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Detection of Immune Response Against Synthetic Polymers of Amino Acids Employing the Plaque-Forming Cell System

IV. Effect of Incorporation of Soluble Polypeptides Into the Plaque Assay Medium

Marianne Egan and Paul H. Maurer
J Immunol September 1, 1970, 105 (3) 710-718;
Marianne Egan
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Paul H. Maurer
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Abstract

Incorporation of homologous polypeptide (10-5 M) into plaque assay plates containing anti-GLA30 (LLL) immune lymph node cells specifically inhibited these PFC. However, at lower levels of antigen incorporation an enhancement of PFC was noted. The enhancement was complement-dependent, did not occur with non-immune lymph node cells and was inhibited by specific anti-allotype serum. Cross-reaction studies suggested that the polypeptide did not enhance by merely optimally coating the erythrocytes. Serum titers were measured on modified localized hemolysis-in-gel plates. Titers were increased when certain concentrations of antigen were incorporated into these plates, thereby implicating the increased hemolytic efficiency of complement or antibody in the presence of excess antigen. Puromycin (5 to 25 µg/ml) did not inhibit the production of unenhanced plaques, but prevented the optimal enhancement of plaques by antigen. These same concentrations of puromycin inhibited (40% to 96%, respectively) the incorporation of 14 C-L-leucine into trichloroacetic acid precipitable protein. The inclusion of polypeptide in the incubation medium increased the above incorporation by 20% to 170%; puromycin in addition to polypeptide prevented this increase, although the radioactivity was above that obtained when puromycin alone was present. A similar effect was noted with chloramphenicol. The results suggest that enhancement of anti-polypeptide plaques by incorporation of polymer in the medium is a complex phenomenon and is probably due to at least two factors: the increased hemolytic efficiency of immunoglobulin or complement in the presence of excess antigen, and the stimulation of protein synthesis in immune lymph node cells in the presence of excess antigen.

Footnotes

  • ↵3 Public Health Service Predoctoral Fellow. Taken from the thesis submitted in partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biochemistry.

  • ↵1 This work was supported by Grants A1-07825 and T1A1-334 from the National Institute of Allergy and Infectious Diseases and Public Health Service Predoctoral Fellowship 1-F1-GM-38,458-01. Paper 34 in a series of “Antigenicity of Polypeptides (Poly-α-Amino Acids).”

  • Received February 2, 1970.
  • Copyright, 1970, by The Williams & Wilkins Company
  • Copyright © 1970 by The American Association of Immunologists, Inc.

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The Journal of Immunology
Vol. 105, Issue 3
1 Sep 1970
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Detection of Immune Response Against Synthetic Polymers of Amino Acids Employing the Plaque-Forming Cell System
Marianne Egan, Paul H. Maurer
The Journal of Immunology September 1, 1970, 105 (3) 710-718;

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Detection of Immune Response Against Synthetic Polymers of Amino Acids Employing the Plaque-Forming Cell System
Marianne Egan, Paul H. Maurer
The Journal of Immunology September 1, 1970, 105 (3) 710-718;
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Print ISSN 0022-1767        Online ISSN 1550-6606