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The present invention relates to an improved process for the qualitative or
quantitative determination of a reactant of an immunologic reaction,
utilizing the known affinity of antibodies and antigens to each other, by
decreasing unspecific linkages. The invention is based on the fact that
such reactions can be recorded more distinctly by means of certain
identification criteria.
In principle, there are two different kinds of immunologic reaction methods
with marked reactants: the direct test and the indirect techniques.
For the direct test, the reactant to be determined, in most cases an
antigen is determined by incubation with a marked second reactant
specifically directed against the antigen, generally an antibody.
For the indirect techniques, the reactant to be determined is incubated
with a second reactant specifically directed against it and, after
separation and elimination of the unbound amount of the second reactant,
the reaction mixture is incubated with a third, generally marked, reactant
specifically directed against the second one.
It is known to those skilled in the art that the direct test is
distinguished by a high specificity. However, its disadvantage resides in
an insufficient sensitivity. On the other hand, the indirect techniques
are known for their high sensitivity but low specificity.
One of the essential factors influencing the specificity in the direct and
indirect tests is supposed to be the unspecific linkage of the antibodies
to the antigen and the structures surrounding it.
An unspecific linkage is mainly caused by sections of the so-called Fc part
of the antibody (=immune globulin) molecule, and this goes also for highly
specified antibodies.
The binding affinity of the Fc parts of an antibody to various structures
of cells or tissues described in the literature as Fc receptors is
increased when a change of conformation occurs in the antibody molecule.
This change takes place on the occasion of spontaneous aggregation of
antibody molecules in antibody preparations, linkage of antibodies to an
antigen (immune complexes), or adsorption of antibody molecules onto
synthetic surfaces, for example latex particles.
Hitherto, various methods for decreasing the unspecific linkage have been
applied, for example elimination of aggregates or immune complexes from
antibody preparations by means of ultracentrifugation, addition of albumin
to the antibody preparations in order to stabilize the proteins or to
prevent aggregate formation.
Additionally, in the direct and indirect tests there have been used with
success such antibody preparations the Fc part of which had been split off
according to known methods without adversely affecting the immunologic
reaction of the remaining fragment F(ab).sub.1 or F(ab).sub.2. These
methods, however, because F(ab).sub.1 or F(ab).sub.2 fragments of the
antibodies have to be specially obtained, are complicated and expensive.
Moreover, the corresponding operations are very difficult to carry out in
some cases, especially when antisera are used in the indirect tests.
In accordance with the present invention, the direct and indirect tests for
the immuno-reaction with marked reactants are altered in a simple manner
and to such an extent that the known high sensitivity is completely
maintained also in the case where natural antibodies or sera containing
such antibodies are used, while the known high unspecificity is reduced to
a minimum.
The present invention is based on the idea that in the direct and indirect
tests the unspecific linkage of natural antibodies due to Fc can be
prevented when the structures of various antigens which react with the Fc
part (in a general sense Fc receptors) are previously saturated by immune
globulin fragments having a binding affinity to these Fc receptors in a
general sense. Such immune globulin fragments should stem from immune
globulins of that species from which either the antibody (direct test) or
the first antibody (indirect techniques) is derived. On the other hand,
all antibodies used must not have any specificity to this immune globulin
fragment. Furthermore, as far as this is required according to the test,
the immune globulin fragments must not fix a complement.
A possible steric hindrance of the specifically immunologic reaction, such
as occurs for example when using natural immune globulins or immune
globulin aggregates instead of immune globulin fragments, is not observed
when immune globulin fragments having an affinity to Fc receptors are
employed.
Furthermore, the application of immune globulin fragments, for example Fc
fragments of different immune globulin classes, allows the use of
antibodies directed against parts of immune globulins, for example the
light chains thereof, the Fd fragment or F(ab) fragments of the
corresponding immune globulin classes, in the direct and indirect tests
without the risk of an immunologic reaction with the Fc fragment.
The present invention relates to a process for the qualitative or
quantitative determination of a reactant of an immunologic reaction
according to the direct test or the indirect techniques, which comprises
contacting an antigen bearing Fc receptors in a general sense with an
immune globulin fragment having a binding affinity to Fc receptors in a
general sense, and subsequently bringing it into contact with antibodies
having no specificity to the immune globulin fragment. Thus, a
considerable improvement in the result of an immunologic determination is
achieved by decreasing unspecific side reactions.
By indirect techniques in accordance with this invention, there are to be
understood all tests in which the intended reactant is detected by more
than one further reactant only. Therefore, indirect techniques in
accordance with this invention are especially the antiglobulin test, the
direct test with complement, and the sandwich test.
