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Патент USA US3035996

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Patented May 22, 1962
Roy T. Fisk, Glendale, and Nancy K. Allen, Les An
geles, Gali?, assignors to Hyland Laboratories, Los
Angeles, Calif., a corporation of California
No Drawing. Filed Ian. 2, 1959, Ser. No. 784,455
5 Claims. (Cl. 167-84.5)
one individual, they agglutinated the red cells of all
other human beings, and consequently did not divide
them into groups. In 1945 Dr. W. C. Boyd discovered
that extracts of some seeds were speci?c for certain blood
group antigens (i.e., they agglutinated only blood of cer
tain blood groups), and thereby established the possibility
of using such extracts in blood grouping. Such a seed
extract reacts speci?cally with a blood cell factor and
behaves very muchlike a speci?c agglutinin of human
This invention relates to compositions which are useful 10 or animal origin. It is believed that this speci?city is
of an accidental nature and that the plant ‘forms the
reagents for the diagnosis and characterization of blood.
agglutinin-like substance without agglutinogenic stimu
Landsteiner discovered that human blood shows in
lation. For this
dividual differences characterized by agglu-tinogens car
“lectins” for the
ried by erythrocytes and by agglutinins present in blood
serum. The agglutinogens which comprise the four major 15 in order to avoid
would imply that
blood groups were identi?ed by means of anti-A and
reason, Dr. Boyd proposed the term
blood-group-speci?c plant agglutinins,
the use of the word “antibody” which
they were the result of immunization,
anti-B agglutinins which are of natural or normal occur
rence in serums of individuals not possessing the corre
which may or may not be the case.
sponding agglutinogen. Group 0 persons lack both A
and B cellular agglutinogens and develop both anti-A
and anti-B. agglutinins early in life. Group A individuals,
however, inheret the A agglutino-gen and develop agglu
tinins only to the B agglutinogen which they lack, while
anti-B, anti-N and anti-O speci?city are known to exist;
Lectins of anti-A, anti-A1 (a sub-group of type A),
and, of these, anti-A and anti-H(O) lectins have been
made commercially available. There is however, no com
mercially available anti~M speci?c leotin. A preparation
of thistype is of considerable practical importance in
View of the well-known di?‘lculty of making good anti-M
group B persons form only anti-A agglutinin. The rarer
AB group develops neither of these agglutinins.
25 agglutinin from immune rabbit serum.
Group B serum is a ready source of anti-A agglutinins
and group A serum affords an anti-B grouping reagent.
The natural occurrence of these agglutinins was Land~
steiner’s key for unlocking the mystery of blood incom
It is an object of this invention to provide a new
source of anti-M lectin. Another object is to provide an
anti-M lectin which is useful in the formulation of diag
nostic compositions which have commercial signi?cance.
patibility which up to that time made transfusion of 30 Another object is to provide an anti-M lectin which is
this important agent hazardous, if not impossible. The
subsequent identi?cation of other important blood agglu
characterized by a high degree of sensitivity and speci
?city. Another object is to provide novel procedures for
tinogens by Landsteiner and by others have made whole
the isolation and preparation of anti-M lectins.
blood transfusion an even safer procedure.
‘objects and advantages, both general and speci?c, will
be apparent from the following detailed description of
In this con
nection, and listed in the order of their historical identi
?cation, are the more important of these agglutinogens:
M, N, P, Rh and Hr.
It should be noted that the discovery of these agglu
the invention.
This invention relates to the preparation and use of
lectins, derived from the seeds of Cruciferae Iberis, as
diagnostic reagents for the identi?cation of human eryth
blood groups because of the lack of naturally occurring 4.0 rocytes of types M and MN.
