FI88403C - MONOCLONAL ANTIKROPPAR, FOERFARANDE FOER FRAMSTAELLNING AV DESSA OCH AFFINITETSRENINGSFOERFARANDE VARI DESSA ANVAENDS - Google Patents

Description

1 88403

FIELD OF THE INVENTION This invention relates to the purification of antigen and antibodies by affinity chromatography. It also applies to monoclonal antibodies and their method of preparation. Background 10 Purification of antigen by affinity chromatography using serum antibodies generated by a host animal in response to the antigen and bound to a solid support acting as an immunoadsorbent is a method that has been used for many years. However, this method also has two serious weaknesses that reduce its applicability. Thus, if antibodies with high affinity are used to separate the antigen from the sample, stringent conditions are required to remove the antigen from the antibodies after the non-adsorbed contaminants have been rinsed out of the immunoadsorbent mass. Conditions required for this, for example pH below 3 or above 11 or concentrated Kao -. * · *. a trope, such as a solution of guanidine or urea, can denature antigen and antibodies, reducing, if not destroying, an- | I immunochemical and / or biological properties of the antigen "/ 25 and shortening the life of the immunoadsorbent.

In order to avoid the problems associated with the use of antibodies with high antigen affinity, it has become common practice to use immobilized antibodies with low affinity as immunoadsorbents. The use of these antibodies makes it possible to elute the antigen from the immunoadsorbent mass using mild, non-denaturing conditions. However, in the necessary column washing step, in which impurities from the bound antigen are eluted, some antigen is also eluted, so much so that the separation efficiency is greatly reduced. In addition, low-affinity antibodies are not able to bind 2 38403 antigens efficiently. in the medium at relatively low concentrations, i.e. less than about 10 ng / ml.

With the development of hybridoma technology, it has become possible to obtain monoclonal antibodies, which have then been proposed for use as immunoadsorbents in the affinity purification of the antigens against which they act. See, e.g., Stenman et al., J. Immunological Methods 46 (1981) 337; Stallcup et al., J. Immunology 127 (1981) 924 and Katzman et al., Proc. Natl. Acad.

10 Sci. USA 78 (1981) 162. These reports suggest that the monoclonal antibodies used should have at most only a moderate affinity for the antigens, which would allow them to be desorbed from the immunoadsorbent under mild conditions. Thus, the experience gained so far with the use of monoclonal antibodies as immunoadsorbents suggests that their properties would be parallel to those of "polyclonal" antibodies of conventional antiserum, i. the use of a low affinity antibody allows for the elution of antigen 20 under mild conditions, whereas the use of a high affinity antibody requires strong conditions to detach the antigen from the antibody.

SUMMARY OF THE INVENTION As is well known, a hybridoma is formed by random fusion of B lymphocytes to myeloma cells in the presence of a fusion-promoting agent. Each hybridoma in the large hybridoma population that can be produced by fusion secretes a different monoclonal antibody. Normally, the hybridoma population is searched for those hybridomas that secrete an antibody with the desired antigen specificity for further cloning to obtain useful amounts of antibody. We have found that in a population of hybridomas that secrete antibodies to a particular antigen, as well as in a subpopulation of 35 hybridomas that secrete antibodies with high affinity for the antigen, a much smaller population of antibodies is secreted. having a high affinity for the antigen in the first environment but a much lower affinity in the second environment, neither of which is destructive to the immunochemical or biological properties of the 5 antigen or antibody, We believe that the presence of these antibodies in high affinity antisera has gone unnoticed because the majority of the high affinity antibodies in the antiserum are those that require stringent conditions to separate the antigen from the antibodies and because these are dominant in the immunochemical properties of the antisera.

Thus, we have found that we are able to screen a hybridoma population that may be the product of multiple fusions and identify those hybridomas that produce a monoclonal antibody with high affinity in the first environment and low affinity in the second environment, and clone at least one of these hybridomas. , to obtain a sufficient amount of the antibody produced by it to be used as a very effective immunoadsorbent in affinity chromatography. In this context, the affinity of an antibody is considered to be high, • 'q when the affinity constant (Ka) is about £ 10 and low when Ka; ; is about S 10.

