Development and evaluation of ELISAs for factor XIIIA and XIIIB subunits in plasma

Abstract

Severe, congenital deficiency of factor XIII is extremely rare. However, a moderate reduction in the plasma level of the functional subunit (factor XIIIA) and also to a lesser extent of the carrier subunit (factor XIIIB), and a decrease in the XIIIA:B subunit ratio, have recently been reported in patients with the inflammatory bowel disorder Crohn's disease, particularly during clinical relapse. In order to accurately monitor patients, sensitive, reliable assays for the two subunits of factor XIII are required. We report here the development and validation of ELISAs for these components. The assays are identical except in respect of the specificity of the polyclonal antiserum used as starting material, both of which are commercially available. The antisera are purified by n-octanoic acid precipitation and portions of these purified immunoglobulins are used as coating antibodies. The remaining portions are biotinylated and used with streptavidin and horse-radish peroxidase as tracer antibodies. A normal range (n=24) was established for factor XIIIA (mean 95 range 60–130 U/dl) and for factor XIIIB (mean 99 range 60–130 U/dl). There were no significant differences between the ELISA and electroimmunodiffusion assays either for factor XIIIA (means ± 1 standard deviation 95 ± 15.9 and 89 ± 22.7 respectively) or for factor XIIIB (99 ± 18.3 and 106 ± 23.4 respectively). These assays have been in routine use for six months, during which time two further antisera purifications and biotinylations have been carried out without significant problems of reproducibility or stability.