Note: Descriptions are shown in the official language in which they were submitted.
12Q99~7
I~ETHOD OF AFFINITY PURIFICATION
EMPLOYING MO~OCLONAL ANTIBODIES
FIELD O~ THE I~VE~TION
This invention relates to the purification of an~igens and
5 antibodîes by affinity chromatography. In another aspec~ it relates
to monoclonal antibod ies.
BA CKGROVND
The purification of an antigen by affinity chromatography
using serum antibodies, produced by a host animal's response to the
10 antigen, that are bound to a solid support as an immunoadsorbent is a
process which has bee~ used for many yçars. This process has,
ho~ever, at least two serious shortcomings which impair its
usefulness. Thuss i~ antibodies of high affi~ity are used to e~tract
the antigen from a sample, harsh conditions are required to dissociate
the ~ntige~ from the antibodies after no~-absorbed impurities have
been washed $rom the bod~ of immunoadsorbent. The conditions required
for this, for example, a pH of less than 3 or greater than 11 or a
conce~trated chaotrope such as guanidi~e or urea solutio~, ca~
denature the a~tige~ and the an*ibodies, diminishi~g, if not
destroying, the immunochemical a~d/or biological propertles of the
antigen and shorte~ing the useful life oi the immunoadsorbent.
To avoid the problems associated with the use of antibodies
having a high affinity for the antigen, it has become common practice
to use immobilized antibodies of low affi~ity as an immunoadsorbent.
Use of these antibodies permits elution of the antigen from the body
o~ immunoadsorbent using mild, non-denaturing conditions. However,
~he requisite step of washing the colum~ to elute impurities from the
bound antigen also elutes some of the antigen, so much so that the
efficiency of separation is greatly reduced. In addition, low
affinity antibodies cannot efficie~tly bind antigens which are present
~ZQ~9~)7
in the media at relatively low concentrations, i.e., less than
about 10 ng/ml.
With the advent of hybridoma technology, it has become
possible to obtain monoclonal antibodies, which subsequently
have been proposed for use as immunoadsorbents in the affinity
purification of the antigens against which they were raised.
See, for example, Stenman et al, J. Immunological r~ethods~ _ ,
337 (1981); Stallcup et al, J. Immunology, 127, 92~ (1981) and
Katzmann et al, Proc. Natl. Acad. Sci. US~, 78, 162 (1981).
These reports suggest that the monoclonal antibodies employed,
at best, had only a modest affinity for the antigens, permitting
their desorption from the immunoadsorbent using mild conditions.
Thus, the experience to date using monoclonal antibodies as
immunoadsorbents suggest that their properties should parallel
those of the "polyclonal" antibodies of conventional antisera,
i.e., the use of low affinity antibody permits elution of the
antigen under mild conditions whereas use of a high affinity
antibody requires harsh conditions to dissociate the antigen
from the antibody.
SUMM~RY OF TXE INVENTION
The invention provides a monoclonal antibody having a
high affinity for the antigen against which it is raised in a
first environment and a low affinity for the antigen in a second
environment, neither environment substantially irreversibly
altering the immunochemical properties of the antigen or the
antibody.
The invention also provides a process for the purifica-
tion of an antigen comprising the steps:
a) selecting an antibody having a high affinity for
the antigen in a first environment and a low affinity for the
antigen in a second environment, neither environment substan-
tially irreversibly altering the immunochemical or biological
-2-
properties of the antigen or antibody,
b) i~nobilizing the antibody on a solid support;
c) binding the antigen to the antibody in the first
environment;
d) separating unbound impurities from the bound anti-
gen; and
e) eluting the antigen in a purified form from the
immobilized antibody using, as an eluant, a medium which is the
second environment.
As is well known, hybridomas are formed by the random
fusion of B-lymphocytes with myeloma cells in the presence of a
fusion promoting agent. Each hyd~idoma of the large population
of hybridomas which can be produced by a fusion secretes a
different monoclonal antibody. Typically, the population of
hybridomas is screened to select for further cloning those that
secrete an antibody of the desired antigenic specificity in
order to obtain useful quantities of antibody. We have found
that, among the population of hybridomas which secrete anti-
bodies against a specific antigen and the subpopulation of those
which secrete antibodies having a high affinity for the antigen,
a very much smaller population secretes antibodies which have a
high affinity for the antigen in a first environment, but a
much lower affinity in a second enviromnent, neither environment
-2a-
~L2Q990~
ing detrimental to the immunochemical or biological properties of
either the antigen or antibodies. We believe that the e~istence of
these antibodies i~ high affinity an~isera has gone unrecognlzed
because the majority of the high af~inity antibodies in the antisera
are ones which require harsh conditions before the antigen can be
separated from the an~ibodies and they d~ominate the immunochemical
properties of the antisera.