· '·' · · 25 Brief Description of the Drawings ·· “Figures 1 and 2 are graphs illustrating the effect of changes in pH on the radiolabeled human growth hormone bound to four solid phase immobilized to a different monoclonal antibody, desorp -. ** · .: 30 thio.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

According to our invention, the purification of the antigen is performed by a method consisting of the following steps.

a) selecting a monoclonal antibody having a high affinity for the antigen in the first environment and a low affinity for the antigen in the second environment, neither of which causes significant 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 the impure antigen in a first environment, wherein the antigen binds to the antibody; d) separating unbound impurities from the bound 10 antigens; and e) eluting the antigen in purified form from the immobilized antibody using a medium other than the eluent.

As already mentioned, antibodies useful in the context of our invention can be obtained by screening for antibodies produced by a hybridoma population obtained by fusing myeloma cells and B lymphocytes together by known methods. B lymphocytes are typically spleen cells taken from a hyperimmunized animal that has been pre-administered with 20 target antigens as an immunogen. Once those hybridomas that produce monoclonal antibodies specific for the desired antigen have been identified, they can be further screened to identify hybridomas that produce antibodies whose affinity changes with environmental changes that do not damage the antigen or antibody. . For example, antigens are usually stable in the pH range of 4 to 10.5. In order to obtain a pH-sensitive antibody for use as an immunoadsorbent, a population of monoclonal antibodies is screened to identify those antibodies which have a high affinity for the antibody, i. Also about 109 and preferably £ 10, at a pH in one of said ranges and a low affinity, i.e. Also by measuring about 10 and 5 88403, after the antibody has been allowed to adhere to the antigen, the amount of antigen desorbed at different pHs can be made, for example, using a radiolabeled antigen and counting the radiation emitted by the solid phase and / or supernatant. Similar screening can be performed to identify antibodies that respond to other types of environmental changes.

According to the present invention, it is preferred to use monoclonal antibodies whose antigen binding capacity is sensitive to changes in pH. For this purpose, an antibody with a high affinity at one pH and a low affinity at a second pH, which may be higher or lower than the first pH, is selected. The first pH is 7 or close to it, usually but not necessarily. However, it is also within the scope of the invention to select antibodies that respond to other changes in environmental conditions. For example, a monoclonal antibody whose affinity changes from high to low can be selected in the presence of a chaotropic solution as the elution medium. Suitable chaotropes include KBr, KI, KSCN, urea, * ··. guanidine and MgCl 2. Thus, monoclonal ... antibodies with Ka '10 in the absence of a chaotrope can be selected,

'Q

but Ka £ 10 in the presence of a particular chaotrope at a concentration not detrimental to the antigen in question. Selection based on chaotropic sensitivity can also be made in buffer solutions with a certain pH. Alternatively, antibodies that are sensitive to changes in pH can be selected when chaotrope is present at a constant concentration.

- ': 30 Monoclonal antibodies with affinity. to the antigen are sufficiently reduced by changes other than changes in pH or chaotropic concentration. Media sensitivities for which antibodies can be tested to select antibodies whose antigen binding capacity is affected by a change in the elution medium include methylmannoside sensitivity and sensitivity to nonionic or 6 38403 ionic detergents and reagents that affect certain amino acids such as Tyrosine-so.

The selected monoclonal antibody for affinity chromatography can be bound to any solid support commonly used in affinity chromatography. These include sepharose, polystyrene, glass, nylon, cellulose, polymethyl methacrylate, silica gel, polyacrylamide and nitrocellulose. The following examples illustrate the application of this invention to the preparation of monoclonal antibodies with high affinity binding affinity to the antigen in the second environment. , of which environments do not rapidly impair the immunochemical properties of the antigen and the utility of such antibodies as immunoadsorbents in affinity chromatography.

Example 1

Spleen cells from 20 Balb / c mice hyperimmunized with human growth hormone (HGH) were fused with polyethylene glycol to mouse myeloma cells (lines NS-1 or SP-2XO). · The resulting hybridomas were cloned and screened for those that secrete HGH-specific antibodies. radioimmunoassay using I-HGH and 25 equine IgG antibody against mouse on sepharose beads.