These methods are described in detail in J. H. Humphrey, R. G. White,
Kurzes Lehrbuch der Immunologie, Stuttgart 1975, pp 257-260; D. M. Weir,
Handbook of Experimental Immunology, Oxford 1973, pp. 18.14-18.16; and G.
Wick, Wiener klinische Wochenschrift (1972), 84.1, pp 2-7, using
fluorescent dyestuffs as identification criteria.
The methodology of the indirect techniques can be extended in such a manner
that all antibodies used are marked and/or that, in addition to a third
reactant, further reactants specifically directed against the preceding
reactant may be employed.
Therefore, all those processes according to which incubation with marked
antibodies is carried out several times are indirect techniques, too.
It is known from the literature that certain sections of the Fc part of
immune globulin have a high affinity to Fc receptors in a general sense.
It is furthermore known that antibody molecules (immune globulin
molecules) are disintegrated by means of proteolytic enzymes to form
numerous fragments. Thus, the proteinases plasmin or papain divide the
immune globulin molecule into two F(ab).sub.1 fragments and one Fc
fragment. Pepsin attacks the molecule at another place and causes the
formation of a bivalent so-called F(ab).sub.2 fragment and further little
peptides, for example pFc. These proteolytic degradation products of the
antibody molecule denominated Fc fragments or pFc can be used in
accordance with this invention as immune globulin fragments having an
affinity to Fc receptors in a general sense.
Examples of preparation of Fc fragment or pFc are described in Porter, R.
P. (1959), Biochem. J. 73, 119; Hershgold, E. J. et al. (1963) Nature 199,
284; and Haupt; H. (1969), Klin Wschr. 47, 270.
The methodology of the direct test and the indirect techniques may be
succinctly described and exemplified as follows:
In the direct test, the antigen containing substrate is contatacted with a
marked reactant, generally a specific antibody, which causes the antibody
to be linked to the corresponding antigen. Thus, the antigen is identified
directly.
According to the indirect techniques, the intended reactant (for example
the antigen) is detected by more than one further reactant.
In the antiglobulin test, for example, the antigen containing substrate is
reacted with a specific antibody which is optionally marked, the excess
antibody is eliminated from the reaction mixture by washing, and the
reaction mixture is reacted with a second marked antibody specifically
directed against the first antibody, and subsequently the amount of
unbound antibody is removed.
In the sandwich test, an antibody is reacted with the antigen specifically
directed against it, the unbound amount of the antigen is eliminated, and
the reaction mixture is reacted with a marked antibody again specifically
directed against the antigen, and the unbound amount of the antibody is
removed.
In the indirected test with complement, the antigen substrate either fixes
complement directly, or it is reacted with a complement-fixing antibody
specifically directed against the antigen; subsequently, unbound antibody
is removed, and the reaction mixture is reacted with complement. After
elimination of the unfixed complement amount, the complement fixed to the
antigen or antigen/antibody complex is detected by means of a marked
antibody specifically directed against complement.
Within the scope of this invention the expert is of course free to use
those identification media known to him, for example marking with
fluorescent dyestuffs, radioactive marking or enzyme marking.
The use of immune globulin fragments having an affinity to Fc receptors in
a general sense according to this invention may be succinctly exemplified
and described as follows for the direct test as well as for the
antiglobulin test.
For the detection or determination of an antigen in accordance with this
invention, the sample to be tested or a dilution series thereof is
contacted with different amounts, beginning for example with 1 mg, of an
immune globulin fragment which has a binding affinity to Fc receptors in a
general sense, for example the Fc fragment of IgG after fission by papain.
Subsequently, the mixture is allowed to stand for a period of from 20
minutes to 20 hours, preferably for about 30 minutes, at a temperature of
from 4.degree. to 30.degree. C., preferably about 20.degree. C., in a
water vapor-saturated atmosphere.
Thereafter, unbound Fc fragment is separated from the reaction mixture, for
example by repeated washing (generally twice), preferably with a
polyionic, isotonic aqueous solution. Subsequently, the reaction mixture
is contacted with a determined amount of the marked antibody specifically
directed against the antigen (direct test) or an antiserum or an unmarked
antibody (antiglobulin test), and the mixture is then allowed to stand for
20 minutes to 20 hours, preferably about 30 minutes, at a temperature of
from 4.degree. to 30.degree. C. in a water vapor-saturated atmosphere.
