溶血プラーク法の一般化

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The present report provides an assay method for detecting immunologically potent cells producing antibodies against protein antigens. The principle involved, is based on the passive immune hemolysis, as well as the efficacy of antiglobulin sera for detection of low-efficiency-hemolysin (7S) producing cells. A protein antigen (human gammaglobulin, HGG) is conjugated to highly fresh sheep-RBC by means of bis-diazotized benzidin (BDB). The standard procedure can be summarized as follows: A bottom layer is made from 1.2 per cent agarose dissolved in Dulbecco's phosphate buffered saline in a Petri dish. Over this layer is poured a top layer of 0.8 per cent agarose in Parker 199 medium containing the antigencoated sheep-RBC, mouse spleen cells (source of antibody-producing cells), and rabbit anti-mouse IgG antiserum. The plates are kept for 40 minutes at room temperature, and thereafter incubated for 2 hours at 37℃ in a humid atomosphere. The complement (1:10 dilution) is added and the plates are incubated for additional 30 minutes. Immediately after this time, the plates are fixed with 2.5 per cent glutaraldehyde solution, because of sheep-RBCs coupled to protein with BDB being liable to spontaneous lysis. The number of plaques formed varies greatly with concentration of anti-IgG serum. An optimal concentration for maximal development of plaque lies in 1:2 to 1:16 dilution of the antiserum, 0.1 ml of which is added to each plate. This modified technique was applied to the time course study of immune response of mice to HGG. Primary immune response of male ddN strain mice to alum-precipitated HGG, injected intraperitoneally, was of slight degree. The number of PFC at the height of the primary response was three order of magnitude lower than the corresponding number of PFC for the secondary response evoked after an interval of 4 weeks in hyperimmunized mice. On the other hand, HGG, when injected with Freund's complete adjuvant elicited a better primary response.
1969-07-30

溶血プラーク法の一般化

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