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Weight Estimates of Rabbit Anti-Human Serum Albumin Based on Antigen-Binding Capacity¹

From the Department of Microbiology, The Johns Hopkins University, School of Medicine, Baltimore, Maryland

Abstract

A quantitative technique is described for weight estimations of anti-human serum albumin (HSA) based on the primary union of the specific reactants rather than on the formation of insoluble aggregates. Trace-labeled I¹²⁵·HSA is added, in quantities adequate to supply a great excess of antigen, to a series of tubes containing a constant volume of rabbit anti-human serum albumin. The globulins are precipitated and washed with half saturated ammonium sulfate, and assayed for I¹²⁵·HSA. These values are converted to weight units of HSA, after appropriate correction, and are in turn used to calculate the absolute amount of antibody in the serum.

The results obtained in these assays are relatively insensitive to such variables as pH, duration of antigen-antibody incubation, and the presence of ethylenediaminetetraacetic acid.

A recovery assay with sheep anti-rabbit -globulin serum gave results similar to those obtained by mABC and by quantitative precipitin analyses.

The specificity of the assay system has been investigated with rabbit antisera to egg albumin, bovine serum albumin, human -globulin, and pneumococcus polysaccharide Type VI.

Footnotes

¹ Submitted to the School of Hygiene and Public Health, The Johns Hopkins University, in conformity with the requirements for the degree of Master of Science.

Support for this investigation has been available, in part, from the National Science Foundation, Grant No. G-6205; The American Cancer Society, Inc., Grant No. T-257; The National Institute of Allergy and Infectious Diseases of the United States Public Health Service, Grant No. AI-03151; and from the Office of The Surgeon General, Department of the Army, under the auspices of the Commission on Immunization of the Armed Forces Epidemiological Board, Contract No. DA-49-193-MD-2468.