JPH1010124A - Reagent for measuring transferrin in urine and early diagnostic method for diabetes-complication - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide the reagent and the kit for measuring the transferrin in urinte for the early diagnosis of diabetes complication and the early diagnosis method for the diabetes complication. SOLUTION: When this reagent comprises the latex suspension of the latex, wherein antihistotransferrin antibody is bonded, the kit for measuring the transferrin in urine is constituted of the latex suspension and buffer solution. Furthermore, in this method, the diagnosis for the diabetes complication comprising the method for measuring the concentration of the transferrin in urine by the reaction of the above-descrived latex suspension and the sample urine is performed.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a reagent and kit for measuring transferrin in urine. More specifically, the present invention relates to a highly sensitive urinary transferrin measuring reagent using latex particles as an early diagnostic agent for diabetic complications, a kit containing the reagent, and a method for early diagnosis of diabetic complications using the above measuring reagent.

[0002]

2. Description of the Related Art Diabetic complications are caused by diabetes and mainly include retinopathy, neuropathy and nephropathy. In particular, diabetic nephropathy is a fatal complication in which kidneys are damaged due to diabetes. Conventionally, diabetic nephropathy has been diagnosed with the appearance of persistent proteinuria using a protein test strip, etc., but even if treatment is performed at the time of appearance of persistent proteinuria, renal lesions are irreversible, so a therapeutic effect is expected. Can not. Therefore, it is necessary to diagnose nephropathy earlier. It is essential that diabetic neuropathy and retinopathy can be diagnosed and treated at an early stage.

[0003] Transferrin has a molecular weight of 7,000 daltons, is slightly larger than albumin, is a protein that carries and transports iron, and is one of the major plasma proteins. Measuring urinary transferrin clinically has not only the same significance as albumin measurement, but also urinary transferrin has recently been used as an index to diagnose diabetic complications such as early diabetic nephropathy and retinopathy. There have been reports of a higher sensitivity than urinary albumin (see Medicine and Pharmacy, Vol. 31, No. 2, 399-404 (1994); and Medicine and Pharmacy, Vol. 31, No. 3, 713-718 (1994)).

[0004]

DISCLOSURE OF THE INVENTION The present invention is intended to improve the above-mentioned conventional method, that is, to quickly diagnose early diabetic complications, especially early diabetic nephropathy before the appearance of persistent proteinuria. Therefore, it has been developed so that urinary transferrin as an index can be measured with high sensitivity and rapidity by applying a latex agglutination reaction.

[0005]

The urinary transferrin measurement reagent of the present invention comprises a latex suspension in which a latex bound with an anti-human transferrin antibody is suspended in a suspension buffer. The transferrin measurement kit is composed of a buffer solution as a first reagent and the latex suspension as a second reagent.

The compositions of the latex suspension and the buffer are as follows.

First reagent: buffer Tris-HCl buffer, phosphate buffer, Good buffer,
A buffer such as a glycine buffer having a pH of about 5 to 10 and a concentration of about 1 mM to 1 M.

Second Reagent: Latex Suspension The latex suspension consists of (a) a suspension buffer and (b) latex, to which the (c) antibody is bound. Store this latex suspension at 2-10 ° C to avoid freezing.

Component (a) of the above latex suspension
The composition or content of (c) is as follows.

(A) Composition of suspension buffer Tris-HCl buffer, phosphate buffer, Good buffer,
A buffer solution such as a glycine buffer solution having a pH of about 5 to 10 and having a concentration of about 1 mM to 1 M and containing proteins, polysaccharides and other stabilizers.

(B) a polymerized styrene resin having a latex particle size of 0.05 to 0.9 μ, a polymethacryl resin having a particle size of 0.07 to 0.9 μ, a particle size of 0.0
5-0.9μ polystyrene, butadiene and styrene copolymer resin, styrene and butadiene carboxylated copolymer, carboxylated polystyrene, amino group-containing carboxylated polystyrene, acrylic acid polymer,
Examples include methacrylic acid polymer, acrylonitrile, a mixed polymer of butadiene and styrene, polyvinyl acetate acrylate, polyvinyl pyridine, and vinyl chloride acrylate.

(C) Antibodies Any antibody can be used as long as it can bind to the above-mentioned latex, such as a purified immunoglobulin fraction of each animal antiserum, and can sufficiently agglutinate human transferrin in a specimen.

For example, in the case of rabbits, goats, sheep, etc., sufficient aggregation can be achieved by purifying the antiserum into an immunoglobulin fraction by salting out and ion exchange chromatography. Even when a monoclonal antibody is used, sufficient aggregation can be achieved by using an antibody that recognizes a plurality of different epitopes.

Immunogen: transferrin isolated from human serum Specificity: reacting antibodies with human transferrin. Traces of contaminating antibodies were removed by solid phase adsorption with human plasma proteins. Antibody specificity was determined as follows: Cross-over immunoelectrophoresis: transferrin precipitation arch was 2
This occurred when 12.5 μl of antibody / cm ² gel area was used for μl of human plasma. Staining: Commassie Brilliant Blue Immunohistochemistry: PAP for formalin-fixed paraffin-embedded human fetal liver, colonic adenocarcinoma, and normal liver
When tested at 1: 200 to 1: 300 dilution by the method, positive staining of fetal liver hepatocytes and pockets of colon adenocarcinoma epithelial cells and weaker staining of normal liver hepatocytes was observed. . No other staining was observed in the test tissues.

