The proposed method was designed to replace the tedious and difficult separation of immunoglobulin M (IgM) from IgG by sucrose gradient sedimentation. In this method, a 250-rd portion of serum diluted 20-fold was passed through a small column of quaternary aminoethyl-Sephadex A-50 ion exchanger. IgG was not retained, but additional washes were required to remove all but 5%. A second buffer-eluting fluid recovered an average of 80% of the original IgM in a defined dilution. The entire operation took 15 min. The efficiency of this process was evaluated by the following: (i) radial immunodiffusion measurements of IgG and IgM; (ii) recovery studies of isohemagglutinins; and (iii) demonstrated removal of interference by the rheumatoid factor. The method was applied successfully to distinguish rubella IgM antibody.
Horse-radish peroxidase and glucose oxidase were each separately conjugated to identical aliquots of goat IgG containing anti-human IgG antibodies. We used a minor modification of the periodate method of Nakane and Kawaoi to covalently bind each enzyme to IgG. Glucose oxidase conjugates proved superior to peroxidase conjugates based on the following qualities. The glucose oxidase conjugates had 1) usable dilutions 2 to 10 times greater than peroxidase conjugates, 2) much lower background or control non-specific activities, and 3) nearly twice the sensitivity as expressed by absorbance change vs. change in antigen. Comparison of the antibody titers showed the glucose oxidase conjugate with 80% and the peroxidase conjugate with 20% of the original goat antibody.
In this two-point Scatchard-plot assay, with which a test of competitive inhibition is combined, the sample is mechanically homogenized in a buffer containing dithiothreitol, ultracentrifuged to obtain a fat-free cytosol, the protein content of which is then adjusted, and free and bound labeled estradiol are separated with dextrancoated charcoal after overnight incubation. We tested the method for precision and reliability by assaying such cytosols from pregnant rabbit uteri before and after dilution with kidney cytosol, and by assaying several other target and nontarget animal and human tissues. The Scatchard plot data were more reliable than tests for percent inhibition of binding by a competitor (diethylstilbestrol). For a tumor tissue to be judged positive it must bind at least 8 fmol of estradiol per milligram of protein and have a Kd of 0.1 to 5 x 10-10 mol/liter. Some nontarget tissues showed less than 70% inhibition by 104-fold concentrations (over labeled estradiol) of inhibitor. Of 19 breast-tumor specimens, seven were found to be positive.
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