U.S. patent number 3,853,472 [Application Number 05/376,248] was granted by the patent office on 1974-12-10 for diagnostic test strip for the detection of components of body fluids.
This patent grant is currently assigned to Boehringer Mannheim GmbH. Invention is credited to Werner Guthlein, Hans-George Rey, Peter Rieckmann, Walter Rittersdorf.
United States Patent |
3,853,472 |
Rittersdorf , et
al. |
December 10, 1974 |
DIAGNOSTIC TEST STRIP FOR THE DETECTION OF COMPONENTS OF BODY
FLUIDS
Abstract
Test strips are provided for detecting even small amounts of
blood or other peroxidatively active substances in body fluids; the
strips comprising a carrier containing a hydroperoxide, a
chromogen, and as an activator, a compound of the formula Wherein
R.sub.1 is a hydrogen atom or a methyl radical and benzene and/or
pyridine rings are fused on at least one of the positions indicated
with c, (d,e), f and g, provided that two adjacent rings must not
simultaneously each contain a cyclic nitrogen atom, and wherein the
aromatic compounds (1), apart from the positions indicated by H and
R.sub.1, can be substituted by lower alkyl radicals which together
can also form a hydroaromatic ring.
Inventors: |
Rittersdorf; Walter
(Mannheim-Waldhof, DT), Rey; Hans-George
(Mannheim-Waldhof, DT), Guthlein; Werner
(Mannheim-Neckarau, DT), Rieckmann; Peter
(Mannheim-Waldhof, DT) |
Assignee: |
Boehringer Mannheim GmbH
(Mannheim, DT)
|
Family
ID: |
5850916 |
Appl.
No.: |
05/376,248 |
Filed: |
July 3, 1973 |
Foreign Application Priority Data
|
|
|
|
|
Jul 18, 1972 [DT] |
|
|
2235152 |
|
Current U.S.
Class: |
436/66; 436/904;
422/420 |
Current CPC
Class: |
C12Q
1/04 (20130101); C12Q 1/28 (20130101); Y10S
436/904 (20130101) |
Current International
Class: |
C12Q
1/04 (20060101); C12Q 1/28 (20060101); G01n
031/22 (); G01n 033/16 (); C07d 033/00 () |
Field of
Search: |
;23/23B,253TP
;252/408,186,4A ;195/13.5R |
References Cited
[Referenced By]
U.S. Patent Documents
|
|
|
3290117 |
December 1966 |
Adams et al. |
3712853 |
January 1973 |
Rittersdorf et al. |
|
Primary Examiner: Wolk; Morris O.
Assistant Examiner: Turk; Arnold
Attorney, Agent or Firm: Burgess, Dinklage & Sprung
Claims
What is claimed is:
1. Test strip for the detection of peroxidatively-active substances
in body fluids, comprising a carrier containing a hydroperoxide, at
least one chromogen and, as an activator, a compound of the formula
##SPC2##
wherein
R.sub.1 is hydrogen or methyl; and
benzene and/or pyridine rings are fused on at least one of the
positions indicated with c, (d,e), f and g, provided that two
adjacent rings must not simultaneously contain a cyclic nitrogen
atom, and
wherein
said compound can be substituted, other than in the positions
indicated by H and R.sub.1, by lower alkyl radicals, which radicals
together can also form a hydroaromatic ring.
2. Test strip as claimed in claim 1, wherein R.sub.1 is formula (I)
is hydrogen.
3. Test strip as claimed in claim 1, wherein R.sub.1 in formula (I)
is methyl.
4. Test strip as claimed in claim 1, wherein said compound contains
a benzene ring fused on at least one of the c, (d,e), f or g
positions.
5. Test strip as claimed in claim 1, wherein said compound contains
a pyridine ring fused on at least one of the f or g positions.
6. Test strip as claimed in claim 1, wherein said compound contains
a benzene ring fused to at least one of the c, (d,e), f or g
positions and a pyridine ring on one of the f and g positions.
7. Test strip as claimed in claim 1, wherein said compound is
substituted in at least one available ring position by a lower
alkyl radical of from one to four carbon atoms.
8. Test strip as claimed in claim 7, wherein there are two such
alkyl radicals linked together to form a 5 or 6-membered
hydroaromatic ring.
9. Test strip as claimed in claim 1, wherein said compound is a
benzo [c] quinoline.
10. Test strip as claimed in claim 1, wherein said compound is a
benzo [f] quinoline.
11. Test strip as claimed in claim 1, wherein said compound is a
benzo [g] quinoline.
