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The isolation of rabbit insulin antibodies.

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(4
The Is01 tion of Rabbit Insulin Antibodies'#'
EDWARD R. ARQUILLA AND CHARLOTTE COBLENCE
Department of Pathotogy, School of Medicine,
University o f Southern California,
Los Angeles, California
This investigation is concerned with the
isolation of tabbit insulin antibodies by
elution from an insoluble insulin complex. The ebtion of antibodies from insoluble anti$en antibody aggregates has
been previously demonstrated (Campbell,
Luescher and Lerman, '51; Isliker, '51).
Campbell, buescher and Lerman ('51)
prepared an insoluble antigen by conjugating bovin serum albumin to a diazobenzene-cellu ose derivative. They were
able to foqm an insoluble antibody
antigen aggaegate and then elute the
bovine serum albumin antibodies from
this preparatibn at a low pH.
In the follbwing experiments the insoluble antibod3 insulin aggregate was prepared by adsorbing antibodies from rabbit
insulin antiserum onto an insoluble insulin complek made by conjugating insulin to an erythrocyte stroma-cellulose
mixture. The antibody insulin aggregate
was isolated from proteins and other
materials in serum by washing with
saline. The e ect of pH, temperature and
salt concentr tion on the dissociation of
antibody from insulin was studied. Berson and Yalow ('59), using Pi-labeled
insulin and whole human insulin antiserum, were llble to estimate the dissociation constants of insulin antibody complex. They qoncluded that insulin has
two antigenic sites, each having different
dissociation onstants. In these experiments the e ct of pH, temperature and
salt concentr tion on the dissociation or"
antibody fro insulin in an isolated system was stu 'ed. It appears that insulin
like other pr tein antigens may have a
multiplicity o antigenic sites, the binding
properties of which are not well understood.
By selectin the optimum pH, temperature and sal concentration for the dis-
1
s"
i
8
ti
sociation of antibody from insulin it has
been possible to purify the insulin antibody from rabbit antiserum about 500fold with approximately a 30% recovery.
METHODS AND MATERIALS
Antigen. The antigen used in preparing the rabbit insulin antiserum was crystalline beef i n ~ u l i n .This
~
is a sample of
zinc crystals of bovine insulin having 27
units per mg. There was some hyperglycemic factor present in this preparation.
Erythrocyte preparations. The sheep
erythrocytes used for the titration of antibody activity were stored as sheep blood
in an equal part of Alsever's solution
(Bukantz, Rein and Kent, '46). This
blood was stored at 5°C and if not used
within three weeks was discarded.
Antiserum. Adult male white rabbits
were immunized with alum-precipitated
insulin as has previously been described
by ArquilIa and Stavitsky ('56). The antiserum obtained was stored in aliquots
at -60°C.
Titration of insulin antibody activity.
The insulin antibody titer of these sera
was measured by the hemagglutination
method, employing sheep erythrocytes to
which insulin was conjugated with bisdiazobenzidine, (ArquiUa and Stavitsky,
'56). The diluent used for titrations was
verond buffered saline, pH 7.4 (Kabat and
Mayer, '48), containing 5 X lo-' M MgClp
M CaCL in which 1.5 mg
and 1.5 X
bovine serum albumin (Armour Co.) per
'This work is supported by U.S.P.H.S. grant
A-3541 (Sl).
* A report on this work was presented to the
Annual Meeting of the American Diabetes Association in Miami Beach, June, 1960.
wish to thank h.Otto K. Bebrens, Eli
Lilly, for the generous supply of crystalline beef
insulin.
203
204
EDWARD R. ARQUILLA AND CHARLOTTE COBLENCE
packed heavy layer and a lighter fluffy
layer. As much of the clear solution as
possible was drawn off with a capillary
pipette. The lighter layer could then be
separated from the heavier layer by gently
rinsing the tube with 0.11 M phosphate
buffer. The remaining precipitate was
also suspended in 0.11 M phosphate buffer. Both portions of the stroma were
then dialyzed against distilled water and
lyophilized. The lighter top fraction of
the sheep erythrocyte stroma (SI) was
used in making the cellulose stroma complex to which insulin was conjugated.
