Process for the determination of peroxidase

The present invention provides a process for the determination of peroxidase by the addition of a peroxide and of a chromogen and measurement of the color resulting from the oxidation of the chromogen, the color formation being stopped after a definite time by the addition of a stop agent, wherein catalase is used as stop agent.

The present invention is concerned with a process for the determination of 
peroxidase. 
The determination of peroxidase is of importance in various technical 
fields. In recent years, the increasing use of peroxidase as marking 
enzyme in the scope of the so-called enzyme immune determination (EIA), 
which has supplanted to a considerable extent the previously usual 
radioimmune determination (RIA), has thereby achieved great importance. 
The enzyme immune determination of haptens, antigens and antibodies is an 
extraordinarily sensitive process which, therefore also places 
correspondingly high demands on the exactitude of the determination of the 
marking enzymes thereby used. In the case of the very frequent use of 
peroxidase as marking enzyme, the enzyme activity determination frequently 
takes place according to the fixed time method by extinction measurement. 
In principle, the process depends upon the face that peroxidase (POD) 
oxidises an appropriate chromogen in the presence of an peroxide with the 
formation of a colour, the speed of the colour formation corresponding to 
the activity or amount of the POD. In the case of the fixed time method, 
after a definite reaction time after addition of the substrate (usually 30 
to 60 minutes), the colour formed is measured against a blank. Since, at 
this point of time, the colour reaction is, in general, not yet concluded, 
it must be ensured that the same incubation times are maintained for all 
standards and samples. This requires that not only the substrate addition 
but also the extinction measurement must be carried out in an exactly 
fixed space of time of, as a rule, 10 to 15 seconds between the individual 
test tubes. This disadvantage, which makes the handling difficult, can be 
avoided by using a stop agent for the reaction. 
In the above-mentioned connection, it is already known from U.S. Pat. No. 
4,234,680 to use an alkali metal bisulphite as stop agent. However, this 
agent has the disadvantage, as reducing agent, of attacking the colour 
formed in the case of the oxidation reaction and of reducing it with 
decolorisation thereof. This applies especially for 
2,2'-azino-di-(3-ethylbenzthiazoline-6-sulphonic acid) salts, usually the 
diammonium salt (ABTS).RTM., which is particularly suitable as a chromogen 
for POD determinations, in the case of which a bleaching of the colour 
takes place within a short period of time. 
Another stop agent for the POD reaction is formaldehyde. However, the 
stability of the colour solution mixed with the agent is here 
unsatisfactory. There is also to be added the troublesome smell and the 
potential danger to health caused by formaldehyde. 
Another known stop agent for POD is oxalic acid. However, this agent 
results in an impairment of the precision of the determination. This 
applies especially when solid bodies (e.g. tubes or spheres) coated with 
antibodies are used for the enzyme immune determination in the case of 
which, in general, the POD bound to the wall after separation of the 
liquid phase from the solid phase is determined in the usual way. In the 
case of the addition of oxalic acid, such protein bound to the solid phase 
can, however, be loosened therefrom and/or denatured. 
A further known stop agent is sodium azide. Here, too, there is a danger of 
a bleaching of the colour. Furthermore, there is a danger of explosion in 
combination with heavy metal salts. 
Finally, surface-active agents, such as secondary alkyl sulphate or dodecyl 
hydrogen sulphate, have been suggested as stop agents. However, they are 
not able fully to suppress the colour formation. 
Therefore, it is an object of the present invention to overcome the 
disadvantages of these known stop agents and to provide a process for the 
determination of peroxidase in the case of which a stopping of the colour 
formation at a definite, predetermined point of time is possible, which is 
immediately effective and which does not result in a disadvantageous 
change of the colour present at the point of time of the addition of the 
stop agent. 
Thus, according to the present invention, there is provided a process for 
the determination of peroxidase by the addition of a peroxide and of a 
chromogen and kinetic measurement of the colour resulting from the 
oxidation of the chromogen, the colour formation being stopped after a 
definite period of time by the addition of a stop agent, wherein catalase 
is used as stop agent. 
