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.