Test aids and method for the preparation thereof

A process for preparing test aids for detecting substances to be analyzed by means of redox reactions containing single component oxidation indicators as the indicator substance in which oxidizable aromatic amine or hydrazino compounds, which are not capable of coupling with themselves, are added as decolorants.

The present invention relates to a method for the preparation of test aids 
for detecting substances to be analyzed by means of redox reactions in 
which a single-component indicator is used as the indicator substance and, 
in addition, in which oxidizable aromatic amino or hydrazinol compounds, 
which are not capable of coupling with themselves, are added to the test 
aid as decolorants. 
The addition of the decolorant prevents the appearance of a blank color 
value of the test aid. The addition of relatively large quantities of the 
decolorant makes it possible to adjust the sensitivity of the test aid 
precisely. 
In recent years, the so-called "color tests" have acquired ever-increasing 
importance in medical diagnostics. The "color tests" are methods for 
detecting substances to be analyzed in which peroxides such as, hydrogen 
peroxide or cumene hydroperoxide play a part. Hydrogen peroxide is 
produced during the oxidation, catalyzed by suitable oxidases, of a 
substance to be analyzed in the presence of oxygen. In these color tests 
either liquid reagents or so-called test strips are used as test aids. 
Possible substances to be analyzed are glucose, glycerol, glycerol 
phosphate, sarcosine, galactose and cholesterol. As appropriate oxidases, 
mention can be made of glucose oxidase, glycerol oxidase, glycerol 
phosphate oxidase, sarcosine oxidase, galactose oxidase and cholesterol 
oxidase. Still other enzyme/substrate pairs are known in addition. 
The cumene hydroperoxide mentioned above is employed to detect substances 
which are peroxidatively active, such as hemoglobin. The detection of 
concealed blood in stool samples is based on this principle. 
All these detection methods have in common the fact that redox equivalents, 
catalyzed by peroxidase or a peroxidatively active substance, are 
transferred from the peroxide to an indicator. These indicators can be 
divided into two groups. 
1. Single-component indicators. 
In these indicators the redox equivalents are transferred to an indicator 
and the resultant oxidation product is colored. As typical representatives 
mention can be made of benzidine, alkylbenzidines, such as tetramethyl-, 
tetraethylbenzidine or tolidine, etc. 
2. Two-component indicators. 
In these indicators one component is oxidized by the transfer of the redox 
equivalents. This colorless oxidation product is capable of reacting with 
a suitable coupling component to form a dyestuff. Probably the most well 
known reaction of this type is the so-called "Trinder reaction" [Trinder, 
P.A., Ann. Clin. Biochem., 6:24-27 (1969)]in which 4-aminoantipyrine is 
the oxidizable component which reacts with a phenol as coupling component 
to form a red quinone imine dyestuff. In a similar system 
3-methyl-2-benzothizolinonehydrazone finds application as the oxidizable 
component. 
The advantage of detection methods which proceed via peroxide is their high 
sensitivity which makes it possible to detect extremely small quantities 
of a substance to be analyzed. The high sensitivity, however, also results 
in a high susceptibility to interference. Thus, before the sample to be 
analyzed is added, the indicators mentioned produce distinct colorations 
even with traces of oxidatively active impurities or with atmospheric 
oxygen during the preparation of test strips or when liquid analyses are 
being carried out. This has the result that before a quantitative analysis 
is carried out a blank has to be measured which has to be subtracted from 
the measured value after the analysis. In particular, low concentrations 
of substances to be analyzed can only be determined imprecisely. To avoid 
these blank values, it would be possible, in principle, to work only with 
highly purified reagents with atmospheric oxygen being excluded, but this 
cannot be implemented technically because of the high costs. 
In DE-OS (German Published Specification) No. 2,716,060 stabilized rapid 
methods of diagnosis are described employing oxidation indicators to which 
aryl semicarbazides are added to avoid the appearance of a blank color 
value. These aryl semicarbazides must, however, be added in large 
quantities [Examples are described in the DE-OS (German Published 
Specifications) No. 3,012,368 and No. 3,406,328]and are, in addition, 
suspected of being carcinogenic, so that their use in test aids is 
inadvisable. 
Attempts to avoid the blank color value of test aids by adding ascorbic 
acid resulted in irreproducible results in relation to the sensitivity of 
the test aid if the ascorbic acid was added in quantities sufficient for 
the above-mentioned purpose. 
It has now been found that reagents, reagent solutions or impregnating 
solutions for preparing test aids which contain single-component oxidation 
indicators can be decolored or the formation of a blank color value can be 
prevented by adding small quantities oxidizable aromatic amino or 
hydrazino compounds, which do not couple with themselves, as decolorants. 
It has further been found that a reduction of the sensitivity and a shift 
in the calibration relationship (intensity of coloration as a function of 
the concentration of the substance to be analyzed) towards higher 
concentrations of the substance to be analyzed can be achieved by adding 
larger quantities of the decolorants. 
The invention relates to a process for preparing test aids for detecting 
substances to be analyzed by means of redox reactions containing 
single-component oxidation indicators as the indicator substance in which 
oxidizable aromatic amino or hydrazino compounds, which are not capable of 
coupling with themselves, are added as decolorants. 
The decolorants are employed for decoloring the test aid in concentrations 
of between 0.05 and 5% by weight referred to the single-component 
oxidation indicator and preferably between 0.1 and 1% by weight. The 
appropriate quantity can readily be determined by titrating the 
impurities. A small excess does not have a measurable affect on the 
calibration relationship. 
In order to reduce the sensitivity of the test aid or to shift the 
intersection of the calibration curve with the abscissa towards higher 
concentrations, the decolorants are used in quantities of between 5 and 
200% by weight, preferably between 5 and 100% by weight, of the oxidation 
indicator. 
The invention further relates to a method for preparing a colorless 
impregnating solution or reagent mixtures which serve to prepare test 
strips and contain the single-component oxidation indicators, said 
solutions or mixtures additionally containing the decolorants. 
The invention further relates to test aids containing single-component 
oxidation indicators and, additionally, the decolorants. Test aids are 
understood to mean reagent mixtures, lyophilization products, reagent 
combinations, liquid reagents, etc. 
In addition, the invention relates to the use of the test aids for 
detecting substances to be analyzed. Suitable substances to be analyzed 
are glucose, cholesterol, triglycerides, galactose, etc. or enzymes such 
as peroxidase or proteins with peroxidative activity such as hemoglobin or 
methemoglobin. 
As possible sample materials mention can be made of body fluids such as 
blood, plasma, liquor, urine and stool samples. 
The decolorants to be used according to the invention are normally employed 
as oxidizable components in two-component indicator systems, but within 
the framework of the present invention are used without any coupling 
component. 
Suitable decolorants are, for example, 4-aminopyrazolinones substituted in 
the 3 position of the general formula 
##STR1## 
where R.sub.1 =C.sub.1 -C.sub.4 alkyl, optionally substituted by --OH, OR, 
OCOR, OCOOR, C.sub.1 -C.sub.4 alkoxy, aryl, aralkyl, R representing 
C.sub.1 -C.sub.4 alkyl, 
R.sub.2 =H, C.sub.1 -C.sub.4 alkyl, 
R.sub.3 =aryl, aralkyl, C.sub.1 -C.sub.4 alkyl, optionally substituted as 
in the case of R.sub.1, H, or 2-benzothiazolinone hydrazones of the 
general formula 
##STR2## 
where R.sub.1 =H, C.sub.1 -C.sub.4 alkyl, 
R.sub.2 =H, SO.sub.3 H. 
4-aminoantipyrine (formula I, R.sub.1 =R.sub.2 =methyl, R.sub.3 =phenyl) or 
3-methyl-2-benzothiazolinone hydrazone (formula II, R.sub.1 =methyl, 
R.sub.2 =H) are preferred.

