EP0105359A4

EP0105359A4 - Method of affinity purification employing monoclonal antibodies.

Info

Publication number: EP0105359A4
Application number: EP19830901671
Authority: EP
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: 1983-04-12
Publication date: 1984-08-20
Also published as: CA1209907A; GB2128630A; IT1219779B; IT8320550A0; FI834530A; ATA901983A; WO1983003678A1; ES8706964A1; FI88403C; DK192191D0; AT393386B; GB2128630B; FI834530A0; ES8506902A1; ES528000A0; CH672028A5; DK192191A; ES521371A0; FI88403B; EP0105359A1

Description

-1-

DESCRIPTION

METHOD OF AFFINITY PURIFICATION EMPLOYING MONOCLONAL ANTIBODIES

Field of the Invention

This invention relates to the purification of anti gens and antibodies by affinity chromatography. I another aspect it relates to monoclonal antibodies.

Background

The purification of an antigen by affinity chromato graphy using serum antibodies, produced by a host animal' response to the antigen, that are bound to a solid suppor as an immunoadsorbent is a process which has been used fo many years. This process has, however, at least tw serious shortcomings which impair its usefulness. Thus if antibodies of high affinity are used to extract th antigen from a sample, harsh conditions are required t dissociate the antigen from the antibodies after non absorbed impurities have been washed from the body o immunoadsorbent. The conditions required for this, fo example, a pH of less than 3 or greater than 11 or concentrated chaotrope such as guanidine or urea solution can denature the antigen and the antibodies, diminishing if not destroying, the immunochemical and/or biologica properties of the antigen and shortening the useful lif of the immunoadsorbent.

To avoid the problems associated with the use o antibodies having a high affinity for the antigen, it ha become common practice to use immobilized antibodies o low affinity as an immunoadsorbent. Use of these anti¬ bodies permits elution of the antigen from the body of immunoadsorbent using mild, non-denaturing conditions. However, the requisite step of washing the column to elute impurities from the bound antigen also elutes some of the antigen, so much so that the efficiency of separation i greatly reduced. In addition, low affinity antibodie cannot efficiently bind antigens which are present in th media at relatively low concentrations, i.e., less tha about 10 ng/ml.

With the advent of hybridoma technology, it ha become possible to obtain monoclonal antibodies, whic subsequently have been proposed for use as im unoadsor bents in the affinity purification of the antigens agains which they were raised. See, for example, Stenman et al, J. Immunological Methods, 46, 337 (1981); Stallcup et al, J. Immunology, 127, ^'924 (1981) and Katzman et al, Proc. Natl. Acad. Sci. USA, 78, 162 (1981). These report suggest that the monoclonal antibodies employed, at best, had only a modest affinity for the antigens, permittin their desorption from the immunoadsorbent using mil conditions. Thus, the experience to date using monoclona antibodies as immunoadsorbents suggest that their proper ties should parallel those of the "polyclonal" antibodie of conventional antisera, i.e. , the use of low affinit antibody permits elution of the antigen under mild condi tions whereas use of a high affinity antibody requires harsh conditions to dissociate the antigen from th antibody.

Summary of Invention As is well known, hybridomas are -formed by the rando fusion of B-lymphocytes with myeloma cells in the presence of a fusion promoting agent. Each hybridoma 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 furthe 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 antibodies against a specifi antigen and the subpopulation of those which secrete antibodies having a high affinity for the antigen, a ve much smaller population secretes antibodies which have high affinity for the antigen in a first environment, b a much lower affinity in a second environment, neith environment being detrimental to the immunochemical biological properties of either the antigen or antibodie We believe that the existence of these antibodies in hi affinity antisera has gone unrecognized because t majority of the high affinity antibodies in the ant sera are ones which' require harsh conditions before t antigen can be separated from the antibodies and th dominate the immunochemical properties of the antiser

Accordingly, we have found that we can screen population of hybridomas, which can be the product o multiple fusions, and identify those, which produce monoclonal antibody having a high affinity in a firs environment and a low affinity in a second environment an clone at least one of the hybridomas to obtain a suffi cient quantity of the antibody it produces to permi its use as a highly effective immunoadsorbent for affinit chromatography. As used herein, an antibody is considere to exhibit a high affinity when its affinity constant (Ka is about >_ 10⁹ and to exhibit a low affinity when its K is about lθ8.

