Detection and quantitation of cleaved and uncleaved high molecular weight kininogen in plasma by ligand blotting with radiolabeled plasma prekallikrein or factor XI.
A method for the quantitative assay of native single chain and kallikrein cleaved two-chain high molecular weight (HMW)-kininogen in plasma is described. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of whole plasma is followed by electrotransfer of the electropherogram to nitrocellulose membranes and detection of the blotted HMW-kininogen with its physiologic ligands, radiolabeled plasma prekallikrein or radiolabeled factor XI. Using unreduced SDS-PAGE cleaved two-chain HMW-kininogen (Mr approximately 107,000 and 95,000), is electrophoretically separated from uncleaved single chain HMW-kininogen (Mr approximately 150,000). Counting the radioactivity of the nitrocellulose pieces corresponding to cleaved HMW-kininogen permits its quantitative measurement by comparison with standards consisting of decreasing amounts of fully dextran sulfate activated normal human plasma. Single chain HMW-kininogen is similarly assayed using reduced SDS-PAGE and unactivated normal human plasma standards. This technique is highly specific and sensitive to about 50 ng of either cleaved or uncleaved HMW-kininogen. Varying amounts of cleaved HMW-kininogen were found in a small series of plasmas from patients suffering from various inflammatory conditions. Higher levels of in vivo cleaved HMW-kininogen were observed during acute attacks of hereditary angioedema due to Cl-inhibitor deficiency. This technique may be useful for the assessment of the degree of in vitro or in vivo activation of the contact system.
Lämmle
B
B
Department of Immunology, Scripps Clinic and Research Foundation, La Jolla, CA 92037.
Zuraw
B L
BL
Heeb
M J
MJ
Schwarz
H P
HP
Berrettini
M
M
Curd
J G
JG
Griffin
J H
JH
D000799:Angioedema / Q000097:blood; D004591:Electrophoresis, Polyacrylamide Gel; D005172:Factor XI; D006801:Humans; D007704:Kininogens / Q000097:blood*; D011288:Prekallikrein; D011869:Radioligand Assay / Q000379:methods*; D012967:Sodium Dodecyl Sulfate
D007704:Kininogens; D012967:Sodium Dodecyl Sulfate; D005172:Factor XI; D011288:Prekallikrein