MXPA99004096A - Diagnostic agent based on tetrazo compounds - Google Patents

Abstract

The present invention relates to a reagent is suitable for measuring the concentration of an analyte in a biological fluid containing hemoglobin, such as whole blood, the reagent comprises enzyme dehydrogenase having specificity for the analyte, nicotinamide adenine dinucleotide, a derivative of nicotinamide-adenine dinucleotide, pyrrolo-quinolinquinone, or a pyrrolo-quinolinquinone derivative, a tetrazolium dye precursor, a diaphorase enzyme or an analogue thereof, and a nitrite salt; the reagent causes dye formation, which is a measure of the concentration of analyte, the nitrite salt suppresses the interference of dye formation caused non-enzymatically by hemoglobin, preferably, the reagent is used on a dry strip to measure ketone bodies such as beta-hydroxybutyrate

Description

DIAGNOSTIC AGENT BASED ON TETRAZOLIO COMPOUNDS CROSS REFERENCE TO PREVIOUS REQUEST This is a continuation in part of the US application.

Serial No. 09/161876, filed on September 28, 1998.

BACKGROUND OF THE INVENTION 1. FIELD OF THE INVENTION This invention relates to diagnostic compositions that allow the measurement of analyte concentrations in biological fluids containing hemoglobin. The compositions are based on tetrazolium dye precursors and include the suppression of their reduction induced by hemoglobin. 2. DESCRIPTION OF THE RELATED TECHNIQUE Adipose tissue is one of the most abundant forms of energy storage in the body. It releases stored fatty acids into the circulatory system to be metabolized mainly by the liver. In the process, fat is consumed and energy is released that is made available to the body. Normally, little fat is consumed, the fatty acids are completely metabolized to carbon dioxide and water, and the conversion does not disturb the body's delicate pH balance. However, if insufficient amounts of carbohydrates are present in the body, for example due to diet, then the consumption of fat and the production of fatty acid can increase to potentially dangerous levels. In addition to dieters, insulin dependent patients are vulnerable due to the deterioration of their carbohydrate metabolism. When excess fatty acid is used to supply a demand for energy from the body, then large amounts of acetoacetate, acetone and beta-hydroxybutyrate are produced. These intermediates are known as ketone bodies, and the condition is known as ketoacidosis. Ketone bodies can normally be recycled in other forms by the body, provided they are not overwhelming. Therefore, a healthy individual accumulates a negligible amount of these analytes. When a large amount of fats is metabolized in a relatively short period, or when most of the energy is derived from fats, massive amounts of ketone bodies are produced. Excessive production of these fat metabolites can cause certain neurogenic disorders if the problem is not corrected quickly. Ketone bodies are present in the blood, and if a certain threshold is exceeded, they are excreted through the urine. They are easily detected by a modern clinical analyzer. On average, the percentages of beta-hydroxybutyrate, acetoacetate and acetone are 78%, 20% and 2%, respectively. Due to its relatively low concentration and high volatility, acetone is rarely measured. Instead, acetoacetate is determined quantitatively by a nitroprusside reaction, and beta-hydroxybutyrate is quantified by an enzymatic method. For decades, acetoacetate test strips have been available. They are based on a coupling reaction of nitroprusside ion with aldehydes and ketones. A sample of urine or an alkaline serum specimen is allowed to react with the nitroprusside for a few minutes, and a purplish color develops. The intensity of the color indicates the concentration of acetoacetate. However, acetone interferes with the test, resulting in higher readings. In addition, as the patient recovers from an episode of ketoacidosis, the level of acetoacetate increases in the urine and in the blood, making the diagnosis difficult. The beta-hydroxybutyrate test is most useful for monitoring the concentrations of ketone bodies. It is based on the oxidation of beta-hydroxybutyrate with the corresponding dehydrogenase in the presence of a nicotinamide-adenine dinucleotide cofactor (NAD). (Strictly speaking, only beta-hydroxybutyrate is naturally present and is oxidized, but the authors hereby omit the "D" for brevity throughout this specification and the appended claims). After oxidation, NADH is produced, and its concentration is measured directly with a UV spectrophotometer. Therefore, the corresponding change of signal in the spectrum is proportional to the concentration of the analyte. Unfortunately, NADH excitation occurs in the UV region; thus, this detection mode is suitable only for laboratory instruments. Another method to monitor beta-hydroxybutyrate is the oxidation of NADH with a tetrazolium compound. In general, tetrazolium compounds are very sensitive to strong bases and light. Therefore, special care must be taken to ensure the integrity of these compounds. However, tetrazolium compounds have played an important role in the studies of tissue metabolism. For example, this class of compounds has been used in the