Note: Descriptions are shown in the official language in which they were submitted.
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22237-366
The occurrence of housedust mites ~Dermatophagoides pteronyssinus,
farinae etc.) has attracted increasing attention in recent times (G. Hoffmann in
"Luftqualit~t in Innenraumen" ["The Quality of Air in Illteriors"] published by
Gustav Fischer, Stuttgart - New York 1982, pages 385 to 401). The reason for
this is that it has been established that a considerable fraction of the popula-
tion - about 5% has been calculated - suffers from asthmatic symptoms which are
attributable to constituents of housedust.
These constituents are principally the feces of the abovementioned
housedust mites and fragments of dead mites. The intestinal tract of the live
and dead mites contains the allergens which induce the disturbances of health
(E. Petri et al. in Allergologie, Vol. 5, No. 3/1982, pages 109 to 119). A live
mite itself contains only about one thousandth of the amount of allergen which
it produces in the course of its life. The feces themselves are frequently
colonized by fungi which, in turn, may likewise produce allergens. The major
amount of allergens is found in dust deposits, even when the original mite popu-
lation has been killed off or reduced by control measures.
The housedust mites reside in upholstered furniture, beds, floors and
walls, especially when they are covered with textile materials. In respect of
area, it is probable that carpets make the greatest contribution.
A very fine dust is raised from floors and upholstered furniture by
being walked on and used, and this frequently collects in inaccessible places
which are essentially free of air draughts, for example behind cupboards. Even
the very process of vacuum cleaning raises some of this very fine dust, and thus
it reaches places which are not themselves colonized by mites. In any event,
when more is raised there is more annoyance for those in the rooms affected.
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Elimination of the causes of housedust allergy presupposes that the
cause is known. For there are also other allergies, such as, for example, to
pollen (hay-fever). However, housedust allergy is a nuisance throughout the
year for those affected, since mite feces and mite residues are present virtu-
ally without interruption. Thus, the physician and patient are repeatedly con-
fronted by the question of whether there is infestation with mites.
Our copending Canadian Patent Applications Serial Nos. 469,364 and
471,104 disclose procedures for the detection of allergen-containing mite resi-
dues. An aromatic diazo compound, for example diazosulfanilic acid, forms a
brick-red coloration in the presence of mite excreta. The intensity of this
colour is, as the applicant has established, proportional to the concentration
of mite feces present in the dust sample.
In continuation of this work, it has now emerged that the way of assess-
ing the concentration of mite feces described in the patent applications is also
suitable for the assessment of the overall allergological activity of a dust
sample. Hence, it is not only possible to carry out qualitative and semi-
quantitative detection of allergen-containing housedust by a color reaction, but
it is furthermore possible to gain information on the pollution by allergens
contained in this housedust.
Accordingly, the present invention provides a procedure for detecting
the presence of allergen-containing dust from housedust mites in a sample of
dust, which procedure comprises suspending the sample of dust in an aqueous-
alcoholic alkali metal hydroxide solution, conducting a color reaction between the
resulting extract and an aromatic diazo compound, and assessing the concentration
of mite residues on the basis of the intensity of the coloration, wherein the
assessment is carried out by measuring quantitatively the color intensity.
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The value of the invention is confirmed by comparison of the results
of measurement in the procedure according to the invention with that test method
which, according to the state of the art, is to be regarded as the most effective
at present, that is to say the radioallergosorbent test ~RAST).
Statistical analysis of the results of the investigations obtained on
the same dust samples both with the RAST and with the procedure described here
~using the color reaction), and the relation of them to the mite population
checked by microscopy has shown a high degree of agreement in the assessment of
the overall allergological activity. The analysis has been carried out by the
Spearman correlation coefficient method (Sidney Siegel, 'iNonparametric Statis-
tics", McGraw Hill Intern. Book Comp. J Auckland (1956)).
