Direct chromogenic substrate immuno-capture activity assay for testing of factor VII-activating protease.

BACKGROUND: The Marburg I (MRI) single nucleotide polymorphism (SNP) of the factor VII-activating protease (FSAP) gene has been associated with thrombophilia and atherosclerotic disease. PCR is used to detect the SNP. Also, the specific FSAP activity to cleave single-chain urokinase-type plasminogen activator (scu-PA) serves as a surrogate for PCR testing. Development of further assays is indicated in order to increase testing opportunities for future studies. METHODS: A direct chromogenic substrate immuno-capture activity assay for FSAP (FSAP dcs activity assay) was established. Performance characteristics of the FSAP dcs activity assay were compared to the FSAP scu-PA activity assay. RESULTS: The FSAP dcs activity assay detects FSAP activity from 25% to 150% of the norm. Total CVs ranged from 6% to 10% for FSAP wild type samples and 9%-18% for MRI samples. Correlation between the FSAP dcs and scu-PA activity assays was low (R=0.7). The FSAP dcs activity determined the presence of the MRI FSAP alloenzyme with a diagnostic sensitivity and specificity of 100% [95% confidence interval (CI): 89.6%-100%] and 96.2% (95% CI: 93.2%-97.4%), respectively, whereas the specific FSAP dcs activity increased specificity to 99.0% (95% CI: 97.2%-99.6%). CONCLUSIONS: The specific FSAP dcs activity represents a reliable method for the detection of the FSAP MRI alloenzyme. Due to the limited correlation between the FSAP dcs and scu-PA activity assays, these different measurands may exhibit different utility in research and clinical applications. Thus, the FSAP dcs activity assay can represent a valuable complement or alternative for FSAP testing in future studies.

Coagulation assays based on the Luminescent Oxygen Channeling Immunoassay technology 1).

BACKGROUND: The Luminescent Oxygen Channeling Immunoassay (LOCI) technology is a well-established homogeneous assay format that allows for fast, accurate, and highly sensitive quantitation of analytes. We set out to develop and prove a novel concept to establish a LOCI format that should principally allow for the determination of the activity of coagulation factors and anticoagulants of clinical relevance. METHODS: The concept is based on the linkage of LOCI nano-beads by a peptide that can be cleaved by a coagulation factor. To prove the principle, we used a peptide that can be cleaved by thrombin. RESULTS: We were able to show that coagulation activation of plasma or whole blood samples that were combined with the LOCI components degraded the thrombin-sensitive peptide and consequently, led to a reduction of the LOCI signal. Signal reduction was proportional to the amount of active thrombin generated. The research prototype assay allowed for the detection of factor deficiencies in both the extrinsic and intrinsic coagulation pathways, and for the quantification of hirudin, a direct thrombin inhibitor. CONCLUSIONS: Taken together, we conclude that the LOCI technology has the potential for extension to functional blood coagulation assays.

Qualitative detection of the Marburg I alloenzyme of factor VII-activating protease by an immunoassay and its comparison to PCR testing.

BACKGROUND: The Marburg I (MRI) single nucleotide polymorphism (SNP) of the factor VII-activating protease (FSAP) gene has been associated with thrombophilia, thromboembolism, atherosclerosis, and the incidence and progression of carotid stenosis. At present, MRI SNP testing is mainly performed using costly nucleic acid analysis. The ratio between FSAP activity and antigen concentrations in citrated plasma has been used to assess the FSAP genotype. METHODS: This article describes the development of a prototype ELISA for the detection of the MRI FSAP alloenzyme, and its correlation to FSAP genotypes to assess whether a positive MRI FSAP ELISA result may be used as a surrogate marker for the presence of the MRI SNP. RESULTS: ELISA results were correlated with FSAP genotypes from 523 blood donors measured using PCR. Diagnostic sensitivity and specificity of the assay for determination of the genotype were 100% (95% confidence interval [CI]: 93.36-100) and 99.79% (95% CI: 98.80-99.96), respectively. Maximum run-to-run, within-run, and total coefficients of variation were 7.8%, 7.9%, and 9.9%, respectively. No cross-reactivities with homologues of the MRI FSAP alloenzyme were observed. Test performance was not affected by typical interfering compounds. CONCLUSIONS: The data demonstrate that an immunoassay applying antibodies specific to the MRI FSAP alloenzyme can provide sufficiently accurate detection of the MRI SNP. This will significantly simplify MRI FSAP testing, particularly in large cohorts.