In the products employed in the practice of this in
agglutinins to them in human blood. Speci?c agglutinins
vention, the seeds of Cruciferae Iberis, a plant more
necessary for identifying certain agglutinogens (M, N. P,
commonly known as common candytuft, variety Iceberg,
and Rh) were obtained by immunizing laboratory ani
are extracted by procedures described in greater detail
mals. Some agglutinogens (H (O) and P) were identi
?ed by analyzing the normal serums of various animals 45 hereinafter. Anti-M lectins ‘suitable for routine use as
anti-M diagnostic reagents are obtained by puri?cation
or by studying agglutinins developed by individuals who
and conceneration of these extracts.
had become immunized or sensitized to a particular blood
The Cruciferae Iberis lectin of this invention is pre
type agglutinogen. Agglutinins from sensitized human
pared by reducing the seeds of the plant to a small, par
beings are at present the only practical source of anti-Rh
50 ticulate form, such as by mechanical attrition. The lectin
agglutinins (anti-C, anti-D, anti-E, anti-c, and anti-e).
is solubilized at reduced temperatures, for example, 2 to
The understanding of blood groups and blood types
10 degrees (3., through the use of a ‘suitable solvent, such
has enlarged considerably since the discovery of the Rh
as an aqueous solution of sodium chloride; and any in
factor by Landsteiner and Wiener in 1940. Recent ob
soluble inert material is discarded. The extract may be
servations contribute to a growing list of blood cell agglu
tinogens, as well as their corresponding agglutinins, and 55 clari?ed by centrifugation and ?ltration through a me
tinogens was much more di?‘icult than those of the major
methods used in detecting them are of interest to those
dium which neither adsorbs nor adversely affects the ac
in allied ?elds of study. These ?ndings are of particular
signi?cance in medicine and laboratory diagnosis, as for
tive principal. The extract is then further puri?ed by
example, in ascertaining blood compatibility prior to
dialysis. The leotin is found in the precipitate which re
sults from the dialysis. This precipitate is solubilized
transfusion and in the ?eld of legal medicine where the
through the use of a suitable solvent such as an aqueous
presence or absence of blood agglutinogens serves as
20% solution of sodium chloride, and the insoluble inert
material is removed and discarded.
It has been observed that the activity of the puri?ed
evidence in disproving parentage. Also, blood group and
type relationships worked out by geneticists have given
anthropologists a valuable instrument for racial studies.
Although blood has been the principal source or" agglu 65 anti-M lectin prepared from Cruciferae Iberis, accord
ing to the methods described herein, can be signi?cantly
tinins for use in blood typing, it is not the only source.
increased in the presence of acacia. Acacia itself has
It has long been known that products of certain bacteria,
no demonstrable anti-M activity. It is apparent, therefore,
a number of organic and inorganic chemical substances,
that the anti-M lectin of this invention and acacia con
and extracts of the seeds of certain plants will cause the
agglutination of red blood cells. None of these, however, 70 stitute a synergistic combination. Compositions which
were of use in blood typing because they were non‘
incorporate this synergistic combination are preferred for
speci?c. That is, if they agglutinated the red cells of
use in the practice of this invention.
The following detailed example will serve to more fully
describe the invention.
Example I
' An anti-M lectin was obtained from Cruciferae Iberis
seeds in the following manner. The seed was ?nely
ground by mechanical means and then extracted in ten
times its weight of ‘0.9% sodium chloride solution.
extraction was continued at room temperature for 8
hours, with occasional mixing, and was continued over
night at a temperature of 4 degrees C. The extraction
was clari?ed by centrifugation and ?ltration through glass
speci?city of the original lectin preparation and provided
stronger and more clear-cut agglutination reactions than
were provided by the lectin preparation which did not
contain acacia.
Furthermore, although a one hour in
cubation period was required for the lectin preparation
of Example I, the improved results obtainable with the
admixture employed in this example, could be realized
in 15 minutes. The 3% acacia solution itself, that is,
without the added presence of lectin, displayed no agglu
tinating properties.
While in the foregoing speci?cation, a detailed descrip
tion of embodiments of the invention has been set forth
wool. This crude extract, which contained the active
for the purpose of illustration, it will be apparent to those
principal, was then dialyzed against distilled water for
skilled in the art that many modi?cations in the details
24 hours in 12 changes of water. Each time the water
of these embodiments may be made without departing
was changed, the extract was thoroughly mixed by invert 15
ing the Visking tubing several times. The insoluble ma
terial which resulted from this dialysis was collected by
centrifugation, and was solubilized by the addition of .
one-fourth volume of 20% sodium chloride solution. '
This 20% sodium chloride solution of the precipitate
was then diluted with a su?icient quantity of 0.9% so
dium chloride solution to provide a volume equal to
one-half the volume of the original crude extract, which
was subjected to dialysis. This solution was then tested
for anti-M activity, using the following technique:
from the spirit and principles of the invention.