analysis of anaerobic oxidation and reduction reactions in cells. In addition, they are commonly used in clinical diagnosis. The compounds are typically colorless or light-colored compounds that undergo a reduction reaction in the presence of a reducing agent, to produce a highly colored formazan. Reductive agents such as ascorbates, sulfhydryls, or variants of NADH, NADPH, and PQQH2 (PQQ-reduced-quinolinequinone-pyrrolo) are capable of forming the dye. In clinical diagnosis, it has been found that these dyes are inappreciable to monitor the formation of NAD (P) H from their compounds of origin, NAD (P) +, in anaerobic reactions. The oxide-reduction reaction is rapid and is not sensitive to oxygen. The color of the resulting dye is very intense and has low solubility in water. In principle, tetrazolium dye precursors can be used to measure ketone bodies and whole blood glucose. However, tetrazolium can be reduced nonenzymatically by hemoglobin (Fe (ll)) to form a colored formazan, if the hemoglobin is not contained within the red blood cells. In this way, free hemoglobin causes serious interference with the measurements. In fact, due to the hemolysis and the resulting abundance of free hemoglobin in relation to the analyte of interest, in a typical measurement of ketone bodies, the interference signal of the hemoglobin could exceed the signal sought. Glucose measurements, particularly at or above normal concentration, are not adversely affected. When the reaction is carried out in samples of high hematocrit, or at a higher temperature, where the oxidation reaction of hemoglobin is faster, interference with glucose measurements is also significant. Since hemolysis of red blood cells, which causes the presence of free hemoglobin, can not be easily avoided, red blood cells should be removed from the samples before the test if tetrazolium is used for analysis. Red blood cells can be removed from the samples by filtering with membranes and filters, by clamping with chemical reagents, or by a combination of both methods. Filtration methods to separate red blood cells from whole blood are expensive and require rather large sample volumes. An example of a ketone test (beta-hydroxybutyrate) in blood that uses filtration to remove red blood cells from a whole blood sample is the KetoSiteR test, available from GDS Diagnostics, Elkhart, Indiana (see "Tietz Textbook of Clinical Chemistry", 2nd edition, edited by C. Burtis et al., WB Saunders Co., Philadelphia, Pennsylvania, 1994, page 974). The "Test Card" card used in that test has two filter layers, which makes the card rather expensive and requires a large sample (25 μl) of blood. In addition, the blood should not be hemolyzed. A combination of filtration and chemical restraint is used in the AmesR Glucometer EncoreT blood glucose strip, available from Miles. That strip uses a layer of filter material and an agglutination aid (potato lectin) to eliminate the interference of red blood cells (see Chu et al., European Patent Application 0 638 805 A2, published February 15, 1995). . Another way to reduce the interference of hemoglobin is the introduction of an oxidizing agent in a system, to oxidize the hemoglobin to methemoglobin. Although it is known that ferricyanides transform hemoglobin into methemoglobin, they also destroy the desired product, NADH.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides a reagent for measuring the concentration of an analyte in a biological fluid containing hemoglobin. The reagent comprises: a) a dehydrogenase enzyme having specificity for the analyte; b) nicotinamide adenine dinucleotide (NAD), a derivative of NAD; pyrrolo-quinolinquinone (PQQ), or a derivative of PQQ; c) a tetrazolium dye precursor; d) a diaphorase enzyme or an analogue thereof, and e) a nitrite salt. The reagent is particularly suitable to be applied as a coating on one or more substrates to form a dry test strip to measure an analyte. A particularly preferred strip comprises: a) a support layer; b) on the support layer, a test pad having a coating comprising: i) a dehydrogenase enzyme having specificity for the analyte; ii) nicotinamide adenine dinucleotide (NAD), a derivative of NAD, pyrrolo-quinolinquinone (PQQ), or a derivative of PQQ; iii) a tetrazolium dye precursor, and iv) a diaphorase enzyme or an analogue thereof, and c) on the test pad, an absorbent top layer that is coated with a nitrite salt.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a perspective view of a test strip of this invention. Figure 2 is a schematic view of another test strip of this invention. Figure 3 is a schematic view of another test strip of this invention. Figure 4 is a graphical representation of the chemistry of a ketone test of this invention. Figure 5 is a graphical representation of the chemistry of a glucose test of this invention. Figure 6 is a graph showing the effect of nitrite as a hemoglobin suppressant in a ketone test. Figure 7 is a graph showing the effect of nitrite as a hemoglobin suppressant in a glucose test.