Accordingly, we have found that we can scr~en a population
of hybridomas, which can be the product of multiple fusions, and
ide~tify those which produce a monoclonal antibody having a high
affinity in a first e~vironment and a low affinity in a second
snvironment and clone at least one o~ the hybridomas to obtaiD a
sufficient quantity of the anti~ody it produces to permit its use as a
highly effective immunoadsorbent for affinity chromatography. As used
herei~, an antibody is considered to exhibit a hlgh affinity ~he~ its
a,finity constant tKa) is abou~ ~ 109 and to exh~bit a low a~finity
whe~ its Ea is a~out < 108.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1 and 2 are graphs of data reflecting the ef~ect of
changes in pH on the desorption of radiolabeled hum~n growth hormone
bound to four different monoclonal antibodies immobilized on a solid
phase.
DESCRIPTION OF PREFERRED EMBODIMENTS
- Accordi~g to our invention, purification o~ an antigen is
accomplished by a process comprising the steps:
a) se~ecting a monoclonal antibody having a high afiinitY
for the antigen in a first environment and a low affinity for the
antibody in a second environment, ne{ther environment causing
substantial, irreversible changes in the desired immunochemical
~roperties of the antigen,.
~Z~.9~7
b) i~mobilizing the antibod~ on a solid support;
c) contacting the immobilized antibody with a sample
containing impure antigen in the first environment to bind ~he antigen
to the antibody;
d) separating unbound impurities from the bound antigen;
and
e) e~uting the antigen in a purified form from the
immobilized antibody using, as an eluant, a medium which is tbe second
enviro~men~.
As already noted, antibodies useful in our invention can be
obtained by scree~ing the antibodies produced b~ a population of
hy~ridomas obtained by the fusion, using known methods, of myeloma
cells ~ith 8-l~mphocytes. The B-lymphocytes are typically spleen
cells taken from a hyperimmunized animal to which the target antigen
has previously been administered as an immunogen. After those
hybridomas that produce monoclonal antibodies whose specificities are
against the desired antigen have bee~ identtfied, they can ~e further
screened to identify those that produce antibodies wh~se--af~inities
vary ~ith chan~es ln enviro~ment which are Dot damaging to the antige~
or antibody. For example, antigens are usually stable i~ solution
withln the pH ra~ge of 4-10.~. To obtain a p~ sensitive a~tibody for
use as an immu~oPdsorbent, the population of monoclonal a~tibodies ijs
screened to identify those which have a high a~finity for the antibody
at one pH within ~he range, i.e., a Ka of about 109 and perferable
2~ 101 and a low affi~ity ~t a second pH, i.e., a Ka of about 108 and
preferably less than 105 within the same range. This kind of
screening can be done b~ immobiliziDg the antibody o~ a solid support
and, after permi~ting it to bind antigen, measuring the e~ten,t of
desorption of the antigen that occurs at different pH levels, a
measurement ~hich can be made, for instance, by employing radiolabeled
antigen and counting the radiation emitted by the solid phase and/or
supernatant. A similar screen can be carried out to identify
antibodies that respond to other kinds of environmental change.
~2Q~19~7
It is presently preferred tG exploit mo~oclonal antibodies
whose capability to bi~d antigen is sensitive to changes in pH. In
this regard, the antibody is selected to have a high affinity a~ one
pH and a low affi~ity at a second pH which may be higher or lower than
3 the first pH. Usually the first pH will be at or near pH 7 although
it ~eed DO~ be. However, it is also wit~in ~he scope of the i~vention
to select antibodies which respond to a dif ferent kind of change in
environmental condition. For e~ample, a monoclonal antibody can be
selected which undergoes a cha~ge from high low affin~ty in the
presence of a chaotropic solutio~ as the eluting medium. Among
suitable chaotropes are EBr, KI, KSCN, guanidine, urea and MgC12.
