JP3518746B2 - Highly sensitive trace protein determination by silver staining - Google Patents

Description

Description: BACKGROUND OF THE INVENTION [0001] 1. Field of the Invention
As one of the simplest and cheapest samples that can be collected without any pain on the human body, urinalysis is regarded as important, and urinary sugar and urine protein are routinely examined at the first consultation and at a resident examination. I have. The present invention relates to a quantitative test of urine protein which is one of them. [0002] In normal humans, the appearance of urine protein in normal adults is said to be 150 mg / L or less per day. This one
The concentration of 50 mg / L is 1% less than the amount of total protein in blood.
/ 1000. About 40% of urine protein is albumin and the remaining 60% is various globulins. [0003] The appearance of pathological urinary protein may be caused by prerenal proteinuria (Bence Jones proteinuria, hemoglobinuria, myoglobinuria) or glomerular proteinuria (glomerulonephritis,
Pyelonephritis, nephrosis, renal sclerosis, pregnancy kidney), circulatory disorders (congestive kidney, shock kidney), other prerenal proteinuria (jaundice, cerebral hemorrhage, cerebral glucosyte, severe anemia, polyemia, diabetes,
Gastritis, intestinal obstruction, heatstroke, hyperthyroidism), tubular proteinuria (Fuanconi syndrome, mercury / cadmium poisoning),
Post-renal proteinuria (inflammation below the renal pelvis, stones, tumors, ulcers) and the like are known. There are qualitative tests and quantitative tests as methods for testing proteins contained in urine. The qualitative test is a method of roughly examining whether or not urine contains a certain amount of protein or more or a large amount of protein, as typified by a urine test paper test. Quantitative tests, on the other hand, are tests in which the protein concentration can be obtained numerically, as typified by the immunoassay and the dye-binding method, and are performed by a medical institution to determine the severity of the patient or to monitor the patient And has been. [0005] Inspection method: [0005] Urine protein inspection methods conventionally used are mainly qualitative inspections such as a sulfosalicylic acid method, a boiling method and a test paper method. In particular, the test strip method is widely used for medical examinations of residents and school children and is well known.
In the urine protein quantitative test, radiolabeled immunoassay (RIA), enzyme-labeled immunoassay (EIA), latex agglutination assay (L
Immunological measuring methods such as APA) and immunoturbidimetry (TIA) are used. These measuring methods are all immunological measuring methods using an antigen-antibody reaction. Immunoassays can be used to measure small amounts of protein, but the use of expensive antibodies makes the assay kit very expensive. The sensitivity of these assays is targeted at approximately 50 mg / L. [0006] Many of these assay kits specifically measure albumin among proteins, and do not measure total protein. As described in the technical field of the invention, albumin in urine accounts for about 40% of the protein, and the remaining 60% is an aggregate of various globulins. If a measurement method for measuring globulin is to be made, a large number of antibodies must be used for various globulins, or the kit must be prepared after examining its specificity. It is too expensive to be practical. After all, the immunological assay is an effective assay for individually measuring each specific protein, and a method for measuring total protein with high sensitivity, simple and inexpensive has not been developed. Generally, the dye binding method is used, but the sensitivity is about 300 m.
g / L. [0007] Urinary proteins include M protein (Bence Jones protein, a protein produced by tumor cells such as multiple myeloma).
And IgG (non-selective glomerular proteinuria) and α1 microglobulin / β2 microglobulin (tubular proteinuria, glomeruli, mixed proteinuria) and other proteins in the globulin region. Doctors are required to make a comprehensive diagnosis while observing the total protein (quantitative value) and urine protein fraction in urine, and to treat them. It was not helpful. [0008] Specific techniques: The silver stain for polyacrylamide gel or agarose gel, which has been conventionally used as a support, binds silver ions to proteins in these gels, and then reduces the amount of formalin or the like. It was a method of reducing silver particles with an agent. Since this conventional silver staining solution could not stain proteins on the cellulose acetate membrane at all,
