Multi-color labeling of different antigens in a biological system

A method of multi-color labeling at least two antigens present in a tissue such as non-liquid tissue or body fluids, with the aid of different antibodies is improved by introducing between the labeling of the first antigen and the labeling of the second antigen, and between labeling of the second and that of any further antigen, at least once a treatment with a non-immune normal serum and optionally other agents blocking any binding sites remaining free in the tissue after a last-preceding labeling step.

BACKGROUND OF THE INVENTION 
This invention relates to a method of multi-color labeling at least two 
antigens being simultaneously present in a common biological system, with 
the aid of a corresponding number of different antibodies. In a second 
aspect the invention relates to a novel kit for carrying out the aforesaid 
method. 
Fluorescene-optical multi-color labeling of different antigens in the same 
biological system, in particular animal, and especially human "tissue" 
(meaning non-liquid tissue as well as blood) is of special importance for 
investigating diagnostic or medical scientific problems. 
For instance, such labeling is useful in the precise identification of 
lymphocyte subpopulations in pathologically and non-pathological tissues 
such as inflammatory myopathies, or auto-immune thyreoiditis, normal and 
pathologically changed lymphoid tissues, infiltrations or inflammatory 
changes in malignant and non-malignant tumors and metastases of malignant 
tumors, blood and cerebral fluid in pathological and non-pathological 
states diagnoses of immunodeficiency syndromes in blood such as AIDS and 
others listed on page 28 of a publication entitled "Ortho-mune (R), 
Monoklonale Antikorper, Zelltypisierung in Peripherblut and lymphatischem 
Gewebe" by Dr. Molter GmbH, D-6903 Neckergemund, Federal Republic of 
Germany, and many other fields of biological investigation. 
Hitherto, it was only possible to effect multi-markings of blood, but not 
of other tissues with very costly and highly complicated laser analyzers 
such as a FACS analyzer or a Spectrum 3 analyzer which are available, due 
to their cost, in only a very limited number of medical institutions. 
Methods published by William E. Gathings, Ph.D. and others in 1977 in the 
European Journal of Immunology 7, page 804 under the title of 
"Immunofluorescent Studies of Development of Pre-B Cells, B-Lymphocities 
and Immunoglobulin Isotype Diversity in Humans" have been reported by 
Becton Dickinson Labor Systems' "Monoclonal Antibody Source Book" 
published subsequently in Heidelberg in the Chapter 
"Methods--Immunofluorescence Staining of Cell Surfaces--Cytocentrifuge 
Preparations" comprise two-color indirect immunofluorescence preparations 
of human peripheral blood with the following reagents: 
(1) Fluorescine(FITC) or rhodamine (RITC) labeled mouse monoclonal antibody 
specific for membrane antigen, as well as, for double-marking: 
(1a) unconjugated or biotin-labeled mouse monoclonal antibody specific for 
membrane antigen, followed by 
(1b) anti-mouse Ig FITC/RITC) or followed by 
(1c) Avidin FITC/RITC. 
I have found that this method will not work for double-marking when (1a) is 
unconjugated and (1b) is anti-mouse Ig FITC/RITC, when the unconjugated 
antibody of (1a) is of the same immunoglobulin class as directly 
conjugated antibody of (1), as this method yields unspecific double 
markings. 
Moreover, biotin-labeled mouse monoclonal antibody of (1a) followed by 
avidin FITC/RITC of (1b) as obtained from Becton and Dickinson fails to 
afford specific double markings. 
However, even when using in the last combination an avidin FITC/RITC of 
different origin, I obtained only weak and quickly fading fluorescent 
markings. The weakness and fading were particularly pronounced in the 
green (FITC) markings. 
Another method of fluorescence double marking of primary antibodies from 
the same animal species has been published by Amersham Buchler GmbH and 
Co. KG, D-3300 Braunschweig in "The biotin-streptavidin system" published 
in April 1985 (penultimate sheet). This method always leads to unspecific 
fluorescence double markings of the first incubation sequence. 
Furthermore, D. Y. Mason, Z. Abdulazig, B. Falini and H. Stein assert on 
page 119 in an article entitled "Double Immunoenzymatic Labelling" 
published in "Immunocytochemistry, Practical Applications in Pathology and 
Biology", by G. Wright, Bristol and Boston, 1983 that it is "not possible 
to perform double labeling by" use of class- or subclass-specific 
antibodies (FIG.7.6) "if the two primary antibodies are of the same 
subclass. Since many monoclonal antibodies are of the Ig Gl subclass this 
represents an important potential limitation" of this known method. 
OBJECTS AND SUMMARY OF THE INVENTION 
It is an important object of the present invention to provide a method 
enabling such multi-labeling, in particular double or triple labeling of 
tissues, which can be carried out not only in laboratories with highly 
expensive detection apparatus such as laser analysers, and by highly 
trained specialists in this field, but in ordinary hospital and even 
individual immunologist's laboratories equipped with a fluorescence 
microscope or, in the case of immunoenzymatic labelling, only a standard 
laboratory microscope by normally trained medical technical assistents, 
and even untrained personnel. 
Another object of my invention is to provide a method and kit for improved 
detection of two, three, four or even more antigens in a tissue, 
completely avoiding any spurious labeling. 
A further object of my invention is to provide a method and kit for 
performing double labeling of two antigens using monoclonal antibodies of 
the same species and even the same subclass, for instance the IgG 1, IgG 
2a, IgG 2b or other subclasses of immunoglobulins. 
A still further object of my invention is to provide a method and kit for 
an especially potent double immunofluorescent labeling suitable for 
detecting and selectively showing small amounts of different overlapping 
or super-imposed imposed antigens on the same tissue structures, such as a 
membrane or introcellular particles. 
