Sensitizers for peroxidative activity tests

Improved test compositions, devices and methods for the detecting of peroxidatively active substances are provided. The test compositions include an indicator capable of being oxidized in the presence of peroxidatively active substances to provide a color change, an oxidizing agent effective to oxidize said indicator, and a sensitizing agent. The sensitizing agent comprises a compound selected from the class of compounds of the general formula: ##STR1## wherein R.sub.1 and R.sub.2 are independently selected from hydrogen and C.sub.1 -C.sub.4 alkyl. Preferred compounds are benzimidazole and 5,6-dimethylbenzimidazole.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates generally to the field of diagnostic tests 
and, more particularly, to those tests useful in qualitative and 
quantitative determination of peroxidatively active substances. 
2. Description of the Prior Art 
The detection of small amounts of peroxidatively active substances, such as 
occult blood, hemoglobin, myoglobin, leukocytes, bacteria, or other 
peroxidatively active analytes, in body fluids and in body excreta has 
long been recognized as an invaluable aid to the medical practitioner in 
the diagnosis of many abnormal conditions. 
Various procedures, compositions and devices are described in the 
literature for the detection of occult peroxidatively active substances. 
For example, Kamlet in U.S. Pat. No. 2,290,436; Nicholls and Fonner in 
U.S. Pat. No. 2,799,660; Fonner in U.S. Pat. No. 2,838,377; and Adams and 
Peterson in U.S. Pat. Nos. 3,012,976, 3,092,463, and 3,092,464, all 
assigned to the instant assignee, illustrate several test compositions 
which have been supplied to meet the need for a simple, reliable test for 
occult blood. These test compositions are based on the peroxidative or 
catalytic activity of the prosthetic groups present in blood. See also 
Schwartz, Zeitschrift fur gerichtlich Medizin (Journal of Forensic 
Medicine) 12: 1928. 
It has been determined by Adams et al., in U.S. Pat. No. 3,290,117 that the 
sensitivity of these occult blood test compositions can be markedly 
improved and potentiated by the addition of quinoline or certain quinoline 
derivatives such as quinine. With the addition of these quinoline 
derivatives to the prior art occult blood compositions it is possible to 
detect 5 to 50 red blood cells (RBC)/microliter (.mu.l) of sample which 
corresponds to a blood dilution as low as 1:1,000,000. Use of the acid 
addition salts or adducts of quinoline and its derivatives as potentiating 
agents is disclosed by Mast et al., in U.S. Pat. No. 3,986,833. Another 
approach, disclosed in U.S. Pat. No. 3,853,472, reports the use of fused 
polycyclic derivatives of quinoline, such as benzoquinolines and 
pyridoquinolines, as potentiating or activating agents with similar 
sensitivities. In U.S. Pat. No. 3,975,161 Svoboda et al., disclose the use 
of isoquinolines as potentiators. 
Compounds other than those in the quinoline family have been found 
effective as well. In U.S. Pat. No. 3,917,452 Ritterdorf et al., disclose 
the use of vinyl pyridine compounds for use as sensitizers. In U.S. Pat. 
No. 4,063,894 Ogawa et al., disclose the use of substituted thiazole 
compounds for this purpose. 
SUMMARY OF THE INVENTION 
In accordance with the present invention, improved test compositions, 
devices and methods for the detecting of peroxidatively active substances 
are provided. The test compositions include an indicator capable of being 
oxidized in the presence of peroxidatively active substances to provide a 
color change, an oxidizing agent effective to oxidize said indicator, and 
a sensitizing agent. The sensitizing agent comprises a compound selected 
from the class of compounds of the general formula: 
##STR2## 
wherein R.sub.1 and R.sub.2 are independently selected from hydrogen and 
C.sub.1 -C.sub.4 alkyl. Preferred compounds are benzimidazole and 
5,6-dimethylbenzimidazole. 
DESCRIPTION OF THE PREFERRED EMBODIMENTS 
Although specific terms are used for clarity, these terms refer only to the 
embodiments selected for illustration, and are not intended to limit the 
scope of the invention. 
The test compositions, which are improved by incorporation of the 
sensitizers described above, further contain at least an indicator and an 
oxidizing agent and may be prepared in a tablet form or incorporated with 
a carrier such as an absorbent matrix. Suitable indicators are capable of 
being oxidized in the presence of a peroxidatively active substance to 
provide a color change and include well known materials such as 
3,3',5,5'-tetramethylbenzidine, o-tolidine, o-toluidine, p-toluidine, 
o-phenylenediamine, N,N'-dimethyl-p-phenylenediamine, 
N,N'-diethyl-p-phenylenediamine, benzidine, p-anisidine, dianisidine, 
o-cresol, m-cresol, p-cresol, alpha-naphthol, beta-naphthol, catechol, 
guaiacol, pyrogallol or those of the heterocyclic azine series for example 
bis-(N-ethyl-quinol-2-one)-azine or 
(N-methylbenzothiazol-2-one)-(1-ethyl-3-phenyl-5-methtriazol-2-one)-azine. 
