JPS59193354A - Composition for detecting peroxide active substance, test means and manufacture of said test means and method of detecting peroxide active substance - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION BACKGROUND OF THE INVENTION Part 4 of the Invention The present invention is useful in general for analytical measurements of peroxide active substances in a sample, and in particular for such measurements, which may be present in a sample. The present invention relates to compositions, test means, test devices and test methods that are resistant to harmful effects from ascorbic acid.

A number of analytical methods are currently available to detect the presence of peroxide-active substances in biological samples such as urine, fecal suspensions and gastrointestinal contents. For example, hemoglobin and its derivatives, the analytes measured by the occult blood test, are representative of peroxide-active substances, as they behave similarly to peroxidase enzymes, and are themselves also referred to as pseudoperoxidases. be exposed. Peroxide-active substances are peroxides or hydroperoxides and indicators that produce a detectable response such as a color change, such as benzinone, o
-) It is similar to an enzyme in that it catalyzes a redox reaction between lysine, 3.3', 5.5'-Te1-lamedelbenzidine, 2,7-diamitufluorene, etc. Therefore, most methods for determining the presence of occult blood in a sample rely on this pseudoveroxidase activity.

Depends on the enzyme-like catalysis of peroxide oxidation by colored Fti indicators.
．． A number of analytical powers θ have been developed to determine peroxide active substances. Firstly, these involve wet chemical or dissolution operations and the so-called dip-reading.
rl-r(!yul) Contains a J-type reagent holding strip device. A typical example of the former is Henry (R,L 1len
ry) et al. Clinical Gemistry Principles Anne I Techniques (C1inical Che)
MistryPrinciples and Tech
niques), 2nd edition 1124-1125 (
Burgerstown, Maryland: 1larp (!r
and Rosy, 1974).

Examples of this operation are glacial acetic acid (buffer), diphenylamine (
indicator) and hydrogen peroxide. Although such wet chemistry methods have proven useful for analysis, these methods have a number of drawbacks, two of which are lack of reagent stability and inadequate sensitivity. There is a drawback.

Another method of measuring peroxide actives, currently preferred by many clinical analysts, utilizes so-called "dip-read" reagent strip devices. Typical reading tools include Miles Laboratories + Inc.
)'s Ames Divison
ion) to trademark Mastics (HEMASTI)
It is commercially available under the trademark X). This test device consists of porous paper 7
It consists of trisox impregnated with a buffered mixture of organic hydroperoxide and indicator and fixed to a plastic strip or handle. When this matrix is immersed in a liquid containing hemoglobin, myoglobin, red blood cells or other peroxide-active substances, i.e. pseudoperoxidase, a blue color appears in the 7-trinox, the intensity of which depends on the concentration of the substance in the sample. Proportional. By comparing the color that appears with a standard color chart, the analyst can semi-quantitatively determine the 7t of the analyte present in the sample.

First, the advantages of such a reagent strip compared to wet chemistry are: 1) Slip type is easy to use;
No reagent production or associated equipment is required; and 2) the reagents are more stable in the strip, improving viscosity, sensitivity, and economy.

Although specific analyzes for peroxide active species have been performed in either of the above methods, there are inherent problems with both. That is,
Interference is caused by the presence of reducing agents in general and ascorbic acid or ascorbate ions in particular in the sample (hereinafter referred to as
For example, in the case of urine analysis, the recent popularity of diets containing high doses of vitamin C (ascorbic acid) has made it difficult to detect certain urine components, such as occult blood. Serious death. -1 without causing the problem of Luheto interference.

[11] Also in 1938, ascorbate-like 1
7 The harmful effects of the drug were recognized. Corn (R.

Kohn) and Watrous (R,M)
), Journal of Biological Chemistry (formerly o1ologic)
al Chemistry), I24:163
~・168 (1938). When performing an occult blood (pseudoperoxidase) analysis, an ascorbate 1-analysis is also performed at the same time to improve the accuracy of the occult blood measurement.
The same problem still plagues this field of diagnostic analysis, as evidenced by the 1979 proposal that it is transparent. Nielsen (1,.

N1clSen), Jörkensen (P,, 1. Jo
rgensen) and Hansen (8, C, trans
en) Author, Ugeskrift for L ae
H(!r, 141, 791-793
(1979).

A test system, for example a system containing a glucose-sensitive reagent.
Numerous attempts have been reported in the literature to remove the 1-interference to Ascol. Regarding the glucose sensitivity analysis,
From the method of filtering off ascorbate before it reaches the ascorbate reagent, 1 minute of ascorbate can be removed on the spot.
? There are various approaches including methods that utilize 1r enzymes.

Dahlqvist's Canadian special '1 No. 844-56.
Specification No. 4 discloses a measuring device for measuring glucose in urine or other media, which has a typical glucose response (in addition to a porous part impregnated with a reagent, urine/sample Accept (= J addition part included. Sample acceptance f93 minutes is the only one to cut the measurement 5jl tool G to the ascol present in the urine sample - [to be absorbed) It is an ion exchanger ♂ (including '1).

American 9' as learned by Danninger and others! No. 4, No. 168.205 Mei 1. ■The first book is
Where to find test reagents! It is suggested that ascol tooxidase, which is one of the most important ingredients, be added. In this case, the ascorbate present in the sample O;, 1 is enzymatically oxidized by ascorbate oxidase +1, and is a compound that does not adversely affect the desired analysis. ＼-1 by 4 t7.

Another approach for dividing the death-1-lethal-interference by 1:1 is described in Japanese Patent Application Laid-open No. 58-55757 (1987) by Fuji Kinkiyaku Co., Ltd. In this publication, various It is disclosed that samples are pretreated using Gantts, e.g. metal gylates of ethylenediaminetetraf!it-acid and diethylenetriminepentaacetic acid, and then analyzed for cholesterol, glucose or other components, e.g. uric acid. There is.

Ku, U.S. Pat. No. 3,41L,887, discloses that ``Ascorbe-1. 1. It is stated that interference should be eliminated.

The ``rl-wrapping system'' relates to ionizable heavy metal mud compounds that, when ionized, drop the oxidation series photopotential between the redox indicator dye and Ascol. Suitable metals are, for example, ':1Hart, iron, mercury and nickel.

No. 4,288,541, duly assigned to Magers et al.
114 mercury ions to impart Ascorbe I resistance to the glucose/glucose oxidase analysis system;
For example, the use of mercuric sarcosinate is described.

In addition to the above-mentioned literature, Ascol-1 associated with glucose testing
The haste regarding the problem is described in the following documents: 1.1.
．． Amer.

Med, 85soc, 178,
I, 19-150 (1961).

2. O'Gorman (P, O'Gorman) et al., B
r i t.

Med,,1. ．． 603-(i06 (1,960)
.

3. Brandt (R, ) et al.
Cl1n, Chcm.

8c1. ), 51, 103-104 (1
974).

4, Funonh (R, 11rantH) Ca, 8m
, J., Cl1n.

l'aLIlol, 68.592~594 (1,9
77).

Similar to Ku's '1 approach mentioned above, other papers use cobalt to make ascorbate 171,
It's oxidized. For example, Burakagnoro (G.

Br(1Bagnolo) i=8nn, C
him, 8pplicL1ta 3 1. 3
50-368 (1941) No. J, As: Z A solution of lupic acid is oxidized in air in the presence of metallic cobalt (
Kokichi U7. Also, Journal of the Chemical
Soliie Uichii Off Japan (Jo former τ1.) 1
of the Cbcm1cat 5ociety o
f, Iapan:), 53, 820-826 (
1942) and Miyazaki Tomokichi Nyott 1Zco (N113)
) 6 Similar activity was reported for C13.

