JP2001021559A - Pretreatment method for measuring protein-coupled saccharified protein by enzyme immunoassay - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a pretreatment method for easily and accurately measuring a protein-coupled saccharified protein in a living body. SOLUTION: For this simple and quick pretreatment method, a proteolytic enzyme such as pronase or protinase-K is used, and an enzyme reaction is effected at a relatively high temperature (about 550 deg.C) so as to reduce the reaction time. In enzyme immunoassay, a pretreatment reagent is used in deactivating residual enzyme activity that may cause damage to an antibody and an antigen, by boiling in boiled water for about ten and several minutes. In the method, the time required to bring about a protein decomposition reaction by means of an enzyme and deactivate the enzyme is two hours or less, and a sample pretreated by the simple method in a short time can be put to precise analysis by anzyme immunoassay.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method of reacting a protein saccharification reaction product with a protease at a high temperature for a short time and then inactivating it in boiling water, and subjecting the specimen to enzyme immunoassay. The present invention relates to a pretreatment method for simply and accurately measuring a protein-bound glycated protein in a living body.

[0002]

2. Description of the Related Art Since the discovery that Maillard heats amino acids and reducing sugars to produce a yellow-brown pigment that affects fragrance and storage, protein saccharification has long been regarded as important in the field of food chemistry. Was. Recently, it has been noted in the medical field that hemoglobin A1c, which is an early product of a protein glycation reaction, has been revealed to be useful as a marker for controlling blood sugar, and that a late product of a protein glycation reaction ( Academic papers suggesting that AGEs are deeply involved in the development and progression of nephropathy and diabetic complications have been published. As an initial reaction product, glycoalbumin and fructosamine are used as indicators of blood glucose control in addition to hemoglobin A1c, and the measurement methods are also high performance liquid chromatography, immunological measurement, colorimetry, affinity chromatography. There is a photographic method. Furthermore, because the components to be detected differ depending on the measurement method and the measurement values vary depending on the measurement facility, the Diabetes Society has been standardizing the work for many years. Late reaction products such as pentosidine, carboxymethyl lysine, pyralin and croslin have been reported, and there is strong evidence strongly suggesting that there is a close correlation between the deposition of these products and various tissue disorders. Many have been reported. Pentosidine has been shown to be deposited in the brain of patients with Alzheimer's disease and in the tubules of patients with renal failure.In addition, the blood level of patients with rheumatoid arthritis and renal failure has been significantly increased. Is strongly correlated with the progress of the disease state, and is expected to be used as a diagnostic marker. Since these late-stage reaction products are found to be deposited on the peripheral nerves, lens, and kidneys of diabetic patients, they are attracting attention as one of the factors for the onset and progression of diabetic complications. It is expected that product control will lead to prevention of complications. Therefore, establishing a measurement method is an urgent issue.

[0003] Since the late reaction products are bound to proteins in vivo, their measurement requires complicated pretreatments and less common measuring instruments. Among the late reaction products, only pentosidine, for which a quantitative method has been established, precipitates plasma proteins with trichloroacetic acid, and after washing twice, hydrolyzes with 6N hydrochloric acid at 110 ° C. for 17 hours under nitrogen gas filling. I do. After that, the hydrochloric acid was evaporated under reduced pressure, dried and removed,
It must be diluted with distilled water and measured by fluorescence high performance liquid chromatography. In the case of carboxymethyl lysine, plasma is precipitated as in the case of pentosidine,
After washing, hydrolysis with 6N hydrochloric acid and removal of hydrochloric acid, followed by measurement by gas chromatography / gas spectrometry or measurement by an amino acid analyzer have been reported.

