Autoimmune haemorrhaphilia XIII/13 (AH13) is an acquired life-threatening bleeding disorder due to anti-factor XIII (FXIII) autoantibodies (auto-Abs). AH13 patients may die of haemorrhage without correct diagnosis and proper treatment because of lack of awareness and the absence of rapid easy-to-use tests specific for this disease. Currently, the definitive diagnosis is established by cumbersome and time-consuming laboratory tests such as dot-blot assays and enzyme-linked immunosorbent assays (ELISA), and therefore these tests are generally not carried out. To save AH13 patients' lives, there is an urgent necessity for developing a rapid test for FXIII auto-Abs. We first generated and characterised mouse monoclonal antibodies (mAb) against human FXIII A subunit (FXIII-A), and then developed a rapid immunochromatographic test (ICT) for detection of anti-FXIII-A auto-Abs using one mAb with a dissociation constant of 9.3 × 10⁻¹¹ M. The auto-Ab-FXIII-A complex was captured by the mAb on a nitrocellulose membrane and visualised by Au-conjugated anti-human IgG Ab. Mixing with healthy control plasma improved the detection of auto-Abs in patients having extremely low levels of FXIII-A. The specificity and sensitivity of the ICT were 87 % and 94 %, respectively. We also detected auto-Abs against activated FXIII (FXIIIa) in three patients by pre-converting FXIII to FXIIIa by thrombin treatment. ICT values were significantly inversely correlated with FXIII activity levels, indicating an association between the quantity of anti-FXIII autoantibodies and AH13. This reliable rapid ICT assay can be applied to a point-of-care test to detect anti-FXIII-A auto-Abs, and will contribute to early diagnosis and treatment of AH13.

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The urinary fibrin/fibrinogen degradation products (FDP), as sensitive indicators of various renal disorders, have been measured by several methods. For their determination, a new and highly sensitive enzyme-linked immunosorbent assay not requiring the urine concentration has been developed. The study comprised 42 patients with nonnephrotic chronic glomerulonephritis (CGN), 23 patients with primary nephrotic syndrome (NS), and 29 healthy adults. The results were as follow: (1) the content of urinary FDP in normal subjects was 10.30 ± 9.08 ng/ ml; (2) the mean level of urinary FDP in both CGN and NS groups was significantly higher than in normal subjects; (3) in the CGN group itself there was a tendency for an increase of urinary FDP during more active forms of the disease, and (4) there was a significant correlation between urinary FDP and urinary protein in the CGN group, whereas no correlation was observed in the NS group. These results suggest that the major part of urinary FDP in the CGN group derives from the increased filtration, while its origin in the NS group is not related to increased filtration only, but may also have involved intraglomerular coagulation abnormalities. The newly developed enzyme-linked immunosorbent assay can detect urinary FDP levels lower than 3.9 ng/ml. Therefore, this method can be of great value in determining the degree of abnormalities of intraglomerular coagulation and fibrinolysis in renal diseases.

ABSTRACT. The cross-reactivity of the anti-human D-dimer antibody 1C9-6F10 to the canine D-dimer was evaluated by western blotting using purified canine fibrinogen, fibrin degradation products (XDPs) and plasma from a dog with suspected disseminated intravascular coagulation (DIC). The antibody cross-reacted with canine XDPs and plasma from the dog with suspected DIC. A latex agglutination assay using 1C9-6F10 measured the canine XDPs in dogs. In conclusion, the antibody 1C9-6F10 cross-reacted to the canine D-fragment with high specificity, but lower affinity compared with the human D-dimer. The latex agglutination assay using the 1C9-6F10 antibody was used to measure canine plasma D-dimer concentrations. KEY WORDS: canine, D-dimer antibody, fibrin degradation product.doi: 10.1292/jvms.12-0270; J. Vet. Med. Sci. 75 (7): [963][964][965][966] 2013 Disseminated intravascular coagulation (DIC) is a lifethreatening condition characterized by abnormal systemic coagulation followed by secondary fibrinolysis. An elevated concentration of fibrin degradation markers indicates the activation of fibrinolysis and is thus one of the diagnostic criteria for DIC in humans and dogs [6,12]. Fibrinogen comprises a central globular E region and two identical D regions that form a D-E-D structure. As such, the cleavage of fibrinogen produces four fragment patterns: In general, most laboratory "FDP" tests measure the degradation products from both cross-linked fibrin and fibrinogen itself, while the "D-dimer" test measures only XDPs. Thus, the "D-dimer" test is thought to be suitable for the evaluation of secondary fibrinolysis. The measurement of D-dimer concentrations in dogs is used to diagnose DIC and other fibrinolysis-related diseases [2-5, 7, 9, 10]. However, the specific cross-reactivity of the canine D-dimer antibodies used in these tests has not been investigated. Therefore, we investigated whether an antibody developed for the human D-dimer that is used in the Factor Auto D-dimer kit (Q-may, Oita, Japan) and is commercially available from Mitsubishi Chemical Medience Corporation can cross-react with canine XDPs. We then evaluated the potential utility of the latex agglutination assay using this antibody to measure canine D-dimer concentrations in plasma.The fibrinogen was prepared from dog plasma, passed through lysine-Sepharose 4B (Pharmacia Biotech AB, Uppsala, Sweden) and gelatin-Sepharose 4B (American Bioscience AB, Uppsala, Sweden), centrifuged three times (12,900 × g for 20 min) and subjected to precipitation cycles with ammonium sulfate (1 M). The final precipitate was dissolved in buffer containing 0.05 M Tris-HCl (pH 7.5) with 0.9% NaCl. The cross-linked fibrin was prepared by incubation with thrombin (0.25 U/ml) and Fibrogammin P (0.11 U/ml) in the fibrinogen at 37°C for 1 hr. The cross-linked XDPs were produced by incubation with both urokinase (0.01 U/ ml) and plasminogen (0.04 U/ml) at 37°C for 1 hr. The digestion was terminated by the addition of aprotinin (51.4 U/ml). Protein concentrations were determine...

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