; Preferably, after the reaction is terminated, the supernatant is collected by centrifugation and analyzed by LC-MS / MS. Preferably, the LC-MS / MS uses a peptide with the amino acid sequence AAPEVYVFATPEK as the basis for quantification; Preferably, the mass spectrometry acquisition time in the LC-MS / MS is 3 min, and the parent ion / daughter ion (Q1 / Q3) parameters for quantitative IgE are 711.369 / 954.493.

[0015] The present invention also provides the application of the hybrid LC-MS / MS-based method for detecting immunoglobulin E in monkey serum / plasma in research on allergic / immune-related diseases, preclinical evaluation of anti-IgE drugs, and evaluation of vaccine immunotoxicity.

[0016] The beneficial effects of this invention are: (1) This invention first uses specific antibodies to perform immunoaffinity capture and enrichment of the target protein (IgE), and then performs enzymatic digestion and LC-MS / MS analysis. It perfectly combines the advantages of immunological methods such as enzyme-linked immunosorbent assay (ELISA) and liquid chromatography-tandem mass spectrometry (LC-MS / MS) technology, which have high specificity and multi-index simultaneous analysis capabilities. It is significantly superior to traditional immunological methods such as enzyme-linked immunosorbent assay (ELISA) for detecting IgE in monkey serum / plasma in terms of specificity (extremely high specificity, based on peptide sequence recognition, with no cross-reactivity) and quantitative accuracy, especially in complex matrix or low concentration analysis.

[0017] (2) The method for detecting immunoglobulin E in monkey serum / plasma based on hybrid LC-MS / MS of this invention selects the conserved peptide segment of IgE heavy chain (AAPEVYVFATPEK) and performs triple confirmation of molecular weight and fragment ions (retention time, parent ion, daughter ion) in mass spectrometry, which can accurately locate the target molecule and avoid cross-reaction. It has a clear metrological basis and can be used as a reference method to calibrate immunoassay results, thereby improving inter-laboratory consistency.

[0018] (3) The method for detecting immunoglobulin E in monkey serum / plasma based on hybrid LC-MS / MS of this invention can use different alternative antibodies as detection antibodies, avoiding the problem of inconsistent results due to antibody differences between different immunoassay platforms, and has excellent accessibility and universality. Based on this feature, if other competitive antibodies or free / bound IgE-specific antibodies are used as capture antibodies, and the same specific peptide is selected, it can be used to detect IgE in different states of monkey serum, such as free and bound IgE, which has important reference value for the preclinical development of anti-allergy drugs. Attached Figure Description

[0019] Figure 1 The standard curve was obtained using a biotin-labeled goat anti-human IgE polyclonal antibody as a capture reagent. Figure 2 This is the limit of quantitation (LOQ) spectrum using biotin-labeled goat anti-human IgE polyclonal antibody as a capture reagent; Figure 3 The standard curve was obtained using a biotin-labeled goat anti-human IgE monoclonal antibody as a capture reagent. Figure 4 This is a spectrum showing the lower limit of quantitation using a biotin-labeled goat anti-human IgE monoclonal antibody as a capture reagent. Detailed Implementation

[0020] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the methods and reagents used in the present invention are common methods and reagents in the art.

[0021] Example 1: Capture and separation of IgE from monkey serum / plasma (1) Preparation of IgE-specific antibody-coated magnetic beads: Add 20 μL of magnetic bead suspension to each well of the low-adsorption deep-well plate, wash the magnetic beads twice with 200 μL of PBST in each well, add 85 μL of coating working solution (containing 100 μg / mL of goat anti-human IgE polyclonal / monoclonal antibody labeled with Biotin) to each well, and incubate at 1300 rpm for 120 minutes at room temperature. Wash the magnetic beads with 200 μL of PBS containing 0.1% Tween 20, add 100 μL of 1×PBS solution containing 3% BSA and 0.05% Tween 20 to each well, and incubate at 1300 rpm for 60 minutes at room temperature. Wash the magnetic beads with 200 μL of PBS containing 0.1% Tween 20.

