ABSTRACT
We determined the conditions for labeling rabbit Fab' with fluorescein isothiocyanate to provide maximal fluorescence intensity compatible with the preservation of integrity of its single thiol residue. We found that we could reproducibly substitute Fab' with two fluorescein moieties in 30 min at room temperature at pH 8.9. We achieved a high level of fluorescence intensity while we preserved the integrity of more than half of the existing thiols.
Subject(s)
Fluoresceins , Immunoglobulin Fab Fragments , Thiocyanates , Animals , Binding Sites , Fluorescein-5-isothiocyanate , Rabbits , Spectrometry, Fluorescence , Sulfhydryl CompoundsSubject(s)
Imidoesters/pharmacology , Immunoglobulin G/metabolism , Animals , Iodine Radioisotopes , Isotope Labeling , Radioimmunoassay , RatsABSTRACT
The IgG subclass distribution of rat antibodies to 13 different antigens was measured. Antibodies to protein and hapten-protein conjugates were predominantly IgG2a. Antigens labeled thymus-independent type 1, based upon responses in mice, stimulated both IgG2b and IgG2c antibodies, but little IgG2a. Polysaccharide and hapten-polysaccharide antigens (thymus-independent type 2) as well as phosphocholine-keyhole limpet hemocyanin, stimulated predominantly IgG2c antibodies. A division of antigens into essentially the same categories has been made on the basis of subclass restriction in mice. Antigens that stimulate IgG2c in rats stimulate IgG3 in mice. Thus, by comparing subclass preference with a variety of antigens, functional analogues among subclasses in different species can be identified.
Subject(s)
Antigens/immunology , Immunoglobulin Allotypes/biosynthesis , Immunoglobulin G/biosynthesis , Mice/immunology , Rats/immunology , Animals , Antibody Formation , B-Lymphocytes/immunology , Epitopes , Female , Immunization , Immunoglobulin G/immunology , MaleABSTRACT
Antigens have been classified previously into three categories, thymus-dependent (TD), thymus-independent type (TI) 1, and TI-2, based upon thymic dependence and ability to stimulate an immunodeficient strain of mouse, CBA/N. Here we demonstrate that the different antigen classes elicit IgG antibodies of different subclasses. TD antigens stimulate predominantly IgG1 antibodies, with smaller amounts of IgG2 and IgG3 being expressed. TI-1 antigens stimulate almost no IgG1 antibodies and equal amounts of IgG2 and IgG3. TI-2 antigens elicit predominantly IgG3 antibodies. Mice expressing the CBA/N phenotype are known to be nonresponsive to TI-2 antigens. This was confirmed in this study. In addition, we demonstrate that the IgG3 component of the response to TI-1 antigens is virtually absent in mice expressing the CBA/N phenotype, which supports our previous finding that the CBA/N defect may be restricted to a B-lymphocyte subpopulation containing most of the precursors of IgG3-secreting cells.