Preparation of the cellulose stroma mixture. Eight grams of Whatman’s cellulose powder (type A ashless standard
grade) were added to 2 gm of S,. This
material was suspended in 50 ml of saline
and homogenized in a ground glass hand
homogenizer so that equal distribution of
the stroma and cellulose could be obtained.
This mixture was diluted to 600 ml with
saline and allowed to stand at 5°C for
24 hours with constant gentle stirring.
The mixture was then washed with saline
an additional 5 times, each wash containing about 600 ml. Hemoglobin was present in the first 4 washes, whereas the last
liter was dissolved. Prior to use the diluent
was incubated at 80°C for one hour and
then cooled to 2°C in a water bath. Much
of the nonspecific agglutination observed
at high dilutions of antiserum was eliminated with this diluent.
Protein determination. Protein concentration was determined by the method of
Lowry, Rosebrough, Farr and Randall
(’51).
Preparation of sheep erythrocgte stroma.
Sheep’s blood stored with an equal part
of Alsever’s solution was washed three
times with saline. The packed erythrocytes were suspended with an equal volume of saline and dialyzed against 20
volumes of distilled water. This suspension was centrifuged at 18,000 g’s in a
Servall angle centrifuge and the precipitate washed with water until the supernatant was a pale pink. The precipitate
was then washed with 0.11 M phosphate
buffer, pH 7.4, at 40,000 g’s for 30 minutes at 2°C in a Spinco ultracentrifuge until the supernatant was very faintly pink.
Finally the precipitate was suspended in
0.11 M phosphate buffer, pH 7.4, and
centrifuged at 18,000 g’s. Two distinct
layers of precipitate formed, a tightly
To 40 mg of stroma cellulose suspension (in 4 ml) add 5 mg
insulin (in 2.5 ml) and 1 ml BDB (1 to 15 with phosphate
buffer pH 7.4). Incubate at room temperature for 20 minutes
while shaking and centrifuge at 2100 g’s.
I
Supernatant
Precipitate
L
&-
Wash with 20 ml saline.
Add 4 ml antiserum and incubate
at room temperature while shaking.
Centrifuge for 30 minutes
at 2100 g’s at 2°C.
Discard
I
Supernatant
J
Precipitate
&
Absorbed antiserum.
Save for titration
and protein determination.
Precipitate
J
Discard
Washed with 20 rnl saline at 2°C
Elute with 12 ml of 5X buffered saline,
pH 3.2, at 37°C for 10 minutes
while stirring.
Centrifuge 30 minutes 2100 g’s at 2°C
I
Dialyzed against
buffered saline pH 7.4
Fig. 1 Procedure for elution of rabbit insulin antibodies.
205
ISOLATION O F INSULIN ANTIBODIES
two washes appeared to be grossly free
of hemoglotfn. The resultant white precipitate (C
was dialyzed against distilled water and lyophilized.
Preparatidn of insulin conjugated
stroma cellulose mixture. The CS, mixture was su pended in saline in a concentration of 1 mg per ml. Crystalline beef
insulin was made up in a solution containing 2 m per ml (fig. 1). To 4 ml of
the evenly uspended CS,, 2.5 ml of insulin solutiop containing 5 mg of insulin
was added pnd evenly suspended. One
ml of bis-diasobenzidine (BDB) was added
to this mixture, and then gently agitated
at room temperature for 20 minutes and
centrifuged for 10 minutes at 2,100 g’s.
The supernatant was discarded and the
button washed twice with 20 ml of saline.
This amount of insoluble insulin complex
(ICSI) was used to adsorb the antibodies
from 4 ml of rabbit insulin antiserum
with the subsequent formation of the insoluble antibody insulin complex (AbICS;).
8
$
RESULTS
Adsorption of antibodies from antiserum
with insoluble insulin complex. The ability of insulin antiserum to agglutinate
insulin-sensitized erythrocytes was markedly decreased following the incubation
of the antisesum with ICSl (table 1).
