Production of monoclonal antibodies capable of neutralizing dermonecrotic activity of Loxosceles intermedia spider venom and their use in a specific immunometric assay.
We have produced 13 mAbs for Loxosceles intermedia crude venom. Twelve were reactive against proteins of 32-35 kDa and one of these Li mAb(7) showed high neutralizing potency for the dermonecrotic activity of L. intermedia venom. This Li mAb(7) showed no cross-reactivity, with Loxosceles laeta (Brazil), L. laeta (Perú) and Loxosceles gaucho venoms. The mAbs were produced by immunization with the crude venom and screened by enzyme-linked immunosorbent assay (ELISA) using L. intermedia whole venom or dermonecrotic fraction (DNF) as antigens coated onto microtitre plates. A sensitive two-site immunometric assay was designed and shown to be useful for identifying and quantifying DNF from L. intermedia in biological samples. The Li mAb(7) coated onto microtitre plates and hyperimmune horse anti-L. intermedia IgGs prepared by immunoaffinity chromatography and conjugated to horseradish peroxidase were used to set up a sandwich-type ELISA. Measurable absorbance signals were obtained with 0.2 ng of L. intermedia crude venom per assay.
Alvarenga
Larissa M
LM
Departamentos de Bioquímica-Imunologia, ICB, Universidade Federal de Minas Gerais, CEP 31270901, Belo Horizonte-MG, Brazil.
Martins
Maria S
MS
Moura
Juliana F
JF
Kalapothakis
Evanguedes
E
Oliveira
Júlio C
JC
Mangili
Oldemir C
OC
Granier
Claude
C
Chávez-Olórtegui
Carlos
C
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology*; D000918:Antibody Specificity; D000997:Antivenins / Q000737:chemistry / Q000494:pharmacology*; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D009500:Neutralization Tests; D011817:Rabbits; D001098:Spider Bites / Q000097:blood / Q000276:immunology; D013111:Spider Venoms / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000276:immunology*; D013112:Spiders
D000911:Antibodies, Monoclonal; D000997:Antivenins; D013111:Spider Venoms