title: Improved detection of proteolytically cleaved high molecular weight kininogen by immunoblotting using an antiserum against its reduced 47 kDa light chain.
abstract: Several ligand blotting or immunoblotting assays for the detection of single-chain and proteolytically cleaved two-chain high molecular weight kininogen (HK) in whole plasma have been described. Since they may suffer from poor sensitivity for the light chain species of cleaved HK on reduced blots, an antiserum against the reduced and alkylated 47 kDa light chain of HK was raised in rabbits allowing improved immunodetection of HK species on blots of reduced electropherograms. This immunoblotting method is highly specific and sensitive, permitting detection of 0.2 ng single-chain HK or the light chains of 2 ng proteolytically cleaved HK in whole plasma. Thus, this immunoblotting technique is at least 50-100 times more sensitive than ligand blotting with radiolabelled factor XI overlay. A similar cleavage pattern was observed following in vitro activation of normal human plasma by dextran sulphate and after plasma kallikrein-induced proteolysis of purified HK. However, bands of different molecular weights were generated after HK had been cleaved by purified leukocyte elastase. During acute attack in a patient with hereditary angioedema, high levels of in vivo cleaved HK were noticed, whereas concentration of cleaved HK in plasma samples and synovial fluids from patients suffering from various inflammatory conditions were not substantially higher than those in normal plasma. During in vitro cold activation of plasma samples of pregnant women concomitant HK cleavage and plasma kallikrein generation were observed.
authors.affiliation: Central Haematology Laboratory, Inselspital, University Hospital of Bern, Switzerland.
authors.forename: R
authors.initials: R
authors.lastname: Bühler
authors.forename: J K
authors.initials: JK
authors.lastname: Hovinga
authors.forename: I
authors.initials: I
authors.lastname: Aebi-Huber
authors.forename: M
authors.initials: M
authors.lastname: Furlan
authors.forename: B
authors.initials: B
authors.lastname: Lämmle
mesh_terms: D000799:Angioedema / Q000378:metabolism; D000818:Animals; D001168:Arthritis / Q000097:blood; D015153:Blotting, Western; D005260:Female; D006801:Humans; D007106:Immune Sera; D007704:Kininogens / Q000032:analysis* / Q000378:metabolism; D008970:Molecular Weight; D010084:Oxidation-Reduction; D011247:Pregnancy; D011654:Pulmonary Edema / Q000097:blood; D011817:Rabbits; D012680:Sensitivity and Specificity; D018805:Sepsis / Q000097:blood; D012772:Shock, Septic / Q000097:blood; D013582:Synovial Fluid / Q000378:metabolism
chemical_list: D007106:Immune Sera; D007704:Kininogens