C. d. terrificus and, B. arietans snake venom representative anti-toxin antibodies will be prepared in BALB/c and C57Bl/6 mice lines. Immunization schedules will be oriented to provide the production of antibodies with different interaction affinities for the respective antigenic epitopes. Antitoxin IgG immunoglobulin will be isolated by successive affinity and immuneaffinity chromatographies, Fab (50kDa) and scFab (28kDa) fragments will be prepared, VL (L1, L2 and L3) and VH (H1, H2 and H3) correspondent regions to the CDRs will be isolated and aminoacid sequences will be determined. Aminoacid sequencing patterns corresponding to low and high affinity anti-toxins IgGs will be determined. Epitopes recognized by anti-toxin peptides will be identified by specific blocking of their enzymatic or biological activities. Splenic B lymphocyte populations will be separated in a high-density microwell plated and lysed in situ. mRNA will be captured on magnetic beads, reverse transcribed and amplified by emulsion VH:VL linkage PCR. The linked transcripts will be analyzed by illumine high-throughput sequencing. After the fidelity of the VH:VL pairs be identified validation the corresponding sequences will be lined and compared with the amino acid sequences from low and high-affinity antivenom antibodies.
These sequences will be submitted to the “ImMunoGeneTics (IMGT´S) annotated data-base of Acs co-crystallized structures registered on “protein Data Bank (IMGT/3Dstructure-DB)”. Experimental/computational obtained sequencing platforms will be adjusted and used to construct anti-toxin multi-peptides. Neutralizing efficiency will be evaluated in vitro and in vivo.
Obtained results will be submitted for publication in specialized magazines (Protein Eng; Proteins) and/or registered as patents by “Britânia – marcas e patentes).
KEY-WORDS: Antibodies; epitopes; toxins; IgG fragments; multi-peptides; molecular interactions.