Since the measurement of late reaction products requires complicated pretreatment and special analytical equipment, a large number of polyclonal and monoclonal antibodies against late reaction products have been obtained for the purpose of simple enzyme immunoassay analysis. However, only some antibodies are used for immunostaining,
It has not been established as an enzyme immunoassay. For this reason, research on protein saccharification reaction products is being conducted in a limited number of research institutes, despite its importance being recognized. Despite the fact that antibodies have been obtained, the biggest reason that enzyme immunoassay has not been established is that the late reaction product of protein glycation crosslinks with protein molecules in vivo, so it is necessary to measure with enzyme immunoassay. Pretreatment for exposing late reaction products to the surface of protein molecules is necessary, but a simple method for that purpose has not been developed.

[0005]

With respect to methods for exposing late reaction products bound to proteins to the surface of protein molecules, hydrolysis methods, proteolytic enzyme methods, alkali treatment methods and the like have been reported. With regard to the hydrolysis method, a method has been reported in which plasma protein is hydrolyzed at 6 ° C. and 110 ° C. under nitrogen gas sealing, neutralized with 3N sodium carbonate, filtered through a microfilter, and then measured by enzyme immunoassay. (Nephrology, Dialyss, Transplantation 14, 576-580, 1999). For the proteolytic enzyme method, plasma proteins were treated with 2% pronase E at 37%.
C. for 24 hours, followed by removal of pronase E by a centrifugal membrane filter and measurement by enzyme immunoassay (Clinical Chemistry Vol. 40, vol.
1766-1773, 1994). The former is the same as the pretreatment method in the case of the liquid chromatography method. The operation is complicated and the hydrolysis reaction takes 17 hours, and the treatment before and after takes a long time. The latter takes 24 hours for the enzymatic reaction. And
Furthermore, both have problems in speed, throughput, and simplicity in that expensive filters are used to remove enzymes, and there are difficulties in practicality. There was a demand for a suitable processing method.

[0006]

The present invention uses a protease as a simple and rapid pretreatment method. In this case, the most important problem is to convert a protein saccharification reaction product bound to a protein into a protein molecule. It is the setting of reaction conditions for complete exposure to the surface, and how to remove or inactivate the enzyme after the reaction. Regarding the setting of reaction conditions, an enzyme reaction temperature, a reaction time, an amount of the enzyme, and an enzyme inactivation method instead of the enzyme removal method were examined. These studies are indispensable conditions in the development of diagnostic agents that require rapidity and simplicity. The enzyme reaction was assayed by a competitive enzyme immunoassay using an anti-pentosidine-rabbit polyclonal antibody and an anti-carboxymethyl lysine-rabbit polyclonal antibody developed by the inventors of the present invention and a high performance liquid chromatography method.

[0007] The anti-pentosidine antibody was obtained by culturing a reaction solution containing lysine, arginine and ribose for 2 months, and separating it by ion exchange resin chromatography and gel filtration. The pentosidine fraction was collected by high performance liquid chromatography and purified, and the pentosidine was bound to the carrier protein, and then administered to rabbits. After 8 weeks, the antiserum obtained by sacrifice was purified by affinity chromatography to obtain antibodies. did. Anti-carboxymethyl lysine antibody was prepared by dissolving sodium cyanide borate and carrier protein in borate buffer, and adding a glyoxylic acid solution while stirring under ice-cooling to administer carboxymethyl lysine to rabbits for 8 weeks. Antibodies were obtained from the antiserum obtained by sacrifice later. For both anti-pentosidine antibody and anti-carboxymethyl lysine antibody, an enzyme immunoassay system was established by a competitive method and used for assaying the reaction of proteolytic enzymes.

[0008] The assay by high performance liquid chromatography was carried out by the following method. The protein in the plasma obtained by adding trichloroacetic acid was evaporated to dryness under reduced pressure, 6N hydrochloric acid was added, the mixture was purged with nitrogen, and then hydrolyzed at 120 ° C. for 24 hours. Further, it was dried under reduced pressure and redissolved in distilled water to obtain a sample for high performance liquid chromatography. Analysis by high performance liquid chromatography was performed under the following measurement conditions: mobile phase: methanol / 7 mM phosphate buffer = 1/99, column: ODS-80TM, fluorescence wavelength: excitation wavelength 335 nm, fluorescence wavelength: 385 nm.