The above-mentioned literature provides extensive coverage of analytical systems for measuring glucose;
No suggestion has been made to solve the problem of As=Zruhe1-interference associated with the measurement of n (-\MokuL1hin). Despite the disclosure in the above-mentioned Koo patent, the literature indicates that metal ions such as Co(Ill) are in fact pseudoperoxidases. For example, Ilft
No. 1 is used commercially to decompose cumene hydroperoxide using a catalyst.

[The Merck Index
Index), 9th edition 311 (1976). )
Low (K,[,ohs,),Monatsber,
■) eut, 81 (ad, Wiss, Be
rlin, 8. 657-659 (196
6) (Chem, Abstracts.

67.120383z, 1967) have reported a series of Go(In) complexes for catalytic decomposition of peroxides. Therefore, measuring peroxide active substances)
The use of one such metal layer in a 111-type analytical preparation, i.e., an analytical preparation containing an oxide and an indicator, can lead to a self-harmful reaction with human peroxide. 1-pseudointestinal" result, or υ
, 1 problem of peroxide 411 substance? 'j, analogy (3 (along 1)
It is clear that a person skilled in the art would think that 1114=Z reacts]A', 2, 7, and therefore cannot be used for such measurements. In fact, occult blood 81 (
Efforts to use one mercury agent, for example mercury sulfuric acid, in the experiment.

jJ-J:lu M xiti? ) fA Merkhall I-(Burkhard I:
) et al rice Fl'(] 't!+H No. 4,310,62
No. 6 details 1'! No. 1 describes the use of ammonium Co (In ) InF in order to reduce the interference of Asml Luhe I with compositions for measuring peroxide active substances in relation to the above-mentioned problem. The q, prefix 1 is an organic hydroperoxide and a suitable indicator, such as 3,3'',
5.5'-tetramethylhensidine with ammonium C
o (iTl) 1iij body, such as especially C:o (
Discloses a composition comprising Nci) e C10. However, these complexes did not provide sufficient resistance to Ascol-1 to be industrially advantageous for occult blood testing.

To Ascol in the analysis and measurement of peroxide active substances-1.
Other approaches to interference are found, for example, in German Patent No. 2,907,628. This German patent relates to the analysis of urine in solution, in which a urine sample is pretreated with one or more oxidizing agents to remove it to Ascor and then contacted with a suitable analytical reagent.

The oxidizing agents disclosed are iodine oxide, sulfurium periodate, calcium hypochlorite, potassium triiodide, sulfurium hypochlorite, chloroamine, and fromosuccinimide. be.

In summary, various approaches to eliminate the interference problem caused by ascorhinic acid in the determination of peroxide active substances include the use of various Co(III) ammonium complexes,
Techniques included pretreatment of samples with oxidizing agents and direct addition of alkali metal iodates to the reagent composition.

To elucidate the mechanism of action of natural peroxidases, such as peroxidase obtained from horseradish or potato, pseudoperoxidases, such as hemoglobin, are often studied as other peroxidase systems. Ascorbic acid has long been known as a typical substrate for peroxidases, and ascorbic acid 1T nelation in the presence of metal chelates that act as pseudoperoxidases is a known phenomenon. 1967 and 1! + 68 <[ni, Khano (M, Khan
) and martyl (to, tlarLcll) are 1.
reported on the kinetic study of the oxidation of az:1 ruhinic acid in the presence of several iron chelates and copper chelates over a pH range of 8 to 3.45 [Kano and Martill, J. , Am, Chem, Soc, , 89
, 4176 (1967); J, Hachim,
Chem, Soc,, 81), 7104 (19
67) ;J, Am, Chem, Soc,, 9
0.3386 (1968). ) In these studies (::2) a large number of kinetic and thermodynamic parameters were studied. The results were the llli position of various chelates depending on their ability to oxidize ascorhinic acid.

Of the four aminobolycarboxylic acids studied by these authors, Fe + Pu's N - < 2 1-1" ml-door, Ni"-!・) Engineering Narency〒′ζ゛22! lIl
+; #, (n E +)-1゛8) Chelate? ii
Q is also a rapid oxidizing agent.
o-1・””-”i”le research and cleaning/l'
(Grinst, cad) Otokoyomo Saraotoko early opening (”
The store j1 study is ``Motel:・Maokiu・Gu upper thread composing the thread, -as】Lu Hin, T, 12 M Kakatsu 1
Thinking about life (Haru [Grinste, Fl, 1. Hat n,
ChemSac, 82.3464 (1960)
3. From the above-mentioned study by Grinste et. Attempts were made to study the Bar No. 42 Cedase Mecha J. Sum. In fact, his author repeatedly uses the term "Motel Bar Oxycuse System-1" in his paper. However, this beautiful
By other similar substances in I and reactivity Q [-
Module 1 Bar A: Since t'sidase activity is indicated, such materials are recommended for use in hydroperoxide/oxygen systems, e.g. for the determination of peroxidase or other peroxide-active substances. Analytical Reagent Composition and Measurement I7 Currently used ζ;
1.(I', I'm expecting a crab./)I'I'd like to.Furthermore, regarding the activity of B.
From the IJI research carried out by the assignee of the invention, it has been found that i[T unstained, e.g. −Ri℃・n(
i9. ) Detection of the presence of oxide active substances using an analytical system (62 typically used, 5 fingers unstained), (11th, 1: -j' , Zl /[/
If' 7 ji and about 2oO times 'Tj < t, y,
', 11, wake up: -J' It turns out that K- and U7 are about to be born. Second, such a peroxide active metal layer 1.
When ter-7 readily oxidizes asnylic acid (to,
(Assumptions made by Kerr 7, Martyr and Cleanse j2 = l), , I'MH, l, - peroxyter--1: reaction with 20 i, ] double culm 1 [
J＜Even if not (K after armpits are done) (7)
p), at least as
Proceeds at the same speed as Vj. Obviously, very reaction I1
When an analyte of 1 is mixed into a true reagent product of 1 which changes color due to the presence of the analyte, a result of 1-false μj (+11) will be obtained. Despite the foregoing teachings and suggestions, it is anticipated that certain peroxide-active metal compounds, and in particular of polycarboxyalkylamines, may be used in the process of the present invention. not only does it not produce the expected "false positive" results in compositions containing reagent systems that measure peroxide actives, but it also avoids actually measuring the analyte in such systems. It is surprisingly advantageous in terms of reliability, stability and sensitivity.

Furthermore, it has been demonstrated that the use of metal chelates according to the invention is particularly advantageous in overcoming inaccuracies caused by interference, particularly from ascorbate ions present in the sample.

The present invention is therefore based on this discovery and, as mentioned above, provides a method for the analytical determination of peroxide-active substances that are generally resistant to Ascol interference and, in particular, to organic hydrocarbons according to the present invention. Peroxide and redox indicators, such as 〇-tolidine or 3゜3', 5. 5'-tel~ramethylhensidine and peroxide 71'i +1
The present invention relates to a detection method that utilizes a composition containing a 1-metal complex. For such closures, the peroxide profile11I analyte is similar to the enzyme peroxidase, so
Catalyzes or participates in the reaction between the indicator and organic hydrogen oxide.

Zui). The reaction produces a color or other analyte response, the intensity of which is an indicator of the concentration of the analyte. Ascorbate ions, when present, cause significant disturbances. It would also be expected that the presence of peroxide-active metals in the composition would interfere with the measurement of peroxide-active analytes.

Nevertheless, the irradiation of peroxide-active substances in the sample
It has now been found that new compositions, test means (and test devices) resistant to the interfering effects of ascorbic acid in a sample can be effectively prepared for the detection of
The test means (and test device) consists of polycarboxyalkylamine iF, also known as "Moulperoxidase".
Contains four metal chelates.