The process according to the present invention has an immediate stop action 
of the colour formation but does not subsequently change this so that it 
is possible, within several hours after the stopping, to carry out a 
measurement of the colour formed at any desired point of time. 
The process according to the present invention can be used for all 
chromogens useful for carrying out POD determinations. Typical examples of 
appropriate chromogens include 
2,2'-azino-di-(3-ethylbenzthiazoline-6-sulphonic acid) diammonium salt 
(ABTS).RTM., o-phenylenediamine, p-phenylenediamine, m-aminosalicylic 
acid, dianisidine, p-aminobenzoic acid, aniline, 4-aminoantipyrine and the 
like. These chromogens are well known for POD determinations and do not 
here require any further explanation. ABTS.RTM. is preferably used. 
o-Phenylenediamine and 4-aminoantipyrine are also preferred. 
The amount of catalase which is added in carrying out the process according 
to the present invention is not critical. However, it must be in excess 
with regard to the possible POD activity present. In the scope of an 
enzyme immune determination, the added amount of POD, of which only a part 
must then be determined, is known. In the case of the determination of POD 
in other connections, in case of doubt, the order of magnitude of POD can 
be determined by a simple preliminary experiment and then the amount 
necessary for the achievement of an excess of catalase can be easily 
ascertained. In general, amounts of from 50 to 1000 U catalase per 
determination batch are used but, for certain cases, smaller amounts of 
catalase can also suffice or still larger amounts are desirable. 
The process according to the present invention is also especially useful 
when the POD to be determined is present in immunologically or chemically 
bound form. This is, for example, the case when using POD in the form of 
conjugates, for example chemically bound to an antibody, an antibody 
fragment, an antigen, a globulin fraction or the like. The process 
according to the present invention can also be very useful in case where 
the POD is present immunologically fixed as such or via a ligand 
chemically bound with it, which can be connected to a solid phase. In the 
case of these embodimental forms of POD determinations, the requirements 
for a stop agent are especially critical since actions of the stop agent 
on the binding partner of the POD can easily result in falsifications of 
the results. Naturally, the process of the present invention is 
particularly useful for the determination of free, dissolved POD. 
In the case of the process according to the present invention, the 
measurement of the colour formed can be carried out without 
disadvantageous influence on the precision of the determination within the 
course of several hours after the addition of the catalase. This is a 
considerable simplification and excludes various possibilities of error.

The following Examples are given for the purpose of illustrating the 
present invention: 
EXAMPLE 1 
Stopping of the colour formation reaction in the ABTS.RTM. system 
The Enzymun-Test.RTM.TSH of Boehringer Mannheim GmbH (order No. 736082) is 
hereby used. This test contains POD, chemically bound in the form of 
antibody-POD conjugate which, during the immune reaction, is bound to the 
vessel wall of a sample tube made of plastic as well as ABTS.RTM. as 
chromogen and sodium perborate as peroxide. 
The test is carried out according to the manufacturer's instructions with a 
TSH standard (50 .mu.U/ml.). Sample serum to be investigated is incubated 
for 60 minutes at pH 6.9 (phosphate buffer) in the same tube, then 
antibody-POD conjugate is added thereto and incubated for 60 minutes. 
Perborate and ABTS.RTM. in phosphate-citrate buffer (pH 4.4) are then 
added thereto. The colour formation reaction 
##STR1## 
is stopped after 60 minutes by the addition of 0.1 ml. of a stop reagent 
consisting of 
250 U/ml. catalase 
50 mmol/l. acetate buffer, pH 5.5 
2% by weight Triton X-405 (non-ionic, surface-active agent). 