As a means for exposing a protein saccharification reaction product present by binding to a protein to the surface of a protein molecule, a protease was used. Known proteolytic enzymes are plasmin, plasminogen, thrombin, etc., which are produced by extracting from plasma, and pronase, proteinase K, etc., which are produced by extracting from microorganisms.
Usually, the reaction temperature of these enzymes is 37 ° C. However, in order to carry out the reaction in a short time, the enzymes and a biological sample such as plasma and urine are added to a Tris-HCl buffer containing calcium chloride, and the reaction is carried out at high temperature (50 (−60 ° C.). By reacting at a high temperature, it became possible to completely expose the product of the protein saccharification reaction to the surface of the protein molecule in only 1-3 hours from the decomposition reaction which usually required one day and night.

Since the protease itself affects the antibody, the protease must be removed or inactivated after the reaction. For example, there is a method in which enzymes are removed by high-speed centrifugation using a microtube equipped with an ultrafiltration membrane.However, these products are expensive, and depending on the amount of protein in plasma, centrifugation is required. In some cases, it cannot be completely removed by separation, and in this case, it causes variation in measured values in the enzyme immunoassay. On the other hand, the enzyme can be easily deactivated in a short time (5 to 15 minutes) by heating in boiling water (95 ° C. or higher). When performing a competitive reaction between the reaction solution after heating and the antibody, it is necessary to perform the reaction at 4 ° C. for a long time with a protein denaturing agent such as a surfactant because the influence on the antibody is concerned. In the present invention, the competitive reaction can achieve its purpose even at 37 ° C. for 1 hour, and does not affect the measured value at all even when used in an enzyme immunoassay system.

[0011]

DETAILED DESCRIPTION OF THE INVENTION Final concentration of 10-50 mg / ml
A reaction solution obtained by adding a biological sample such as plasma to a Tris-HCl buffer solution containing proteolytic enzyme and calcium chloride and distilled water is allowed to react at a high temperature for a short time, inactivated, and then subjected to enzyme immunization. In the present invention, the term “high temperature, short time” means 50-60 ° C., preferably 55 ° C., which is higher than the normal reaction temperature.
Perform the reaction for 1-3 hours in a water bath at ℃ C. Immediately after the completion of the enzymatic reaction, the enzyme is inactivated by immersing it in boiling water at 80 ° C. or higher, preferably 95 ° C. or higher. Thereafter, excess calcium is chelated by adding ethylenediaminetetraacetic acid, and then subjected to an enzyme immunoassay system, whereby glycated protein bound to the protein can be easily and quickly measured. In addition, a reaction solution consisting of a protease and a Tris-HCl buffer containing calcium chloride excluding the biological sample from the above reaction system is freeze-dried in advance, and distilled water and a biological sample are added before use. Is also possible.

[0012]

EXAMPLES The present invention will be described below in detail with reference to examples, but these examples do not limit the present invention in any way.

[0013]

Example 1 A small test tube with a lid was added with Tris-HCl buffer containing 20 mg / ml or 40 mg / ml proteinase K or Tris-HCl buffer solution containing calcium chloride in pronase and plasma to make 300 μl, 0.5, 2.0 Time 5
After incubation at 5 ° C., the reaction was stopped by boiling the tubes for 10 minutes. Table 1 shows the results of measuring pentosidine in the reaction stop solution by the competitive enzyme immunoassay. Both enzymes decomposed blood proteins, and it was possible to measure pentosidine in blood by enzyme immunoassay.

