RESUMO
We report the production of a neutralizing monoclonal antibody able to recognize the venoms of three major medically important species of Loxosceles spiders in Brazil. The mAb was produced by immunization of mice with a toxic recombinant L. intermedia sphingomyelinase D {SMases D isoform (rLiD1)} [1] and screened by enzyme-linked immunosorbent assay (ELISA) using L. intermedia, L. laeta and L. gaucho venoms as antigens. One clone (LiD1mAb16) out of seventeen anti-rLiD1 hybridomas was cross-reactive with the three whole Loxosceles venoms. 2D Western blot analysis indicated that LiD1mAb16 was capable of interacting with 34 proteins of 29-36kDa in L. intermedia, 33 in L. gaucho and 27 in L. laeta venoms. The results of immunoassays with cellulose-bound peptides revealed that the LiD1mAb16 recognizes a highly conserved linear epitope localized in the catalytic region of SMases D toxins. The selected mAb displayed in vivo protective activity in rabbits after challenge with rLiD1. These results show the potential usefulness of monoclonal antibodies for future therapeutic approaches and also opens up the perspective of utilization of these antibodies for immunodiagnostic assays in loxoscelism.
Assuntos
Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Diester Fosfórico Hidrolases/imunologia , Venenos de Aranha/enzimologia , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/imunologia , Reações Cruzadas , Mapeamento de Epitopos , Hibridomas , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Dados de Sequência Molecular , Testes de Neutralização , Coelhos , Proteínas Recombinantes/imunologia , Venenos de Aranha/imunologia , Aranhas/enzimologiaRESUMO
The venom of Loxosceles intermedia (Li) spiders is responsible for cutaneous lesions and other clinical manifestations. We previously reported that the monoclonal antibody LimAb7 can neutralize the dermonecrotic activity of crude Li venom. In this study, we observed that this antibody recognizes several proteins from the venom dermonecrotic fraction (DNF), including LiD1. Identifying the epitope of such a neutralizing antibody could help designing immunogens for producing therapeutic sera or vaccination approaches. To this aim, two sets of 25- and 15-mer overlapping peptides that cover the complete amino acid sequence of LiD1 were synthesized using the SPOT technique. None of them was recognized by LimAb7, suggesting that the epitope is discontinuous. Then, the screening of four peptide phage-display libraries yielded four possible epitope mimics that, however, did not show any obvious similarity with the LiD1 sequence. These mimotopes, together with a 3D model of LiD1, were used to predict with the MIMOP bioinformatic tool the putative epitope region (residues C197, Y224, W225, T226, D228, K229, R230, T232 and Y248 of LiD1) recognized by LimAb7. This analysis and the results of alanine-scanning experiments highlighted a few residues (such as W225 and D228) that are found in the active site of different SMases D and that may be important for LiD1 enzymatic activity. Finally, the only mimotope NCNKNDHLFACW that interacts with LimAb7 by SPOT and its analog NSNKNDHLFASW were used as immunogens in rabbits. The resulting antibodies could neutralize some of the biological effects induced by crude Li venom, demonstrating a mimotope-induced protection against L. intermedia venom.
Assuntos
Anticorpos Neutralizantes/sangue , Antitoxinas/sangue , Aracnídeos , Epitopos/imunologia , Venenos de Aranha/antagonistas & inibidores , Vacinas de Subunidades Antigênicas/imunologia , Animais , Mapeamento de Epitopos , Feminino , Biblioteca de Peptídeos , Perciformes , Coelhos , Venenos de Aranha/toxicidadeRESUMO
Mutalysin-II (mut-II) from Lachesis muta snake venom is an endopeptidase with hemorrhagic activity. A mAb against mutalysin-II that neutralized the hemorrhagic effect was produced previously. To identify the mAb epitopes, sets of 15-mer overlapping peptides covering the mut-II amino acid sequence were synthesized using the SPOT method and tested but failed to react with the mAb. Using a phage-display approach seventeen clones reactive with mAb were identified. Additional immunoassays with the peptides and mAb identified the QCTMDQGRLRCR, TCATDQGRLRCT, HCFHDQGRVRCA, HCTMDQGRLRCR and SCMLDQGRSRCR sequences as possible epitopes. Immunization of rabbits with these peptides induced antibodies that recognize mut-II and protected against the hemorrhagic effects of Lachesis venom.
