Development of Monoclonal Antibodies for Identifying Plant-Parasitic Nematodes

Four nematode antigens were prepared by grinding masses of eggs (for root-knot nematode [RKN; Meloidogyne incognita] races 3 and 4) and egg bearing cysts (for soybean cyst nematode [SCN; Heterodera glycines] HG types 0 and 1.2.3.5.6.7) in liquid nitrogen using mortar and pestle. Since grinding eggs and cysts into powder was impossible, suspensions were made from pulverized nematode material, which were then withdrawn using 29-gauge, U-100 insulin syringe (Smiths Medical International Ltd., Keene NH, USA) to immunize mice. Immunization, cytometry and single-cell RNA-seq (scRNA-seq) were performed at the Interdisciplinary Center for Biotechnology Research (ICBR), University of Florida (Cytometry, RRID:SCR_019119; Monoclonal Antibody – RRID:SCR_019147; Gene Expression and Genotyping Core, RRID:SCR_019145; NextGen DNA Sequencing Core Facility, RRID:SCR_019152).

Three mice (seven- to eight-weeks-old female BALB/c) were used per antigen group. Before the first immunization, an initial blood sample was collected from a facial vein of each mouse. A subcutaneous injection of 50 μg of nematode extract in Imject Freund's Incomplete Adjuvant (FIA; Thermo Scientific, Rockford IL, USA) was used for immunization, which was repeated three, six, 13 and 17 weeks later. One week after the second and those subsequent immunizations, blood was collected, and serum anti-nematode antibody titers were evaluated using indirect ELISA. Four weeks after the fifth immunization, mice were injected with 25 μg of nematode extract intraperitoneally. Four days later, the mice were sacrificed to harvest their spleens for B-cell enrichment and sorting. Animal use protocols were approved by the University of Florida Institutional Animal Care and Use Committee (IACUC #10682 and #09723).

Spleens were processed into single-cell suspensions by grinding the tissues between frosted ends of two glass slides and passing the suspension through a 70-μm filter (Genesee Scientific, San Diego CA, USA) to remove debris. Red blood cells were lysed by incubating the suspension in Ammonium-Chloride-Potassium buffer (ACK; NH₄Cl 8,024 mg/L, KHCO₃ 1,001 mg/L, EDTA·Na₂·2H₂O 3.722 mg/L) on ice for 15 min, quenching with PBS, and collecting nucleated cells by centrifugation (300 × g for 5 min at 4°C). CD19⁺ cells were isolated using Mouse CD19 magnetic MicroBeads and LS columns (Miltenyi Biotec, Auburn CA, USA) according to the manufacturer's protocol.

Cells were resuspended at 1 × 10⁶ concentration in 1 mL solution containing 100 μL BD Brilliant Staining Buffer Plus (BD Biosciences, Franklin Lakes NJ, USA) and a mixture of dye-labelled rat anti-mouse antibodies (CD20, CD3, CD19, BC45R/B220a, IGD and IGG) for cell sorting using flow cytometry. The suspension was incubated at room temperature in the dark for 30 min, after which 1 mL of BD Brilliant Staining Buffer was added and centrifuged at 100 × g for 5 min. Cells were resuspended in 500 μL of the staining buffer and run-on BD FACSAria IIU/III upgraded cell sorter (BD Biosciences, San Jose CA, USA) with FSC/SSC and 15 detectors. The run was performed using a 70-μm integrated nozzle. Each sample had 40 U/mL of Ribolock added to protect intact cells from RNAases.

For single cell sequencing, 10x single cell V(D)J libraries were prepared following the 10x Chromium (10x Genomics, Pleasanton CA, USA) Next GEM Single Cell V(D)J user guide. Briefly, 5,000–12,000 FACS-sorted B220+ cells were loaded at 700–1,200 cells/μL onto the 10x Chromium instrument. After breakdown of the Gel Beads-in-emulsion (GEMs), the barcoded cDNAs were purified with DynaBeads. This was followed by 13 cycles of PCR amplification (98ºC for 45 sec; [98ºC for 20 sec, 67ºC for 30 sec, 72ºC for 1 min] × 6; 72ºC for 1 min). The mouse V(D) J were enriched by amplification with Mouse B cell Mix 1 for six cycles of PCR (98ºC for 45 sec; [98ºC for 20 sec, 67ºC for 30 sec, 72ºC for 1 min] × 5; 72ºC for 1 min), followed by eight cycles of PCR amplification using Mouse B cell Mix 2. After library enrichment involving fragmentation, adaptor ligation and index PCR, followed by a DynaBead cleanup, scRNA-seq was performed on an Illumina HiSeq 3000 platform by pair-end (PE)-sequencing on a single lane at 100 PE (i.e., 2 × 100). The sequences generated were processed using the Chromium Single Cell Gene Expression software suite (San Francisco CA, USA) for demultiplexing, barcode processing, gene counting and visualization.

Selected mAbs were synthesized in vitro using a NEB PURExpress CFPS kit (New England Biosciences, Ipswich MA, USA). For this purpose, heavy- and light-chain DNA templates were modified as outlined in Ojima-Kato et al (2017), fulfilling the template requirements for the PURExpress system. From 5′- to 3-end, DNA templates contained: six Ns; T7 promoter; five Ns; a ribosomal binding site (AAGGAG); five Ns; start codon (ATG); SKIK codons; the mAb sequence (N-terminal to C-terminal; 5′ to 3′); Leucine zipper A or B (for heavy or light chain, respectively); stop codon (TAA); and spacer (six Ns) followed by T7 terminator sequence. Antibodies were synthesized in 2-μL PCR tubes using a GenAmp thermal cycler (Applied Biosystems, Waltham, MA, USA). The reaction mixture was prepared following the recommended protocol for PURExpress, and included NEB disulfide bond enhancers 1 and 2 (1.2 μL each) and 300 ng each of the heavy- and light-chain DNA templates. Protein synthesis was carried out on the thermocycler at 37°C for three hours. Sequence information for these mAb is provided in supplementary document S1.

Indirect ELISA was used to evaluate mAbs synthesized in vitro. Antigen suspensions were diluted to ~20 μg/mL in PBS, of which 50 μL was used for ELISA plate coating overnight at 4°C. The plate was washed three times using 200 μL of PBS, and blocked by filling the wells with 200 μL blocking buffer and incubating overnight at 4°C. The plate was then washed, and 100 μL of primary antibody was added to all wells containing antigens. Four wells were used as negative controls for the four antigens. After overnight incubation at 4°C, the plate was washed and 100 μL of secondary antibody (Peroxidase-conjugated AffinPure Goat Anti-Mouse IgG+IgM (H+L); Jackson ImmunoResearch; West Grove PA, USA) was added to all wells at 1.6 μg/μL and incubated at 4°C overnight. For detection, the plate was washed and 100 μL of TMB (3,3′,5,5′-tetramethylbenzidine; ThermoFisher, USA) substrate was added to all wells and incubated for 30 min at room temperature. The reaction was stopped by adding 100 μL of stop solution (2 M H₂SO₄).