Differential sensitivity of CD20 epitopes to detergent lysis.

(A) Raji B cells were lysed in either 1% Triton X-100 or 1% digitonin and cleared lysates were layered on top of 5% to 40% linear sucrose density gradients and centrifuged to equilibrium. Fractions were collected from the top of the gradient and probed by immunoblot using a polyclonal antibody raised against a cytoplasmic CD20 peptide (anti-CD20N). Densitometry analysis was performed and the results plotted to generate the graphs shown. Molecular weight standards were run in parallel samples (arrows). (B) Immunoprecipitation of CD20 by mAbs 1F5, 2H7, and B1 from Raji B cells lysed in 1% Triton X-100. The mAbs were added to cleared (13 000g) lysates as indicated to immunoprecipitate CD20. Samples were analyzed by anti-CD20N immunoblot. (C) Immunoprecipitation of CD20 by mAbs 2H7 and B1 from Raji B cells lysed in either 1% Triton X-100 or in 1% digitonin. The mAbs were added to cleared (13 000g) lysates as indicated. Anti-CD20N immunoblots were analyzed by densitometry and the relative density of the CD20 bands was expressed as the percent of the amount precipitated by B1. (D) As in panel C, except that the lysates were cleared at 100 000g for 1 hour. Note the loss of 2H7-precipitable CD20 from Triton lysates.