T~us, monoclonal antibodies can be selected havi~g a Ka > 109 i~ the
absence of chaotrope but which has a Ka of < 108 in the preseDce of
the partlcular chaotrope whose concentratioD is not detrimented to the
15 antigen in quPstion. The selection ior chaotrope sensitivi~y can also
be made in buffers at a specific pH. Alternatively, antibodies ca~ be
selected which are sensitive to changes in pH in the prese~ce of a
constant concentration o~ a chaotrope. ~~-
Monoclonal antibodies ~hose affinity for an antigen is
adequately lowered by changes other than pH or coDcentration ofchaotrope can also be selected. Among the ki~ds of media sensitivity
for which the antibodies can be screened to select those whose a~tigen
binding ability is a~fected by a change in eluting medium can include
borate sensiti~ity, methylmannoside sensitivity and sensitivity to
non-ionic or ionic detergents and reagents which affect specific amino
acids such as tryptophan and t~rosine.
For use in affinity chromatography, a selected mo~oclo~al
antibody can be bound to any of the solid supports commonly used in
~ Sepharose ~)
f~inity chromatography. These include ~ =L~CS4, polystyrene, glass,
3~ nylon, cellulose, polymet~ methacrylate, silicagel, polyacrylamide
and nitrocellulose.
- ~Z~$~7
.
The follo~ing e~amples illustrate the application o~ the
present in-~eDtion to obtaining monoclonæl antibodies whose binding
affinity for an antigen vPries from a high affinity in a first
environment to a low affinity in a second environmen~, neither
environment causiDg damage to the immunochemical properties of th~
a~tigen and their usefulness as immunoadsorbents for affiDity
chromatography.
E~ample 1
Spleen cells taken from Balb/c mice hyperimmunized with
human gro~th hormone (HGH) were fused using polyethylene glycol with
mouse myeloma cells (NS-1 or SP-2/0 lines). The resulting hybridomas
were cloned and screened to determine those secreting antibody
specific for ~GH by a radioimmunoassay employing 12~I-HGH a~d horse
a~ti-mouse IgG on sepharose beads. The hybridomas produ ing anti-HGR
monoclonal antibodies were further screened to identify those
producing antibdies having a Ka of at le~ st about 109 at pH 7. These
were further screened to identify those whose affinitie~~-were
se~sitive to changes in pH over the range from 4 to 10. ~. Data
reflecting the pH sensitivity of four mo~oclonal antibodies is shown
in Tables 1 and 2 and Fig~. 1 and 2. The individual antibodie~ are
desig~ated by the letters A, 3, C a~d D, respectivel~. These da~a
were obtai~ed in the following way: HGH labeled with 125I was bound
at pH 7 to each antibody which had previously been immobilized on
pol~styrene ballsO Each ball contai~ed appro~imately 1 ng of antigen
and 10,000 cpm. Three of eacb were incubated in 1 ml of PBS in 10%
horse serum for four hours at the pH indicated. Tbe adjustments in pH
were made bv the addition of either a buffer of sodium carbo~ate (10%
in horse serum) to obtain pH ~ 7 or by the addition of sodium acetate
buffer (10~ in horse serum) to obtain pH < 7. After incubation, 800
ul of the supernatant was counted. The counts of desorbed antigen at
each pH are recorded in Tables 1 and 2 and plotted in Figs. 1 and 2.
Table 1 ~ 7
,
Counts/Minute ~ 10-3 of Desorbed HGH*
PH AD tibody A Antibody B
3.0 4.810 2.442
3- 5 4. 62~ G. 803
4.0 4.1~6 0.357
4 . 5 - 1 . 608 0 . 2~8
5. 0 0. 44~ 0. 206
. 5 0. 176 0. 162
~.0 0.220 0.17~
6 . 5 0 . 336 0 . 167
7.0 0.328 0.1~2
Average of three supernatants
Table 2
Count /Minute z 10-3 of ~esorbed HGH*
.
Antibody C Antibody D
7.0 0.236 0.1~7
7.:~ . 0.208 0.336
8.0 0.240 0.321
8~ ~ û. 6~6 û. ~77
9 . 0 0 . 401 0 . 23?