Quantification of coagulation factor XIII activity by a thio-NADH based assay using factor XIII immuno-depleted plasma as a diluent for calibration.

BACKGROUND: Accurate determination of factor XIII (FXIII) activity is crucial for replacement therapy. FXIII activity is typically determined using a coupled enzymatic reaction that measures nicotinamide adenine dinucleotide hydride (NADH) consumption at 340 nm. METHODS: Here, we describe the development of a prototype for a novel FXIII activity assay for detection at 405 nm by replacing NADH with thio-NADH, and the application of FXIII immuno-depleted plasma as a diluent for calibration. RESULTS: Performance data show up to two-fold lower susceptibility of the prototype assay to interferences from hemolyzed, icteric, and lipemic samples when compared to a NADH assay format. In addition, the use of FXIII immuno-depleted plasma as diluent for calibration improved recovery almost two-fold in the lower measurement range. The novel prototype assay correlates well with a conventional assay (r=0.98, y=0.99·x+2.17% FXIII, n=173). CONCLUSIONS: The described prototype assay has the potential to (a) increase trueness of measurement of low levels of FXIII, (b) improve robustness due to reduction from interferences, and (c) can be used on a broad range of coagulation instruments due to its detection at 405 nm.

Tests for the measurement of factor VII-activating protease (FSAP) activity and antigen levels in citrated plasma, their correlation to PCR testing, and utility for the detection of the Marburg I-polymorphism of FSAP.

BACKGROUND: The single nucleotide Marburg I (MRI) polymorphism of the factor VII-activating protease (FSAP) gene, the prourokinase-activating activity of FSAP, and antigen levels of FSAP in plasma have been associated with incidence and progression of carotid stenosis and venous thromboembolism. However, more information on the extent of these associations, potential further ones, and respective clinical utilities remain to be determined. At present, testing is performed mainly by PCR assays based on probes or SYBR Green I. Some studies include testing for antigen levels of total FSAP and its ability to activate prourokinase. To test large cohorts, it is beneficial to rely on assays that are cost-effective, reliable, easy to use, rapid to perform, and that may eventually be automated. In addition, it appears advantageous to use functional tests or tests that determine antigen levels as they may relate more closely to the phenotype than the genotype does. METHODS: Tests for the measurements of antigen levels of FSAP and its prourokinase-activating activity were improved and performance characteristics assessed. To determine the FSAP genotypes, an amplification created restriction site (ACRS) PCR test was developed. RESULTS: Key performance characteristics of the FSAP activity and antigen tests were as follows: measuring range: 350-1400 mPEU/mL and 1.8-120 ng/mL, total coefficients of variation (CV): 5%-20% and 5%-14%, within-run CV: 4%-11% and 2.3%-12%, and run-to-run CV: 2%-17% and 4.3%-8.3%, respectively. The ratio of the activity and antigen level of FSAP correctly identified the FSAP genotypes of 126 samples tested. CONCLUSIONS: The ACRS PCR test is useful for laboratories that do not have the equipment to perform probe or SYBR Green I based real-time PCR. Furthermore, the tests developed for the determination of FSAP activity and antigen levels are convenient for determining clinical correlations, even for large population studies. The ratio of activity and antigen level of FSAP appears to be a promising and efficient alternative to molecular diagnostic techniques to detect the MRI polymorphism of FSAP.