Immunological preparations

An immunological preparation of an antigenic material in combination with a major histocompatibility complex antigen, which is itself in the form of complex with a protein with which it is normally associated in nature or with a modified form of such protein which retains the epitope thereof intact, said antigenic material being attached to the protein of the complex through antibody to that protein, is disclosed as being useful for the production of an immunogenic response in human or veterinary use.

This invention relates to immunological preparations, particularly those 
containing antigenic materials. 
Immunisation, for example against infectious diseases, is widely used in 
both a human and a veterinary context. It is often the case, however, that 
the antigenic material contained in the immunological preparation is not 
as immunogenic as could be desired, necessitating the use of a plurality 
of injections of the preparation. As an alternative, or in addition to the 
use of repeated injections, an adjuvant may be incorporated in the 
immunological preparation in order to increase the immune response 
provoked by the antigenic material. However, the effective adjuvants at 
present available, for example Freund's incomplete and complete adjuvants, 
have disadvantages particularly for human use. For some time, therefore, 
attempts have been made to discover new forms of adjuvant which would 
simplify immunization procedures and reduce the amounts of antigenic 
material required or amplify the response to a given quantity of antigenic 
material. Such an effect is applicable not only for protective 
immunization but also for the production of therapeutic or diagnostic 
immunological and serological reagents. 
In U.K. Pat. Application No. 7838177 there is described and claimed a novel 
form of immunologically active preparation based upon the discovery that 
members of a group of naturally occurring substances have the effect of 
increasing the immunogenicity of antigenic materials when administered in 
combination therewith. Such preparations comprise an antigenic material in 
combination with an MHC (Major Histocompatibility Complex) antigen. 
I have now developed a new approach to the production of such combinations 
of antigenic material with MHC antigen which has particular advantages as 
compared with the modes of preparation described in U.K. Patent 
Application No. 7838177. 
Accordingly the present invention comprises an antigenic material in 
combination with an MHC antigen which is itself in the form of a complex 
with a protein with which it is normally associated in nature, said 
antigenic material being attached to the protein of the complex through 
antibody to that protein. 
MHC antigens have been found in all animal species (including man) where 
they have been sought, being carried on the surface of nucleated cells of 
tissues, and constitute a particularly polymorphic system within any 
species. Therefore, although it is necessary in order for the MHC antigen 
to enhance the immunogenicity of the antigen material with which it is 
combined, that the MHC antigen should contain an antigenic determinant or 
determinants foreign to the recipient, the extreme polymorphism of these 
MHC antigens is generally sufficient to ensure that this is the case 
between one member of a species and another, except in the extreme case of 
members of a species which are genetically identical. The MHC antigen may 
therefore be from either the same species or another species, for example 
a phylogenetically similar species such as a primate in the case of 
preparations for human administration. MHC antigen from the same species 
may often be preferred, however, unless there are reasons, particularly an 
enhancement of effect, for using MHC antigen from another species. 
One major group of MHC antigens to which the present invention is 
applicable comprises the HLA antigens which are found in man and the 
analogous antigens of other species. Such MHC antigens are referred to by 
many workers as SD (Serologically Determined) antigens. A second group of 
MHC antigens of some interest which may be distinguished from these 
Serologically Determined MHC antigens, are those antigens such as the Ia 
antigens of mice and their equivalents in man (DRW) and other animal 
species. Examples of the MHC antigens analogous to HLA in other species 
are RLA in rabbits and H-2 in mice, etc. The MHC antigens of a combination 
according to the present invention may be of various specificities. 
Moreover, they may comprise not only the whole molecule which occurs in 
nature but also a portion thereof which retains the epitope intact, for 
example such derivatives as the papain-solubilised MHC antigens. 