Therefore, the composition of the present invention comprises an organic hydroperoxide,
a peroxide active substance and an indicator capable of producing a detectable response, e.g. a color change, in the presence of peroxide, and also of the general formula: [wherein R1 is hydrogen or a straight chain having 2 to 3 carbon atoms] represents a branched chain alkyl alcohol or an alkylcarboxylic acid group, and l'(p, R3, Rx and R
y&;l is the same or different and represents a linear or branched argyl alcohol or argyl carboxylic acid group having 2 to 3 carbon atoms, R1, R2, R3, Rx or R
At least two of y are the above-mentioned alkylcarboxylic acid groups, Rp and Rq are the same or different, and are straight or branched alkylene groups having 1 to 3 carbon atoms or 6 to 9
represents a divalent 1,2-alicyclic group having carbon atoms,
n is an integer having a numerical value of O to 1, ni is an integer having a numerical value of 0 to 2, and M is Fe.
including.

A preferred compound is one in which m is 0 and rl is 0 or aCt
1, and Rp is an ethyl/one group.

Metal chelate haf of polycarboquine alkylamine derivative with alkylcarphonic acid group or -Cll2COOtl 1! i Otoko 4-cho is very nice.

t(4-Kin-kiki tz-1 is N-(2-human p4-syl)ethylenediaminetriacetic acid iron-kyl-1-(I・(
・-III formula DTΔ).

In a preferred embodiment of the invention, the iJl composition is incorporated into a support 7Trinox, water-absorbent paper to form a test means, and the test means is fixed to an inert support. , it is difficult to form one tool).Furthermore, 71 steps of the test (
and testing devices) are also provided by the present invention.

According to the present invention, the present invention includes the following. sesame), asnilubate interference, as well as good storage and high temperature stability and r (lh
Compositions, test means (and test devices) are provided that have unexpectedly advantageous stability so that the lack of a "positive" result can be confirmed experimentally.

Early wet chemistry experiments during the development of this invention revealed that N-(2-
Hydroxyethyl) ethylenediaminetriacetic acid iron salt
1-(Hereinafter, it is written as Fe-HE DTA, but this is used for convenience and does not mean the existence of a covalent bond between the gold mud ion and the polycarboxyalkylamine derivative. ), ascorbic acid and IN
% agent and confirmed the rapidity of ascorbic acid oxidation in the presence of this KIRE-1, a result that would be expected from the above-mentioned report of Kerr 7 and Martill. Therefore, in a peroxidase-containing composition, it has been discovered how quickly TMB is oxidized in proportion to the oxidation of ascorbic acid by such a composition. , e.g.
J1 product GJ is extremely unstable and rapidly yields 1-1 false positives
It was expected that results would occur.

However, further experiments revealed that such a metal
Suitable for the detection of peroxide active substances, including - suitable for the dry solid state type, during production and! Ii'' shows good reagent stability in storage and is ``false positive'' when in contact with known hemoglobin Ijhin-negative urine,! It has been found that it is possible to prepare compositions that do not target fruit. The present invention thus achieved, as described above, is based on a metal beam 1, for example, F.
The above literature suggests that e-11 EDTA may be used as a [model baroxidase] and therefore would be unsuitable for use in compositions for determining the concentration of peroxide-active analytes. contradictory.

Urine, 1. Biological 11ν Occult blood (OB) in the body, immediately! Test means (and test 1) that could be produced from the novel composition of the invention for the detection of E.
-1) was found to be C-resistant due to the abnormally high concentration of ascorbic acid in the urine. As mentioned above) 7. This is caused by ascorbic acid! r−1,,tt−, while dwarfed, especially considering that urine samples (about 25% of TI can be expected to exhibit higher As:I rupic acid d? degrees than lQ mg/dl−1). Some types of ascorheb6.'j;49IyCommonly used OB detectors, which do not include prolongation, are usually 511W l (] I, l1ltl- by l1ltl- to a lesser extent Am9z Ruhinic acid?5 degrees ( That is, ＼Not too sharp f+ for the existence of Mogrohin
It turns out that it was lost in the J. However, the test device made according to the present invention is different from the conventional test device ζ.
In this company, Ascorhe I - a liquid with extremely high interference resistance 1 (I) containing azoluhinic acid at a relatively low concentration, i++i 5 ng/dl, is present. Therefore, the present invention provides a composition for the detection of peroxide-active substances in biological fluids such as urine. , provide a test means (and test device).The compositions, test means (and test device) of the present invention enable the detection of peroxidase, myoglobin, red blood cell, and pseudoperoxidase of Hemog II, for example. be able to detect other peroxide-active substances, including

The present invention provides derivatives of polyalpoxyargylamines which reduce the deleterious effects of As:Iruhine in analyzes performed on biological fluids.
; 2-4-1 "1-(: 1. Please use the 1-cell 3-kisitase set al+ai\Rero Metallic Cleaner I. From your point of view, Metallic Cleaner 1 is suitable for such liquids. It has the effect of promoting the oxidation of the ester rupine ions that may be present in it.

The present invention () Silk composition test means (and test devices) are substantially insensitive to disturbances and have a culm density of 0.3 mg/dL or 6J There are traces of ``1'Nige 11Hini・'' and ζ
When detected with the naked eye or with one or more instruments, for example, the response of 71 colors can be detected every 4L.

The organic compound used in the compositions of the present invention can be selected from a number of well-known hydroperoxides. However, since the detectable response C1
1, e.g. a change in color or absorption by the test compound; H,
If it's 1, then there is no (゛). Among the hydroperoxides, two suitable for samurai are cumene 1'l peroxy 1-1t-phylloxy 1-' r-+ peroxide, diisopropylbenzene 2 peroxide, and 2,5-dimethylhexane-2°5-hydroperoxide. ni-l-roba-oxide, varamentan-hi (wa-per-oxy) and other well-known hydroperoxides suitable for oxidizing the indicators used, or mixtures of these compounds.
Peroxide is most preferred.

as long as it is capable of reacting to produce a detectable response in the presence of organic hydroperoxide and peroxide active substances.
Many indicators are suitable for use in the compositions of the invention. The indicator is, for example, a so-called "henshisin type F"compound;hensidine;o-1-lysi:/;3,3',5
．． 5'-tetra(lower alkyl)benzidine, 2.7-
diamitsufluorene; and mixtures thereof or mixtures of various other indicators. The term used in this specification is “
"Lower alkyl" refers to methyl, ethyl, n-propyl and isopropyl groups, and the various butyl, pentyl and hexyl isomers having 1 to 1 carbon atoms.
There are 6 alkyl groups. 3 + 3 which is an indicator
' + J + 5"-tetramethylhenzidine (
T M +3) is particularly preferred.

Use - 3 ゛ (selected person)! ] Constant metal sharpness
The qualification of 1. is that fi which promotes the oxidation of scorhinic acid
The compatibility with the other components of the composition is determined not only by the L force but also by the compatibility with the other components of the composition. Metal chelates of alkylamine derivatives were found to include metal chelates of alkylamine derivatives: ftf: , -+, metal chelates found to be suitable for use in the present invention include, for example, N-
Hill;
Hexylenecyaminetetraacetic acid iron-1□ (Fe-CD
TA) 1,n l-I2E iron acetate-1・(Fc-
N TΔ), iron iminodiacetate chelate (Fe-IMDA
), ethylenecyamine dipropionate iron chelate (Fe -F D D P , α-type and β-type),
and iron hydroxyethyliminodiacetate) (Fe
-HI MDA) and mixtures thereof. Forms of Fe-Hr-1~ are preferred in general 6, and the most preferred compounds are Fe-Hr>I) T A and Fe-E I)
The most desirable one is Fe-HEDTA. However, as will be apparent to those skilled in the art having read specification 7), a large number of suitable chelates in addition to those specifically listed above fall within the scope of the present invention.