12. Test strip as claimed in claim 1, wherein said compound is a
dibenzoquinoline.
13. Test strip as claimed in claim 12, wherein said
dibenzoquinoline is dibenzo [c,f] quinoline.
14. Test strip as claimed in claim 12, wherein said
dibenzoquinoline is dibenzo [c,d,e] quinoline.
15. Test strip as claimed in claim 1, wherein said compound is a
pyridoquinoline.
16. Test strip as claimed in claim 15, wherein said pyridoquinoline
is a pyrido [2,3-f] quinoline.
17. Test strip as claimed in claim 16, wherein said pyridoquinoline
is a pyrido [3,2-f] quinoline.
18. Test strip as claimed in claim 16, wherein said pyridoquinoline
is a pyrido [2,3-g] quinoline.
19. Test strip as claimed in claim 1, wherein said compound is
phenanthridine.
20. Test strip as claimed in claim 1, wherein said compound is
selected from the group consisting of
2-methyl- or 2-ethyl-phenanthridine
benzo [f] quinoline
1,2-tetramethylenebenzo [f] quinoline
benzo [g] quinoline
4-methylbenzo [g] quinoline
dibenzo [c,d,e] quinoline
dibenzo [c,f] quinoline
pyrido [3,2-f] quinoline
3-methyl-4,7-phenanthroline
pyrido [2,3-f] quinoline
3-methylbenzo [f] quinoline
1,3-dimethylbenzo [f] quinoline
2,4-dimethylbenzo [g] quinoline
6-methylphenanthridine
3,8-dimethyl-4,7-phenanthroline
2,8-dimethyl-1,7-phenanthroline, and
2,7-dimethyl-1,6-anthrazoline
21. Test strip as claimed in claim 1, wherein the carrier is an
absorbent material impregnated with the reagents.
22. Test strip as claimed in claim 1, wherein the carrier is a
water-stable film with the reagents incorporated therein.
23. Test strip as claimed in claim 1, wherein the reagent solution
used for the production thereof contains 0.05-1 percent of
activator per 100 ml. of solution.
24. Test strip as claimed in claim 1, wherein the reagent solution
used for the production thereof contains 0.2-0.5 percent of
activator per 100 ml. of solution.
25. Test strip as claimed in claim 1, wherein the reagent solution
used for the production thereof contains 0.05-5 g. of chromogen per
100 ml. of solution.
26. Test strip as claimed in claim 1, wherein the reagent solution
used for the production thereof contains 0.2-1.0 g. of chromogen
per 100 ml. of solution.
27. Test strip as claimed in claim 1, wherein the reagent solution
used for the production thereof contains a buffer and or a complex
former, a thickener or a wetting agent.
28. Method of detecting small amounts of blood in a body fluid
which method comprises contacting a test sample of the body fluid
with a test strip as claimed in claim 1 and observing the color
formation thereon as an indication of the absence or presence of
blood in said saple.
29. Method of detecting small amounts of peroxidatively active
substances in a body fluid which method comprises contacting a test
sample of the body fluid with a test strip as claimed in claim 1
and observing the color formation thereon as an indication of the
absence or presence of peroxidatively active substances in said
sample.
Description
The present invention is concerned with a test strip for the
sensitive and rapid detection of very small amounts of blood and of
other peroxidate-active substances in body fluids.
The detection of small amounts of blood, which are invisible to the
naked eye, in urine, feces or vomit is very important for the
diagnosis of hemorrhages in the stomach, intestines and urinary
tract. Such hemorrhages are caused, for example, by tumors, ulcers
and inflammations of the corresponding organs. Furthermore, free
hemoglobin can also occur in the urine and plasma due to the
influence of certain hemolytic toxins in the blood. Blood and
hemoglobin are peroxidate-active, i.e., they liberate oxygen from
hydroperoxides and transfer it to certain acceptors. Other
peroxidate-active, substances occur in leukocytes and bacteria. The
detection of these substances is important for the diagnosis of
diseases and infections of the kidney and urinary tract. Myoglobin,
which is also perioxidate-active, is found in the urine, for
example, after a cardiac infarct. Furthermore, blood occurs
especially frequently in the urine when calculi are present in the
bladder or kidneys.
