Note: Descriptions are shown in the official language in which they were submitted.
-- 1 --
The present invention is concerned with an improved
device for the de-tection of the presence of an allergy
and for the specific detection of the allergen respon-
sible for the allergy.
This application is a division of Canadian Patent
Application No. 497,664, filed December 13, 1985.
The device can be used for the diaqnosis of other
diseases, in the pathomechanism of which a stimu-
lus-induced liberation of mediators from mast cells and
basophils participates. Furthermore, the device can be
used for an automatable screening process for pharma-
ceuticals which suppress the stimulus-lnduced mediator
liberation from basophils, mast cells and phagocytes and
thus can be used for a therapy of allerglc or inflam-
matory dlseases.
In the case of allergy patients, an increased IgF
;~ level is Eound in the blood. Furthermore, IgE is found
bound to mast cells and basophils. The allergic reac-
tion is initiated by the blnding of the specific aller-
gen on to cell-bound IgE molecules specific therefor.
It is frequen~tly assumed that this bindlng of the
stimulating allergen to I~gE via the so-called bridging
of neighbouring IgE brings about, in a complex reacti.on~
the degranulation of basophilic leukocytes. As is
known,~ the degranulation liberates histamines, pro-
teases, metabolites of arachidonic acid, phosphatases
and other en~ymes.
In Federal Republic of Germany Patent Specification
No. 31 47 763, for the diagnosis of allergic diseases,
-- 2
it is su~g~sted to separate the blood o~ possibl~
aller~ic persons into a plasma la~er containing the
white blood corpuscles and into a lager with the red
blood corpuscles, to recover the plasma la~er, to mix
with an indicator solution and to retain a part as
control and to mix a part with the aller~en(s) to be
tested. A~ter a more or less lon~ incubation time~ the
indicator reaction is determined either directl~ or
photometricallg after the addition of further reagents,
lo th~ enz~me activit~ being determined by comparison
with the initial values or the value of the control
solution~ As enz~mes possibl~ to be measured, there
are mentioned proteases a~d al~aline ~hosphatase which
are to be activated b~ the aller~ic reaction, ~ifficult-
ies are caused in the case of this process because
allergens, which themselves displa~ a protease or
phosphatase activit~ 9 cannot be detected with this test
and, on the other hand, even the serum of health~
subaects displa~ a certain degree of protease and phos-
phatase activit~ and the contral solution thereb~ also
displays a reaction which must be deducted fro~ that of
the measure~ent solution in order to obtain the value
specificall~ brought about b~ the allerg~ which, as is
know~, leads to a considerable breadth o~ error.
'~herefore, in ~ederal Republic of German~ Patent
Specification No~ 32 11 254, it is su~gested, ln analogy
to the known fluorimetric histamine liberation test,
-- 3 --
to separate the leukocytes from the serum, to remove
adhering ser-um residues by repe~ed washi~ with an
appropriate buffer solution and to resuspend the cells
again in such a buffer. The so obtained suspen~ion î~
therea~ter mixed with an aller~en or with a solution of
anti-I~E antibodies and, in a ~urther step, by the
addition of a calcium chloride solution, the liberation
of histamine or protease is stimulatedO A~ter a definite
incubation time, the reaction is stopped b~ the addition-
of a solution o~ ethglenediamine-tetraacetic acid (~DTA),
the cells are sedi.mented and in the supernatant~ instead
o~ the previously Xnown, laborious fluorimetric measure-
ment o~ ~istamine, by the addition of an indicator
solution1 the protease activit~ liberated b~ the allergen
is measured '~hus, the process can:be carried out without
apparatus or with the use of simple photometer. Although
this process is substantially le~ subject to disturb-
anoe than the ~luorimetric hi~tamine measurement, due to
the necessary number of process ~teps, it pro~es to be
laborious for a routine testO
Therefore, the present invention seeks to provide
a device ~or the d-agnosis of allergic diseases by
means of which the above-mentioned cellular reaction
on allergens can be carried out more simply and
quickly, and, if possible, the sensitivity of the
detection reaction is also increased. Therefore, the pro-
cess principle should also be suitable for the diagnosis of
~ ,7~ ~ ~
other diseases, for example, of rheumatic arthritis,
of pharmaceutical supersensitivity (e.g. penicillin
incompatibility) or of incompatibility in the case of
patients requiring dialysis, in the pathomechanism of
which a s-timulus-induced mediator liberation from
basophils and mass cells participates.
