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816
SCIENTIFIC PAPER
NATURE VOL. 333 30 JUNE 1988
Human basophil degranulation triggered by very
dilute antiserum against IgE
E. Dayenas, F. Beauvais, J. Amara*, M. Oberbaum*, B. Robinzon t, A. Miadonna t, A. Tedeschit, B. Pomeranz§,
P. Fortner§, P. Belon, J. Sainte-Laudy, B. Poitevin & J. Benveniste||
INSERM U 200, Universite Paris-Sud, 32 rue des Carnets. 92140 Clamart, France
* Ruth Ben Ari Institute of Clinical Immunology, Kaplan Hospital, Rehovot 76100, Israel
t Department of Animal Sciences, Faculty of Agriculture, PO Box 12, The Hebrew University of Jerusalem, Rehovot 76100, Israel
t Department of Internal Medicine, Infectious Diseases and Immunopathology, University of Milano, Ospedale Maggiore Policlinico,
Milano, Italy
§ Departments of Zoology and Physiology,* Ramsay Wright Zoological Laboratories, University of Toronto. 25 Harbord Street,
Toronto. Ontario M5S 1Al, Canada
To whom correspondence should be addressed.
When, human polymorphonuclear basophils, a type of white blood cell with antibodies of the immunoglobulin E (IgE) type on its
surface, are exposed to anti-IgE antibodies, they release histamine from their intracellular granules and change their staining properties.
The latter can be demonstrated at dilutions of anti-lgE that range from 1 x 1012 to 1 x 10120; over that range, there are successive peaks of
degranulation from 40 to 60% of the basophils, despite the calculated absence of any anti-lgE molecules at the highest dilutions. Since
dilutions need to be accompanied by vigorous shaking for the effects to be observed, transmission of the biological information could be
related to the molecular organization of water.
THE antibodies responsible for human immediate hypersensi-
tivity belong to the IgE isotype1. The most salient feature of IgE
is its capacity to bind to mast cell and polymorphonuclear
basophil membranes through receptors with high affinity".
Human basophils are specifically challenged by immunological
stimuli such as allergens or anti-lgE antiserum that can bridge
IgE molecules in membrane,. This process triggers trans-
membrane and intracellular signals followed by granule
exocytosis with the release of histamine and loss of meta-
chromatic staining of basophil granules by a basic dye such as
toluidine blue. Optical basophil degranulation is well corre-
lated with other in vitro and in vivo procedures for the
diagnosis of allergy4-7.
In preliminary experiments, degranulation of human baso-
phils contained in leukocyte suspensions was induced not only
by the usual concentration of anti-lgE antibody (1 x 103 dilution
of anti-lgE antiserum, corresponding to 2.2 x 10-9 M anti-lgE
antibody in the assay), but also by very low concentrations of
this antibody (2.2 x 10-16/18 M), where the number of IgG anti-
lgE; molecules in the assay is supposedly too low to trigger the
process. We then further explored this phenomenon.
Serial tenfold dilutions of goat anti-human IgE (Fc) anti-
serum (1 mg specific antibody per ml) were prepared in
HEPES-buffered Tyrode's solution containing human serum
albumin (USA) down to 1 x 1060, dilution, corresponding to a
2.2 x l0-66 M theoretical concentration (th) in the assay (see
Fig. 1 legend for methods). The expected basophil degranula-
tion, which was assessed by counting cells with metachromatical
properties, was observed after exposure of leukocyte prepara-
tions to low antiserum dilutions with a maximum at ~ 1 x 103
dilution. Successive peaks of degranulation varying between 40
and 60% were then found down to 1 x 1060 dilution, with periods
of 6 to 9 tenfold dilutions (Fig. 1a). In other experiments, the
antiserum was serially diluted a hundred-fold down to 1 x 10120
(to give 2.2 x l0-126 M th in the assay) and similar results were
obtained (Fig. 16). Degranulation induced by high dilutions of
anti-lgE antiserum was observed in ten experiments on the full
range of dilutions down to 1 x 1060, when at least 70 similar
results were obtained at one or the other part of the high dilution
scale in the participating laboratories (Toronto, preliminary
results). As controls, goat antihuman IgG (Fc) antiserum (Fig.
