Anti-gamma Globulin Research Articles

IMMUNE CYTOLYSIS: ELECTRON MICROSCOPIC LOCALIZATION OF CELLULAR ANTIGENS WITH FERRITIN-ANTIBODY CONJUGATES

Immune gamma globulin has been coupled to ferritin by the diisocyanate method of Singer. The final product ("ferroglobulin") had approximately 13 per cent of its gamma globulin coupled to ferritin, and roughly half of its ferritin coupled to gamma globulin. The uncoupled gamma globulin could be removed by ultracentrifugal sedimentation of the free ferritin and the ferritin-antibody conjugates. The characteristics of the native antibody were retained by the ferritin-antibody conjugates, for they could be precipitated by anti-gamma globulin antisera, and when used as antibodies, they reacted specifically with soluble and cellular antigens. Ferroglobulin preparations made from rabbit antisera against whole ascites tumor cells were incubated with the cells, and the location of ferritin determined by electron microscopy of thin-sectioned material. It was found that the immune ferroglobulins localized specifically on antigens of the cell membrane. Some of the ferritin label entered the cells by pinocytosis, but the ferritin-antibody units did not appear able to pass directly through the cell membrane into the cytoplasmic matrix. When cells were incubated with ferritin-labeled antibody and complement, antibody could be located in the cytoplasmic matrix, and it therefore appeared that complement action was required before antibody could pass directly through the cell membrane. This finding was consistent with previous observations that the plasma membrane of an antibody-complement treated cell becomes permeable to large molecules. In broken cell preparations incubated with ferroglobulin, antibody combined with amorphous material and with structures derived from cell membranes and from smooth membranes of the endoplasmic reticulum. The data favor the concept that antigens contained within the membranous structures are most important in the formation of cytotoxic antibodies. The reported experiments support the view that cytotoxic antibodies fix primarily to surface antigens of the cell membrane. The subsequent action of complement establishes the permeability defect that induces the osmotic lysis of the cell and permits antibody to pass into the cell where it may act in a similar fashion on intracellular organelles.

Active components of antiglobulin sera reacting with red cells sensitized with natural and immune anti-A haemagglutinins.

Four antihuman globulin sera (AHG) were prepared by immunizing rabbits with (1) purified gamma globulin and (2) anti-A haemagglutinins purified by elution from sensitized cells namely, (a) natural anti-A from a male who had received no known antigenic stimulus, (b) immune anti-A + B after delivery, and (c) immune anti-A from the cord blood of an infant with mild ABO haemolytic disease. The highest dilution of AHG sera agglutinating haemagglutinin-sensitized cells was essentially the same with the natural (2a) and immune (2b) sera with titers from 1:40 to 1:320 while with the immune (2c) serum the titers ranged from 0 to 1:20. The antihuman gamma globulin (AHGG) serum did not agglutinate cells sensitized with the natural anti-A and showed a titer of 1:2560 with cells sensitized with immune anti-A+B.Micro-Kjeldahl analysis of the four AHG sera precipitated with gamma globulin showed marked differences in μg nitrogen per ml of gamma globulin and of antigamma globulin and in the ratio of antibody to antigen at maximum precipitation. There appeared to be a quantitative relationship between the activity of serum in the antiglobulin test and the ability of serum to combine specifically with gamma globulin. Quantitative analysis snowed that the amount of gamma globulin in μg nitrogen required for maximum precipitation of the natural AHG serum was the same as required for neutralization of the serum agglutinating natural anti-A-sensitized cells but only one-third was required for serum agglutinating cord anti-A-sensitized cells. The gamma globulin (1) serum, which was inactive with cells sensitized with natural anti-A, restored the agglutinating activity of the gamma-globulin-neutralized natural AHG (2a) serum.Electrophoretic studies showed that normal rabbit serum, natural AHG rabbit serum, and gamma globulin AHGG contained the same globulin components except for the alpha one fraction. Both the AHG sera were characterized by a new alpha component while the gamma globulin AHG showed an additional new alpha component. The significance of these findings is discussed.