There is disclosed herein a process
for the detection of the presence of
an allergy and for the specific detection of an
allergen responsible for an allergy comprising:
incubating leukocytes of a sample to be investigated,
said sample having a leuXocyte concentration of 1~ to
101 per litre,;' with a stimulation factor in the
presence of a solution containing 0.5 to 5 x 10
mol/l. of calcium ions and 10 to 10 3 mol/l. of
chromogen, said solution containing a buffer effective
to establish a pH of from 6.3 to 9 and being isotonic
to said sample, reacting any liberated protease with
said chromogen to produce a chromophor, and deter-
mining the increase in chromophor concentration as ameasure of protease activity.
The stimulation factor or stimuIant functions to
induce pro-tease liberation.
In a particular embodiment the
leukocytes of the sample to be investigated are
incubated with a natural or synthetic allergen or with
another stimulation factor, for example? anti-IgE
antibody, RNA, DNA or immune complexes, in an aqueous
medium, the liberated protease is reacted with the
chromogen and the resulting chromophor .is determined,
the allergen or another stimulant being mixed in a
buffer with a pH of 6.3 to 9 with the calcium ions
and chromogen in isotonic solution and mixed with the
leukocytes to be investigated, the protease activity
being measured kinetically after an incubation period
by the increase of the chromophor concentration.
Analogously, for the detection of a rheumatic
arthritis, there is used the liberatlon of protease
after the additionof cell nucleus parts of eukaryotic
cells, especially DNA or RNA, or immune complexes to
the leukocyte suspen~ion.
~ ~5~
. -- 6
Furthermore, there is disclosed a
rea~ent for carrying out the above process~ which com-
prises a buffer with a pH of from 6~3 to 9, an allergen
or other ~timulant in an amount of from 0.01 to IC0 mgO/l,,
a source of calcium ions, for exa~ple a calcium salt, in a
concentration of from 0.5 to 5 x 10 3 moltl. ~nd chro~ogen
in a concentration of 10-4 to 10-3 mol/l.
In accordance with the invention there is provided a
device for the detection of allergies or correspon~ng st~lation
factors, as well as for the determination of anti-
allergic substance~, comprising a microtitre plate
whicn contains, arranged in rows, di~ferent reagents
as defined hereinbefore with anti-IgE anti-
bodies or various allergens or other sti~ulants in
various concentrations, the same reagent mixtures
optionallg being provided in several positions for
obtaining average values
There is also ~isclosed a process
for the determination of antiallergic and antiinflamma-
tor~ substances, wherein a reagent according to thepresent invention is mixed,in a process according to
the present invention, with a pharmaceutical and leuko-
cytes are added thereto which are sensitive towards the
:~ allergen or further sti~ulants (anti-Ig~ antibodies,
opsonised z~osan) and the reaction weakened in compar-
ison with a control is measured~ ~
7~3
7--
Since onl~ small amounts of reagent and test
substances (OD2 to l ml~ per sample) are nee~ed for
the reaction, the reaction can preferabl~ be carried
out on a microtitre plate, the reagents thereby p~efer-
abl~ being present in a solid and especiallv l~ophil-
ised form and in a definite amount in the compartments
of the microtitre plate, the reactio~ solution being
reconstituted b~ the addit1on of the leukocgte suspen
sion~ The reagents can aIso be impregnated on to an
absorbent carrier, for example, a paper, s~nthetic resin
or ~lass fibre fleece, a swellable s~nthetic resin ~ilm
or a gel and introduced in this form into a reaction
vessel. However 7 the impregnated carrier can al~o be
moistened directlg with the leukoc~te suspension and
brought to reaction in the manner usual for other dr~
chemical reactlons~ The chromogenic reaction can be
measured either visually or, in the case of higheP
re~uirements for exactitude, with a photometer For the
throughput of larger numbers of samples, this process
can, of course, be automated in known manner. Therefore,
the process is also especiall~ appropriate for the
; pharmaceutical screening of antialler~ic compounds
which inhibit the stimulus-induced degranulation of
mast cells and basophils. ~he same test principle can
also be used for the detection of enzyme liberation
from phagoc~ting cells after the addition of~ for example,
s`~
opsonised z~mosan and thus for the discover~ of
inflammation-inhibiting substances~
It is surprising that it is possi~le to place all
the reagents necessar~ for the liberation and detection
o~ a protease in a uniform mixture and merel~ to add
thereto the leukocyte suspen~ion in a simple pipetting
step since, o~ the one hand~ the indicators in the
hitherto u~ual concentration of 10 2 to 10 3 mol/l.