In their natural form, MHC antigens are normally bound to a protein 
molecule, for example HLA and the equivalent MHC antigens of other 
non-human species being bound to B2-microglobulin (B2M) of the appropriate 
species. The nature of the binding between the two molecules in such a 
normal association of MHC antigen and protein is not yet known for any 
species, although present indications are that it is not of the form of a 
covalent bond. However, the bonding is strong enough so that in several 
species, including man, B2M is co-isolated with MHC antigen under normal 
circumstances. It will be appreciated, therefore, that the reference made 
hereinbefore to "an MHC antigen which is itself in the form of a complex 
with a protein with which it is normally associated in nature" refers to 
this type of protein and not to a protein with which the MHC antigen may 
be associated only under certain particular circumstances, for example on 
occurrence of a pathological condition. Moreover, the B2M or other similar 
protein component of such a complex may be the whole protein or a suitable 
modification thereof which retains the epitope since it is the epitope 
which is necessary for attachment of the antibody and thus the antigenic 
material to the complex. Thus, the protein,for example a B2-microglobulin, 
present in a combination according to the present invention may be not 
only the whole protein found in nature but also a portion thereof which 
retains the epitope intact. 
The present invention derives from the appreciation that significant 
advantages accrue from attaching the antigen whose immune response it is 
desired to increase to an antibody against the protein which is in turn 
attached to the protein of the MHC-protein complex. Such a procedure 
enables the antigenic material and the MHC-protein complex to be combined 
through an immunological rather than a chemical reaction with all the 
advantages which this implies by way of specificity, lack of 
de-naturation, and consequent high yield etc. In particular, it is 
possible by this method to obtain a product of a very high state of 
purity. 
For the sake of convenience only, further discussion of the preparation of 
a combination as defined above will be presented in terms of a combination 
of the type antigenic material/anti-B2M/B2M/MHC althrough it will be 
appreciated that the discussion is generally applicable to combinations 
incorporating other than B2M proteins. 
The antibody which forms a vital part of a combination according to the 
present invention is often of the IgG type but this is not necessarily 
always the case. It is usually desirable that the MHC/B2M complex and thus 
the antibody thereto, is specific for the intended human, mammalian or 
avian recipient of the immunological preparation. Commonly, therefore, a 
complex of HLA or the equivalent bovine, horse, pig, sheep, chicken etc., 
LA with the appropriate species B2M is used. However, although it is the 
case, for example, that for the treatment of cattle and pigs bovine LA and 
porcine LA are preferred as the MHC antigen, there may in some instances 
be an advantage in using the MHC antigen of a closely related species in 
combination with the B2M of that species so that, for example, the use of 
bovine LA in pigs and of porcine LA in cattle may be considered in 
addition to the more usual intraspecies usage. 
Production of the antibody is conveniently effected through one of the 
standard immunological techniques which are described in the literature of 
this art. A preferred method involves the use of B2M of the appropriate 
species as the antigen in the established techniques of monoclonal 
antibody production. This technique involves the immunisation of a 
suitable animal host, for example a mouse or a rat of a suitable strain 
for the cell line used, with the B2M protein followed by fusion of spleen 
or other immunocyte cells from the animal with cells of a suitable cell 
line, such as the mouse P3-X63-Ag8 or NS1/1-Ag4-1 and the rat 21ORC Y3-Ag 
1.2.3 cell lines of Milstein, to produce a hybrid cell line capable of 
anti-B2M production. The hybrid cells may then conveniently be inoculated 
into the appropriate animal host to induce myeloma growth therein, and the 
antibodies subsequently isolated from serum or ascites. As B2M is an 
immuno-dominant species it is possible simply to use lymphocytes as a 
source thereof for injection into the animal host. A source of isolated 
B2M is not therefore necessary for this purpose. Such material is, 
however, available in the use of human B2M from the urine of patients with 
renal failure and is of value in purification of the anti-B2M prepared in 
vivo since such purification may conveniently be effected by passing the 
material obtained, for example from serum or ascites, through a column of 
B2M coupled to a suitable support material, for example a Sepharose such 
as that marketed under the designation S4B. The antibody immunologically 
bound to the immoblised B2M may conveniently be eluted with a suitable 
aqueous acidic medium, for example aqueous glycine hydrochloride. An 
alternative technique for producing the antibody from the hydrid cells 
comprises their culture in vitro which will in general give a purer 
product but may present more problems with regard to the production of a 
significant quantity of antibody. 
