Cross Reactivity of Rabbit Anti-Recothrom (Human Recombinant Thrombin) Antibodies with Molecular Variants of Human Thrombin, and Various Bovine Thrombin Preparations

Abstract 4418

Bovine thrombin preparations are widely used as topical hemostatic agents. While earlier bovine thrombin preparations were reported to produce immunologic responses upon repeated exposure, the refined bovine thrombin and recombinant human thrombin preparations (Zymogenetics, Seattle, WA, USA) are claimed to be less immunogenic. It was hypothesized that recombinant human thrombin despite its reduced immunogenic nature, upon repeated administration may result in the generation of antibodies and that may cross react with bovine and human thrombin. To test this hypothesis, groups of rabbits were challenged repeatedly with human recombinant thrombin over a 6 month period. Purified IgG preparations were obtained using protein G columns.To generate specific antisera in rabbits, the Recothrom (Human recombinant thrombin) conjugated with keyhole lymphocyte hemocyanin(KLH) as a carrier were administered intravenously to individual groups of rabbits (n=3) at a dosage of 100 μ g using standard immunologic methods. Preimmune blood and antiserum were collected from each rabbit in 0,30,50,90,120,150,180 days. In order to determine their cross reactivity with 3 preparations of bovine thrombin with differing purity, namely thrombin 4A, thrombin 4B, and bovine prothrombin were evaluated by immunoblotting techniques using the rabbit IgG fractions. In addition, to determine the relative cross reactivity of anti-Recothrom antibodies with human-α and γ- thrombin, one batch of each was evaluated by immunoblotting techniques using the rabbit IgG fractions. Gel electrophoresis and Western blotting were carried out. In brief, samples were subjected to electrophoresis through 4% to 20% gradient Tris-4-(2-hydro-xyethyl)-1-piperazineethanesulfonic acid (HEPES)-sodium dodecyl sulfate (SDS) polyacrylamide mini gels and then electrotransferred onto nitrocellulose membranes. The membranes were blocked with 5% milk and probed with anti-Recothrom IgG. The membranes were then washed and incubated with horseradish peroxidase conjugated donkey anti-rabbit IgG (1:20000 dilutions).After extensively washing; immunoreactive bands were detected with SuperSignal West Pico Chemiluminescent Substrate followed by film exposure for 1- to 5 minutes. The results from these studies suggested that anti-Recothrom antibodies cross react with human thrombins (α and γ), Evithrom brand of human thrombin, and bovine thrombin in a time dependent fashion. Although the Recothrom was not immunogenic in rabbit up to 90 days, antibodies harvested on day 150 cross-reacted with human α and γ thrombin as well as Evithrom. Visible cross reactivity, immunoblots around 36 KDa with human thrombins, bovine crude thrombin, and bovine thrombin 4A and 4B were observed with anti-Recothrom IgG collected from day 180. Bovine prothrombin did not exhibit any cross reactivity with anti-recothrombin antibodies at any time period. Thus, this data suggest that it is likely that antibodies against human recombinant thrombin may also cross-react with human thrombin and bovine thrombin. Therefore, the use of Recothrom may not be recommended in patients who have been previously exposed to human thrombin and bovine thrombin preparations.