Unbound antibodies are removed from the reaction mixture by repeated
washing of the latter (generally to 2 to 6 times), preferably with a
polyionic, isotonic aqueous solution. Subsequently, in the case of the
antiglobulin test, the reaction mixture is contacted with a determined
amount of a marked antibody specifically directed against the first
antibody, and then incubated and washed under the conditions as described
for the antibody used first.
Thereafter, depending on the kind of antigen used, the reaction mixture is
spread onto a slide (for example cell suspension) or it is already fixed
to a carrier (histological section).
Examination is then carried out using apparatus for qualitative and
quantitative measurement and observation known to those skilled in the art
and marketed by corresponding specialized companies, such as fluorescence
microscopes with direct light or transmitted light excitation,
fluorescence photometers or radiation measuring devices.
The optimum amount of immune globulin fragment having a binding affinity to
Fc receptors, for example of the Fc fragment, in order to prevent
unspecific linkage can be easily evaluated by a preliminary test for any
specific antigen.
Suitable antigens are for example viral antigens (for example rubella,
measles or hepatitis antigens), bacterial antigens (for example
Salmonella, Escherichia coli or staphylococci antigens), carbohydrate
antigens (for example blood group substances), protein antigens (for
example plasmoproteins such as immune globulins or complement factors),
glycoprotein antigens (for example .alpha..sub.1 -antitrypsin,
caeruloplasmin), or lipoprotein antigens (for example .alpha..sub.1
-lipoprotein). They may be isolated, dissolved, suspended, linked to
carriers such as water-insoluble polymers, for example chemically
cross-linked carbohydrates, be present in or on cells, or in histological
tissue sections.
The immune globulin fragments required for obtaining immune globulin
fragments having a binding affinity to Fc receptors in a general sense are
prepared in known manner from the blood or serum of man or animal;
however, synthetic immune globulin fragments may also be used within the
scope of this invention.
The decrease of unspecific linkage of specific antibodies, although their
sensitivity in the direct and indirect tests remains unchanged, which
decrease is achieved in accordance with this invention by using immune
globulin fragments having a binding affinity to Fc receptors, for example
Fc fragments, is proved by the following tests, the results of which are
partially listed in the following Tables:
Table 1 shows results proving the influence of Fc fragments of human IgG on
the unspecific linkage of rabbit gamma globulin to the surface of
lymphocytes of two donors (columns A and B), and the dependence thereof on
a preliminary incubation of the lymphocytes with neuraminidase (columns C
and D). As already described (Seiler et al. (1974) Behring Inst. Mitt. 55,
258), a neuraminidase treatment of lymphocytes increases the amount of
cells bearing Fc receptors.
Table 1 (columns B and D) shows that the unspecific linkage of rabbit
gamma-globulin to lymphocytes according to the double antibody method is
substantially reduced by a preliminary treatment with Fc fragments, as
compared to cells not treated with Fc fragments (columns A and C).
When in a further example frozen sections of different organs, for example
liver or kidney of mouse, rat, rabbit or man, are spread onto glass slides
according to known methods, subsequently incubated for 30 minutes in a
moisture chamber at 20.degree. C. with about 0.1 ml of a 1% or 0.1%
solution of Fc fragments of human IgG, then washed gently in isotonic
saline solution, and when thereupon a double antibody test is carried out
in known manner (indirect method: G. Wick (1972) Wein.klin. Wschr. 84,
2-7) with the use of human antisera as first antibody and a second
antibody not cross-reacting with the Fc fragment, there results a lower
rate of unspecific linkage of the antibodies to the frozen section than in
the case of a frozen section not treated with Fc fragments as described
above.
TABLE 1:
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Inhibition of unspecific linkage of normal rabbit gamma
globulin to human peripheric lymphocytes by Fc fragments of
human IgG in the indirect method
A B C D
VCN - - - - + + + +
Fc - 1.sup.+
10 100 - 1 10 100
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Donor I
rabbit 1.sup.+ 8.sup.++
3 1 1 55 5 2 5
.gamma.-globulin
4 6 4 2 1 34 4 2 3
donor II
rabbit 1.sup.+ 11 2 3 3 12 4 5 5
.gamma.-globulin
4 5 2 6 1 17 14 3 17
16 4 6 2 8 9 12 10 7
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VCN = Vibrio cholerae neuraminidase 25 U/5 .times. 10.sup.7 cells, 30 min
37.degree. C.
Fc = Fc-fragment of human IgG, 1% stock solution. O.1 ml of each dilution
on 5 .times. 10.sup.6 cells (sediment) for 30 min. at room temperature,
subsequently 2 washings.