These objects and others that will become apparent from the further 
description hereinafter are attained, in accordance with a first aspect of 
my invention by the improvement in a method of multi-color labeling at 
least two different antigens being simultaneously present in a common 
biological system, with the aid of a corresponding number of different 
antibodies, comprising: 
(a) adding to an animal tissue containing at least one first antigen (AG I) 
and a second antigen (AG II), a first primary antiserum being a first 
solution, in an aqueous, antigen- and antibody-compatible solvent, of at 
least one primary antibody, active against said first antigen, in an 
amount effectively immunologically reacting with said first antigen, free 
from a moiety labeling any antigen in an optically detectable manner, 
(b) adding to the tissue resulting from Step (a) at least one secondary 
antiserum, being a solution in the same kind of solvent, of at least one 
secondary antibody immunologically reacting with said at least one antigen 
I-specific primary antibody, 
at least one such secondary antiserum containing at least one of said 
secondary antibodies being conjugated with a first detecting moiety 
selected from 
(i) a first labeling radical conjugated directly with said secondary 
antibody, and 
(ii) a first labeling radical conjugated with said secondary antibody via a 
first labeling effect-enhancing bridge member; thereby labeling said first 
antigen in an optically detectable manner, 
(c) adding to the resulting treated tissue from Step (b) a second primary 
antiserum of a random animal species and being a second solution, in an 
aqueous, antigen- and antibody-compatible solvent, of at least one antigen 
II-specific primary antibody immunologically reactive with said second 
antigen (AG II), 
said at least one different primary antibody being completely free from 
activity against any antibodies and any normal sera introduced previously 
into said tissue, and being conjugated with a second detecting moiety 
selected from 
(i) a second labeling radical conjugated directly with said antigen 
II-specific primary antibody, and 
(ii) a second labeling radical conjugated with said antigen II-specific 
primary antibody via a second labeling effect-enhancing bridge member; 
said second detecting moiety affording a second labeled antigen (AG II) 
being readily optically distinguishable from the first-labeled antigen (AG 
I) obtained with said first detecting moiety; which comprises 
(d) the at least one secondary antibody in said at least one secondary 
antiserum of Step (b) containing free binding sites in excess of those 
becoming occupied by said at least one primary antibody of Step (a) 
said second detecting moiety being free from cross reactivity with said 
first detecting moiety, and 
(e) adding to the tissue after Step (b) and prior to Step (c), still 
containing free binding sites being capable of acting against any 
subsequently introduced antibodies or against subsequently introduced 
bridge members, from at least one specific non-immune fraction up to the 
entire non-immune normal serum, of the same animal species as said second 
primary antiserum of Step (c), in an amount sufficient for blocking all 
those free binding sites remaining from the last-preceding step; and 
when any of said free binding sites are capable of acting against any 
subsequently introduced bridge members, adding further a blocking agent 
for blocking said last-mentioned sites; 
with the PROVISO that Step (e) follows Step (c) only when all antibodies of 
Steps (a), (b) and (c) are free from mutual cross reactivities, and 
with the further PROVISO that when the animal species from which said first 
primary antiserum added in Step (a) is produced is a first subclass of a 
determined species such as the mouse, and 
the first secondary antibody contained in said secondary antiserum from 
said second animal species, added in Step (b) is conjugated with a first 
labeling radical via a first labeling effect-enhancing bridge member, and 
the animal species yielding the antigen (AG II)-specific antibody contained 
in the second primary antiserum added in Step (c) is an animal such as a 
mouse from another subclass of the said one or another animal species than 
mouse, and said last-mentioned antibody is free from a detecting moiety, 
then follows the step of: 
(f) adding to the tissue resulting from Step (c) being free from any cross 
reactivity a third secondary antiserum containing a third secondary 
antibody being reactive with said antigen (AG II)-specific antibody, said 
third secondary antibody being conjugated with said second labeling 
radical directly or via said second labeling effect-enhancing bridge 
member, said second bridge member being free from reactivity with any 
previously or subsequently added bridge member, 
and Step (e) is omitted. 
"Biological systems" as used in this specification and claims, are animal, 
and particularly human, "tissues" including non-liquid tissues as well as 
blood and other body fluids. 
Sera and antisera of animal species most readily available in commerce and 
recommended for use in the method according to the invention are those of 
the mouse, rat, goat, sheep and rabbit. For instance, as the first 
non-immune normal serum used in Step (AA), infra, there is recommended, 
for instance, the use of goat serum or a mixture of goat and rabbit, or 
goat and human serum. 
As the antibody of the second species used in Step (B), infra, there is 
recommended a mouse monoclonal antibody suitable for reacting with a 
determined antigen. 
Antigen- and antibody-compatible solvents are, for instance, 
phosphate-buffered salines of a pH of about 7.3 to 7.6 such as the 
phosphate-buffered saline PBS of pH 7.4, or tris-buffered saline TBS, of 
pH 7.6, described on page 20 in "Monoclonal Antibodies" published by DAKO 
Corporation, Santa Barbara, California 93103 in January 1984. 
I did not obtain any better results by adding to the aforesaid salines an 
albumin buffer solution recommended by earlier investigators, when 
labeling lymphocyte surface antigens in cryostat sections of inflamed 
skeletal muscle tissue. 
By "primary" are meant those being specifically reactive with an antigen, 
while "secondary" antibodies mean antibodies which are specifically 
reactive with another antibody previously added to the tissue being 
labeled. 
By a "detecting moiety" there is meant a chemical grouping being linked 
with an antibody molecule by a chemically conjugated bond. 
Such detecting moiety consists either of a labeling radical alone and 
directly bonded to the remainder of the antibody molecule; or they can 
consist of a labeling-effect enhancing bridge member which is conjugated 
directly with the said antibody molecule and with which there is in turn 
conjugated the aforesaid labeling radical. 
Such labeling radical can be the radical of a fluorochrome such as 
fluorescin isothiocyanate or rhodamine. Only three of the presently known 
fluorochromes excited by ultraviolet light are easily optically 
distinguishable in a fluorescence microscope or in a laser microscope, 
built by Zein AG, D-7082 Oberkochen, Fed. Rep. of Germany, by the use of 
excitation, band-path, emission filters etc. 
On the other hand, the labeling radical can be that of an enzyme being 
capable of catalizing a specific substrate reaction in which the resulting 
product can be made visible via a colored product obtained in turn at the 
site of the antigen. Such enzymes are, for instance, peroxidase, alkaline 
phosphatase, glucosoxidase gelactosidase and others. 