Anti-HGH-producing hybridomas were further screened to identify those that produce antibodies with Ka 9 of at least about 10 at pH 7. These were further screened to identify those with an affinity sensitive to pH changes in the pH range of 4 to 10.5. Data depicting the pH sensitivity of the four monoclonal antibodies are given in Tables 1 and 2 and Figures 1 and 2. The different antibodies are denoted by the letters A, B, C and D, respectively. This information was obtained as follows: I-labeled HGH was bound at pH 7 to each antibody pre-immobilized on polystyrene beads. Each bead contained approximately 1 ng of 7 88403 antigen and had a radioactivity of 10,000 cpm. Three beads of each type were incubated in 1 ml of PBS containing 10% horse serum for four hours at the indicated pH. The pH was adjusted by adding either sodium carbonate buffer (10% in horse serum) to adjust the pH to seven or by adding sodium acetate buffer (10% in horse serum) to adjust the pH to seven. After incubation, a count of 800 supernatant was counted. The calculated results for the desorbed antigen at each pH are given in Tables 1 and 2 as curves in Figures 1 and 2.

Table 1 - 3 *

Cpm x 10 for desorbed HGH

pH Antibody A Antibody B

15 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 20 1 5.5 0.176 0.162 6.0 0.220 0.176 6.5 0.336 0.167 i V 7.0 0.328 0.162 * V 25 * Average of the three supernatants 8 88403

Table 2

Cpm x 10 3 for desorbed HGH *

pH Antibody C Antibody_D

7.0 0.236 0.187 5 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 10.0 3.030 0.364 10.5 4.809 0.584 11.0 5.508 3.460 * Mean of the three supernatants 15 The data in Table 1, especially shown as a curve in the figure 1, show that the uptake of antibody B into HGH was approximately independent of pH changes in the pH range of 3.5 to 7, but that the uptake of HGH into antibody A decreased significantly in the pH range of 4.5 to 4.0 , indicating that the antibody would not effectively bind antigen at pH 4.0.

On the other hand, the data given in Table 2 and Figure 2 show that the adhesion of antibody D to HGH was approximately independent of pH changes in the pH range of 7.0 to 25.5.5, while the adhesion of antibody C to HGHs decreased significantly in the range of pH 9.5 to 10.5, indicating that the antibody would not effectively bind antigen at pH 10.5.

Supernatants eluted from antibodies A and C at pH 4.0 and 10.5 were added to PBS buffer (10% equine serum) where the samples were pooled and the pH was adjusted to seven.

• Combined samples were incubated with antibodies A, B, C

3a D and two other polystyrene beads coated with anti-HGH monoclonal antibody.

Each of these antibodies recognizes different regions of the HGH molecule. The immunoreactivity of the antigen eluted at either pH 4 or pH 10.5 with five of the six of the 88,8403 subjects studied, including antibodies A, B, C, and D, was not impaired and was only slightly reduced with the sixth. These data indicate that elution of the antigen at either pH 4.0 or pH 10.5 did not impair its immunochemical properties.

Example 2

High-affinity monoclonal antibody (Ka = 5 x 10) against prostatic acid phosphatase (PAP), a highly labile enzyme, obtained by screening hybridomas producing monoclonal antibodies against PAP obtained by fusion with PAP: 11a hyperimmunized Balb / c mouse spleen cells and mouse myeloma cells according to Example 1, sensitivity in the ability to bind antigen was observed in the pH range 6.0 to 4.0. The antibody was attached to 15 sepharose beads using CNBr technique at a concentration of 1 mg antibody / 1 ml packaged sepharose beads and used to purify PAP as follows. A 170 μl sample of semen containing 0.912 mg / ml PAP as determined immunoradiometrically using a TANDEM assay device for 20 PAPs manufactured by Hybritech, Inc., San Diego, Ca., was diluted to 5 ml with acetate buffer ( 10% sodium acetate in horse serum (relative to 0.15 M NaCl) to give a solution of 31 μg PAP / ml solution at pH 6.

V 25 The PAP solution was passed through a column containing 1.5 ml of Sepharose beads at a rate of 1 ml / h and the column was washed

7.5 ml of 11a buffer solution. Immunometric examination of the eluate (5 ml of sample and 7.4 ml of washing liquid) showed that 99.3% of the PAP was adsorbed on the column. PAP was eluted with 0.1M

With acetate buffer, pH 4, relative to 0.15M NaCl. Three 1 ml fractions were collected and dialyzed overnight against 50 mM citrate solution, pH 6.0. Combined and [* '. the amount of PAP contained in the dialyzed fractions was determined. immunoradiometrically 54% of the total amount fed to the column. The purity of the dialyzed material was determined · / .; sodium dodecyl sulfate and Ornstein-Davis PAGE.

10 88403

In both cases, one line was observed. Enzyme activity measurements were performed and showed that the enzymatic activity of the purified PAP was maintained.