bring about a spontaneous liberation of protease from
leukoc~tes if this reaction is not previously inhibited
by the addition of complex-forming agents, such as ~D~A,
and, on thë other hand, it was known that it is not
po~ible to add the calcium or magnesium ions neces~ar~
for the Iibe~ration of the protease from the basophilic
leukoc~tes before the addition of the stimulating aller-
gens to the leukocyte solution since the calcium ions,~
n this case, bring about a change of the leukoc~te
membrane which inhibits the attack of the allergen.
~ owever, as we have now found, the addition of the
chromo~ens in an amount of from 10 4 to 10 ~ mol~
either~does;not bring about a non-specific, spontaneous
liberation of~protease or onl~ such a small liberatlon~
that it does~not dlsturb the test. On the other hand,
b~ means of this addition, the protease liberated b~
the aller~en is, surprisin~l~, stabilised in an as ~t
unknown manner so that, in the case of the addition o~
'~.
9 -
the same amount of allergen, a markedl~ hi~h protease
activity is measured in comparison with the previously
known reactions. It is assumed that, in the case of the
simultaneous adaition of allergen and calcium ions, the
allergen binds SO quickly to the active places of the
cell membrane th~t a reduction of the reactivitg due to
the calcium ions does not occur and these merel~ catalyse
the desired protease transport through the cell membranes.
The following statements for the carr~ing out of the
process apply generall~ to all its variants. ~he~ are
illustrated using the example of the use of the process
for the diagnosis of an allergy. For the process according
to the present invention, depending upon the nature of
the allergen and the severity of the allergic reaction,
the allergens are used in an amount of from 0.01 to
100 mg./lO and especially of from 1 to 10 mg./l. of
reaction solution.
Calcium ions, as well as possibly magnesium ions,
are used advantageously in a concentration o~ from 0~5
to 5 x 10 3 moljl. and e~pecially of from ~ to 4 X 10 3
mol/l. ~rhese ions are usually employed in the form of
water-soluble salts and especially as chlorides,
sulphates, acetates or citr~esv
mhe reaction is carried out in a solution buffered
to a pH of ~.3 to 9, preferably of 7.G to 8 and especi-
ally of 7.5 ~he buffer used is preferably the same
buffer which is used for washing and suspending the
~75i~
--10--
leukoc~tes, i.e. a ph~siological buffer which, b~ the
addition of sodium chloride, potassium chloride, glucose
or other physiologically compatible substances, i5 made
isotonic and has a concentration of about 5 x 10 3 to
10 I mol/l. of buffer substance. ~he buffer substances
which can be used include tris buffer, phosphate buf~er,
gl~cine buffer and other ph~siologicallg compatible
buffers, or possibl~ also mixtures of buffers, the
buffer capacit~ of which can be adjusted to the above-
mentioned pH range and which, in the above-given con-
centrations, do not form insoluble salts with the calcium
or magnesium ionsO In the scope of the present invention,
these buffer s~stems are also called "buffers"
For the stabilisation of the leukoc~tes, this
solution can additionally also contain gelatine or
other macromolecular materials with cell-stabilising
properties, such as are well known.
he reaction is usuall~ carried out bet~een ambient(2ooc)
temperature and 37C., the physiological ~onditions
corresponding to-a temperature o~ 35 to 37C~ being
preferred. In the case o~ thi temperature, after 15
to 60 minutes incubation, normally so much proteinase
ic liberated that the measurement can take place~
However, in the case of a smaller allerOen concentration
and a weaker liberation reaction, the incubation time
can be increased. In this case 9 the measurement reaction
is pre~erabl~ carried out at ambient temperature since
~_~ 75j~3L~ ~_
~11--
the enz~mes, as we have ascertained9 are stable at
this temperature even over comparativel~ long periods
of time (over 100 hours) and give a linear reaction
kinetic~ In the case of a stronger liberation reaction~
care must be taken that no substance impoverishment
occurs because otherwise the colour reaction no longer
proceeds linearl~ In these c~ses~ a measurement at
37C~ accelerates the enzgme reaction. ~owever, measure-
ments can also be advantageously carried out at ambient
lo temperature
Because of the simple carryin~ out of the process,
which onlg re~uires one pipetting step for the addition
of the Ieukoc~tes to the finished reagent mixture, the
process according to the present invention is especiall~
suitable for a large series of ex~eriments in which the
sensitivitg of a given leukoc~te suspension towards
various allergens or other stimuli i~ to be tested
and, on the other hand~ in oraer to test unkno-~n sub-
stances for an antiallergic action These substances
are thereby admixed either with the reagent or wlth
the leukoc~te ~uspension in approprlate concentration
and the reduction of the protease liberation is deter-
mined in comparl~on with a control solution not mixe~
with the substance in question.
