GB2154735A - Reagent for determining blood glucose content - Google Patents

Abstract

A stable and ready to use liquid reagent for determining the glucose content in blood comprises a glucose-oxidase, (free from catalse), and a nonionic surface-active agent, such as, for example, the polyoxyethylene derivatives, hydroxypolyethylene ethoxydodecane, or the lauryl ether of polyoxyethylene-glycol, as a stabilizer.

Description

SPECIFICATION Reagent for determining blood glucose content The present invention relates to a ready to use liquid reagent for determing the glucose content in blood. More particularly, the invention relates to a stable liquid reagent, in a single kit ready to use form, comprising a glucose-oxidase free from catalase and a non ionic surface-active agent.

It is known that the simplest and most used methods for determining the glucose content in blood (plasma or serum), are founded on the Trinder method or on variations of the same.

The glucose content in blood can be determined by means of the glucose-oxidase and the coloured Trinder reaction according to the following steps:

-D-g1ucopyranose + H20 (64S )

glucono- 6-lactone.H2O2.

3) 2H202*phenol+4-aminophenazone

(p-benzoqulnonemonoimino)-phenazone + 4H20.

The reagent used in the above consists of two components: a) A suitably buffered enzymatic component (glucose-oxidase-peroxidase); b) A chromogen, specifically 4-aminophenazone pluse phenol (the latter can be replaced by its derivatives, hence the variations on the original method introduced by Trinder, who first used phenol).

All the reagents according to this method must be reconstituted at the moment of their use in a single operating reagent, having, when reconstituted, limited stability. Thus, it should be noted that such a reagent, when reconstituted, presents the disadvantage of a guaranteed expiration date not longer than 60 days and of about 30 days on average. Such a reagent cannot therefore really be regarded as a ready to use reagent due to its limited stability.

At present, two methods for reconstituting the operating reagent are known: I) By adding water to a premixed enzyme-chromogen system; II) By mixing the enzymatic component and the chromogen, which, in this case, have been kept apart.

However, in all cases, the user is required to manipulate the product. This operation, even if it is simple, frequently causes mistakes because in the reagent's preparation some technical problems affecting the system's validity can arise.

In case I, when the operation reagent is reconstituted, high-purity distilled water is required, while only deionized water, not completely free from chlorine, is available in laboratories for clinical tests (a common inconvenince, particularly in summer time).

In case II during the pouring off step a loss of one of the two reagents frequently occurs due to outflow of one of them, or, more frequently, due to the incomplete dripping of one of the reagents from its bottle. In such cases, the operating reagent often has a final concentration different from the desired one.

Disadvantages occur also when the adopted procedure prevents the obtaining of a complete mixture of the solute and the solvent, as frequently occurs if the enzyme is provided as an anhydrous powder together with the buffer, which often being a phosphate buffer, is only slightly soluble.

When powdered reagents are used, as frequently occurs, it must be taken into account that the powdered ingredients proportions can be stated within a very broad range and that incomplete homogeneity can derive therefrom, even within the same preparation lot. This fact is even clearer when lyophilized enzymes are used, because, as is known, the lyophilization process may often heterogeneously reduce enzymatic activity (the single bottle's position, with respect to its place on the plates during the freezing step, is surely an influence), so that the final concentrations (as enzymatic activity) can be very different from each other.

In addition to these technical inconveniences affecting the system's reliability, the reagents so far employed have very high production costs.

The present production methods include two possibilities: a) anhydrous powders b) lyophiles.

In cases a) production requires a special humidity-controlled room and expensive plants where operators are exposed to serious risks due to hard work conditions. Thus, the use of such enzymes as powders, and especially the presence of sodium azide and of the various buffers, can cause damage, sometimes irreversible, to the respiratory organs of operators if they are exposed for a long time. Moreover, it has been noted that some people, after some years of such work, become sensitized to said enzymes and subject to consequent injuries.

In cases b), the use of lyophilizers calls for large amounts of electrical energy and water, in addition to the plants extinction costs.

It is therefore clear that it would be most important to have available a reagent for determining glucose content in blood which avoids the delicate reconstitution operations mentioned before and the expensive production techniques and the dangers for staff and the long lead-times (minimum times for reagent set-up).

The present invention is based upon the surprising discovery that these problems may be solved by using as a reagent a glucose-oxidase (GOD), completely free from catalase, combined with stabilizers consisting of nonionic surface-active agents.

This combination provides a stable unitary liquid reagent ready to use. The homogeneous concentration of the single components' enzymatic activities appears to be particularly favourable, because the reaction rate, which can be expressed as AA At of the coloured chromogen (A = absorbance or extinction), is particularly related to the ratio u.i/litre of glucose-oxidase, where u.i. is, as is know, the amount of enzyme which oxidases 1 mole of glucose per min at 25"C and pH = 7.0 (sodium phosphate buffer).

On this subject it is to be observed that sometimes remarkable differences in enzymatic activities can be found in reagents at present on the market, even amongst bottles from the same lot. Consequently there are some problems in the technique known as "Fixed Time", which can, in contrast, be properly carried out with a reagent linke that according to this invention whose invariability between bottles is substantially assured.

