Method for the immunolocalization of antigens with the use of antibodies directed against epitopes of non-glucidic nature

A method is disclosed for the immunolocalization of epitopes of non-glucidic nature, in cellular cultures, in tissue slices, or immobilized on such supports as nitrocellulose, by means of the incubation of the sample with specific antibodies for the antigen to be immunolocalized, belonging to the class of the IgM's, and sequential treatment of concanavalin A and with an enzymatic marker capable of binding with concanavalin A, such as, e.g., peroxidase. The method comprises, before the incubation, a suitable treatment of the sample under test, e.g., with periodic acid, in order to prevent concanavalin A from binding with the carbohydrates present in the sample.

The object of the present invention is a method for the 
immonoidentification or immunolocalization of epitopes of non-glucidic 
nature. 
In particular, the method according to the present invention, which is 
based on the capability of concanavalin A of very strongly binding with 
the immunoglobulins of IgM type, can be used for the immunolocalization of 
epitopes of non-glucidic nature in the cellular cultures and in slices of 
tissue, and also for the identification of determined antigens in the 
biological fluids and in tissular or cellular extracts, after the 
preliminary immobilization of such substances on an inert support. 
The novel method makes it possible the system of detection of the 
antigen-antibody complex to be considerably simplified. The possibility of 
identifying particular antigens, or of localizing their presence in 
determined tissues by means of a highly antigen-antidbody reaction, is an 
extremely useful means both for an as precise and early diagnosis as 
possible of such pathologic forms, and from the viewpoint of an increase 
in basic knowledge relevant to the genesis of such pathologic forms; and, 
finally, for a verification of the efficacy of the therapy carried out. 
The routine methods used in immunochemistry provide, in case the (so-said 
primary) antibodies specific for the investigated antigen are not directly 
"marked", for the use of rather complex detection systems, wherein a 
second, species-specific antibody (i.e., a "secondary antibody") is always 
present, which is conjugated with a so-said "marker" (fluorescent 
substances, enzymes, radioisotopes, biotin, and so forth). Such detection 
systems represent a critical point as regards both the good outcome of the 
investigation, in that possible aspecific reactions would completely 
distort the work, and the cheapness of the investigation system, in that 
to carry out a full set of operations of-whether primary or 
secondary-antibody modification, if one takes into account the cost 
thereof, and the extremely low yields to pure product, is not economically 
feasible. 
Many attempts of use of lectins in the diagnostic field were made in the 
past. 
For example, U.S. Pat. No. 4,298,689, discloses a test for Neisseria 
gonorrhoeae, which is carried out by using a lectin showing a high 
affinity for N-acetylglucosamine. In practice, according to such a patent, 
the sample to be analysed in placed into direct contact with the lectin 
and the possible presence of the reaction product on the cellular surface 
of the microorganism is used as an evidence of the presence of the same 
microorganism. 
In a similar way, in U.S. Pat. No. 4,389,392, particular lectins are used, 
which combine with determined end saccharides, in order to evidence the 
presence of glycoproteins associated with certain tumor forms. The use is 
furthermore known of concanavalin A and of other lectins (LCH, PHA, RCA, 
and so forth), in a method for the differential diagnosis of cancer 
(EP-A-0 171 243), based on the determination of certain glycoantigens 
antigens present in the biological fluids of some cancer forms. The 
present of the different glyco-antigens is detected by means of the 
capability shown by these latter, of binding or less with the different 
lectins. In all these cases, anyway, no selective immunologic reactions 
are concerned, and such methods are therefore for an extremely limited 
use. 
A different approach to the use of lectins is that of U.S. Pat. Nos. 
4,289,747 and 4,371,515, wherein a lectin is conjugated with an 
immunoglobulin, and the affinity of lectin for the sugars is exploited, in 
order to immobilize the conjugate, and insolubilize it. 
Recently, in patent literature (EP-A-41426 and DD-155082) methods of 
detection have beend disclosed, which use, as the either primary, or 
secondary, antibody, an antibody conjugated with a lectin, and, in 
particular with concanavalin A, and, as the detection system, peroxidase. 
As previously stated, the preparation of such an antibody-lectin conjugate 
presupposes the reaction, in one or two separate steps, of the antibody 
and of the lectin with a coupling agent, such as, e.g., glutaraldehyde or 
p-benzoquinone, and the purification of the obtained product, both which 
reaction involve a considerably decrease in yields. 