Brief Description of the Drawings

Figures 1 and 2 are graphs of data reflecting th effect of changes in pH on the desorption of radiolabele human growth hormone bound to four different monoclona antibodies immobilized on a solid phase.

Description of Preferred Embodiments

According to our invention, purification of a antigen is accomplished by a process comprising th steps: a) selecting a monoclonal antibody having a high affinity for the antigen in a first environment and a low affinity for the antibody in a second environment, neither environment causing substantial, irreversible changes in the desired immunochemical properties of the antigen; b) immobilizing the antibody on a solid support; c) contacting the immobilized antibody with a sample containing impure antigen in the first environment to bind the antigen to the antibody; d) separating unbound impurities from the bound antigen; 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 already noted, antibodies useful in our invention can be obtained by screening the antibodies produced by a population of hybridomas obtained by the fusion, using known methods, of myeloma cells with B-lymphocytes. The B—lymphocytes are typically spleen cells taken from a hype immunized 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 been identified, they can be further screened to identify those that produce antibodies whose affinities vary with changes in environment which are not damaging to the antigen or antibody. For example, antigens are usually stable in solution within the pH range of 4-10.5. To obtain a pH sensitive antibody for use as an immunoadsorbent, the population of monoclonal antibodies is screened to iden¬ tify those which have a high affinity for the antibody at one pH within the range, i.e., a Ka of about 10⁹ and preferably > 10l0 and a low affinity at a second pH,

f Q i.e., a Ka of about 10⁸ and preferably less than 10 within the same range. This kind of screening can be don by immobilizing the antibody on a solid support and, afte permitting it to bind antigen, measuring the extent o desorption of the antigen that occurs at different p levels, a measurement which can be made, for instance, b employing radiolabeled antigen and counting the radiatio emitted by the solid phase and/or supernatant. A simila screen can be carried out to identify antibodies tha respond to other kinds of environmental change. It is presently preferred to exploit monoclona antibodies whose capability to bind antigen is sensitiv to changes in pH. In this regard, the antibody is se lected to have a high affinity at one pH and a low af finity at a second pH which may be higher or lower tha the first pH^'. Usually the first pH will be at or near p 7 although it need not be. However, it is also within th scope of the invention tb select antibodies which respon to a different kind of change in environmental condition For example, a monoclonal antibody can be selected whic undergoes a change from high low affinity in the presenc of a chaotropic solution as the eluting medium. Amon suitable chaotropes are KBr, KI, KSCN, guanidine, urea an MgCl2» Thus, monoclonal antibodies can be selecte having a Ka > 10-**¹ in the absence of chaotrope but whic has a Ka of _< 10*** in the presence of the particula chaotrope whose concentration is not detrimented to th antigen in question. The selection for chaotrope sen sitivity can also be made in buffers at a specific pH. Alternatively, antibodies can be selected which are sensitive to changes in pH in the presence of a constant concentration of a chaotrope.

Monoclonal antibodies whose affinity for an antigen is adequately lowered by changes other than pH or concen¬ tration of chaotrope can also be selected. Among the kinds of media sensitivity for which the antibodies can be screened to select those whose antigen binding ability i affected by a change in eluting medium can include borat sensitivity, methylmannoside sensitivity and sensitivit to non-ionic or ionic detergents and reagents whic affect specific amino acids such as tryptophan and tyro sine.

For use in affinity chromatrography, a selecte monoclonal antibody can be bound to any of the soli supports commonly used in affinity chromatography. Thes include sepharose, polystyrene, glass, nylon, cellulose polymethyl methacrylate, silicagel, polyacrylamide an nitrocellulose.

The following examples illustrate the application o the present invention to obtaining monoclonal antibodie whose binding affinity for an antigen varies from a hig affinity in a first environment to a low affinity in second environment, neither environment causing damage t the immunochemical properties of the antigen and thei usefulness as immunoadsorbents for affinity chromato graphy. Example 1