For correct setting up of such a technique it is necessary that the glucose oxidation by "GOD" follow psuedo-first-order kinetics with respect to glucose.

This causes a series of coupled reactions, to which the kinetics principle of fixed time measurements can be applied.

The above-mentioned technique can be applied to the reagent according to this invention, making it particularly suitable for fast analyzers that allow a considerable saving in time.

It is therefore a specific object of this invention to provide a ready to use liquid reagent for determining the glucose content in blood which comprises a glucose-oxidase free from catalase and a nonionic surface-active agent as a stabilizer.

In a preferred embodiment according to the invention a glucose-oxidase amount of from 9,000 to 40,000 u.i/litre and a nonionic surface-active agent amount of from 5 mg/litre to 50 g/litre are employed.

A polyoxyethylene (POE) nonionic surface-active agent is preferably employed, such as, for example, POE-tridecyl alcohol, POE-nonylphenol, etc. (RENEX), POE-octylphenol (TRITON) and POE-lauryl-, cetyl-, stearyl-, oleyl-alcohol (BRIJ).

Similarly, very good results are obtained by employing, as a surface-active agent, hydroxypolyethylene-etoxydodecane (C,2H25-(0-2J4)n-OH) or the lauryl ether of polyoxyethylenglycol (TH ES IT).

It is quite clear and must be pointed out that the absence of catalase was not taken into account either by Trinder or by other authors.

For illustrative and not for limitative purposes the following compositions are now described: COMPOSITION A Amounts referred to 1000ml of reagent GOD 9,000-40,000 u.i.

Peroxidase 300-3,000 u.i.

4-aminoantipyrine 0.1-1.5 mmoles Phenol 1.5-15.5 mmoles Phosphate buffer pH 6.5-11.4 POE-lauryl alcohol (Brij 35), at a concentration of from 5 mg/litre, was employed in this composition as a surface-active agent.

COMPOSITION B Amount referred to 1,000 ml of reagent GOD 9,000-40,000 u.i.

Peroxidase 300-3,000 u.i.

4-aminoantipyrine 0.1-1.5 mmoles phenol 1.5-15.5 mmoles Phosphate buffer pH 6.5-11.4 POE-octylphenol (Triton-X100), at a concentration of from 5 mg/litre to 50 g/litre, was employed in this composition as a surface-active agent.

As can be seen from Fig. 1 of the accompanying drawings, wherein the absorbance values are plotted vs. the reaction times, the resulting reaction kinetics for the compositions A and B, measured on Perkin-Elmer, are exactly alike even when the surface-active agent employed is changed .

In order to demonstrate the unalterability of product quality, Fig. 2 of the accompanying drawings depicts the absorbance of a reagent sample of the kind used in composition A prepared in February 1 983 but not tested until January 1 984. Besides noting how the curve is in accordance with theory, even at high concentration (500 mg/dl), it should be noted that the reaction is completely accomplished within an estimated 10 minutes.

This result was obtained by using a reagent lot as herein before disclosed which was stored, during the noted period, under stress test conditions (in fact a transparent vessel was used instead of the usual opaque vessel, which vessel was often opened and closed). It is further to be noted that the absorbance of white against H2O is only 0.1 28, that is quite an acceptable value from the moment that, moreover, such a reagent increases its absorbance under the direct action of the light.

A graphic demonstration pertinent to the product used in obtained the results of Fig. 2 is given in Fig. 3 of the accompanying drawings, wherein the absorbance (A) is plotted as a function of the concentration (c.St.) of the standard employed (abscissa). This diagram clearly shows the perfect linearity of the reagent up to a maximum concentration of 500 mg/litre. This straight was line plotted by computer utilizing a series of standards of 25, 50, 100, 200, 400, 500 mg/litre. The proportion of reagent to sample was 1,000 litre/10 litre; the reaction time was 10 minutes. The colour remains stable for about 1 hour.

The present invention has been disclosed with particular reference to some specific embodiments, but it is to be understood that modifications and changes can be introduced in the above disclosure by those skilled in the art without departing from its true spirit and scope.

Claims (7)

1. A ready to use liquid reagent for determining the glucose content of blood comprising a glucose-oxidase substantially free from catalase and a nonionic surface-active agent as a stabilizer.

2. A reagent as claimed in claim 1, wherein the glucose-oxidase is present in an amount of from 9,000 to 40,000 u;i./litre and the nonionic surface-active agent is present in an amount of from 5 mg/litre-50 g/litre.

3. A reagent as claimed in claim 1 or claim 2, wherein the nonionic surface-active agent is a polyoxyethylene surface-active agent.

4. A reagent as claimed in claim 3, wherein the non ionic polyoxyethylene surface-active agent is POE-lauryl alcohol or POE-octylphenol.

5. A stable ready to use liquid reagent for determining the glucose content in blood and substantially as hereinbefore described.

6. A ready to use liquid reagent for determining the glucose content in blood and substantially as hereinbefore described either as "Composition A" or as "Composition B".

7. The use of a liquid composition containing a glucose-oxidase substantially free from catalase and a nonionic surface-active agent in the analysis of glucose content in a sample, e.g.

in a blood sample.