The procedure for diagnosing diabetic complications by measuring urinary transferrin using the urinary transferrin measuring reagent or kit thereof of the present invention will be described below using diabetic nephropathy as an example.

Measurement Principle The measurement of transferrin in urine of the present invention is based on the so-called latex agglutination turbidimetry. That is, when the sample and the latex suspension are reacted, the transferrin in the sample and the anti-transferrin antibody adsorbed on the latex particles start an antigen-antibody reaction, and the latex particles start to aggregate.
When this agglutination reaction is considered as a change in absorbance, the amount of change is proportional to the transferrin concentration in the sample.
A calibration curve is prepared using a standard having a known concentration, and the transferrin concentration in the sample can be determined.

Measurement procedure 1) Prepare a test sample as fresh as possible, and if it is not to be measured immediately, avoid freezing and store it refrigerated. If suspended matter is found in the urine, centrifuge it and use the supernatant as the specimen.

2) A buffer such as Tris buffer is added to the sample, the latex suspension of the present invention is mixed, and the absorbance or scattered light intensity at 340 to 800 nm of the mixture is measured before the start of the reaction.

3) When the transferrin in the sample and the anti-transferrin antibody adsorbed to the latex particles initiate an antigen-antibody reaction, the latex particles begin to aggregate. The absorbance or the scattered light intensity at a wavelength of 340 to 800 nm of this agglutination reaction product is measured, and the change from the absorbance in the above 2) is calculated.

4) Since the amount of change is proportional to the transferrin concentration in the sample (the effect of bilirubin, hemoglobin, glucose, ascorbic acid, etc. is hardly recognized), a calibration curve is determined using a standard having a known concentration. And determine the transferrin concentration in the sample.

Criteria for determination of measurement results Reference normal value of urinary transferrin is 0.9 mg / gC
Since it is r or less, if it is significantly higher than this value, it can be diagnosed as diabetic nephropathy.

[0022]

EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to the examples.

Example 1 Non-insulin dependent diabetes patient group (45 males, 3 females)
2 people, average age 53 ± 16 years old, and a group without any abnormalities in blood biochemistry and renal function by periodic examination (26 males, 9 females, average age 49 ± 5 years old) (hereinafter group of healthy subjects) Abbreviated)
And urinary transferrin was measured. The average value ± SD was 9.1 ± 22.7 mg / gCr in the patient group, which was significantly increased as compared with 0.27 ± 0.15 mg / gCr in the healthy person group. Further, of this group of patients, patients 45 who could not be diagnosed as having nephropathy by
When urinary transferrin was measured for each of the names, the average value ± SD was 1.76 ± 3.15 mg / gCr, which was significantly increased with respect to the healthy group. Therefore, in these patient groups, nephropathy could be detected earlier than before, and treatment of nephropathy could be started earlier.

Example 2 A group of 15 patients with retinopathy which is one of the three major complications of diabetes among non-insulin-dependent ischemic urine patients (15 males,
Urinary transferrin was measured in a group of 7 women and a mean age of 60 ± 13 years old and no group (24 men and 18 women, mean age 54 ± 16 years old). The mean value of urinary transferrin ± SD was 15.81 ± 27 in the group with retinopathy.
65 mg / g Cr, 2.4 in the group without retinopathy
± 3.68 mg / g Cr. Comparing the two, urinary transferrin in the retinopathy group was significantly increased.
Thus, measurement of urinary transferrin was also useful in diagnosing retinopathy.

Example 3 Urine transferrin was measured in 26 patients with neuropathy, one of the three major complications of diabetes, and 44 without, among non-insulin-dependent isuria patients. Mean ± SD is 2.45 ± 2.00 m for group with neurosis
1.28 ± in the group without neurosis against g / gCr
At 1.09 mg / g Cr, urinary transferrin was significantly increased in the group with neurosis compared to the group without neurosis. Thus, measurement of urinary transferrin was also useful in diagnosing neurosis.

[0026]

The main complications of diabetic complications are diabetic nephropathy, retinopathy and neuropathy, and in particular, renal disorders are characterized by irreversible course. Therefore, it is important that these complications such as diabetic nephropathy are detected and treated at an early stage, and if the time is missed, the subsequent treatment tends to be meaningless.

According to the urinary transferrin diagnostic agent of the present invention, urinary transferrin as an indicator can be measured with high sensitivity and rapidity by using latex agglutination reaction.
It can quickly diagnose other diabetic complications, including early diabetic nephropathy before the appearance of persistent proteinuria, and can start treatment at an earlier stage.

Claims (5)

[Claims] 1. A urinary transferrin measurement reagent comprising a latex suspension containing a latex bound with an anti-human transferrin antibody. 2. The urinary transferrin measurement reagent according to claim 1, which is used for early diagnosis of diabetic complications. 3. A kit for measuring transferrin in urine, comprising the latex suspension according to claim 1 and a buffer. 4. The urine transferrin measurement kit according to claim 3, which is used for early diagnosis of diabetic complications. 5. A method for early diagnosis of diabetic complications, comprising reacting a latex suspension containing a latex to which an anti-human transferrin antibody is bound with a sample urine and measuring the concentration of transferrin in the sample urine. .