[Immunoenzyme assay for histamine].

Abstract

In order to produce monoclonal antibody to histamine, mice are immunized with histamine conjugated to bovine serum albumin (BSA) by the 1,4-benzoquinone method. After an initial screening using ovalbumin (OVA) and histamine-OVA conjugate as antigens to identify monoclonal antibody secreting clones, the hybridomas are isolated by limiting dilution cloning and grown in ascites. The specificity of selected monoclonal antibody (D22) is studied using a direct enzyme immunoassay and an ELISA-inhibition test. D22 antibody reacts with histamine-protein conjugates prepared by the 1,4-benzoquinone coupling procedure. On the contrary, this antibody is unreactive with native proteins, 1,4-benzoquinone treated proteins or various amine-protein conjugates. Free unconjugated histamine significantly inhibits antibody binding to histamine-OVA and 50% inhibition (IC50) is recorded at 5 X 10(-3) M. On a histamine molar concentration basis a much more lower inhibitory potency of free histamine is recorded, as compared to histamine-benzoquinone derivative (IC50 = 2 X 10(-8) M) and to histamine-OVA (IC50 = 7 X 10(-10) M). It is our interpretation that for the D22 antibody, the main epitope encompasses the 2-histaminyl-1,4-benzoquinone moiety and that this points to the importance of polyvalent ligands for efficient bivalent binding of the IgG antibodies. Using the D22 antibody we set up a competitive enzyme immunoassay for measuring histamine in various biological samples. In this assay, the histamine to be quantified is chemically modified by 1,4-benzoquinone and allowed to compete with a histamine-peroxidase conjugate for binding to a limited amount of monoclonal antibody which is used to coat the wells of a microtitration plate.(ABSTRACT TRUNCATED AT 250 WORDS)