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a reagent for measuring the concentration of analyte in biological fluids containing hemoglobin (such as whole blood), producing a concentration of the reduced form of a cofactor, such as NADH, NAD (P) H, or PQQH2, which it is a measure of analyte concentration. The inclusion of nitrite in the reagent overcomes the interference of hemoglobin with the measurement of the reduced cofactor concentration. It is particularly useful for measuring ketones and glucose, but is not limited to them. Figure 1 represents a test strip 10, typical of the invention, consisting of a test pad 12 fixed on a support 14. The support can be a sheet of plastic-eg, polystyrene, nylon, or polyester- or metallic, or any other material known in the art. The test pad is coated with a reagent that reacts with the analyte to cause a color change. The test pad preferably comprises an absorbent material, such as filter paper or polymer membrane. However, since the reaction does not require oxygen, the test pad may be of a non-absorbent material, such as plastic film. The reagent includes an enzyme that is specific for the analyte, a hydride transfer agent, a tetrazolium dye precursor, a suitable enzyme cofactor, and a hemoglobin suppressant. Optionally, a shock absorber and a stabilizer are included for greater stability. As shown in Figure 2, the test strip can also be a multi-layer construction, with the top layer 16 covering the test pad 12. In that construction, the reagent can be divided between the two layers. For example, the hemoglobin suppressant can be applied as a coating on the optional top layer 16 and the remainder of the reagent can be applied as a coating on the test pad 12. Preferably, the top layer 16 is absorbent and serves as an extension layer. and as an absorbent layer to absorb the excess sample. The sample is applied to the upper layer 16 and passes completely to the test pad 12. The analyte concentration is determined by measuring the color change through the support layer 14 or, if the layer 14 is not transparent where it adjoins with the reaction area, through the optional past window or hole 18. In the alternative embodiment shown in Figure 3, the separator 20 separates the top layer 16 and the test pad 12. The separator 20 is preferably a film of non-absorbent plastic having an adhesive coating (not shown) on both sides. The channel 22 in the separator 20 provides a capillary path for the sample to flow from the opening 24 to the measurement area 26. The flow depends on the air ventilation between a surface of the test pad 12 and an adjacent layer or alternatively, through optional ventilation 18. The color change in the measurement area 26 is monitored through the optional window / vent 18. The entire reagent can be on the test pad 12 or, alternatively, it can be divided between the test pad and one or both non-absorbent layers 14 and 16. In this way, a first part of the reagent can be on the test pad and a second part of the reagent can be on one or both non-absorbent layers. When reference is made here to the reagent being applied as a "coating" or "on" a layer, it is intended to include the possibility that the reagent is absorbed into the layer, particularly if it is absorbent. Enzymes which are suitable for tests with this invention and the corresponding analytes are: alcohol dehydrogenase for alcohol, formaldehyde dehydrogenase for formaldehyde, glucose dehydrogenase for glucose, glucose-6-phosphate dehydrogenase for glucose-6-phosphate, glutamate dehydrogenase for glutamic acid , glycerol dehydrogenase for glycerol, beta-hydroxybutyrate dehydrogenase for beta-hydroxybutyrate, hydroxysteroid dehydrogenase for steroids, L-lactate dehydrogenase for L-lactate, leucine dehydrogenase for leucine, malate dehydrogenase for malic acid, and pyruvate dehydrogenase for pyruvic acid. A suitable enzyme cofactor is required to activate the enzyme. Depending on the enzyme, these cofactors can be used: beta-nicotinamide adenine dinucleotide (beta-NAD), beta-nicotinamide adenine dinucleotide phosphate (beta-NADP), thionicotinamide adenine dinucleotide, thionicotinamide dinucleotide phosphate -adenine, nicotinamide-1 dinucleotide, N-6-ethenoadenine, nicotinamide-1 dinucleotide phosphate, N-6-ethenoadenine, and pyrrolo-quinolinquinone (PQQ). In the presence of the enzyme, the analyte reduces the cofactor. The next step in the dye formation process is the separation of the hydride from the reduced cofactor. It can be carried out by means of a diaphorase, such as lipoic dehydrogenase, ferredoxin-NADP reductase, lipoamide dehydrogenase, or by means of an analog such as phenazine methosulfate (PMS) or Meldola blue. The kinetics of the reaction and stability are the main factors for selecting a hydride transfer agent or "attractant". For example, PMS is the universal hydride attractant, because it has a relatively rapid reaction kinetics with most of the tetrazolium compounds mentioned below. For that reason, it is preferred when the cofactor is PQQ. However, PMS is more sensitive to light than enzyme-based hydride attractants. Diaphorase is more stable and for that reason it is preferred when the cofactor is NAD. The captured hydride is transferred to a tetrazolium compound (dye precursor) to form a colored formazan. The tetrazolium compounds that are most suitable for this device are: 2- (2'-benzothiazolyl) -5-styryl-3- (4, -phatalylhydrazidyl) tetrazolium (BSPT), 