Assuntos
Anticorpos Monoclonais/imunologia , Venenos de Crotalídeos/imunologia , Hemorragia/prevenção & controle , Metaloendopeptidases/imunologia , Peptídeos/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/metabolismo , Epitopos , Feminino , Hemorragia/imunologia , Metaloendopeptidases/metabolismo , Dados de Sequência Molecular , Biblioteca de Peptídeos , Peptídeos/metabolismo , Coelhos , VacinaçãoRESUMO
Antibodies raised against recombinant Loxosceles intermedia dermonecrotic protein isoform 1 (rLiD1) display neutralizing capacity for the L. intermedia whole venom. We previously found that an immunodominant continuous B-cell epitope, recognized by these antibodies corresponds to a region of the protein known to be involved in the active site. In this study, we extend previous work by preparing a 27-residue synthetic replica of this epitope ((25)NLGANSIETDVSFDDNANPEYTYHGIP(51)) and using it as an immunogen in mice and rabbits. The immunization process induced antibodies that protected mice from a lethal dose of L. intermedia crude venom and rabbits against the dermonecrotic effects of rLiD1. An Ala scan of the epitope indicated that 4 residues, E44, Y45, T46 and Y47, are essential (over 70% decrease in binding upon replacement with alanine) for antibody recognition. The possible mechanisms of neutralization are discussed in light of these findings.
Assuntos
Antígenos/química , Antígenos/imunologia , Antivenenos/farmacologia , Hemorragia/induzido quimicamente , Fragmentos de Peptídeos/imunologia , Venenos de Aranha/imunologia , Venenos de Aranha/toxicidade , Sequência de Aminoácidos , Animais , Antivenenos/biossíntese , Edema/induzido quimicamente , Edema/patologia , Ensaio de Imunoadsorção Enzimática , Epitopos , Feminino , Hemorragia/sangue , Esquemas de Imunização , Dose Letal Mediana , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Necrose/induzido quimicamente , Necrose/patologia , Testes de Neutralização , Coelhos , Proteínas Recombinantes , Dermatopatias/induzido quimicamente , Dermatopatias/patologia , Venenos de Aranha/antagonistas & inibidoresRESUMO
The usefulness of a synthetic peptide in the serodiagnosis of Taenia solium human neurocysticercosis (NC) has been evaluated. Phage-displayed peptides were screened with human antibodies to scolex protein antigen from cysticercus cellulosae (SPACc). One clone was found to interact specifically with anti-SPACc IgGs. The corresponding synthetic peptide was found to be recognized in ELISA by NC patient's sera. The study was carried out with sera from 28 confirmed NC patients, 13 control sera and 73 sera from patients suffering from other infectious diseases. A 93% sensibility and a 94.3% specificity was achieved. Figures of 89% and 31.4% of sensibility and specificity were obtained in a SPACc-based ELISA. Immunoblotting of SPACc with anti-peptide antibodies revealed a single band of approximately 45 kDa in 1D and four 45 kDa isoforms in 2D-gel electrophoresis. A strong and specific immunostaining in the fibers beneath the suckers, at the base of the rostellum, and in the tissue surrounding the scolex of cysticerci was observed by immunomicroscopy. Our results show that a peptide-based immunodiagnostic of neurocisticercosis can be envisioned.
