Polyclonal antibodies to tropoelastin and the specific detection and measurement of tropoelastin in vitro.
Because tropoelastin is difficult to purify, most antibodies to elastin are raised against the insoluble form of the molecule. While these antibodies cross-react with tropoelastin, antigenic differences between insoluble and soluble elastin suggest that antibodies raised directly against tropoelastin might provide a more sensitive and specific reagent for evaluating tropoelastin production in elastin-producing systems. Using an improved method for purifying tropoelastin from tissue culture explants, we describe the generation and characterization of an antibody to bovine tropoelastin. This antibody was used to develop a sensitive, direct-binding immunoassay capable of quantifying small levels of tropoelastin in conditioned medium from cultured cells. This assay takes advantage of the propensity of tropoelastin to adsorb to vinyl microtiter plates, even in the presence of serum proteins. This property, in combination with the increased sensitivity obtained using antibodies to tropoelastin, provides for a direct-binding immunoassay that detects nanogram quantities of tropoelastin directly in cell culture medium, avoiding sample preparation steps that result in extensive loss of tropoelastin. In addition, this direct-binding assay is ten- to 30-fold more sensitive than the existing competitive ELISA assays.
Prosser
I W
IW
Department of Medicine, Jewish Hospital, Washington University Medical Center, St. Louis, Missouri.
Whitehouse
L A
LA
Parks
W C
WC
Stahle-Bäckdahl
M
M
Hinek
A
A
Park
P W
PW
Mecham
R P
RP
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D001769:Blood; D002417:Cattle; D002478:Cells, Cultured; D003470:Culture Media / Q000032:analysis; D004549:Elastin / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D007118:Immunoassay; D007150:Immunohistochemistry; D007163:Immunosorbent Techniques; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000032:analysis; D012995:Solubility; D014333:Tropoelastin / Q000032:analysis* / Q000235:genetics / Q000276:immunology
D000906:Antibodies; D003470:Culture Media; D012333:RNA, Messenger; D014333:Tropoelastin; D004549:Elastin