Their peroxidate action is especially suitable for a sensitive
detection of all of these substances. The oxygen liberated from a
hydroperoxide is hereby transferred to a chromogen which is
oxidized to a colored substance and thus the presence of the
perioxidate-active substance is indicated. This reaction has been
used for quite a long time in medicinal and forensic analysis,
especially for the detection of blood. The reaction is, as a rule,
carried out in a test tube or as a spot test, hydrogen peroxide
usually being employed as the hydroperoxide. As chromogen, there is
preferably used benzidine, o-tolidine or leuko malachite green.
Rapid tests are usually absorbent carriers, preferably papers,
which have been impregnated with all of the reagents necessary for
the detection reaction. After simply dipping them into a body
fluid, they show a color reaction. Because of the great importance
which rapid tests have recently achieved, they have been developed
in various ways for the detection of blood in body fluids.
Since, for the detection of blood, the sensitivity of the rapid
test is of decisive importance and, furthermore, it is also
desirable, if possible, to include leukocytes and bacteria which
are less peroxidate-active, various attempts have already been made
to increase the sensitivity of the known detection reactions by
means of additives. Thus, for example, German Pat. No. 1,242,905
describes test papers which, as activating additives, contain
certain quinoline derivatives, preferably quinine. It is certainly
asserted that the sensitivity can be considerably increased with
the mentioned additives but, acccording to our own investigations,
it is practically impossible also to detect leukocytes and bacteria
with the test strips improved in this manner.
The activating action of quinoline per se on the blood detection
reaction has been known for a long time (see Zeitschrift f.
gerichtl. Med., 12, 216/1928); however, this, as well as its simple
derivatives, are liquid or volatile and, therefore, cannot be used
for a rapid test.
The instant invention provides test strips for the detection of
peroxidate-active substances with a hitherto unachievably high
sensitivity.
Essentially, the present invention comprises test strips comprising
as the activator, an aromatic compound of the general formula:
##SPC1##
wherein R.sub.1 is a hydrogen atom or a methyl radical and benzene
and/or pyridine rings are fused on at least one of the positions
indicated with c, (d,e), f and g, provided that two adjacent rings
must not simultaneously each contain a cyclic nitrogen atom, and
wherein the aromatic compounds (1), apart from the positions
indicated by H and R.sub.1, can be substituted by lower alkyl
radicals which together can also form a hydroaromatic ring.
Those compounds of general formula (1) are preferred in which
R.sub.1 is a hydrogen atom. By lower alkyl radicals, there are to
be understood alkyl radicals containing up to four carbon atoms
and, when two alkyl radicals together form a hydroaromatic ring, 5-
and 6-membered rings are preferred.
In the following, there are given examples of the most important
groups of compounds coming within the scope of general formula (1);
the individual compounds are all known from the literature:
1. Benzoquinolines
1.1 benzo [c] quinoline (phenanthridine)
2-methylphenanthridine
6-methylphenanthridine
2-ethylphenanthridine
1.2 benzo [f] quinoline
3-methylbenzo [f] quinoline
1,3-dimethylbenzo [f] quinoline
1,2-tetramethylenebenzo [f] quinoline
1,2-trimethylenebenzo [f] quinoline
1.3 benzo [g] quinoline
4-methylbenzo [g] quinoline
2,4-dimethylbenzo [g] quinoline
2. Dibenzoquinolines.
2.1 dibenzo [c,f] quinoline (benzo [a] phenanthridine)
2.2 dibenzo [c,d,e] quinoline (4-azapyrene, thebenidine)
3. Pyridoquinolines
3.1 pyrido [2,3-f] quinoline (1,7-phenanthroline)
2-methyl-1, 7-phenanthroline
2,8-dimethyl-1,7-phenanthroline
3.2 pyrido [3,2-f] quinoline (4,7-phenanthroline)
3-methyl-4,7-phenanthroline
3,8-dimethyl-4,7-phenanthroline
1,3,4,8-tetramethyl-4,7-phenanthroline
3.3 pyrido [2,3-f] quinoline (1,6-anthrazoline)
2,7-dimethyl-1,6-anthrazoline
Consequently, according to the present invention, there is provided
a test strip for the detection of peroxidate-active substances in
body fluids, comprising a carrier containing a hydroperoxide, at
least one chromogen and, as activator, a compound of general
formula (I).
It was not to have been foreseen that the modification according to
the present invention, of the known activator quinoline would lead
to such an extraordinary increase in effectiveness. It is important
for the present invention that only anelation of certain positions
of the quinoline system enables an increase of activity to be
achieved. Thus, for example, in the case of anelation of the [b]-
and [h]-positions of the quinoline, i.e., in the case of acridine
or of benzo [h] quinoline, the activity is reduced in comparison
with quinoline. The b and h positions being the next logical sides
of the compound of general formula (I).
