U.S. patent number 3,917,452 [Application Number 05/526,661] was granted by the patent office on 1975-11-04 for diagnostic agent for the detection of peroxidatively active substances.
This patent grant is currently assigned to Boehringer Mannheim GmbH. Invention is credited to Werner Guthlein, Hans-Georg Rey, Peter Rieckmann, Walter Rittersdorf.
United States Patent |
3,917,452 |
Rittersdorf , et
al. |
November 4, 1975 |
Diagnostic agent for the detection of peroxidatively active
substances
Abstract
Test strips for the detection of peroxidatively active
substances in body fluids, e.g., for the detection of blood in
urine, comprising a carrier containing A. a hydroperoxide; B. at
least one chromogen; and C. as an activator a compound of the
formula: ##SPC1## Wherein R.sub.1 is pyridyl or phenyl, optionally
substituted by lower alkyl or alkoxy, and R.sub.2 is hydrogen or
lower alkyl.
Inventors: |
Rittersdorf; Walter
(Mannheim-Waldhof, DT), Guthlein; Werner
(Mannheim-Neckarau, DT), Rey; Hans-Georg
(Mannheim-Waldhof, DT), Rieckmann; Peter
(Mannheim-Waldhof, DT) |
Assignee: |
Boehringer Mannheim GmbH
(Mannheim, DT)
|
Family
ID: |
5901291 |
Appl.
No.: |
05/526,661 |
Filed: |
November 25, 1974 |
Foreign Application Priority Data
|
|
|
|
|
Dec 20, 1973 [DT] |
|
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2363344 |
|
Current U.S.
Class: |
436/66; 435/805;
435/28; 436/904; 422/420 |
Current CPC
Class: |
C12Q
1/28 (20130101); C12Q 2326/00 (20130101); Y10S
436/904 (20130101); Y10S 435/805 (20130101) |
Current International
Class: |
C12Q
1/28 (20060101); C07D 213/24 (); G01N 031/22 ();
G01N 033/16 () |
Field of
Search: |
;23/23B,253TP ;195/103.5
;252/408 ;260/24D,24E |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Reese; Robert M.
Attorney, Agent or Firm: Burgess, Dinklage & Sprung
Claims
What is claimed is:
1. Test strip composition 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: ##SPC3##
wherein
R.sub.1 is pyridyl or a phenyl optionally substituted by lower
alkyl or alkoxy; and
R.sub.2 is hydrogen or lower alkyl.
2. Test strip as claimed in claim 1 and wherein R.sub.1 in the
formula of said activator compound is pyridyl.
3. Test strip as claimed in claim 1 wherein R.sub.1 in the formula
of said activator compound is phenyl.
4. Test strip as claimed in claim 1 wherein R.sub.1 in the formula
of said activator compound is pyridyl substituted by methyl or
methoxy.
5. Test strip as claimed in claim 1 wherein R.sub.1 in the formula
of said activator compound is phenyl substituted by methyl or
methoxy.
6. Test strip as claimed in claim 1 wherein R.sub.2 in the formula
of said activator compound is hydrogen.
7. Test strip as claimed in claim 1 wherein R.sub.2 in the formula
of said activator compound is methyl.
8. Test strip as claimed in claim 1 wherein said activator compound
is 2-(phenylvinyl)-4-methylpyridine.
9. Test strip as claimed in claim 1 wherein said activator compound
is pyridyl-2-pyridyl-4-ethylene.
10. Test strip as claimed in claim 1 wherein said activator
compound is 2-(4-methylphenylvinyl)-pyridine.
11. Test strip as claimed in claim 1 wherein said activator
compound is 2-(4-methoxyphenylvinyl)-pyridine.
12. Test strip as claimed in claim 1 wherein said activator
compound is di-(pyridyl-2)-ethylene.
13. Test strip as claimed in claim 1 wherein said activator
compound is pyridyl-2-pyridyl-3-ethylene.
14. Test strip as claimed in claim 1 wherein said activator
compound is .alpha.-styryl-pyridine.
15. Test strip as claimed in claim 1 wherein said hydroperoxide is
diisopropyl-benzene hydroperoxide or
2,5-dimethylhexane-2,5-dihydroperoxide.
16. Test strip as claimed in claim 1 wherein said indicator is of
the benzidine series or of the heterocyclic azine series.
17. Test strip as claimed in claim 1 wherein said composition also
contains a small amount of a complex forming agent.
18. Test strip as claimed in claim 1 wherein said composition also
contains a thickening agent.
19. Test strip as claimed in claim 1 wherein said composition also
contains a wetting agent.
20. Test strip as claimed in claim 1 comprising
citrate buffer
ethylenediamine-tetraacetic acid, disodium salt
dodecyl-benzene-sulfonic acid sodium salt
2. 5-dimethylhexane-2,5-dihydroperoxide
phosphoric acid trimorpholide
o-tolidine
o-stilbazole.
