Agonistic properties of anti-B cell antibodies purified on staphylococcal protein A may be due to contaminating protein A.
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.
Jakobson
E
E
Department of Immunology, Stockholm University, Sweden.
Axelsson
B
B
Paulie
S
S
D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000502:physiology; D015703:Antigens, CD / Q000276:immunology; D000944:Antigens, Differentiation, B-Lymphocyte / Q000276:immunology; D001402:B-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D019013:CD40 Antigens; D002118:Calcium / Q000378:metabolism; D006801:Humans; D051917:Leukosialin; D008213:Lymphocyte Activation / Q000187:drug effects; D009419:Nerve Tissue Proteins / Q000276:immunology / Q000494:pharmacology; D014066:Palatine Tonsil; D012795:Sialoglycoproteins / Q000276:immunology; D012995:Solubility; D013205:Staphylococcal Protein A / Q000737:chemistry* / Q000276:immunology
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D000944:Antigens, Differentiation, B-Lymphocyte; D019013:CD40 Antigens; C029152:G-substrate; D051917:Leukosialin; D009419:Nerve Tissue Proteins; C465014:SPN protein, human; D012795:Sialoglycoproteins; D013205:Staphylococcal Protein A; D002118:Calcium