Had not been used. SUMMARY OF THE INVENTION The present invention is a method for quantifying a small amount of urine protein with high sensitivity and a large number of samples easily and without using an immunological method. It is now possible to provide an inexpensive measurement method without using expensive antibodies. As described in the "prior art", an immunoassay using an expensive antibody is usually used to determine a concentration of about 50 mg / L. The present invention is characterized in that a trace amount of protein can be measured at low cost without using an antigen-antibody reaction. The silver staining method conventionally used for high sensitivity uses a polyacrylamide gel or agarose gel. In other words, the silver staining method requires a gel-like support such as a polyacrylamide gel or agarose gel. Not only is it difficult to dye the target protein well, but the operation is very complicated and requires a long time. Conventionally, protein staining solutions used for cellulose acetate membrane electrophoresis include nigrosine, pyrogallol red, and acidlet, but these have low sensitivity for protein quantification. [0012] The present invention is not only capable of dyeing and fixing a trace amount of protein on a cellulose acetate membrane, which has been difficult in the past, but also using a buffered polymer by using a silver staining solution which does not adsorb to the support itself. When silver staining was performed using a porous membrane, the buffer interfered with the protein, making it difficult for silver to adhere to the protein, making it impossible to quantify the protein. In order to use this silver staining method for quantification, it was necessary to stably immobilize the protein on the support, and also required that the support itself be not stained. Furthermore, in order to optically measure the concentration, the silver colloid that has been dyed must be stably fixed without being dissolved even if it is made transparent or translucent. Means for Solving the Problems The conventional silver staining method as described in the prior art is applied to a cellulose acetate membrane which is much cheaper and easier to operate than when using polyacrylamide gel or the like. When used, silver was not fixed and consequently could not be used. In the present invention, in order to prepare a staining solution for use in a cellulose acetate membrane, silver ions are previously reacted with a reducing agent based on ferrous sulfate to form a silver colloid, and then the protein and silver colloid in the cellulose acetate membrane are bound and stabilized. It was possible to quantify it by using the method of quantification. According to the present invention, a fixed amount of a sample is directly spotted on a cellulose acetate membrane in a dry state without buffering, the protein is fixed in this state with sulfosalicylic acid and trichloroacetic acid, and then washed thoroughly with a diluted acetic acid solution. After washing, the problem was solved by staining the protein with the silver staining solution described above. The use of a dried cellulose acetate membrane not only increases the sensitivity but also significantly improves the uniformity and accuracy compared to the use of a cellulose acetate membrane that is in a wet state with a buffer solution, water or other liquid. I was able to do it. Further, an alkaline fixing solution comprising sodium hydrogen carbonate and sodium carbonate is used as a "silver-protein conjugate".
Was more completely fixed. As a result, it was possible to invent a method in which the "silver-protein conjugate" was not dissolved in decalin, liquid paraffin, and the clarifying reagent used for clarifying the cellulose acetate membrane, and was stable, and the precision was significantly improved. DETAILED DESCRIPTION OF THE INVENTION In the present invention, silver sulfate, silver lactate, etc. can be used in addition to silver nitrate as a silver staining solution. Further, in addition to ferrous sulfate, sodium sulfite, formalin, tin chloride (divalent) and the like can be used as a reducing agent. As an alkaline fixing agent, a glycine-sodium hydroxide solution, a trisaminomethane-sodium hydroxide solution, or the like can be used in addition to a sodium hydrogencarbonate-sodium carbonate solution. EXAMPLE 1 In FIG. 1, compartments (2) are formed at equal intervals on a cellulose acetate membrane (1) in a dry state without buffering with a ball-point pen or the like. Accurately measure the standard solution and patient urine sample with a micropipette.
Dotting application (3) is performed in μL. The cellulose acetate membrane coated with this sample is first immobilized with a fixing solution consisting of 1 volume of 0.2 g / dL sulfosalicylic acid and 1 volume of 9.8 g / dL trichloroacetic acid. After fixation, the plate is sufficiently washed with 1% acetic acid, and the protein is stained with a silver staining solution shown in Table 1. After dyeing, wash with water and fix further with an alkaline fixing solution (PH: 9.0) consisting of 0.1 M sodium bicarbonate and 0.1 M sodium carbonate. After drying, the solution is clarified with decalin or clarified with an electrophoresis buffer of a clarified reagent solution, the absorbance is measured with a colorimeter (densitometer), and the concentration is determined from a calibration curve. In FIG. 1, (4), which is surrounded by a frame, is a protein of a known concentration used for a calibration curve, which is silver-stained. The lighter protein is the leftmost, and the concentration increases each time the protein moves to the right. ing. Dots (5) not surrounded by the frame (4) are spotted and fixed with 