It is not necessary for the combination to contain the whole of the 
antibody and, if desired, the antibody obtained by the procedures as 
described above may be treated by conventional methods to separate a 
sub-fraction thereof which is combined with the antigenic material rather 
than the whole antibody. The antibody, for example anti-B2M, present in a 
combination according to the present invention may thus be the whole 
antibody or an active fragment thereof. Thus, the F(ab').sub.2 fragment or 
particularly the Fab fragment may be used, these being obtained, for 
example, by pepsin and papain hydrolysis, respectively. 
As regards the antigenic material, the present invention is widely 
applicable to a whole variety of antigenic materials which are of use in 
vaccines or in other contexts, for example in therapeutic or diagnostic 
immunological and serological reagents. The term "antigenic material" as 
used herein thus covers any substance that will elicit a specific immune 
response when formulated in combination with an MHC antigen, and includes 
antigenic determinants such as peptides (small or large), oligo or 
polysaccharides, alloepitopes, haptens and the like. 
In the context of vaccines for human administration a variety of microbial 
antigens may be used as the antigenic material. Examples include bacterial 
antigens, for instance toxins such as Staphylococcus enterotoxin and 
particularly toxoids such as diphtheria and tetanus toxoid as well as 
antigens useful in the treatment of conditions such as caries, and viral 
antigens such as those derived from the influenza and the rabies viruses. 
The invention is also of interest, however, in relation to vaccines 
against other forms of pathogen such as protozoa, for example in the 
treatment of schizotrypanosomiasis, and fungi and also in relation to the 
field of both human and animal contraception, for example in vaccination 
against HCG when a hormone peptide is used as the antigenic material, as 
well as in the immunotherapy of cancer when a tumour specific antigen may 
be used as the antigenic material. The invention is also of considerable 
interest and of similar wide applicability in the context of veterinary 
vaccines for both mammalium and avian administration, for example in the 
treatment of the viral foot and mouth disease in cattle and pigs. 
The antigenic material is conveniently attached to the whole anti-B2M or a 
fragment thereof by means of a covalent bond. Various techniques are 
described in the literature for attaching antigens or haptens to proteins 
and these usually include the use of some form of coupling agent, for 
example agents such as chromic chloride, divinyl sulphone, cyanogen 
bromide, bis-diazotized benzidine (BDB) and especially glutaraldehyde, 
tannic acid and carbodiimides, for example 
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). One 
method which has recently attracted some interest is that based upon a 
procedure described by Kitagawa and Aikawa in the Journal of Biochemistry, 
1976, 79, 233, which involves the use of a heterobifunctional coupler or 
cross-linking agent for use with proteins. Such a coupler contains two 
different coupling groups which react with different types of functional 
groups; in the present case, one which will bond to the antigenic material 
and a different group which will bond to the antibody. 
An example of such a heterobifunctional coupler which has been used with 
some success is N-(m-maleimidobenzoyloxy)succinimide (MBS). This coupler 
will couple with free amino groups through active ester acylation by the 
m-maleimido-benzoyl groups and also with sulphydryl groups, for example of 
cysteine, by addition at the double bond of the maleimido group. For the 
sake of simplicity the symbol MBS has also been used hereinafter in 
identifying products linked through the use of this reagent although such 
products will, or course, only contain a residue of the reagent. 
Conveniently, the N-(M-maleimidobenzoyloxy)succinimide is coupled first to 
the antibody through free amino groups therein, the antibody not 
containing sulphydryl groups. Although such coupling may reduce the 
antibody activity, the extent of this reduction is not generally 
sufficient to have any substantial effect upon the subsequent anti-B2M/B2M 
coupling. The MBS/anti-B2M complex may then be treated with the antigenic 
material to effect combination therewith through reaction of the MBS 
moiety with sulphydryl groups naturally present in the antigenic material 
or inserted therein for this purpose. The formation of antibody not 
containing sulphydryl groups will often similarly require modification of 
the natural antibody by treatment with a reagent such as N-ethyl maleimide 
to block sulphydryl groups present therein.) It will be appreciated from 
the foregoing that the antigenic material/anti-B2M combination is a key 
intermediate in the formation of the final combination as described above. 