1b, n = 4) or Tyrode's solution containing HSA (n = 5) were
diluted down to 1 x 10120 and 1 x 1030, respectively. Cells
incubated in conditions identical to those with anti-lgE anti-
serum gave no significant degranulation. The repetitive waves of
anti-IgE-induced degranulation were reproducible, but the
peaks of degranulation could shift by one or two dilutions with
every fresh sequential dilution of anti-lgE and depended on the
blood sample. The waves of basophil degranulation were also
seen with substances other than anti-lgE anti-serum at high and
low dilutions, such as monoclonal anti-human IgE antibodies,
specific antigen in allergic patients or in peroxidase-immunized
rabbits, phospholipase A2 from bee venom or porcine pancreas,
the Na+ ionophore monensin (up to 90% degranulation at 1 x
10-30 M th) and the Ca2+ ionophores A23187 and monomycin (1 x
10-38 M th). The specificity of the observed effects at high dilu-
tions (already noted when comparing antiserum against IgE
with antiserum against IgG) was further strikingly illustrated in
the ionophore experiments, because removing the correspon-
ding ion from the cellular environment blunted basophil
Blind experiments: test tubes were randomly coded twice by two independent pairs of observers and assayed. The codes were simultaneously broken at the end of all
experiments. Dilutions of anti-lgE antiserum were performed as described in legend to Fig. 1.
* Uncpded additional tubes for negative (Tyrode's-HSA) or positive (algE 1 x 10 ~*) controls, t Data represent the mean 1 s.e. of basophil number actually counted in
triplicate (see legend to Fig. 1 for methods). % Number in parenthesis indicates percentage degranulation compared with Tyrode's-HSA.
NATURE VOL. 333 30 JUNE 1988
Fig. 1 Human basophil
degranulation induced either
by anti-IgE anti-serum (•)
diluted tenfold from 1 x 102
down to 1 x l060 (a) or
hundredfold down to 1 x
10120 (b) or by anti-IgG anti-
serum (O) diluted hundred-
fold from 1 x 102 down to 1
x 10120 (representatives of
at least 10 experiments for
anti-IgE and 4 experiments
for anti-IgG). The signifi-
cant (P < 0.05) percentage
of degranulation was 15%
(a) and 20% (b). (....) rela-
tion to the number of
counted basophils from
control wells'''.
Methods Goat anti-human
IgE (Fc) antiserum or as a
control, goat anti-human
IgG (Fc) antiserum (Nordic
Immunology, The Nether-
lands) was serially diluted as
indicated above in HEPES-
buffered Tyrode's solution
(in g 1-1 : NaCl, 8; KCl.
0.195;HEPES,2.6;EDTA-
Na4. 1.040; glucose, 1
human serum albumin (HSA), 1.0; heparin. 5000 U per 1; pH 7.4). Between each
dilution, the solution was thoroughly mixed for 10 s using a Vortex. Given the
molecular weight of IgG molecules (150,000). the 1 x 1060 and 1 x 10120 dilutions
correspond in the assay to 2.2 x 10-66 M(th)and 2.2x l0-126 M (th) respectively.
Venous blood (20 ml) from healthy donors was collected using heparin (1 U per
ml) and a mixture of 2.5mM EDTA-Na4/2.5 mM EDTA-Na, (final concentrations)
as anticoagulants and allowed to sediment. The leukocyte-rich plasma was
recovered, twice washed by centrifugation (400g, 10 min) and finally resuspended
in an aliquot of HEPES-buffered Tyrode's solution. The cell suspension (10 μl)
was deposited on the bottom of each well of a microtitre plate containing 10 μl
CaCl2, (5 mM final) and 10 μl of either of anti-IgE or anti-IgG antiserum dilutions.