Four ml of rabbit insulin antiserum
were added to an insoluble insulin complex made with 10 mg of CSI and 5 mg
of insulin. This mixture was incubated
for 30 minutes at room temperature with
constant stirring, and then centrifuged
(fig. 1 ) . The anti-insulin activity in the
supernatants from 5 different antisera
treated in this manner were compared
with the activity of aliquots from the
same antisera which had not been incubated with ICSl (table 1).
TABLE 1
Adsorption of insulin antibodies from antiserum
incubated unith insoluble insulin complex
Antiserum
A
B
-
,. r
D
E
Titer
antiserum
not
incubated
Titer
antiserum
after.
incubation
1/3200
1/420
1/5120
1/50
1/25
1 /40
1/10
1J40
Most of the insulin antibodies were
adsorbed out of the rabbit antiserum by
the insoluble insulin complex. Following
this adsorption, the insoluble antibodyinsulin complex (AbICSI) was washed
twice with 20 ml of saline. Most of the
weakly bound serum proteins were removed with the first saline wash. There
were a small amount of insulin antibodies
present in the first saline wash. No detectable antibodies were present in the
second saline wash. The small amount of
insulin antibodies consistently observed in
the first saline washes probably represent
insulin antibodies in the residual serum
which was in the liquid phase of the
AbICS, (table 2).
TABLE 2
Effectiveness of saline washes on insoluble
antibodg insulin complex
Titer
Antiserum
Saline wash 1
Saline wash 2
<
1/6400
1/8
I/1
Protein
concentration
mglml
83.0
1.26
0.038
About 80 to 85% of the total serum
proteins were recovered in the adsorbed
antiserum. Between 2 and 3% of the
total proteins were present in the first
saline wash, and about 0.5% in the
second saline wash. Between 0.05 and
0.1% of the total proteins were recovered
as purified insulin antibodies. What portion of the unaccountable (15 to 20% )
total serum proteins were still bound to
insoluble aggregate is unknown.
Factom influencing dissociation of antibody from the antibody insulin complex.
The effect of pH, temperature, and salt
concentration on the elution of insulin
antibodies from AblCS, was investigated.
The pH of the solution employed for eluting the antibodies was found to be the
most critical of these three parameters.
At pH levels above 3.5 the amount of
antibodies dissociated decreased markedly.
Variations in salt concentration and temperature had minor effect on the amount
of insulin antibody activity recovered in
the eluate (fig. 2).
Optimal conditions for the dissociation
of antibodies from AbIC& were obtained
206
EDWARD R. ARQUILLA AND CHARLOTTE COBLENCE
EFFECT OF IONIC STRENGTH
pH 3.2
TEMP. 0.C
512
256
i
128
W
L
.-
&
c
64
32
0
IX
ox EX
TIMES SALINE
2x
5x
EFFECT OF TEMPERATURE
pH 3.2
IONIC STRENGTH 5 X SALINE
with a solution containing 9 parts of 0.77
M NaCl and one part of 1 M citric acid,
pH adjusted to 3.2 with NaOH (fig. 1).
A volume of this eluting solution equal to
three times the amount of antiserum was
added to the washed AbICSl and incubated
at 37°C for 10 minutes with constant
gentle shaking. The aggregate was then
centrifuged, the supernatant decanted and
adjusted to pH 7 with NaOH and dialyzed
against saline (fig. 1).
Recovery o f antibody. Using the above
conditions, it was possible to recover approximately one-third of the insulin antibody activity in the eluate which contained
about 1/1400th of the protein concentration of the serum from which the antibody
was obtained (table 3).
TABLE 3
The elution of rabbit insulin antibodies
512
Titer
Protein
1/12800
80,000
1/50
1/48001
61,000
19
r/ml
Antiserum
Absorbed
antiserum
Eluate
256
128
Q)
L
.c
c
'The observed titer was multiplied X 3 since
the elution volume was 3X the volume of the
antiserum used.
