BRIEF COMMUNICATION

Standardization and stability of

immunoglobulin E (IgE)
Michael Bazaral, Ph.D., and Robert N. Hamburger, M.D. La Jolla, Cdif.

World Health Organizalion Reference Standard 68-541 for immzlnoglobuEQ E (IgE)

was found to contain, 6.46 ng. per unit relative to pwrified myeloma IgE. IgE is
relatively stable under various conditions of storage and promsing.

Standardization of IgE assays has been. relative to a purified myeloma

IgE’ or relative to the IgE Reference Standard 68-341,2 consisting of pooled
high-titer human serum which contained 10,000 U. per milliliter by definition.3
The use of two different standards makes comparison of results among labora-
tories impossible, and additional confusion results from the unwarranted as-
sumpGoon that 1 ng. of IgE is 1 U. of IgE. In an attempt to minimize this
problem, it has been suggested that IgE levels be reported in units relative
to Standard 68-34L3 We have additionally studied the quantitative relation-
ship between nanograms of IgE and units.
MATERIALS AND METHODS
IgE assays
IgE assays were done as previously described.5 The method consists of a radioimmunosor-
bent assay in which IgE in an unknown sample is measured by its blocking of the reaction
between 1251 PS myeloma IgE and anti-ND myeloma IgE bound to bromacetyl cellulose
(BAC). All samples were assayed in duplicate. Radioactive IgE (0.82 ng. in.0.4 ml. of 1 per
cent human serum albumin phosphate-buffered saline [HgA-PBS] containing 10 Pg per
milliliter IgG) was added initially to each tube. Serum samples or PS myeloma IgE (0.5 ml.,
diluted as required in 5 per cent HSA-PBS containing 50 cg per milliliter of IgG) were added.
Anti-ND-BAC (0.1 ml.), at a concentration previously determined to bind 55 per cent of the
1251 IgE in the absence of additional IgE, was added. The reaction tubes were capped and
vertically rotated at room temperature. The binding of IgE to the anti-ND-BAC under these
conditions was a first-order reaction with a half-life of 85 minutes. Therefore, samples were
routinely rotated for 16 hours to approach equilibrium. At the end of 16 hours, the assay
tubes were centrifuged (2000 x G at room temperature for 40 minutes) and 0.5 ml. of each

From the PBdiatria Immunology and Allergy Division, Department of Pediatrics, Universitj of
California, San Diego.
Supported by United States Public Health Service, National Institutes of Health Grants
HD03015 and AM29966.
Received for publication July 14, 1971.
Reprint requests to: Dr. Robert Hamburger, Department of Pediatrics, P.O. 109, University of
California, San Diego, La Jolla, Calif. 92037.
Vol. 49, No. 3, pp. 189-191
190 Bazaral and Hamburger J. ALLERGY CLIN. IMMUNOL.
MARCH 1972

supernatant was withdrawn and counted for 20 minutes, so that a minimum of 9,000 counts
were accumulated for each sample.
The amount of blocking for each sample was determined relative to 5 per cent HSA-lgG-
PBS diluent (0 per cent block) and a sample tube in which anti-ND-BAC was replaced by 0.1
ml. of PBS (100 per cent block) according to the formula:

sample (c.p.m.) - diluent (c.p.m.) x 100 = per cent block.

(no anti-ND) (c.p,m.) - diluent (e.p.m.)

The per cent block for duplicates was averaged and converted t,o units of IgE by reference
to a standard curve. Secondary standard curves were generated by nominal concentrations of
1.5, 4.5, 15, and 45 U. of PS my&ma IgE included in duplicate with each assay run.

IgE purification
PS myeloma serum, containing approximately 20 mg. per milliliter IgE, was fractionated
by DEAE-cellulose column chromatography with the use of gradient elntion. The IgE peak was
further purified on a Sephadex G-206 column. A single symmetrical peak was obtained, and the
central fractions were pooled. This material gave a single band on Ouchterlony immunodiffusion
with monospecific anti-IgE and gave no visable reaction with commercial antihuman IgG Fe.
When labeled with 1251, 98 per cent of the IgE protein counts bound to monospecific anti-PS
IgE coupled to Sepharose.
Protein nitrogen was determined by a micro-Kjeldahl technique, with a standard deviation
of 2 per cent on three determinations relative to an ammonium sulfate internal standard. A
conversion factor of 6.25 mg. of protein per milEgram of nitrogen is assumed in subsequent
calculations. The extinction coefficient of the purified IgE was 14.1 (Et’&) at 280 my.