The use of fluorochrome radicals is preferred by me because antigen 
superimposed in different strata of the same structure or located adjacent 
each other, but so closely that, when they are labeled 
immunoenzymatically, they can not be seen separately in a standard light 
microscope, cannot safely identified by the observer's eye, leading to 
erroneous interpretations of the results. 
However, when they are labeled with fluorochromes which are of sufficiently 
different emissions separable by filters, they can be readily 
distinguished in a fluorescence microscope or a laser microscope; even 
antigens being located one above the other in different superimposed 
strata can be made selectively visible after dyeing, for instance, one of 
them green and the other red, and then viewing the structure through the 
fluorescence microscope using first an appropriate filter for green and 
then another for red emission. 
When photographing by double exposure such structure, being selectively 
filtered in the fluorescence microscope, there is obtained a photograph in 
which the superimposed green and red spots yield a yellow spot, all spots 
in which the two antigens are not superimposed, appearing green or red. 
This permits to distinguish in the tissue being investigated three 
different types of cells or the like biological units, one bearing a first 
antigen, another bearing a second antigen, and a third one bearing both 
antigens superimposed with one another. 
Labeling of three antigens simultaneously present in the same structure 
permits identification of up to six different types of biological units. 
The only instance known to me, where such fluorochrome identification of to 
antigens in the same structure has been achieved, was done in the Mayo 
Clinic in Rochester, Minnesota, with a not generally applicable method of 
carrying out the incubation steps, as reported by Arahata and Engel in 
"Annals of Neurology" Vol. 16 No. 2, pages 193-208 and ibidem, pages 
209-215 (August 1984). This known method, however, suffers from the 
drawback of a very weak primary fluorescence of the obtained labeled 
tissue sites which can be improved by very sophisticated and complicated 
measures requiring a highly specialized technology not to be found in 
normal hospital and similar laboratories. 
"Binding sites" of antibodies are capable of immunologically reacting with 
antigens or antibodies at epitopes which are more or less reaction-avid 
sites of the antibody molecule generally not by means of covalent chemical 
bonds but, for instance, Van der Waals bond and hydrogen bridges. 
"Binding sites" of labeling effect-enhancing bridge members are capable of 
entering with a very strong affinity into bonds with active sites on 
another molecule, closely resembling that of chemical bonds. 
Blocking agents capable of blocking such free binding sites of the 
above-mentioned bridge members are, for instance, haptens such as avidin 
which can block biotin (Vitamin H), p-aminobenzoyl glycine, p-aminobenzoyl 
glutamic acid, arsanilic acid and others. 
There will be described hereinafter preferred modes of carrying out the 
method according to the invention in practice. 
According to a first such mode, applied to a tissue which contains besides 
the first and second antigens, also at least one n-th antigen different 
from the former ones; the Step (e) is carried out following one of Steps 
(b) and (c), and the improvement according to the invention further 
comprises: 
(f) adding to the tissue resulting from the later-applied one of Steps (c) 
and (e), an n-th primary antiserum, other than the first and second 
primary sera, containing, dissolved in a solvent as defined, a primary 
antibody specially active against an n-th antigen other than said first 
and second antigens; 
the tissue resulting from said last applied Step being completely free from 
binding sites active against the antigen n-specific primary antibody and 
against an subsequently introduced defecting system; 
the n-th antigen-specific primary antibody being conjugated with an n-th 
detecting moiety selected from (i) an labeling radical conjugated directly 
with the last-mentioned antibody, and (ii) a labeling radical conjugated 
with the last-mentioned antibody via a labeling effect-enhancing bridge 
member; 
said labeling radical of said n-th detecting moiety affording an n-th 
antigen labeled in an optically distinguishable manner from all antigens 
labeled previously with an earlier-introduced labeling agent. 
Preferably, the first primary antiserum of Step (a) and the non-immune 
normal serum of Step (e) are obtained from the same animal species. 
In a more preferred mode of operation, the improved method according to the 
invention comprises the incubation steps of 
(AA) adding to the animal tissue containing the at least two different 
antigens, in an effective amount, a first non-immune, heat inactivated 
normal serum of an animal of a first animal species, different from the 
species from which the primary antibody is produced, and 
(A) adding to the resulting serum-treated tissue from Step (AA) the first 
primary antiserum of Step (a) contains an antigen (AG I)-specific primary 
antibody produced in an animal of at least one determined immuno-globulin 
class of a second animal species and being free from reactivity with the 
first normal serum, 
in an amount effectively reacting with a first one of the different 
antigens. 
The more preferred mode of operation further comprises 
(B) adding to the tissue resulting from Step (A) a secondary antiserum of 
another than the second animal species and containing at least one first 
secondary antibody being free from reactivity with the first normal serum 
of Step (AA), and being active against the first antibody of the second 
animal species; 
(D) adding to the tissue resulting from Step (B) a second secondary 
antiserum of a random animal species and containing at least one further 
secondary antibody active against the first secondary antibody added in 
Step (B); 
at least one of the secondary antibodies introduced by Steps (B) and (D), 
and preferably both, being conjugated with a first detecting moiety, 
and at least one of the said secondary antibodies having free binding sites 
in excess of those occupied by the first primary antibody, and being 
capable of binding at least one antigen II-specific primary antibody. 
The first secondary antibody of Step (B) can also be free from conjugation 
with a first detecting moiety, but then the further secondary antibody of 
Step (D) must be conjugated with such first detecting moiety. 
In the most preferred mode of operation, the tissue obtained from Step (A) 
is treated further with Step (B), supra but not with Step (D), and at 
least one of the secondary antibodies in the antiserum of (B) must be 
conjugated with the said first detecting moiety, and at least one of the 
secondary antibodies must have free binding sites in excess of those 
occupied by the first primary antibody, which free binding sites are 
capable of binding antigen II-specific primary antibodies; the resulting 
tissue is then treated further by: 
(E) adding to the tissue from Step (B) an agent consisting essentially of 
from at least one inactive immunoglobulin subclass up to the entire 
non-immune normal serum, of the same animal species as the second primary 
antiserum of Step (c), supra, in an amount sufficient for blocking all 
those free binding sites remaining from the last-preceding Step (B); and 
when any of the free binding sites are capable of acting against any 
subsequently introduced bridge members, a blocking agent for blocking the 
last-mentioned sites must also be added; 
(C) adding to the resulting treated tissue from Step (E) a second primary 
antiserum of a random animal species and being a second solution, in an 
aqueous, antigen- and anti-body-compatible solvent, of at least one 
antigen II-specific primary antibody immunologically reactive with the 
second antigen (AG II). This at least one antigen II-specific primary 
antibody should be completely free from activity against any antibodies 
and any normal sera introduced previously into the tissue, and must be 
conjugated with a second detecting moiety which affords a second labeled 
antigen (AG II) being readily optically distinguishable from the first 
labeled antigen (AG I) obtained with the first detecting moiety. 
