Receptor antibody time‐resolved A2 phospholipase bead immunochromatography and its application in idiopathic membranous nephropathy

Abstract Objective To detect phospholipase A2 receptor (PLA2R) antibody by established time‐resolved fluorescent bead immunochromatographic assay. Methods The reaction time of coupling, pH of the reaction, and coupling ratio of the label to PLA2R were determined. The EDC method was used to covalently couple PLA2R to time‐resolved fluorescent beads, which were sprayed onto a bonding pad. PLA2R and rabbit anti‐PLA2R antibody sprayed onto a nitrocellulose membrane were used as detection and quality control lines, respectively. Immunochromatographic test strips were prepared to enable rapid detection of PLA2R antibodies. Various technical indicators were evaluated, and the correlation among this method, enzyme‐linked immunosorbent assay (ELISA), and serum analysis was examined. Results The pH suitable for labeling was 6.5. The optimal mass ratio of PLA2R protein to fluorescent beads was 0.08:1, and the reaction time of coupling was at least 1.5 hours. The appropriate spray film size of the coupled fluorescent bead was 5 μL/cm, and the appropriate staining concentration of the test line was 0.28 mg/mL. Further, 80 µL of sample was required for the test, and the result was obtained in only 15 minutes. The measurable range of this method was 5‐1500 RU/mL. Intra‐ and inter‐assay coefficients of variation were 7.61% and 11.07%, respectively, with an average recovery rate of 93.77%. The method showed a good correlation with ELISA, with a correlation coefficient of 0.936. Conclusions This method could better meet the clinical demand for idiopathic membranous nephropathy (IMN) detection.

Kidney disease occurs in 10.8% of Chinese adults. Nephrotic syndrome is a group of clinical syndromes involving increased glomerular basement membrane permeability and high levels of proteinuria, hypoproteinemia, edema, and hyperlipidemia. Among these diseases, membranous nephropathy (MN) is a common cause of adult nephrotic syndrome. 1,2 Membranous nephropathy is a group of diseases characterized by glomerular basement membrane epithelial cell immune complex deposition with diffuse thickening of the glomerular basement membrane. MN is often insidious. Approximately 20% of patients have asymptomatic, non-renal disease proteinuria. Some patients realize their condition only after learning that they have proteinuria during routine physical examination. However, obtaining a proper diagnosis by renal puncture is difficult, and thus, the condition may be worsened before disease detection. MN can be divided into idiopathic membranous nephropathy (IMN) and secondary membranous nephropathy (SMN). IMN accounts for approximately two-thirds of cases, whereas SMN accounts for approximately one-third of cases and includes autoimmune diseases, infections, tumors, and drugs. 3,4 The characteristic pathological changes in IMN are the deposition of a large number of immune complexes on the glomerular capillary epithelial side. The clinical progress is slow, and some patients can spontaneously recover, but 30%-40% of patients eventually develop final-stage renal disease or die. 5 IMN is mainly diagnosed by renal puncture and histological examination. 6 However, this method has limitations such as a long inspection time, high trauma risk, and difficulty in carrying out the procedure in ordinary hospitals. Phospholipase A2 receptor (PLA2R) was first discovered in 2009 by Beck and others. 4 This receptor is the target antigen of adult IMN and is detected by Western blotting. Antibodies for this receptor are observed in the serum of 70% of patients with IMN. 4 A PLA2R-lgG ELISA Kit was developed to simplify antibody detection that shows an IMN detection rate between 40% and 70%. [7][8][9] However, the detection time is more than 2 hours and is not individualized. To overcome these limitations, we developed a time-resolved fluorescent bead immunochromatographic technique to establish a rapid detection method for PLA2R antibody on an individual patient basis. We also evaluated various technical indicators and explored whether this method can be applied in clinical trials.

| Main materials and instruments
PLA2R antigen, rabbit anti-PLA2R antibody, and the PLA2R-IgG standard were provided by KeyGen Biotechnology Co., Ltd.

| Preparation of sealing solution
Bovine serum albumin (1.25 g) was dissolved in 25 mL of 0.01 mol/L phosphate buffer (PB, pH 7.5).

| Preparation of scribing buffer
Trehalose (0.2 g) was mixed with 0.6 mL of methanol and dissolved in 9.4 mL of 0.01 mol/L PB (pH 7.5).

| Preparation of suspension
Bovine serum albumin (1 g) and trehalose (5 g) were mixed. Next, 25 μL of Tween-20 was added and this mixture was dissolved in 100 mL of 0.01 mol/L PB (pH 7.5). The solution was mixed by stirring.

