Jakobson E, Axelsson B, Paulie S
Department of Immunology, Stockholm University, Sweden.
J Immunol Methods. 1992 Jul 31;152(1):49-57. doi: 10.1016/0022-1759(92)90088-b.
Some antibodies directed to cell surface receptors may mimic the physiological ligands by inducing the transmission of activation or growth signals. Such agonistic antibodies have proven very useful when studying functional properties of various receptor molecules on, e.g., lymphoid cells. However, while investigating the agonistic effects on tonsillar B cells of the anti-CD43 monoclonal antibody (mAb) D4B11 and the anti-CD40 mAb S2C6, we made some observations which emphasize the need for caution when using antibodies purified by protein A affinity chromatography. Both antibody preparations were found to elicit changes in the intracellular free calcium concentration ([Ca2+]i) as well as promoting proliferation of phorbol ester activated cells. However, a closer analysis showed that the increase in [Ca2+]i could be attributed to soluble staphylococcal protein A (SpA) desorbed during antibody purification. By using pure soluble SpA, we were able to show that nanogram amounts were sufficient to increase [Ca2+]i by a mechanism that involved both a mobilization from intracellular stores and an influx across the B cell membrane. A similar effect on cytosolic Ca2+ in B cells was also noted for streptococcal protein G (protein G), another bacterial component used for antibody purification. However, in contrast to SpA, protein G had little effect on cell proliferation. These observations suggest that the presence of trace amounts of SpA or protein G in antibodies purified on these bacterial components may lead to incorrect interpretations of the agonistic properties of such antibodies. When the above findings were taken into account, it was found that the CD43 mAb D4B11, like the CD40 mAb S2C6, stimulated growth of B cells without causing any measurable increase in [Ca2+]i. Both CD40 and CD43 may thus be coupled to signalling pathways which do not involve breakdown of membrane phosphoinositides.
一些针对细胞表面受体的抗体可通过诱导激活或生长信号的传递来模拟生理配体。事实证明，此类激动性抗体在研究例如淋巴细胞上各种受体分子的功能特性时非常有用。然而，在研究抗CD43单克隆抗体（mAb）D4B11和抗CD40 mAb S2C6对扁桃体B细胞的激动作用时，我们有一些观察结果，强调了在使用通过蛋白A亲和层析纯化的抗体时需要谨慎。发现这两种抗体制剂均可引起细胞内游离钙浓度（[Ca2+]i）的变化，并促进佛波酯激活细胞的增殖。然而，进一步分析表明，[Ca2+]i的增加可归因于抗体纯化过程中解吸的可溶性葡萄球菌蛋白A（SpA）。通过使用纯可溶性SpA，我们能够表明纳克量就足以通过一种涉及从细胞内储存库动员和跨B细胞膜内流的机制来增加[Ca2+]i。链球菌蛋白G（蛋白G）是另一种用于抗体纯化的细菌成分，对B细胞胞质Ca2+也有类似作用。然而，与SpA不同，蛋白G对细胞增殖几乎没有影响。这些观察结果表明，在这些细菌成分上纯化的抗体中存在痕量的SpA或蛋白G可能导致对此类抗体激动特性的错误解释。考虑到上述发现，发现CD43 mAb D4B11与CD40 mAb S2C6一样，可刺激B细胞生长，而不会引起[Ca2+]i有任何可测量的增加。因此，CD40和CD43可能都与不涉及膜磷酸肌醇分解的信号通路偶联。
