CA1209907A

CA1209907A - Method of affinity purification employing monoclonal antibodies

Info

Publication number: CA1209907A
Application number: CA000425555A
Authority: CA
Inventors: Richard M. Bartholomew; Daniel E. Beidler; Gary S. David
Original assignee: Hybritech Inc
Current assignee: Hybritech Inc
Priority date: 1982-04-12
Filing date: 1983-04-11
Publication date: 1986-08-19
Also published as: GB2128630A; IT1219779B; IT8320550A0; FI834530A; ATA901983A; WO1983003678A1; ES8706964A1; FI88403C; DK192191D0; AT393386B; GB2128630B; FI834530A0; ES8506902A1; ES528000A0; CH672028A5; DK192191A; ES521371A0; FI88403B; EP0105359A4; EP0105359A1

Abstract

ABSTRACT
Described herein is a process for affinity purification of antigens and antibodies that employs monoclonal antibodies having a high affinity for the antigen in a first environment and a low affinity in a second environment, the environments being ones in which the immunochemical properties of the antigens and antibodies are not adversely affected. Also described is a process for fractionating antisera to obtain an antibody fraction having similar antigen binding properties as exhibited by the environmentally sensitive monoclonal antibodies.

Description

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-,~

- ~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.

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. 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 immunochemi-cal properties of the antigen or the antibody.

2. A monoclonal antibody according to claim 1 wherein the binding constant for the antibody to the antigen in the first environment is about ? 109 and the binding constant for the antibody to the antigen in the second environment is about ?
108.

3. A monoclonal antibody according to claim 2 wherein the binding constant in the first environment is ? 1010 and the binding constant in the second environment is ? 106.

4. A monoclonal antibody according to claim 1 wherein the first environment is a liquid medium having a first pH and the second environment is a liquid medium having a second pH.

5. A monoclonal antibody according to claim 2 wherein the first environment is a liquid medium having a first pH and the second environment is a liquid medium having a second pH.

6. A monoclonal antibody according to claim 3 wherein the first environment is a liquid medium having a first pH and the second environment is a liquid medium having a second pH.

7. A monoclonal antibody according to claim 4, 5 or 6 wherein the pH of the first environment and the pH of the second environment is within the range of about 4 to about 10.5.

8. A monoclonal antibody according to claim 1 wherein the second environment is a solution containing a chaotropic agent.

9. A monoclonal antibody according to claim 2 wherein the second environment is a solution containing a chaotropic agent.

10. A monoclonal antibody according to claim 3 wherein the second environment is a solution containing a chaotropic agent.

11. A monoclonal antibody according to claim 8, 9 or 10 wherein the chaotropic agent is selected from the group consist-ing of urea, guanidine, KSCN, KBr, KI and MgC12.

12. A hybrid monoclonal antibody having a dual specificity, one specificity being against a first antigen and the second specificity being against a second antigen, the specificity against the first antigen having a high affinity in a first environment and a low affinity in a second environment, neither environment substantially irreversibly altering the immunochemi-cal properties of the first antigen or the antibody and wherein the affinity of the hybrid antibody for the second antigen is not substantially different in said first and second environ-ments.

13. A hybrid monoclonal antibody according to claim 12 wherein the antibody has a high affinity for the second antigen in the first environment and a low affinity for the second anti-gen in a third environment.

14. A hybrid monoclonal antibody according to claim 13 wherein the first environment is a liquid medium having a first pH, the second environment is a liquid medium having a second pH and the third environment is a liquid medium having a third pH.

15. A monoclonal antibody according to claim 1 wherein the antibody is bound to a solid support.

16. A monoclonal antibody according to claim 2 wherein the antibody is bound to a solid support.

17. A monoclonal antibody according to claim 3 wherein the antibody is bound to a solid support.

18. A monoclonal antibody according to claim 12 wherein the antibody is bound to a solid support

19. A monoclonal antibody according to claim 13 wherein the antibody is bound to a solid support.

20. A monoclonal antibody according to claim 15, 16 or 17 wherein the solid support is selected from the group consisting of Sepharose, polystyrene, glass, nylon, cellulose, polymethyl methacrylate, silica gel, polyacrylamide and nitrocellulose.