| Selection conditions for NC film coating
Different concentrations of PLA2R antigen were plated at 1.0 μL/cm onto the NC membrane as a detection line (T line). At the same time, the rabbit anti-PLA2R antibody was also drawn at 1.0 μL/cm with a spectator and used as a quality control line and to judge whether the reaction is complete. The NC membrane was dried at room temperature for 12 hours.

| Selection of PLA2R protein labeling conditions
Time-resolved phospholipase beads were added to a centrifuge tube, an equal volume of pH 5.0, 0.1 mol/L MES buffer was added, and the solution was mixed. Ultrasonic cleaning was performed for 5 minutes, followed by centrifugation at 18894.2 g for 15 minutes.

| Selection of spray film conditions
Different amounts of labeled fluorescent microspheres were sprayed onto the bonding pad. After drying, the test card was assembled with the lined NC film.

| Assembly of fluorescent immunochromatographic test strips
In an environment of less than 35% humidity and stable temperature of 20-25°C, the NC film with the T line and C line, and the bonding pad with labeled fluorescent microspheres were adhered to the polyvinyl chloride substrate. The sample pad and absorbent paper were attached to form a microfiltration system, which was cut to a width of 0.4 cm. The test strip was prepared and placed in the card case to form a strip. The aluminum film bag was sealed and stored.

| Test method
A double-antigen sandwich method was used to establish an assay for detecting PLA2R antibody; 80 µL of standard or test serum was added to the well and incubated at room temperature for 15 minutes. The test strip was inserted into the card insertion port of the fluorescence detecting device, and readings were obtained using the instrument. The fluorescence count was determined, and the corresponding concentration was obtained according to the set standard curve.

| Minimum detection limit
The minimum concentration that could be reliably identified from the background of the test was determined, and the average (x) and standard deviation (s) were obtained from 10 sets of standard curve zero tube counts. The value of (x + 2s) was calculated, and the corresponding concentration on the standard curve was detected.

| Specificity
To investigate the degree of cross-reactivity between the antibody and test substance and measure the specificity of the test, we used high concentrations of hemoglobin, bilirubin, and triglyceride as samples.

| Recovery rate
A high-concentration standard was added to a sample of known concentration, and the sample was measured according to the detection method. The ratio of the measured value to the theoretical value was calculated.

| Precision
The quality control of the low and high medium groups was tested 10 times, and the mean value, standard deviation, and coefficient of variation (CV) were calculated.

| Comparison with PLA2R-IgG ELISA kits
Thirty samples were simultaneously tested using ELISA Kits and time-resolved fluorescent microsphere immunochromatographic test strips. Correlation analysis was performed between the two methods.

| Effect of pH on labeling
Labeling was performed using 0.1 mol/L MES buffer at pH 5.0, 5.5, 5.8, 6.0, 6.2, 6.5, 6.8, 7.0, 7.2, and 7.5, and 1-μg microspheres were labeled with 80 μg protein and centrifuged after the reaction. The concentration of residual protein in the supernatant was determined by BCA protein assay (Figure 1). When the pH condition of labeling was between 6.0 and 6.8, the protein concentration in the supernatant was low, and most of the protein was labeled onto the beads. Thus, pH 6.5 was chosen as the optimum pH for labeling.

| The ratio of PLA2R protein to fluorescent beads during labeling
According to the labeling method, 1 mg of fluorescent beads was reacted with 30-100 μg of PLA2R proteins at pH 6.5. After labeling, the residual protein concentration in the supernatant was determined by BCA protein assay. The results are shown in Figure 2.
As shown in Figure 2, when using 1 mg fluorescent beads and an amount of loaded PLA2R protein higher than 80 μg, the residual amount of protein in the supernatant began increasing, indicating that the protein level was excessive. Using PLA2R protein-to-beads ratio of 0.08:1 allowed labeling of as many proteins as possible on the fluorescent beads.

| Optimum duration for PLA2R protein labeling
According to the labeling method, at pH 6.5, 1 mg of beads and 80 μg of PLA2R protein were mixed and reacted for different durations following the standard steps, and the amount of residual protein in the supernatant was measured. As shown in Figure 3, when the labeling duration was more than 1.5 hours, the proteins were mostly labeled onto the beads. Thus, the minimum duration for labeling proteins onto the fluorescent beads was 1.5 hours.