Retention of 46% PAP on the column is likely to be, at least in part, the result of non-specific binding and the use of a large excess of antibody, leading to "dragging" of the antigen from the column. The former can be reduced by pretreating the column with a sample under elution conditions and washing thoroughly to remove any eluting material. The latter can be reduced by lowering the amount of antibody attached. Finally, Sepharose is not the best possible matrix due to the pore size heterogeneity of affinity chromatography, which leads to diffusion and steric problems.

15 Example 3

Purification of chlamydia-associated antigen is difficult because it is difficult to reconstitute. However, it can be dissolved in various detergents. Hybridomas producing monoclonal antibodies against chlamydia-related antigens obtained by fusing spleen cells from hyperimmunized Balb / c mice to mouse myeloma cells according to Example 1 were examined for their detergent concentration sensitivity. The effect of detergents on the binding of four such antibodies is shown in Tau-25, Chapter 3. The detergents used were deoxycholate (DOC), sodium dodecyl sulfate (SDS), and octylphenoxypolyethoxyethanol, sold under the name Nonidet P-40 (NP-40). The Ehrlich abdomen was used as a reference.

The data given in Table 3 were obtained by coating 30 microplates with antigen and incubating them with a buffer solution of each antibody at the detergent concentration according to the table. After incubation, the plate was washed and reacted with sheep polyclonal antibodies against mice labeled with horseradish peroxidase (HPR) at room temperature. The plate is washed again and reacted with a solution of orthophenylenediamine (ODP), a chromagen substrate of 11 88403 HRP. The absorbance of each well was measured at 490 nm and is given in Table 3.

Table 3 5 Effect of detergents on binding by anti-chlamydial monoclonal antibodies

Antibody- 0.D.1 O.D.l O.D.l O.D.l O.D.l reaction Ehrlich— Antibody Antibody Antibody Antibody mixture Water abdomen 1234 10 ö

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 15 0.1% SDS 0.05 1.17 1.20 1.30 1.05 1. OD measured at wavelength 490 is the mean of 2 samples with a standard deviation of 0.05.

2. The aqueous buffer is Autopow tissue culture medium-20 containing 8% equine serum and 2% fetal calf serum. All detergents used in the experiment were dissolved in this buffer.

: ***: 3. Used as a reference.

: These data show the effect of different detergents and detergent concentration on the binding of selected monoclonal antibodies. Antibody # 1 and Antibody # 2 had a relatively high affinity for chlamydia in aqueous buffer, which was not affected by any of the detergents except 2% NP-40. The affinity of antibody 3 was low in 2% DOC solution, but its high affinity was maintained in other media. The affinity of antibody 4 was low in 2% DOC solution and 2% NP-40 solution, but high in other media.

In other experiments, antigen-coated micro-plates were first incubated with detergents at the concentrations given in Table 3 for one hour and then washed. Anti-chlamydial antibodies were then incubated in the wells and then, after washing, HRP-labeled anti-mouse antibodies. This incubation was followed, after washing, by incubation with the enzyme substrate.

5 The optical densities measured from each well compared to the results obtained from wells pretreated with aqueous buffer indicated that the detergents were not harmful to the antigen. Thus, monoclonal antibodies could be used for affinity purification of Chlamydia antigen by dissolving the antigen in a detergent suitable for antibody binding and applying the preparation to a column containing immobilized antibody, whereby the antigen binds. The antigen is then released by eluting the column with another detergent composition in which the antibody does not bind the antigen.

From the foregoing, it will be apparent to those skilled in the art that efficient purification of antigen by affinity chromatography using a monoclonal antibody as an immunoadsorbent can be performed under conditions that do not denature the antigen. Specific applications of this method include its use for the purification of antigens contained in samples in which the antigen occurs naturally and for the purification of radiolabeled antigen that has degraded during storage. One particular application is the purification of recombinant protein products. Such products include insulin and human growth hormone. Isolation of complex proteins, for example factor V and factor VIII, from serum is possible using the method of this invention.

It is also possible to reverse the method and purify the monoclonal antibody using the immobilized antigen as an immunoadsorbent. For example, a radiolabeled antibody used in an immunoradiometric measurement that has degraded as a result of storage can be purified in this manner. Monoclonal antibody 13 88403 can also be recovered from aqueous humor or culture medium using immobilized antigen as an immunoadsorbent.