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 21 wherein the carrier is a
water-stable film with the reagents incorporated therein.
23. Method of detecting a peroxidatively active substance in a body
fluid which method comprises contacting a sample of said body fluid
with a test strip composition as claimed in claim 1 and observing a
color change in said test strip composition as an indication of the
absence or presence of the peroxidatively active substance.
24. Method as claimed in claim 23 wherein said peroxidatively
active substance is blood.
25. Method as claimed in claim 23 wherein said body fluid is urine.
Description
The present invention relates to a test strip for the detection of
very small amounts of blood and of other peroxidatively-active
substances in the body fluids.
The detection of small amounts of blood, which cannot be seen by
the naked eye, in urine, feces and 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
or inflammations in the organs in question. Furthermore, due to the
influence of certain hemolytic toxins, free hemoglobin can also
occur in the urine and plasma. Blood and hemoglobin are
peroxidatively-active, i.e., they liberate oxygen from
hydroperoxides and transfer it to certain acceptors. Myoglobin,
which is also peroxidatively-active, is found, for example, in the
urine after a cardiac infarct. Furthermore, blood occurs especially
frequently in the urine when calculi are present in the bladder or
kidneys.
For a sensitive detection of all of these substances, their
peroxidate action is especially suitable. The oxygen liberated from
a hydroperoxide is thereby transmitted to a chromogen which is
oxidized to a color material and thus indicates the presence of the
peroxidatively-active substances. This reaction has been used for
quite a long time in medicinal and forensic analysis, especially
for the detection of blood. As a rule, the reaction is carried out
in a test tube or as a spot test, hydrogen peroxide usually being
employed as the hydroperoxide. As chromogen, there is usually
employed benzidine, o-tolidine or leucomalachite green.
Rapid test compostions are absorbent carriers, usually papers,
which are impregnated with all the reagents needed for the
detection reaction and which, after simply dipping into the body
fluid to be tested, show a color reaction. Because of the great
importance which these rapid tests have recently achieved, they
have also 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, various attempts have already been
made to increase the sensitivity of the known detection reactions
by means of additives. Thus, for example, German Pat. Spec. 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 by means of these additives but, according to our own
investigations, it is practically impossible, with the so improved
tests strips, to detect, for example, blood in a concentration of 5
erythrocytes /mm.sup.3, which would be necessary for a
differentiation between normal and pathological values.
We have now found that test papers for the detection of
peroxidatively-active substances with a previously unachievably
high sensitivity are obtained by utilizing certain vinylpyridine
compounds as activators.
The vinyl pyridine compounds showing this efficacious action are of
the formula ##SPC2##
wherein
R.sub.1 is pyridyl or phenyl, optionally substituted by lower alkyl
or alkoxy, and
R.sub.2 is hydrogen or lower alkyl.
Preferred compounds of general formula (I) are those in which
R.sub.2 is hydrogen. The lower alkyl and alkoxy radicals can
contain up to 4 carbon atoms, the methyl and methoxy radicals being
preferred.
Thus, according to the present invention, there is provided a test
strip for the detection of peroxidatively-active substances in body
fluids, comprising a carrier containing a hydroperoxide, at least
one chromogen and, as activator, at least one compound of general
formula (I).
Surprisingly, the compounds of general formula (I) do not increase
the sensitivity of peroxidases of vegetable origin, for example
horse radish peroxidase, but act specifically on
perodidatively-active substances of human and animal origin. It is
thus possible selectively to detect blood and blood components in
feces or in vomit in the presence of vegetable peroxidases.
Myoglobin is thereby detected with about the same degree of
sensitivity as, for example, hemoglobin.
The sensitivity of the detection reaction is increased by some of
the compounds of general formula (I) to such an extent that it is
even possible to detect individual erythrocytes in urine, which are
visible on the test paper as colored dots. Thus, for example, with
the use of .alpha.-styryl-pyridine, it is possible to produce a
test paper with which it is possible to detect 5
erythrocytes/mm.sup.3 urine. This corresponds to a blood dilution
of 1:10.sup.6.
Of course, not all of the compounds 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
following compounds in the given order:
2-(phenylvinyl)-4-methylpyridine
Pyridyl-2-pyridyl-4-ethylene
2-(methylphenylvinyl)-pyridine
2-(4-methoxyphenylvinyl)-pyridine
di-(pyridyl-2)-ethylene
pyridyl-2-pyridyl-3-ethylene
.alpha.-styryl-pyridine.
The activating agent used according to the present invention can be
employed in amounts of about 0.05 - 1.0g, preferably of 0.2 - 0.5g,
per 100 ml. of 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 conventional adjuvants.