21. A monoclonal antibody according to claim 18 or 19 wherein the solid support is selected from the group consisting of Sepharose, polystyrene, glass, nylon, cellulose, polymethyl methacrylate, silica gel, polyacrylamide and nitrocellulose.

22. A process for the purification 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 properties of the antigen or antibody;
b) immobilizing 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.

23. A process according to claim 22 wherein the antibody is a monoclonal antibody.

24. A process according to claims 22 or 23 wherein the binding constant for the antibody to the antigen in the first environment is about ? 109 and the binding constant for the antibody to the antigen in the second environment is about ?
108.

25. A process according to claim 22 or 23 wherein the bind-ing constant in the first environment is ? 1010 and the binding constant in the second environment is ? 106.

26. A process according to claim 22 wherein the first environment is a liquid medium having a first pH and the second environment is a liquid medium having a second pH.

27. A process according to claim 23 wherein the first environment is a liquid medium having a first pH and the second environment is a liquid medium having a second pH.

28. A process according to claim 26 or 27 wherein the pH
of the first environment and the pH of the second environment are within the range of about 4 to about 10.5.

29. A process according to claim 22 wherein the second environment is a solution containing a chaotropic agent.

30. A process according to claim 23 wherein the second environment is a solution containing a chaotropic agent.

31. A process according to claim 29 or 30 wherein the chao-tropic agent is selected from the group consisting of urea, guanidine, KSCN, KBr, KI and MgC12.

32. A process according to claim 23 wherein the monoclonal antibody is a hybrid monoclonal antibody having a first speci-ficity against the antigen to be purified and a second specifi-city having a high affinity against a second antigen, the second antigen being immobilized on the solid support and providing the means whereby the antibody is immobilized on the solid sup-port, the affinity of the antibody for the second antigen not being substantially lowered in the second environment.

33. A process according to claim 32 wherein the antibody has a low affinity for the second antigen in a third environment.

34. A process according to claim 33 wherein the first environment is a liquid medium having a first pH, the second environment is a liquid having a second pH and the third environ-ment is a third liquid having a third pH.

35. A process according to claim 22 wherein the antibody is an antiserum fraction.

36. A process for fractionating serum antibodies to obtain a fraction having a high affinity for an antigen 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 anti-bodies, comprising immobilizing the antigen on a solid support, contacting the antigen with the serum antibodies in the first environment to bind the antibodies to the antigen and eluting a fraction of the antigens using, as an eluant, a medium which is the second environment, the eluted antibodies having a low affinity for the antigen in the second environment.

37. A process according to claim 36 wherein the first environment is a liquid medium having a first pH and the second environment is a liquid medium having a second pH.

38. A process according to claim 37 wherein the pH of the first environment and the pH of the second environment is within the range of about 4 to about 10.5.

39. A process according to claim 36 wherein the second environment is a solution containing a chaotropic agent.

40. A process for the purification of an antibody having a high affinity for an antigen in a first environment and a low affinity for the antigen in a second environment, neither environment substantially irreversibly altering the immunochemi-cal properties of the antigen or antibody, comprising the steps:
a) immobilizing the antigen on a solid support;
b) binding the antibody to the antigen in the first environment;
c) separating the unbound impurities from the bound antibody; and d) eluting the antibody in a purified form from the immobilized antigen using, as an eluant, a medium which is the second environment.

41. A process according to claim 40 wherein the antibody is a monoclonal antibody.

42. A process according to claim 40 wherein the first environment is a liquid medium having a first pH and the second environment is a liquid medium having a second pH.

43. A process according to claim 42 wherein said first pH
and said second pH are within the range of about 4 to about 10.5.

44. A process according to claim 40 wherein the second environment is a solution containing a chaotropic agent.

45. A process according to claim 44 wherein the chaotropic agent is selected from the group consisting of urea, guanidine, KSCN, KBr, KI and MgC12.