RESUMEN
Gizzerosine (GZ), a derivative of histamine, is a biogenic amine found in fish meal, and one of the causative agents of black vomit, a poultry disease. We describe here the preparation of anti-idiotype antibodies to the anti-GZ monoclonal antibody (anti-GZ 3H4) and their possible application to an immunoassay. BALB-c mice were immunized with anti-GZ 3H4 antibody coupled to hemocyanin from Concholepas concholepas. Using somatic cell fusion between NSO/2 cells and splenic lymphocytes from the immunized mice, we obtained 34 potential anti-idiotype antibodies. They were characterized by passive agglutination with supernatants from hybridoma cultures and latex particles conjugated to the idiotype. Anti-idiotype antibodies were analyzed by a competitive RIA, to determine their ability to dissociate the interaction between (125)I-GZ and the anti-GZ 3H4-idiotype antibody. They were also characterized by GZ inhibition of latex passive agglutination assay. Three anti-idiotypes named 2D11, 2H6, and 3A12, all of the IgG isotype, were obtained. They were evaluated by a competitive ELISA, in which GZ competes with the tracer (HRP-idiotype). All presented sensitivity in the range of 0.1-10 microg/mL of GZ; and the 3A12 anti-idiotype antibody showed the best performance. An ELISA was developed using the idiotype bound to the solid phase and the anti-idiotype 3A12-HRP as the tracer. The assay showed a similar sensitivity and cross-reactivity with histamine was only observed at concentrations over 10 microg/mL. Lysine and histidine did not interfere with the assay up to 500 microg/mL. An experiment was conducted with fish meal contaminated with synthetic GZ. The results are promising, and showed that no other compounds of the fish meal interfere with the ELISA system; however the extraction procedure of the sample needs to be improved. From the results presented here, we conclude that the idiotype anti-idiotype ELISA would be an appropriate method to determine GZ in fish meal.
Asunto(s)
Alimentación Animal , Anticuerpos Monoclonales/inmunología , Epítopos/inmunología , Peces , Imidazoles/inmunología , Animales , Anticuerpos Antiidiotipos/inmunología , Ensayo de Inmunoadsorción Enzimática , Epítopos/química , Ratones , Ratones Endogámicos BALB CRESUMEN
This study is the first report of the development of monoclonal antibodies (MAbs) against gizzerosine (GZ), one of the causative agents of black vomit, a serious poultry disease. Balb/c mice were immunized with different GZ conjugates; the most immunogenic conjugate in experimental animals was determined by enzyme-linked immunoadsorbent assays (ELISA). Somatic fusions were carried out using splenic lymphocytes from GZ-immune mice and the NSO/2 myeloid cell line. Primary selection of hybridomas secreting antibodies to GZ was done using a direct ELISA, with GZ bound to bovine serum albumin (BSA), GZ directly bound to maleinimide preactivated plates and histamine bound to BSA, a GZ related biogenic amine present in fish meal. Four MAbs--3H4, 3H10, and 5B1 of the IgG1 isotype, and 8G7 of the Ig2a isotype-were specific to GZ and did not cross-react with histamine. Only monoclonals 3H4 and 8G7 bound GZ in solution by means of a competitive ELISA. Finally, to determine the performance of the competitive ELISA developed with the MAbs, experiments were conducted with GZ in solution (0 to 10 microg/ml) and with GZ labeled with horseradish peroxidase (HRP) as the tracer; the antibody complex was captured by using rabbit anti-mouse IgG preactivated ELISA plates. These experiments showed that monoclonal anti-GZ-3H4 generates a more sensitive assay close to linearity in the range about of 0.1 to 10 microg/ml of GZ. No cross-reaction was observed with histamine, histidine, or lysine at all concentrations tested.
Asunto(s)
Aminas Biogénicas/análisis , Ensayo de Inmunoadsorción Enzimática/métodos , Imidazoles/análisis , Alimentación Animal/análisis , Anticuerpos Monoclonales , Aminas Biogénicas/inmunología , Aminas Biogénicas/toxicidad , Harina de Pescado/análisis , Hibridomas , Imidazoles/inmunología , Imidazoles/toxicidad , Toxinas Biológicas/análisisRESUMEN
A homologous RIA for the determination of toxic gizzerosine activity in commercial fish meals has been developed. Three polyclonal antibodies (GR316, GR415, and GR418) were produced in female rabbits and extensively characterized for their potential use in individual RIA. The RIA had lower detection limits of 0.048, 0.78, and 0.39 ng/mg using the three respective antibodies. Because gizzerosine is derived from lysine and histidine, crossreactivity with these amino acids, and with histamine was examined. The antibodies crossreacted with histamine from 21 to 100%. No crossreactivity with histidine or lysine was observed for any of the three antibodies. Antibody GR415 was chosen for determination of gizzerosine in extracted fish meal samples because crossreactivity of histamine using this antibody was only present at high concentrations, and the Ka value for gizzerosine was 10-fold greater than for histamine. A mild buffer extraction procedure was used, resulting in 98% gizzerosine recovery. Displacement curves from extracted and serially diluted fish meal samples were parallel with gizzerosine standard. Inter-and intra-assay coefficients of variation were 11 and 15%, respectively. We used the RIA for determination of gizzerosine activity in a pool of 23 fish meal samples of known gizzerosine scores (determined with a chick bioassay), and histamine content. The partial correlation coefficient between gizzerosine content determined by the RIA and gizzerosine scores from the bioassay was high (0.83), and significant (P < 0.01). There were also significant correlations between gizzerosine scores and histamine content of the fish meals (0.63, P < 0.01), and between histamine content and gizzerosine levels determined by the RIA (0.59, P < 0.01). The application of the homologous RIA for the determination of gizzerosine activity in commercial fish meals could be of importance for the prevention of gizzard erosions in the poultry industry, and for studying gizzerosine-induced pathology and metabolism.