Immunogenicity and specificity of collagen. 13. Preparation and definition of soluble collagen-anticollagen complexes by gel filtration.

Since antisera to human soluble collagen are non-precipitating and can only be defined by passive haemagglutination, low molecular weight immune complexes of collagen antibodies and hydroxamated collagen were prepared according to the inhibiting concentration of antigen, as observed in haemagglutination inhibition. The existence of these immune complexes could be demonstrated by incubating anti-collagen sera with hydroxamated collagen and passing the reaction mixtures through columns of superfine Sephadex G-100 saturated with buffer containing a given concentration of hydroxamated collagen. By this procedure it could be shown, in comparison to control runs, that the non-reacted hydroxamated collagen was eluted at a distinctly different position from that of hydroxamated collagen eluted in combination with gamma globulin. Controls with hydroxamated collagen alone or together with normal rabbit serum showed the elution pattern of non-reacted antigen. This way of isolation opens the possibility of obtaining defined soluble collagen—anti-collagen immune complexes for further research.