Spleen cells taken from Balb/c mice hyperimmunize with human growth hormone (HGH) were fused using poly ethylene glycol with mouse myeloma cells (NS-1 or SP-2/ lines). The resulting hybridomas were cloned and screene to determine those secreting antibody specific for HGH b a radioimmunoassay employing 125ι-HGH and horse anti mouse IgG on sepharose beads. The hybridomas producin anti-HGH were further screened to identify those producin antibodies having a Ka of at least about lO-**' at pH 7 These were further screened to identify those whose affin ities were sensitive to changes in pH over the range from to 10.5. Data reflecting the pH sensitivity of fou monoclonal antibodies is shown in Tables 1 and 2 and Figs 1 and 2. The individual antibodies are designated by th letters A, B, C and D, respectively. These data wer obtained in the following way: HGH labeled with 125l w bound at pH 7 to each antibody which had previously be immobilized on polystyrene balls. Each ball contain approximately 1 ng of antigen and 10,000 cpm. Three each were incubated in 1 ml of PBS in 10% horse serum f four hours at the pH indicated. The adjustments in pH we made by the addition of either a buffer of sodium carbona (10% in horse serum) to obtain pH 7 or by the addition sodium acetate buffer (10% in horse serum) to obtain pH After incubation, 800 *.l of the supernatant was counte The counts of desorbed antigen at each pH are recorded Tables 1 and 2 and plotted in Figs. 1 and 2.

Table 1

Counts/Minute x 10-3 of Desorbed HGH*

VH Antibody A Antibody

3.0 4.810 2.442

3.5 4.625 0.803

4.0 4.156 0.357

4.5 1.608 0.248

5.0 0.449 0.206

5.5 0.176 0.162

6.0 0.220 0.176

6.5 0.336 0.167

7.0 0.328 0.162

Average of three supernatants

Table 2 Counts/Minute x 10"³ of Desorbed HGH* pH Antibody C Antibody D

7.0 0.236 0.187 7.5 0.208 0.336

8 .0 0.240 0.321

8 .5 0.666 ^' 0.277

9 .0 0. 401 0 .237

9 .5 1.038 0 .287 10 .0 3 .030 0 .364

10.5 4.809 0 .584

11.0 5.508 3 .460

* Average of three supernatants

The data in Table 1, particularly as plotted in Fig 1 , show that the binding of HGH by antibody B was essen tially insensit ive to changes of pH over the range p 3 . 5-7 but that the bind ing of HGH to antibody A wa s ignificantly decreased in the range pH 4.5-4. 0 indicatin that the antibody would not effectively bind the antige at pH 4. 0.

The data in Table 2 and Fig. 2 on the other hand show that the binding of HGH by antibody D was essentiall insensitive to changes in pH over the range pH 7.0-10. whereas the binding of HGH by antibody C was significantl decreased in the range of pH 9.5-10.5 indicating that th antibody would not effectively bind the antigen at p 10.5.

The supernatants eluted from antibodies A and C at p 4.0 and 10.5 were added to PBS buffer (10% in horse serum and adjusted to pH 7 and the samples pooled. The poole samples were incubated with polystyrene balls coated wit antibodies A, B, C and D and two other monoclonal anti bodies against HGH. Each of these antibodies recogniz different areas of the HGH molecule. The immunoreactivit of the antigen eluted at either pH 4 or pH 10.5 with fiv

of the six antibodies, including antibodies A, B, C and had not been affected and was only slightly diminish against the sixth. These data indicate that elution the antigen at either pH 4.0 or pH 10.5 did not adverse affect its immunochemical properties.

Example 2

A high affinity monoclonal antibody (Ka = 5 x 10-¹ against prostatic acid phosphatase (PAP) , an extreme labile enzyme, obtained by screening hybridomas produci anti-PAP monoclonal antibodies derived from fusions spleen cells taken from a Balb/c mouse hyperimmunized wi PAP and mouse myeloma cells as described in Example 1 w found to exhibit antigen binding sensitivity in the range 6.0-4.0. The antibody was bound to sepharose bea using the CNBr technique at a concentration of 1 mg antibody per 1 ml of packed sepharose beads and used purify PAP from seminal fluid as follows. A 170 JΛ ! samp of seminal fluid containing 0.912 mg/ml of PAP as dete mined by an immunoradiometric assay, using a TANDE assay kit for PAP manufactured by Hybritech, Inc., Sa Diego, Ca. , was diluted to 5 mis with acetate buffer (10 sodium acetate in horse serum containing 0.15 M NaCl) t obtain a solution of 31 x.g PAP/ml of solution having a p of 6.