64
32
The antibodies in the eluate were apparently
not altered to any detectable degree
0 25 37 56
by the method of elution. The antibodies
TEMPERATURE
were still capable of agglutinating insulinsensitized erythrocytes. If complement
was added to the system, hemolysis of the
EFFECT OF pH
insulin-sensitized erythrocytes took place.
IONIC STRENGTH S X SALINE
Both the hemolysis and agglutination of
TEMP. 0.C
the insulin-sensitized erythrocytes by the
purified antibodies were inhibited with
crystalline insulin.
512
Residual binding of antibodies to in256
soluble insulin aggregate. In order to
determine whether residual antibodies to
I28
L
insulin still remained attached to the inc
sulin complex after elution, the complex
c 64
was labeled with fluorescent anti-rabbit
32
gamma globulin antibody (fig. 3).
It was necessary to minimize the degree
I
0'
of nonspecific staining of the insulin complex by adsorbing the fluorescent conjuPH
gate with 10 mg/ml ICW. The adsorbed
Fig. 2 Parameters for the elution of rabbit in- fluorescent conjugate still contained antibodies to rabbit gamma globulin, whereas
sulin antibodies.
n
-
I
Q)
.-
-
ISOLATION OF INSULI N ANTIBODIES
207
Fig. 3 Residual binding of antibody to insoluble insulin aggregates as demonstrated
with fluorkscent anti-rabbit gamma globulin.
the antibodiels to stroma and much of the
fluorescent npaterials which attached nonspecifically tQ the insulin cellulose stroma
complex werR removed by this prior adsorption.
This ads bed fluorescent anti-rabbit
gamma glob lin conjugate was then incubated for 20 minutes at room temperature
with AbICS, which had been subjected to
three elution similar to those described
previously. he complex was then centrifuged and ashed twice with saline. A
smear of the utton was made on a microscopic slide nd examined by fluorescent
microscopy (fig. 3). There was marked
staining of th aggregate by the fluorescent
anti-rabbit gamma globulin antibody. It
therefore applears that rabbit antibody remains attached to the insulin complex in
spite of repealted elutions with highly concentrated salU solutions at pH 3.2.
There is sohne question whether the fluorescent conjogate was staining only antibodies attached to insulin. The possibility
that rabbit amma globulin other than
insulin antib dy was stained by the fluorescent antibody cannot be eliminated.
7
4
1
L
i
DISCUSSION
The use of a stroma cellulose mixture
for the preparation of an insoluble antigen
has versatility since once prepared it is
quite stable and within a relatively short
period of time almost any antigen can be
conjugated to it with bis-diazobenzidine.
The diazobenzene derivative of cellulose,
as described by Campbell, Luescher and
Lerman ('51 ) requires the preparation of
the complete antigen and therefore can
be used only for the isolation of one species of antibodies. The amount of stroma
protein which will contaminate preparations of eluted purified antibody is an
unknown but a real consideration when
using AbICS1. Theoretically this danger
does not exist with the use of the diazobenzene derivative of cellulose.
The insoluble insulin aggregate was
found to be very effective in adsorbing
out antibodies from insulin antisera. In
these experiments antiserum was used in
excess so as to insure a complete saturation of all available insulin. Therefore,
there were residual antibodies in the anti-
208
EDWARD R . ARQUILLA A N D CHARLOTTE COBLENCE
sera adsorbed with ICS,. In other experiments it has been possible to remove all
of the demonstrable antibodies in antisera
by employing slightly larger quantities of
ICS, to adsorb the antibodies from the
antisera.
The purified antibody which was eluted
from the aggregate was stable at 0°C for
at least two weeks. Dialysis against water
and subsequent lyophilization resulted in
complete loss of anti-insulin activity as
measured by the hemagglutination reaction. Acetone precipitation of the eluate
also caused the loss of the antibody activity. In preliminary experiments it has
been possible to concentrate the eluate
at least 100-fold by dialysis against 25%
polyvinylpyrrolidone in water. Preliminary
studies on the homogeneity of the isolated
antibody indicate that there are three
components which can be isolated by
column chromatography. The characteristics of the various components are presently being investigated.