~ - chromogenic substances according to the pre~nt
invention~ there can be used all those which react in
the p~ range used accordin~ to the present invention
7 ~
-12-
with protease and produce a visuall~, photometrically
or also fluorimetricall~ detectable product. Subst.ates
which can be used include, for example, N-benzoyl-
arginine-~-nitroanilide~ N (3 carbox~propionyl)-phen~l~
alanine-~ nitroanilide or p-nitroanilides substituted
b~ peptides, such as are described, for example 7 in
Federal Republic o~ Germany P~tent Specificatio~s Nos,
25 27 932; 25 52 570; 2629 067 and 32 11 254~ Other
reagents with chromophoric or fluorescing residues,
which are substituted by peptides which can be ~plit
of~ are described in ~ederal Republic of German~ Patent
~pecifications Nos 29 36 543; 30 17 721 and 28 54 987
and in ~uropean Patent Specification No. 0,078,703.
B~ chromogen in the sense of the present invention,
there is to be understood not only the substrate itself
;: :
but also the other rea~ents possibly also necessar~ for
the ~ormation or c~olour formlng~ such as are described,
for example, ln the above-mentioned Patent Specificat1ons
Especiall~ mentioned are reaction accelerators, stabil-
isers, oxidation adjuvants9 colour couplers, contrast
colour~agents~ viscosit~-regulating materials and
wetting a~ents. ~
he;~ollowing ~amples are given for the purpose
of illu~trating the present invention:-
. .
1~1 Obtainin~ leu ocy~
60 ml~ of venous blood from a donor were carefull~mixed with 30 ml. of a dextran mixture (dextran with an
~,. .
~. :
~5
-13-
average molecular weight of 75,00C 27~; D-~lucose 2
~D~A 20 mmol/l.; gelatine 0~07~) in a pol~eth~lene
beaker and transferred to a ~iliconi~3d separating
funnel. A~ter leaving to stand for 50 to 90 minutes at
ambient temperature, the supernatant containing the
leukocytes (about 50 ml.) was separated off and mixed
with 50 ml. tris buffer of p~ 7 6 (tris 22 mmol/l~;
sodium chloride 0~l2 mol/l.; potassium chloride
5 mmol/1 ; ~elatine 0.05~; ~D~A 1 mmol/l.; D-gluco~e
0 1~) and centrifu~ed for 15 minutes at 800 g. A~ter
decanting, the cells were washed twice in that, i~ each
case, theg were a~ain suspended ~ith 50 ml. of the same
buffer mixture of pH 7,6 and, in each case, centri`fuged
for 15 minutes at 800 g ~he cells were suspended in
~10 m~ of the same buffer mixture and, b~ dilution ~ith
further buffer~ adjuqted to 107 cells/ml.
:
~ 1.2 Reage~t mixture
: :
10 ml Anti-Ig~ solution (Behring Werke I~o. 0~ 04/05
diluted 1:500 with tris buffer of pH 7.6 as
in 1.1)
10 ml. calcium~ch1oride solution (4 mmol/l~ in isotonic
sodium~chloride solution, pH 7.6)
20 ml. chromo3yme ~H ~olution 0.75 mmol/l (one bottle
Boehringer Mannheim GmbH, Order No. 199664
; dissolved in 10 ml. water) were thoroughl~
~ mixed and ctored until used~
: :
14
1~3 Proteinase stimulation with anti-I~E antibodies
0.5 ml9 amounts of leukocgte suspension ~ere added
to 0,5 ml. of the rea~ent mixture, incubated at 37C.
and the extinction mezsured at 405 nm after 457 90 and
180 minutesS
In order to determine the non-specific decomposition,
there was carried out a corresponding control wlthout the
addition of anti-Ig~ but with all the other components
of the reagent solution. ~rom the extinction differences
l~ bet~een measurement and control solution, there can be
determined the proteinase liberation from the leu~ocvtes
by the qtimulant.
a) Blood from non-aller~ic sub,iects
average valuec from 45 min ; 90 min 180 min.