Finally, in EP-A-166 623 application, a method is disclosed for the 
determination of glycoproteins, glycopeptides, carbohydrates or 
glycolipids by means of a first reaction through the immunologic reaction 
with an immobilized antibody and hence a determination by means of the 
non-specific reaction with a lectin conjugated with a marker. In this 
latter case too, the lectin is conjugated, by means of a true chemical 
reaction, with the marker before being used in the determination, with the 
problems relevant to the preparation of the conjugate, as above said. In 
both these latter references, the lectin is anyway used in order to 
determine the antigens containing a glucidic residue. 
The present applicant has found now that, when in an immunochemical assay, 
specific antibodies are used, which belong to the class of IgM's, it is 
possible to detect the possible presence of the antigen-IgM antibody 
complex, in a very simple and cheap way, by treating the complex with 
concanavalin A, which forms a strong bond with the IgM, and then with a 
suitable enzymatic marker, containing an .alpha.-D-glucose or 
.alpha.-D-mannose group able to bind with concanavalin A, capable of 
producing, in the presence of a suitable enzymatic substrate, a 
characteristic coloured precipitate, easily appreciable by the naked-eye 
on inert supports, or as regards the tissue slices or the cellular 
cultures, under the microscope. 
In this case, concanavalin A is hence used in the first step of the 
detection process, and interacts with the antibody, and not with the 
antigen, thus offering wide possibilities of use of this system in the 
determinazion of many antigens. 
Extremely good results were obtained by using peroxidases as the marker, 
but according to an alternative route, other enzymes endowed with the 
above-reported requisites are regarded as suitable for such an use, with 
analogous results. Anyway, the use of the peroxidase yields the additional 
advantage that it has been deeply studied and optimized, so that from the 
relevant literature many methods are known in order to detect this enzyme 
(e.g., the classic method of diamino-benzidine (DAB), described by R. C. 
Graham and M. J. Karnovsky, in J. Histochem. Cytochem., 14 291-302 (1966), 
or the method as described bu J. S. Hanker et al. in Histochem. J., 9, 
789-92 (1977), which uses p-phenylene-diamine and pyrocathecol as the 
reactant, as well as the method of 3-amino-9-ethyl-carbazole (AEC), 
described by R. C. Graham et al., in J. Histochem. Cytochem., 13, 150-2 
(1965). 
But, as known, inasmuch as concanavalin A has a great affinity for 
carbohydrates (J. Roth, Exp. Path. Suppl. 3, (1978), and B. A. J. Ponder, 
Immunocytochemistry, practical applications in pathology and biology, 
129-42, John Wright & Sons (1983)), in order to prevent non-controllable 
interferences between the detection system and the carbohydrates possibly 
present in the test sample, the preliminary removal or masking these 
latter is necessary. 
This can be carried out more advantageously by treating the sample with 
periodic acid, according to the methodology described by N. Beccari and V. 
Mazzi in "Manuale di Tecnica Microscopica (Handbook of Microscopy 
Technique)", pages 240-1, in order to oxidate the carbohydrates present in 
the test sample. 
Alternative treatments can be developed by those skilled in the art, on the 
basis of the common knowledge in this field, and of the particular 
requirments of the technique used. The treatment with periodic acid makes 
it anyway possible, in case in order to detect concanavalin A peroxidase 
is used, the endogenous peroxidase to be also removed from the sample. 
In case the presence of a particular antigen in a proteinic blend obtained 
from a biological fluid, or in the proteinic isolates obtained by 
fractionating such a blend, has to be detected, the elimination of the 
possible interferences between concanavalin A and the possible present 
carbohydrates, can be alternatively obtained by causing the proteinic 
blend to flow over a column containing immobilized concanavalin A, and 
subsequently analysing the effluent mixture, possibly fractionated and 
suitable immobilized on an inert solvent. 
This method necessarily uses specific antibodies belonging to the class of 
the immunoglobulins IgM's, in that only the IgM's form a strong enough 
bond with concanavalin A, such as to secure the extremely good 
experimental results obtained. 
Between the IgG's and concanavalin A, in fact, a high affinity does not 
exist (cfr. J. Arends, Methods in Enzymology, Vol. 73, 166-175) and, in 
that case, it is necessary to conjugate the IgG's with concanavalin A by 
means of a coupling agent (reference is made to the above cited patents). 