As oxidizing agents there can be used, for example, cumene hydroperoxide, 
diisopropylbenzene hydroperoxide, paramenthane hydroperoxide, 
2,5-dimethylhexane-2,5-dihydroperoxide and other well known oxidizing 
agents effective to oxidize the indicators. 
In a preferred embodiment, the improved test compositions of this invention 
are incorporated on or with a carrier and utilized as a dip and read test 
device. The test device may be prepared by various well known methods 
which include impregnating an absorbent carrier material with a solution 
or solutions of the test composition and thereafter drying the impregnated 
matrix, thus adhesively incorporating within the matrix a finely divided, 
intimate mixture of the ingredients. The concentration range of 
sensitizing agent which can be used for impregnation solutions is 
generally from about 0.05 gram (g)/100 ml to about 1.0 g/100 ml and, 
preferably, from about 0.1 g/100 ml to about 0.5 g/100 ml. The term 
carrier refers to matrices which are insoluble in and maintain their 
structural integrity when exposed to physiological or other liquids to be 
tested. Suitable matrices which may be used include paper, cellulose, 
wood, synthetic resin fleeces, glass fiber, nonwoven and woven fabrics, 
various organic polymers, such as polypropylene, and other organic 
materials well known as film formers to those skilled in the art. 
Alternatively, the carrier may take the form of a pressed or molded tablet 
containing conventional carrier material. For convenience, the carrier can 
be suitably attached to an insoluble support or handle member which can be 
made from polystyrene. 
The test device is advantageously used by momentarily dipping it in a test 
sample or by otherwise introducing a test sample into the carrier matrix, 
whereby a detectable color change results when the peroxidatively active 
analyte is present. The volumetric capacity of the carrier serves to limit 
the amount of sample absorbed thereby and to which the test composition 
incorporated therewith is exposed. Any excess sample can be removed by 
washing or blotting the carrier to thereby limit the amount of sample 
tested to the volume thereof which has actually entered the carrier 
matrix. The test device can be used in the same way when samples of 
plasma, serum or other body fluids are tested. 
Test devices in the form of treated carrier matrices are often stored for 
considerable periods of time before use, and it is therefore desirable 
that the reagents chosen are not easily auto-oxidizable in air. Advisably, 
the test devices should be protected from exposure to light and in some 
cases it is desirable to keep them sealed in a moisture repellent package 
which is opened only for removal of one or more test devices shortly 
before use. 
Reflectance readings of color produced by reaction with the peroxidatively 
active analyte present in the sample can be obtained from commercially 
available spectrophotometers such as Beckman DK-2 Spectrophotometer, 
Beckman Instruments, Inc., Fullerton, California or Spectrocolorimeter 
SCF-1, Israel Electrooptical Industry Ltd. (distributed in the U.S. by 
Broomer Research Corporation, Plainwell, Long Island, N.Y.). 
The following example illustrates preferred embodiments of the invention.

EXAMPLE I 
This example reports tests performed to compare the sensitizing agents 
according to the invention with a known sensitizing agent, 6-methoxy 
quinoline. 
Four impregnation solutions were prepared as follows: 1. The following 
compounds were added to 41.67 milliliters (ml) of distilled water in the 
following order. Each ingredient was dissolved before the next was added. 
sodium citrate--1.78 grams (g) 
citric acid--2.31 g. 
triethanolamine borate--5.56 g. 
versene--0.055 g. 
methyl sulfone--5.56 g. 
sodium lauryl sulfate--0.833 g. 
Then, 41.67 ml N,N-dimethylformamide was added to this solution and then 
the following compounds were added: 
6-methoxy quinoline--0.333 g. 
cumene hydroperoxide--1.667 g. 
o-tolidine--0.444 g. 
2. Same, except no 6-methoxy quinoline 
3. Substitute 0.124 g. benzimidazole for 6-methoxy quinoline 
4. Substitute 0.153 g. 5,6-dimethylbenzimidazole for 6-methoxy quinoline. 
A 10 inches.times.4 inches piece of Eaton & Dikeman No. 237 paper was 
impregnated to saturation with the first solution and dried 11 minutes at 
95.degree.-97.degree. C. Identical pieces of paper were then each 
impregnated with one of the remaining solutions. They were then cut into 
pieces 0.2 inch.times.0.2 inch and attached to plastic handles. 
Solutions of hemoglobin were prepared and the devices prepared above were 
tested both fresh and after stress storage of 1 week at 60.degree. C. The 
lowest detectable levels of hemoglobin in urine are reported as milligrams 
(mg)/deciliter (dl) in Table I. 
TABLE I 
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Lowest Detectable Levels of Hemoglobin in Urine 
1 wk 60.degree. C. 
Sensitizer Fresh (mg/dl) (mg/dl) 
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1. 6-methoxy quinoline 
0.016 0.064 
2. none 0.322 &gt;0.805 
3. benzimidazole 0.064 0.322 
4. 5,6-dimethylbenzimidazole 
0.032 0.161 
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These results show that the compounds used in accordance with the invention 
are effective as sensitizers in the detection of peroxidatively active 
substances, such as occult blood. 
Although the invention has been described with a certain degree of 
particularily, numerous changes may be resorted to without departing from 
the scope of the invention.