.sup.+ = reciprocal titer of the antibody or Fc dilution
.sup.++ = unspecifically dyed lymphocytes in %.
The example of following Table 2 proves that saturation of Fc receptors on
lymphocytes, for example by means of Fc fragments, does not result in a
steric hindrance of adjacent antigen structures, in contrast to the use of
immune globulin aggregates.
In this Example, lymphoblastoid culture cells of B type lymphocytes/RPMI
1788) were incubated with an antiserum (serum 196602), which hinders the
so-called mixed lymphocyte culture (MLC). Because of the congruence of
antigen structures of B type lymphocytes in the MLC and the lymphoblastoid
culture cell, antibodies of this antiserum are linked to the
lymphoblastoid cell. These antibodies can be detected by a marked second
antibody directed against the first one (FITC anti-IgM or FITC
anti-k+anti-l). It is known that unspecific reactions of the Fc part of
the immune aggregates or immune complexes present in the antibody
preparations give falsely positive results. A preliminary saturation of
the so-called Fc receptors on the cells by means of aggregates of immune
globulin G makes it impossible for practically all antibodies to be linked
to the cell, (see column C and the corresponding literature) possibly
because of steric hindrance. When Fc fragments are used instead of immune
globulin aggregates, such inhibition of the antibodies does not occur
(column D), although Fc fragments saturate the Fc receptors, which is
proved by the fact that subsequently used immune globulin aggregates are
not linked to the cell any more (column E).
On account of these observations, the process of the invention is therefore
especially suitable for improving an immunologic reaction by decreasing
unspecific linkage in the determination processes of cytoplasmic or cell
membrane antigens (especially MLC determinants), or of nucleus antigens,
antigens in the tissue or on carriers, or antibodies which are dissolved.
TABLE 2:
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Inhibition of linkage of aggregated IgG by Fc fragments
of IgG.
Target cell: RPMI 1788
IgG Serum FITC FITC
Column
Fc aggr. 196602 Anti-IgM
Anti-k + Anti-1
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A - - - 18.sup.+
22.sup.+
B - - + 28 40
C -+ + 0 0
D + - + 35 43
E + + + 31 40
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.sup.+ % dyed lymphoblasts
The following example of detection of cell membrane-linked MLC determinants
on lymphocytes illustrates the invention.
EXAMPLE
According to known methods, human peripheric lymphocytes are isolated from
venous whole blood. The lymphocyte suspension so obtained is a cell
mixture consisting substantially of B type and T type lymphocytes. The T
type cells are separated as far as possible according to known methods,
and rejected. The remaining lymphocytes enriched with B type cells are
washed twice in an isotonic saline solution. A number of cell sediments,
1.times.10.sup.6 lymphocytes each, are resuspended each in 0.1 ml of a 1%
solution of Fc fragment of IgG (this rate has proved to be the optimum
according to preliminary tests) and incubated for 30 minutes at room
temperature. The batches are then washed twice with isotonic saline
solution. Each batch (cell sediment) is resuspended in 0.1 ml of a human
serum to be tested and incubated at room temperature. In this Example, 9
test sera and one negative control serum (AB) are used. Subsequently, the
reaction mixture is washed 3 times with an isotonic saline solution.
The sediment is resuspended in 0.05 ml of a fluorescin marked rabbit
antibody directed against the light chains of human immune globulins (1%
protein solution), incubated for 30 minutes at room temperature, and
subsequently washed 3 times with an isotonic saline solution.
Subsequently, the sediment is resuspended in 0.05 ml of bovine serum
albumin solution (2% w/v), the suspension is spread onto a slide,
air-dried and embedded in glycerol according to known methods.
By means of a fluorescence microscope, the amount of fluorescing cells in
the totality of cells is counted and indicated in percent.
Result: The number of positive cells after treatment with Fc fragment is
indicated in column II of the following Table 3. It is evident that, as
compared to the values obtained with the same antibodies but without
treatment with Fc fragment (column I), there is a decrease of positive
cells, which decrease differs depending on the serum used. Thus,
differentiation between positive and negative sera is improved.
TABLE 3:
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Reduction of unspecific linkage of antigens to lymphocytes
by preliminary Fc fragment incubation
Sera without Fc with Fc
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20075 23.sup.+ 12
20138 19 14
20330 26 11
10376 20 10
20377 18 17
20379 23 9
20380 27 12
20381 17 10
20630 13 11
AB 11 6
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.sup.+ % of positive lymphocytes
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