Assuntos
Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/imunologia , Testes Imunológicos/métodos , Neurocisticercose/imunologia , Peptídeos/imunologia , Taenia solium/imunologia , Animais , Anticorpos Anti-Helmínticos/imunologia , Especificidade de Anticorpos , DNA de Helmintos/química , DNA de Helmintos/genética , Eletroforese em Gel Bidimensional , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Neurocisticercose/sangue , Neurocisticercose/diagnóstico , Neurocisticercose/parasitologia , Biblioteca de Peptídeos , Reação em Cadeia da Polimerase , Taenia solium/isolamento & purificaçãoRESUMO
Antibodies (Abs) that block factor VIII (FVIII) activity occur in hemophilia A patients treated with FVIII replacement therapy and severely impair treatment. In this work, we designed and synthesized ten peptides whose sequences are found in putative epitopes at the surface of a1 and C2 domains of the FVIII molecule. These peptides were screened for their ability to inhibit the binding of anti-FVIII Abs from plasmas of hemophilia A patients to FVIII. All peptides were efficient in inhibiting anti-FVIII Abs in plasma from patients with inhibitors, with however different efficiencies. It was found that each tested patient's plasma had a different profile of reactivity with peptides, consistent with an individual anti-FVIII Ab specificity. The profile of recognized peptides was also changing during the treatment of the patients. Three peptides were used in an affinity chromatography assay to attempt to remove anti-FVIII Abs from patients' plasma. Anti-FVIII IgGs were significantly captured by the peptide-Sepharose affinity matrixes as assessed by enzyme-linked immunosorbent assay. However, due to the low level of Abs in the plasma samples, other methods (Chromogenic and Bethesda assays) were not sensitive enough to properly detect the reduction of inhibitors.
Assuntos
Autoanticorpos/metabolismo , Epitopos/metabolismo , Fator VIII/imunologia , Hemofilia A/imunologia , Fragmentos de Peptídeos/metabolismo , Reações Antígeno-Anticorpo , Ligação Competitiva , Cromatografia de Afinidade/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Epitopos Imunodominantes/metabolismo , Masculino , Fragmentos de Peptídeos/imunologiaRESUMO
We isolated cDNA sequences coding for dermonecrotic/sphingomyelinases factor proteins from the brown spider Loxosceles intermedia, here named Loxtox proteins. The amino acid sequences based on cloned cDNA of several Loxtox proteins revealed at least six distinct groups of proteins expressed in the venom gland. The level of similarity among the toxins varied from 99% to 55%. The finding of several isoforms of Loxtox in the venom of this spider may reflect an evolutionary adaptation for different prey types and reinforces the idea of an efficient mutational mechanism in the venom gland of spiders.
Assuntos
Diester Fosfórico Hidrolases/química , Esfingomielina Fosfodiesterase/metabolismo , Venenos de Aranha/química , Aranhas/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Regulação da Expressão Gênica , Dados de Sequência Molecular , Diester Fosfórico Hidrolases/metabolismo , Filogenia , RNA Mensageiro/genética , Esfingomielina Fosfodiesterase/genética , Venenos de Aranha/metabolismoRESUMO
Mutalysin II (mut-II), a 22.5kDa zinc endopeptidase isolated from bushmaster (Lachesis muta muta) snake venom, is a direct acting fibrin(ogen)olytic proteinase. It induces monoclonal and polyclonal antibodies which efficiently neutralize the hemorrhagic effect of L. muta and several Bothrops whole venoms. To characterize epitopes of protective antibodies we have used the Spot method of multiple peptide synthesis to prepare 64 overlapping dodecapeptides frameshifted by three residues, covering the complete amino acid sequence of mut-II. The rabbit anti-mut-II antibodies binding pattern to peptides revealed several continuous antigenic regions: one in the N-terminal part, two in the central region and the other in the C-terminal of mut-II. By using homology modelling, a three-dimensional model of mut-II was built which showed that epitopes are surface exposed. Anti-peptide antibodies were raised against three peptides (one representative of each epitope region) covalently coupled as a mixture to keyhole limpet hemocyanin. Purified IgG from the resulting anti- peptide antibodies cross-reacted with mut-II and induced a dose-dependent inhibition of the mut-II catalyzed proteolysis of fibrinogen.