[0022] (2) IgE capture and separation: Add 15 μL of STD / QC / BL / sample and 200 μL of PBS containing 0.1% Tween 20 to each well of a low-adsorption deep-well plate containing magnetic beads. Vortex to mix and shake the mixture at room temperature for 2 hours. Then wash the magnetic beads with 200 μL of PBS containing 0.1% Tween 20, elute with 30 μL of 0.1 M glycine (pH 2.7), and neutralize with 1 M Tris-HCl (pH 9) to obtain separated IgE from monkey serum / plasma.

[0023] Example 2: Denaturation, Reduction and Enzymatic Digestion of IgE in Monkey Serum / Plasma Denaturation of IgE in monkey serum / plasma: Add 10 μL of 0.1% Rapi Gest to each well of a low-adsorption deep-well plate containing IgE in monkey serum / plasma, seal the plate, shake at 800 rpm for about 5 minutes, and then place the mixture in a 95℃ PCR incubator for 1 hour.

[0024] Reduction of IgE in monkey serum / plasma: 10 μL of 100 mM DTT was added and incubated at 60°C for 30 min. Then, 10 μL of 100 mM IAA was added, and alkylation was performed at 30°C and 1000 rpm for 30 min in a preheated thermostat. Enzymatic digestion of IgE in monkey serum / plasma: 20 μL of trypsin (sequencing-grade trypsin, 50 μg / mL) was added to each well, and digestion was performed overnight at 37°C and 1000 rpm in a preheated thermostat. Then, 15 μL of 20% formic acid solution was added to each well, and the mixture was shaken at 800 rpm for 10 min at room temperature. The reaction was terminated by adding 40 μL of acetonitrile containing the internal standard. After centrifugation at 4000g for 10 min, 120 μL of the supernatant was transferred to a 96-well analytical plate for LC-MS / MS analysis.

[0025] Example 3: LC-MS / MS Analysis Because IgE shares structural similarities with other immunoglobulins, especially in its heavy chain region, homology is present. Therefore, it is necessary to screen for stable characteristic peptides for quantification, while ensuring their representativeness across different individuals. Finding suitable peptides is crucial, ensuring the absence of interference, high signal strength, and good stability. Inappropriate peptide selection can easily lead to cross-reactivity, affecting the specificity of the results. This invention, through sequence analysis, alignment, and repeated experimental verification, screened the conserved peptide AAPEVYVFATPEK on the IgE heavy chain, and further obtained the corresponding liquid chromatography and mass spectrometry conditions through repeated experiments.

[0026] The 96-well analytical plate containing IgE enzyme hydrolysate from Example 2 was analyzed by LC-MS / MS. The liquid chromatography conditions are shown in Table 1, and the mass spectrometry conditions are shown in Table 2.

[0027] Table 1 Liquid Chromatography Conditions

[0028] Table 2 Mass Spectrometry Conditions

[0029] The standard curve was obtained based on the test results of the standard samples. The regression model of the standard curve is shown in Table 3. Table 3 Standard Curve Regression Model

[0030] Example 4: Method validation of the detection method for immunoglobulin E in monkey serum / plasma based on hybrid LC-MS / MS The reliability of the IgE detection method of this invention was verified by analyzing sample residue, matrix interference, analyte interference and internal standard interference during the detection process.

[0031] (1) The residual status of the batch sample detection process was evaluated by examining the blank sample injected after the upper limit of the standard curve of each analytical batch (carryover). The results are shown in Table 4.

[0032] Table 4 Residues from the Batch Sample Testing Process

[0033] Conclusion: The peak area of ​​the carryover sample at the analyte retention time did not exceed 20.0% of the average peak area of ​​the standard curve limit of quantitation samples that met the acceptance criteria in the same analytical batch; the peak area of ​​the internal standard retention time did not exceed 5.0% of the average peak area of ​​the internal standard in the standard curve limit of quantitation samples that met the acceptance criteria in the same analytical batch. In this method validation, the residues of both the analyte and the internal standard were 0%, which met the acceptance criteria.

[0034] (2) The interference of blank biological matrix analytes and internal standards in 6 different individuals was investigated. The results are shown in Table 5.

[0035] Table 5. Selective Study (Endogenous Interference - Alternative Matrix)

[0036] Conclusion: The interference of the matrix on the analyte and internal standard meets the following requirements: the response value of the interfering component in the blank matrix at the analyte retention time does not exceed 20.0% of the analyte response value in the LLOQ sample prepared with the same individual blank matrix; the response value of the internal standard at the internal standard retention time does not exceed 5.0% of the internal standard response value in the LLOQ sample prepared with the same individual blank matrix. Therefore, the interference of the matrix on the analyte and internal standard in this validation meets the acceptance criteria.

