Novel monoclonal antibodies against house dust mite allergen Der p 21 and their application to analyze allergen extracts

Production of recombinant MBP-fused Der p 21 in E. coli

The synthetic gene coding for Der p 21 was obtained from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA). The DNA fragment of 378 bp was digested with BamHI and XhoI restriction endonucleases and cloned into pET28-MBP-TEV vector (a gift from Zita Balklava & Thomas Wassmer, Addgene plasmid #69929; Addgene, Watertown, MA, USA) (Currinn et al., 2016). The resulting plasmid pET28-MBP-TEV-Der p 21 included the Der p 21 fused to the maltose binding protein (MBP) coding sequence. The synthesis of rDer p 21 with N-terminal MBP in E. coli was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). After 2.5 h of cultivation at 37 °C, the cells were separated by centrifugation at 1811 x g for 15 min at 4 °C, washed with rinse buffer (20 mM TRIS, 200 mM NaCl, pH 7.4) and collected by additional centrifugation.

Purification and cleavage of rMBP-Der p 21

E. coli biomass was suspended in MBP binding buffer (20 mM NaH2PO4, 200 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), pH 7.4) with 1 mM phenylmethylsulfonyl fluoride (PMSF). The cells were disrupted by sonication. The soluble fraction was separated by centrifugation at 15,000 x g for 20 min at 4 °C. The rMBP-Der p 21 was purified by MBP-affinity chromatography using ÄKTA start chromatography system (Cytiva, USA) and MBPTrap™ HP one mL column (Cytiva, USA). Bound proteins were eluted with MBP elution buffer (MBP binding buffer containing 10 mM maltose). Concentration of rMBP-Der p 21 was determined with NanoDrop 2000c (Thermo Fisher Scientific, Waltham, MA, USA) spectrophotometer (extinction coefficient 80,790 M⁻¹ cm⁻¹). The fused protein rMBP-Der p 21 contained TEV protease cleavage site between the allergen and MBP. Hydrolysis reaction was performed at room temperature (RT) overnight (ON) with TEV and rMBP-Der p 21 mass ratio 1:20, adding 3 mM reduced glutathione and 0.3 mM oxidized glutathione. Since MBP and TEV had hexahistidine sequence inserted at the N-terminus, rDer p 21 was purified by immobilized metal affinity chromatography using ÄKTA start. The hydrolysis reaction mixture was buffer exchanged to Ni-NTA binding buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8) and loaded on the HisPur™ HP one mL column (Thermo Fisher Scientific, Waltham, MA, USA). rDer p 21 was found in the sample application flow-through fractions. The concentration of rDer p 21 was measured with NanoDrop 2000c (extinction coefficient 12,950 M⁻¹ cm⁻¹) and adjusted with ImageJ software (National Institutes of Health, Bethesda, USA).

Generation of monoclonal antibodies against rDer p 21

Three 6–8 week old female BALB/c mice were selected for immunization. Sample size was based on laboratory practice and hybridoma technology protocol, no control group, randomization or blinding were applied. Mice were immunized three times every 28 days by a subcutaneous injections with 50 µg rDer p 21, using the complete Freund’s adjuvant (Thermo Fisher Scientific, Waltham, MA, USA) for the primary and incomplete Freund’s adjuvant (Thermo Fisher Scientific, Waltham, MA, USA) for the secondary immunization (final volume 200 µL). Third immunization and the booster dose were injected without adjuvant. Blood samples were collected by tail bleeding before each immunization and 28 days after the last immunization. Samples were analyzed by an indirect enzyme-linked immunosorbent assay (ELISA) and a titer of rDer p 21-specific IgG was determined. Boosted dose was injected three days before the hybridization. All three immunized mice were euthanized by cervical dislocation as this method was in accordance with the requirements laid down in Annex IV to DIRECTIVE 2010/63/EU. The killing of animals was completed by confirmation of the onset of rigor mortis. No surviving mice were left at the conclusion of the experiment. The hybridization was performed as described by Köhler & Milstein (1975). Briefly, the spleen cells of the immunized mouse were fused with mouse myeloma Sp2/0 cells (CRL-1581, ATCC, USA) (ratio 6:1) in the presence of polyethylene glycol solution (PEG-4000; Sigma-Aldrich, Darmstadt, Germany). Fused cells were cultivated in Dulbecco’s modified Eagle’s growth medium (Merck, Rahway, NJ, USA) supplemented with 15% fetal bovine serum (Merck, Rahway, NJ, USA), hypoxanthine-aminopterin-thymidine (Sigma-Aldrich, Darmstadt, Germany) and 200 µg/mL gentamicin (Carl Roth, Karlsruhe, Germany). After 2 weeks, production of rDer p 21 specific antibodies by hybridoma cells was tested by indirect ELISA. Then selected clones were cultivated and cryoconservated in liquid nitrogen.