In order to obtain stronger labeling especially in the case of the labeling 
radical of the first detecting moiety being a fluorochrome a second 
secondary antiserum of Step (D) is added to the tissue resulting from Step 
(B), which second secondary antiserum contains at least one further 
secondary antibody conjugated with a first detecting moiety having the 
same color as the first detecting moiety conjugated with the first 
secondary antibody of Step (B); the further secondary antibody of (D) 
being free from reactivity with the normal serum of Step (AA) and being 
capable of immunologically reacting with the first secondary antibody of 
Step (B). 
In the further treatment of the tissue 
(E.sub.1) the non-immune normal serum of Step (e) can be added to the 
tissue resulting from Step (D), and the further secondary antibody of (D) 
should be free from reactivity with said normal serum of Step (AA). 
Alternatively, 
(C.sub.1) the second primary antiserum containing the at least one antigen 
II-specific antibody of Step (c) can be added to the tissue resulting from 
Step (E.sub.1); or 
(E.sub.2) the non-immune normal serum of Step (e) can be added directly to 
the tissue resulting from Step (B), before or concurrently with the second 
secondary antiserum of Step (D). 
When the secondary antiserum of Step (D) is added to the tissue resulting 
from treatment with Step (E.sub.2), the further secondary antibody of (D) 
should be free from reactivity with the non-immune normal sera of Steps 
(AA) and (E), and be reactive with the first secondary antibody of Step 
(B) as well as being conjugated with a first detecting moiety of the same 
color as any first detecting moiety conjugated with the first secondary 
antibody of Step (B). 
In a further variation 
(C.sub.2) the second primary antiserum containing the at least one antigen 
II-specific antibody of Step (c) which is non-reactive with any normal 
sera and any antisera added previously to said tissue, can be added to the 
tissue resulting from Step (D). Or, according to the variation, 
(C.sub.3) the second primary antiserum of a random animal species 
containing the at least one antigen II-specific antibody is added to the 
tissue resulting from Step (E.sub.2). 
In a particularly simple mode of operation, the antigen I-specific primary 
antibody of Step (A), the normal serum of Step (E) and the antigen 
II-specific primary antibody from Step (C) can all be produced from the 
same animal species of a Type I; moreover, the normal serum of Step (AA) 
and the first secondary antibody of Step (B) can both be produced from the 
same animal species, but of a Type II being different from that of type I. 
Results of clear labeling are further greatly improved by 
(H) washing the treated tissue after each of the preceding steps (a) 
through (f) or any partial steps explained hereinbefore or hereafter with 
an aqueous buffered saline solution having a pH-value of about 7.3 to 7.6. 
In all those cases where only double labeling is to be achieved, the method 
according to the invention will end with Step (c) and Step (g) can be 
dispensed with. 
However, when a tissue contains more than two antigens to be labeled, then, 
subsequent to the above-explained Steps (a) to (e) of the method according 
to the invention, there should follow the additional steps of 
(F) adding to the tissue resulting from one of Steps (C), (C.sub.1), 
(C.sub.2) and (C.sub.3), at least one specific non-immune fraction up to 
the entire non-immune normal serum of a random animal species, in an 
amount sufficient for blocking any free binding sites remaining from Stage 
(C), and 
(G) adding, in the case of at least one different antigen (AGx) other than 
the first and second antigens being present in the tissue, to the tissue 
resulting from the Step (F) at least one further antiserum of the same 
animal species as that from which the normal serum of (F) is produced, the 
last-mentioned antiserum being a solution, in an aqueous antigen- and 
antibody-compatible solvent, of a further antigen (AGx)-specific antibody, 
different from any other previously added antibody, of a random animal 
species, in an amount sufficient for effectively reacting with the further 
different antigen (AGx); the further antigen (AGx)-specific antibody must 
be active against the different antigen (AGx) and must also be free from 
reactivity with any other previously added antibody and must be conjugated 
with a further detecting moiety affording a color different from those of 
the first and second detecting moieties, and being selected from 
(i) a different labeling radical conjugated directly with the antigen 
(AGx)-specific antibody, and 
(ii) a different labeling radical conjugated with the antigen 
(AGx)-specific antibody via a bridge member enhancing the labeling effect 
of the said different labeling radical, 
While any previously introduced bridge members of the first and second 
detecting moieties must be free from reactivity with the last-mentioned 
bridge member or such reactivity must have been blocked prior to Step (G). 
When the animal species from which the first primary antiserum added in 
Step (A) is obtained is a first subclass of the mouse, and the first 
secondary antibody contained in the secondary antiserum from the second 
animal species, added in Step (B) is conjugated with a first labeling 
radical via a first labeling effect-enhancing bridge member; moreover, the 
animal species yielding the antigen (AG II)-specific antibody contained in 
the second primary antiserum added in Stage (c) is a mouse from another 
subclass than the first one or another animal species than the mouse, and 
the last-mentioned antibody is free from a detecting moiety, then there 
can follow the step of further treating the tissue by: 
(K) adding to the tissue resulting from Step (c) being free from any cross 
reactivity a third secondary antiserum containing a third secondary 
antibody being reactive with the antigen (AG II)-specific antibody, the 
third secondary antibody being conjugated with the second labeling radical 
directly or via the second labeling effect-enhancing bridge member, the 
second bridge member being free from reactivity with any previously or 
subsequently added bridge member, while Step (E) can then be omitted. 
