BIOCHEMICAL MEDICAL DIAGNOSTIC KIT

Abstract

Disclosed is a deamidation rate measurement kit, which includes a stock solution of the compound Boc-Asn-Gly-OEt in deionized/distilled water, and a stock solution of the compound Boc-Asp-Gly-OEt in deionized/distilled water.

Claims

1. A deamidation rate measurement kit comprising a stock solution in deionized/distilled water containing Boc-Asn-Gly-OEt compound and a stock solution in deionized/distilled water containing Boc-Asp-Gly-OEt compound.

2. A kit according to claim 1, wherein the stock solution in deionized/distilled water containing the Boc-Asn-Gly-OEt compound is in the concentration range of 0.000001-1000 mM.

3. A kit according to claim 2, wherein the concentration is 0.25 mM.

4. A kit according to claim 1, wherein the stock solution in deionized/distilled water containing Boc-Asp-Gly-OEt compound is in the concentration range of 0.000001-1000 mM.

5. A kit in accordance with claim 4, wherein the concentration is 0.25 mM.

6. A rate measurement method made with a deamidation rate measurement kit in accordance claim 1, comprising the steps of: preparation of stock solution and serial dilutions of Boc-Asn-Gly-OEt compound in deionized/distilled water; preparation of stock solution and serial dilutions of Boc-Asp-Gly-OEt compound in deionized/distilled water; creation of a calibration curve on the instrument to be measured using serial dilutions of the Boc-Asn-Gly-OEt stock solution; creation of the calibration curve on the instrument to be measured using serial dilutions of the Boc-Asp-Gly-OEt stock solution; aliquoting the biological sample whose effect on the deamidation rate will be tested in equal volumes in three different tubes; adding Boc-Asn-Gly-OEt stock solution to one of the sample tubes, Boc-Asp-Gly-OEt (as an internal standard) stock solution to the other, and deionized/distilled water to the last one as the blank, in the range of 1/1000-1/2 of the final reaction volume; making measurements from all three tubes at the beginning of the measurement; measurement of all three tubes after incubation; subtracting the measurement results in the tubes with added Boc-Asn-Gly-OEt and Boc-Asp-Gly-OEt from the measurement in the tube with added deionized/distilled water; determination of the deamidation rate by measuring Boc-Asp-Gly-OEt, which is formed as a result of spontaneous deamidation in the tube with added Boc-Asn-Gly-OEt.

7. The method according to claim 6, wherein the measurements are made by LC-MS method.

8. The method according to claim 6, wherein the reaction volume is 1/10.

Description

FIGURES TO HELP EXPLAIN THE INVENTION

[0015] FIG. 1: Structure of the Boc-Asn-Gly-OEt compound

[0016]

TABLE-US-00001 Explanation of References 1 Boc- 2 Asn 3 Gly 4 -Oet 5 Region being deamidated

DETAILED EXPLANATION OF THE INVENTION

[0017] In this detailed explanation, the deamidation rate measurement kit, which is the subject of the invention, is explained only for a better understanding of the subject and without causing any limiting effect.

[0018] The deamidation rate measurement kit, which is the subject of the invention, in its most basic form; contains stock solution in deionized/distilled water containing the compound Boc-Asn-Gly-OEt (preferably in the range of 0.000001-1000 mM, more preferably at the concentration of 0.25 mM) and stock solution in deionized/distilled water containing the compound Boc-Asp-Gly-OEt (preferably 0.000001-1000 mM) mM, more preferably at a concentration of 0.25 mM).