The PAP solution was passed through a column con taining 1.5 ml of the sepharose beads at the rate of ml/hour and the column washed with 7.5 mis of the startin buffer. Immunometric assay of the eluant (5 mis of sampl and 7.4 ml of wash liquid) demonstrated that 99.3% of th PAP had adsorbed to the column. The PAP was eluted wit 0.1 M acetate buffer, pH 4 containing 0.15 M NaCl. Thre 1 ml fractions were collected and dlalyzed overnight vs 50 mM citrate, pH 6.0. The PAP content of the pooled an dialyzed fractions was determined by immunoradiometri assay to be 54% of the total applied to the column Purity of the dialyzed material was determined by sodiu dodecyl sulfate and Ornstein-Davis PAGE. A single ban was observed in each case. Enzymatic activity measure ments were done and documented that the purified PA retained its enzymatic activity.

The retention of 46% of the PAP on the column i likely the result, at least in part, of non-specifi binding and the use of a large excess of antibody whic results in antigen "trail^w from the column. The forme can be reduced by pretreating the column with sample unde the conditions at which elution will be accomplishe followed by extensive washing to remove any materia which will elute. The latter can be reduced by lowerin the_. concentration of bound antibody. Finally, sepharos is not an ideal matrix for affinity chromatography becaus of the heterogeneity of pore size, resulting in diffusio and steric problems.

Example 3

The purification of the antigen associated wit chlamydia is complicated because it is difficult t solubilize. However, it can be solubilized in a variet of detergents. Hybridomas which produce monoclona antibodies against chlamydial antigen, obtained by fusin spleen cells from hyperimmunized Balb/c mice with mous myeloma cells as described in Example 1, were screened fo

- sensitivity to detergent concentration. The effect o detergents on the binding of four such antibodies is se forth in Table 3. The detergents used were deoxycholat (DOC), sodium dodecyl sulfate (SDS) and octylphenoxypoly ethoxyethanol sold as Nonidet P-40 (NP 40). Ehrlic ascites was used as a control.

The data in Table 3 were obtained by coating th antigen on microtiter plates and incubating it with solution of each of the antibodies in a buffer at th concentration of the detergent indicated in the tabl After incubation, the plate is washed and reacted wi polyclonal sheep anti-mouse antibodies labeled wi horse radish peroxidase (HRP). Incubations were for 1 h at room temperature. The plate is washed again a reacted with a solution of orthophenylenediamine (ODP), chromagen substrate for HRP. Absorbance in each well w measured at 490 nm and is reported in Table 3.

Table 3 Effects of.Detergents on Binding By Anti-Chlamydia Monoclonal Antibodies Antibody O.D.¹ O.D.¹ O.D.¹ O.D.¹ O.D.I Reaction Ehrlich Antibody Antibody Antibody Antibody Mixture Ascites 1 2 3 4

Aqueous****

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 as an average of 2 sample with a standard deviation of 0.05.

2. Aqueous buffer is Autopow tissue culture media wit 8% horse serum, 2% fetal calf serum. All detergent used in the experiment were diluted in this buffer

3. Used as a control.

These data show the effect of different detergent and detergent concentration on the binding of the selecte monoclonal antibodies. Antibody No. 1 and Antibody 2 hav a relatively high affinity for Chlamydia in aqueous buffe that was not affected by any of the detergents except 2 NP-40. Antibody 3 had a low affinity in 2% DOC, ye retained its high affinity in the other media. Anti body 4 had a low affinity in 2% DOC and 2% NP-40 but high affinity in the other media. In other experiments, the antigen coated microtite plates were first incubated with detergents in the concen trations shown in Table 3 for 1 hr. and then washed. Thereafter, the antichlamydia antibodies were incubated i the wells followed, after washing, by an incubation with the HRP labeled anti-mouse antibodies. This incubation, after washing, was followed by an incubation with th enzyme substrate. The optical densities measured in each well compared to wells which were pretreated with th aqueous buffer suggested that the antigen was not harmed by the detergents. Accordingly, the monoclonal antibodies could be used for the affinity purification of the Chlamy¬ dia antigen by solubilizing the antigen in a detergen compatible with antibody binding and passing the prepara- tion over a column of immobilized antibody to bind the antigen. Subsequently, the antigen is released b eluting the column with another detergent composition i which the antibody does not bind to the antigen.

From the foregoing, it will be clear to those skille in the art that efficient purifiction of an antigen by means of affinity chromatography using a selected mono¬ clonal antibody as the immunoadsorbent can be accomplished under conditions which do not denature the antigen. Specific applications of this process include its use to purify antigens in samples where they occur naturally and to purify radiolabeled antigen which has degraded upon storage. A particular application is the purification of protein products obtained by recombinant DNA technology. Among such products may be mentioned insulin and huma growth hormone. The isolation of complex proteins from serum, for example Factor V or Factor VIII, is possible using the process of this invention.