The effectiveness of the compounds used as activators according to
the present invention is difficult to explain; if, as might be
regarded as being obvious, it were due to complex formation with
the peroxidate-active substances, then, for example,
o-phenanthroline, which is known to be a strong complex former,
should also have a strong activating effect. However, this is not
the case, whereas m- and p-phenanthroline are very effective
activators.
Surprisingly, the compounds of general formula (I) do not increase
the sensitivity of peroxidases of vegetable origin, for example
horseradish peroxidase, but act specifically on peroxidate-active
substances of human and animal origin. It is thus possible
selectively to detect leukocytes, blood and blood components and
bacteria in feces or in vomit in the presence of vegetable
peroxidases. In this case, myoglobin is detected with about the
same degree of sensitivity as hemoglobin.
The sensitivity of the detection reaction is increased by some of
the compounds of general formula (I) to such an extent that even in
urine it is possible to detect individual erythrocytes which are
visible on the test paper as colored dots. Thus, for example, by
means of phenanthridine, it is possible to produce a test paper
with which it is still possible clearly to detect 5 erythrocytes
per mm.sup.3 urine. This corresponds to a blood dilution of
1:1,000,000.
It is, of course, obvious that not all of the compounds coming
within the scope of general formula (I) possess activating
properties of the same degree. Thus, it is possible to adjust the
sensitivity of, for example, a blood test in accordance with
practical requirements. For example, test strips of increasing
activity are obtained when, as activator, there are used the
compounds set out in the following and in the given order:
m-phenanthroline<p-phenanthroline<benzo-(f)quinoline<phenanthridine.
The sensitivity can be further modified by alkyl substitution;
thus, for example, by means of a methyl substituent in a
.alpha.-position to a cyclic nitrogen atom, the activity is
somewhat reduced, whereas otherwise it usually leads to an increase
of the activation.
Thus, for example, the sensitivity limit of a test strip which
contains a weak activator, for example 2,
8-dimethyl-1,7-phenanthroline, is about 50 erythrocytes/mm.sup.3
urine.
The activation agents according to the present invention can be
used in amounts of about 0.05-1.0 percent, preferably of 0.2-0.5
percent, per 100 ml. impregnation solution.
Further components of a rapid test for blood are an organic
hydroperoxide, an oxidation indicator (chromogen), a buffer and a
surface-active agent, as well as, if desired, a phosphoramide, for
example phosphoric acid trimorpholide, for stabilization, as well
as other conventional adjuvants.
As hydroperoxides, there can be used, for example, cumol
hydroperoxide or 2,5-dimethyl-hexane-2,5-dihydroperoxide, and as
indicators those of the benzidine series, for example, o-tolidine,
or those of the heterocyclic azines series, for example
bis-(N-ethyl-quinol-2-one)-azine or
(N-methylbenzthiazol-2-one)-(1-ethyl-3-phenyl-5-methyltriazol-2-one)-azine
(see German Pat. No. 1,648,840).
The indicators can be used in amounts of from 0.05-5 g., preferably
of 0.2-1.0 g., per 100 ml. of impregnation solution.
As buffer, there can be used, for example, a citrate, phosphate,
phthalate or succinate buffer, the pH value and capacity being so
chosen that, after dipping the test strip into a body fluid, a pH
value of 4-7, preferably of 5-6, is obtained thereon.
It is also advantageous to add to the formulation small amounts of
about 0.05-0.5 g. per 100 ml. of a complex former, for example
sodium metaphosphate or ethylene-diamine-tetraacetic acid, falsely
positive reactions, which could be due to traces of metals, thereby
being avoided.
Since the test strips, due to the relatively large amounts of
water-soluble substances present therein, could tend to bleed, it
is of practical importance to add a thickening agent to the
formulation, for example methyl cellulose and, in particular,
gelatine, preferably in an amount of about 0.5-5 g. per 100 ml.
As wetting agent, there is preferably used a long-chained organic
sulphate or sulphonate, for example sodium dodecyl-benzene
sulphonate, dioctyl sodium sulphosuccinate or sodium lauryl
sulphate, which, as is known, stabilizes radical cations, such as
oxidized o-tolidine. The wetting agents can be added to the
impregnation solution in amounts of 0.5 to 5 percent, preferably of
1 - 3 percent.