2-benzothiazolyl- (2) -3,5 -diphenyl tetrazolium (BTDP), 2,3-di (4-nitrophenyl) tetrazolium (DNP), 2,5-diphenyl-3- (4-styrylphenyl) tetrazolium (DPSP), tetrazolium blue-tetrahydroxy (DS-NBT), S. S'-rAS'-dimethoxy- (1,1-biphenyl) -4,4, -diyl] bis [2- (4-nitrophenyl) -5-phenyl (2H-tetrazolium (NBT), 3- (4, 5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium (MTT), 2-phenyl-3- (4-carboxyphenyl) -5-methyltetrazolium (PCPM), tetrazolium blue (TB), thiocarbamylnitroazole tetrazolium (TCNBT), tetrazolium tetrazolium blue (TNBT), tetrazolium violet (TV), 2-benzothiazothiazolyl-3- (4-carboxy-2-methoxyphenyl) -5- [4- (2-sulfoethylcarbamoyl) phenyl] -2H- methoxyphenyl) -5- [4- (2-sulfoethylcarbamoyl) phenyl] -2H-tetrazolium (WST-4), and 2,2'-dibenzothiazolyl-5-5, -bis [4-di (2-sulfoethyl) carbamoylphenyl] - S.S'-SS.S'-dimethoxy-4-biphenylenetetrazolium, disodium salt (WST-5). WST-5 is preferred because it dissolves rapidly in an aqueous medium, which is more compatible with biological samples. In addition, the resulting formazan compound exhibits strong spectral absorption in the purple-blue region, thus reducing the need to correct the base signal of hemoglobin. Finally, a hemoglobin suppressant is present in the reagent to restrict the inconvenient reaction of dye formation between the hemoglobin and the tetrazolium compound. The function of the hemoglobin suppressor is to oxidize hemoglobin to methemoglobin, which does not react with tetrazolium or formazan. Surprisingly, nitrite salts such as sodium nitrite, potassium nitrite, and their derivatives, are very effective in suppressing hemoglobin, without destroying the reduced cofactor (such as NADH or PQQH2). Nitrites are also effective at high temperature and with samples of high hematocrit. Sodium nitrite is preferred because it has high solubility in water, is non-toxic and is relatively inexpensive. Although the reagent of this invention can be used in a wet chemical mode such as in a cuvette, in preferred embodiments, the invention provides dry strips for analyzing beta-hydroxybutyrate or glucose in whole blood. A strip consists of a test membrane pad, preferably of nylon, which is placed between a support and an upper layer. The support is preferably made of polyester sheet. The top layer can be any absorbent material known in the art. A preferred material is a porous polyethylene treated with sodium methyl oleoyltaurate, available from Porex Corp. of Fairbum, Georgia E.U.A. In the present description, this material is referred to as "Porex". The test pad contains a reagent comprising beta-hydroxybutyrate dehydrogenase (or glucose dehydrogenase), NAD (or PQQ), diaphorase (or PMS), and WST-5 (Table 1 (or 3) below). The upper layer of Porex contains a nitrite reagent (Table 2). During the operation, a user applies a drop of whole blood to the upper surface of the top layer of the Porex. As whole blood or lysed blood comes into contact with Porex, sodium nitrite is reconstituted and reacts with available free hemoglobin, thus making the hemoglobin safe for testing. The resulting sample, substantially free of hemoglobin, is transferred to the test pad downwards, by capillary action or gravitational force. On the test pad, the sample initiates the reaction in cascade to produce a dye, whose concentration is proportional to the concentration of analyte in the sample and can be determined directly with a photometer. Figure 4 represents the reaction for beta-hydroxybutyrate, using enzyme, NAD and diaphorase. Figure 5 depicts the reaction for glucose, using enzyme, PQQ and PMS. Figure 6 represents the change in optical density of the blood samples over time, all having 55% hematocrit and containing 0 to 15 mg / dl of beta-hydroxybutyrate, both with nitrite and without nitrite. The NAD system was used and the nitrite concentration was 5 g / dl. In the absence of nitrite, hemoglobin reduces tetrazolium to form a continuously increasing dye concentration, with a corresponding increase in optical density. By removing hemoglobin (by oxidation), nitrite limits the formation of color to that which originates only from the ketone bodies (ie, beta-hydroxybutyrate) in the sample. The preparation of the strip that was used to generate the data represented in the graph is described later in Example 1. Figure 7 shows the effect of nitrite on the color formation reaction in the glucose / PQQ system. The blood samples, which all had 60% hematocrit, contained 0 or 100 mg / dl of glucose and 0 or 5 g / dl of nitrite. Glucose-free samples were developed at 35 ° C. The graph shows that this system is effective at temperatures of up to 35 ° and hematocrit of up to 60%. The preparation of the strip that was used is described below in example 2. The following example demonstrates a preferred embodiment of the present invention, in which the analyte is beta-hydroxybutyrate and the enzyme is beta-hydroxybutyrate dehydrogenase. The composition can be easily modified for application to other enzyme-analyte combinations mentioned above (see, for example, "Tietz Textbook of Clinical Chemistry, 2nd edition, edited by C. Burtis et al., W. B. Saunders Co., Philadelphia, Pennsylvania, 1994, page 976-978 and 1174-1175). The example is not constructed to be limiting in any way.