Although we do not wish to be bound by any particular theory, the change in Ka as the pH changes is likely to result from protonation of histidine residues or deprotonation of lysine or possibly tyrosine or arginine residues in either the antibody, antigen, or both, which alters the antigen and the ability of the substance to form a complex with each other. The individual residues affected may or may not be located in the adhesive regions of the molecules.

Based on our finding that antibodies produced by an animal's immune response to an antigen contain antibodies with varying sensitivity to environmental changes, it is within the scope of our invention to fractionate polyclonal antisera to obtain an antibody mixture in which the antibodies behave in the same manner as the present invention. environmentally sensitive monoclonal antibodies. This fractionation can be performed by contacting the immobilized antigen with an excess of antiserum under the first desired environmental conditions, after which the immunoadsorbent is washed to remove unbound material. This step is followed by contacting the immunoadsorbent with a medium acting as a second medium. to elute antibodies that do not elute in the first medium. For example, if it is desired to obtain antibodies with high affinity at pH 7 and low affinity at pH 4, the immobilized antigen is contacted with an excess of antiserum at pH 7 and the immobilized antigen is washed with a medium having a pH of 7. , to remove antibodies with low affinity at pH 7. The immunoadsorbent is then eluted; at pH 4 to remove antibodies with low affinity at pH 4. The eluate contains a fraction of antibodies whose binding to the antigen is sensitive to changes in pH of 14 to 88403 in the pH range of 7-4. Similar fractionation can be performed with urea and other antigen-antibody binding inhibitors. The resulting antibody populations may require further fractionation to further eliminate those antibodies that elute due to their inherently low affinity rather than Ka-coupling.

It is also possible to use hybrid Divasta monoclonal agents with dual specificity for affinity purification. A method for producing hybrid monoclonal antibodies is described in Martin et al. Canadian Patent No. 1,213,229, "Antibodies Having Dual Specifications, Their Preparation And Uses Therefor," which is incorporated herein by reference.

15 A hybrid monoclonal antibody has two specificities that may be for different antigens. The hybrid antibody to be used is selected to have pH or other environmental sensitivity in specificity for the antigen for which it is to be used as an immunoadsorbent in affinity chromatography. The second specificity of the hybrid is selected to have a high affinity for the second antigen attached to the solid support. When the hybrid antibody is applied to a solid support, the second antigen binds it to the support. The affinity of the hybrid for the second antigen is not: of course, it must be significantly lower under the environmental conditions in which elution of the first or target antigen is possible. However, the binding of the second antigen to the antibody is preferably sensitive to a different environmental state than that which allows the target antigen to elute from the immunoadsorbent. For example, the hybrid antibody can be selected such that the affinity for the target antigen decreases as the pH decreases and the affinity for the other antigen decreases. 35 nee as the pH rises. This allows the hybrid monoclonal antibody to be easily desorbed from a solid is 88403 support when desired because the support is contaminated with impurities or for other reasons that reduce the usability of the support.

The environmentally sensitive antibodies of our invention can also be used in solution to store unstable antigens in the solid phase. For example, a radiolabeled antigen can be bound to an immobilized antibody for storage and desorbed if necessary. Prior to desorption, the immunoadsorbent can be washed to remove any degradation products formed during storage. The opposite method is also possible, i. the antigen can be used to maintain an unstable antibody in the solid phase. For example, radiolabeled antibodies used in radiometric studies can be stored as an antigen-antibody complex and desorbed as needed.

The foregoing is a description of the presently preferred embodiments of our invention, which is limited only by the appended claims.

Claims (26)