According to another aspect of the invention, there is provided a kit for 
carrying out a method of multi-color labeling at least two different 
antigens, namely a first antigen (AG I) and a second antigen (AG II), 
being simultaneously present in a common biological system, with the aid 
of a corresponding number of different antibodies, which kit comprises as 
components: 
(AA) a non-immune normal serum from a first animal species; 
(A) a first primary antiserum from a second animal species and being a 
first solution, in an aqueous, antigen- and antibody-compatible solvent, 
of at least one (AG I)-specific primary antibody, which antibody is free 
from a moiety labeling any antigen in an optically detectable manner; 
(B) a secondary antiserum from a random animal species, being a solution, 
in an aqueous, antigen- and antibody-compatible solvent, of at least one 
secondary antibody acting against the at least one (AG I)-specific primary 
antibody, at least one such (AG I)-specific primary antibody being 
conjugated with a first detecting moiety; 
(E) a non-immune normal serum from an animal species other than the first 
one; 
(C) at least one second primary antiserum from the same species as the 
component (E) and containing at least one (AG II)-specific antibody, free 
from immunological reactivity with any of components (AA), (A) and (B), 
and being conjugated with a second detecting moiety being free from 
reactivity with the first detecting moiety; and 
(H) an aqueous physiologically acceptable buffer solution having a pH of 
about 7.3 to 7.6. 
In preferred kits, one or several of the following details are recommend: 
(1) The second primary antiserum (C) is of the second animal species. 
(2) The at least one antibody contained in Component (B) is polyclonal and 
the antibodies of Components (A) and (C) are monoclonal. 
(3) The antisera of Components (A) and (C) are of the same species and 
monoclonal. 
(4) The antibodies in Components (A) and (C) are free from the mouse, 
Component (AA) is a non-immune normal serum selected from goat serum, 
rabbit serum, sheep serum, human serum and mixtures of at least two 
thereof; and the secondary antiserum of Component (B) is a goat antiserum, 
rabbit antiserum or a sheep antiserum. 
(5) The antiserum of Component (B) is of the first animal species other 
than human antiserum. 
(6) The normal serum (AA) contains from 0 to about 98% by weight of a 
physiologically acceptable buffer having a pH-value of about 7.3 to 7.6, 
and there is present in the undiluted state per kit unit at least about 
one millimeter of this serum. 
(7) The first primary antiserum (A) contains from 0 to about 90% of a 
physiologically acceptable buffer having a pH-value of about 7.3 to 7.6, 
and, in the undiluted state, per kit unit, at least about 50 micrograms of 
antibody in about 1.0 milliliter phosphate buffered saline containing 
about 0.2% gelatin and about 0.1 sodium azide, the saline having a 
pH-value of about 7.3 to 7.6. 
(8) The secondary antiserum (B) contains from about 0 to 90% of a 
physiologically acceptable buffer having a pH-value of about 7.3 to 7.6, 
and, in the undiluted state, per kit unit, at least about 100 micrograms 
of antibody in 1.0 milliliter of physiologically acceptable liquid medium. 
(9) The second primary antiserum (C) is of a random animal species and 
contains from about 0 to 90% of a physiologically acceptable buffer having 
a pH-value of about 7.3 to 7.6, and, in the undiluted state, per kit unit, 
at least about 50 micrograms of antibody in about 1.0 milliliter of 
physiologically acceptable liquid medium. 
(10) The non-immune normal serum (E), of the same animal species as the 
donor of the second primary antiserum (C), contains from about 0 to 90% by 
weight of a physiologically acceptable buffer having a pH-value of about 
7.3 to 7.6, and, in the undiluted state, per kit unit at least about one 
milliliter. 
(11) The Component (B) comprises 
(B') as a first sub-component thereof, a first secondary antiserum of 
another than the second animal species as defined under (B), supra, and 
containing at least one first secondary antibody being active against said 
first primary antibody of Component (A), and, 
(D) as a second sub-component thereof, a second secondary antiserum of a 
random animal species and containing at least one further secondary 
antibody active against the first secondary antibody of the first 
secondary antiserum of (B'); 
at least one of the subcomponents (B') and (D) containing the antibody 
therein conjugated with the said second detecting moiety. 
(12) The first secondary antiserum of (B') is of an animal species other 
than the second species, and the second secondary antiserum is produced 
from an animal species other than the first and second species, 
each of the above first and second secondary sera containing from 0 to 
about 95% of a physiologically acceptable buffer having a pH-value of 
about 7.3 to 7.6, and, in undiluted state, per kit unit at least about 100 
micrograms grams of antibody in 1.0 milliliter of physiologically 
acceptable liquid medium. 
(13) (G) a third primary antiserum of a random animal species containing a 
third antigen (AG III)-specific antibody, free from any immunological 
reactivity with any of components (AA), (A), (B), (B'), (C), (D), and (E) 
present in the kit, and being conjugated with a third detecting moiety 
free from reactivity with the first and second detecting moities. 
(14) (J) a fourth primary antiserum of a random animal species containing a 
fourth antigen (AG IV)-specific antibody, free from any immunological 
reactivity with any of components (AA), (A), (B), (B'), (C), (D), (E) and 
(G), present in the kit, and being conjugated with a fourth detecting 
moiety free from reactivity with the said first, second and third 
detecting moieties. 
In the two last-mentioned kits according to the invention, from one to at 
most three of the detecting moieties contain a fluorochrome radical, and 
remaining moieties are enzyme radicals affording different optically 
visible labeling. 
In another preferred ambodiment of the last-mentioned three types of kits, 
Component (C) is constituted by 
(C') a first subcomponent being a second primary antiserum containing at 
least one (AG II)-specific primary antibody free from immunological 
reactivity with any of components (AA), (A), and (B) and being 
unconjugated with any detecting moiety, this second primary antiserum of 
(C') being from a different immunoglobulin class or subclass of the second 
animal species or from an animal species different from the second 
species; and 
(F) as a second subcomponent, another secondary antiserum from an animal 
species other than that from which the antiserum (C') is produced, this 
last-mentioned secondary antiserum containing a different secondary 
antibody being specifically reactive with the respective class, subclass 
or species of the (AG II)-specific primary antibody and being conjugated 
with a second detecting moiety non-reactive with, and being optically 
distinguishable from the first detecting moiety. 
the Components (A), (B), (C) and (F) as far as present in this kit being 
free from cross reactivities therebetween.