Accordingly, the present invention further comprises an antigenic material 
in combination with antibody as defined above, for example anti-B2M. 
The combination of antigenic material and anti-B2M may be purified simply 
by gel filtration on a material such as Sephadex G200 to effect a 
fractionation on the basis of molecular weight. However, a preferred 
procedure employs, as an initial purification step, the passage of the 
mixture obtained from the reaction of antigenic material and antibody over 
an immobilised solid phase to which B2M is attached. This solid phase may 
comprise one of a variety of forms of polymeric material suitable as the 
basis of an immunoadsorbent. Such materials are described in the 
literature in relation to gel filtration chromatography of immunoglobulins 
and include carbohydrate materials based upon dextran, agar or agarose and 
also polyacrylamide based materials, cross-linking conveniently being used 
to impart the required degree of exclusion of molecules dependent upon 
size and shape. Examples of such materials are marketed under the trade 
name Sepharose (agarose based), Sephadex (dextran based) and Sephacryl 
(polyacrylamide based). The dextran material marketed under the trade name 
Sepharose 4B has, for example, been found to be very suitable. The B2M may 
be attached to the solid phase through the use of one of the several 
methods described in the literature by which proteins or other materials 
may be attached to these insoluble solid phase supports. The chemical 
reactions described effect covalent bond attachment, but by a 
non-denaturing methodology so that destruction of the attached labile 
substances is minimised. Examples of suitable linking agents among such 
agents described above are glutaraldehyde and particularly cyanogen 
bromide. 
The use of such a B2M column enables unreacted antigenic material (which is 
often used in excess to effect optimum reaction of the anti-B2M) and any 
other materials not possessing the ability to bind immunologically to B2M 
to be separated from the bound antigenic material/anti-B2M combination by 
eluting the column with a suitable eluant, for example a suitable 
irrelevant protein solution such as 10% v/v foetal calf serum in phosphate 
buffered saline. The required combination of antigenic material and 
anti-B2M is then eluted from the column, together with any unreacted 
anti-B2M in the form used in the original reaction, for example 
MBS/anti-B2M, by the use of a further suitable eluant. Such second eluant 
may be a suitable acidic medium such as a 0.5% v/v aqueous acetic acid 
solution, or a solution of a chaotropic agent, for example of lithium 
chloride or iodide or of ammonium thiocyanate at neutral pH. 
The initial immunosorbent purification step is conveniently followed by a 
gel filtration step to effect a separation on the basis of molecular 
weight. Broadly similar types of solid phase may be used in this step to 
those used in the previous one but best results are obtained in this 
instance with materials such as those marketed under the trade names 
Sephadex G-200 and Sephacryl S-300. In a preferred aspect of this 
purification procedure, an acidic medium is used for the final elution of 
the immunosorbent column. The acidic eluate may then conveniently be 
passed directly from the immunosorbent column to the gel filtration 
column. This latter column may conveniently be made up and equilibrated in 
a medium such as phosphate buffered saline which is then also used to 
elute both the immunosorbent and gel filtration columns in series. The 
amount of acidic medium used to elute the former column usually does not 
need to exceed its bed volume and on the second column the acidic eluate 
is rapidly separated into its acid and protein components which differ 
widely in molecular weight. Further elution with a medium such as 
phosphate buffered saline will continue the separation according to 
molecular weight and result in elution from the latter column of antigenic 
material/anti-B2M followed by anti-B2M, for example as MBS/anti-B2M, and 
finally by the acid, for example acetic acid. This procedure results in a 
rapid separation giving a recovery of the desired antigenic 
material/anti-B2M combination in purified form with its constituent 
components having been minimally exposed to denaturing conditions. 