3 measurements
,~ _ _
control 0,OlSO.C42 0.097
.
anti IgE 0~0620.170 0.382
E405 C,0~-0.l28 ~.2e~
b) ~lood from aller ic sub~iects (ha~ fever)
20avera~e values ~rom 45 min.90 min 180 min
3 measurements .
__ ~
GontrOl o~ 0250,058 0.122
anti-IgE 0.3050.857 1,7~
~ _ .. .: .. . _. _
~ E405 0,2800,799 1.62l
-15-
1~4 Protease stimulation with grass aller~ens
Under the same conditions as under 1,3 above butwith a reagent which contains 10 ml. aller~en extract
from grass pollen (10 5 ~/ml, aller~en in tris.buffer
of pH 7.6 according to 1,1 above), blood samples from
healthy and aller~ic subaects were compared.
_ _ _
average values from 45 min. 90 min. 180 min.
3 measurements
. _
control 0~018 0,037 0,092
_ _ ~
allergen 0.037 0.045 0.109
_ _
~ E405 ¦ 0.019 0.008 0,017
b) Blood from aller~ic sub1ects (ha2_fever)
_ ; _ ___
average values from l~,5 ~in 9 min, 130 min
3 mea~urement~
_ , _
control C,020 0,G48 1 0,082
_ _ I .
aller~en 0~180 0.424 ¦ 0.864
__ __
L~ C5 ~,0,160 Oo376 I C.782
~ terpretatio~
: 20 It is shown from a co~ari~on of the reaction~
that the non-specific coloured material formation
brought about by the buffer is clearlg smaller than
that brought about bg liberated protease~
~x~
-16-
~ nti~ antibodies also bring about a certain
protease liberation with the leukoc~tes of healthg .
subjects which, however, is weaker -than in the case of
aller~ic ~ubaects ~o that this test can be used as a
non-~peci~ic test ~or aller~ic react;ions~ `
The weak reaction of normal leukoc~tes towards
aller~ens differs, in turn, si~nificantl~ from the
strong reaction of allergised leukocytes, which makes
possible a satis~actor~ determination of the allergensO
Example 2.
~ uence o~ the additlon of calcium ions on the
histamine and proteinase liberation~
For the demonstration of the influence of the
sequence o~ the addition of the reagents, the test
described in Example 1~3 a) was repeated but the re-
agent components accordin~ to Example 1~2 were added
in t he followin~ sequence:
a) 0.5 ml. of cell suspension accordin~ to Example 1.1
was mixed with 0 125 ml. calcium chloride solution
and, after 10 m1nutes, 0 125 ml. anti-IgE solution
was added thereto.
b) 0,5 ml~ cell suspension was mixed with a mixture o~ ~ :
: :: ~ : :
- 0.125 ml. calcium chloride solution-and 0~125 ml~
: anti-IgE solution.
- c) 0.5 ml. cell suspension ~as mixed with o. 12S ml
anti-IgE~reagent solution and, a~ter 1 minute,
0 125 ml. calcium chloride solution added thereto.
::
:
-17-
As control, in each case~, 0.5 ml. of tris buffer
wa~ mixed in the same ~Ja~ ith the rea~ent ~olutions,
All the batches were incubated .~or 60 minutes at
37C,, the protein~se liberation l~a~ stopped by the
addition of 0.5 m~ D~A solution (0~1 mol/l. in
0.15M tris buffer of pH 7.6) and the cells were
centrifu~ed off, In the cell-~ree supernatant, the
liberated histamine was measured by automated fluori-
metry and, in a separated part, by the addition of
chromoz~me '~H (5 x 10 4M), there was measured photo-
metricall~ the protease activity liberated after a
further 50 minute~ at 37C', ~he values set out in the
following ~able were determined from 3 to 6 measurements~
,- ~ -
hlstamine proteinase (~ ~405) ~ :
;~ ~ : ~ of totaI ~ ontent
: : a b c a b c
: ~ _ _
control 7 4 6 0.040 0~016 0.017
:: ~., ~ .
ample : 14 40 35 0.084 0,271 0.227
dif~èrence ~ 7 36 29 0~044 O.Z55 0,210
___ ~. :--- , _ ~ ~
his experiment clearly ~shows that a premature
; addition of cslcium ohloride lnhibits the liberation9
~:: In~uence of the_chromogen~addition of the histami~e
:
and proteinase liberation,
~ ~ ,
,~i;1
.