Assuntos
Antivenenos/farmacologia , Epitopos/imunologia , Metaloendopeptidases/antagonistas & inibidores , Venenos de Víboras/antagonistas & inibidores , Animais , Epitopos/química , Feminino , Imunoglobulina G/farmacologia , Metaloendopeptidases/química , Metaloendopeptidases/imunologia , Modelos Moleculares , Estrutura Terciária de Proteína , Coelhos , Venenos de Víboras/química , Venenos de Víboras/imunologiaRESUMO
In the present study the recombinant form (recLiD1) of a dermonecrotic protein present in the Brazilian brown spider Loxosceles intermedia venom was expressed in Escherichia coli cells and purified by reversed-phase HPLC using a C8 Vydac column. About 25.8mg of purified recLiD1 was produced from a litre of bacterial culture. SDS/PAGE and immunoblot analysis of the recombinant protein revealed an apparent molecular weight of 32-35kDa. The later result was confirmed by mass spectrometry (32,758Da). recLiD1 displayed dermonecrotic and platelet aggregation activities which were qualitatively similar to that displayed by the crude L. intermedia venom. However, very low sphingomyelinase D enzymatic activity and complement-dependent haemolytic activities were observed. recLiD1 immunized BALB/c mice developed an antibody response. Anti-recLiD1 antibodies recognized L. intermedia venom in an indirect enzyme-linked immunosorbent assay (ELISA) and cross-reacted with crude venoms from L. intermedia, L. gaucho and L. laeta. An in vivo protection assay carried out 5 weeks after the end of the immunization protocol showed that 75% of the vaccinated mice could resist the challenge by 2.5LD(50) of L. intermedia venom. To characterize epitopes associated with protective antibodies, we prepare sets of immobilized synthetic 15 mer overlapping peptides covering the complete amino acid sequences of the recLiD1. Antibodies revealed one antigenic region in the N-terminal part of the toxin. The amino acid sequence of this epitope was found in several dermonecrotic proteins and some of its residues have been implicated with the active site of the toxin.
Assuntos
Diester Fosfórico Hidrolases/toxicidade , Proteínas Recombinantes/toxicidade , Serina Endopeptidases/toxicidade , Pele/efeitos dos fármacos , Venenos de Aranha/toxicidade , Animais , Epitopos/química , Hemólise/efeitos dos fármacos , Humanos , Técnicas In Vitro , Espectrometria de Massas , Camundongos , Peso Molecular , Necrose/induzido quimicamente , Necrose/patologia , Diester Fosfórico Hidrolases/análise , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/imunologia , Agregação Plaquetária/efeitos dos fármacos , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Serina Endopeptidases/química , Serina Endopeptidases/imunologia , Pele/patologia , Venenos de Aranha/química , Venenos de Aranha/imunologiaRESUMO
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.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Hibridomas/metabolismo , Modelos Moleculares , Venenos de Escorpião/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/metabolismo , Encéfalo/citologia , Linhagem Celular Tumoral , Fracionamento Químico , Ensaio de Imunoadsorção Enzimática , Epitopos/genética , Glutaral , Hibridomas/química , Camundongos , Dados de Sequência Molecular , Testes de Neutralização , Venenos de Escorpião/genética , Venenos de Escorpião/metabolismo , Soroalbumina Bovina , Baço/citologia , Sinaptossomos/metabolismoRESUMO
Overlapping pentadecapeptides covering the complete amino acid sequence of TsII, TsVII and TsIV toxins from the venom of scorpion Tityus serrulatus (Ts), were prepared by use of the Spot method of multiple peptide synthesis. Horse anti-Ts antisera for therapeutic use were tested for their binding to peptides. All nine antisera tested showed reactivity with several peptides from the three toxins. Three antigenic regions, one in the very N-terminal, the second in the central part and the other in the C-terminal part of the three toxins were frequently, but not constantly recognized, with an intensity that seemed to be related to the neutralizing potency of the tested antivenom. Thus the corresponding peptides (residues 1-15 and 48-62 of TsII; residues 1-15, 16-30 and 48-62 of TsIV and residues 1-15 and 47-61 of TsVII) were synthesized, coupled to KLH and used as antigens to coat the microtitration plates to determine any relationship between their ELISA reactivity with therapeutic horse antivenoms and the neutralizing potential of these antivenoms. The mixture of the N-terminal peptide of TsII, of the N-terminal TsVII peptide and of the C-terminal of TsIV was found to give a linear relationship with the neutralizing titer of horse serum of low neutralizing potency (< or =1 mg/ml). However, high neutralizing antivenoms did not show the expected response in peptide ELISA. This observation is discussed in the context of the occurrence of continuous and discontinuous epitopes on toxins.