Typically, monoclonal IgM's are used. In fact, it is knopwn that the IgM's 
are the immunoglobulins which are obtained as the primary response to the 
introduction of the antigen in the body, viz., it is the immunoglobulins 
which are obtained during the first steps of the immunization (F. A. 
Murphy et al., J. Infect. Dis., 116, 99 (1966)) and are therefore present 
in an extremely reduced amount in the serum of the animal. Therefore, it 
is not economically advantageous to obtain these specific antibodies by 
immunizing the animal, in that the animal must be sacrificed when its 
antibody response is still extremely reduced. 
The techniques for the production of monoclonal antibodies are anyway 
widely known (see, e.g., C. Milstein et al., in Nature, 256, 495-497 
(1975); C. Milstein et al., in Cell. Bio. Int. Rep., 3, 1-16 (1979); C. 
Milstein, in Sci. Amer., 243, 56-64 (1980); and A. J. McMichael et al., in 
"Monoclonal Antibodies in Clinical Medicine", Academic Press (1982), and 
it is not necessary to repeat them herein; furthermore, some of such 
specific antibodies are available on the market. 
The method according to the present invention is generally applicable in 
the immunochemical, immunocytochemical and immunohistochemical techniques. 
Obviously, it cannot be used in the determination of antigens in case the 
antibody is directed against an epitope of glucidic nature, but, on the 
contrary, it can be also used in the determination of some antigens of a 
different type. It could be used, e.g., for the determination of some 
"haptens", such as, e.g., the non-peptidic steroid hormones, e.g., 
progesterone, estradiol testosterone, and so forth, the thyroid hormones, 
e.g., thyroxine and triiodothyronine, some antibiotics, vitamins, 
pesticides, and so forth, on condition that against them monoclonal 
antibodies belonging to the class of IgM's are obtained, and that the 
preliminary treatment designed to eliminate the undesired concanavalin A 
binding does not alter the structure thereof, such as to eliminate also 
the antigen-antibody binding. 
In practice, the method of immunolocalization of epitopes of non-glucidic 
nature according to the present invention can be advantageously used on 
cryostatic slices of tissue on slices of tissue fixed in formalin or in 
another fixative agent, and included inside paraffin, on suitably fixed 
cellular smears or preparations from cytocentrifuge, on proteinic blends 
immobilized in inert supports, such as nitrocellulose, or on proteinic 
isolates obtained by fractionating such blends. Inasmuch as the primary 
use of such a method is anyway in the field of immunohistochemistry, in 
the following the procedure is disclosed in greater detail, which is 
advantageously followed for immunolocalization of antigens in tissue 
slices. 
On the basis of the information supplied in the following, and of what is 
known from the technical literature on this subject, anyone having an 
average skill in this field will be surely capable of applying the 
immunochemical determination method according to the present invention to 
different substrates. 
For the preparation of the slices of tissue to be submitted to the 
immunochemical test, the tissues are submitted to the conventional steps 
of fixation and inclusion, according to the methods known in the art. 
According to the method of the present invention, after removing paraffins 
from the slices, or, in case of cryostatic tissue slices, after 
re-hydrating them, the tissue slices are submitted to a treatment with a 
diluted solution of periodic acid, having the purpose of oxidating the 
sugars possibly present in the tissue, and thus preventing concanavalin A 
from binding with them. In this step, a 1% solution of periodic acid can 
be advantageously used, with the treatment being carried out at room 
temperature, and being continued for about 15 minutes. Of course, the 
concentration of the acid can be increased, or decreased (e.g., a 
concentration comprised within the range of from 0.5 to 1.5% can be used), 
and the treatment time can be proportionally extended or shortened. In any 
case, adapting the treatment to the type of sample under test is 
necessary. 
In practice, it is advantageous to verify, before carrying out the 
immunolocalization, the efficacy of the treatment on the investigated 
material, by testing different concentrations of periodic acid, or 
different treatment times on several samples of the same material, and 
varifying--by means of a treatment of concanavalin A and peroxidase--that 
the binding of concanavalin A is actually eliminated. The sample is then 
thoroughly washed with water, and then with a buffer solution, suitable 
for causing the immunologic reaction to take place, generally a phosphate 
buffer-saline (PBS) having a pH value of 7.2-7.4. The sample is then 
treated with the antibody belonging to IgM class, specific for the 
angtigen which one wants to determine, under such conditions as commonly 
used in immunohistochemical methods, which are the suitable conditions for 
enabling the immunologic reations between both partners to occur. 