A method for producing a monoclonal antibody having a high affinity for the corresponding antigen in the first environment and a low affinity for the antigen in the second environment, neither of which substantially alters the immunochemical properties of the antigen or the antibody irreversibly, characterized in that 10) a) screening the hybridomas and selecting a hybridoma population that produces monoclonal antibodies specific for the antigen, b) screening the population of step (a) and selecting a subpopulation of hybridomas that produce monoclonal antibodies with high affinity for the antigen c) screening the subpopulation of step (b) and selecting a subpopulation of hybridomas that produce monoclonal antibodies with low affinity for the antigen in the second environment, wherein neither environment is le detrimental to the immunochemical or biological properties of the antigen and not of the antibodies, and; d) cloning at least one of the hybridomas selected in step (c) to obtain a sufficient amount of monoclonal antibodies. A method according to claim 1, characterized in that the first medium is a liquid medium having a first pH and the second environment is a liquid medium having a second pH. A method according to claim 2, characterized in that the pH of the first environment and the pH of the second environment are in the range of about 4 to 10.5. The method of claim 1, characterized in that the binding constant between the antibody and the antigen 17 88403 in the first environment is α109 and the binding constant between the antibody and the antigen in the second environment is α108. A method according to claim 4, characterized in that the binding constant in the first environment is ä 1010 and the binding constant in the second environment is s 10®. The method of claim 1, characterized in that the first medium is a liquid medium having a first detergent concentration and the second medium is a liquid medium having a second detergent concentration. Process according to Claim 6, characterized in that the first and second detergents are ionic or nonionic detergents. A method for purifying an antigen, comprising the steps of: a) immobilizing an antibody prepared according to any one of claims 1 to 7 which has a high affinity for the antigen in the first environment and a low affinity for the antigen in the second environment. : in an environment in which neither of the environments substantially changes the immunochemical or biological properties of the antigen or antibody irreversibly, 25 b) binding the antigen to the antibody in the first environment; c) separating unbound impurities from the bound antigen; and d) eluting the antigen in pure form from the immobilized antibody using a medium other than the medium as eluent. A method according to claim 8, characterized in that the first medium is a liquid medium having a first pH and the second medium a liquid medium having a second pH. 88403 BC A method according to claim 9, characterized in that the first and second pH are between about 4 and 10.5. A method according to claim 8, characterized in that the first medium is a liquid medium having a first detergent concentration and the second medium is a liquid medium having a second detergent concentration. A method according to claim 11, characterized in that the first and second detergents are ionic or non-ionic detergents. The method of claim 8, characterized in that the binding constant between the antibody and the antigen in the first environment is> 15 109 and the binding constant between the antibody and the antigen in the second environment is <108. A method of purifying an antibody prepared according to claim 1 having a high affinity for the antigen in the first environment and a low affinity for the antigen in the second environment, neither of which substantially irreversibly alters the immunochemical or biological properties of the antigen or antibody. comprising the steps of: a) immobilizing the antigen on a solid support; b) binding the antibody to the antigen in the first environment; c) separating unbound impurities from the bound antibody; and d) eluting the antibody in purified form from the immobilized antigen using a medium other than the medium as eluent. A method according to claim 14, characterized in that the first medium is a liquid medium having a first pH and the second medium is a liquid medium having a second pH. 19 88403 The method of claim 15, characterized in that the first and second pH are between about 4 and 10.5. 16 88403 A method according to claim 14, characterized in that the first medium is a liquid medium having a first detergent concentration and the second medium is a liquid medium having a second detergent concentration. A method according to claim 17, characterized in that the first and second detergents are ionic or non-ionic detergents. The method of claim 14, characterized in that the binding constant between the antibody and the antigen in the first environment is> 109 15 and the binding constant between the antibody and the antigen in the second environment is s 10®. 20. A method of fractionating serum antibodies to obtain a fraction having a high affinity for the antigen in the first environment and a low affinity for the antigen in the second environment, neither of which is substantially altered. immunochemical properties of the antigen and not of the antibodies or:. biological properties irreversibly, emotional | immobilizing the antigen on a solid support, contacting the antigen with serum antibodies in a first medium to bind the antibodies to the antigen, and eluting the antibody fraction using a medium other than the medium as the eluent, wherein the eluted antibody has a low affinity. -: 30 commissioned antigen in another environment. The method of claim 20, characterized in that the first medium is a liquid medium having a first pH and the second medium is a liquid medium having a second pH. 20 8 8403 The method of claim 21, characterized in that the pH of the first environment and the pH of the second environment are in the range of about 4 to 10.5. The method of claim 20, characterized in that the first medium is a liquid medium having a first detergent concentration and the second medium is a liquid medium having a second detergent concentration. A method according to claim 23, characterized in that the first and second detergents are ionic or non-ionic detergents. The method of claim 20, characterized in that the binding constant between antibody and antigen in the first environment is> 109 and the binding constant between antibody and antigen in the second environment is <108. 26. A monoclonal antibody having a high affinity for its corresponding antigen in a first environment and a low affinity for that antigen in a second environment, neither of which substantially irreversibly alters the immunochemical properties of the antigen or the antibody, characterized in that that it is prepared by a method according to any one of claims 1 to 7. 2i 38403