Therefore, Fe-HF, DTA and Fe-EDT
A, in particular, Fe-HED TA gives excellent results in the compositions, test means (and test devices) of the invention, and most satisfactorily produces ascorbic acid interference resistance (detects hemoglobin) in the occult bleeding test. It has been experimentally found that the reagents exhibit good reagent stability before use and do not produce false positive results. Additionally, the other metals mentioned, as well as numerous other metals, will work satisfactorily. However, one can expect significant variation in the rate at which peroxide actives can be detected when using such other compounds. This is because the rate of oxidation of ascorbic acid varies. Therefore,
Suitable metal chelates for use in the present invention may be selected from those which are polycarboxydialkylamine derivatives within the above-mentioned classes of compounds, such that the ZC compound is removed and the ascorbic acid is removed. 111 acid salt to l front foot,
However, it is possible to do the following and fall within the scope of the present invention. However, many of them act slowly in terms of energy and are therefore less practical and less preferred in p7t-like applications.

Metal chelates suitable for use in the present invention include Al 1-
"Rich Chemical Synthesis" - (8 Idrich Chem
icalCo, ), Sigma Chemical Company (SigmaCh+!n+1cal Company)
) or polycarboxylic dialkylamine derivatives commercially available from similar suppliers. For example, the metal chelate Fe-(tED' i-8 is commercially available HF, I
) TA and FeCl3 ・61L+0 are mixed in an aqueous solution in equimolar amounts to form an iron: chelate of 1 (mol:
It can be produced by mole by mole. Other dissolved concentration ratios of metal:chelate can thus be easily adjusted simply by varying the respective concentrations of the mixing solutions. The best results in overcoming ascorbic acid interference are obtained when the concentration of the metal ion and polycarboxyalkylamine derivative in the compound is approximately 1:1 (mole:mole). Power (It turns out.

Various fruits of the present invention 71! ! ! In embodiments, the preferred concentration range for a given metal chelate-I varies widely. For example, Fe-1-TP. In the case of D 'T', organic hydroperoxy 1- and Te).U(lower alkyl)
When used in compositions containing hensidine indicators, a preferred concentration range is from about 0.5 millimolar (mM) to about 50
It is mM. This range is approximately 50cm/”d urine sample width.
It has been determined that it is optimal for resisting L ascol to -1.concentrations. Furthermore, in experiments, appropriate iron sharpness = 1
- can be used at relatively low concentrations to detect hemoglobin relatively quickly, but using the same Kir-1- or other Kir-1 at high doses is not very effective. These apparently anomalous results are discussed in more detail below; these results demonstrate that there is no general correlation between KIRE-1 concentration and function or suitability in the compositions of the present invention. prove that.

The majority of suitable metal chelates that work satisfactorily in the compositions of this invention have a structural alkylamine group or amine center group and a carboxylic acid group -- Cll2COOII
has. However, other compounds which do not have such characteristics or which belong to the general range of compounds described above are generally effective and satisfactory in overcoming the asteroid interference. It exhibits sensitivity and stability 471 and is therefore well suited for use. In use, the sex candy of the specific embodiments of the compositions, test means (and test devices) based on the general idea of the invention are numerous. depends on different factors.

A representative urine sample from a person who takes polyr117scorhinic acid (hitamin C) (■) often contains 25 to 100 mg/d of As.
Contains L or above. ? The reference ascorbate concentration for the iJf study was chosen to be approximately 50 μ/dL. Preferred embodiments of the ascorbate resistance compositions, test means (and test devices), and test methods of the present invention not only contain peroxide actives in such samples at a reference concentration of about 50 mg/dL; It was found that various other ascorbate concentrations could also be detected. In most cases, muscorbate concentrations much higher than 1 degree will cause a delay in response time. As below 1. [Lag time], i.e., the time until an observable response occurs, is 50 n:H
, /(]l As'': Experiments with respect to preferred embodiments of the present invention with varying chelate concentrations tested for Ijhika detecting MoI lohine in urine in the presence of I route. Approximately 1/2 from 29173 minutes 20 short ■)
Over the course of an hour, the i- lighted up 1'11.

In a preferred embodiment, the composition of the invention is used (
- Manufacture test means (and test devices) for the determination of peroxide active substances. In such preferred embodiments,
l'J! The substitute can be incorporated into a suitable support matrix to form the test means. The support matrix can take the form of polyamides, such as those disclosed in U.S. Pat.
May be sewn with felt, porous ceramic 1 lip and woven or cloaked glass fibers). 7 described in U.S. Patent No. 3,552,928.
Trinox (wood sticks, cloth, sponge materials and clay 5jT materials) are also suitable. British Patent No. 1.369,
No. 139, No. 11 II, (Synthetic resin 71 north and glass fiber felt 1- as support 71-I knock)
It is suggested that you use Also, UK 11! ,
4'1 1.349. 623+; 3 specification 7) is below H
j Thin filaments l as a coating on the paper matrix
We propose the use of optical transparency characteristics. france '17i
``Q'l No. 2'', No. 170.623 'f5 discloses polyamide +- fibers. Trinox has been used in the past and is used in the present invention.1.
A preferred and suitable material is a water-absorbing material such as filter paper. However, the support body 71-
Nox may be in a variety of physical and other forms as described above, and all such forms are suitable for use in the present invention.

In manufacturing the test means of the invention, the components of the composition can be incorporated into the support matrix in a variety of ways. For example, the components can be dissolved or suspended in water or other suitable solvents, preferably organic solvents such as ethanol, acetone or cymedylpolamide (DMF) and mixtures of these and other solvents. I'll come. This solution or +LL suspension can then be used to impregnate a water-absorbing filter paper, eg, as an ink printed with the reagents onto a suitable matrix. Alternatively, the support matrix may be soaked in the composition.
1 or coated with the composition using, for example, a doctor blade.

A currently preferred method of incorporating the components of the composition into the support matrix is to impregnate the water absorbent filter iJE with a solution or suspension of two or more of the components. Impregnation is accomplished by dipping the filter paper 11 into such a solution or suspension two or more times, drying the soaked paper in an oven after each dipping. The test means thus formed is laminated to one side of a piece of double-sided adhesive tape, the uncut piece is cut into strips, and
Each strip is secured to a long sheet of plastic backing material (e.g. polystyrene), which is then cut parallel to the shorter dimension with the impregnated paper at one end. to form a rectangular test device to serve as the other end or handle. The test device thus formed consists of a twice-dried, impregnated test strip secured at one end to one flat side of a long plastic support which constitutes a convenient handle.

4. A preferred method of making the test means of the invention is, for example, to prepare the 111E l) i''8 together with the organic human 1:I peroxide in an aqueous first immersion prior to the addition of the 111 indicator. !paper 111. That is, first, the filter paper is mixed with one or more suitable solvents and/or buffers, such as triethanolamine hol-1- and ) Aminomethane-Maroney 1-
(hereinafter written as T': 1-Lis-Maroney I) and - together with F
Impregnated with an aqueous solution of e-HED-FΔ and hydroperoxide, dried, impregnated again with a second and soaking solution of the indicator in a suitable solvent, e.g. ethanol, and dried a second time.
). In this way, paper is first impregnated with metal chelate-1
It has been found that the two-fold method, followed by impregnation with other active reagents, yields test devices exhibiting 1 m+'k and storage stability.

