Monoclonal Antibodies Against the Heparin- Dependent Protein C Inhibitor Suitable for Inhibitor Purification and Assay of Inhibitor Complexes

Abstract

summaryTwo different monoclonal antibodies against the heparin- dependent inhibitor of human activated protein C were produced, using cleaved modified inhibitor for immunization and partially purified inhibitor for screening of the hybridomas, One of the antibodies recognized free and complexed forms of the inhibitor in immunoblotting experiments. The other antibody was used to develop an assay for A PC -PCI inhibitor complexes. Using the assay the formation of complexes was studied in plasma, both in the presence and absence of heparin. The rate of complex formation was similar to that reported previously for the loss of activated protein C amidolytic activity in plasma. The same antibody was also immobilized on Sepharose and used to purify the inhibitor from fresh human plasma. The purified material appeared as two narrowly spaced bands with Mr about 57,000 in SDS-PAGE. The average yield from 1 liter of fresh plasma was 1 mg of inhibitor, The purified inhibitor formed SDS stable complexes with activated protein G and urokinase that could be identified in immunoblots using specific antibodies.

Protein C inhibitor in human body fluids. Seminal plasma is rich in inhibitor antigen deriving from cells throughout the male reproductive system.

“…The monoclonal anti-PCI (M1 1-5) was previously reported to be unsuitable for Western blot analysis (25), and dilutions in buffer of blood and seminal plasma resulted in parallel curves, when assayed with the PCI IRMA. We thus concluded that the IRMA recognized the same antigenic epitope in the two fluids.…”

“…After 60 min, the samples were analyzed simultaneously parathyroid glands, kidney, brain, adrenal gland. A negative for APC-PCI complexes as described (25 shown). This suggests that the PCI-coding transcripts syntheother tissues is shorter than expected from the previously charsized in the male reproductive glands and other tissues may be acterized cDNA from the liver (5) although this transcript identical with the transcript obtained from human liver.…”

“…The antibody fraction was prepared by ammonium sulfate precipitation of the antisera followed by DEAE Sepharose chromatography (26). The production of the monoclonal antibodies against PCI (M 1 1-5 and M 1-12) has been described previously (25). Rabbit antibodies against mouse immunoglobulins and alkaline phosphatase conjugated rabbit immunoglobulins against mouse immunoglobulins were from DAKOPATTS, Copenhagen, Denmark.…”

“…Complexes of APC-PCI were prepared and assayed as previously reported (25). PCI-depleted plasma was prepared by passing aliquots of fresh plasma over an immunoaffinity column with immobilized monoclonal anti-PCI antibodies (M 1 1-5).…”

Protein C inhibitor is expressed in tubular cells of human kidney.

“…Plasma PCI was first described by Marlar and Griffin as an inhibitor of activated protein C (4) and later purified and characterized by several groups (1,(5)(6)(7). Urinary PCI, purified and characterized by Stump et al, was initially described as a new urokinase (uPA) inhibitor and therefore named plasminogen activator inhibitor-3 (PAI-3) (8).…”