[0037] (3) The study was conducted using three samples containing only a single analyte and without an internal standard at the upper limit of quantitation concentration. The results are shown in Table 6.

[0038] Table 6 Selectivity Study (Interference of the Analyte on the Internal Standard)

[0039] Conclusion: The interference of the analyte on the internal standard meets the requirement that the average peak area of ​​the sample containing only the analyte at the retention time of the internal standard is no greater than 5.0% of the average peak area of ​​the internal standard in the samples meeting the lower limit of quantitation of the standard curve in the same analytical batch. The interference of the analyte on the internal standard is 0%, which meets the acceptance criteria.

[0040] (4) The results were examined using three samples containing only a single internal standard and without any analyte. The results are shown in Table 7.

[0041] Table 7. Selective Study (Interference of Internal Standard with Analyte)

[0042] Conclusion: The interference of internal standards on the analyte meets the requirement that the average peak area at the retention time of the analyte is no greater than 20.0% of the average peak area of ​​the samples meeting the lower limit of quantitation of the standard curve in the same analytical batch. This study verified that the interference of the three internal standards on the analyte was 0%, meeting the acceptance criteria.

[0043] By testing the intra-batch / inter-batch accuracy deviation and coefficient of variation of three batches in 18 replicates, as well as by plotting the standard curve and calculating the concentration, the linear range of the standard curve was determined, validating the precision, accuracy, and stability of this method. The results are shown in Table 8-10.

[0044] Table 8 Precision and accuracy of in-batch sample testing

[0045] Table 9 Precision and accuracy of in-batch sample testing

[0046] Table 10 Precision and accuracy of intra-batch and inter-batch sample testing

[0047] Precision and accuracy were assessed using three validation batches (LLOQ QC, LQC, MQC, and HQC, with six replicates for each concentration). The conclusions were: the deviation of the mean measured concentration of each concentration level control sample from its theoretical concentration was within ±15.0% (±20.0% for LLOQ QC), and the coefficient of variation did not exceed 15.0% (not exceeding 20.0% for LLOQ QC). In this validation, the intra-batch and inter-batch accuracy deviations for all three batches did not exceed ±15.0%, and the intra-batch and inter-batch coefficients of variation did not exceed 10%, demonstrating that the precision and accuracy of this detection method are extremely high, and it exhibits excellent stability and consistency.

[0048] Table 11 Summary of Standard Curve Parameters

[0049] Table 12 Back-calculation concentration from standard curve

[0050] Conclusion: The linear range of the standard curve was 0.100–50.0 μg / mL, and the goodness of fit R² was greater than 0.98 for all values. Example 5: Consistency Analysis of Different Capture Reagents Biotin-labeled goat anti-human IgE polyclonal antibody and monoclonal antibody were used as capture reagents to detect IgE concentrations in standards (1000 ng / mL and 6000 ng / mL, respectively). The standard curve obtained using the biotin-labeled goat anti-human IgE polyclonal antibody as the capture reagent showed good linearity. Figure 1 As shown in the figure, the lowest limit of quantitation spectrum is as follows: Figure 2 As shown. Using biotin-labeled goat anti-human IgE monoclonal antibody as the capture reagent, the obtained standard curve showed good linearity, as shown in the figure. Figure 3 As shown in the figure, the lowest limit of quantitation spectrum is as follows: Figure 4 As shown in the figure. The test results are shown in Table 13.

[0051] Table 13. Consistency analysis of different antibodies

[0052] The above results demonstrate good consistency in detection outcomes using different antibodies. Although the affinity of different antibodies may vary, this does not affect the accuracy of the present invention's method for detecting immunoglobulin E in monkey serum / plasma based on hybrid LC-MS / MS. This method can use different alternative antibodies as capture reagents, making it accessible and universal.