It is also possible to reverse the process and to purify the monoclonal antibody by using immobilized antigen as an immunoadsorbent. For example, radiolabeled antibody used in an immunoradiometric assay which has degraded as a result of storage can be purified in this

P way. The monoclonal antibody can also be recovered fro ascites fluid or culture medium by using the immobilize antigen as an immunoadsorbent.

While we do not wish to be bound by any particula theory, the change in Ka with changes in pH is the likel effect of protonation of histidine residues or deprotona tion of lysine or possibly tyrosine or aginine residues i either the antibody, the antigen or both which alters th ability of the antigen and antibody to complex with eac other. Specific residues affected may or may not li within the binding regions of the^"molecules.

Based upon our discovery that the antibodies produce by an animal's immune response to an antigen includ antibodies that vary in their sensitivity to changes i environment, it is within the scope of our invention t fractionate polyclonal antisera to obtain a mixtur of antibodies which behave in a manner similar to th environmentally sensitive monoclonal antibodies of this invention. This fractionation can be accomplished b contacting the immobilized antigen with an excess of th antisera in a first desired environmental condition followed by washing the immunoadsorbent to remove unbound material. This step is followed by contacting the immno- adsorbent with a medium which is the second environment to elute antibodies which are not eluted under the first environmental condition. For example, 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 antisera 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 antigen 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 antibodies 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 speci¬ ficities for affinity purification. A process for ob¬ taining hybrid monoclonal* antibodies is described in the concurrently filed application of Martinis et al, "Anti- bodies Having Dual Specificities, Their Preparation And Uses Therefor", Serial No. —, (Lyon and Lyon Docket 162/98), the disclosure of which is incorporated by reference.

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 environmental sensitivity in the specificity for the antigen for which it is to be used as an immunoadsor¬ bent in an affinity chromatography. The other specificity exhibited by the hybrid is selected to have a high affinity against a second antigen which is bound to a solid support. When the hybrid antibody is applied to the solid support, it is bound to the support by the second antigen. Of course, the affinity of the hybrid for the second antigen must not be substantially lower 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 immuno¬ adsorbent. For example, the hybrid antibody may be selected so that the affinity for the target antigen is reduced by a lowering of pH and the affinity for the second antigen reduced by increasing the pH. This permits the hybrid monoclonal antibody to be desorbed readily from a solid support when it is desirable to do so because the support has become contaminated by impurities or oth reasons which impairs its usefulness.

The environmentally sensitive antibodies of ou invention can also be used to store antigens in a soli phase that are unstable in solution. For example, radio labeled antigen can be bound to the immobilized antibod for storage and desorbed as needed. Desorption can b preceded by washing the immunoadsorbent to remove an products of degration that arose during storage. Th reverse process is also possible, i.e., the antigen can b used to store unstable antibody in a solid phase. Fo example, radiolabeled antibody used in a radioassay can b stored as the antigen: antibody complex and desorbe as required. The foregoing is a description of the presentl preferred embodiments of our invention which is to b limited only by the appended claims.

ϊJRE

Claims

Clai s:

1. A monoclonal antibody having a high affinity for the antigen against which it is raised in a first en¬ vironment and a low affinity for the antigen in a second environment, neither environment substantially irrever¬ sibly altering the immunochemical 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 >_^ lO¹*** and the binding constant in the second environment is v lθ6.

4. A monoclonal antibody according to Claim 1, 2 or 3 wherein the first environment 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 4 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.

6. A monoclonal antibody according to Claims 1, 2 or 3 wherein the second environment is a solution containing a chaotropic agent.

7_* A monoclonal antibody according to Claim 6 wherein the chaotropic agent is selected from the group consisting of urea, guanidine, KSCN, KBr, KI and MgCl2- 8. A hybrid monoclonal antibody having a dual speci ficity, one specificity being against a first antigen an the second specificity being against a second antigen, th specificity against the first antigen having a hig. affinity in a first environment and a low affinity in second environment, neither environment substantiall irreversibly altering the immunochemical properties o the first antigen or the antibody and wherein the affinit of the hybrid antibody for the second antigen is no substantially different in said first and second environ ments.

