Herrin et al. 10.1073/pnas.0711619105.
Fig. 5. A method for producing antigen-specific monoclonal VLR-B antibodies. (A) Lamprey were immunized (i.p.) every 2 weeks for 4-8 weeks. After immunization, buffy coat lymphocytes were harvested from blood samples by centrifugation. VLR-B cDNAs were amplified from RNA extracted from lymphocytes by PCR with primers specific for constant portions of the VLR-B gene and cloned into a mammalian expression vector to create a VLR-B cDNA library. Plasmids were purified from individual bacterial colonies and transfected into HEK-293T cells. Each well of HEK-293T cells was transfected with plasmid derived from one bacterial colony and therefore represented a single VLR-B clone. Two days after transfection, supernatants were screened for antigen-specific clones by ELISA or flow cytometry. Plasmids of clones that were positive for antigen binding were retransformed into bacteria, purified, transfected into HEK-293T cells, and screened for antigen binding a second time to verify clonality. (B) Time-based comparison of methods used to produce mouse mAbs versus lamprey monoclonal VLR-B antibodies.
Fig. 6. VLR-B antibody clones expressed in HEK-293T cells are secreted as multimers similar to the VLR-B antibodies in lamprey plasma. (A) Western blot of lamprey plasma with anti-VLR-B mAb (4C4) in the presence (+) or absence (−) of the reducing agent 2-mercaptoethanol (2-ME). (B) Western blot of supernatant from HEK-293T cells transfected with a lamprey VLR-B cDNA clone in the presence (+) or absence (−) of 2-ME. (A and B) Reduced and nonreduced samples were run on separate gels to prevent diffusion of 2-ME into nonreducing lanes; vertical black lines divide samples derived from separate gels.
Fig. 7. Sequence alignment of BclA-CTD of B. anthracis and B. cereus T. Solvent-exposed amino acid differences (black shading) and buried amino acid differences (gray shading) are indicated.
Fig. 8. Multiple sequence alignment of BclA-CTD-specific VLR-B antibodies. Sequences start at the N terminus of the mature protein and stop at the beginning of the invariant stalk region. Amino acid positions with 100% sequence identity are shaded black, amino acids chemically similar to the consensus residue at a given position are shaded green, and positions that either lack a consensus residue or do not match the consensus are shaded white. *, Hypervariable amino acid positions.
Fig. 9. Purification of VLR4 antibody by antigen affinity chromatography. (A) VLR4 transfectant supernatant was incubated with BclA-CTD-conjugated Sepharose beads, washed with PBS, and then tested for elution with the following: SDS-2ME (positive control); 0.1 M glycine, pH 2.5; 0.1 M HCl, pH 1.5; 0.1 M triethylamine (TEA), pH 11.5; 0.1 M NaOH, pH 12.5; 50% ethylene glycol (EtGlycol), pH 8.0; 3 M MgCl2, pH 8.0; and 5 M LiCl, pH 8.0. After elution, samples were Western blotted with anti-VLR-B mAb (4C4) to detect eluted VLR4. (B) Supernatant from large-scale culture of VLR4 stable transfectants were passed through a BclA-CTD-Sepharose column and purified VLR4 was eluted with 0.1 M triethylamine, pH 11.5. Purified VLR4 antibody was separated by nonreducing SDS-PAGE and visualized with Gelcode Blue staining.
Fig. 10. B. anthracis spore agglutination assay. Purified VLR4 and anti-BclA-CTD mouse mAb (EA2-1) (IgG2b) were serially diluted in 10-fold increments starting at a stock concentration of 0.5 mg/ml (100). The diluted antibodies were added to 1 ´ 106B. anthracis spores overnight at room temperature. The following day, spores were visually inspected by light microscopy and an agglutination score from 0 to 4 was assigned (0, single spores; 1, clusters of 2-3 spores; 2, clusters of 4-6 spores; 3, clusters of 7-10 spores; 4, large clusters of >10 spores).
Fig. 11. VLR4 truncated at the GPI cleavage site is secreted as a low-affinity monomer. (A) Nonreducing Western blot of WT and GPI-stop VLR4. (B) ELISA of WT and GPI-stop VLR4 antibodies with BclA-CTD-coated plates.