The final step in the preparation of the active component of immunological 
preparations according to the present invention conveniently consists of 
combining the antigenic material/anti-B2M combination with a B2M-MHC 
complex. The MHC-B2M complex required may conveniently be obtained from 
natural sources using one of the procedures for doing this which are 
described in the literature. The HLA antigens, for example, may 
conveniently be obtained from solubilised human spleens or by extraction 
of the membranes of human lymphoblastoid cells cultured in a suitable 
medium to provide sufficient quantities thereof for extraction. 
Alternatively, a human non-lymphoid cell line may be used as the source. 
Conveniently, however, the HLA-B2M complex may advantageously be purified 
by use of some of the anti-B2M which has been prepared. Thus a column of 
the anti-B2M coupled to a suitable support material, for example a 
Sepharose such as that marketed under the designation S4B, may be used and 
the HLA-B2M complex passed down this column, when it will be retained on 
the column by attachment to the anti-B2M. Elution with free B2M, not 
associated with HLA, will then competitively displace the HLA-B2M complex 
from the column in purified form. Conveniently the B2M may then also be 
recovered from the column, for example by elution with a suitable aqueous 
acidic medium such as aqueous glycine hydrochloride, and the column may be 
restored to a state ready for further repeated use by washing, for example 
with phosphate buffered saline. Following elution of the HLA-B2M not 
associated with HLA, the two materials may be separated by gel filtration 
and the recovered B2M used again. 
Coupling of the antigenic material/anti-B2M combination with the B2M-MHC 
complex may usually be achieved by simple admixture in an appropriate 
aqueous medium, for example in phosphate buffered saline. This is a simple 
procedure and, indeed, it may be possible to carry it out just prior to 
administration of the resulting antigenic material/anti-B2M/B2M/MHC 
combination. However, in order to restrict coupling to that occurring by 
an immunological route with the substantial avoidance of chemical 
coupling, it is possible if desired to block sulphydryl groups in the MHC 
antigen, for example by the use of N-ethyl maleimide, to prevent binding 
to these through any residual activity resulting from the coupling agent 
used in effecting the combination of antigenic material and anti-B2M. 
It will be appreciated that the present invention further includes an 
immunologically active preparation for human or veterinary use comprising 
an antigenic material in combination with an MHC antigen which is itself 
in the form of a complex with a protein with which it is normally 
associated in nature, said antigenic material being attached to the 
protein of the complex through antibody thereto, together with a 
physiologically acceptable diluent or carrier. 
The amounts of antigenic material incorporated as the immunologically 
effective agent to provide pharmaceutical preparations according to the 
present invention may be similar to those used in existing vaccines 
incorporating such material, but it may be possible in view of the 
increased immunogenicity to reduce these amounts. Similarly, although 
additional adjuvants may be incorporated into the preparations, this may 
likewise be unnecessary. The proportion of MHC antigen to antigenic 
material may be varied according to the particular circumstances. However, 
as a guide it may be indicated that in primates three dosages containing 
about 10, 10 and 5 micrograms of HLA have proved effective and that 
broadly similar unit dosage levels are generally applicable in humans, for 
example in the range from 5 to 100 micrograms, particularly from 10 to 50 
micrograms, for example about 25 micrograms. A similar basis may be used 
for veterinary applications with due consideration for variation in body 
weight. 
In other respects, the preparations may be formulated in a similar manner 
to conventional vaccines, for example in a medium such as isotonic saline, 
and may be administered similarly, often by a parenteral route, for 
example intravenously, intramuscularly or subcutaneously. 
The present invention thus includes a method for the immunization of a 
mammal or a bird which comprises administering thereto as an 
immunologically effective agent an antigenic material in combination with 
an MHC antigen which is itself in the form of a complex with a protein 
with which it is normally associated in nature, said antigenic material 
being attached to the protein of the complex through antibody to that 
protein. 
It should be noted that the HLA antigens, which were originally designated 
in the literature as Human Lymphocyte Antigens, have more recently been 
designated by some workers as Human Lymphocyte System A antigens. 