Compared with RAST, the procedure according to the invention is more
straightforward to carry out, since it is merely necessary to extract the rele-
vant dust sample, and it is then possible directly to measure the color intensity
in the extract. Even the design of the RAST method is more elaborate, and its
individual steps are subject to the problems appropriate to them; thus, attempts
have been made to abandon the use of radioactive substances for indication (in
this context, see D. Nolte "Allergiediagnostik" ["The Diagnosis of Allergies"]
DustriVerlag, Karl Feistle, Munich-Deisenhofen (1981), in particular pages 29
et seq., and M. Werner and V. Ruppert "Praktische Allergiediagnostik" ["Practi-
cal Diagnosis of Allergies"] published by Georg Thieme, Stuttgart (1979), pages
96 et seq.).
These new findings by the applicant on the assessment of the color
reaction have prompted the present extension of the main patent, namely a
quantitative measurement.
Some preferred embodiments of the procedure according to the invention
are described below:
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It is preferable for constituents which interfere with the detection
in the dust sample which is to be investigated to be removed beforehand by
treatment with a heated aqueous-alcoholic solvent mixture.
The constituents relevant to the detection are removed from the re-
maining dust sample by extraction with an aqueous-alcoholic alkali metal hydro-
xide solution. The following should be specifically noted for carrying out the
detection procedure according to the invention. The alcohol used for the aqueous-
alcoholic solution is preferably an alcohol which is infinitely miscible with
water, such as, for example, lower monohydric aliphatic alcohols. Methanol is
particularly preferred for this; however, it is also possible to use ethanol.
The weight ratio of alcohol to water in the aqueous-alcoholic solution can vary,
and it is generally (10 to 95) : (90 to 5). A weight ratio of alcohol to water
of (50 to 90) : (50 to 10), in particular of (75 to 90) : (25 to 10), has proved
particularly useful.
The alkali metal hydroxide used in the aqueous-alcoholic solution is
preferably potassium hydroxide and sodium hydroxide. However, it is also poss-
ible to use lithium hydroxide. The amount of the alkali metal hydroxide in the
aqueous-alcoholic solution can vary; it is generally 0.1 to 50% by weight. An
alkali metal hydroxide content of 0.1 to 20% by weight is advantageous, and 0.1
to 10% by weight is preferred. In a very particularly preferred manner, the
alkali metal hydroxide content in the aqueous-alcoholic solution is about 4 to
5% by weight.
In principle, the compounds suitable for the color reaction are all
those which give a color reaction with the dust containing the mite residues.
The reaction used in the present case is based on the ability of the mite
residues (residues of mite feces and fragments of dead mites) to give a color
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reaction with aromatic diazo compounds. Thus, a suitable aromatic diazo com-
pound is every aromatic diazo compound which undergoes a color reaction with
the extraction solution (in the case of the presence of housedust mite residues)
and whose resulting dyestuff is soluble in the extraction solution. Diazo-
sulfanilic acid (4-diazobenzenesulfonic acid), which gives a brick-red colora-
tion, has proved particularly useful as the aromatic diazo compound. The
aromatic diazo compound can be used in the solid form (where appropriate sta-
bilized in a customary manner) or in the form of an aqueous, in particular
saturated, solution.
In the detection procedure according to the invention, it is advan-
tageous to proceed in such a manner that a dust sample which has been obtained
is (after removing the coarse fractions) impregnated with the aqueous-alcoholic
alkali metal hydroxide solution. For this, it is possible to vary the volume
ratio of the dust sample to the extraction solution. In general, this volume
ratio of the dust sample to the extraction solution is (0.1 to 2) : (1). A
volume ratio of 0.5 : 1 has proved particularly useful.
An aromatic diazo compound, in particular diazo-sulfanilic acid (4-
diazobenzenesulfonic acid), is added to the dust sample to which the extraction
solution has been added. This is carried out by, for example, addition of the
aromatic diazo compound in the solid form which is, where appropriate, stabi-
lized. However, in place of this it is also possible to meter in the aromatic
diazo compound in the dissolved form, in particular as a saturated aqueous
solution (a few drops). The whole is then thoroughly mixed by shaking or stir-
ring. The resulting extract suspension is allowed to stand, whereupon the dust
generally settles out of the extraction solution (a few minutes).