[0053] Example 6: Detection of endogenous IgE in monkey serum / plasma using the hybrid LC-MS / MS method of this invention and a commercially available ELISA kit to detect IgE from monkey serum / plasma of different sources. Table 14. Detection of endogenous IgE concentration by different methods

[0054] While specific embodiments of the invention have been described, those skilled in the art will recognize that various changes and modifications can be made to the invention without departing from its scope or spirit. The terminology used herein is chosen to best explain the principles, practical applications, or improvements to technology in the market, or to enable those skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for detecting immunoglobulin E in monkey serum / plasma based on hybrid LC-MS / MS, characterized in that, The method uses immunoaffinity to capture immunoglobulin E in monkey serum / plasma, and after denaturation, reduction, and enzymatic digestion, generates stable characteristic peptides. The target ion pairs are then specifically detected by LC-MS / MS to achieve absolute quantification of immunoglobulin E (IgE).

2. The detection method according to claim 1, characterized in that, The detection method specifically includes the following steps: (1) Capture IgE in monkey serum / plasma using IgE-specific antibodies; (2) After adding the IgE captured in step (1) of the Rapi Gest denaturation process, further denature it at high temperature to obtain denatured IgE; (3) Add the denatured IgE from step (2) of DTT reduction; (4) Add the reduced IgE from the IAA alkylation step (3) to obtain alkylated IgE; (5) Alkylate IgE in enzymatic hydrolysis step (4) to obtain IgE hydrolysis product; (6) After pretreatment of IgE enzymatic hydrolysis products, LC-MS / MS analysis was performed to obtain the IgE concentration in monkey serum / plasma based on the analysis results.

3. The detection method according to claim 2, characterized in that, In step (1), magnetic beads coated with IgE-specific antibodies are used to capture IgE in monkey serum / plasma.

4. The detection method according to claim 2 or 3, characterized in that, The IgE-specific antibody is a monoclonal antibody or a polyclonal antibody.

5. The detection method according to claim 2 or 3, characterized in that, The IgE-specific antibody is a biotin-labeled goat anti-human IgE antibody.

6. The detection method according to claim 2 or 3, characterized in that, The IgE-specific antibody is an antibody that specifically binds to free IgE or specifically binds to drug-bound IgE.

7. The detection method according to claim 2 or 3, characterized in that, The capture conditions are as follows: add the test sample and PBS containing 0.1% Tween 20 to magnetic beads coated with IgE-specific antibodies, vortex mix, and then shake at room temperature.

8. The detection method according to claim 7, characterized in that, The magnetic beads were washed with PBS containing 0.1% Tween 20 after oscillation.

9. The detection method according to claim 8, characterized in that, The washing process was followed by elution with 0.1 M glycine, and then neutralization.

10. The detection method according to claim 2, characterized in that, In step (2), the high-temperature denaturation condition is incubation at 95°C for 1 hour.

11. The detection method according to claim 2, characterized in that, The reduction conditions in step (3) are 100 mM DTT incubated at 95°C for 30 minutes.

12. The detection method according to claim 2, characterized in that, In step (4), the alkylation conditions are 100 mMIAA at 30°C and 1000 rpm for 30 minutes.

13. The detection method according to claim 2, characterized in that, In step (5), trypsin is used to hydrolyze alkylated IgE.

14. The detection method according to claim 2, characterized in that, The enzymatic hydrolysis conditions in step (5) are 37°C and 1000 rpm overnight.

15. The detection method according to claim 2, characterized in that, In step (6), the pretreatment involves adding formic acid, shaking, and then terminating the reaction with acetonitrile.

16. The detection method according to claim 15, characterized in that, The formic acid is a 20% formic acid solution, and the shaking conditions are shaking at 800 rpm for 10 minutes at room temperature.

17. The detection method according to claim 15, characterized in that, After the reaction was terminated, the supernatant was collected by centrifugation and analyzed by LC-MS / MS.

18. The detection method according to claim 2, characterized in that, The LC-MS / MS used peptides with the amino acid sequence AAPEVYVFATPEK as the basis for quantification.

19. The detection method according to claim 2, characterized in that, The mass spectrometry acquisition time in the LC-MS / MS was 3 min, and the parent ion / daughter ion parameters for quantitative IgE were 711.369 / 954.

493.

20. The application of the method for detecting immunoglobulin E in monkey serum / plasma based on hybrid LC-MS / MS as described in any one of claims 1-19 in research on allergic / immune-related diseases, preclinical evaluation of anti-IgE drugs, and evaluation of vaccine immunotoxicity.

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