A particularly preferred method of preparing the test means of the invention is to introduce the reagents other than the metal filler and the indicator into the filter paper by immersing it in a first solution of the reagents as described above and then drying the paper. Then, after drying, an indicator is added by dipping the paper into a solution of a stain-resistant and thickening agent, e.g. polyvinylvinyl coliton, in a suitable solvent, followed by a second drying. That's true.

In addition to the test composition reagents and other ingredients described above, other ingredients,
For example, various thickeners, wetting agents, buffers, emulsifiers and well-known auxiliaries may be added to the compositions, test means (and test devices) of the invention.
You can also join us. For example (516, as a thickener,
In addition to or in place of polyhinylpyrrolidone, various +)f)"l, such as gelatin, atone, carrageenan, casein, albumin, menalcel I:I-
You can use Let's say wet l leap j yen, then (,
Well, sodium dodecyl sulfate is used (preferably 1. Squid, long chain organic sulfates and sulfonic acids, such as sodium dodecyl sulfosuccinate, or t;
Decylhense: 7.'sulfonic acid sulfonate, sulfonate and lithium are not used. Buffer systems include triethanolamine porate and Tris-Maroney I.
lSitrite, phosphate, phthalate 11. ), quesoate,
! 1li1' salts, succinates or other buffers can be used. i, rt composition at a p of about 6.0-7.0
It is preferable to buffer the H value of 6. As the emulsifier, polyhinyl alcohol, arahiacom, carboxyvinyl polymer, etc. can be used. 1 inch indicator chair
'53 J- Useful organic solvents: J is most non-reactive and volatile solvents such as ethanol, acetone,
1) MF, chloropol J4, ethylene dichloride,
Includes Hensen, ethyl acetate and the like. Of course, the selection of other suitable solvents is within the skill of those skilled in the art.

When using a test means (or test device), it can be immersed in the liquid to be tested or a liquid suspension of the substance to be tested7 and then removed directly, or The sample can be applied to the test means (or test device). −
The presence of peroxide actives in the sample causes the test composition to produce a color change or other detectable response. If the response is color, this can be compared to a pre-calibrated color standard to assess the quantitative amount of peroxide active contained in the sample. Intact peroxide-active substances, such as intact red blood cells, appear as colored spots on an otherwise unpigmented matrix. The hemolyzed peroxide active substance can evenly color the matrices. In addition to visual comparison, various instrumental measurements can be used to measure the amount of color or other response exhibited, increasing the accuracy of the test by eliminating the subjective measurements of the human eye. can.

The following examples merely illustrate the concepts and advantages of the present invention?
], and does not limit the scope of the present invention. 1% like this! Determination is determined by the scope of the claims.

To the Examples, Examples of Test Compositions [---Fe-HE DTA Peroxidase or α in a Sample: (Preparation of Compositions of the Invention in Which the Presence of Another Peroxide Active Substance and in particular Hemoglucose) can be Determined An experiment was conducted in which the composition was -)'
Skolbe 1-rihohai! ￥N-(2-oxyethyl)ethylenecyamine Sanishi 1:1 as a laxative
(denoted as mol dimol, MUM) Iron chelate (Fe-1
-([Lr)′d゛Δ) is something that respects. HI Mi D
Dissolve 0.278 g of TΔ in 1'00 ml of distilled water to make 10 m old I:DTA/8 solution.110 m old T
E l) F(ICIa ・61bOO) in TΔ solution
, 270g by 6 to dissolve Fe-II E
DTΔkil/-1- was produced. Ascorbic acid at tj of 5mM was added to the composition in an amount sufficient to produce a 50 micromolar concentration in a final volume of 1. The components of the composition and ascorbic acid were mixed in the order and amounts shown in the table below. The final composition solution contains a 100 micromolar (μM) concentration of Fe-HEDTA, which, as well as the concentrations of other components present, is a significantly lower concentration than used in similar compositions for incorporation into solid test means. Tita.

0.2 mol (M) citric acid) lium buffer
9.5 m, 110mM F
e-14Er)TA 0.1m110
g/dL dodecyl sulfate norium O, 1mlI
M Kumenhito [Coperoxide 0.1ml110
mM 3.3',5.5'-tetramethylhenzidine 0.1ml 15mM
Des:2lupic acid 0.1 ml The composition of the invention thus prepared was added with hydrated blood so as to give a final concentration of 0.1.39 μ per dN in solution. When it was observed to form a blue color, this! ,:l:, AscoγLeherl-concentration of the sample is 5
This shows that the composition is capable of detecting hemoglobin present at 0 μM.

Example If...Fe -E DTA F+co-II ICD TA, but iron-1-(Fe-ED TA) of ethylenediaminetetra-111' acid
The experiment of Example I was repeated, except that a 10 mM 1'S solution was used. Dissolve 0.292 g of 17D1'A in 100ml of distilled water.
Fe-L':D i'Δ was produced in substantially the same manner several times with the addition of eCl3 .61hO. The composition thus prepared was similar to Example I, with a hemoglobin concentration of 0.13'9 mg/di, and a hemoglobin level of 0.13'9 mg/di. A blue color was formed in the presence of 50 μM.

(JIII [・ ・ ・ Fe-CI go AFe
fth E T) 1” not 8, but cyclohexylene diamine 4i”! 11'9 t loss) - Le) - (Fe
The experiment of Example I was repeated except that a 10 mM solution of -CDTA) was used. Dissolve 346 g of cDTAo in 10 (1 ml) of distilled water and add Fe as described in Example ①.
By adding Cl3 61120, Fe
-CD TΔ warm solution was prepared. For example, as in Example I, hemoglobin 0.1:39 m
g/dL and the presence of 50 μM ascorbate formed a blue color.

Example IV The experiment of Example r was repeated, except that a 10 mM solution of iron chelate of iminoniacetic acid (Fe-IMDA) was used instead of Fe-I MDA Fe-H'EDTA. IMDAo
, 133 g in distilled water and the addition of FeCh 61120 as described in Example I. The composition thus produced, similar to Example I, contained hemoglobin 0,
A blue color was formed in the presence of 1.39 μM/lj+ and 50 μM ascorbate.

Example V - Fe-NTA The experiment of Example 1 was repeated, except that a 10 mM solution of iron chelate of nitrilotriacetic acid (Fe-NTA) was used instead of Fe-HEDTA. NTAO, I 91
gt-1 by dissolving in 100 ml of distilled water and adding FeCl3.6H20 as described in Example ①.
Shinoko produces 7e-N TA 7g liquid. The composition thus produced contained 0.139 mg of hemoglobin/
A reddish color was formed in the presence of dL and ascorbe-1.50 μM.

Example Vl...l'e E Dr: + P, xft
J-II Er'l TA is not an iron chelate of α-ethylenediamine-acetic acid dipropionic acid (Fe-Rimi D).
The experiment of Example 1 was repeated except that D 10 mM/811k was used.
o, dissolve 320 g into 100 ml of distilled water/8, Example ■
[?e-ELI Dry solution was prepared by adding P(IC13. A blue color was formed in the presence of 139+ng/'dl- and asgorol-1-50pM.

Example 7-°1°-E 1)I) P(3 1-1!-)IEDTAtebanaku, α-ethylenediaminedi1IllI acid dipropionic acid 1/-1.
(Fe-E DD l) ρ) lomM? Example 1') Repeat the experiment, except for using the liquid as a guest.F I)
D Io (30・, 320 g to 10 Qmif
This melting U7, as described in Example ■, FeCl3
・Adding 6 +120 to l (by r, Fc amount is 31) DPcl volume was completed. 3 go L-ko'
SDi Announcement Roku” The first item is the same as Example I.
．． 139■, /d[, and ゛r゛Skolhe 1-50
A blue color was formed in the presence of 1 tM.