According to such conditions, an incubation is carried out at a temperature 
comprised within the range of from 4.degree. C. to room temperature, for a 
time normally comprised within the range of from 10 minutes to 48 hours, 
according to the titre and the dilution of the antibody. 
After a further washing with the buffer, the tested sample is washed with a 
diluted solution of concanavalin A. As the solvent for concanavalin A, the 
same buffer solution is advantageously used, which is used in the previous 
step, preferably with the addition of some salts of bivalent metals 
(calcium, magnesium and manganese), which serve to secure a good operation 
of concanavalin A, each sub-unit of which contains a Ca.sup.++ ion and an 
Mn.sup.++ ion. Normally, besides these bivalent ions, which will be 
present in extremely small amounts (5-15 .mu.mol/liter), the buffer 
solution will further more contain also extremely small amounts 
(.ltoreq.1%) of a non-ionic surfactant. 
Extremely good results can be obtained by using concentrations of 
concanavlin A comprised within the range of from 5 to 20 .mu.g/ml. 
In general, an incubation of 1 hour at room temperature is enough in order 
to achieve the complete binding of concanavalin A to IgM. Both the 
treatment time and the concentration can be anyway changed and adjusted 
according to the requirements, anyway taking into account that higher 
concentrations than as disclosed can originate an undesired background. 
At the end of the incubation, a further washing is carried out in order to 
remove unbound concanavalin A, and the same is then treated with a dilute 
solution of the enzyme used as the marker, preferably peroxidase. 
Inasmuch as concanavalin A is a tetramer, and therefore has four binding 
sites for the glucidic residues, only one of which is engaged in the 
binding with the IgM, still three sites for binding the peroxidase are 
available per each molecule of concanavalin A. Good results were obtained 
by using solutions of peroxidase in the same buffer system as used for 
concanavalin A, having a concentration comprised within the range of from 
15 to 60 .mu.g/ml. In this case too, the same considerations as regards 
the concentration and the treatment time, as above stated for concanavalin 
A, hold true. 
Also in this case, an incubation time of approximately one hour is 
generally enough in order to have the binding of the peroxidase to the 
free sites of bound concanavalin A. 
The conventional washes, in order to remove the unbound enzyme, and then 
the incubation with a suitable enzyme substrate makes it furthermore 
possible the antigen to be immunolocalized, by means of the formation of a 
coloured precipitate. 
The results which are obtained by means of the method according to the 
present invention are comparable to those which are obtained by means of 
the most sophisticated and sensitive immunochemical methods known from the 
relevant literature, or available from the market, such as the method of 
the indirect immunoperoxidase, which, after the specific antibody, uses a 
second species-specific antibody conjugated with the peroxidase, or the 
ABC (avidin-biotin complex) commercial system which, after the specific 
antibody, sequentially uses a second species-specific antibody conjugated 
with biotin, then a pre-constituted complex of avidin and biotinylated 
peroxidase, thus the detection and amplification of the signal being 
obtained. 
If desired, after the incubation of the sample with concanavalin A, the 
signal can be detected or amplified by means of any know technique, which 
involves the use of avidin, such as, e.g., in the ABC commercial system, 
in that avidin is a glycoprotein with which concanavalin A can bind.

The greater simplicity of the method according to the present invention, 
which, on the contrary, uses one single specific antibody, as compared to 
the methods known from the prior art, such as, e.g., the above discussed 
methods, is immediately evident from the following examples. 
EXAMPLE 1 
Immunolocalization of Vimentin 
Vimentin is a protein having a molecular weight of 58,000 daltons, which 
constitutes the intermediate filaments of the tissue of mesenchymal 
origin, excluding the muscular cells. The identification of vimentin makes 
it possible the mesenchimal origin of melanomas, histiocytomas and 
sarcomas to be attributed. Vimentin is a cytoskeletal marker, the 
synthesis of which is acquired by all the epithelial cells in culture. 
Furthermore, epithelial tumors can express vimentin and cytokeratins. 
The Antibody 
The monoclonal anti-vimentin antibody is a supernatant from a hybridoma 
culture, belonging to IgM class, and is a product traded by Sclavo. 
The Immunohistochemical Test 
Slices of the tissue, fixed in formalin, and included in paraffin, are 
deprived of paraffin by means of the conventional methods (three changes 
of 10 minutes in xylene, rehydration in a series of alcoholic solutions, 
from 95 to 50.degree., then washing with distilled water). 