To a control well were added 10 μl CaCl2, and 10 μl Tyrode's but no anti-IgE or
anti-IgG antiserum, plates were then incubated at 37°C for 30 min. Staining
solution (90 ml; HX) mg toluidine blue and 280 μl glacial acetic acid in 100 ml 25%
ethanol, pH 3.2 — 3.4) was added to each well and the suspension thoroughly
mixed. Specifically redstained basophils (non-degranulated basophils) were
counted under a microscope using a Fuchs-Rosenthal haemocytometer. The
percentage of basophil degranulation was calculated using the following formula:
Basophil no. in control - basophil no. in sample/ basophil no. in control x 100.
Between 60 arid 120 basophils were counted in cell suspensions from control wells
after incubation either in the absence of anti-IgE antiserum, or in the presence of
anti-IgG antiserum.
degranulation.
To confirm these surprising findings, four blind experiments
were carried out (Table 1). In all cases the results were clear-cut,
with typical bell-shaped degranulations at anti-IgE dilutions
from 1 x 1032 to 1 x 1037. The replicates were usually very close
and of high significance (ANOVA test). In a fifth experiment, 7
control tubes and 3 tubes containing a dilution previously
determined as active (1 x 1034) were counted blind: basophil
degranulation was 7.7 ± 1.4% for the controls, and 44.8, 42.8
and 45.7% for the tubes containing diluted anti-IgE. The
random chance in all these experiments was 2% and therefore
the cumulative results statistically confirm the measured effect.
Two further blind experiments were performed using the
usual dilution procedure: of the 12 tubes used in the first
experiment (Table 2), 2 tubes contained goat anti-human anti-
serum IgE at 1 x 102 and 1 x 103 dilutions, 6 tubes contained
dilutions from 1 x 1032 to l x 1037, and 4 tubes buffer-HSA alone.
The tubes were then randomly coded twice by three parties, one
of which kept the two codes. The 12 tubes were each divided into
4. Three batches of 12 tubes were lyophilized, one of which was
used for gel electrophoresis, one for assay of monoclonal anti-
bodies, and the last (with the unlyophilized sample) for gel
electrophoresis and basophil degranulation. By comparing the
results of the different tests it was easy to identify the tubes
containing IgE at normal concentrations compared with the
tubes containing highly diluted IgE and the control tubes. When
the codes were broken, the actual results exactly fitted those
predicted, but HSA and its aggregates were present in all solu-
tions and complicated interpretation of the gel electrophoresis.
•SCIENTIFIC PAPER
817
Fig. 2 Electrophoresis (polyacrylamide 7-15%. bands revealed by silver staining):
samples numbered 1 to 5 are standards for the blind experiments a. c. e, h, m, p.
Lane 1. Molecular weight standards for electrophoresis; lane 2, monoclonal IgG
added with human serum albumin; lane 3. Tyrode's buffer without human serum
albumin; lane 4.1 x 102 anti-IgE dilution; lane 5.1 x 103 dilution. Samples tested
Wind: a and c, buffer; e, 1 x 1036 anti-IgE dilution: h. 1 x 102 anti-IgE dilution; m,
1 x 103 anti-IgE dilution; p. 1 x 1035 anti-IgE dilution.
So we performed another almost identical experiment, using 6
tubes containing unlyophilized samples and buffer without
HSA. Pour tubes contained antibody at 1 x 102, 1 x 103, 1 x 1035
and 1 x 1036 dilutions, and 2 contained buffer alone. These
tubes were coded and assayed according to the above protocol.
The decoded results were clear-cut, high basophil degranulation
being obtained with 1 x 102, 103, 1035 and 1036 dilutions, but no
anti-IgE activity or immunoglobulins were detected either in the
control tubes or in assays containing the 1 x 1035 and 1036
dilutions (Tables 2 and 3 and Fig. 2). Thus there is no doubt that
there was basophil degranulation in the absence of any detec-
table anti-IgE molecule.
