Background staining in immunoblot assays reduction of signal caused by cross-reactivity with blocking agents

Certain serum samples produce high background in Western and direct immunoblot assays that detect human serum IgG against specific antigens. We determined that this was due to a reaction between endogenous IgG and the membrane blocking agent (we refer to this as blocking-specific background). Using...

Ausführliche Beschreibung

Gespeichert in:
Bibliographische Detailangaben
Veröffentlicht in:Journal of immunological methods 1993-01, Vol.158 (1), p.67-76
Hauptverfasser: Craig, Wendy Y., Poulin, Sue E., Collins, Marilyn F., Ledue, Thomas B., Ritchie, Robert F.
Format: Artikel
Sprache:eng
Schlagworte:
Online-Zugang:Volltext
Tags: Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
Beschreibung
Zusammenfassung:Certain serum samples produce high background in Western and direct immunoblot assays that detect human serum IgG against specific antigens. We determined that this was due to a reaction between endogenous IgG and the membrane blocking agent (we refer to this as blocking-specific background). Using milk as blocking agent, we screened 107 sera by Western immunoblot or checkerboard immunoblot assays, and found that 6.5% of sera had background intensities sufficient to interfere with the interpretation of final results. Blocking-specific background was also observed using bovine serum albumin and other animal protein-based blocking agents. As the primary antibody in these immunoblot assays was human IgG, we investigated human serum albumin as a blocking agent; this approach eliminated the problem of blocking-specific background.
ISSN:0022-1759
1872-7905
DOI:10.1016/0022-1759(93)90259-A