As hydroperoxides, there can be used, for example,
diisopropyl-benzene hydroperoxide or
2,5-dimethylhexane-2,5-dihydroperoxide, and as indictors, there can
be used, for example, those of the benzidine series, such as
o-tolidine, or those of the heterocylic azine series, such as
bis-(N-ethyl-quinol-2-one)-azine or
(N-methyl-benz-thiazol-2-one)-(1-ethyl-3-phenyl-5-methyltriazol-2-one)-azi
ne (see German Pat. Spec. No. 1,648,840).
The indicator (chromogen) can be used in amounts of from 0.05 - 5
g., preferably of 0.2 - 1.0 g., per 100 ml. 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 5 - 6, is obtained thereon.
It is also advantageous to add to the formulation small amounts
(for example about 0.05 - 0.5 g. per 100 ml.) of a complex-forming
agent, for example, sodium metaphosphate or
ethylenediamine-tetraacetic acid, in order to avoid falsely
positive reactions which could be caused by traces of metals.
Since the test papers can, because of the relatively large amounts
of water-soluble substances present, tend to "bleed", it is
practical to add to the formulation a thickening agent, for
example, methyl cellulose and, in particular, polyvinylpyrrolidone,
preferably in amounts 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-benzenesulphonate, dioctyl sodium sulphosuccinate or sodium
lauryl sulphate, which, as is known, stabilizes radical cations,
such as oxidized o-tolidine. The wetting agent can be added to the
impregnation solution in amounts of from 0.5 - 5%, preferably of 1
- 3%.
For the production of the test strips according to the present
invention, absorbent carriers, for example, filter paper, cellulose
or synthetic fibre fleeces, can be impregnated with solutions of
the reagents in readily volatile liquids. This is preferably
carried out in two or three separate steps. Thus, for example,
impregnation is first 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).
After drying, the test strips according to the present invention
are cut up into strips and preferably sealed between a synthetic
resin film and a fine-mesh material in the manner described in
German Pat. Spec. No. 2,118,455.
For the detection of peroxidatively-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 German Pat. Spec. No. 1,598,153.
This has the advantage that the surface of the test strip can, for
reading off the color reaction, be cleaned by simply wiping it.
The following Examples are given for the purposes of illustrating
the present invention:
EXAMPLE 1:
Filter paper is successively impregnated with the following
solutions and dried at 40.degree.C.:
Solution 1
______________________________________ 1.2 M citrate buffer of pH
5.25 35.0 ml. ethylenediamine-tetraacetic acid, disodium salt 0.1
g. dodecyl-benzene-sulphonic acid sodium salt 0.2 g.
2,5-dimethylhexane-2,5-dihydroperoxide (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.
o-stilbazole 0.2 g. toluene ad 100.0 ml.
______________________________________
A white test paper is obtained which, upon dipping into
blood-containing urine, becomes blue-green colored after about 5 -
20 seconds. If the erythrocytes are intact, then the papers are
blue-green speckled. If hemolysis has taken place or if free
hemoglobin was initially present in the urine, then the papers are
uniformly blue-green colored. The sensitivity is about 5
erythrocytes/mm.sup.3 or the corresponding amount of hemoglobin. A
lower 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.
Example 2:
Filter paper is impregnated with the following solutions and dried
at 40.degree.C.:
Solution 1
______________________________________ 1.2M citrate buffer of pH
5.25 40.0 ml. ethylenediamine-tetraacetic acid disodium salt 0.1 g.
distilled water ad 100.0 ml.
______________________________________
Solution 2
______________________________________ dioctyl-sodium
sulphosuccinate 0.5 g. polyvinylpyrrolidone 0.5 g.
diisopropyl-benzene hydroperoxide (about 50%) 3.5 g. phosphoric
acid trimorpholide 12.7 g. methanol ad 100.0 ml.
______________________________________
Solution 3
______________________________________ o-tolidine 0.25 g. Compound
I 0.25 g. toluene ad 100.0 ml.
______________________________________
Test papers are obtained with practically the same properties as
those described in Example 1. The sensitivety is between about 5
and 20 erythrocytes/mm.sup.3, decreasing with the compound (I) used
in the given order:
Compound I used:
______________________________________ pyridyl-2-pyridyl-3-ethylene
di-(pyridyl-2)-ethylene 2-(4-methoxyphenylvinyl)-pyridine
2-(4-methylphenylvinyl)-pyridine pyridyl-2-pyridyl-4-ethylene
2-(phenylvinyl)-4-methylpyridine.
______________________________________
All the above-mentioned compounds are known from the
literature.
It will be understood that the foregoing specification and examples
are illustrative but not limitative of the present invention
inasmuch as other embodiments within the spirit and scope of the
invention will suggest themselves to those skilled in the art.
* * * * *