EXAMPLES 
The following non-limitative examples illustrate the method according to 
the invention further. Percentages given therein are by weight unless 
expressly stated otherwise. Temperatures are given in degrees Celsius 
(centigrades); all examples are carried out at 20.degree. C. unless 
another temperature is mentioned. 
EXAMPLE 1 
A cryostat section from a human skeletal muscle, musculus gastrocnemius, is 
placed on a glass object support lamina and left uncovered, then fixed in 
ice-cold acetone for 10 minutes, air-dried and then into a slide-carrier 
glass cuvette filled with phosphate buffer saline (PBS) for 2 minutes. The 
slide is then taken out, residual liquid is removed by careful shaking, 
and the area on the slide around the section is completely cleaned with a 
cloth of any liquid. The section is not covered with a cover slip. 
STEP AA: The thus prepared slide is then placed on an object carrier and 
introduced horizontally into a dark humidity box. The section on the slide 
inside the dark box is then covered completely with 30 microliters (mcl) 
of a non-immune normal goat serum sold by ORTHO Diagnostic Systems GmbH, 
D-6903 Neckergemund, Germany (a), which serum is diluted to 4% 
weight/volume and the slide is then left for 30 minutes in the closed box. 
The slide is than taken out of the box, excess liquid is removed, and the 
slide is placed in a PBS-filled cuvette for 2 minutes, excess buffer is 
removed, the slide is cleaned around the section and placed again 
horizontally into the humidity box. (Intermediary Treatment). 
STEP A: The section on the slide is then covered with 30 mcl of a solution 
of commercially available mouse-anti-human monoclonal antibody OKT 8 
(meaning: against the ORTHOKung-T-lymphocyte No. 8) in PBS prepared by me 
in a volume ratio of 1:10. After 30 minutes incubation in the dark 
humidity box, the slide is taken out and the above-described intermediary 
treatment is repeated. 
STEP B: The slide is then returned to the dark humidity box, the section on 
the slide is covered therein with 30 mcl of a 1:20 (volume ratio) diluted 
solution in PBS of fluorescein isothiocyanate (FITC)-conjugated ORTHO 
goat-anti-mouse (GAM) serum commercially available from (a), supra, and 
incubated in the dark box for 30 minutes. The intermediary treatment of 
the slide removed from the dark box is repeated as described hereinbefore. 
STEP E: In the dark humidity box, the section is then covered with 30 mcl 
of an undiluted non-immune normal mouse serum, commercially available from 
Camon Labor Service GMbH, D-6200 Wiesbaden, Germany, and incubated in the 
dark for 30 minutes. The slide is then taken out of the box in the dark, 
excess liquid is removed, and the slide is then placed, still in the dark, 
into PBS in a cuvette and left therein for 30 minutes. The slide is then 
cleaned about the section in the light, and is then placed again into the 
dark humidity box. 
STEP C: The section is then covered in the dark with 30 mcl of 
phycoerythrin (PE)-conjugated monoclonal mouse-anti-human antibody 
(against the antigen) Leu 3a, commercially available from Becton Dickinson 
Labor System, D-6900 Heidelberg, Germany (c), diluted by me with PBS to a 
volume ratio of 1:10. 
While the above-described intermediary washing treatment is carried out for 
2 minutes in the dark, a fresh covering agent consisting of 1 mg of 
p-phenylenediamine in 1 ml of glycerol gelatin is prepared, and the 
section on the slide is covered with a small amount of this agent (having 
a temperature of 37.degree. C.) and then with a cover slip. 
When observed under a fluorescence microscope with oil immersion, the 
antigen OKT 8 showed green fluorescence and the other antigen, Leu 3a, 
showed red-orange fluorescence, while the background was not completely 
dark but showed a greyish-brown hue; this permits judging of the 
morphology of the unlabeled tissue, which is not possible when the 
background tissue is completely black. In fact, the black background 
resulting in several known labeling methods was the reason why 
immunoenzymatic labeling was preferred to fluorochrome labeling up till 
now. 
When the slide is then stored at -85.degree. C., the fluorescence of the 
preparation will remain unchanged for at least four months or even much 
longer. Observation of the 4-month old slide stored at the said 
temperature, showed in a fluorescence microscope fully satisfactory 
colors, and fading under excitation with ultraviolet light is less then 
when the slide is observed immediately after preparation. The improved 
fluorescence of both labeled antigens can be observed already when storing 
the freshly prepared section overnight at a temperature below 0.degree. 
C., and especially at -85.degree. C. 
Examples 2-5 
The table shown hereinafter presents further examples of double labeling 
sections of the same human skeletal tissue and by the same method steps as 
described in detail in Example 1. Commercially available agents used in 
the following examples are sold by the companies designated by either (a), 
(b) or (c) listed supra, or by 
(d) DAKO Corporation, Santa Barbara, Calif. 93103; 
(e) Behring-Werke AG, D-6800 Mannheim, Germany; 
(f) Amersham Buchler GmbH and Co. Kg, D-3300 Braunschweig, Germany; 
TRITC means rhodamine triisocyanate. 
__________________________________________________________________________ 
STEP AA STEP A STEP B STEP E STEP C 
dilu- dilu- dilu- dilu- dilu- 
tion tion tion tion tion 
incub. incub. incub. incub. incub. 
Exam- time time time time time 
ple No. 
serum 
(min.) 
serum (min.) 
serum (min.) 
serum 
(min.) 
serum (min.) 