This invention is illustrated by the following Examples, a general 
procedure being described in Examples 1 and 2 and a specific preparation 
relating to the antigenic material ovalbumin in Examples 3 and 4.

EXAMPLES 
(Gel filtration and immunosorbent columns are run at room temperatures 
under peristaltic pumping so as to achieve optimal resolution as defined 
by the manufacturer.) 
EXAMPLE 1 
PREATION OF ANTIGENIC MATERIAL/ANTI-B2M COMBINATION 
(a) Preparation of anti-B2M 
Using the general procedure of Kohler and Milstein (European Journal of 
Immunology, 1976, 6, 511), a mouse hybridoma is obtained by the fusion of 
spleen cells from a BALB/c mouse immunised with human B2M, together with 
the cells of the mouse cell line NS1/1-Ag4-1. During the isolation of the 
desired clone, hybridoma cells producing anti-B2M antibody are identified 
by the ability of culture supernatants to bind to the wells of a plastic 
plate previously coated with pure B2M. Thus, to wells of the plate is 
first added 50 .mu.l of a solution of pure B2M at 10-40 .mu.g protein per 
ml of phosphate-buffered saline. Following incubation of the covered plate 
at 37.degree. C. for 1 hour, unadsorbed protein is aspirated and the wells 
washed thrice with 100 .mu.l of a solution containing 10% v/v foetal calf 
serum. Plates may then be stored covered at 4.degree. C. until required. 
In the assay an aliquot of 25 .mu.l of culture supernatant is incubated in 
a well for 1 hour at 37.degree. C. Following washing thrice with 10% v/v 
foetal calf serum, attachment of mouse immunoglobulin having anti-B2M 
activity is revealed by the ability of the plate well to bind 
immunopurified radio-iodinated sheep anti-mouse immunoglobulin [The latter 
reagent is prepared by immunising sheep with DEAE-cellulose purified mouse 
IgG. The specific antibody in the serum of immunised animals is purified 
on a column of mouse IgG prepared by the cyanogen bromide technique (March 
et al, Analystical Biochemistry, 1971, 60, 149), eluting with 0.1 
M-glycine hydrochloride pH 3.0, and immediately neutralising the eluted 
protein.] Having identified cultures which are actively secreting antibody 
to B2M, the cells are cloned to homogenity using the "limit dilution" 
protocol defined by Kohler and Milstein. Cells thus cloned may be 
preserved in liquid nitrogen using established methodology. When 
quantities of anti-B2M are required, ampoules of frozen anti-B2M producing 
hybridoma cells are thawed from liquid nitrogen and about 3.times.10.sup.6 
cells are injected subcutaneously into BALB/c mice. Serum from such mice 
soon shows a high titre of monoclonal mouse antibody. Specific antibody is 
isolated from the serum by use of an immunosorbent column of pure human 
B2M by the following protocol. B2M is isolated from the urine of patients 
with renal disease following Berggard and Bearn, Journal of Biological 
Chemistry 1968, 243, 4095. The purified protein is covalently attached to 
Sepharose-4B by the cyanogen bromide technique (March et al., ibid) at a 
final concentration of about 4 mg/ml. Antibody is eluted from the column 
with 0.1 M glycine hydrochloride at pH 3 or using 0.5% v/v aqueous acetic 
acid. The eluted protein is immediately neutralised with 
tris(2-amino-2-hydroxymethylpropane 1,3-diol) base and stored frozen at 
-20.degree. C. until required. Characterisation of the antibody by 
SDS-gels or isoelectric focusing establishes its monoclonality. 
(b) Fab fragment of antibody 
Antibody protein at 10 mg/ml in aqueous solution is incubated with 3% by 
weight of crystalline papain in 0.01 M-cysteine at pH 7 for 16 hours at 
37.degree. C. The Fab fragment is isolated using a similar column of 
Sepharose 4B-B2M as described above for purification of the intact 
antibody. In this way only Fab fragments which have retained B2M-binding 
activity are selected. Elution is with 0.5% v/v acetic acid followed by 
immediate neutralisation and storage of the Fab protein at -20.degree. C. 