| Selection of bead amount sprayed onto the membrane
The PLA2R protein-conjugated fluorescent beads at a concentration of 0.2 mg/mL were sprayed onto the conjugate pad at concentrations of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 μL/cm and dried with the marked NC film overnight. The testing strip was then assembled.
First, highly concentrated serum was tested. As shown in Figure 4, when the amount of spray was less than 5 μL/cm, the amount of the Thus, the amount of spray on the membrane should be higher than 5 μL/cm.

| Concentration at T line
The PLA2R protein was diluted with capture antibody buffer solution at concentrations of 2.3, 1.15, 0.575, 0.28, 0.20, and 0.14 mg/ mL, immobilized onto the nitrocellulose membrane with a dispenser at 1.0 μL/cm, and dried at room temperature for 12 hours. The C-line antibody concentration was 1 mg/mL. Using the high-concentration positive sample, the appropriate T-line concentration was selected.
When the T-line concentration was below 0.28 mg/mL, the measured value of the high-concentration sample increased with the T-line concentration, as shown in Figure 5. When the concentration was higher than 0.28 mg/mL, the measured value of the high-concentration sample was stable, indicating that the optimum concentration was 0.28 mg/mL.

| Sensitivity
The average value (x) and standard deviation (s) were calculated from 10 sets of standard curve zero counts, and x + 2s was calculated. The corresponding concentration on the standard curve was considered as the sensitivity and showed a value of 0.6 RU/mL.

F I G U R E 4 Relationship between spray amount of PLA2R
protein labeling solution and the tested concentration of highconcentration samples

| Specificity
Samples of 3 mg/mL hemoglobin, 2 mg/mL bilirubin, and 10 mg/mL triglyceride were tested, and the measured concentrations were all lower than 0.6 RU/mL, indicating that these substances did not affect the reaction. Thus, the reaction was highly specific.

| Recovery rate
A high-concentration standard sample was added to a known lowconcentration sample (1:9, V/V), and the sample was tested. The ratio of the measured value to the theoretical value was calculated, and the average recovery rate was 93.77%, as shown in Table 1.

| Precision rate
The low, medium, and high-concentration quality control groups were tested 10 times, with 3 sets of reagents. The intra-assay CV was 7.61%, and the inter-assay CV was 11.07%.

| Comparison with PLA2R-IgG ELISA Kit
Thirty samples were simultaneously tested using an ELISA Kit and time-resolved fluorescent immunochromatography test strips, and 7 samples with concentrations lower than the minimum sensitivity were excluded. Correlation analysis was performed between the two methods. As shown in Figure 8, the linear correlation between the two methods was y = 0.9302x − 1.0105 and R 2 = .8773. At a cutoff value of 14 RU/mL, the positive and negative results given by the two methods exhibited 100% agreement, as shown in Table 2.

| D ISCUSS I ON
PLA2R autoantibodies are an ideal marker for diagnosing MN and determining its activity, treatment timing, drug selection, and efficacy. Western blotting was first used to detect PLA2R autoantibodies 4 but showed limited sensitivity and is mainly used for qualitative or semi-quantitative analysis. The protocol is complicated, and the amount of sample that can be evaluated in each test is limited, making this method unsuitable for large-scale testing. The detection rate of IMN using PLA2R-IgG ELISA reagents is between 40% and 70%. [7][8][9] Although the protocol is less complicated than Western blotting, the detection rate is lower. In recent years, immunoassay methods have been developed rapidly, among which dissociation-enhanced time-resolved fluoroimmunoassay (TRFIA) can amplify the fluorescence signal by one-million-fold, and thus is more sensitive than both Western blotting and ELISA. The measurement range is also more comprehensive. The detection rate of IMN is 89%, which is much higher than that of the PLA2R-IgG ELISA reagent. 10,11 However, the ELISA or TRFIA reactions require more than 2 hours, and they cannot  time. 13 In this study, nano-fluorescent beads coated with the rare earth ion EU were used as markers rather than traditional colloidal gold particles. EU not only has a strong fluorescence signal under ultraviolet excitation but also has a long decay time. Because the NC membrane also exhibits a fluorescent signal under ultraviolet excitation, we detected the time delay after excitation. We only measured the fluorescent signal of rare earth ions after the background fluorescence had disappeared, eliminating background interference and improving detection sensitivity. Previously, PLA2R antibody was detected by a two-step method. 10,11,14,15 To make the reaction more convenient and rapid, we used the double-antigen sandwich method to achieve a one-step method. By determining and optimizing the conditions such as labeling time, reaction pH, and amount of spray, we developed PLA2R TRFIA immunochromatography. The time- WJ and LP collected the data. ZH and T.W designed the study, purchased reagents, and revised the article. All the authors approved the submission.