IgE reference standard

A sample of IgE Reference Standard 68-341s was obtained from Dr. D. S. Rowe. An
additional sample was obtained from Dr. J. Fahey at the regional World Health Organization
Reference Center for Immunoglabulins, National Cancer Institute, National Institutes of
Health, Bethesda, Maryland. The lyopholyzed preparation was reeonstitut.ed&.o contain 9,346
U. of IgE per milliliter.
WHO Standard 68-341 was diluted to l/50 and l/100 to contain 187 and 93.5 U. per
milliliter. The values obtained in duplicate determinations relative to the secondary standard
curve were divided into the known values, and the resulting quotients were averaged to obtain
a correction factor. The IgE levels (relative to the secondary standard) determined for
duplicate 500 ng. per milliliter and 250 ng. per milliliter samples of pure PS myeloma IgE
were multiplied by this correction factor, and the averaged value of the ratio of units to
nanograms was computed. The values obtained from three separate runs were 0.395 MU. per
nanogram, 0.459 U. per nanogram, and 0.386 U. per nanogram, resulting in a mean value of
0.413 U. per nanogram + 8 per cent standard d&&ion. Thus, one WHO Reference Standard
unit equals 2.42 ng. of IgE.
Measurements of the stability of IgE under various conditions were also performed to
verify that the preparations were sufficiently stable under the conditions employed to obtain
a valid result. PuriAed PS myelonta IgE stored for 2 days at- room temperature at a eoncen-
tration of 11 pg per milliliter in phosphate-buffered ,saline (0.05M sodium phosphate 0.1&i
NaCl, pH 7.35) or in Sephadex column buffer (I.(fM NaCl, 0.3M tris, pH 8.0) lost 10 + 8
per cent of the initial antigenie activity, and a sample stored in DEAE buffer (O&65&$ sodium
phosphate, pH 8.0) lost 21 t 8 per cent of the antigenie activity. These determinations were
done in duplicate reIative to a sample of pure PS my&ma~IgE stored at a coneentra~ion of
0.22 mg. per milliliter in phosphate-buffered saline at 4” C. By comparison, the purification
conditions were less severe: IgE remained at room temperature in DEAE buffer for 6 hours
at concentrations no less than 50 pg per milliliter and in Sephadex buffer for 24 hours at
concentrations no less than 100 pg per milliliter.
Additionally, a sample of Standard 68841 reconstituted 6 months previously and main-
VOLUME 49 Standardization and stability of IgE 191
NUMBER 3

tained at 4’ C. in a l/10 dilution in 5 per cent human serum albumin in phosphate-buffered

saline retained 92 + 2 per cent of its initial activity (relative to a newly reconstituted sample
of Standard 68-341). Three duplicate determinations were done on each of three separate
occasions.
Finally, no loss of activity (+ 4 per cent) was observed in a sample of blood, from an
allergic patient, carried about at 32” C. for one day prior to separation, and the serum held
at room temperature for 5 more days and measured relative to an aliquot of the original sample
maintained at 4” C. These conditions simulate the worst handling to which a clinical sample
is likely to be subjected.

DISCUSSION
The ratio of ng PS myeloma IgE to Reference Standard IgE 68-341, 0.413
U. per nanogram t 8 per cent, observed in this work is in agreeement with
a previous determination, 0.46 U. per nanogram + 6 per cent, with the use of
similar methodology.5 It is unlikely that this ratio is affected by loss of anti-
genie activity of the PS myeloma. IgE during purification, since the antigenic
activity of IgE is relatively stable under a variety of conditions including
storage in dilute solution at room temperature in the buffers used during puri-
fication. It is also unlikely that quantitative errors result from using myeloma
protein PS, since the assay system is specific only for antigens shared by PS
and ND myeloma proteins.
IgE levels should continue to be reported in units until several independent
measurements are in agreement on the ratio of units to nanograms of IgE.
The technical assistance of Donna Mason and Carolyn Ashcraft are gratefully acknowl-
edged.

REFERENCES
1 Johansson, 8. G. O., Bennich, II., and Wide, L.: A new class of immunoglobulin in human
serum, Immunology 14: 265, 1968.
2 Rowe, D. S., and Wood, C. B. 8.: The measurement of serum immunoglobulin E levels in
healthy adults and children and in children with allergic asthma, Int. Arch. Allergy Appl.
Immunol. 39: 1, 1970.
3 Rowe, D. S., Tackett, L., Bennich, H., Ishisaka, K., Johannson, S. G. O., and Anderson,
5. G.: A research standard for human serum immunoglobulin E, Bull. W. H. 0. 43: 609,
1970.
4 Ishizaka, K., Tomioka, H., and Ishizaka, T.: Mechanism of passive sensitization, J.
Immunol. 106: 1459, 1970.
5 Bazaral, M., Orgel, H. A., and Hamburger, R. N.: IgE levels in normal infants and children
and an inheritance hypothesis, J. Immunol. 107: 794, 1971.