-18~
In order to demonstrate the influence o~ the
chromo~en addition, the following experiments were
carried out:
a) 0.5 ml. cell suspension was mixed with a mixture of
0 125 ml. calcium chloride solution and 0~125 ml
anti-Ig~ solution. After incubation for 60 minutes
at 37C. 7 the reaction was ~topped with 0.125 ml.
~DTA solution. A~ter centrifugin~, the histamine
and proteinase content was determined in the s~lper~
natant. ~he protein~se was measured by the addition
of 0.125 ml. chromozyme ~H solution, further incub-
ation for 60 minute~ at 37C. and measurement of
the extinction difference at l~05 nm.
b) 0.5 ml~ cell suspension was mixed with 0.5 ml.
rea~ent according to Example 102, incubated for 60
minutes a~ 37C., the histamine content was deter-
mlned in a cell-free part and a first extinction
value was measured at 405 nm. A~ter further incubation
at 37C. for 60 minutes, a secand extinction was
measured and the proteinase content determined ~rom
; ~ the extinction inc~rea~e~
c)~In a~further batch, the operation ~as as described
under a) but~the cells were not separated fron the
sample serving for the proteinase determination~
:: :
: :
' '
,~
.
- ~ ~ 7
-19-
_ _ _ histamine ~ proteinase (~ E405)
_ . __ __ __
a b c a b c
__ , ____ _ _
4 ll 6 0,0~ 0,07 0,03
sampIe24 40 35 0~18 ~.5~ 0~20
_ _ , ~ , .. .
difference 20- 29 29 a. I6 0,4~ 0.17
_ _ . . _ _ ,
It is shown from a comparison o~ the above measure-
ment values that test variant b) ~ves a clearlg
stron~er proteinase measurement ~i~nal than i~ the case
of c), whereas the histamine liberation is about egually
high, It is c~ncluded ~rom this that, in the presence of
the chromogenic substrate, the liberated proteinase is
stabilised or is protected from inactivation by other
test components.
Exam~ple 4.
- . :
om~arison of the aIIer~en- and anti-I~E-induced protein-
ase liberatlon ~rom leukoc~te ~uspensions of various
~ubaects with suspected aller~.
Measurement example in microtitre plate: 0.3 ml. batches
x 10~7 cells/ml. (microtltre plates Nunclon 96U).
Concentrations: various aller~ens 10 ~ /ml,; anti-
E antlbody solution: 1:8000 dilution~ other rea~entsas in Example ~c
Protein qe activit~: absolute ~ easured 50 and
~05
110 minutes after the addition of the-stimuli wi-thout
cellseparation ~ith a Dynatech llicroelisa ~utoreader
MR 580,
,
-20 ~ ~
i ~ __ _ _ _ C _ ._ _ .
0.25 .170 .147 .OS8 .165 .030 .OlS .085 .033 .025 .033 .022
. _ ~ ~ .... ~_ ..... _
.032 .180 .163 .070 .155 .040 ¦ .018 .095 .043 .02B .039 .027
. ~ __ ' _ _ _ . _ _
.018 .177 .154 .066 .168 .037 1 .013 .098 .037 .029 .027 .029
. _ _ . _ . .. _ __ _
.039 .187 .170 .Oa4 .175 .054 .022 .087 .033 .031 .026 .015
. .... . _ _ ,,_ _ .
.037 .llo .042 .100 .080 .030 ~ .2?0 .170 .015 .130 .240
.027 .105 .040 .Og5 .090 .025 .017 .205 .185 .025 .135 .235
_ _ _ _ __
.OqS .103 .026 .092 .095 .038 .019 .235 .165 .022 .~38 .224
_ _ _ _ _ . _ ._ _ _
.047 .122 .045 087 ~094 .217 .175 .027 .125 ,238
.
