Evaluation of an in vitro method for the measurement of specific IgE antibody responses: the rat basophilic leukemia (RBL) cell assay. (1)
As IgE antibody is present in the serum at concentrations that are orders of magnitude lower than other isotypes of immunoglobulin including IgG, precautions must be taken against the blocking effects of or cross-reactivity of reagents with such antibody. It is therefore necessary to employ steps that either deplete of non-IgE isotypes, or that are selective for IgE antibody.
Mouse monoclonal anti- DNP IgE antibody diluted to various extents with PBS (10–0.001 ug/ml) was injected (30 ul) into the dermis of the ears of naive recipient mice.Two days later, 0.25 mg of DNP–albumin conjugates, together with Evans Blue dye (1.25 mg), in 0.25 ml of PBS were injected intravenously.
Relatively low levels of serotonin release were recorded at high concentrations of anti-DNP IgE (2000–500 ng/ml), with RBL cell degranulation increasing to maximal levels between 250 and 7.8 ng/ml of anti-DNP IgE. At concentrations of 3.9ng/ml and below, serotonin release approached background levels. The prozone shape of the dose–response curve was unaffected by the challenge concentration of hapten–protein conjugate, with a bell-shaped profile recorded for each dose used.
Conjugates with substitution ratios of 32–16 mol of DNP per mole of albumin induced typical profiles of serotonin release, with optimal RBL cell degranulation achieved at concentrations of between 250 and 31.25 ng/ml anti-DNP IgE.
All five conjugates provoked the maximal response when tested against 10,000 ng/ml anti-DNP IgE.
Exposure to 2% OVA induced positive PCA reactions with serum dilutions of 1/8, 1/16 and 1/32, with cutaneous responses also recorded in one assay only at a 1/64 dilution of serum, equating to anti-OVA IgE titers of 1/32, 1/32 and 1/64.
The PCA is more sensitive than the RBL assay for the detection of both high and low titer polyclonal IgE antibody. RBL cell degranula- tion is affected markedly by antibody affinity, such that a combination of low affinity and low epitope density fails to cross link FcR effectively on the surface of RBL cells.
Under a marked prozone effect (bell-shaped dose–response curve), which may be circumstances where IgE antibody is saturating, there will be insufficient conjugate available to crosslink receptor bound IgE and optimal degranulation will not be induced, resulting in a potential underestimation of IgE levels.