__________________________________________________________________________ 
2 goat 100% 
anti OKT 3 
1:5 GAM-FITC 
1:20 
mouse 
1:10 
mouse 1:10 
20 mouse- 20 (a) 20 20 Leu 2a-PE 
20 
3 rabbit 
1:10 
anti OKT 4 
1:10 
GAM-FITC 
1:30 
mouse 
1:2 mouse 1:5 
30 mouse 30 30 30 Leu 2a-PE 
30 
4 sheep 
1:10 
mouse 1:8 rabbit 1:15 
mouse 
1:10 
mouse 1:10 
40 anti OKT 11 
60 AM-FITC 
40 40 Leu 15-PE 
30 
5 human 
1:5 rabbit 1:20 
goat 1:40 
rabbit 
100% 
rabbit 1:20 
20 anti-human 
30 anti-rabbit- 
20 30 anti-neuron- 
30 
myoglobin (d) 
FITC (b) specific 
enolase (d) 
+ biotin- 
1:20 
streptavi- 
30 
din Texas Red (f) 
6 bovine 
100% 
sheep-anti- 
1:10 
donkey 1:50 
sheep 
1:30 
sheep 1:20 
serum- 
30 human immuno- 
30 anti-sheep 
40 40 anti-human 
30 
albumin globulin Ig G-TRITC Ig M-FITC 
BSA (e) (Ig) G (b) (b) (b) 
__________________________________________________________________________ 
The rabbit anti-neuron specific enolase, commercially available from Dako 
Corp. is conjugated with an equivalent amount of Biotin and the resulting 
conjugate is then further reacted with an equivalent amount of 
Streptavidin-Texas Red. 
Example 7 
A cryostat section from a human lymphnode tissue is prepared and mounted in 
the same manner as described in Example 1. 
Step AA is carried out with fetal calf serum (b) concentration: undilutes, 
treatment time 30 minutes. 
Step A is carried out with mouse-anti-human Leu 11, commercially available 
from Becton-Dickinson Labor Systems, (c), supra, diluted 1:10, with an 
incubation time of 30 minutes. 
Step B is carried out with goat-anti-mouse (GAM)-FITC (a), diluted 1:20, 
for 30 minutes. 
Step D: The slide is then placed in the humidity box and the section 
thereon is covered with 30 microliters of biotinylated rabbit-anti-goat 
antibody (b), diluted 1:20, for 20 minutes, and after PBS-washing, the 
slide is again covered with streptavidin-FITC (f), diluted 1:20, for 30 
minutes. 
In Step E, mouse normal serum is used 1:2 for 30 minutes. 
After the same washing step as in Example 1, the slide is further treated 
in the same manner, but with mouse-anti-Leu 1 phycoerythrin (PE) diluted 
1:10 for 30 minutes. 
The weak antigen LEU 11 is labeled a satisfactorily strong green, and Leu 1 
a pronounced red-orange. 
Example 8 
Example 3 is repeated and the still uncovered slide resulting from Step (C) 
is washed in PBS and reintroduced into the dark humidity box. 
STEP G: The slide is then covered with 30 mcl of 1:5-diluted biotinylated 
mouse-anti-Leu 4 antibody for 30 minutes and further incubated in the dark 
humidity box with 30 mcl of 1:20-diluted streptavidin-Texas Red. 
The finished section shows on the tissue changed by inflammation the first 
antigen, OKT 4, green-fluorescent, the second antigen, Leu 2a, red-orange, 
and the third antigen, Leu 4, red fluorescent, which are distinguishable 
by using appropriate filters, including a Texas Red-filter, manufactured 
by Carl Zeiss AG, D-7082 Oberkochen, Germany. 
Example 9 
A cryostat section of an inflamed human muscle, biceps brachii, is prepared 
and treated in a Step (AA) in the same manner as described in Example 1, 
with following washing with PBS. 
The slide is then treated in exactly the same manner as described in Step A 
of Example 1, wherein the antibody against OKT8 is IgG. 
Step B of Example 1 is then repeated, however the antiserum used therein is 
biotinylated sheep-anti-mouse Ig F(ab').sub.2 fragment (f), diluted 1:20, 
for 30 minutes. 
The slide is then covered in the dark humidity box with 30 mcl of 
streptavidin Texas Red solution (f), diluted 1:20, and also treated for 30 
minutes. 
STEP D: After washing with PBS, the slide is then incubated in the dark 
humidity box with 30 mcl of rat-anti-human mono-clonal immune-answer 
associated (Ia-like) antigen Ig M-antibody (b), diluted 1:10, treatment 
time 30 minutes, followed again by PBS-washing. 
The washed slide is then covered in the dark humidity box with 30 mcl of 
FITC-conjugated sheep-anti-rat anti-Ig M antiserum (b) diluted 1:20, the 
treatment lasting 30 minutes. The resulting double labeled slide could be 
examined in a fluorescence microscope. The first antigen OKT8 is labeled 
red fluorescent, and the second antigen Ia is labeled green fluorescent. 
However, the treatment of the slide is to be continued in order to label a 
third antigen present in the tissue. Following Step E, the slide is 
incubated in the dark humidity box during 30 minutes with 30 mcl of 1:10, 
diluted PE-conjugated monoclonal mouse-anti-human Leu 3a antibody solution 
commercially available from Becton-Dickinson GmbH (c). 
The triple-labeled slide is then finished as described in Example 1. 
The labeled third antigen, Leu 3a, shows orange-red fluorescence. 
Example 10 
A cryostat slide from the same human tissue treated in Example 1 is used 
and 
Step AA was applied as described in that Example, but in the light. The 
biologically active substance used in Step AA and B as well as other 
intermediate treatment stages were taken from an "Ortho-OKT-Kit" sold by 
Ortho Diagnostic Systems (a). 
Step A: The slide obtained from Step AA and washed is reintroduced into the 
light humidity box and was covered for 30 minutes with 30 mcl of 1:10 
diluted monoclonal mouse-anti-human anti-actin (a cytoskeletal protein) 
(f). 
Step B.sub.1 : The PBS-washed slide was then covered in the light humidity 
box with 30 mcl of a 1:20-diluted solution of peroxidase-conjugated 
F(ab').sub.2 -sheep-anti-mouse immunoglobulin (f) incubated for 30 minutes 
and then washed with PBS. 
Step B.sub.2 : A substrate solution for reaction with the enzyme is 
prepared by adding to 
a solution of diaminobenzidine (DAB) in PBS having a concentration of 0.5 
mg of DAB per milliliter PBS, 
a sufficient amount of hydrogen peroxide affording a concentration of 0.02% 
H.sub.2 O.sub.2 in the substrate solution, which is then filtered. 
The slide obtained from Step (B.sub.1), supra, is then covered with 30 mcl 
of the aforesaid filtered substrate solution and a brown precipitate is 
formed at the site of the first antigen being labeled, actin. The 
precipitation reaction is arrested by placing the slide in distilled 
water, and the slide is then washed with PBS in a cuvette for 2 minutes. 