~ 7~;~ L7P~ '?L
~1--
inhe above Table shows the mea~urement values of
4 subject~ (blocks A - D), lines l - 4 and 5 - 8 in
each case showing parallel measurements. Columns l
and 7 ~how the control values ~lith buffer soIution~
columns 2 and 8 the inducin~ with anti-Ig~ anti-
bodies~ columns 3 and 9 with allergen extract from
grass pollen (Allergopharma Ganzer KG, Order No. 006
columns 4 and lO ~llergen extract from hazelnut
(~llergopharma G~nzer KG, Order No, 012), columns 5
and ll allergen extract from birch pollen ~A~lergo-
pharma Ganzer hG9 Order ~o, 013) and columns 6 and 12
allergen extract from ~ites ~Allergopharma Ganzer hG,
Order No~ 725). Due to the reaction with anti-Ig~
antibodies, in the case of subjects A, B and D, there-
is given an indication of an existing allerg~7~
~ ubaect ~- shows a clearly alle~ic reaction with
grasses and birch pollen. Subject B shows a reaction
of avera~e ~tren~th with both tree pollens. Subject D
reacts sensitl~ely to grasses and birch and very
stron~ly to mite allergens. Only in the case of
hazelnut i9 no si~nificant reaction to be seen.
~ he ~uspected allergy from the anti Ig~ reaction
is confirmed ~y the allergen-specific reactions of
subjects A~ B and D~ ~
Subject C reacts clearlv more weakly with anti-
Ig~ and practically not at all with allergen~ ~o that
he is not an allergic subaect.
PJ
, "t~,
-22-
Example 5.
Inhi~ition of the anti~ induced_pr~te}~y ~ _
_rom ~ pooled leukoc~te su~pen~ion of health~ sub~ects
(n = 4) with various_~tandard su~stan~
In a microtitre plate (Munclon 96U) with 8 x 12
vessles, in each case there was placed 0.1 mlO leuko-
cytes prepared according to ~xample 1.1 (about 1.2 x 107
cells/ml.). and in each case 4 vessels were supplied with
.1 ml~ of the substrate solution to be tested in the
concentrations ~i~en below. ~hereafter, 0.1 ml~ reagent
solution of 5 x 10 4M chromoz~me ~H and 3 x 10 ~M calcium
chloride, 0.15 M tris buffer of p~ 7.6 in i~otonic solu~
tion of sodium chloride was added thereto, incubated at
37C and, after 45 minutes and 140 minutes, without
cell separation, the extinction was measured with a
Dynatech MicrOelisa Autoreader MR 580 at 405 nm.
C0ncentrations of the substrates raferred to the total
batch:
theo~h~lline: 10 3M, 2 x 1 0 4M~ 4 x 10 5M
: 10 ~M, 2 x 10 5M, 4 x 10 6M
p~ 5 x~ 10 4M, 10-4M~ 2 x 10 5M
iodoacetate: 5 x 10 ~M, 10 3~,2 x 10 5M
~t~i~ 5 x 10 5M ~ 10--sM
E~ 5 x 10 5M, 10 5M, 2 x 10 M
:
- :
r~
--23--
buf er
I 2 3 cr~cate 2, 4-D~nit~phenol
--2 ~ L ~ 2
, , ~ 1~, l
. D 5 . 0 i; . 0 _ .1 r2 .15 D .1 'S.1 _ .115 .170 _ . 0~ iS ¦ .1 ;5
__ __ ___ _ _
.025 .037 .069 .178 .170 .195 .1~5 .1~1 .16S .la2 .0s5 .135
.ola .02~ .06~ .111~ .1~9 .1~!17 I .ltU .1-6 .17~ .19S ,061 .147
_ _ _ I- _ _
.022 OZ9 .062 .laZ .192 .laa I .109 .1~ .las .193 .070 .1~,~
_ _ _ =_ =_ =--
.185 .0~5.06i7 .lao .077 .17l .20~ .190 .029 .09l .160 .02l
.1~5 .025.069 .171 .O~i7 .161 .202 .lao .027 .071 .177 .oza
.~00 .0~0.oe2 .17~ .05~ .15~ .13< .1~5 .03l .077 .169 .03l
.18~ .0~7 .076 .165 .05S .163 .209 .176 .022 .079 ~172- .015
_ l _ I _ ~- l . . _ _ _ _ r _ _ _ _
. 'I T2 ~ Tl T2 t~ I t2 '
p""p~ ,7~, Pic~
I ~d OacK~ b~ f~s
ont ~-1~
_
:
7~
-2~-
It is shown that, as antiallergicall~-active known
sub~tances, also in the new test arrangement, a clearl~,
partl~ concentration-dependent inhibition action is
obtained, In the same wa~, ener~ metabolism blocXer~,
such as dinitrophenol or iodoacetate~ also promote the
proteinase liberation but not, according to expectation~
the ~-stimulators isoprena~ine or cromoglycate,. mhe test
is thus well suited for the serial investigation of such
substance~.