(c) F(ab').sub.2 fragment of antibody 
Antibody protein in aqueous solution is incubated with 10% of its weight of 
crystalline pepsin at pH 4 and 37.degree. C. for 20 hours. The 
F(ab').sub.2 fragment is isolated on a column of Sepharose 4B-B2M exactly 
as described above for the Fab fragment and with the same elution 
procedure. 
(It has been established in small scale trials that the Fab and 
F(ab').sub.2 fragments may be repeatedly absorbed to and eluted from 
Sepharose 4B-B2M columns without undergoing extensive degradation during 
the acid elution procedure.) 
(d) Linkage of anti-B2M to antigenic material 
The monoclonal antibody (which is usually IgG, for example IgG2) or Fab or 
F(ab').sub.2 fragment thereof in aqueous solution as obtained in (a), (b) 
or (c) above, is treated with 0.1 M N-ethylmaleimide (NEM) for 1 hour at 
room temperature to block any reactive sulphydryl groups and is then 
re-isolated by Sephadex gel filtration eluting with 0.05 M tris chloride. 
The protein is incubated at 5 mg/ml in 0.05 M tris chloride of H 7.3 with 
an approximately 5 molar excess of N-(m-maleimidobenzoyloxy)succinimide 
(MBS) for 1.5 hours at room temperature. The acylation reaction is 
terminated by the addition of an excess of a 20 mg/ml aqueous solution of 
lysine and the m-maleimidobenzoyl derivative of the antibody 
(MBS/anti-B2M) is separated by Sephadex G25 using phosphate-buffer saline. 
The MBS/anti-B2M is reacted with a molar excess of the antigenic material 
at a concentration of this material of 5-10 mg/ml in phosphate-buffered 
saline for 5-16 hours at room temperature. The resulting combination of 
antigenic material/MBS/anti-B2M is then purified by gel filtration on 
Sephadex G200 in phosphate-buffered saline. The desired complex of 
antigenic material/MBS/anti-B2M is clearly separated from unreacted 
MBS/anti-B2M by virtue of its larger size. As an alternative, an 
immunosorbent purification procedure as described in Example 3 may be 
employed. 
B2M binding capacity may be tested for by incubation with radio-iodinated 
B2M, the mixture being examined for the change in mobility of labelled 
protein, a new peak corresponding to a radio-labelled protein of 
appropriately higher molecular weight indicating binding of the labelled 
B2M. 
EXAMPLE 2 
PREATION OF ANTIGENIC MATERIAL/ANTI-B2M/B2M/HLA COMBINATION 
(a) Preparation of B2M-HLA 
Whole B2M-HLA is prepared by a method similar to that of Robb and 
Strominger (Journal of Biological Chemistry, 1976, 251, 5427) but using a 
column of mouse anti-human B2M prepared as described in Example 1, eluting 
with pure human B2M (prepared as described in Example 1) in phosphate 
buffered saline at a concentration of about 1 mg/ml. The purified B2M-HLA 
thus obtained is separated from the B2M by gel filtration on Sephadex G75 
eluting with phosphate buffered saline. 
The complex may optionally be treated with N-ethylmaleimide as described 
for the antibody in Example 1(a) to block sulphydryl groups. 
(b) Reaction of B2M-HLA with antigenic material/anti-B2M combination 
Having characterised the antigenic material/anti-B2M combination as 
described in Example (1) above, both by molecular weight and by its 
ability to react with B2M chains, the desired complex of B2M-HLA with 
antigenic material/anti-B2M is achieved by a simple mixing of these two 
materials in equivalent proportions in phosphate buffered saline followed 
by a brief incubation. The resulting material may be purified by gel 
chromatography on Sephadex G75 eluting with phosphate buffered saline or 
alternatively may be administered directly. 
