Immunochemical Studies on Proteins Important in Blood Coagulation^[*]

Summary

This report draws attention to the fact that only minute quantities of purified serum proteins (1 —15 μg/kg body weight) are required to stimulate antibody production. Injection of amounts greater than 1 mg/kg either is a waste or favors the production of non-specific antisera. Potent antisera can be produced in rabbits with a single intramuscular injection using either Al(OH)₃ gel or Amphojel as an adjuvant.

A number of purified plasma protein preparations which appear homogenous by ultra centrifugation or biological activity studies actually contain several antigenic components and are immunologically heterogeneous, when checked with highly specific antisera.

Fibrinogen prepared by alcohol fractionation and tannic acid purification was found to be free of prothrombin, antihemophilic factor and Profibrinolysin. Both Immunoelectrophoresis and double diffusion in agar indicated a homogeneous preparation.

Fibrinogens prepared from human, canine and bovine plasmas exhibited a low species specificity.

^* Aided by a research grant from the Michigan Heart Association and research grant H-4712 from the National Heart Institute, National Institutes of Health, United States Public Health Service.

^** Present address Ortho Research Foundation, Raritan, N. J.

• References

• 1 Freund J. Some aspects of active immunization.. Ann. Rev. Microbiol. 1947; 1: 291
• 2 Cohn M. Production of antibodies in experimental animals.. Meth. med. Res. 1952; 5: 271
• 3 Ware A. G, Guest M. M, Seegers W. H. Fibrinogen: With special reference to its preparation and certain properties of the product.. Arch. Biochem. 1947; 13: 231
• 4 Blombäck B. Studies on fibrinogen: Its purification and conversion into fibrin.. Acta physiol. scand. 1958; 43: 1
• 5 Simonetti C, Casillas G, Pavlovsky A. Purification du facteur VIII antihémophilique (FAH).. Hémostase 1961; 1: 57
• 6 Anderson G. F. Prothrombin synthesis in the dog studied with a fluorescent antibody.. Doctor of Philosophy Thesis. Wayne State University; Detroit, Michigan: 1961
• 7 Lewis M. L, Ware A. G. A simple procedure for separation of prothrombin and accelerator globulin from citrated human plasma.. Proc. Soc. exp. Biol. (N.Y.) 1953; 84: 636
• 8 Nordschow C. D, Robinson J. N, Carter J. R. Personal communication. Iowa State University; Iowa City, Iowa.:
• 9 Seegers W. H. The purification of prothrombin.. Rec. Chem. Prog. 1952; 13: 143
• 10 Kabat E. A, Mayer M. M. Experimental Immunochemistry. Charles C. Thomas Springfield; Illinois: 1948
• 11 Grabar P, Williams C. A. Méthode permettant l’étude conjuguée des propriétés électrophorétiques et immunochimiques d’un mélange de protéines. Application au sérum sanguin.. Biochim. biophys. Acta 1953; 10: 193
• 12 Wilson M. W, Pringle B. H. Experimental studies of the agar plate precipitin test of Ouchterlony.. J. Immunol. 1954; 73: 232
• 13 Wilson M. W. Experimental records. Medical Library, Wayne State University, Detroit, Michigan. 1958 1957
• 14 Goodman M, Ramsey D. S, Simpson W. L, Remp D. G, Ba-sinski D. H, Brennan M. J. The use of chicken antiserum for the rapid determination of plasma protein components.. J. Lab. clin. Med. 1957; 49: 151
• 15 Seegers W. H, Casillas G, Shepard R. S, Thomas W. R, Halick P. Some properties of thrombin preparations.. Canad. J. Biochem. 1959; 37: 775
• 16 Scheiffarth F, Götz H, Soergel K. Immunoelektrophoretische Studien über heterologe Präzipitinreaktionen.. Int. Arch. Allergy 1957; 10: 82
• 17 Melcher L. R, Masouredis S. P, Reed R. The immunological relationship between human and bovine serum albumin.. J. Immunol. 1953; 70: 125
• 18 Vigliani E. C, Pernis B. Immunological factors in the pathogenesis of the hyaline tissue of Silicosis.. Brit. J. industr. Med. 1958; 15: 8
• 19 Farr R. S, Dixon Jr. F. J. The effect of antigen concentration on the initiation of detectable antibody synthesis in rabbits.. J. Immunol. 1960; 85: 250
• 20 Schwick G, Schultze H. E. Immunchemische Untersuchungen mit Prothrombin und Thrombin. Colloq. Sint. Jans Hospital, Brügge, Belgien, 1958.. Clin. chim. Acta 1959; 4: 26