Chemical: 3,3′,5,5′-Tetramethylbenzidin (TMB) was from Suo Laibao Technology Co., Ltd (Beijing, China);
Horseradish peroxidase (HRP) was from Sigma-Aldrich (Shanghai, China);
Human mononuclear cell separation solution (product ID: 25171004) was from Dongfang Huahui Technology Co., Ltd (Shanghai, China);
Mem-PER™ Plus Membrane Protein Extraction Kit (product ID: 89842) was from ThermoFisherScientific (Shanghai, China);
Nitrocellulose filter membrane (NC membrane) was from Bio-Rad Laboratories, Inc (Shanghai, China);
Protein-G column chromatography was from GE Healthcare (Shanghai, China);
Horseradish peroxidase (HRP)-conjugated rabbit secondary antibody (Goat Anti-Rabbit IgG, HRP) was from Zhuangzhi Biotech, Xi'an, China;
Horseradish peroxidase (HRP)-conjugated mouse secondary antibody (Rabbit Anti- Mouse IgG, HRP) was from Zhuangzhi Biotech, Xi'an, China;
MrgX2 standard protein (Recombinant Human GPCR MRGX2 protein, ab165129) was from Abcam (Shanghai, China);
Washing solution (composed of PBS and Tween 20; homemade);
Coating solution (composed of sodium carbonate and sodium bicarbonate; homemade);
Blocking solution (composed of gelatin, sucrose and BSA; homemade);
Diluents solution (consisting of PBS, Tween 20 and BSA; homemade);
Stop solution (2M H2SO4; homemade).
2.1. Preparation of human MrgX2 peptide (T1) immunogen
The amino acid sequence of human MrgX2 was sourced from the GenBank database and DNAStar software predicted a strong antigenicity and high hydrophilicity for the MrgX2 peptide (T1). T1 was synthesized by the manual solid-phase Fmoc method, and purified by reversed-phase high-performance liquid chromatography (HPLC). Purity of the T1 was tested by analytical HPLC (Agela C18-10×250 mm, flow rate: 1 mL/min). The chemical structure of T1 was characterized by MALDI-TOF mass spectrometry. T1 was bound to the carrier protein keyhole limpet hemocyanin (KLH) by a cysteine residue added to the N-terminus of the T1 chain, and the glutaraldehyde method.
2.2. Preparation of anti-human MrgX2 peptide (T1) antibodies
Animal experiments were conducted in accordance with the "Administrative Measures for Experimental Animals” (Ministry of Science and Technology) and as approved by the Animal Ethics Committee at Xi'an Jiaotong University, Xi'an, China (Permit Number: XJTU 2011-0045).
Anti-MrgX2 polyclonal antibodies (Pabs) were produced by immunizing rabbits (3 pairs) with human MrgX2 peptide (T1). The T1-KLH conjugate (4.2 mg/pair) was dissolved in Freund's complete adjuvant (1:1 volume ratio). Take subcutaneous sub-point injections, spray alcohol on the animal's dorsal midline to avoid areas with immune swelling. One injection was divided into four injections and injected into 4 different points. Freund's incomplete adjuvant booster injections were given every 2 weeks. After each booster injection, serum was collected from the ear vein, and antibody production was detected by Dot Blot method. Then the antiserum was purified by saturated ammonium sulfate precipitation and protein-G column chromatography, and then titrated by Dot Blot method.
Anti-MrgX2 monoclonal antibodies (Mabs) were produced by immunizing mice (10 pairs) with human MrgX2 peptide (T1). The T1-KLH (0.9 mg/pair) conjugate was dissolved in Freund's complete adjuvant (1:1 volume ratio). Take subcutaneous sub-point injections, spray alcohol on the animal's dorsal midline to avoid areas with immune swelling. One injection is divided into four injections and injected into 4 different points. Freund's incomplete adjuvant booster injections were given every 2 weeks. After each booster injection, the serum was collected from the tail vein of mice, and antibody production was detected by indirect ELISA. After cell fusion, subcloning and antibody affinity purification (using saturated ammonium sulfate and protein columns), the purified monoclonal antibodies were obtained. Finally, indirect ELISA was used to determine the titer of purified antibodies.
1 mg of each antibody was labeled with biotin (Biotin labeling method) for ELISA detection. Dilute each antibody with 0.1 mol/L sodium bicarbonate buffer (pH 8.0) to 1 mg/mL. Alternately use 0.1 mol/L sodium bicarbonate buffer (PH 8.0) to fully dialyze the antibody. Using 1 mL DMSO to dissolve biotin succinimide Ester (NHSB) (1 mg), and add 120 μL of NHSB solution to 1 mL of antibody solution, keep stirring at room temperature for 2 h; add 9.6 μL of 1 mol/L NH4Cl (per 25 μg NHSB plus 1 μL), stir at room temperature for 10 min. The labeled antibodies were then diluted in 50% glycerol and stored at -20 °C until further use.
2.3. MrgX2 antibody titer by indirect ELISA
Screening of monoclonal antibody producing cell lines and antibody identification was performed by indirect ELISA (96-well plates). Aliquots of 100 μL of T1 were diluted in coating solution and added onto a microtiter plate (final concentration: 50 ng/well) and incubated at 4°C overnight. The plate was then washed 3x with 300 μL/well of washing solution (manual washing), before 200 μL/well of blocking solution and incubated at 37°C for 2 h. The plate was washed 3x and 100 μL/well of antibody (1:1000) diluted with diluents solution or 100 μL/well of cell supernatant was added and incubated at 37°C for 1 h. The microtiter plate was washed 3x with washing solution and 100 μL/well HRP-conjugated anti-rabbit secondary antibody (1:1000) or anti-mouse secondary antibody (1:1000) diluted with diluents solution were added, and incubated at 37°C for 1 h. The plate was washed 3x, and 100 μL/well of TMB substrate was added at 37°C for 5 min. The reaction was stopped by adding 50 μL/well 2 M H2SO4 and the absorbtion was determined at a wavelength of 450 nm (Flexstation 3, Meigu MolecularDevices, Shanghai, China).