9. A hybrid monoclonal antibody according to Claim wherein the antibody has a high affinity for the secon antigen in the first environment and a low affinity fo the second antigen in a third environment.

10. A hybrid monoclonal antibody according to Claim 9 wherein the first environment is a liquid medium having first pH, the second environment is a liquid medium having a second pH and the third environment is a liquid mediu having a third pH.

11. A monoclonal antibody according to Claims 1, 2, 3, 8 or 9 wherein the antibody is bound to a solid support.

12. A monoclonal antibody according to Claim 11 wherein the solid support is selected from the group consisting of Sepharose, polystyrene, glass, nylon, cellulose, poly¬ methyl methacrylate, silica gel, polyacrylamide and nitrocellulose.

13_* A process for the purification of an antigen comprising the stepsr a) selecting an antibody having a high affinity for the antigen in a first environment and a low affinity

O P for the antigen in a second environment, neither environ¬ ment substantially irreversibly altering the immuno chemical 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 antigen; and e) eluting the antigen in a purified form from th immobilized antibody using,, as an eluant, a medium which is the second environment.

14. A process according to Claim 13 wherein the anti body is a monoclonal antibody.

15. A process according to Claims 13 or 14 wherein th binding constant for the antibody to the antigen in the first environment is about 10⁹ and the binding con¬ stant for the antibody to the antigen in the second environment is about lO^****.-

16. A process according to Claims 13 or 14 wherein the binding constant in the first environment is >_ 10¹*-* and the binding constant in the second environment is - 10⁶.

17. A process according to Claims 13 or 14 wherein the first environment is a liquid medium having a first pH and the second environment is a liquid medium having a second pH.

18. A process according to Claim 17 wherein the pH of the first environment and the pH of the second environmen are within the range of about 4 to about 10.5.

OMP 19. A process according to Claims 13 or 14 wherein th second environment is a solution containing a chaotropi agent.

20. A process according to Claim 19 wherein the chao tropic agent is selected from the group consisting o urea, guanidine, KSCN, KBr, KI and MgCl2.

21. A process according to Claim 14 wherein the mono clonal antibody is a hybrid monoclonal antibody having first specificity against the antigen to be purified and second specificity having a high affinity against a secon antigen, the second antigen being immobilized on the solid support and providing the means whereby the antibody is immobilized on the solid support, the affinity of the antibody for the second antigen not being substantially lowered in the second environment.

22. A process according to Claim 21 wherein the anti¬ body has a low affinity for the second antigen in a third environment.

23. A process according to Claim 22 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 environment is a third liquid having a third pH.

24. A process according to Claim 13 wherein the anti¬ body is an antiserum fraction.

25. 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 antibodies, comprising immobilizing the antigen on a solid support, contacting the antigen with the serum antibodies in the first environment to bind the antibodie to the antigen and eluting a fraction of the antigen using, as an eluant, a medium which is the second environ ment, the eluted antibodies having a low affinity for th antigen in the second environment.

26. A process according to Claim 25 wherein the firs environment is a liquid medium having a first pH and th second environment is a liquid medium having a secon pH.

27. A process according to Claim 26 wherein the pH o the first environment and the pH of the second environmen is within the range of about 4 to about 10.5.

28. A process according to Claim 25 wherein the secon environment is a solution containing a chaotropic agent.

29. A process for the purification of an antibod having a high affinity for an antigen in a first environ ment and a low affinity for the antigen in a secon environment, neither environment substantially irrever sibly altering the immunochemical properties of th antigen or antibody, comprising the steps: a) immobilizing the antigen on a solid support; b) binding the antibody to the antigen in th first environment; c) separating the unbound impurities from th bound antibody; and d) eluting the antibody in a purified form fro the immobilized antigen using, as an eluant, a mediu which is the second environment.

30. A process according to Claim 29 wherein the anti body is a monoclonal antibody. 31. A process according to Claim 29 wherein the firs environment is a liquid medium having a first pH and th second environment is a liquid medium having a secon pH.

32. A process according to Claim 31 wherein said firs pH and said second pH are within the range of about 4 t about 10.5.

' 33. A process according to Claim 29 wherein the secon environment is a solution containing a chaotropic agent

34. A process according to Claim 33 wherein the chao tropic agent is selected from the group consisting o urea, guanidine, KSCN, KBr, KI and MgCl2»

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