Assuntos
Epitopos/genética , Soros Imunes/imunologia , Soros Imunes/metabolismo , Peptídeos/metabolismo , Venenos de Escorpião/genética , Escorpiões/química , Sequência de Aminoácidos , Animais , Ensaio de Imunoadsorção Enzimática , Cavalos/sangue , Soros Imunes/genética , Imunoensaio , Dados de Sequência Molecular , Testes de Neutralização , Peptídeos/genética , Venenos de Escorpião/imunologia , Escorpiões/genéticaRESUMO
In the present investigation we used native and recombinant TsNTxP to elicit antibodies in three different animal models (mouse, rabbit and sheep). Differences among anti-TsNTxP antibodies were analyzed using sets of overlapping pentadecapeptides of the TsNTxP amino acid sequence and also modified peptides to reveal key residues in antibody-peptide binding. Despite the identification of similar peptides by the antibodies in the C and N-terminal, peculiarities of each system were observed including the level of reactivity and also the number and type of key residues in the continuous epitopes of TsNTxP. In addition, in vitro neutralization assays indicated that sheep are an alternative and efficient model for the production of anti-Tityus serrulatus venom.
Assuntos
Mapeamento de Epitopos , Venenos de Escorpião/metabolismo , Escorpiões , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Sítios de Ligação de Anticorpos , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Testes de Neutralização , Peptídeos/genética , Coelhos , Venenos de Escorpião/imunologia , OvinosRESUMO
The possibility of raising a humoral immune response capable of inducing in vivo protection against the lethal effects of Tityus serrulatus (Ts) scorpion venom was evaluated in the mouse model. An immunogen was prepared that consists of a toxic fraction (TstFG(50)) of the Tityus venom (this G(50) chromatography fraction represents most of the toxicity of the crude venom) conjugated to bovine serum albumin (BSA) with glutaraldehyde. TstFG(50) coupled to BSA yielded a thoroughly detoxified immunogen. BALB/c and C57BL/10 mice were immunized with this preparation and all developed an antibody response. In vivo protection assays one week after the last immunization showed that vaccinated mice could resist the challenge by twice the LD(50) of the TstFG(50), a dose which killed all control non-immune mice. The protective effect persisted nine weeks after the end of the immunization protocol. To characterize epitopes of protective antibodies we used the Spot method of multiple peptide synthesis to prepare sets of immobilized 15 mer overlapping peptides, covering the complete amino acid sequences of the main Tityus toxins, TsII and TsVII (both beta-type toxins) and TsIV, an alpha-type toxin that is the major lethal component of the venom. Antibody binding to peptides, revealed one major antigenic region in the C-terminal part of the three toxins and another region in the helical part of TsII and TsIV toxins. It is likely that these epitopes correspond to neutralizing epitopes since they correspond to regions of the toxins that are known to be involved in the active site of the toxins.
Assuntos
Anticorpos/genética , Formação de Anticorpos/imunologia , Imunização , Camundongos/imunologia , Venenos de Escorpião/imunologia , Escorpiões/metabolismo , Sequência de Aminoácidos , Animais , Ensaio de Imunoadsorção Enzimática , Epitopos/genética , Glutaral/metabolismo , Imunoensaio , Dose Letal Mediana , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Modelos Moleculares , Dados de Sequência Molecular , Venenos de Escorpião/metabolismo , Venenos de Escorpião/toxicidade , Alinhamento de Sequência , Soroalbumina Bovina/metabolismo , Testes de Toxicidade Aguda , Vacinas Sintéticas/imunologiaRESUMO
We have used the Spot method of multiple peptide synthesis to prepare sets of immobilized overlapping peptides of uniform size (15 mer), covering the complete amino acid sequences of TsNTxP a non-toxic and immunogenic protein and TsIV, an alpha-type toxin that is the major lethal component of the venom of scorpion Tityus serrulatus. Anti-TsNTxP antibodies binding to peptides, revealed three antigenic regions, one in the N-terminal, the second in the central part and the other in the C-terminal part of TsNTxP. One peptide epitope in the C-terminal part of TsIV was identified with anti-TsIV neutralizing rabbit antibodies. Anti-peptide antibodies were raised against these four peptides all together covalently coupled to keyhole limpet hemocyanin (KLH) and found to neutralize in vitro the toxic effects of the T. serrulatus venom. Quantities of venom equivalent to 13.5 LD(50) were effectively neutralized by 1ml of the anti-peptide serum. The antigenic specificities of the anti-peptides were compared by an indirect enzyme-linked immunosorbent assay (ELISA) using synthetic peptides and crude venoms from T. serrulatus, T. bahiensis, T. cambridgei, T. stigmurus, Androctonus autralis Hector and Centruroides sculpturatus to coat the microtitration plates. The anti-peptide antibodies had a comparable high reactivity with the crude venom of T. serrulatus, moderate binding to T. bahiensis, T. cambridgei, T. stigmurus and Centruroides sculpturatus venoms but were unable to recognize the venom of Androctonus autralis Hector. These results show that by using peptides derived from the sequence of scorpion toxins, the generation of anti-peptide antibodies able to neutralize the cognate venom appears to be an alternative strategy for the easy preparation of antivenoms.