Mice for the immunization were obtained from Vilnius University, Life Sciences Center, Institute of Biochemistry, Lithuania (Vet. Approval No LT 59–13-001, LT 60–13-001, LT 61–13-004). The permission to use BALB/c mice in the experiment was obtained from the Lithuanian State Food and Veterinary Agency (Permission No G2-117, issued 11 June 2019). Mice were supervised daily, housed under controlled conditions (temperature 22 ± 1 °C, humidity 55 ± 3%, 12 h light-dark cycle, cardboard enrichment), given constant access to standard rodent food and water ad libitum. The maintenance of mice and experimental procedures were performed by qualified staff and conformed to Directive 2010/63/EU requirements. No anesthesia or analgesia was given in any procedures based on hybridoma technology protocol and bioethics permission. Criteria for euthanizing mice prior to the planned end of the experiment were severe signs of distress or heavy weight loss, but no such incident has happened.

Purification of the MAbs

MAbs were purified by Protein A affinity chromatography using ÄKTA start as previously described (Sližiene et al., 2022).

Conjugation of the MAbs to horseradish peroxidase

The MAbs were conjugated with horseradish peroxidase (HRP) using the periodate method, as previously described (Stravinskiene et al., 2019).

Serum specimens

A collection of blood serum specimens of allergic patients was used. Informed written consent was obtained from all involved subjects. The study was approved by Vilnius Regional Biomedical Research Ethics Committee (approval no. 158200-17-926-430). The levels of IgE specific to Der p 21 were determined by Alex² (MacroArray Diagnostics GmbH, Austria) test and ranged from 1.9 to 51.1 kU_A/L.

Western blot

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) fractionated antigen samples were transferred to a polyvinylidene difluoride membrane (Carl Roth, Karlsruhe, Germany). The membrane was blocked with phosphate buffered saline (PBS) containing 2% powdered milk. Then membrane was incubated with purified MAbs (0.2 µg/mL) or allergic patients’ sera diluted 1:50 in PBS with Tween-20 (PBS-T) with 2% powdered milk for 1 h at RT. Next, the membrane was incubated with antibody conjugates (goat anti-mouse IgG (H+L)-HRP (Bio-Rad, Hercules, CA, USA, catalogue number #1721011), or mouse anti-human IgE Fc-HRP (SouthernBiotech, Birmingham, AL, USA, catalogue number #9160-05)) 1:4000 and 1:1000 dilution respectively in PBS-T with 2% powdered milk for 1 h at RT. 1-Step™ Tetramethylbenzidine (TMB)-Blotting Substrate Solution (Thermo Fisher Scientific, Waltham, MA, USA) was used and the enzymatic reaction was stopped by washing the membrane with water.

ELISA

Sandwich ELISA

Quantification of Der p 21 was performed by sandwich ELISA. A set of 96-well plates were coated with capture MAb 4D4 (0.5 µg/mL, 100 µL/well). The wells were blocked with ROTI^®block (Carl Roth, Karlsruhe, Germany). Then the plates were incubated for 1 h with allergen extract or standard antigen rDer p 21 in serial dilutions from 10–300 µg/mL for extracts; from 500 or 600 ng/mL for rDer p 21 (100 µL/well). Next, the plates were incubated for 1 h with detection conjugated MAb 1A8 (1:5,000 dilution, 100 µL/well). TMB was added (100 µL/well) followed by 3.6% H2SO4 (50 µL/well). The concentration of Der p 21 in allergen extracts was determined by fitting a logistic function.

Inhibition ELISA

The reactivity of rDer p 21 with HDM-specific IgE was additionaly analyzed by inhibition ELISA. Blood serum sample (no. 7) of patient with diagnosed HDM allergy was analyzed with four different HDM extracts from different manufacturers (MNF #2-5). Firstly, 96-well plates were coated with rDer p 21 (1 µg/mL, 50 µL/well) and blocked with 2% BSA. Serum sample (1:20 dilution) was preincubated with HDM extract for 1 h for inhibition of Der p 21-specific IgE. The total protein amount of HDM extract from MNF #2 was chosen for preincubation, based on sandwich ELISA results, to contain 2:1 mass ratio of nDer p 21 in the extract (100 ng) vs rDer p 21 in a plate well (50 ng). For HDM extracts from MNF #3-5 containing lower or undetectable amounts of Der p 21, the total protein amount for preincubation was chosen to be the same as for the extract from MNF #2. After the blocking step, the preincubated samples were added to the wells (100 µL/well) and incubated for 1 h, then the conjugate anti-human IgE Fc-HRP (1:1,000 dilution, 50 µL/well) was added and incubated for 1 h. The enzymatic reaction was developed and stopped as described above. The inhibition efficiency was calculated from the OD450 values of the positive control (serum preincubation with purified rDer p 21) and negative control (serum preincubation without HDM extract).