(・51 Ham! '-'Pc-II F to IMD
e - If E
MDA) 10mM/8? The experiment of Example 2 was repeated except that g was used. HI Li D, to 0.
177 g of Geirusui 100 rivers 1
Thus, a Fe-III M DΔ solution was prepared. The composition thus prepared was similar to Example I, with a concentration of 0.139 + ng/dlJ3.
A blue color was formed in the presence of 1 l\-150 pM.

Example IX-X 1. , PA1 0.2 M-wolfberry,/acid on each side! Use 9.4 ml of aluminum buffer instead of 9.5 ml for each iron cleanser.
1 in 0.1ml (: 0.2ml instead of 1゛.
, δself1. ((Noko yora (1 ko experiment sharp 1
B) According to the invention, the product manufactured in C.11 is announced. Konoj,):t?・'l-1i1, substitute medium ((
;' OOIt MO) thick (f,: (7'> 71
Sharp h G existence ii”,'=,” ”k]j yyoshiki-
Become. Each J9-combination 6. -1200μN・1 iron II・-I・Composition 4 as above・-IQ,
: Rit: ! ! 1 Kazuheko, and the All compound substitute\moku (J Hin O, 139■/d1. and Asuni+,
11-. - l.50 If M is blue, the form I
Tomo-1-ru no tsu...view': And this happened during trial O1. One piece is q<-4,1・゛)・
1.0 (l p M iron cut-IN
11 generation 11++) when i experienced 1i1411: (
There was a difference between 41- 'Jp': ts, time 0) in color formation; , '5+1 soshi 1 color) Suginari time ('-) Geta - 1' no, 1 (1 iris)
B)) E The following Table C1 is shown. Time 1 ↑ Old 5 [above, l, ゛) C5 2, 1 it-It product of the present invention 1 Nishigawa-4-i 1 Company-affiliated Ki L- (・, for example) 1; i description 6 (Concentration of one type of Ki body and its composition Ryosuji, l,!
Resists ibe 1 interference, peroxide active 1''! Detection of G5- is a general relationship or 4.I between G5- and G5-
There is no relationship between IV and IV.

113, Test Device Example This test device also contained the composition of the invention described herein, except that the concentrations of the components were changed to eM ('f-') in the solid state test port (Example 1, above). The paper support consists of 7 trisox.
υ Kinseiji 10) / J'Ii Formation number of people and test equipment, 1-
2 Using the following procedure Example N - (2-Hydroxyethyl) Oude I, Njiamin - Nishi 11j Boiled 1.39g"g Dissolved in 100 liters of distilled water 7
, then -L- to this solution 1 ec]x 61h O
By adding 1,35 g ζN-(2-hi):'
+:+ :+diethyl) Egg 1/Nnn-Amine Sanzu Vinegar M
1:1 (mol:mol)ij+lzt(Fe-H
E 1) TA) ni'J 50mM? Yong l (coined l)
C2 Zoko.

Two types of solutions (resulting in compositions) were produced. : Approximately 15.24 cm (6 inches) x 1 to manufacture the test means!
1. Whatman 3 MMi, 5i, lt' with dimensions I 6cIn (4 inches)
The IEM was also impregnated with the reagent solution so that the paper was fully loaded with the reagent composition after the second dip. The operation used was to impregnate the paper by dipping it in a second solution, drying the impregnated paper, and then immersing the dried paper in a second solution to impregnate it. dry properly. Drying is
It i Q 5° after the second impregnation in a forced draft drying mold
C for about 8 minutes, and after the second impregnation, at 50℃ for about 5 minutes.
I did it.

The first reagent solution was prepared by mixing the following components.

Distilled water 74.0mlIM
)・Lis=Ma IJ Nee 1~Light TJ'R agent pl-16,5
10.0m1 5.5M cumene hydroperoxide 4.0...11
0 g/di-, sodium dotecyl sulfate 2.0
A 10.0 ml second reagent solution (prepared as described above) was prepared by mixing the following components:

2 “Tano Tsuki no 79.4m
16=Methoxyquinoline, free form 0.6m12
0% (w/v) polyvinylpyrrolidone (hydrated) (min 7i40,000) 20.0
m13.3", 5.5'-tetramedyl..., Ncidine 0.6g
), oyster y61y, impregnated paper, 3λ4, company
(S↑2Piit+I, formerly nn, 55]44).9. One side of the double-sided adhesive transfer plate? , this laminate L7. Next, add the laminate to approximately 5.24t:m (
It was cut to dimensions of 6 inches x 0.51 cm (0.2 inches). Take these on the unused fence.''
Legal 8.89cm (3.5 inches) x 15, 24. c.
, M is combined with the Boristine 1/2 sheet, and the pile I
-Cut the object parallel to the short side of the J' method, and put a piece of paper with a non-soakable U on one end and a handle on the other end to form a rectangle of 8.89 cm. Boristile: A test device consisting of a strip was formed.

Example
When tested with a sample containing -1, it was found that 1 + L for various hemoglobin concentrations was easily distinguishable, and the paleochrome, blue, and chloride samples were tested. ]Kaoi J
It was found that the test device was unable to detect any hemoglobin present, even though it contained ascorbic acid at a temperature of 100.degree.

Example X■ The experiment of Example X■ was repeated except that 10.0 ml of 50mM Fe-EDTA solution was used instead of Pe-HETJ)TA in the first reagent solution.

The test device prepared according to Example A monkey blue color appeared.

Example XIX In the first reagent solution, instead of Fe-HEDTA,
The experiment of example

The test device prepared according to Example A bright blue color appeared.

Example XX In the first reagent IH solution, add Fe −] (E I) i゛G instead of Δ, 5 [1mM Pc −I MDA? 8 liquid to 10. On+I use 1; J remove, 1, example X ■t
The experiment was repeated.

The test device manufactured according to Example
When tested as described above, urine samples containing 50 mg/dL of Ascol.
−・ru occurred.

Example XXI In the first reagent solution, C1 instead of Fe-HE D TA
', 50mM Fe-NTA? Example 1 The experiment of Example XVII was repeated using 10.0 ml of S solution.

The test device prepared in accordance with Example XXI was tested as described above with urine samples containing various 97 degrees of hemoglobin and 50 mg/dL of ascoluate, which corresponded to various hemoglobin concentrations. It produced an easily distinguishable blue color.

C1 Ascol Test Device - 1 Interference Resistance and Safety of the Test Device Manufactured as Described in Example X
Further experiments were performed to evaluate the ability to detect hemoglobin in urine in the presence of ascorbate after stress simulating long-term storage. Experiments were conducted on newly manufactured test devices and test devices that had been stored for long periods of time under high temperature conditions. Specifically, the test devices were tested and compared for performance immediately after manufacture at ambient temperature (23°C) and after 10 and 28 days of "heat stress" at 50°C during drying cycle.

Test stock solutions containing various hemoglobin concentrations were prepared.

50mg/d1. Two hemoglobin solutions were also prepared containing Ascol at a concentration of .

Distilled whole blood with distilled water, hemoglobin 15 per 100ml of water
．． Hemoglobin 15 by diluting to a concentration of 4.
．． A stock solution containing 4 mg/dL was prepared.

The hemoclohin content of whole blood was previously determined by conventional techniques. Pooled, previously screened urine samples negative for hemoglobin and ascorbic acid were co-located in a blinded manner. The blood solution was then pipetted into the pooled urine to give a hemoglobin concentration of 0.015.0.031.
．． Test solutions were prepared by making stock solutions containing 0.062 and 0, ] 39 mg/dL. The stock solutions containing hemoglobin at 0.031 and 0.062 + ng/dL were separated into separate containers, and AsJ1 Ruhinic acid was added to the test tube 111 to bring the As:1 rubate concentration of the solution to 50.
■/d1. I made it.