9.~ 1.038 0.287
1 0 . ~ 3 . 030 0.364
10 . 5 4 . 809 0. 58~
1 1 . 0 5 . ~08 3 . 460
* AYerage of three supernatants
The data in Table 1, particularly as plotted in Fig. 1, show
that the bi~ding of HGH by antibody B uas essentially insensitive to
changes of pH o~er the range pH 3. 5-7 but that the biDding o~ HGH to
an~ibody A ~as significantly decreased in the range pH 4.5-4.0
indicating that the a~tibody would ~ot effectively bind the antigen at
p~ 4~0.
- ~2~907
The data iD Table 2 and Fig. 2 on the other hand, sho~ that
the binding of HGH by antibody D was essentially insensitive to
changes in pH over the range pH 7.0-10.5 whereas the binding of HGH by
antibody C was significantly deoreased in the range pH 9.5-10.5
indicating tkat the antibody would not e~fec~ively bind th~ antigen at
pH 10.5.
The supernatants eluted from ntibodies A and C at pH 4.0
and 10.~ were added to PBS buffer (10% in horse serum) and adjusted to
pH ? and the samples pooled. The pooled samples were incubated with
polystyrene balls coated ~ith antibodies A, B, C and D and two other
monoclonal antibodies against ~GH. ach of these antibodies recognize
different aréas of the HGH molecule. The immuDoreactivity of the
antigen elu~ed at either p~ 4 or pH 10.~ with five of the si~
antibodies, including antibodies, ~., B, C and D, had not been ~ffected
and was o~l~ slightly diminished agaiDst the si~th. These data
indicate thaS elution o~ the antige~ at either pH 4.0 or pH 10.5 did
not adversely affect its immunochemical properties. ~~-
E~am~le 2
A high affi~ity monoclonal antlbody (Ka = ~ x 101~) agai~st
prostatic acid phosphatase (PAP), an extremely labile enzyme, obtai~e~by screening hybridomas produciDg anti-PAP monoclonal antibodies
derived from fusions of spleen cells taken from a Balb/c mouse
hyperimmu~ized with PAP and mouse myeloma oells as described in
Example 1 was found to e~hiblt antige~ binding sensitivity in the pH
range 6.0-~.0~ The antibody was bound *o sepharose beads using the
C~Br technique at a concentration of 1 mg of antibody per 1 ml of
packed sepharose beads and used to purify PAP from seminal fluid as
follows. ~ 170 ul sample of seminal fluid containing 0.912 mg/ml of
PAP as de~ermined by an immunoradiometric assay, using a
9~907
~ TA~DE~ assay kit for PAP manufactured by Hybritech, Inc., San D~ego,
/~
Ca., ~as dilu~ed to 5 mls ~ith acetate buffer (1Oa sodium acetate i3
horse serum contaiQing 0.15 M NaCl) to obtain a solution of 31 ug
PAP~ml of solu.ion having a pH of 6.
.
The P~P solution was passed through a column contain~ng 1.
ml of t~e sepharose beads at the rate of 1 ml/hour a~d the colum~
~ashed ~ith 7.5 mls of the st~rting buffer. Immunometric assav of the
eluant (5.mls of sample P~d 7.4 ml of wash liquid) demo~stra~ed that
~9.3~ of the PAP had adsorbed to tbe column. The PAP waS eluted ~ith
0.1 ~ acerate buffer, pH 4 contaiDing 0.1~ N NaClO Three 1 ~1
,ractions ~ere collected and dialyzed overnight vs. 5~ mM citrate, p~
ÇØ The PA~ coDtent of the pooled aDd dialyzed fractions wa~
determined by immunorædiometric assæy to be ~4Z of the totP1 applied
to the column. Purit~ o~ the dialyzed ma;erial was determi~ed by
sodi~n àodec~l sul~ate and Qr~st~in-Davis PAGE. A single band w~s
o~served in each case. Enzymatic artivity meæsuremel~ts were done a~d
documented that the purified PAP retained its enzymati~- ~ti~it~.
The reteDtion o~ 46Z of the PAP on ~he column is likely *he
result, at least in part, of DoD-specific binding aDd the use of a
large e~cess of aDtibody uhich results in antigen "trail" from the
coluMn. The fo.mer can be reduced by pretreating the colum~ ~ith
sample unde~ tne conditions at which elution ~ e accomplished
followed by e~tensive ~ashing to remove ~nv material ~hich will
elute. The latter can be reduced bv loweriDg the concentration o~
2~ bound ~ntibod~. ~inally, sepharose is not an ideal matri~ lor
af inity chromato~raphv because of the heterogenei-y of pore si~e,
resul~ing in àif,usioD and steric problems.