These results may be related to the recent double-blind
clinical study of Reilly et al.8 which showed a significant reduc-
tion of symptoms in hay-fever patients treated with a high
dilution (1 x 1060) of grass pollen versus placebo, and to our ex
vivo experiments in the mouse". We have extended these
experiments to other biological systems: using the fluorescent
probe fura-2, we recently demonstrated changes in intra-
cellular Ca2+ levels in human platelets in the presence of the
Ca2+ ionophore ionomycin diluted down to 1 x 10-39 M th (F. B.
et al., unpublished results).
Using the molecular weight of immunoglobulins and
Avogadro's number, we calculate that less than one molecule
of antibody is present in the assay when anti-IgE antiserum is
diluted to 1 x 1014 (corresponding to 2.2 x 10-20 M). But in the
experiments reported here we have detected significant
basophil degranulation down to the 1 x l0120 dilution. Specific
effects have also been triggered by highly diluted agents in
other in vitro and in vivo biological systems8-11, but still remain
unexplained. The valid use of Avogadro's number could be
questioned, but we are dealing with dilutions far below the
Avogadro limit (1 x 10100 and below). It could be argued that
our serial dilution procedure is subject to experimental error,
but this is ruled out because: (1) pipette tips and glass micro-
pipettes were discarded between each dilution (performed
under laminar flow hood). (2) The c.p.m. in tubes containing
serially diluted radioactive compounds decreased in propor-
tion to the degree of dilution down to the background (data
not shown). (3) Contamination would not explain the succes-
sive peaks of activity that evoke a periodic phenomenon and
not a monotonous dose-effect curve, as usually observed when
concentration of an agonist decreases. (4) To eliminate the
possibility of contaminating molecules present in the highly
diluted solutions, we carried out two series of experiments which
can be summarized as follows. An Amicon membrane with
molecular weight cut-off 10K retained the basophil degranu-
lating IgG (150K) present at low dilutions (1 x 102 1 x 103) in
anti-IgE antiserum. By contrast, the activity present at high
dilutions (1 x 1027 1 x 1032) was totally recovered in the 10K
Amicon filtrate. Anion or cation exchange chromatography,
818
•SCIENTIFIC PAPER
NATURE VOL. 333 30 JUNE 1988
Blind experiments and dilution protocols as in Table 1. —, Lack of strained
bands. ND, not determined. A faint band corresponding to IgG appeared after
reduction by 2-mercaptoethanol.
* Basophil degranulation tests I, II, III were performed using 3 different blood
samples (see Fig. 1). Percentage basophil degranulation induced by aIgE, as
compared to Tyrode's HSA, was calculated from duplicates.
t Electrophoresis (polyacrylamide 7-15%, revealed by silver staining) was carried
out in Rehovot (A) and at INSERM U 200 (B).
$ Uncoded additional tube for positive control.
§ + +, + Bands correspond to IgG present in large or small amounts.
according to the type of resin used and the pH, did or did not
retain the anti-IgE IgG at low dilutions, whereas the same
activity at high dilution was always excluded from the columns
and fully recovered in the first eluate. These filtration and ion-
exchange experiments demonstrated that the activity of the
antiserum at high dilution cannot result from contamination of
the highly diluted solution with the starting material. They
showed, in addition, that the high-dilution activity does not
present in space the steric conformation of an IgG molecule as it
acts like a 150K charged molecule, but is not retained by the 10K
filter or by a charged chromatography column.
