Establishment of time‐resolved fluoroimmunoassay of IgG4 based on magnetic microspheres

Abstract Background The abnormal increase in serum IgG4 level is an important clinical symptom of IgG4‐related disease (IgG4‐RD), and the detection of serum IgG4 level is a powerful tool for the diagnosis of IgG4‐RD. This study was conducted to establish a simple and rapid immunoassay for the determination of human serum IgG4 levels. Methods Based on the competition method, a novel immunoassay was established for the determination of human serum IgG4 using a combination of time‐resolved fluoroimmunoassay (TRFIA) and magnetic microspheres. IgG4 was coupled with magnetic microspheres and competed with IgG4 in the samples to bind the Eu3+‐labeled anti‐IgG4 antibody. The immunocomplex was separated and washed in a magnetic field, and the fluorescence counts were measured according to the number of dissociated europium ions. Results The analytical sensitivity of IgG4‐TRFIA based on magnetic microspheres was 0.006 g/L, and the detection range was 0.006–20 g/L under optimal conditions. The precision, recovery, and specificity of this immunoassay were demonstrated to be acceptable. The clinical application of IgG4‐TRFIA based on magnetic microspheres was evaluated and compared with that of immunonephelometry. The results showed that the two detection methods had a good correlation, with a correlation coefficient of .9871. Conclusion IgG4‐TRFIA based on magnetic microspheres has the advantages of high sensitivity, wide detection range, and short analysis time and has the potential to become a useful tool for the diagnosis of IgG4‐RD.


| INTRODUC TI ON
Immunoglobulin G (IgG) is a type of immunoglobulin secreted by plasma cells; it has the highest content in serum (75%-80%). IgG can be divided into four subtypes according to its structure: IgG1, IgG2, IgG3, and IgG4. Different IgG subtypes have different contents and functions in the body. IgG4 accounts for 1%-7% of total IgG, and its content is the lowest among the four subtypes. Abnormal increases in serum IgG4 levels frequently occur in IgG4-RD such as autoimmune pancreatitis (AIP), Mikulicz disease, and autoimmune hepatitis. [1][2][3] Recently, a study 4 proposed that IgG4-RD can be divided into "proliferative" type and "fibrotic" type based on clinicopathologic characteristics. Most patients with "proliferative" type IgG4-RD manifested high IgG4 levels, and the probability of detecting autoantibodies in these patients was higher.
IgG4-RD is an immune-mediated chronic fibrotic inflammatory disease that has been discovered in recent years. The disease was first proposed by Kamisawa et al. 5 and was officially named in 2010. 6 The disease is complex and can involve various organs and tissues, such as the pancreas, kidneys, lymph nodes, and thyroid. It is often accompanied by elevated serum IgG4 levels and IgG4-positive cell infiltration of various organs. [7][8][9][10] The comprehensive diagnostic criteria for IgG4-RD generally include clinical, serological, and histological examinations. 11 The "International Consensus Guidelines for the Management and Treatment of IgG4related Diseases" suggests that the detection of serum IgG4 levels is an important tool for the diagnosis of IgG4-RD, and a previous study 12 suggested that serum IgG4 levels can serve as one of the serological indicators for early diagnosis of AIP. Moreover, dynamic monitoring of serum IgG4 levels may provide guidance for efficacy detection, recurrence prediction, and prognostic judgment in AIP.
The detection of serum IgG4 levels is the most effective method for patients with relative contraindications for biopsy, as it can effectively exclude tumors or other diseases with clinical and pathological features similar to those of IgG4-RD. 13,14 Therefore, the detection of serum IgG4 still has important clinical value; however, current traditional detection methods have certain limitations.
Here, we propose a simple and rapid immunoassay method for the detection of serum IgG4 by combining the technical advantages of TRFIA and magnetic microspheres.

| Preparation of IgG4-coated magnetic microspheres
Thirty microliters of NHS (10 mg/ml) and 50 μl of EDC (10 mg/ml) were added to 10 mg of Fe 3 O 4 microspheres, which had diameters of 2 μm, and the solution was mixed at 25℃ for 30 min. The mixture was magnetically separated and washed three times with 0.05 mol/L MES buffer (pH = 5.0) and was then suspended with the above buffer solution to make a magnetic particle suspension with a concentration of 100 mg/ml. Then, 1 ml of 0.05 mol/L MES buffer (pH = 5.0) and 50 μg of IgG4 were added to 100 μl of magnetic particle suspension, mixed well, and incubated overnight at 25℃. Then, the supernatant was removed after magnetic separation, and protein concentration was measured. The retained magnetic microspheres were washed three times with 0.05 mol/L Tris-HCl buffer (containing 5% BSA, pH = 7.2) and blocked at 25℃ for 30 min. Finally, magnetic microspheres were washed three times with Tris-HCl buffer (0.5% BSA, 0.1% NaN 3 , pH = 7.2), resuspended, aliquoted, and stored at 2-8℃.

| Preparation and purification of IgG4 antibody labeled with Eu 3+
Anti-human IgG4 antibody labeling was performed following the instructions of the Eu 3+ labeling kit. Anti-IgG4 antibody (1 mg) was dissolved in 50 mmol/L Na 2 CO 3 -NaHCO 3 buffer (containing 0.155 mol/L NaCl, pH=8.5). Then, 0.2 mg of Eu 3+ -N 2 -[p-isocyanatebenzyl]-DTTA (Eu 3+ -DTTA) was added to 1 mg of anti-IgG4 antibody solution, and the mixture was incubated with shaking at 25℃ for 20 h. The next day, the reaction solution was transferred to a Sephadex-G50 column pre-equilibrated with 80 mmol/L Tris-HCl buffer (pH = 7.8) for purification. The fractions from the first peak containing the highest Eu 3+ fluorescence counts were pooled and characterized. The anti-human IgG4 antibody labeled with Eu 3+ was diluted with elution buffer containing 0.2% BSA and stored at −20℃ until use.