12~907
Example 3
The purification of the antigen associated with chlamy-
dia is complicated because it is difficult to solubilize. However,
it can be solubilized in a variety of detergents. Hybridomas which
produce monoclonal antibodies against chlamydial antigen, obtained
by fusing spleen cells from hyperimmuniæed Balb/c mice
wi~h mouse myeloma cells as described in Example 1, were screened
for sensitivity to detergent concentration. The effect of deter-
gents on the binding of four such antibodies is set forth in Table
3. The detergents used were deoxycholate (~o~, sodium dodecyl
sulfate (SDS) and octylphenoxypolyethoxyethanol sold as Nonidet~
P-40 (NP 40). Ehrlich ascites was used as a control.
-
The data in Table 3 were obtained by coating the antigenon microtiter plates and incubating it with a solution of each of
the antibodies in a buffer at the concentration of the detergent
indicated in the table r After incubation, the plate is washed and
reacted with polyclonal sheep anti-mouse antibodies labeled with
horse radish peroxidase (HRP). Incubations were for 1 hr. at room
temperature. The plate is washed again and reacted with a solu-
tion of orthophenylenediamine (ODP)~ a chromagen substrate for
HRP. Absorbance in each well was measured at 490 nm and is re-
ported in Table 3.
12~19~7
Table 3
Effect of Detergents on Binding By
Anti-Chlamydia Monoclonal Antibodies
Antibody O.D.l o.D.l o.D.l O.D.l o.D.l
ReactionEhrlich Antibody Antibody Antibody Antibody
MixtureAscites 1 2 3 4
, _
~ueous
Buffer 0.00 1.06 1.15 1.02 0.95
2% DOC 0.02 1.10 0.70 0.11 0.25
2%NP-40 0.00 0.20 0.11 0.80 0.06
0.5~DOC 0.03 1.37 1.36 1.22 1.25
0.1%SDS 0.05 1.17 1.20 1.30 1.05
1. O.D. at 490 nm obtained ~s an average of 2 samples with a s~andard
deviation of 0.05.
2. Aqueous buffer is Autopow tissue culture media with 8% horse serum,
2% fe~al calf serum. All detergents used in the experiment were
diluted in this buffer.
3. Used as a control.
These date show the effect of different detergents and de-
texgent concentration on the binding of the selected monoclonal
antibodies. Antibody No. 1 and Antibody No. 2 havea relatively
high affinity for Ch~amydia in aquesus buffer that was not affected
by any of the detergents except 2% NP-40. Antibody 3 had a low
affinity in 2% ~OC, yet retained its high affinity in the other
media. Antibody 4 had a low affinity in 2% DOC and 2% NP-40 but
a high affinity in the other media.
In other experiments, the antigen coated microtiter plates
were first incubated with detergents in the concentrations shown in
Table 3 for 1 hr. and then washed. Thereafter, the antichlamydla
antibodies were incubated in the wells followed, after washing, by
an incubation with ~he HRP labeled anti-mouse antibodies. This
12~9~7
incubation, after washing, was followed by an incubation with the
enzyme substrate. The optical densities measured in each well
compared to wells which were pretreated with the aqueous buffer
suggested that the antigen was not harmed by the de~ergents.
AccordingLy, the monoclonal antibodies could be used for the
affinity purification of the Chlamydia antigen by solubilizing
the antigen in a detergent compatible with antibody binding and
passing the preparation over a column of immobilized antibody to
bind the antigen. Subsequently, the antigen is released by
eluting the column with another detergent composition in which
the antibody does not bind to the antigen.
From the foregoing, it will be clear to those skilled in
the art that efficient purification of an antigen by means of af-
finity chromatoyraphy using a selected monoclonal antibody as the
~2~S907
i~munoadsorbent can be accomplished under conditions which do not
denature the a~tigen. Specific applications of this process include
i~s use to purify antigens in samples where they occur naturally and
to purify radiolabeled antigen which has degraded upon storage. A
particular ~pplication is the purification of pro~ein products
obtained by recombinant DNA technology. Among such products m~y be
men~ioned insulin and human growth hormone. The isolation of complex
proteins from serum, for e~ample Factor V or Factor VIII, is possible
using the process of this invention.