Step E: The slide is then treated as described in Step E of Example 1, 
including the final PBS-washing for 30 minutes. 
Step C: The slide is then treated in the light humidity box by covering it 
with 30 mcl of a biotinylated monoclonal mouse-anti-human antibody against 
Leu 2a (c), diluted 1:10 and incubating it for 30 minutes. 
The PBS-washed slide is then covered with 30 mcl of a 1:20 solution, in 
PBS, of streptavidin--galactosidase complex (f) for 30 minutes. 
The slide is then washed in PBS and then covered with a substrate solution 
prepared by adding to a solution of 5-bromo-4-chloro-3-indoyl-galactoside 
(BCIG) dissolved in PBS and having a concentration of 0.5 
milligram/milliliter BCIG, containing further, per milliliter of the 
solution, one milli-Mol of magnesium chloride, 3 milli-Mol of potassium 
ferricyanide and 3 milli-Mol of potassium ferrocyanide; the solution which 
has a pH-value of 7.4, is used without further dilution and the section is 
incubated therein for 20 minutes. 
A blue precipitate is formed at the sites of the second antigen, Leu 2a. 
The slide is then placed in distilled water and covered with a cover slip 
in an aqueous mounting medium. 
Example 11 
Example 10 was repeated up to and including Step E: 
Step C: The slide is then treated in the dark humidity box with a 
1:10-diluted solution of PE-conjugated monoclonal mouse-anti-human 
antibody against Leu 2a, the incubation time being 30 minutes. 
The second antigen, Leu 2a, is labeled a fluorescent red-orange. The slide 
is then washed with PBS for 2 minutes and then covered with a cover slip 
using as mounting medium a mixture of p-phenylenediamine with 
glycerol-gelatin in a concentration of one mg per milliliter of the 
mixture. 
The simultaneous observation of the red orange-labeled Leu 2a-sites and the 
brown actin sites under ultraviolet light with concurrent 
transillumination with appropriately adjusted bright-field light is made 
possible by the fact that the labeled actin shows no noticeable 
auto-fluorescence under ultraviolet light. 
This example is, therefore, recommended for tissues in which one of the 
antigens is of low concentration. 
Example 12 
Human blood is freshly drawn and heparinized. An amount of 10 ml of this 
blood is brought into a test tube charged with 4 to 5 ml of plastic beads 
coated with one spoon tip of iron powder, thereby absorbing the 
macrophages and granular leucocytes on the beads. The contents of the test 
tube are then incubated at 37.degree. C. in a shaking bath. 
A dextran solution is obtained by dissolving 5 g of dextran in 100 ml of 
physiological sodium chloride saline, and an amount of 6 ml of the dextran 
solution is then added to the test tube and the mixture is allowed to 
settle for 60 minutes with the test tube being held inclined at an angle 
of 45.degree. while incubating the contents at 37.degree. C. The 
supernatant is removed and carefully layered on to a sterile density 
gradient material (e.g. Ficoll Hypaque, sold by Pharmacia, D-7800 Freiburg 
im Breisgau, Germany) in a proportion of one part of Ficoll to 2 parts of 
the cell suspension; the resulting supernatant contains only lymphocytes. 
The mixture of density gradient and cell suspension is then centrifuged at 
700 G for 20 minutes. 
The band layer between the Ficoll layer and the sera layer is then 
carefully removed with a Pasteur pipette and the band layer containing the 
lymphocytic cells is washed with PBS for 5 minutes. A lymphocyte 
shape-stabilizing agent (e.g., Media 199, sold by GIBCO, D-7514 Eggenheim, 
Germany) is then added to the washed cell suspension, and the 
concentration of the mixture is adjusted with PBS to 4 million cells per 
milliliter suspension. 
STEP A: 200 mcl of the adjusted stabilized cell suspension are passed into 
another test tube and 5 mcl of undiluted monoclonal mouse-anti-human 
antibody (against) OKT 8 are added thereto; the resulting mixture is 
incubated for 30 minutes in and ice/water bath (about 0.degree. C.) while 
shaking briefly every ten minutes. 
Centrifuging Step: 2 ml of the stabilizing agent, supra, are added to the 
incubated suspension, and the latter is then centrifuged at 300 G for 5 
minutes at +4.degree. C., the suspension is then briefly shaken to 
re-suspend the cells, and centrifuging is repeated. 
STEP B: After re-suspension of the cells, 100 mcl of FITC-conjugated 
goat-anti-mouse serum are added and the resulting suspension is placed in 
a dark space at 20.degree. C. The centrifuging step is then repeated and 
then, 100 mcl of 1:5 diluted non-immune normal mouse serum are added and 
then incubated in the dark for 30 minutes. The centrifuging step is then 
repeated. 
STEP C: 5 mcl of an undiluted primary solution of phycoerythrin-conjugated 
monoclonal mouse-anti-human antibody (against) Leu 3a, sold by (c), are 
added to the suspension which is then shaken briefly and incubated in the 
dark for 30 minutes in an ice/water bath, the brief shaking being repeated 
every 10 minutes. The centrifuging step is then repeated, the supernatant 
is separated from the sediment, and the latter is suspended in a small 
amount of the lymphocyte shape-stabilizing agent. 
A drop of the resulting suspension is placed on a slide and covered with a 
cover slip, and the labeled lymphocytes are then counted in a fluorescence 
microscope using FITC-adjusted and PE-adjusted filters. 
In general, labeled membranes in a tissue being investigated such as 
lymphocyte membranes cannot be made visible by the described method, if 
the tissue is formaldehyde-fixed and embedded in paraffin. Therefore, 
cryostat sections of tissue shock-frozen in iso-pentane floating in liquid 
nitrogen are preferably used in the method according to the invention. 
Double labeling with the aid of fluorochromes affords particularly 
satisfactory results on tissues which contain little connective tissue, 
because the latter tissue becomes itself fluorescent even when no antigens 
are labeled therein. This auto-fluorescence may disturb the labeling of 
antigens present on such connective tissue. Therefore, double labeling of 
such tissue is better done using immunoenzyme techniques in the method 
according to the invention.