There is claimed:
1. In a method of preparing an agglutinin which is
speci?c for the M agglutinogen of blood, the step of par
ticulating seeds of Cruciferae Iberis, mixing the particu
lated seeds with an aqueous solvent to dissolve the agglu
tinin portions thereof, discarding the insoluble portions
of the particulated seeds, removing inactive material from
the said agglutinin portions by dialysis, and combining
the residual agglutinin with acacia.
2. In a method of preparing an agglutinin which is
(1) Place two drops of the lectin preparation in each
of three suitable, e.g., 10 x 75 mm., test tubes.
(2) Add to one of these test tubes one drop of a 2%
speci?c for. the M agglutinogen of blood, the step of par-'
ticulating seeds of Cruciferae Iberis, mixing the particu
lated seeds with an aqueous solvent to dissolve the agglu
by volume suspension of erythrocytes of M speci?city, 30 tinin portions thereof, discarding the insoluble portions
of the particulated seeds, and removing the inactive ma
and 0.9% sodium chloride solution, and mix. Repeat
this procedure for each of the other two test tubes, using
terial from the seed agglutinin portions by dialysis.
erythrocytes of MN and N speci?city,prespectively.
3. In a method of preparing an agglutinin which is
(3) Prepare negative controls, each with two drops of
speci?c for the M agglutinogen of blood, the steps of
sodium chloride solution and one drop of erythrocytes 35 particulating seeds of Cruciferae Iberis, mixing the par
of M, MN, and N speci?city, respectively.
ticulated seeds with an aqueous electrolyte solvent to
(4) Allow the test tubes to remain at room tempera
dissolve the agglutinin portions thereof, discarding that
ture for one hour.
portion of the particulated seeds, which is insoluble in
(5) Resuspend the sediment which forms in the tubes
the aqueous electrolyte solvent, removing dialyzable, in
and centrifuge for one minute at 1700 r.p.m.
active material from the agglutinin portions by dialysis,
(‘6) Shake the tubes gently and observe for macro
and thereafter recovering the non-dialyzable water-in
scopic agglutination.
soluble agglutinin.
4. In a method of preparing an agglutinin which is
(7) vExtracts which contain lectin speci?c to the
erythrocyte agglutinogens produce visible ?oculation of
speci?c for the M agglutinogen 0 blood, the steps of
the erythrocytes, whereas a smooth suspension is ob
particulating seeds of Cruciferae I eris_ mixing the par
served in a negative reaction.
ticulated seeds with an aqueous electrolyte solvent to
dissolve the agglutinin portions thereof; discarding that
When the above test procedure was employed, the
portion of the particulated seeds which is insoluble in the
lectin preparation of this example caused a visible agglu
tination of the cells which were known to possess M and 50 aqueous electrolyte solvent; removing dialyzable, inactive
material from the agglutinin portions by dialysis; recover
MN agglutinogens. This lectin preparation did not cause
a visible agglutination of the cells which were known
ing the non-dialyzable, water-insoluble agglutinin; and
combining the agglutinin with acacia.
to possess vonly the N agglutinogen. These results
5. The agglutinin-acacia product resulting from the
demonstrate the presence of an M speci?c lectin in the
seeds of Cruciferae Iberis. They further demonstrate 55 process of claim 1.
that this lectin can be extracted from these seeds and
References Cited in the ?le of this patent
puri?ed in a manner that will provide a commercially
acceptable lectin preparation.
Example I1
One volume of the puri?ed lectin preparation of Ex
ample I was admixed with two volumes of an aqueous
3% acacia solution. This admixture was tested for lectin
activity according to the procedures set forth in Example
Eldon ______________ __ Nov. 13, 1956
Boyd: Archives of Biochemistry & Biophysics, March
1955, pp. 226-234.
Dictionary of Antibiosis, Columbia U. Press, New
I. It was observed that the admixture retained the M 65 York, 1951, p. 147.
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