A test device manufactured in the same manner as Example X■ and Fe-HE',
I) Control test devices prepared as described in that example, but without TA, were tested blind and with each hemoglobin solution. The test device was momentarily immersed in each solution, taken out, and after 1 minute, iJ color formation was observed on the test device. Regarding the relative intensity of the formed color 1 minute after immersion,
A visual comparison was made with another test device and standard color chart 1. Color ranges from colorless (blind) to dark green-blue at 0.139 µ/(1 L hemocrohine/8 fluid).

The results of this test are shown in the table below. When tested after 10 days of exposure, it is shown to produce a color response to the presence of hemoglobin similar to the response of the test device used to test urine samples free of Ascol. Similar tests of control devices without A showed that the response of the device was almost completely impaired by ascorbate in the sample. The test device made according to this preferred embodiment of
It was demonstrated that hemoglobin at a concentration of 62/d (7) could be easily detected. Therefore, the presence of Fe-HEDTA was reduced for the purpose of interfering with Ascor.

(See margin below) The results shown in the table above demonstrate that there is little difference in reactivity between newly manufactured test devices of the present invention and test devices stored at 50° C. for 10 or 28 days. This result predicts that the reaction of iron chelates and hydroperoxides on the same test device will reduce the reactivity of the test device at a faster rate under hot chloride than when stored at ambient temperature. This contradicts the results obtained. This statement attests to the good stability and advantageous "shelf life" of the compositions and test devices of the present invention. As can be seen from the data, organic hydroperoxide and Pc-HEDTA,
Between Fe-HEDTA and the indicator, or other substances and other strip components, even after long-term storage at high temperatures,
There were no or few apparent incompatibilities. Additionally, test devices containing the metal chelate (Fe-HEDTA) and test devices similarly made without Fe-HEDTA are substantially similar in sensitivity to the presence of hemoglobin in test solutions that do not contain Ascor. showed no "false positive" results, indicating excellent compatibility of the reagents. However, test devices prepared without Fe-HE I) T8 were actually completely inhibited by the presence of Ascolhe I in the test solution, whereas Fe-111-D
The strip of the present invention containing TΔ
In fact, for mogrohin concentration IQ- of the order of 0.03] and iJ' 0.062 mg/dL, the appearance of a color discernible to the naked eye after 1 minute C ,-Yoge C3 has been able to respond.

To further demonstrate the advantages of the present invention, tests similar to those described above were carried out using test devices prepared as in Example X, ie containing Fe-11E D TΔ. However, 7. Since we use naked eye technology, we can't see it without seeing it.

(-・Scanner (Rapid 5canner)
Color formation was followed using an apparatus known as J. This device is a scanning reflection spectrometer with a laboratory microcomputer interposed. This device is used to quickly measure the reflectance spectrum in the visible range. A computer enables storage of spectrometers and performs dJ calculations. Measuring the performance of, for example, has the following advantages over visual observation of the same strip: 1. The light source and the conditions surrounding the sample are held constant.

In macroscopic observation, the light source can vary not only in wavelength but also with respect to the position of the observed strip.

2. Detector characteristics are held constant. In naked eye observation,
The detector (ie, the observer's eyes) can vary from person to person, and even from day to day for the same person.

3.Rapid scanners can quantify data in a more detailed manner than by visual observation, which allows for more objective comparison of results.

The Rapid Scanner device was manufactured by the Miles Laboratories Manufacturing Co., Ltd., Eltakha, Inch., and complete information regarding its construction and performance characteristics is obtained therefrom. Genshau (M,
A, Gen5t+aw) and Rogers (R,W
, ROgerS), 8na1. Chem, Vo
l.

53: 194.9-1952 (1981).

Using the tristimulus values from the Lapis 7D scanner,
rCommission TnternaLional
Addendum 2 Publication No. to 1 Michelage (Paris, France)
15, Colorimeter, (E, -1,3,1) 1971 j
The color difference value (ΔF) was calculated based on the trial decision contained in the following. Therefore, the data from this device is recorded below as ΔE or color difference units.

Using the procedure described above, a test strip device according to the invention containing F(!-HE r,)
The ability to detect hemoglobin concentrations of dl and 0.062 mg/dL was tested. Some test devices were tested with urine samples containing 50 μ/dL of Ascol He-1, some were tested with similar samples that did not contain Ascol He-1, and some were newly manufactured at ambient temperature. After testing, some were tested at ambient temperature and 50'C for 11 and 2 days.
It was tested after being stored for 8 days.

Color difference fl' provided by rabbit scanner
The j position (Δl) corresponds to the concentration of various hemoglobins.
．． The results are shown in the table below.

Newly manufactured test device urine sample and hemoglobin Ascol iku I upper two test tubes 0.
03] 0 21.890.031
50 15.750, 062 0
29.780.062 50 2
2. Test device stored at 28B at 84 ambient temperature - Cosmetic hemoglobin Ascol 3 e11' - Two people + -
1/Vic acid ±Knee's processing AE) - Uni Otsushiri 11 Erosion υ ------- Surplus [,,,,---0,03
1015,50 0,0315013,29 0,062026,31 0,0625018,20 Test device stored at 50°C for 11 days Urine delivery Hemoglobin Ascol Puku Jl---Kudo-Yanbic acid Shiney0211 Limbs μfe Tanka-(-Yui/-
dj, )----Kotzuochi---Koshibe Needle---
----0,031020,86 0,0315012,36 (1,G 62 0 30.040,0
G 2 50 27.28 at 2650℃
Test specimen urine stored for 1 day, test Zhu L hemoglobin Ascol -Pdan LL upper Z-7-Yarn-Hinnic acid length--41 Zhan-η length-(-Aj up-N-=(-■-101J--11- Buichie β-I, -L-
-----------Fushitanichi---m=--(1, (1310
10,31 0,031507,78 0, (162020,86 0,0625016,38 It shows a significant elimination of ascorbate-1 interference in such test devices even after long-term storage and storage at elevated temperatures.

Further visual testing was performed on test devices prepared according to the present invention, ie, containing Fe-EDTA, as described in Example X, above. After the test devices were manufactured, they were tested with urine samples containing and without hemoglobin at various concentrations, as well as samples with two hemoglobin concentrations, including 50 mg/dL ascorbate.

Some test devices are sold at
After storage for 28 days at
/A, r corresponds to the color value seen by the naked eye obtained by comparison with a standard color chart. All testing of these test devices was conducted as described above.

Test of the present invention containing Fe-EDTA 2-A No.: L group stability -------1-
−−−=−−−−−−−−−−−−−−−−−−−−
- Submergence ---------1----------Hemoglobin Vle at ambient temperature 2 at 50°C
8 (■/dL) (P-acid stored for 1 day after being manufactured) -LIJ-1 person-
Nutoner 7-γ tool-0, 1010 (1, O1512' 110, 031
20 15 (1,0623022 0, l 39 3,'] 3
0(-1 scorbic acid-5, -()-+fallen, //!'!Leeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee-0
031 20 14E1.06
2 30 22 The results of these latter tests confirm the stability, lack of false positive results, and significant resistance to Ascol interference of the test device made according to this embodiment of the invention.

It will be apparent that many modifications and variations may be made from the particularly preferred embodiments of the invention as disclosed without departing from the spirit and scope of the invention.

Accordingly, the invention is limited only by the scope of the claims.