Because of the presence of the aromatic diazo compound, for example
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diazosulfanilic acid, when mite excreta are present a brick-red coloration
forms, and the intensity of this is, as the Applicant has established, proport-
ional to the concentration of mite feces present in the dust sample.
In order to make the color detection clearer, it is possible to re-
move the supernatant extraction solution ~over the sedimented dust) and to
assess the color intensity in this form. However, it is also possible to im-
merse a small strip of white filter paper of defined size (for example 0.5 cm2)
in the color solution and to evaluate the coloration remaining on the paper.
The detection procedure, according to the invention itself is based
on some specific findings, which are to some extent surprising:
1) In order, using the aqueous-alcoholic extraction solution at room
temperature, to extract rapidly and exhaustively the feces constituents which
are relevant for the color detection, an alkali metal hydroxide solution (for
example of KOH or NaOH) in the indicated concentration is necessary (a sodium
carbonate solution of corresponding concentration, which would actually be
necessary according to number 2) below, is unsuccessful).
2) The color reaction with the aromatic diazo compound, in particular
with diazosulfanilic acid, would per se take place best only in aqueous solution
which is alkaline with sodium carbonate. (In only aqueous alkali metal hydro-
xide solution, for example of 4-5% by weight KOH or NaOH, the desired color
reaction does not take place - as the Applicant has established). However,
starting from this particular problem in respect of designing a straightforward
detection procedure, it has now been found, surprisingly, that, when an extract-
ion solution of an alkali metal hydroxide in a solvent mixture of alcohol and
water, in particular in a mixture in which the weight ratio of alcohol to water
is (50 to 90) : (50 to 10), is used, on the one hand the feces constituents
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relevant for the color detection are extracted virtually quantitatively and, on
the other hand, the desired color reaction with the aromatic diazo compound, in
particular diazosulfanilic acid, takes place in the same extraction solution.
Moreover, the alcohcl improves the wetting of the dust samples (and, at the
same time, brings about a rapid and uniform distribution of color on the filter
paper which is, where appropriate, immersed).
Further details of the detection procedure according to the invention
are indicated below. The found analytic procedure for infestation of housedust
with allergen-containing residues from housedust mites is capable of a multipli-
city of embodiments. This applies not only to the sampling of the dust, but also
to the mixing of the dust with the extraction solution and to the course of
the color reaction.
With the aim of particularly efficient collection of dust, a filter
(in particular a fabric or paper filter) can be placed in the suction tube of
a conventional vacuum cleaner, for example in the form of a small cloth (in
particular of a handkerchief). This has the advantage that the fine dust is
specifically and concentratedly collected. During the sampling itself, account
has to be taken of the fact that floors which are frequently vacuum-cleaned may
possibly no longer contain a sufficient amount of dust for the detection; in
this case, the vacuum-cleaning is advan~ageously carried out at less accessible
places. The dust collection is carried out particularly advantageously by
vacuum cleaning upholstered furniture and beds.
Particularly suitable for the extraction of the dust samples are small,
closable glass and plactic containers having a capacity of a few milliliters
(for example 1 to 10 milliliters), which are provided with gradations for mea-
suring the volume of the dust sample and the extraction solution. However, in
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specific cases, it is also possible for the extraction of the dust sample to
be carried out on, for example, a watchglass.
Using a detection paper, for example according to Patent Application
Serial No. 471,104, it is possible to establish, in a preliminary test on a
sample of the resulting extract, the color intensity which is to be expected.