| Statistical analysis
Each serum sample was tested in duplicate. A commercial human IgG4 detection kit (immunoturbidimetry) was used to verify the clinical reliability of the established method, and the correlation between the two methods was evaluated using Pearson's correlation. were used for data analysis.

| Optimization of the concentration of IgG4coated magnetic microspheres
Because detection was carried out on the fully automated TRF-1000 time-resolved immunofluorescence analyzer, the separation and washing times of the magnetic microspheres were fixed, and the effects of washing and separation were best when using the universal magnetic microspheres with diameters of 2 μm. Therefore, these microspheres were used for subsequent experiments. IgG4 (50 μg) was coupled with magnetic microspheres (10 mg). After magnetic separation, the protein content of the supernatant was 2.76 μg, which indicated that 47.24 μg of IgG4 had been coupled to magnetic microspheres, and the coupling rate was 94.48%, with an average

| Optimization of the concentration of Eu 3+anti-IgG4 antibody
The optimal dilution of the Eu 3+ -anti-IgG4 antibody was deter-

| Analytical sensitivity and detection range
The standard curve was obtained by repeating the detection

| Precision and recovery
The accuracy of the established method was evaluated using the intra-and inter-assay coefficients of variation (CV). Three qualitycontrolled samples with different concentrations (low, medium, and high) were detected, 10 wells were set for each qualitycontrolled sample, and the experiment was repeated three times.
The range of intra-assay CV was 3.70-5.09%. The range of interassay CV was 4.89-6.26%, and the intra-and inter-assay CVs of each quality-controlled sample were ≤10%. The recovery rate was evaluated by adding a high-concentration IgG4 standard to samples of known concentration at a ratio of 1:9. The recovery rate was calculated as recovery rate (%) = (measured concentration/theoretical concentration) × 100%. The range of recovery rates was 99.39-107.90%, indicating that the precision of this method was good. Because this assay method involved a largeratio dilution of samples, in order to evaluate the matrix effect of assay buffer on serum, a serum sample containing a high concentration of IgG4 (15.82 g/L) was diluted with assay buffer at different dilution ratios for detection, and the difference between the expected and measured concentrations was within 10.00%, indicating that the matrix effect of assay buffer met the requirements (Table 1).

| Specificity
The specificity of the assay method was evaluated by detecting four possible interfering substances: IgM, IgG1, IgG2, and IgG3.
The results showed that the measured concentrations of IgM, IgG1, IgG2, and IgG3 were between 0.01-0.03 g/L, indicating that IgG4-TRFIA based on magnetic microspheres has high specificity for IgG4 and can be used for the clinical detection of serum IgG4.

| DISCUSS ION
To date, a variety of assay methods have been developed for the detection of serum IgG4 levels, including radio immunodiffusion (RID), 15 enzyme-linked immunosorbent assay (ELISA), 16 immunonephelometry, 17 and other methods. In addition, a previous study described a method based on IgG subclass-specific tryptic peptides and liquid chromatography tandem-mass spectrometry (LC-MS/MS) for quantification of IgG subclasses. 18 This method can avoid antigen excess, and the analytical measurement range is similar to that of immunonephelometry. However, complicated pre-processing of samples, such as serum or plasma, is usually required to perform LC-MS/MS analyses and requires experienced technicians for operators. RID is the earliest assay method used to detect serum IgG4, but is seldom used now due to the high consumption of serum, long detection times (48-72 h), and low sensitivities involved. ELISA can be used for qualitative or semiquantitative analysis; however, the influencing factors of ELISA vary, reagent stabilities are poor, and reaction times are relatively long (4 h). Currently, serum IgG4 levels are routinely quantified using immunonephelometry. Compared with RID and ELISA, immunonephelometry has the advantages of automatic operation, good accuracy, and reproducibility; however, clinical use is limited because of the relatively high instrument costs involved. In addition, in some cases this method is not very effective. For example, at present, the detection range of the human IgG4 determination kit is 0.052-3.3 g/L, and the detection range is relatively narrow.
The serum IgG4 level in most patients with IgG4-RD is higher than 3.3 g/L; therefore, the serum sample needs to be diluted 1:2000 to make the concentration within the range of the calibration curve. Such a large-ratio dilution will increase detection error and the time required to produce results. On the other hand, due to the presence of a large number of complexes and proteins in serum samples, these interfering substances produce non-specific scattered light, which affects the sensitivity of detection. In contrast, the assay method for serum IgG4 established in this study   This improved the analysis sensitivity, and the reaction time was significantly shortened (to 12 min). As IgG4 is coupled with magnetic microspheres by chemical groups (the coupling rate was 94.48%), the loss of IgG4 due to washing is reduced, and the precision of detection is improved.

TA B L E 1 Matrix effect in serum samples
In conclusion, we established a novel immunoassay method for the detection of human serum IgG4. This method combines the advantages of TRFIA and magnetic microspheres and has the advantages of simple operation, short detection times, wide detection ranges, and high sensitivities. Moreover, the method established has good agreement with immunonephelometry and provides a useful new technique for the detection of human serum IgG4.

CO N FLI C T S O F I NTE R E S T
The authors declare no conflicts of interest.

E TH I C A L S TATEM ENT
The study protocol was approved by the Medical Ethics Committee of Tong Ren Hospital, Shanghai Jiao Tong University School of Medicine (2020-35).

DATA AVA I L A B I L I T Y S TAT E M E N T
All the data that related to this study are available from the corresponding author upon reasonable request.