Claims (1)

Claims: (1) detecting the presence of a peroxide-active substance in a sample, including an organic hydroperoxide and a peroxide-active substance and an indicator capable of producing a detectable response in the presence of the peroxide; In the composition, the general formula [wherein (a) R1 represents hydrogen or a linear or branched alkyl alcohol or alkyl carboxylic acid group having 2 to 3 carbon atoms, R2, R3, Rx and Ry is the same or different and represents a linear or branched alkyl alcohol or alkylcarboxylic acid having 2 to 3 carbon atoms, and at least two of R3, R2, R31, Rx or Ry are is an alkyl carboxylic acid group;
(b) Rp and Rq are the same or different and are a straight or branched alkylene group having 1 to 3 carbon atoms or G
Divalent 1,2-alicyclic j having ~911^1 carbon atoms
(c) n is an integer having a value of 0 to 1, m is 0 to 2
and when m is greater than O, the repeated Rp and repeated Rq groups may be the same or different; (d) M 4', l: F (U de A polycarboxyalkylamine derivative having a metal complex 1
- A composition for detecting the presence of a peroxide active substance, the q-mouth sign of containing. (2) (aim is O, and (b) Rp is an ethylene group. Claim 1
Compositions as described in Section. (3) At least two of R+, R2, R3, Imax and Ry are alkylcarphonic acid groups -C1b COOI
The composition according to claim 1, which is I. (4) The indicator is benzidine, 0-tolidine, 3゜3“1
5.5"-tetra(lower alkyl)benzidine, 2,7
-Diamitsufluorene or a mixture thereof. (5) Metallic acid-I is N-(2-hydroxyethyl)
Ethylenediaminetriacetic acid, ethylenediaminetetraacetic acid, cyclohexylenediaminetetraacetic acid, nitrilotriacetic acid, imi'noniacetic acid, α=ethylenecyamine dil! ! i1. I'
2. The composition of claim 1, wherein the composition is selected from the group consisting of iron chelates of 1-nibropionic acid, β-ethylenecyaminedioic acid, acetic acid dipropionic acid, hydroxyloxetyl aminodiacetic acid, and mixtures thereof. . (6) The composition according to claim 1, wherein the metal chelate is an iron chelate of N-(2-hydroxyethyl)ethylenecyaminetriacetic acid. (7) The composition according to claim 1, wherein the metal chelate is an iron chelate of ethylenediaminetetraacetic acid. (li) (r+25's hill'roba-oxide to cumene, droperoxide, f-butylhydroperoxy)
ζ, diisopropyl Hensen human IU perogyside, 2
The composition according to claim 1, which is selected from the group consisting of 5-dimethylhexane-2,5-dihydroperoxy I, paramenthane 1-0 peroxide and mixtures thereof. . (!) Ti'tll hydroperoxide or cumench = Roberoxyl, the indicator is 3.3", 5.
The composition according to claim 1+:1, which is 5'-tetramedylhenzidine. (10) The indicator is 3.3°, 5.5”-tetranotyl-nsisine, and the metal chelate is N-(2-human
: ] Gyethyl) ethylenecyaminetriacetic acid iron compound L--
1.1, and the organic human l-1 peroxide is cumene human L'l peroxide. (11) organic hydroperoxides, peroxide active substances, indicators capable of producing a detectable response in the presence of peroxides, and general formulas [wherein (a) R, + is hydrogen or 2 to 3 carbon atoms] Represents a linear or branched alkyl alcohol or alkyl carboxylic acid group having atoms, R2, R3, Rx and Ry
are the same or different and represent a linear or branched alkyl alcohol or alkyl carboxylic acid group having 2 to 3 (llil) carbon atoms, and at least two of R1, R7, R3, Rx or Ry is the alkylcarboxylic acid group described above; (1) lqp and Rq are the same or different and are a straight or branched alkylene group having 1 to 3 carbon atoms or having 6 to 9 carbon atoms; Bivalent 1,2-
Represents an alicyclic group; (cl n is an integer having a numerical value of 0 to 1, m is O to 2
and if m is greater than O, the repeated Rp and repeated Rq groups are the same or 5
a support matrix incorporating a composition for detecting the presence of a peroxide-active substance comprising a polycarboxyalkylamine derivative I. Test means for detecting the presence of a peroxide active substance in a sample comprising: (13) Test means for detecting the presence of peroxide active substance M'3 in the sample. In order to equip a test means for detecting the presence of an oxidic active substance, a) organic human 11 peroxyside, and -. Represents a chain or branched alkyl alcohol or alkyl carboxylic acid group, and R2, R3, Rx and Ry are the same.
or differently, represents a straight-chain or branched alkyl alcohol or alkyl carboxylic acid group having 2 to 3 carbon atoms, and at least two of R7, R2, R3, RX or RV are the above-mentioned alkyl carboxylic acid groups. And; (i
i) Rp and R''q are the same or different, 1 to 3 (straight or branched alkylene group having circular carbon atoms or 6 to 9
represents a divalent 1.1-alicyclic group having carbon atoms; (iii) n is an integer having a numerical value of 0 to 1; m is O to 2;
(iv) if m is greater than 0, the repeated Rp and repeated Rq groups may be the same or different, and (iv) M is F preparing a first reagent solution containing a carboxyalkylamine derivative metalloxy-1; b) wetting a support matrix with the first reagent solution; - incorporating a first reagent solution into the support matrix by c) i'sl! Dry the moistened matrix and apply metal 4N.
d) producing a first 1 reagent melt 71v containing an indicator and a solvent capable of producing a detectable response in the presence of peroxide and peroxide active substance; e) 7trinox Add 1 g of the second reagent to the dry support matrix by wetting it with the second reagent solution.
Incorporate k, ``)■ Dry Torinox and make metal clean 1, human 1
A one-stage test molding method for detecting the presence of a peroxide active material, comprising the steps of: leaving a mixed residue containing a peroxide and an indicator; (14)(・+)m is O and (bl Rp is an ethylene group) Claim 1
The method described in Section 3. (5) R+, R2, R3, Rx and F<V at least 4) 2 (17i1 is an alkylcarboxylic acid group -C112C(1011). The method according to claim 13. (16) The indicator is benzidine, o-tolidine, 3°3'
, 5.5'-tetra(lower alkyl)benzidine, 2.
14. The method according to claim 13, which is 7-diamitsufluorene or a mixture thereof. (17) Gold 1m chelate is N-(2-hydroxyethyl)ethylenediaminetriacetic acid, ethylenediaminetetraacetic acid a,
Consisting of cyclohexylene diaminetetraacetic acid, nitrilotriacetic acid, iminodiacetic acid, α-ethylenecyamine diacetate, dipropionic acid, β-ethylenediamine diacetate, dipropionic acid, hydroxyethylaminodiacetic acid, and mixtures thereof. 14. The method of claim 13, wherein the method is selected from the group. (18) The method according to claim 13, wherein the metal chelate-1- is an iron chelate of N-(2-hydroxyethyl)ethylenediaminetriacetic acid. (19) The method according to claim 13, wherein the metal chelate-1- is an iron chelate of ethylenediaminetetraacetic acid. (20) Organic hydroperoxides include cumene hydroperoxide, t-butylconomeroxy 11, pyrubenzene hydroperoxide, 5-dimethylhexane-2,5-dihydronomeroxide, paramenthane 17conomeroxy F, and these. Claim 13 selected from the group consisting of mixtures of
Section description/brother's method. (21) The method according to claim 13, wherein the organic hydroperoxide is cumene hydroperoxide, and the indicator is 3+3'+5=J"-tetramethylbenzidine. (22N indicator is 3.3' , 5.5=-tetramethylbenzidi, and the metal-killing-t-kaN-(2-hyperlj'l:+xyethyl)ethylenecyaminetriacetic acid is (23) The method according to claim 1311' in which the sample is a peroxide. (23) The test means according to claim 11 A method of detecting the presence of a peroxide-active substance at low concentrations, comprising contacting a soil culm with a soil culm and observing a detectable response.