The slides are dipped in (or on the slices drops are added of) a solution 
of periodic acid at 1% in distilled water at room temperature. Ten minutes 
later, the samples are washed a plurality of times with distilled water 
and then with PBS (Phosphate Buffer-Saline) (a solution in distilled water 
containing Na.sub.2 HPO.sub.4 (0.9 g/l), KH.sub.2 PO.sub.4 (0.2 g/l), NaCl 
(8 g/l), and KCL (0.2 g/l)). 
A solution of the antibody in the reconstitution buffer (PBS) at a dilution 
comprised within the range of from 1:20 to 1:50 is added, and is allowed 
to incubate for 30-60 minutes at room temperature. 
The samples are then washed three times with PBS containing calcium, 
magnesium and manganese ions at a 10-.mu.M concentration (PBS.sup.++) and 
0.5% of Triton.sup.(R) .times.100. 
The samples are then incubated for one hour at room temperature with a 
solution of concanavalin A at a concentration of 10 .mu.g/ml in PBS++. 
The samples are washed three times with PBS ++ and are subsequently 
incubated for one hour at room temperature with peroxidase (horseradish 
peroxidase (type VI)--by Sigma Chemical Company) at a concentration of 
50.mu.g/ml. 
The samples are washed three times with small aliquots of PBS++ in order to 
eliminate any non-bound peroxidase. The incubation with a solution of 
3amino-9-ethyl-carbazole and H.sub.2 O.sub.2 in DMF according to the 
method by Graham, Ludholm and Karnovsky for 10-20 minutes yields, in the 
presence of vimentin, a coloured precipitate clearly detectably under the 
microscope. 
EXAMPLE 2 
The above disclosed technique can be also applied in order to detect the 
antigen in Western blot. 
In this case, the mixture which contains the antigen is denaturated, and 
the polypeptides which constitute it are separated by electrophoresis on a 
polyacrylamide gel containing sodium dodecyl-sulphate. At the end of the 
run, the material is transferred by electrophoresis from the gel to a 
nitrocellulose sheet. The nitrocellulose sheet is submitted to a treatment 
with a solution of periodic acid at 1% for approximately 30 minutes, and 
is then saturated with a solution of bovine serum albumin (BSA) at 3% in 
PBS; then a procedure analogous to that reported in Example 1 is followed, 
by incubating for 2-3 hours with an anti-vimentin antibody belonging to 
IgM class diluted at a 1:5 dilution ratio, then with a solution of 
concanavalin A, with a solution of peroxidase as the enzymatic marker, and 
then with the suitable enzymatic substrate. The methodology used in this 
latter step is the same as used for detecting the glycoproteins on 
nitrocellulose (see J. S. Clegg, Analytical Biochemistry, 127, 389-94, 
1982). 
Example 3 
Immunolocalization of a specific antigen of the respiratory cilium 
The vibratile cilia are constituted by at least 200 different polypetides; 
the absence of one, or of a group, of them can cause pathologic 
alterations in the same cilium. 
The methods used in Examples 1 and 2 for the determination of vimentin are 
substantially repeated by using, instead of the anti-vimentin antibody, an 
A.M. 3.12 monoclonal antibody, belonging to the class of the IgM's, 
directed against a polypetide having a molecular weight of approximately 
300,000 daltons, specific of the respiratory cilium (Mencarelli and 
co-workers, in "Biology of Reproduction and Cell Motility in Plant and 
Animals", Compilers: M. Cresti and R. Dallai, 1986, pages 75-80). 
A further object of the present invention is a kit for the 
immunolocalization of antigens of non-glucidic nature, characterized in 
that it contains the following compounds: 
(a) an antibody belonging to the class of IgM's, specific against the 
antigen to be determined, 
(b) concanavalin A, 
(c) an enzymatic marker containing a glucidic residue capable of being 
recognized by concanavalin A, and 
(d) a suitable substrate for the detection of the enzymatic marker used. 
According to a preferred form of practical embodiment, such a kit will 
furthermore contain periodic acid, to be used in the initial 
pre-treatment, aiming at preventing concanavalin A from binding with 
molecules of glucidic nature present in the sample. 
According to a still more preferred form of practical embodiment, such a 
kit will contain peroxidase as the enzymatic marker.