Production and purification of recombinant globin and antisera B. marinus adult and tadpole globins (GenBank Accession numbers EL342145 and EU877979, respectively) were amplified using the following full length primer sets: adult globin sense 59-ATGGTCCATTTGACAGATCAC -39, and antisense 59-TTA-GTGGTAACCCTTGCCAAG -39 (444 bp), or tadpole globin sense 59-ATGGTTCATTGGACCGCTGAAGA -39, and antisense 59-TTAGAAATAGCCATGGCTCAGG -39 (444 bp). The fragments were cloned into the bacterial expression vector pDEST17 and expressed as His 6 -tagged proteins in E. coli BL21-AI cells (Invitrogen). Cultures were grown overnight (37uC) in LB supplemented with antibiotics, then diluted 100-fold and grown to an OD of 0.6 (600 nm). L-arabinose (Sigma) was added (0.2% final conc.) to induce protein production and incubation continued for 3-5 h. Bacteria were harvested by centrifugation, rinsed and resuspended in Tris-buffered saline (TBS: 50 mM Tris, 500 mM NaCl; pH 7.5), disrupted by freeze/thaw cycles and centrifuged at 10,0006 g for 30 min. The pellet was solubilised in TBS containing 8 M Urea for 30 min and then centrifuged at 20,0006 g for 30 min to remove insoluble materials. His 6 -tagged proteins were purified in the denatured state using Ni 2+ NTA agarose (Qiagen), washed via imidazole-containing steps (TBS+20, 30 or 40 mM imidazol) and eluted in TBS+500 mM imidazole. Size-based secondary purification was then achieved by continuous-elution electrophoresis (Model 491 Prep Cell, Bio-Rad). Globin proteins were dialysed against amphibian Ringers solution [4.89 g NaCl, 0.298 g KCl, 0.265 g CaCl 2 .2H 2 0, 0.197 g MgSO 4 .7H 2 0, 1.495 g NaHCO 3 , 0.127 g NaH 2 PO 4 .H 2 O and 1.982 g glucose per litre dH 2 O] overnight at 4uC and concentrations determined using the Bio-Rad Protein Assay. Proteins were separated by polyacrylamide gel electrophoresis (SDS-PAGE) (15% gels) using the Bio-Rad Protean II System and visualised with Coomassie brilliant blue. Purified recombinant globin proteins were used as immunogens for the generation of rabbit antiserum. Briefly, two rabbits per immunogen were injected 10 days apart with vaccine containing 50 mg of either adult (rAdglob) or tadpole (rTadglob) recombinant globin. Two weeks later a third dose was administered if a boost to the antibody response was required. Each dose was prepared in CSIRO triple adjuvant (60% v/v Montanide; 40%, v/v rHb [combined with Quil A, 3 mg/mL, and DEAE-dextran, 30 mg/mL]), in water.