Peroxidase conjugated rabbit anti mouse igg

One young adult male L. glama, with food and water ad libitum, was immunized at fortnightly intervals seven times with 100 μg of each BthTX-I and BthTX-II [19 (link)] via subcutaneous injections. Animal immune response was monitored by enzyme immunoassay. The assay was performed in triplicate. For this, microtiter plates (Nunc-MaxiSorp) were coated with 1 μg BthTX-I or BthTX-II in PBS/well and incubated overnight at 4°C. Wells were washed with PBS/ 0.05% Tween-20 (PBST) and unspecific sites blocked with blocking solution (BS – 3% of BSA plus 1% of skimmed milk in PBS) for 3 h. Subsequently, serial dilutions (1:10²; 1:10³;1:10⁴; 1:10⁵) of the serum, collected each week, were added to the wells and the plates were incubated for 12 h in BS at 4°C. After washing with PBST, rabbit anti-llama IgG₂/IgG₃ [21 (link)] at a 1:12000 dilution in BS was added. Excess antibody was removed by washing, and peroxidase conjugated mouse anti-rabbit IgG (Sigma Aldrich) was incubated at a 1:40000 dilution for 2 h in BS. TMB-Ultra (Millipore) was used to reveal the reaction. Absorbances were measured at 450 nm in a microplate reader (BioTek-Synergy HT). The negative control was performed using the llama pre-immune serum.

SDS-PAGE gels (12%) were routinely used to analyze protein samples. For analysis of ExeD secretin, 3–8% Criterion pre-cast polyacrylamide Tris-acetate gradient gels (Biorad) were used. Gradient gel samples were standardized to 0.01 OD₆₀₀ per lane. For immunoblotting, the proteins were transferred to PVDF membranes (GE Healthcare Life Sciences). Visualization of ExeD was achieved by incubation with the appropriate rabbit antiserum followed by incubation with peroxidase-conjugated mouse anti-rabbit IgG (Sigma). The signal was developed with a chemiluminescent substrate kit (GE Healthcare Life Sciences).

Recombinant nucleoprotein ARAUV prNΔ₈₅[24] (link) was obtained from Instituto Carlos Chagas/Fiocruz Paraná. Home-made anti-IgG_2,3 was produced by immunizing rabbits with previously isolated Lama glama IgG₂/IgG₃. Peroxidase conjugated Mouse anti-rabbit IgG was purchased from Sigma Aldrich. E.coli TG1 (Stratagene, La Jolla, USA) and HB2151 (provided by Dr. Gerhard Wunderlich, USP, Brazil) strains were used, respectively, for the cloning of VHH and the expression of the selected clones. The pHEN1 phagemid provided by Dr. Pierre Lafaye (Institute Pasteur, Paris, France) was used to clone and produce the VHH immune library after the insertion of the 6xHis-tag sequence by site-directed mutagenesis PCR (pHEN1-6xHis), and the M13KO7 helper phage purchased from New England Biolabs (Ipswich, USA) was used to produce the secondary immune library. Mouse monoclonal antibody (Mab 432/6BF) is an IgG1κ antibody against the nucleoprotein (rNΔ₈₅) of the Araucaria hantavirus strain (ARAUV) [27] (link). Rodent serum was collected during fieldwork of epidemiology surveillance for hantavirus in General Carneiro, Paraná, Brazil in 2006 and 2010. These samples were previously tested for the presence of hantavirus IgG antibodies, and when positive, sequencing was performed to identify the virus genotype [34] (link).