[0019] The Boc-Asn-Gly-OEt compound, a sample view of which is given in FIG. 1, is used both as a calibrator and analytical standard in measurements, and as a deamidated element by being added to biological samples. Boc- (1) and -Oet (4) are for blocking Asparagine (Asn) (2) and glycine (Gly) (3) dipeptides respectively, as details shown in the FIGURE. In the deamidated region (5) shown in the FIGURE, the NH2 structure is removed by deamidation, while the OH structure is attached instead. Thus, the Boc-Asn-Gly-OEt compound turns into the Boc-Asp-Gly-OEt compound and its molecular weight increases by about 1 (0.98) g/mol. Boc- (1) and -OEt (4) structures-in the structure of the Boc-Asn-Gly-OEt compound in solutionallow to block the binding of Asn (2) and Gly (3) dipeptides with unwanted chemical groups during the process. The Asn (2) and Gly (3) dipeptides are one of the structures with the fastest spontaneous deamidation known. For this reason, it is used in measurements as as the calibrator/analytical standard or the compound subject to deamidation after adding to biological samples. Since Boc-Asn-Gly-OEt is converted into Boc-Asp-Gly-OEt compound as a result of spontaneous deamidation, Boc-Asp-Gly-OEt compound is used as Calibrator and analytical standard in drawing the calibration curve. The Boc-Asp-Gly-OEt compound is not commercially available but has been specially synthesized. Deionized/distilled water allows to dissolve Boc-Asn-Gly-OEt and Boc-Asp-Gly-OEt compounds and is used as analytical blank.

[0020] In a sample application of the invention, the deamidation rate measurement kit developed includes the following steps of process: Preparation of stock solution and serial dilutions of Boc-Asn-Gly-OEt compound in deionized/distilled water; Preparation of stock solution and serial dilutions of Boc-Asp-Gly-OEt compound in deionized/distilled water; Creation of the calibration curve on the instrument to be measured using serial dilutions of the Boc-Asn-Gly-OEt stock solution; Creation of the calibration curve on the instrument to be measured using serial dilutions of the Boc-Asp-Gly-OEt stock solution; aliquoting the biological sample whose effect on the deamidation rate will be tested in equal volumes in three different tubes; additionpreferably in the range of 1/1000-1/2 of the final reaction volume (more preferably in the range of 1/10)of Boc-Asn-Gly-OEt and Boc-Asp-Gly-OEt (as an internal standard) stock solutions to different tubes, and of deionized/distilled water to another tube as blank; at the beginning of the measurement, making measurements from all three tubes, preferably by LC-MS method; after incubation making measurements of all three tubes, preferably by LC-MS method; subtraction of the measurement results in the tubes with added Boc-Asn-Gly-OEt and Boc-Asp-Gly-OEt from the measurement in the tube with deionized/distilled water; determination of deamidation rate by measuring Boc-Asp-Gly-OEt resulting from spontaneous deamidation in the tube with added Boc-Asn-Gly-OEt.

[0021] The present invention is based on the principle of spontaneous deamidation of Boc-Asn-Gly-OEt compound in solution into Boc-Asp-Gly-OEt compound and measuring the resulting Boc-Asp-Gly-OEt compound by an appropriate analytical method. Boc-Asn-Gly-OEt solution is added to any sample whose effect on the spontaneous deamidation rate is desired to be measured. Spontaneous deamidation of Boc-Asn-Gly-OEt in this mixture, which is kept at a temperature between 1-100? C. for 1 minute-1 month, is ensured. During the incubation period, as a result of the deamidation of the Boc-Asn-Gly-OEt dipeptide in the mixture, theNH.sub.2 group [FIG. 1:(5)] will spontaneously be released from the molecule, while the asparagine in the compound will be converted to isoaspartate or aspartate with the addition of the OH group, and a molecular weight increase of approximately 1 (0.98) g/mol mass will occur. This change in mass, that is, the resulting Boc-Asp-Gly-OEt and Boc-lsoasp-Gly-OEt can be measured by mass spectrometry, as well as with all kinds of optimized methods based on the different chemical properties of the NH.sub.2 group in the asparagine of the compound and the OH group of the newly formed aspartate in the compound.

[0022] By measuring the effect of the biological samples from individuals/patients on the deamidation rate by using the deamidation rate measurement test, which is the subject of the present invention, and by examining the correlation of individual differencesto be detected and that affect the deamidation ratewith aging and age-related diseases, and if diseases characterized by an increase in deamidation rate are detected, the test is provided to be used as a marker to determine the susceptibility to these diseases and for their early diagnosis.