We then investigated the physical chemical nature of the
entity active at high dilution. Our results can be summarized as
follows. (1) The importance of agitation in the transmission of
information was explored by pipetting dilutions up and down ten
times and comparing with the usual 10-s vortexing. Although the
two processes resulted in the same dilution (degranulations at
1 x 102 and 1 x 103 were superimposable whatever the dilution
process), degranulation did not occur at high dilution after
pipetting. Ten-second vortexing was the minimum time
required, but vortexing for longer (30 or 60 s) did not increase
high-dilution activity. So transmission of the information
depended on vigorous agitation, possibly inducing a sub-
molecular organization of water or closely related liquids. (2)
The latter is possible as ethanol and propanol could also support
the phenomenon. In contrast, dilutions in dimethylsulphoxide
did not transmit the information from one dilution to the other,
but increasing the proportion of water in dimethylsulphoxide
resulted in the appearance and increment of the activity at high
dilutions. (3) Heating, freeze-thawing or ultrasonication
suppressed the activity of highly diluted solutions, but not the
activity of several active compounds at high concentrations. A
striking feature was that molecules reacted to heat according to
their distinctive heat sensitivity, whereas all highly diluted solu-
tions ceased to be active between 70 and 80°C. This result
suggests a common mechanism operating at high dilution,
independent of the nature of the starting molecule.
Therefore we propose that none of the starting molecules is
present in the dilutions beyond the Avogadro limit and that
specific information must have been transmitted during the
dilution/shaking process. Water could act as a 'template' for
the molecule, for example by an infinite hydrogen-bonded
network12, or electric and magnetic fields13,14. At present we can
only speculate on the nature of the specific activity present in the
highly diluted solutions. We can affirm that (1) this activity was
established under stringent experimental conditions, such as
blind double-coded procedures involving six laboratories from
four countries; (2) it is specific for the ligand first introduced, as
illustrated when goat antiserum (IgG) anti-human IgE, but not
goat IgG anti-human IgG supported this phenomenon. The link
between high and low anti-IgE dilutions is shown as we could not
detect basophil degranulation at high dilutions if it did not occur
within the classical range. High dilutions of histamine, but not of
its carboxylated precursor histidine, inhibited IgE-dependent
basophil degranulation. Finally, ionophores at high dilution did
not work when the specific ion was removed from the cell
suspension (F.B., unpublished results). (3) Using six bio-
chemical and physical probes, we demonstrated that what
supports the activity at high dilutions is not a molecule. (4)
Whatever its nature, it is capable of 'reproducing, subtle molec-
ular variations, such as the rearrangement of the variable region
of an IgG (anti-€ versus anti-y) molecule.
The precise nature of this phenomenon remains unex
plained. It was critical that we should first establish the reality
of biological effects in the physical absence of molecules.
The entities supporting this 'metamolecular' biology can only
be explored by physical investigation of agitation causing inter-
action between the original molecules and water, thus yielding
activity capable of specifically imitating the native molecules,
though any such hypothesis is unsubstantiated at present.
We thank Professor Z. Bentwich from Ruth Ben Ari Insti-
tute for supervision of experiments conducted in Rehovot. The
participation of J. Geen (Univ. Toronto), B. Descours and
C. Hieblot (INSERM U 200) in experiments and of V. Besso in
editing is gratefully acknowledged. This work is dedicated to the
late Michel Aubin, who played a decisive role in initiating it.
Received 24 August 1987; accepted 13 June 1988.
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11 Poitevin. B. Davenas. E. & Benveniste. J. Br. J. clin Pharmac. 25.439 444(1988)
12. Stanley, H. E . Teixeira. J. Geiger. A. & Blumherg. R L Physics 106A. 260-277 (1981).
13. Frohlich. H. Adv. Electron. A Electron. Phys. 53. 85- 1/2 (1980).
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Editorial reservation
READERS of this article may share the incredulity of the many referees
who have commented on several versions of it during the past several
months. The essence of the result is that an aqueous solution of an
antibody retains its ability to evoke a biological response even when
diluted to such an extent that there is a negligible chance of there being a
single molecule in any sample. There is rfo physical basis for such an
activity. With the kind collaboration of Professor Benveniste, Nature
has therefore arranged for independent investigators to observe repeti-
tions of the experiments. A report of this investigation will appear
shortly. •
