Bryant R E, Chamovitz B N, Morse S A, Apicella M A, Morthland V H
J Clin Microbiol. 1983 Jun;17(6):1050-3. doi: 10.1128/jcm.17.6.1050-1053.1983.
The specificity of the enzyme-linked immunosorbent assay(s) is thought to depend on the specificity of the antibody used in the assay system. Therefore, the association of broadly reactive antigens like endotoxin with enzyme conjugates or other enzyme-linked immunosorbent assay reagents has the potential of altering the specificity of reactions in the enzyme-linked immunosorbent assay. Using the Limulus amoebocyte lysate assay, we demonstrated that commercially prepared conjugates of goat anti-human immunoglobulin G peroxidase, goat anti-rabbit immunoglobulin G alkaline phosphatase, rabbit anti-human immunoglobulin G, and other enzyme conjugates contained endotoxin. Furthermore, the staphylococcal protein A, horseradish peroxidase, and bovine alkaline phosphatase used to prepare enzyme conjugates also contained endotoxin. Commercially obtained bovine alkaline phosphatase contained as much as 1.0 microgram of endotoxin per ml of enzyme solution. Both commercially prepared enzyme conjugates and those prepared by us contained endotoxin as determined by their absorption to immobilized monoclonal antibody to lipid A or to immobilized Limulus amoebocyte lysate. The results of this study further suggest that the endotoxin was associated with the enzyme component of the conjugate. In a competitive inhibition enzyme immunoassay, 10 micrograms of lipid A per ml inhibited binding of the enzyme conjugate to adsorbed Limulus amoebocyte lysate, thereby confirming that endotoxin mediated the binding of the conjugate in that system. The potential significance of endotoxin bound to enzyme conjugates may be far reaching because of the ubiquity of endotoxin in conjugates and the prevalence of antibodies to endotoxin in mammalian serum.
酶联免疫吸附测定法的特异性被认为取决于测定系统中所用抗体的特异性。因此，诸如内毒素等具有广泛反应性的抗原与酶结合物或其他酶联免疫吸附测定试剂的结合，有可能改变酶联免疫吸附测定中反应的特异性。通过使用鲎试剂法，我们证明了市售的山羊抗人免疫球蛋白G过氧化物酶结合物、山羊抗兔免疫球蛋白G碱性磷酸酶结合物、兔抗人免疫球蛋白G以及其他酶结合物均含有内毒素。此外，用于制备酶结合物的葡萄球菌蛋白A、辣根过氧化物酶和牛碱性磷酸酶也含有内毒素。市售的牛碱性磷酸酶每毫升酶溶液中含多达1.0微克内毒素。通过其与固定化的抗脂质A单克隆抗体或固定化的鲎试剂的吸附作用测定，无论是市售的酶结合物还是我们制备的酶结合物均含有内毒素。本研究结果进一步表明内毒素与结合物的酶成分有关。在竞争性抑制酶免疫测定中，每毫升10微克脂质A可抑制酶结合物与吸附的鲎试剂的结合，从而证实内毒素在该系统中介导了结合物的结合。由于内毒素在结合物中普遍存在且哺乳动物血清中存在抗内毒素抗体，与酶结合物结合的内毒素的潜在意义可能影响深远。