52 patient urine samples and stained with silver. FIG. 2 is a diagram showing a portion surrounded by a frame (4) in FIG. 1 in which dots for a calibration curve are read by a colorimeter. The absorbance (OD) is plotted on the vertical axis, and the known protein concentration (mg / mg) for the calibration curve is plotted. L) is a calibration curve plotted on the horizontal axis. Next, the absorbance (6) obtained by reading a silver-stained dot (5) of a urine sample of unknown concentration with a colorimeter is applied to the vertical axis of the calibration curve in FIG. 2, and a perpendicular line is drawn at a point where the calibration curve intersects. The concentration crossing the horizontal axis was read and the concentration of total protein in urine samples of unknown concentration could be measured. This method of obtaining the concentration could be obtained instantaneously by using a computer or the like. The concentration of trace urine protein determined by this was only 2.5 mg /
It could be measured from the concentration of L. [0023] Example 2 Using an automatic micropipette capable of depositing a number of standard solutions for a calibration curve and a large number of cerebrospinal fluids of a patient at regular intervals on a non-buffered, dry cellulose acetate membrane, 3 μL of FIG. Is applied in the same manner as above. Thereafter, in the same manner as in Example 1, the cellulose acetate membrane coated with this sample was first fixed with a fixing solution consisting of 1 volume of 0.2 g / dL of sulfosalicylic acid and 1 volume of 9.8 g / dL of trichloroacetic acid.
Wash thoroughly with% acetic acid and stain proteins with silver stain. Then, it is further fixed with an alkaline fixing solution (PH: 9.0). After drying, the solution was clarified with decalin or clarified with a buffer solution for electrophoresis of a clarified reagent solution, the absorbance was measured with a colorimeter (densitometer), and the concentration was determined from a calibration curve. This method of obtaining the concentration could be obtained instantaneously by using a computer or the like. As a result, the protein concentration in the trace cerebrospinal fluid was small.
It could be measured from a concentration of 2.5 mg / L. In Example 2, it was shown that cerebrospinal fluid can be quantified. However, the same operation can be carried out for a protein having a low protein concentration, for example, a diluted sample of tears, saliva, plasma proteins, and trace proteins of other animals and plants. Each can be quantified. According to the present invention, a trace amount of protein can be easily and inexpensively determined from 2.5 mg / L without using an expensive antigen-antibody reaction. As described in the background of the related art, it is possible to quantify individual specific proteins such as albumin by the antigen-antibody measurement method, and the present invention is the first measurement method capable of measuring total protein from 2.5 mg / L. . By utilizing the present invention, it becomes possible to accurately and accurately diagnose patients with nephropathy, especially in the early stage of renal impairment, and to utilize the results for the treatment. In addition, renal damage as a side effect due to administration of a drug (for example, an anticancer drug or an antibiotic) can be found at an early stage. In the field of screening for schoolchild screening, resident screening, and medical checkup, abnormalities can be detected earlier. [0028] Early detection can delay the onset of the disease or treat it early, thereby helping to maintain the health of the individual and consequently reducing medical expenses, thereby greatly contributing to society. In general, if the sensitivity of the initial total protein detection is poor in a screening or an initial examination in a hospital, it is overlooked as normal and consequently it is virtually impossible to carry out the next step of the examination. It is. According to the present invention, the ability to detect the appearance of a small amount of urine protein provides information for judging whether the protein that has appeared is abnormal or normal and whether to perform the next test. (The following test means electrophoresis or immunoelectrophoresis or other immunological quantification.)

[Brief description of the drawings] FIG. 1: Dot blot stained according to the invention FIG. 2 is a diagram of a calibration curve [Explanation of symbols] (1) Support (2) Frame (3) Dots that stain the spotted specimen (4) Range of calibration curve (5) Dots that stain patient samples (6) Absorbance of unknown concentration sample (7) Measurement value of unknown concentration sample

────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-3-190898 (JP, A) JP-A-5-209888 (JP, A) JP-A-58-205855 (JP, A) 502615 (JP, A) (58) Field surveyed (Int. Cl. ⁷ , DB name) G01N 33/68 C07K 17/12

Claims (1)

(57) [Claim 1] A silver-protein conjugate immobilized on a cellulose acetate membrane is converted into a more stable colloidal state with an alkaline fixing solution, whereby silver is stained. Sensitive trace protein determination.