EXAMPLE 3 
PREATION OF OVALBUMIN/ANTI-B2M 
The Fab fragment of anti-B2M is prepared as described in Example 1(b). It 
is then treated in aqueous solution with 0.1 M N-ethyl maleimide (NEM) for 
1 hour at room temperature to block any reactive sulphydryl groups and is 
then re-isolated by Sephadex gel filtration eluting with phosphate 
buffered saline. The NEM Fab is concentrated by membrane filtration to a 
concentration of 4.61 mg/ml (Lowry) and 1.5 ml (total protein 6.9 mg). To 
this solution in phosphate buffered saline is added 0.15 .mu.l of dioxane 
containing 1.14 mg MBS (molar ratio: 3.63 .mu.mol MBS/0.14 .mu.mol 
protein=26.3). The mixture is maintained at room temperature for 1.5 hours 
and is then chromatographed on a small column (1 cm.times.10 cm) of 
Sephadex G50 in phosphate buffered saline. The NEM Fab/MBS peak emerges in 
the void volume and is identified by UV absorption. Protein containing 
fractions (about 2 ml total) are pooled and 10 mg (0.22 .mu.mol) of 
crystalline ovalbumin is added and mixed to effect solution. The mixture 
is left overnight at room temperature and is then applied to a column (8 
cm.times.7 cm) of Sephadex G200 in phosphate buffered saline. Two peaks 
are obtained of molecular weight 100,000 (ovalbumin/anti-B2M complex) and 
50,000 (unreacted ovalbumin plus NEM Fab/MBS). Yields of up to 50% based 
on the NEM Fab starting material are obtained. 
Proof of the presence of both covalently bound Fab and covalently bound 
ovalbumin in this complex are obtained as follows. 
Radio-iodinated B2M is mixed with the complex in phosphate buffered saline, 
incubated for 1 hour at 37.degree. C. and chromatographed on Sephacryl 
S300, all radio-activity is then eluted at a position corresponding to a 
molecular weight of at least 110,000 indicating binding of the B2M by the 
complex. Control chromatograms containing radio-iodinated B2M and 
irrelevant protein show B2M elution and the anticipated unbound molecular 
weight of about 12,000. 
The presence of ovalbumin in the complex is shown by double diffusion in 
gels between the complex and on established rabbit anti-ovalbumin serum, 
or by incubating complex with radio-iodinated B2M followed by a rabbit 
anti-ovalbumin serum when all radio activity moves to a position on 
Sephacryl S300 chromatography corresponding to a molecular weight in 
excess of 250,000. 
In a variant of the procedure described above, the reaction mixture from 
the NEM Fab/MBS-ovalbumin reaction is passed through a column (2.times.25 
cm) of Sepharose 4-B2M which is then washed through with phosphate 
buffered saline until no further UV absorbing material emerges. The column 
effluent tubing is then connected to the inlet port of a 2.times.90 cm 
column of Sephacryl S300 previously equilibrated with phosphate buffered 
saline 0.20 ml of 9.5% v/v acetic acid is then passed through the two 
columns connected in tandem, followed by phosphate buffered saline until 
all protein and acid has emerged from the system. Two protein containing 
peaks of molecular weight 100,000 (complex) and 50,000 (unreacted NEM 
Fab/MBS) are obtained. 
EXAMPLE 4 
PREATION OF OVALBUMIN/ANTI-B2M/B2M/HLA 
B2M-HLA is prepared as described in Example 2 and is incubated with 
ovabumin/anti-B2M prepared as described in Example 2(b) for 1 hour at 
37.degree. C. 
Combination between the two complexes from ovalbumin/anti-B2M/B2M-HLA is 
confirmed by carrying out the reaction with radio-iodinated B2M-HLA and 
chromatographing the resultant reaction mixture on Sephacryl S300. All 
radio activity is eluted in a position corresponding to a molecular weight 
in excess of 150,000 in contrast to the situation with unreacted 
radio-iodinated B2M-HLA when the radio-activity is eluted in a position 
corresponding to a molecular weight of 45,000.