Purification of human basophils using mouse monoclonal IgE.
A method was developed to purify human blood basophils using monoclonal mouse IgE. Enriched suspensions of basophils were sensitized with IgE anti-trinitrophenol (TNP) hapten which was prepared from hybridoma culture supernatants. Rosettes were induced with TNP-coupled red cells and separated from contaminating cells on Percoll gradients. Basophils were dissociated from red cells using cold hypotonic lysis. Using this technique the percentage of basophils was increased from less than 1% to an average of 83.5 +/- 7.8% with a recovery of 58.0 +/- 12.7%. Final purities were highest when enriched sensitized suspensions contained greater than or equal to 10% basophils. Using a radioenzymatic assay, purified basophils were shown to contain an average of 1.3 +/- 0.3 pg histamine. Anti-human, but not anti-mouse IgE, released histamine at an optimal dilution. Using this technique, in 3-4 h, we are able to prepare highly purified suspensions of basophils for further study.
Schroeder
J T
JT
Department of Pathology, East Carolina University School of Medicine, Greenville, NC 27834.
Hanrahan
L R
LR
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001491:Basophils / Q000166:cytology / Q000276:immunology; D002021:Buffers; D002469:Cell Separation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D006636:Histamine Release; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007073:Immunoglobulin E / Q000276:immunology*; D007958:Leukocyte Count; D051379:Mice; D012397:Rosette Formation; D014301:Trinitrobenzenes / Q000276:immunology
D000911:Antibodies, Monoclonal; D002021:Buffers; D014301:Trinitrobenzenes; D007073:Immunoglobulin E