[0014]

Example 2 Tris-HCl buffer containing calcium chloride and plasma were added to pronase at concentrations of 10, 20, and 30 mg / ml to make 300 μl, and 0.5, 1.0, 1.
Incubate at 55 ° C. for 5, 3.0, 5.0 hours,
The reaction was stopped by boiling for 10 minutes. For comparison, the reaction was carried out at a reaction temperature of 10 mg / ml at 37 ° ^C. for the same time. Table 2 shows the results of measuring pentosidine in the reaction stop solution by the competitive enzyme immunoassay. Pronase concentration 10-30 mg / ml, reaction time 1.5
-Decomposition reaction is approximately 10 hours at a reaction temperature of 55 ° C for 3.0 hours.
Although the reaction progressed by 0%, it became clear that a longer reaction was required during the reaction at a reaction temperature of 37 ° C.

[0015]

Example 3 In a state in which the activity of the protease is remaining, it affects the enzyme immune system. Therefore, it is essential to remove or inactivate the protease. Therefore, the condition of deactivation by heating was examined. A test tube containing a reaction solution to which Tris-HCl buffer solution containing pronase 10 mg / ml and calcium chloride and a physiological saline solution were added was placed in boiling water (95 ° C) at 0,1.
The reaction was stopped by soaking for 0, 20, 30, 40 and 60 minutes. The reaction stop solution was added to the competitive enzyme immune system to carry out the reaction, and the effect on the antigen-antibody reaction system was examined using physiological saline as a blank. The results are shown in Table-3. It was revealed that the proteolytic activity of pronase was inactivated by immersion in boiling water for 10-30 minutes and had no effect on the antigen-antibody system.

[0016]

Example 4 Pentosidine and carboxymethyl lysine in the plasma of 10 healthy subjects, chronic renal failure patients and end-stage renal failure patients were measured by competitive enzyme immunoassay. Tris-HCl buffer containing each plasma, calcium chloride and pronase 10mg /
After incubating 1.5 ml of the reaction solution at 55 ° C. for 1.5 hours, the reaction was stopped with boiling water (15 minutes). The reaction stop solution was added to a 96-well plate on which each antigen was immobilized, and a rabbit polyclonal antibody was added to each plate to carry out an enzyme immunoreaction. The measurement was performed using a microplate reader at a wavelength of 450 nm / 630 nm. The results are shown in Table-4. Both the levels of pentosidine and carboxymethyllysine in plasma increased markedly with a decrease in renal function. That is, it becomes clear that the protein-linked glycated protein in plasma can be measured by enzyme immunoassay by pretreating the plasma with proteolytic enzyme for a short time (105 minutes including reaction termination). Was.

[0017]

According to the present invention, a protein saccharification reaction product which is present in a living body by binding to a protein is heated to a higher temperature (55 ° C.) than normal (37 ° C.) using a protease such as pronase or proteinase K. For a short time (30-120 minutes)
Thereafter, the objective is to easily measure a protein saccharification reaction product contained in plasma, urine, or the like by enzyme immunization by inactivating in boiling water. The plasma pentosidine value measured by the enzyme immunoassay of the sample pretreated by the method of the present invention shows a high correlation with the value measured by the high performance liquid chromatography method, and the measured values by both methods are close to each other. Was. Further, the pentosidine value in plasma measured by the method of the present invention, for example, is significantly increased in patients with reduced renal function, shows a high correlation with plasma creatinine value, useful for the diagnosis of nephropathy is there.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Mikiko Nakajima 104, Yoshinari, Ojin-cho, Tokushima, Tokushima Pref. Inside the Fushimi Pharmaceutical Works Tokushima Plant (72) Inventor Hiroaki Miyatake 1-29 Akashi-cho, Chuo-ku, Tokyo Famille Tsukiji 202 Fushimi Pharmaceutical Co., Ltd.Tsukiji Branch (72) Inventor Tomoyoshi Hosokawa 104 Address Co., Ltd.Fushimi Pharmaceutical Works Tokushima Plant

Claims (1)

[Claims] 1. A method for measuring a protein saccharification reaction product present by binding to a protein, comprising:
A pretreatment method for a specimen, comprising reacting at a high temperature and inactivating it, and then subjecting it to an enzyme immunoassay.