Subsequently, the extinction of the solution is measured at 405 nm every 60 
minutes for 3 hours. The following extinctions (referred to the addition 
of the solution (t=0, E=100%)) are thereby obtained: 
TABLE I 
______________________________________ 
time (min.) 0 60 120 180 
extinction 100% 99.8% 100% 98.5% 
______________________________________ 
EXAMPLE 2 
Stopping of the colour formation reaction in the o-phenylenediamine system 
Commercially-available CEA-EIA test (Abbot, order No. 586324) is used. This 
reagent contains POD chemically bound as antibody-POD conjugate which is 
bound during the immune reaction to synthetic resin spheres and 
o-phenylenediamine as substrate. The test is carried out, according to the 
manufacturer's instructions, with 2 CEA samples (concentration 1 or 5 
ng/m/l CEA). 
The oxidation (colour formation) of o-phenylenediamine with POD and 
hydrogen peroxide is stopped after 30 minutes by the addition of 2 ml. of 
a stop reagent according to Example 1 which, however, had been diluted 
with water in the ratio of 1:20 v/v. For the 2 samples, numbered in 
increasing concentration, there are obtained the following extinctions and 
referred to the point of time of stopping, t=0: 
TABLE II 
______________________________________ 
sample time = 0' 
time = 90' 
______________________________________ 
1 100.0% 100.0% 
2 100.0% 103.1% 
______________________________________ 
EXAMPLE 3 
Comparison of the stop action of catalase and formaldehyde 
The reagents according to Example 1 are used. For comparison, instead of 
the stop agent of Example 1, there is used a 25% by weight solution of 
formaldehyde. In each case, the stopping of the colour formation reaction 
took place with 0.2 ml. of stop reagent. The following extinctions were 
thereby measured at 405 nm (referred to the point of time of the stopping 
t=0): 
TABLE III 
______________________________________ 
Formaldehyde as stop reagent 
______________________________________ 
time 0' 60' 120' 180' 
extinction 
100% 98.1% 91.3% 89% 
Stop reagent according to Example 1 (catalase) 
time 0' 60' 120' 180' 
extinction 
100% 98.8% 99.8% 100.1% 
______________________________________ 
EXAMPLE 4 
Comparison of the precision in the case of using catalase and oxalic acid 
as stop reagent 
The reagents according to Example 1 are used. The experiment is carried out 
analogously to Example 1 but, instead of the there-described stop reagent, 
there is used 0.2 ml. of a 7% by weight oxalic acid solution. A comparison 
experiment with catalase is carried out according to Example 1. The stop 
action of oxalic acid can be seen from the following Table IV: 
TABLE IV 
______________________________________ 
time 0' 60' 120' 180' 
extinction 
100% 112.6% 117.0% 114.0% 
______________________________________ 
The extinction of the solution is measured at 405 nm (point of time of the 
stopping t=0) for 10 different batches with catalase and oxalic acid. The 
results obtained are given in the following Table V: 
TABLE V 
______________________________________ 
0' 60' 120' 180' 
______________________________________ 
VC with 1.1 0.7 1.5 2.7 
catalase 
VC with 2.0 3.5 6.0 8.5 
oxalic acid 
______________________________________ 
It can be seen that the precision of the determination with oxalic acid as 
stop reagent is considerably impaired. 
EXAMPLE 5 
Stopping of the colour formation reaction in the ABTS.RTM. system 
(non-wall-bound POD conjugate) 
Solution 1: thyroxin-POD conjugate, activity: 0.6 mU/ml., dissolved in 
phosphate buffer (40 mM) 
Solution 2: phosphate-citrate buffer, 100 mMol/l., hydrogen peroxide, 3 
mMol/l. ABTS.RTM., 1.6 mMol/l. 
Solution 3 (stop reagent): acetate buffer, 50 mMol/l. catalase, 250 U/ml. 
Triton X-405, 2% 
8 .mu.l. of solution 1 are added to 1 ml. of solution 2. The colour 
formation reaction: 
##STR2## 
is stopped after 30 minutes by the addition of 100 .mu.l. of solution 3 
and the extinction measured at 405 nm immediately (T=0') and after 60 
minutes. The results obtained are given in Table VI: 
TABLE VI 
______________________________________ 
T = 0' 
T = 60' 
______________________________________ 
E.sub.405 100% 102% 
______________________________________