It is difficult to accept the concept of
Berson and Yalow (’59) that insulin has
two antigenic sites, and that to each of
these sites there is a single species of
antibodies and that each has a different
dissociation constant. It appears that insulin antibodies can be dissociated from
an isolated antigen antibody aggregate at
various pH’s, temperatures, and salt concentrations to varying degrees. In spite
of repeated elutions at conditions where
t h e optimum amounts of antibodies could
be dissociated from the insoluble insulin
complex, only 30% of the antibody activity was recovered and there appeared to
be considerable amounts of rabbit gamma
globulin remaining adherent to the antibody insulin complex as was demonstrated
by the fluorescent antibody technique.
Whether insulin has only two antigenic
sites is still open to question. The insulin
molecule does have a considerable number of amino acids (e.g., aspartic, glutamic and phenylalanine, etc.) which according to the studies of Landsteiner (‘45)
are good antigenic determinants. Landsteiner (’45) has also demonstrated a
number of antibodies with different binding capacities against a single antigenic
site. It therefore appears likely that insulin, like other proteins, is an antigen
which has a multiplicity of antigenic sites
and to which there are a multiplicity of
antibodies with varying binding capacities
to each of these sites.
SUMMARY
1. It has been possible to prepare an
insoluble insulin aggregate by conjugatinq
insulin to a cellulose stroma complex with
bis-diazobenzidine.
2. This insoluble insulin aggregate is
effective in adsorbing antibodies from rabbit insulin antiserum.
3. It has been possible to isolate the
insulin antibodies by elution from this
aggregate with approximately a 500-fold
purification.
4. The effect of pH, temperature, and
salt concentration on the dissociation of
rabbit antibody from insulin permits the
postulate that there are a multiplicity of
antibodies to the insulin molecule which
have a multiplicity of binding characteristics.
ACKNOWLEDGMENTS
We wish to thank Mr. James Alexander
for his technical assistance.
Dr. Arquilla wishes to acknowledge the
kindness and consideration he received
from Dr. Normand L. Hoerr during the
7 years he was associated with the
Department of Anatomy at Western Reserve University. It is his feeling that
without Dr. Hoerr’s direction it would not
have been possible for him to complete
his education and continue his academic
life .
LITERATURE CITED
Arquilla, E. R., and A. B. Stavitsky 1956 The
production and identification of antibodies to
insulin and their use in assaying insulin. J.
Clin. Invest., 35: 458-466.
Berson, S. A., and R. S. Yalow 1959 Quantitative aspects of the reaction between insulin
and insulin-binding antibody. Ibid., 38: 1 9 9 6
2016.
Bukantz, S. C., C. R. Rein and J. R. Kent 1946
Studies in complement fixation. 11. Preservation of sheep’s blood in citrate dextrose mixtures (modified Alsever’s solution) for use
in the complement fixation reaction. J. Lab.
Clin. Med., 31: 394.
Campbell, D. H., E. Luescher and L. S. Lerman
1951 Immunologic adsorbents. I. Isolation
of antibody by means of a cellulose-protein
antigen. Proc. Nat. Acad. Sci. U.S., 37: 575578.
ISOLATION OF INSULIN ANTIBODIES
i.
Isliker, H. C. 1953 Purification of antibodies
by means
antigens linked to ion exchange
resins. A n N. Y. Acad. Sci., 57: 225-238.
Kabat, E. A., and M. M. Mayer 1948 Experimental Im unochemistry. C. C Thomas,
Springfield, Illinois, p. 107.
.
209
Landsteiner, K. 1945 The Specificity of Serological Reactions, rev. ed. Harvard University
Press, Cambridge, Massachusetts.
Lowry, 0. H., M. J. Rosebrough, A. L. F a n and
R. J. Randall 1951 Protein measurement
with folin phenol reagent. J. Biol. Chem.,
193: 265-275.
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