EXAMPLE 1 
To prepare a glucose test film the following mixture is prepared: 
9 mg of glucose oxidase (250 U/mg), 
50 mg peroxidase (80 U/mg) and 
1 mg 4-aminoantipyrine dissolved in 
1.7 ml of citrate buffer (1 m, pH 5.5). 
This solution is dispersed in the form of fine droplets in 
3.4 ml of chloroform which contains 
0.34 g of Na dodecylbenzensulphonate 
by shaking. The dispersion produced is added to a mixture of 16 ml of a w/o 
dispersion with chloroform as the oil phase, a w/o ratio of 1:1 and 13.1% 
by weight of polyacrylamide [preparation described in DE-OS (German 
Published Specification) No. 3,434,822, Example 8]and 34 ml of a 7.5% 
solution of cellulose acetobutyrate [n.sub.rel ](19% in acetone: 
ethanol=19:1)=200]in chloroform and 0.6 g of 3,3', 
5,5'-tetramethylbenzidine. The mix is vigorously shaken. During this 
process a strong green coloration of the mix first appears, which 
disappears again, however, after 30-90 seconds. 
The mix is deposited by means of a doctor blade on polyester film (180 
.mu.m wet coating) and dried by blowing with warm air at 24.degree. C. in 
the dark. A white test film is obtained which is cut into strips 0.5 cm 
wide. 
For testing, 30 .mu.l of complete blood is applied to a test area with a 
size of 0.5 cm .times.1 cm and wiped off after 15 seconds. The color which 
forms in accordance with the glucose content of the blood sample is 
measured in a reflection photometer at 640 nm and the K/S value 
[K/S=(1-R).sup.2 /2R; where R=reflection, K=adsorption coefficient and 
S=scattered light coefficient]is calculated. The plot of K/S against the 
glucose concentration (calibration curve) determined by means of above 
test strips is shown in FIG. 1. It can be seen that the curve has a K/S 
value of zero at the concentration zero. The coloration produced by a 
blood sample containing 20 mg/dl glucose can be distinguished visually 
from the color of the untested strip. 
COMISON EXAMPLE 1 
If the procedure is as in Example 1, but without 4-aminoantipyrine being 
added, a green colored mixture and a bright green colored test strip are 
obtained. A calibration curve drawn up by means of these test strips has a 
K/S value different from zero at a glucose concentration of zero. The 
coloration produced by a blood sample containing 20 mg/dl of glucose 
cannot be distinguished visually from the blank color value of the test 
strip. 
EXAMPLE 2 
A mixture is prepared according to Example 1, but using 2.6 ml of citrate 
buffer which contains a total of 100 mg of 4-aminoantipyrine in addition 
to the enzymes. The test strip prepared with this mixture as in Example 1 
is tested with blood samples of various glucose concentrations as in 
Example 1. The calibration curve obtained in this process is shown in FIG. 
2. A shift of the abscissa intersection to approximately 30 mg/dl glucose 
and a substantially lower slope of the calibration curve between 30 and 
200 mg/dl glucose in comparison to FIG. 1, i.e., a lower sensitivity, can 
be seen. 
EXAMPLE 3 
A mixture is prepared according to Example 1, but using 2 mg of 
3-methyl-2-benzothiazolinone hydrazone hydrochloride instead of 
4-aminoantipyrine. As in Example 1, a coloration of the initially green 
mixture is observed. The test strips prepared by means of this mixture 
yield the same calibration curve as the test strips from Example 1 (FIG. 
1). 
Obviously, many modifications and variations of the invention as 
hereinbefore set forth can be made without departing from the spirit and 
scope thereof and therefore only such limitations should be imposed as are 
indicated by the appended claims.