Glucose 6 phosphate dehydrogenase kit

Abstract

The invention relates to a kit for measuring activity of Glucose 6 phosphate dehydrogenase (G6PD) enzyme, suitable for use in hospital biochemistry laboratories.

Description

[0001] GLUCOSE 6 PHOSPHATE DEHYDROGENASE KIT

[0002] Technical Field

[0003] The invention relates to a kit for measuring activity of Glucose 6 phosphate dehydrogenase (G6PD) enzyme, suitable for use in hospital biochemistry laboratories.

[0004] State of the Art

[0005] Glucose 6 phosphate dehydrogenase (G6PD) is the most commonly enzyme deficiency in the world. Approximately 400 million people worldwide have low levels of the enzyme. Since G6PD is an enzyme that protects blood cells from oxidative stress, when this enzyme deficiency is present, erythrocytes become unprotected against oxidants formed by certain drugs and certain foods such as beans. As a result, erythrocytes break down easily. This case, called intravascular hemolysis, leads to consequences ranging from chronic anemia to life-threatening cases.

[0006] There are various kits measuring G6PD activity that are used in the technical field. However, the G6PD kits have some disadvantages.

[0007] Kit In laboratory, autoanalyzer kits usually comprise 2 separate reagents and they are liquid. There is no need for any dilution. However, current G6PD kits either require dilution or contain more than two reagents and require manual mixing before use. This wastes time and makes standardization difficult. the kits available in the technical field, the samples taken from the patient cannot be studied easily by giving them directly to the device, it is necessary to prepare hemolysate from the sample with lyse solution, and G6PD level can be measured only after this pre-treatment. This causes loss of enzyme activity, loss of time and additional cost, and also leads to a departure from standard measurement.

[0008] Interference Preventing Chemical Many kits included in the state of the art do not use inhibitors to eliminate faulty measurements.

[0009] Result Calculation Kits available in the technical field have a complex way of calculation. A patent and literature search for the state of the art identified a patent application CN108893521 A. The application describes a kit and detection method for the detection of erythrocyte glucose- 6-phosphate dehydrogenase. The negative aspects and differences of the method are summarized below:

[0010] • In this method, preliminary sample processing is necessary. The sample must be hemolyzed with distilled water before measurement.

[0011] • There are 3 reagents in this method. These are R1 , R2 and buffer. Two sets of kits are created, as R1 -buffer and R2-buffer, and two separate measurements are made with the two sets of kits and the results obtained are subtracted from each other.

[0012] • The kit described in the document indirectly proves the lack of activity of the G6PD enzyme. G6PD deficiency is attempted to be proved by comparing it to the 6PGD enzyme, which is in the same pathway as the G6PD enzyme and whose deficiency is very rare. As a result, the kit does not give a quantitative result, and it is assumed that the other enzyme is always intact, and it is tried to prove whether there is a G6PD enzyme deficiency compared to it.

[0013] • The kit described in the document is a kit that is not applied to an autoanalyzer and is manually run on a spectrophotometer and that the results obtained must be calculated manually.

[0014] • The kit described in the document is a manual screening kit that is manually run using large kit volumes and provides qualitative results as deficiency is present or absent.

[0015] As a result, a development in the relevant technical field has become necessary due to the above- mentioned drawbacks and the inadequacy of the existing solutions.

[0016] Object of Invention

[0017] The present invention relates to a programmable kit te measuring the level of the Glucose 6 phosphate dehydrogenase (G6PD) enzyme, which meets the above-mentioned requirements, eliminates all disadvantages and brings some additional advantages.

[0018] The invention is inspired by present situations and aims to solve the above-mentioned drawbacks.

[0019] The main object of the invention is to develop a kit that does not require both kit preliminary preparation and sample preliminary preparation steps. In the inventive kit, blood taken from the patient can be measured directly in the autoanalyzer by means of a ready-to-use kit. In order for the kits to be used directly ready-to-use without the need for dilution or mixing, a number of preservative chemicals in different concentrations have been added to extend the expiry date. In the kit, the sample preliminary preparation step is not done outside manually and using separate chemicals. This process is done with reagent number 1 , which is used for measurement purposes. Reagent number 1 contains both the chemicals needed for measurement and the chemicals needed to hemolyzing the blood. Therefore, there is no need to hemolyzing the blood outside the device and using different chemicals.

[0020] The reaction chain takes place from glucose 6 phosphate to ribulose 5 phosphate

[0021] Another object of the invention is to develop a kit to precisely measure the level of the glucose 6- phosphate dehydrogenase (G6PD) enzyme. As a result of the reaction of the inventive kit, 6- Phosphogluconate (6-PG) is formed as a product. This product is the substrate of the next step in the Hexose Monophosphate pathway. This step also produces NADPH like the step realized in the invention. In the invention, it is given the result according to the amount of NADPH produced in the G6PD step. In the kits used in the technical field, the amount of NADPH from this step causes inaccurate measurement. In order to eliminate this mistake, the enzyme catalyzing this step must be inhibited. In the inventive kit, a specific inhibitor (malemide) of 6-phosphogluconate dehydrogenase (6-PGDH), the enzyme of this step, is used. Thus, it eliminates the interference that will come from the next step.

[0022] In the principle of using the inventive kit, the whole blood sample is given directly to the device and our R1 reagent acts both as a reagent and a lyse. When hemolysate is formed outside the device with Lyse solution, the G6PD enzyme activity in the hemolysate starts to decrease in a short period of half an hour. This creates the need for rapid measurement. This requirement to work in a short time cannot always be fulfilled within the routine intensity of the laboratories and leads to incomplete measurement results. However, since there is no need for hemolysate preparation in the inventive kit, these activity losses are also not occurred.