2.4. Dot Blot for antibody titer determination
Screening and identification of polyclonal antibodies was performed using the Dot Blot. 100 ng/well of T1 (100 μL/well) was added to the nitrocellulose filter membrane and incubated at 37°C for 30 min. The nitrocellulose filter membrane was washed 5x with washing solution (manual washing), and 1 mL/well coating solution was added containing a size of 1 cm2 and incubated for 1 h. Wash the nitrocellulose filter membrane 5x with washing solution, and add 1 mL/well of the polyclonal antibodies dilution (1:1000) and incubate for 2 h. Wash the membrane 5x with washing solution, add 1 mL/well of horseradish peroxidase (HRP)-conjugated rabbit secondary antibody (1:1000) and incubate for 1 h on the shaker. The nitrocellulose filter membrane was washed 5x with washing solution, and finally the processed nitrocellulose filter membrane is developed in a dark room using developer and fixer in accordance with conventional development method.
2.5. Establishment of double antibody sandwich MrgX2-ELISA
The optimal concentrations of Mab capture antibody and biotin-Pab detection antibody were determined by checkerboard titration and the reaction conditions for each step of the MrgX2-ELISA were optimized. The human MrgX2-ELISA was developed using the reagents described above. 100 μL/well of Mab (4 μg/mL, diluted with coating solution) was added to a microtiter plate (96-well plates) and incubated at 4°C overnight. The coating solution was then removed and the plate was washed 3x with 300 μL/well washing solution and the plate was pat dried. Then, 200 μL/well of blocking solution were added and incubated at 37°C for 2 h. The plate was washed 3x and the MrgX2 standard protein (0.02 μg/μL) was diluted to 0.5 μg/mL with dilution solution, 100 μL/well was added and incubated at 37°C for 1 h. The plate was washed 3x and 100 μL/well Biotin-Pabs (0.5 μg/mL, diluted with diluents solution), was added, and incubated at 37°C for 30 min. The plate was washed 3x before 100 μL/well of avidin-HRP (1:500 in diluents solution) was added and incubated at 37°C for 30 min. The plate was washed 3x and 100 μL/well of TMB was added and the color was developed for 15 min. The reaction was stopped by adding 50 μL/well of stop solution and the absorbtion was determined at a wavelength of 450 nm (Flexstation 3, Meigu MolecularDevices, Shanghai, China).
2.6. Optimizing the sandwich MrgX2-ELISA to blood samples
The above described sandwich MrgX2-ELISA was used to compute the standard curve, detection limit, limit of quantification, inter-assay precision, intra-assay precision, accuracy, stability and specificity. The stability of the MrgX2-ELISA was tested after 7 days storage at 37°C. The sensitivity of the MrgX2-ELISA was calculated using the guidelines provided by the National Committee for Clinical Laboratory Standards (NCCLS) evaluation protocol.
2.7. Study design of the CU trial
This study was registered at the Chinese Clinical Trail Registry (# ChiCTR1900025723), the ethical approval was given by Ethics Committee at Xi’an Jiaotong University (Xi’an, China). All specimens in this study were obtained after individual signed informed consent of each participant.
This study design was a single-center, random sampling, case-control study. Human whole blood samples of CU patients (n=75, age 10-70 yrs, median age 40 yrs) were obtained from the Department of Dermatology in the Second Affiliated Hospital of Xi’an Jiaotong University (Xi’an, China), and human whole blood samples of control group (n=75, age 18-76 yrs, median age 47 yrs) were sourced from the Department of Physical Examination in the Second Affiliated Hospital of Xi’an Jiaotong University (Xi’an, China).
2.8 Study subjects
The principle of case and control was applied to sample collection.
Sample collection criteria for CU patients:
- CU was diagnosed as: at least three episodes of symptoms such as weekly wheals and pruritus over a period of more than 6 weeks.
- Age 6-80 years.
- No recent history of other diseases except CU.
- Not pregnant.
Sample collection criteria for control group:
- No history of CU.
- No recent history of other diseases.
- No family history of CU.
- Age 6-80 years.
- Not pregnant.
Patients who had at least one of the following indicators were excluded from the study:
- Unclear symptoms, and inability to confirm CU.
- Patients with confirmed physical urticaria.
- Patients who received systemic treatment of anti-histamines or glucocorticoids within two weeks prior to sample collection.
2.9. Clinical application of the sandwich MrgX2-ELISA
Leukocytes were isolated and purified from fresh whole blood samples using human mononuclear cell separation solution in a final volume of 1 mL according to the instructions. Mem-PER™ Plus Membrane Protein Extraction Kit was used to lyse the cells and extract leukocytes membrane proteins according to the instructions. The MrgX2-ELISA method was used to detect the expression of MrgX2 protein in leukocytes protein extracts from CU patients (n=75) and healthy subjects (n=75).
The expression levels of MrgX2 protein in the whole blood (1 mL whole blood) of CU patients and healthy subjects were compared. The frequency distribution data of 75 healthy subjects was determined, the cutoff value of CU patients was derived, and the ROC curve was constructed based on the results of CU patients and healthy subjects.
2.10. Data analysis
GraphPad Prism 7 software (GraphPad Software, San Diego, California) was used for ELISA standard curves. For each group, the median, 25th percentile, 75th percentile, and interquartile range of MrgX2 were determined. Two-tailed unpaired Student's t-test was applied by GraphPad Prism 7 software, and p value of < 0.05 was considered as statistically significant.