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Radioimmunoassay on polycarbonate membranes: A sensitive and simplified method for the detection and quantitation of antibody
VOLUME48 NUMBER1
Abstracts 7
control subjects were assayed by the I~MCT to determine the quantitative relationship of the I g E antibody level of the sera and the dose of specific antigen required to induce morphologic changes in the mast cells. These changes consisted mainly of palisading of the granules around the periphery of these cells and vacuolization of these granules within the cytoplasm of the cells~ and they were observed microscopically. The percentage of altered cells versus the total cell population was recorded. The titers of 8 of the allergic sera were also assayed by Prausnitz-Kiistner passive transfer tests performed on normal recipients and compared with results obtained by the RMCT. The results indicated that each of the sera tested exhibited a maximum response t h a t was dependent upon the antigen concentration. There was an inverse ratio between the antigen concentration and the titer of the allergic serum as determined by P-K tests. Sera with the highest titer of I g E antibody required the lowest concentration of antigen to elicit a maximum response. By means of antigen titration, it was also possible to d~stinguisb the allergic group of patients from the control group. The authors also studied the changes in I g E levels in 2 untreated ragweed patients during and a f t e r the ragweed season and found t h a t significant amounts of I g E antibodies wer~ detectable for 7 months or more after the season ended. I n other studies it has been found that I g E antibody levels in allergic sera as determined by the I~MCT antigen titration method are comparable to those computed by the t~AST technique. There also appears to be a correlation between RMCT and direct skin tests. The authors hesitate to suggest " t h a t the antigen-antibody relationship necessary to produce histamine release from human basophils may be operative for rat mast cells."
M.M.A. Radioimmunoassay on polycarbonate membranes: A sensitive and simplified method for the detection and quantitation of antibody. Green, R. L., Scales, R. W . , a n d K r a u s , S. J . : A p p ] . M i e r o b i o l . 27: 475, ] 9 7 4 . The authors describe a new radioimmunoassay technique, equivalent in sensitivity to the indirect fluorescent antibody test, whereby whole bacteria (type I gonococci) or soluble antigens (rabbit albumin) are coupled to Sepharose beads by cyanogen bromide, and layered over the surface of polycarbenate membranes held in a sampling manifold. This technique permits simultaneous filtration of up to 30 individual ]0-ml. samples. Washing and suctioning off of excess fluid is a simple procedure, and the bead-coupled antigens adhere to the membranes. Antisera, e.g., goat a n t i r a b b i t IgG or human antigonococcus, then are able to react with the membrane surface-bound antigen and in turn react sequentially with radiolabeled indicator antibody. An important advantage in the use o f polycarbonate membranes over cellulose acetate membranes is that there is less likelihood of nonspecific protein adhering to the polycarbonate system. This makes possible l~AST-like assay for detection of antibody other than IgE. Day-to-day reproducibility of results was judged to be good, with only minimal variability between duplicate samples. To date there is still considerable overlap between gonorrhea patients and control sera in the type I gonococcus experiment, indicating that there is as yet no workable immunologic test for gonorrhea. Pitfalls in the use of whole bacterial antigens are described. D.S.
Anaphylaxis The enhanced emcacy of disodium cromoglycate {DSCG) in DSCG predosed rats. J o h n s o n , H. G., a n d V a n H o u t , C. A~: P r e c . Soc. E x p . B i o l . M e d . 143: 427, 1973. Disodium cromoglycate (DSCG) has been shown to inhibit the passive cutaneous anaphylaxis (PCA) reaction in the rat. I n the present study it was shown t h a t in sensitized
Abstracts 7
control subjects were assayed by the I~MCT to determine the quantitative relationship of the I g E antibody level of the sera and the dose of specific antigen required to induce morphologic changes in the mast cells. These changes consisted mainly of palisading of the granules around the periphery of these cells and vacuolization of these granules within the cytoplasm of the cells~ and they were observed microscopically. The percentage of altered cells versus the total cell population was recorded. The titers of 8 of the allergic sera were also assayed by Prausnitz-Kiistner passive transfer tests performed on normal recipients and compared with results obtained by the RMCT. The results indicated that each of the sera tested exhibited a maximum response t h a t was dependent upon the antigen concentration. There was an inverse ratio between the antigen concentration and the titer of the allergic serum as determined by P-K tests. Sera with the highest titer of I g E antibody required the lowest concentration of antigen to elicit a maximum response. By means of antigen titration, it was also possible to d~stinguisb the allergic group of patients from the control group. The authors also studied the changes in I g E levels in 2 untreated ragweed patients during and a f t e r the ragweed season and found t h a t significant amounts of I g E antibodies wer~ detectable for 7 months or more after the season ended. I n other studies it has been found that I g E antibody levels in allergic sera as determined by the I~MCT antigen titration method are comparable to those computed by the t~AST technique. There also appears to be a correlation between RMCT and direct skin tests. The authors hesitate to suggest " t h a t the antigen-antibody relationship necessary to produce histamine release from human basophils may be operative for rat mast cells."
M.M.A. Radioimmunoassay on polycarbonate membranes: A sensitive and simplified method for the detection and quantitation of antibody. Green, R. L., Scales, R. W . , a n d K r a u s , S. J . : A p p ] . M i e r o b i o l . 27: 475, ] 9 7 4 . The authors describe a new radioimmunoassay technique, equivalent in sensitivity to the indirect fluorescent antibody test, whereby whole bacteria (type I gonococci) or soluble antigens (rabbit albumin) are coupled to Sepharose beads by cyanogen bromide, and layered over the surface of polycarbenate membranes held in a sampling manifold. This technique permits simultaneous filtration of up to 30 individual ]0-ml. samples. Washing and suctioning off of excess fluid is a simple procedure, and the bead-coupled antigens adhere to the membranes. Antisera, e.g., goat a n t i r a b b i t IgG or human antigonococcus, then are able to react with the membrane surface-bound antigen and in turn react sequentially with radiolabeled indicator antibody. An important advantage in the use o f polycarbonate membranes over cellulose acetate membranes is that there is less likelihood of nonspecific protein adhering to the polycarbonate system. This makes possible l~AST-like assay for detection of antibody other than IgE. Day-to-day reproducibility of results was judged to be good, with only minimal variability between duplicate samples. To date there is still considerable overlap between gonorrhea patients and control sera in the type I gonococcus experiment, indicating that there is as yet no workable immunologic test for gonorrhea. Pitfalls in the use of whole bacterial antigens are described. D.S.
Anaphylaxis The enhanced emcacy of disodium cromoglycate {DSCG) in DSCG predosed rats. J o h n s o n , H. G., a n d V a n H o u t , C. A~: P r e c . Soc. E x p . B i o l . M e d . 143: 427, 1973. Disodium cromoglycate (DSCG) has been shown to inhibit the passive cutaneous anaphylaxis (PCA) reaction in the rat. I n the present study it was shown t h a t in sensitized