Assuntos
Venenos de Escorpião/imunologia , Escorpiões , Vacinação , Sequência de Aminoácidos , Animais , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Epitopos/imunologia , Feminino , Dose Letal Mediana , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Testes de Neutralização , Peptídeos/síntese química , Peptídeos/imunologia , Coelhos , Venenos de Escorpião/farmacologiaRESUMO
As bites by Bothrops atrox and Lachesis muta muta snakes are frequent in the north of Brazil and elicit similar clinical symptoms, an ELISA assay was developed to identify objectively the circulating antigen in the serum of accidentally bitten patients. Antigens common to the two venoms were removed by immunoaffinity techniques and the 'individual component' of each venom used as immunogen to raise rabbit IgGs. Each of these antibodies specifically recognized one venom, and they were used to set up a sandwich-type ELISA. The specificity of the assay was demonstrated by its capacity to identify correctly the circulating antigen in mice experimentally poisoned with either of the two venoms. The ELISA was further used to identify the circulating antigen in the sera of humans bitten by B. atrox or L. muta muta and to follow the kinetics distribution of antigens in experimentally envenomed mice or accidentally bitten patients. The assay is specific, and could therefore be valuable both to clinicians and to epidemiologists.
Assuntos
Antígenos/análise , Venenos de Crotalídeos/análise , Mordeduras de Serpentes/diagnóstico , Animais , Especificidade de Anticorpos , Antivenenos/uso terapêutico , Reações Cruzadas , Venenos de Crotalídeos/imunologia , Venenos de Crotalídeos/toxicidade , Ensaio de Imunoadsorção Enzimática , Feminino , Peroxidase do Rábano Silvestre/análise , Humanos , Imunoglobulina G/análise , Masculino , Camundongos , Coelhos , Mordeduras de Serpentes/imunologia , Mordeduras de Serpentes/terapiaRESUMO
The antigenic properties of alpha-type and beta-type toxins purified from Tityus serrulatus (Ts) venom were analysed by radioimmunoassay, using rabbit antibodies raised against Ts VII, the main beta-type toxin in the venom, and against Ts IV, an alpha-type toxin. The anti-Ts VII serum did not recognize either the other beta-toxins Ts I and Ts II or the alpha-toxin Ts IV; the anti-Ts IV serum did not bind any of the three beta-toxins Ts I, Ts II or Ts VII. Thus, Tityus toxins display at least three distinct antigenic reactivity patterns.
Assuntos
Antígenos/imunologia , Venenos de Escorpião/imunologia , Ligação Competitiva , Reações Cruzadas , RadioimunoensaioRESUMO
The possibility of raising a humoral immune response able to induce protection from the lethal effects of scorpion toxins was evaluated in the mouse model. A toxic fraction from the venom of the scorpion Tityus serrulatus was entrapped in sphingomyelin-cholesterol liposomes which yielded a conveniently detoxified immunogen. After three injections of this immunogen, all but three of a group of 18 mice developed an IgG response which was shown to be both specific and of good affinity for the toxic antigen. In vitro neutralization assays indicated that pre-incubation of a lethal dose of the toxic fraction with immune sera strongly diminished its toxicity. In vivo protection assays showed that mice with the highest levels of circulating anti-toxin antibodies could resist the challenge by double the normal LD50 of the toxic fraction, which killed all control non-immune mice. The protection was, however, found to be limited both in its duration and its effectiveness against higher amounts of toxin.