Oligonucleotides sequence.

<p>Oligonucleotides sequence.</p

Pro and anti-inflammatory molecule expression in B1KO and wild type animals.

<p>All molecule expressions were measured by real-time PCR at 24 hours of reperfusion. B1KO group had lower pro-inflammatory molecule expression (T-bet and IL-1β) (A and B) and higher anti-inflammatory response (GATA-3, IL-4 and IL-10) (C, D and E). Statistical analyses were performed using the t-test.* B1KO <i>versus</i> B1B2WT, p<0.05.</p

Renal IRI and bradykinin receptors expression.

<p>Bradykinin receptors were analyzed by real-time PCR. In B1B2WT, receptors expressions were cross-modulated (A). In B2KO, B1R expression was increased at 4 and 24 hours (B). Statistical analyses were performed by ANOVA. *B1R <i>versus</i> B2R, p<0.05. # B2KO <i>versus</i> B1B2WT, p<0.05.</p

Cell death modulation under B1R-knockout.

<p>Apoptosis was estimated by Bcl-2 and Bad expression and caspase-3 activity, at 24 hours of reperfusion. Bcl-2 and Bad expression were measured by real-time PCR, and caspase-3 activity by fluorimetric assay. B1KO animals presented higher Bcl-2 expression (A) along with lower Bad expression (B) and caspase-3 activity (C), indicating a lower degree of apoptosis. Statistical analyses were performed using ANOVA.* B1KO <i>versus</i> B1B2WT and B2KO, p<0.05.</p

Pro and anti-inflammatory molecules expression after B1R antagonist and agonist treatment.

<p>All molecule expressions were estimated by real-time PCR at 24 hours of reperfusion. B1R antagonism (R-954) resulted in lower pro-inflammatory molecule expression (T-bet, IL-1β and MCP-1) (A, B and C) and higher anti-inflammatory response (GATA-3, IL-4, IL-10 and HO-1) (D, E, F and G). Molecule expression after B1R agonism (DABK) were similar to non-treated mice. Statistical analyses were performed using ANOVA.*IR+HOE-140 <i>versus</i> IR, p<0.05.</p

Novel Camelid Antibody Fragments Targeting Recombinant Nucleoprotein of <i>Araucaria hantavirus</i>: A Prototype for an Early Diagnosis of Hantavirus Pulmonary Syndrome

<div><p>In addition to conventional antibodies, camelids produce immunoglobulins G composed exclusively of heavy chains in which the antigen binding site is formed only by single domains called VHH. Their particular characteristics make VHHs interesting tools for drug-delivery, passive immunotherapy and high-throughput diagnosis. Hantaviruses are rodent-borne viruses of the Bunyaviridae family. Two clinical forms of the infection are known. Hemorrhagic Fever with Renal Syndrome (HFRS) is present in the Old World, while Hantavirus Pulmonary Syndrome (HPS) is found on the American continent. There is no specific treatment for HPS and its diagnosis is carried out by molecular or serological techniques, using mainly monoclonal antibodies or hantavirus nucleoprotein (N) to detect IgM and IgG in patient serum. This study proposes the use of camelid VHHs to develop alternative methods for diagnosing and confirming HPS. Phage display technology was employed to obtain VHHs. After immunizing one <i>Lama glama</i> against the recombinant N protein (prNΔ₈₅) of a Brazilian hantavirus strain, VHH regions were isolated to construct an immune library. VHHs were displayed fused to the M13KO7 phage coat protein III and the selection steps were performed on immobilized prNΔ₈₅. After selection, eighty clones recognized specifically the N protein. These were sequenced, grouped based mainly on the CDRs, and five clones were analyzed by western blot (WB), surface plasmon resonance (SPR) device, and ELISA. Besides the ability to recognize prNΔ₈₅ by WB, all selected clones showed affinity constants in the nanomolar range. Additionaly, the clone KC329705 is able to detect prNΔ₈₅ in solution, as well as the native viral antigen. Findings support the hypothesis that selected VHHs could be a powerful tool in the development of rapid and accurate HPS diagnostic assays, which are essential to provide supportive care to patients and reduce the high mortality rate associated with hantavirus infections.</p></div

Surface plasmon resonance analysis to show specific binding of VHH and monoclonal antibody.

<p>Sensorgrams obtained after injection of purified VHHs (KC329704–329708) at concentrations of 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.063, 0.0313, 0.0156, 0.00781, 0.0039 µM.</p

Monitoring the llama immune response by ELISA.

<p>The animal showed a rapid and strong response against the prNΔ85 protein after the second immunization (i.e., by Day 21; see immunization schedule <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108067#pone-0108067-t001" target="_blank">Table 1</a>). A sharp decrease in the humoral response was observed at day 28, when the third immunization was carried out. The final bleed performed on day 35, showed an antiserum titre of 1.0×10⁻⁶. The arrows indicate the immunization days.</p

Llama immunization schedule.

<p>The <i>Lama glama</i> was immunized with the recombinant nucleoprotein ARAUV, strain HPR 02-72. The blood was collected from the jugular vein seven days after the third immunization.</p><p>Llama immunization schedule.</p

Kinetic analysis and ranking of selected llama anti-prNΔ₈₅ VHH clones; ranking by highest to lowest affinity.

<p>k_on - association rate constant; k_off - dissociation rate constant; K_D - equilibrium dissociation constant; R_max - response at saturation; Chi² - mean squared of the signal noise.</p><p>Kinetic analysis and ranking of selected llama anti-prNΔ₈₅ VHH clones; ranking by highest to lowest affinity.</p