A novel assay to diagnose hereditary angioedema utilizing inhibition of bradykinin-forming enzymes

Abstract

Background: Hereditary angioedema types I and II are caused by a functional deficiency of C1 inhibitor (C1-INH), leading to overproduction of bradykinin. The current functional diagnostic assays employ inhibition of activated C1s; however, an alternative, more physiologic method is desirable. Methods: ELISAs were developed using biotinylated activated factor XII (factor XIIa) or biotinylated kallikrein bound to avidin-coated plates. Incubation with plasma was followed by detection of bound C1-INH. Results: After standard curves were developed for quantification of C1-INH, serial dilutions of normal plasma were employed to validate the ability to detect known concentration of C1-INH in the plasma as a percent of normal. Hereditary angioedema (HAE) types I and II were then tested. The level of functional C1-INH in all HAE types I and II plasma tested was less than 40% of our normal control. This was evident regardless of whether we measured factor XIIa-C1-INH or kallikrein-C1-INH complexes, and the two assays were in close agreement. By contrast, testing the same samples utilizing the commercial method (complex ELISA, Quidel Corp.) revealed the levels of C1-INH between 0 and 57% of normal (mean, 38%), and 42 samples were considered equivocal (four controls and 38 patients). Conclusions: Diagnosis of HAE types I and II can be ascertained by inhibition of enzymes of the bradykinin-forming cascade, namely factor XIIa and kallikrein. Either method yields functional C1-INH levels in patients with HAE (types I and II) that are clearly abnormal with less variance or uncertainty than the commercial method.

C1 inhibitor function using contact‐phase proteases as target: evaluation of an innovative assay

“…Methods using contact‐phase proteins had already been introduced in the literature. Recently, an assay took advantage of an ELISA method using biotinylated active enzymes (FXIIa or KK) . Its rationale was founded on the formation of FXIIa–C1Inh and KK–C1Inh complexes after incubation with plasma and detection of bound C1Inh.…”

“…However, the primary mediator of swelling in angioedema patients is BK, whose production is controlled by plasma KK. A recent approach used biotinylated active enzymes (FXIIa or KK) and took advantage of the abundance of enzyme–C1Inh complex as a readout of the C1Inh function in plasma . The authors demonstrated high sensitivity using FXIIa, while C1Inh targeted only 60% of the protease when using KK, presumably due to its inhibition by α2M.…”

Hereditary and Acquired Angioedema: Heterogeneity of Pathogenesis and Clinical Phenotypes

“…A novel assay for the diagnosis of HAE types I and II based on the inhibition of BK-forming enzymes has been recently proposed. It seems to perform better than both the commonly used ELISA and the chromogenic assay as well [66].…”

Kininogen Cleavage Assay: Diagnostic Assistance for Kinin-Mediated Angioedema Conditions

“…Diagnosis of nC1Inh-AE is still controversial: detection of F12 gene mutation is pertinent only to FXII-AE but cannot explain why many carriers are asymptomatic [ 13 , 14 ]. Enzymatic tests have been performed to investigate increased kinin-forming conditions [ 26 ], C1Inh-protease complexes [ 27 ] or defective kinin catabolism [ 13 , 28 ]. While some of these tests are already used in AE classification [ 2 ], their consensual use is still pending on pre-analytical and enzymatic considerations [ 29 ].…”