It is also possible ~o reverse the process and to purify the
monoclonal antibod~ by using immobilized antigen as a~
immunoadsorbent. Por e~ample, radiolabeled antibody used in an
immuDoradiometric assay which has degraded as a result of storage can
be purified in this ~ay. The monoclonal antibody can also be
recovered from ascites fluid or culture medium by using the
immobilized antigen as a~ immunoadsorbent.
While we do not wish to be bound by any particular theory,
the change i~ Xa with changes in pH is the likely e~fect of
proto~ation of histidine residues or deprotonation of lysine or
20 possibly tyrosi~e or arginine residu~s in either the antibody, the
antigen or both which alters the ability of the antige~ aDd Qntibody
to comple~ with each other. Speci~ic residues af~ected may or may ~ot
lie ~ithin the binding regions of tbe molecules~
Based upon our discovery that the antibodies produced by an
2~ a~imal's immune response to an anti~en include antibodies that vary in
their sensitivity to changes in environment, it is within the scope of
our inventlo~ to fractionate polyclonal antisera to obtaiD a mixture
of antibodies which behave in a manner similar to the environmentally
sensitive monoclonal antibodies o~ this invention. This fractionation
can be accomplished by contacting the immobilized antigen ~ith an
1~ 7
excess of the antisera in a first desired environmental condi-
tion followed by washing the immunoadsorbent to remove unbound
material. This step is followed by contacting the immunoadsor-
bent with a medium which is the second environment to elute
antibodies which are not eluted under the first environmental
condition. For e~ample, if one wishes to obtain antibodies
which exhibit a high affinity at pH 7, and a low affinity at pH
4, the immobilized antigen is contacted with an excess of anti-
sera at pH 7 and the immobilized antigen washed with a medium
at pH 7 to remove antibodies which exhibit a low affinity at pH
7. The immunoadsorbent is then eluted at pH 4 to remove anti-
bodies which exhibit a low affinity at pH 4. The eluant will
contain the fraction of antibodies whose binding with the anti-
gen is sensitive to changes in pH over the range pH 7 to pH 4.
Similar fractionation can be done with urea and other inhibitors
of antigen-antibody binding. The resulting populations of anti-
bodies may require further subfractionation to further remove
those antibodies which elute due to an intrinsic low affinity
rather than a Ka "switch".
It is also within the scope of our invention to employ
hybrid monoclonal antibodies having dual specificities for
affinity purification. A process for obtaining hybrid monoclonal
antibodies is described in Canadian patent application serial
no. 425,558 of Martinis et al.
The hybrid monoclonal antibody has two specificities
which may be for different antigens. For use in our invention,
the hybrid antibody is selected to exhibit pH or other environ-
mental sensitivity in the specificity for the antigen for which
it is to be used as an immunoadsorbent in an affinity chromatog-
-14-
,~
- ~2~ )7
raphy. The o~her specificity e~hibited by the hybrid is selected to
have a high affinity against a second antigen uhich is bound to
solid support~ When the hybrid antibody is applied to the solid
support, it is bou~d to the support by the second an~igen. Of course,
5 the affinity of tbe hybrid for the second antigen must not be
substantially l~wer in the environmental condition which will permit
elution of the first, or target antigen. Preferably, however, the
binding of second antigen to the antibody is sensitive to a different
environmental condition than that which permits the target antigen to
be eluted from the immunoadsorbent. For e~ample, the hybrid antibod~
may be selected so that the affinity for the target antigen is reduced
by a lowering of pH a~d the affini~y for the second antigen reduced by
increasi~g the pH. This permits the hybrid monoclonal anti~ody to be
desorbed readily from a solid support when it is desirable to do so
because the support has become co~taminated by impurities or other
reasons which impairs its usefulness.
The environmentally sensitive antibodies of our invention
ca~ also be used to store antigens i~ a solid phase that are unstable
in solution. For e~a~ple, radiolabeled antigen can be bound to the
20 immobilized antibody for storage and desorbed as needed. Desorption
ca~ be preceded by washing the immunoadsorbent to remove any products
of degration that arose during storage. The reverse process is also'
possible, i.e., the antigeD can be used to store unstæb~e ~ntibody i~
a solid phase. For e~ample, radiolabeled antlbody used in a
radioassay can be stored as the antigen:antibody comple~ and desorbed
as required.
The foregoing is a description of the presen~l~ preferred
embodiments of our invention which is to be limited only by the
- appended claims.
