Guesdon J L, Chevrier D, Fadel R, Avrameas S
Laboratoire des Sondes Froides, Institut Pasteur, Paris.
Allerg Immunol (Paris). 1988 Nov;20(9):336-8, 340-2.
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)
为了制备抗组胺单克隆抗体，通过1,4 - 苯醌法用与牛血清白蛋白（BSA）偶联的组胺对小鼠进行免疫。在最初使用卵清蛋白（OVA）和组胺 - OVA偶联物作为抗原筛选以鉴定分泌单克隆抗体的克隆后，通过有限稀释克隆分离杂交瘤并在腹水中培养。使用直接酶免疫测定和ELISA抑制试验研究所选单克隆抗体（D22）的特异性。D22抗体与通过1,4 - 苯醌偶联程序制备的组胺 - 蛋白质偶联物反应。相反，该抗体与天然蛋白质、经1,4 - 苯醌处理的蛋白质或各种胺 - 蛋白质偶联物无反应。游离未偶联的组胺显著抑制抗体与组胺 - OVA的结合，在5×10⁻³ M时记录到50%抑制（IC50）。与组胺 - 苯醌衍生物（IC50 = 2×10⁻⁸ M）和组胺 - OVA（IC50 = 7×10⁻¹⁰ M）相比，基于组胺摩尔浓度，游离组胺的抑制效力要低得多。我们的解释是，对于D22抗体，主要表位包含2 - 组胺基 - 1,4 - 苯醌部分，这表明多价配体对于IgG抗体有效二价结合的重要性。使用D22抗体，我们建立了一种竞争性酶免疫测定法来测量各种生物样品中的组胺。在该测定中，待定量的组胺通过1,4 - 苯醌进行化学修饰，并使其与组胺 - 过氧化物酶偶联物竞争结合有限量的用于包被微量滴定板孔的单克隆抗体。（摘要截短至250字）
