Evaluation of an in vitro method for the measurement of specific IgE antibody responses: the rat basophilic leukemia (RBL) cell assay.
The evaluation of allergenic potential is a key parameter in the safety assessment of novel proteins, including those expressed in genetically modified crops and foodstuffs. The majority of allergic reactions to food proteins are immediate type hypersensitivity reactions in which the principal biological effector is IgE antibody; the accurate measurement of specific IgE antibody is therefore a critical factor in experimental systems designed to characterize protein allergenic potential. Due to the presence of much higher concentrations of other immunoglobulin isotypes, the assessment of specific serum IgE antibody poses substantial technical challenges. We have examined the utility of the rat basophilic leukemia (RBL) cell line for the measurement of murine IgE responses. RBL cells were sensitized with mouse monoclonal anti-dinitrophenyl (DNP) IgE antibody and challenged with DNP-albumin conjugates with various hapten substitution ratios (SR). Polyclonal anti-OVA IgE antisera were also assessed for activity in the RBL assay. Results were compared with titers measured in homologous passive cutaneous anaphylaxis (PCA) assay. Marked degranulation of RBL cells was induced by conjugates with SRs of between 16 and 32, whereas conjugates with lower SRs (of 10 or 3) failed to elicit significant serotonin release. All conjugates were able to induce mast cell degranulation in vivo in a PCA assay. Anti-OVA antisera with PCA titers of 1/32 to 1/64 failed to stimulate RBL cell degranulation, whereas high titer antibody (1/2048 to 1/4096 by PCA) induced a positive RBL cell response. Successful stimulation of RBL cell degranulation requires not only appropriate epitope densities but also high affinity antibody. These data indicate that this assay is inappropriate for the routine analysis of specific polyclonal IgE antibody responses such as those that are induced by exposure to complex protein allergens.
Dearman
R J
RJ
Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire SK10 4TJ, UK. rebecca.dearman@syngenta.com
Skinner
R A
RA
Deakin
N
N
Shaw
D
D
Kimber
I
I
D000485:Allergens / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D001491:Basophils / Q000276:immunology; D015550:Cell Degranulation / Q000276:immunology; D045744:Cell Line, Tumor; D004136:Dinitrobenzenes / Q000276:immunology*; D004305:Dose-Response Relationship, Drug; D005260:Female; D007073:Immunoglobulin E / Q000096:biosynthesis* / Q000097:blood / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D010047:Ovalbumin / Q000276:immunology*; D010323:Passive Cutaneous Anaphylaxis; D051381:Rats; D012709:Serum Albumin / Q000276:immunology; D013047:Specific Pathogen-Free Organisms
D000485:Allergens; D000911:Antibodies, Monoclonal; D004136:Dinitrobenzenes; D012709:Serum Albumin; D007073:Immunoglobulin E; D010047:Ovalbumin