Microarray immunoassay

To analyze rDer p 21 antigenic properties using serum samples and to determine the specificity of the MAbs, rDer p 21, thermally denatured rDer p 21 and rMBP-Der p 21 (0.3 mg/mL) were printed on 2D-Epoxy glass slides (PolyAn GmbH, Berlin, Germany). For determination of antibody reactivity with rDer p 21 and Der p 21 in HDM extracts, a serially diluted rDer p 21 with a concentration ranging from 100 to 0.00305 µg/mL and six different HDM extracts diluted 16 times from their stock concentrations were printed on glass slides. Human serum albumin, rMBP, HRP and PBS were printed as negative controls. Each allergen and control were printed as a single droplet (∼450 pL/drop) in three replicates onto glass slides using sciFLEXARRAYER SX microarray printer (SCIENION GmbH, Berlin, Germany). The slides were blocked with PBS-T containing 2% BSA (blocking buffer) for 30 min at RT. After that, the slides were incubated for 2 h with either serum samples (1:4 dilution with blocking buffer, 80 µL/well) or MAbs (0.5 µg/mL for specificity analysis and 0.2 µg/mL for reactivity of the MAbs analysis, diluted with blocking buffer, 80 µL/well). Then the slides were incubated for 30 min with the mouse anti-human IgE Alexa Fluor^® 647 (SouthernBiotech, USA, catalogue number #9160-31) or goat anti-mouse IgG Fc Alexa Fluor^® 647 (SouthernBiotech, USA, catalogue number #1033-31) (1 µg/mL, diluted with blocking buffer, 80 µL/well). The slides were scanned using InnoScan 710 AL microarray scanner (Innopsys, France). The images were analyzed with MAPIX software (Innopsys, France).

Statistical analysis

The antigenicity analysis data was evaluated by the GraphPad Prism software (Dotmatics, Boston, MA, USA). The results were presented as mean ± standard deviation (SD), n = 3. The results of microarray immunoassay experiments were presented as average mean ± SD of median fluorescence intensity (MFI) values, n = 3. The correlation between results obtained by Alex² and ELISA, and between Alex² and microarray immunoassay was determined by calculating the Pearson correlation coefficient using the GraphPad Prism software. Calculations of the apparent Kd were performed using the OriginPro 8 software (OriginLab, Northampton, MA, USA), and the results were presented as mean ± standard error of mean (SEM), n = 3. The standard curve parameters R² and the limit of detection for the optimized sandwich ELISA were calculated using OriginPro 8 software and according to (Armbruster & Pry, 2008), respectively. Calculations of Der p 21 concentration in allergen extracts were performed using the SoftMax^® Pro software (Molecular Devices, San Jose, CA, USA), and the results were presented as mean ± SD, n = 3.

Expression, purification and analysis of rDer p 21

rMBP-Der p 21 (calculated molecular weight of 58.79 kDa) was produced in E. coli and was found in a soluble fraction of the cell lysate. rDer p 21 was purified by 2-step affinity chromatography (Fig. S1). Firstly, rMBP-Der p 21 was purified by MBP-specific affinity chromatography. The yield of rMBP-Der p 21 was about 1.6 mg from 1 g of wet E. coli biomass. To detach MBP from Der p 21, different conditions of the cleavage by TEV protease were analyzed: TEV and rMBP-Der p 21 mass ratio 1:50 and 1:20; temperatures 4 °C and RT. The mass ratio 1:20 and temperature 4 °C were found to be optimal for the cleavage. After hydrolysis, the second purification step by Ni-affinity chromatography was applied. The rDer p 21 (14.74 kDa), which has no hexahistidine tag, was found in the flow-through fractions and the MBP was found in the eluted fractions. The yield of the purified rDer p 21 was 0.34 mg from 1 g of wet biomass. However, a small amount of MBP was left in the final sample of the purified rDer p 21, possibly due to its partial degradation and loss of the hexahistidine tag. According to calculations using the ImageJ software, the amount of MBP in the rDer p 21 sample was 20.7% (Fig. S1C).

To investigate the antigenic properties of the purified rDer p 21, its reactivity with blood serum specimens of patients with diagnosed HDM allergy was analyzed by an indirect ELISA and the microarray immunoassay. The sera were previously tested by the commercial multiplex test Alex² on its different allergen components, including rDer p 21. The results of ELISA and microarray immunoassay based on the use of our rDer p 21 were compared with those obtained in the allergy diagnostics platform Alex². The purified rDer p 21 was recognized by HDM-specific IgE both in ELISA and the microarray test (Fig. 1) thus confirming the proper antigenic structure of the purified rDer p 21. High correlation between Alex² results with the in-house rDer p 21-based tests—ELISA and microarray immunoassay—was observed with the values of Pearson correlation coefficient 0.9470 (p < 0.0000001) and 0.8220 (p < 0.0001), respectively (obtained values from different tests were converted to percentages in accordance with the serum with highest kU_A/L value (Alex² test) being 100%).