For the production of the test strips according to the present
invention, absorbent carriers, for example filter paper, cellulose
or synthetic resin fleeces, can be impregnated with solutions of
the reagents in readily volatile solvents. This is preferably
carried out in two separate steps. First, impregnation is carried
out with a solution which contains a hydroperoxide, wetting agent,
buffer and optionally a thickening agent. Thereafter, impregnation
is carried out with a solution of an indicator and of an activator
of general formula (I).
The test strips according to the present invention are, after
drying, cut up into strips and preferably sealed between a
synthetic resin film and a fine-meshed material in the manner
described in German Pat. No. 2,118,455.
For the detection of peroxidate-active substances in feces, it is
also possible to incorporate the activators according to the
present invention, together with the reagents, in a water-stable
film in the manner described in U.S. Pat. No. 3,630,957. This has
the advantage that the surface of the test strip can, for reading
off the color reaction, be cleaned simply by wiping it.
The following Examples are given for the purpose of illustrating
the present invention:
EXAMPLE 1
Filter paper is successively impregnated with the following
solutions and dried at 40.degree.C.:
Solution 1 1.2M citrate buffer, pH 5.25 35.0 ml.
ethylenediamine-tetraacetic acid, disodium salt 0.1 g. dioctyl
sodium sulphosuccinate 2.0 g. 2,5-dimethylhexane-2,5-dihydro-
peroxide (about 70%) 1.6 g. phosphoric acid trimorpholide 12.7 g.
ethanol 30.0 ml. distilled water ad 100.0 ml. Solution 2 o-tolidine
0.3 g. phenanthridine 0.2 g. toluene ad 100.0 ml.
A white test paper is obtained which, upon dipping into a
blood-containing urine, becomes green colored after about 5 to 20
seconds. If the erythrocytes are intact, then the paper is green
flecked. If hemolysis has taken place or if free hemoglobin is
present in the urine, then the paper becomes uniformly green
colored. The sensitivity is about 5 erythrocytes/mm.sup.3 or the
corresponding amount of hemoglobin. A smaller number of intact
erythrocytes can, under certain circumstances, still bring about
individual green dots on the test paper. The sensitivity with
regard to myoglobin corresponds to that for hemoglobin.
Leukocytes and bacteria are also detected when intact, by flecking,
or when lysed, by a uniform coloration.
EXAMPLE 2
When, in Solution 1 of Example 1, instead of
2.5-dimethyl-hexane-2,5-dihydroperoxide, there is used an equimolar
amount of diisopropyl-benzene hydroperoxide and the phenanthridine
in Solution 2 is replaced by one activator listed hereinafter, then
test papers are obtained, the sensitivity of which towards blood,
leukocytes and bacteria, is of approximately the same order as the
test paper of Example 1:
2-methyl- or 2-ethyl-phenanthridine; benzo [f] quinoline;
1,2-tetramethylenebenzo [f] quinoline; benzo [g] quinoline;
4-methyl-benzo [g] quinoline; dibenzo [c,d,e] quinoline; dibenzo
[c,f] quinoline; pyrido [3,2-f] quinoline;
3-methyl-4,7-phenanthroline; or pyrido [2,3-f] quinoline.
EXAMPLE 3
Filter paper is impregnated with the following solutions and dried
at 40.degree.C.:
Solution 1 1.2M nitrate buffer, pH 5.25 40.0 ml.
ethylenediamine-tetraacetic acid, disodium salt 0.1 g. dioctyl
sodium sulphosuccinate 2.0 g. gelatine 2.0 g.
2,5-dimethylhexane-2,5-dihydro- peroxide (about 70%) 1.6 g. ethanol
30.0 ml. distilled water ad 100.0 ml. Solution 2 o-tolidine 0.3 g.
3-methylbenzo [f] quinoline 0.2 g. toluene ad 100.0 ml.
The test paper obtained is about ten times less sensitive than the
test papers according to Examples 1 and 2 (about 50-100
erythrocytes/mm.sup.3 in 30-60 seconds).
Test papers of similar sensitivity are obtained when, instead of
3-methylbenzo [f] quinoline, there is used an equimolar amount of
one of the following activators: 1,3-dimethylbenzo [f] quinoline;
2,4-dimethylbenzo [g] quinoline; 6-methylphenanthridine;
3,8-dimethyl-4,7-phenanthroline; 2,8-dimethyl-1,7-phenanthroline;
or 2,7-dimethyl-1,6-anthrazoline.
It will be understood that the specification and examples are
illustrative but not limitative of the present invention and that
other embodiments within the spirit and scope of the invention will
suggest themselves to those skilled in the art.
* * * * *