Molecular characterization of a neutralizing murine monoclonal antibody against Tityus serrulatus scorpion venom.
Monoclonal antibodies (mAbs) against Tityus serrulatus venom were obtained by the fusion of SP2/0 murine myeloma cells and spleen cells from BALB/c mice immunized with a toxic fraction (TstFG50) of the Tityus venom (this G50 chromatography fraction represents most of the toxicity of the crude venom) conjugated to bovine serum albumin (BSA) with glutaraldehyde. From the initial screening of over 200 hybridoma fusion wells, a panel of 9 anti-TstFG50 secreting hybridomas was established. The capacity of mAbs to neutralize the TstFG50 toxic fraction toxic was determined by in vitro neutralization assays and by inhibition of the binding of 125I-TsVII to its site on rat brain synaptosomes. Only mAbTs1 neutralized 50% of the toxic effects produced by scorpion venom and showed 35% inhibition of the binding of 125I-TsVII at 10(-7) M. To map the epitope recognized by the protective mAbTs1, we prepared a comprehensive series of overlapping 15-mer synthetic peptides covering the amino acid sequences of the four Tityus proteins. MAbTs1 reacted with peptide 26 of TsIV (KKSKDKKADSGYSYW), peptide 30 of TsVII (KKGSSGYSAWPASYS) and peptide 31 of TsNTxP (KKGSSGYSAWPASYS). MAbTs1 was not reactive with any peptide from TsII. The N-terminal lysine residue from the epitope was found to be critical for mAbTs1 binding. The epitope was positioned on the available three-dimensional structure of TsVII together with the recently identified residues from the pharmacophore of beta-scorpion toxins. The neutralizing properties of mAbTs1 might be explained by spatial vicinity of epitope residues with pharmacophore residues.
Alvarenga
L M
LM
Departamentos de Bioquímica-Imunologia, ICB, Universidade Federal de Minas Gerais, CP: 486; CEP: 31270901, Belo Horizonte-MG, Brazil.
Machado de Avila
R A
RA
Amim
P R
PR
Martins
M S
MS
Kalapothakis
E
E
de Lima
M E
ME
Santos
R G
RG
Granier
C
C
Chávez-Olórtegui
C
C
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D001921:Brain / Q000166:cytology; D045744:Cell Line, Tumor; D005591:Chemical Fractionation; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000235:genetics; D005976:Glutaral; D006825:Hybridomas / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D012604:Scorpion Venoms / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D012710:Serum Albumin, Bovine; D013154:Spleen / Q000166:cytology; D013574:Synaptosomes / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D012604:Scorpion Venoms; D012710:Serum Albumin, Bovine; D005976:Glutaral