The preferred method of using a detection paper takes the form of treating a
paper strip, for example filter paper, with the aromatic diazo compound (for
example with about 5% by weight of diazo compound relative to the paper) and of
immersing the paper strip thus treated directly in the supernatant extraction
solution, whereupon the color reaction takes place on the paper. The use is
comparable with that of pH indicator paper. Under defined detection conditions,
the color developed can be compared with a color scale provided. Then, by means
of a comparison of this type with a standardized color scale, conclusions can be
drawn about the intensity of housedust mite residues in the housedust.
In the case of the procedure mentioned, that is to say the treatment
of a paper strip with the aromatic diazo compound, it has proved to be particu-
larly advantageous to apply the aromatic diazo compound together with starch
(amylum) to the paper strip. By this means, it is possible to achieve in the
color reaction an even more uniform coloration on the paper strip treated in this
manner. The amount of starch used together with the aromatic diazo compound
can vary; however, it is generally about 50% by weight of the amount of the
aromatic diazo compound. In this connection, the applicant has established, in
a surprising manner, that a paper strip treated with the aromatic diazo compound
(for example a paper strip treated with about 50 mg of diazosulfanilic acid per
g of paper, and with about 25 mg of starch per g of paper) can be stored stable,
without hazard and without any risk over a prolonged period (for several months);
in view of the known properties of aromatic diazo compounds, this is ~horoughly
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unexpected.
Preferably the amount of the solution is restricted to a minimum in
the interest of the subsequent color development. The alkalinity of the
strongly alkaline extract is subsequently greatly reduced by dilution with water,
so that the measurement is conducted under improved conditions for formation of
the azo dyestuff which is the product of the colour reaction. Sodium carbonate,
preferably in excess can be added to optimize the formation of the azo dyestuff.
Depending on the intensity, an aliquot of between 1 and 10 ml of the
extract is removed, and sodium carbonate solution, and then a suitable aroma-
tic diazo compound, for example diazosulfanilic acid, are added.
After about 20 minutes for color development, an aqueous-alcoholic
solvent mixture is added, and the colored solution thus obtained is measured in
a spectrophotometer with a blank sample as reference.
The results of measurement are evaluated using a guanine calibration
curve. Preferably the curve has been obtained by dissolving defined amounts of
guanine in the manner carried out according to the invention for the treatment
of the dust sample, and carrying out the color reaction of these calibration
solutions, likewise in the manner described according to the invention for the
extract obtained from the dust samples.
The detailed designing of the procedure described here can be carried
out by any specialist analyst concerned with it. The following way is mentioned,
merely as an example, without restricting the invention:
Example
1. Amount weighed
Amount weighed: 250 mg of allergen-containing housedust.
2. Cleaning
The dust sample is digested with 5 ml "Solution I" ~see below), which has
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previously been heated to 60C, in a 10 ml capacity centrifuge tube. After
centrifugation and decanting off, the process is repeated.
3. Extraction
The precleaned dust sample which is still moist is mixed with 3 ml
"Solution II" ~see below) in a centrifuge tube, and is then centrifuged.
Rinsing is carried out with 3 ml "Solution I".
4. Color development
The detection solution thus obtained, including the wash solution, is filter-
ed and made up to 100 ml with distilled water in a graduated flask. 1 to 10
ml of this solution (depending on the color intensity which is to be expect-
ed on the basis of the preliminary test) is pipetted into a 25 ml graduated
flask, 3 ml of 20% strength sodium carbonate solution and 2 ml of 0.05%
strength diazosulfanilic acid solution are added. After 20 minutes, 10 ml
of "Solution I" are added, and the mixture is then made up to the mark with
distilled water.
5. Color measurement
The measurement is carried out at 490 nanometers with a blank sample as
reference. Evaluation is carried out using a calibration curve with guanine.
(For this purpose, various solutions of guanine (in "Solution II"), con-
taining between 20JUg and 100Jug, are prepared in several 25 ml graduated
flasks. The color development is carried out in accordance with the method
indicated in section 4 for the dust sample~.
Solution I: 20 parts by volume of methanol and 100 parts by volume of
water;
Solution II: 1.9 g of KOH in 200 ml of "Solution I".
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