[0023] The inventive kit contains the maleimide chemical which is not included in many kits and eliminates faulty measurement and which is a specific 6-Phosphoglucanote dehydrogenase (6PGD) inhibitor. This eliminates interference from the next reaction step.

[0024] In the inventive kit, the result obtained from the device alone is calculated by automatically dividing the patient's hemoglobin value in the LIS (Laboratory Information System) environment and the result is given as lU / g hemoglobin.

[0025] The differences of the invention from the document in the state of the art are summarized below:

[0026] • The kit subject to our application can be run directly without preliminary sample processing.

[0027] • In the kit subject to our application, a single measurement is sufficient for the result.

[0028] • The kit subject to our application has features that can be applied to all biochemistry autoanalyzers, in which a large number of samples can be serially finalized, and results can be calculated automatically.

[0029] • The kit used in our application and autoanalyzers with sample volumes of very low microliters automatically and serially proves the quantitative enzyme activity.

[0030] The structural and characteristic features and all advantages of the invention will be more clearly understood with the detailed description given below, and therefore the evaluation should be made by taking into account the detailed description.

[0031] Detailed Description of the Invention

[0032] In this detailed description, the preferred embodiments of the inventive kit are described only for the purpose of a better understanding of the subject matter. The invention relates to a kit for measuring the level of the Glucose 6 phosphate dehydrogenase (G6PD) enzyme, suitable for use in hospital biochemistry laboratories.

[0033] In the production method of the inventive kit, a pH=pKa tris buffer is prepared at room temperature using trisma acid and base chemicals. The pH of the prepared tris buffer is measured. After the measurement is performed, the buffer is divided into 2 different volumetric flasks. In the next process step, the substances required for the first reagent are prepared in appropriate molarities (MgSO4, albumin, Triton-X, NADP, maleimide, sodium azide, respectively) and added to one of the volumetric flasks. After adding these substances, the volumetric flask is stirred in a magnetic stirrer preferably at room temperature for 1 hour. The substances required for the second reagent are prepared in appropriate molarities (Glucose-6-phosphate, albumin, Triton-X, sodium azide, respectively) and added to the other volumetric flask. After adding these substances, the volumetric flasks are stirred in a magnetic stirrer preferably at room temperature for 1 hour. In the final step, the product is now ready by separately placing the produced Reagent- 1 and Reagent- 2 to plastic kit boxes.

[0034] The reagents obtained by the inventive method are preferably stable for 1 year at +2-8 G. The environment in which the sample and reagents will be mixed and the measurement will be performed is preferably standardized to 37 G.

[0035] The components included in the inventive kit and the functions of the components are given below.

[0036] • Tris Buffer: provides the appropriate pH environment for the enzyme to carry out its chemical reaction. Enzymes can catalyze reactions at certain pH levels. Tris buffer has also been used to ensure this.

[0037] • NADP: Some coenzymes are needed for enzymes to carry out their reactions quickly. The NADP coenzyme used here has been used to provide this speed. • MgS04: Enzymes need some cofactors to catalyze their reactions. Without these cofactors, the reaction cannot take place at optimum speed. MgSO4 is the cofactor used to ensure this optimum speed.

[0038] • Albumin acts as a detergent and reduces the surface tension and ensures that the reaction is more efficiently performed.

[0039] • Triton X-100 is used to stabilize the ionic strength by acting as a detergent.

[0040] • Na Azide prevents the growth of fungi and bacteria in the kits and ensures a longer reagent life and prevents contamination.

[0041] • Glucose 6 Phosphate is substrate in this reaction. Enzymes are specific for their substrates. This substrate allows us to measure the activity of the correct enzyme.

[0042] • Maleimide: This chemical is a specific inhibitor of the 6-Phosphogluconate dehydrogenase (6PGD) enzyme. By means of this inhibitor, the production of NADPH from this step is stopped and only the activity of the G6PD enzyme is measured.

[0043] In different embodiments of the invention, different detergents such as Brij instead of Triton X- 100 can be used. However, this situation may reduce the efficiency of the activity. It may require a number of trials to standardize this.

[0044] In the embodiment of the invention, whole blood samples must be homogenized very well before being studied. For this, it is sufficient to invert the tubes 4-5 times without shaking. If the number of samples is high, a roller device for mixing can be used. Erythrocytes in whole blood samples settle to the bottom over time in the tubes with EDTA. This can cause false results in the kit that measures G6PD in erythrocytes. To avoid this, samples should be well homogenized and should be run without waiting.

Claims

CLAIMS1 . A kit for measuring level of Glucose 6 phosphate dehydrogenase enzyme, characterized in that the kit is obtained by a method comprising following process steps:• preparation of tris buffer,• dividing the tris buffer into 2 volumetric flasks,• adding MgSO4, albumin, Triton-X, NADP, maleimide, sodium azide respectively for the first reagent to one of the volumetric flasks and then mixing,• adding Glucose-6-phosphate, albumin, Triton-X, sodium azide respectively for the second reagent to the other volumetric flask and then mixing,• placing the first reagent obtained from the first volumetric flask and the second reagent obtained from the other volumetric flask in separate boxes.

2. The kit according to claim 1 , characterized in that the kit is prepared at room temperature in such a way that pH=pKa using acid and base chemicals in the preparation of tris buffer step.

3. The kit according to claim 1 , characterized in that the kit is prepared using magnetic stirrer for mixing processes.

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