EXAMPLE 1 A 0.8 μm nylon membrane, obtained from Cuno (Meriden, Connecticut, E.U.A.) in the reagent of Table 1, was immersed until saturation. The excess reagent was removed by scraping gently with a glass rod. The resulting membrane was suspended to dry in an oven at 56 ° C for 10 minutes. Porex (0.6 mm thick) was soaked in the nitrite solution of Table 2 and then suspended to dry in an oven at 100 ° C for ten hours. Finally, the membrane was laminated between a polyester handle (0.4 mm Melenex® polyester from ICI America, Wilmington, Delaware, USA) and Porex impregnated with nitrite.

TABLE 1 Reagent for the test pad TABLE 2 Nitrite reagent TABLE 3 Reagent for a glucose test pad

Claims (10)

NOVELTY OF THE INVENTION CLAIMS

1. - A reagent for measuring a concentration of an analyte in a biological fluid containing hemoglobin, comprising: a) a dehydrogenase enzyme having specificity for the analyte; b) nicotinamide adenine dinucleotide (NAD), a derivative of NAD; pyrrolo-quinolinquinone (PQQ), or a derivative of PQQ; c) a tetrazolium dye precursor; d) a diaphorase enzyme or an analogue thereof; and e) a nitrite salt.

2. The reagent according to claim 1, characterized in that the analyte is beta-hydroxybutyrate and the enzyme is beta-hydroxybutyrate dehydrogenase.

3. The reagent according to claim 1, characterized in that the analyte is glucose and the enzyme is glucose dehydrogenase

4. A dry test strip to determine the presence and amount of an analyte in a biological fluid containing hemoglobin, which comprises a support layer on which is a test pad having a coating of the reagent of claim 1.

5. A dry test strip for determining the presence and amount of an analyte in a biological fluid containing hemoglobin, which it comprises a support layer on which is a test pad, and an upper layer covering the test pad, in which a first part of the reagent of claim 1 is on the test pad, and a second part of the reagent is on the support layer and / or the top layer.

6. The strip according to claim 5, further characterized in that it comprises a separator and a channel between the upper layer and the test pad, to provide a capillarity route between the upper layer and the pad.

7. The strip according to claim 5, characterized in that the analyte is beta-hydroxybutyrate and the enzyme is beta-hydroxybutyrate dehydrogenase.

8. The strip according to claim 5, characterized in that the analyte is glucose and the enzyme is glucose dehydrogenase.

9. The strip according to claim 5, characterized in that the tetrazolium dye precursor is the disodium salt of 2,2'-dibenzothiazolyl-5,5, -bis [4-di (2-sulfoethyl) carbamoylphenyl ] -3,3 '- (3,3, -dimethoxy-4,4, -biphenylene) ditetrazolium (WST-5).

10. A dry reactive test strip for determining the presence and amount of an analyte in a biological fluid containing hemoglobin, comprising: a) a support layer; b) on the support layer, a test pad having a coating comprising: i) a dehydrogenase enzyme having specificity for the analyte, i) nicotinamide adenine dinucleotide (NAD), a derivative of NAD, pyrrolo- quinoxyquinone (PQQ), or a derivative of PQQ, iii) a tetrazolium dye precursor, and iv) a diaphorase enzyme or an analogue thereof; and c) on the test pad, an absorbent top layer that is coated with a nitrite salt.