Detection of Leukocyte Antibodies by Means of 1131-Labelled, Purified Anti-Human-Globulin Antibody. Problems of Nonspecific Adsorption of Globulin by Leukocytes

By ROBERT E. ANDERSON AND Ro L. WALFORD T HE PRESENT study forms part of a continuing effort to develop quantitative methods for detecting antigen-antibody reactions involving nucleated cells as antigen. Here we shall present information regarding the preparation of purified, 1131-labelled anti-human-globulin antibody (AHG antibody) by means of adsorption and elution from normal human globulin, the latter having been rendered insoluble by binding to cellulose via the method of Campbell et al.1 The uptake of this labelled, purified antibody by white blood cells previously “sensitized” by exposure to the anti-leukocvte antibodies of certain patients’ sera will be detailed. In evaluating “specific” uptake of antibody globulin by “sensitized” leukocvtes, consideration must simultaneously be given to the known tendency of leukocytes to adsorb globulin in a “nonspecific” fashion. Such “nonspecific” binding of globulin by leukocytes has presented technical problems in applying both the fluorescent antibody technic and the classical antiglobulin consumption method to the immunology of white blood cells.7’8 The labelled antibody method herein detailed also faces this problem. Quantitative information will therefore also be set forth concerning this “nonspecific” adsorption of human globulin and of rabbit globulin by nonexposed (i.e., nonsensitized) leukocytes, and particular attention will be paid to the degree of removal of such “nonspecifically” adsorbed globulin that can he attained with various washing procedures. The J131 method herein detailed reflects an extension of previous experience with the classical anti-human-globulin consumotion test.7’8 In the consumption test intact or Ivophilized leukocytes are incubated with putatively immune serum, thoroughly washed and then allowed to “adsorb” anti-human-globulin (Coombs) serum. Significant adsorption of the Coomhs serum by the white blood cells is reflected by a fall in titer of the serum. Such a fall is taken to indicate that the leukocytes have been “sensitized” by globulin antibody present in the putatively immune serum. The fall in Coombs titer, called “antiglobuun consumption,” is measured with a reference system of anti-Rh-sensitized