Generation and characterization of anti-Der p 21 MAbs

After fusion of spleen cells of the immunized mouse with myeloma cells, hybrid clones were screened on rMBP-Der p 21 and then retested both on rDer p 21 and MBP (negative control) to exclude their reactivity against MBP. In total, five hybridoma clones producing rDer p 21-specific MAbs were generated (Table 1). Isotype-specific ELISA showed that the MAbs are of IgG1 subtype (κ light chain). The affinity of MAbs was analyzed by measuring the apparent Kd by indirect ELISA (n = 3). The Kd ranged from 61.1 ± 3.32 pM to 115.7 ± 8.26 pM, which indicates high-affinity binding.

The reactivity of the MAbs with denatured rDer p 21 and their potential cross-reactivity with irrelevant bacterial proteins was analyzed by WB (Fig. S2) using E. coli BL21 Star™ (DE3) lysate as a negative control. The MAbs were reactive with rDer p 21 and rDer p 21-MBP. No reactivity with rMBP or any other proteins from the lysate was observed. The reactivity and specificity of the MAbs were also tested by the microarray immunoassay with different antigens—rDer p 21 (Fig. 2), rMBP-Der p 21, rMBP and the denatured rDer p 21 (Fig. S3). The MAbs were reactive with rDer p 21, denatured rDer p 21 and rMBP-Der p 21, while no reactivity with rMBP used as a negative control was observed. The results indicate that all five generated hybridoma clones secrete high-affinity rDer p 21-specific MAbs, that can be applied for Der p 21 detection by different immunoassays.

Development of sandwich ELISA for the quantification of Der p 21

To develop sandwich ELISA for Der p 21 detection, pairs of non-competing MAbs that recognize distinct Der p 21 epitopes were selected. All MAbs were conjugated with HRP and tested in a competitive ELISA. Two groups of MAbs were identified that recognize non-overlapping epitopes—group 1 (MAbs 2G4, 4D4) and group 2 (MAbs 1A8, 2F3). Based on the results of the competitive ELISA, different combinations of MAbs from groups 1 and 2 were tested in sandwich ELISA format. The MAb 8C3 was excluded from testing. The highest detection sensitivity was observed using 4D4 as capture MAb, and 1A8 as detection MAb. These MAbs showed the highest affinity to rDer p 21 (Table 1). Different ELISA conditions were evaluated to optimize the assays: plate type, capture MAb 4D4 concentration, blocking solution, detection MAb 1A8 dilution. The optimized sandwich ELISA showed excellent correlation (R² = 0.99973) between rDer p 21 concentration and the OD450 value (Fig. 3) with the calculated 27.25 pg/mL limit of detection.

Application of the MAbs for the analysis of HDM extracts

The optimized sandwich ELISA was applied to analyse six D. pteronyssinus allergen extracts from different manufacturers (MNF #1-6). Der p 21 was detected only in three of the analyzed allergen extracts. Its mass fraction ranged from 0.51 to 15.17 µg/mg, or mass percent from 0.05 to 1.52% respectively (Table 2). Allergen extracts were also analyzed by WB with MAb 1A8 and by microarray immunoassay with MAb 2G4 (Fig. 4). The analysis of allergen extracts with other MAbs showed similar results (Fig. S4).

The results of the microarray immunoassay (its scheme represented in Fig. S5) are in agreement with the results of sandwich ELISA, since Der p 21 was detected in the same three allergen extracts (MNF #1-3). The concentration of Der p 21 is much lower in the extract from MNF #3 (0.51 ± 0.03 µg/mg) compared to MNF #1 and #2 (15.17 ± 2.26 and 10.85 ± 0.42 µg/mg respectively). In contrast, Der p 21 was not detected in the allergen extract from MNF #3 by WB, possibly due to lower sensitivity of this assay. Der p 21 was not detected by any of these methods in the allergen extracts from MNF #4-6.

To validate the proper folding of rDer p 21 and to confirm the results of HDM extract analysis, inhibition ELISA was performed using HDM IgE-positive blood serum sample (no. 7) and four different HDM extracts (MNF #2-5). HDM extract from MNF #2 containing the highest amount of Der p 21 caused a complete inhibition (>100%) of binding of Der p 21-specific IgE to rDer p 21, while extracts from MNF #3, #4 and #5 with lower amounts of Der p 21 caused only the partial inhibition (43, 33 and 26%, respectively) of anti-Der p 21 IgE binding to rDer p 21 (Fig. S6). These results indicate the proper folding of rDer p 21, since Der p 21-specific IgE competes with natural Der p 21 in HDM extracts for binding to rDer p 21.