Detec~ion of aller~ens with test_str ps.
~ 'ilter paper (schleicher & ~chull Mo. 2352) was
impregnated ~ith the ~ollowing mixture~ dried at 60~o
: and cut up into 10 mm~ wide ~rips,
15:mmol tris buffer (pH 8.0)
: 130~mmol sodium chlor1de
0,2~g. gelatine (M~ about 10,000)
: 50 m~aller6en mixture (gra~ses~ trees~, rnites)
:: 2 mmol calcium chloride
,
~ : 6 mmol glucose : : : ~ ::
:
1 mmol.indicator (dissolved in 10 ml. acetone)
;: 5 mmol pho~phorio acid trimorpholide : : ~ ;
2:g, decanol
ad 1 lltre water.~
A~ indicators;, 3-(N~succln~ L-alanyloxgindole,
~ :3-(N-toluene~ulphonyl.~-alan~loxy)-indole~and
':
~toluene-4-sulphonyl)-L-valyloxy)-indole a~e used with the
same result.
These strips were so sealed in, b~ means of hot
rollers, between a 60 mm~ wide band o:E melt wax-coated
polyester foil and a 20 mm wide band of polgester
fleece (15 g /m2) th~t the middle o~ the test paper
came to lie 6 mm. ~rom the lower ed~e of the polgester
band and under the middle of the fleece band, the hot
rollers used having a recess at the position o~ the
test paper. I'he finished sealed band was then cut
transversel~ into 6 mm. wide strips
When these strips are dipped into a suspension
accordin~ to ~xample 1 containing about lQ7 leuko-
cgtes/ml., then, in the case of leukoc~tes ~rom
allergic subjects, a~ter 15 to 30 minutes, blue to
dark blue coloured papers are obtained. With leukoc~tes
:
of healthy subjects or with pure buffer solution, the
papers do not colour or o~l~ achieve a pale blue colour.
he coloration can possibly aIso be measured bg
remission photometr~.
::~::: : : :
~:
; :: ~ ~ .:
:~ ~r
~7Si~
- 26 -
The Patent Specifications referred to herein are
more particularly identified as follows: -
Federal Republic of Germany Patent 2527932, Lars
Gundro Svendsen, assigned Pentapharm AG, open to public
inspection January 22, 1976;
Federal Republic of Germany Patent 2552570, Lelf
Erik Aurell, assigned AB Xabi, open to public inspection
June 10, 1976;
Federal Republic of Germany Offenlegungsschrift
2629067, Leif Erik Aurell et al, assigned AB Kabi,
open to public inspection January 13, 1977;
Federal Republlc of Germany O~fenlegungsschrift
2854987, Dieter Berger, assigned Boehringer Mannheim
GmbH, open to public inspection June 26, 1980;
Federal Republic of Germany Offenlegungsschrift
2936543, Hermann E~. Karges,~open to public inspection~
Ap~rll 19, 1981
Federal Rep:ublic of Germany Offenlegungsschrift
301:7721~, Dieter Berger,~ddsigned Boeh~rlnger~Mannheim ~ ~ :
GmbH,:open to publ:ic~indpection Novembe~r 26, 1981;
~ :: Fede~ral Republic of Germany:~Offe~nlegungsschri~t ~:
;3l4~7:7~63~Helmut Stickl, open to public inspection June
; Federal:Repu~blic of Germany~Of~enlegungsschrift
3~211254, Otto-Henni~ng Wllhelmd, asdlgned Bodhringer
Mannheim, open to public inspection September 9, 1983;
~:
:~
~5~
- 27 -
European Published Patent Application 0,078,703,
J. W. Ryan et al, assigned University of Miami,
published May 11, 1983.
: ::
:
:: ~
,
:
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