JP6150161B2 - High blood pressure detection reagent - Google Patents

Description

The present invention relates to a method for assisting diagnosis of hypertension and metabolic syndrome and a detection reagent thereof. In addition, it includes therapeutic tools and medicines.

Traditionally, the diagnosis of hypertension has been physical measurement of blood pressure with an instrument. Also, since there was no easy way to see tissue damage associated with high blood pressure, it was unclear how much the vascular tissue was damaged. Therefore, at present, regardless of whether or not a vascular tissue disorder accompanying hypertension occurs, it is easy to administer an antihypertensive agent uniformly for hypertension. Some antihypertensive drugs have been shown to have adverse effects on kidney microvessels over many years.

Although hypertension is thought to have caused vascular tissue damage, there has been no detection method so far. If a tissue disorder can be diagnosed, it will be further useful as an index of the effect in therapeutic drug development and an index of observation of the history of hypertension treatment.

Means for solving the problem

The present invention provides a method and a reagent capable of detecting not only hypertension but also a tissue disorder caused by metabolic syndrome by using type IV collagen, a constituent region thereof, or a constituent peptide thereof, as well as a treatment assisting method and a therapeutic agent. is there.
Type IV collagen is a major component of the biological tissue basement membrane and is composed of three α chains. There are six types of α chain from α1 chain to α6 chain, and the type and amount of α chain are biased depending on the tissue. The α1 chain and α2 chain are distributed throughout the body, but the glomerular basement membrane is rich in α3 to α6 chains. To extract type IV collagen from living tissue, the placenta, kidney glomerular basement membrane, and eyeball, which are known to be abundant, are used as starting materials.
The type IV collagen constituent region is divided into three regions: N-terminal 7S, central helical region, and C-terminal NC1. The amino acid sequences constituting type IV collagen are similar if the α chain is the same even if the animal species is different. On the other hand, even in the same animal species, the amino acid sequences differ greatly if the α chain is different. When, for example, synthetic peptides of 20 residues each are produced using the same sequence order of different α chains, such as α3 chain and α1 chain, several amino acids are often different.

About the relationship between type IV collagen and diseases.
In anti-glomerular basement membrane (GBM) antibody nephritis, which is a rare nephritis, an antibody against the NC1 region (NC1) of type IV collagen (anti-NC1 antibody) increases in the blood. It is used. On the other hand, the inventor of the present application pointed out that anti-NC1 antibody is frequently present in the serum or urine of healthy individuals or those with high urinary albumin (cell 35: 150-154, 2003). However, it has not been clarified what kind of disease is related to an example that shows a high value in serum, not limited to the present inventor. Thereafter, the inventor synthesized that anti-NC1 antibody is present in urine of nephritis in general (medicine and pharmacy 51: 567-572, 2004) and that α5 chain reacts with IgG in nephritis patient urine. Peptides are shown (medicine and pharmacy 57: 605-613, 2007).

This time, the present inventor further discovered the following facts.
The following type IV collagen is used for the experiment. The placenta or kidney cortex is used as a starting material for type IV collagen, and a triple-stranded helical region or NC1 region is used as a constituent region. The α1 chain and α5 chain were used as representative of the α chain, and the synthetic peptide chains yp12, yp08 and others were used.
One of the results revealed by the measurement results is that the anti-NC1 antibodies in urine and blood differed in the origin of antigen NC1. When human and rat anti-NC1 antibodies are measured, there is no correlation between anti-NC1 antibodies in blood and anti-NC1 antibodies in urine.
IgG in the urine of nephritis patients reacted with α1 chain NC1, and IgG in the blood of hypertensive patients reacted with NC1 of α1 and α2 chains. The α5 chain is abundant in the kidney, and the α1 chain and α2 chain are distributed in the basement membrane of the whole body including the basement membrane of blood vessels and skin. In nephritis, the basement membrane of glomeruli, tubules, and Bowman's sac responsible for the filtration function of the kidney is damaged, and NC1 in the damaged kidney tissue basement membrane becomes an antigen and produces anti-NC1 antibody in urine. . In hypertensive patients and metabolic syndrome, damage is caused in the basement membrane of the brain and other microvessels and tissues, and NC1 in the damaged basement membrane becomes an antigen and produces anti-NC1 antibody in the blood. In fact, anti-NC1 antibody was not detected in urine in patients with early hypertension and metabolic syndrome.

Antibodies against peptides derived from each α chain.
An experimental animal, here a rat, was used for detailed examination. Three systems derived from WKY and WKY. Hypertensive rats (SHR) that cause hypertension, stroke rats (SHRsp) that cause stroke with abnormal hypertension, and rats with metabolic syndrome (SHRcp).
The anti-NC1 antibody in urine correlates with antibodies against a plurality of peptide chains (20 residues) -yp05, yp08, yp12- derived from α5 chain NC1 (anti-yp05 antibody, anti-yp08 antibody, anti-yp12 antibody). Conversely, anti-NC1 antibody in blood does not correlate with yp05 antibody and yp08 antibody. Therefore, the anti-NC1 antibody in urine contains anti-α5 chain NC1 antibody, and the anti-NC1 antibody in blood does not contain anti-α5 chain NC1 antibody. If anti-NC1 antibody is detected in the blood, if it is hypertension, especially hereditary hypertension, and if it is a stroke family, an immunosuppressive agent is used from a trace amount. As a result, if the anti-NC1 antibody in the blood decreases, the therapeutic effect can be determined.

The measurement method uses an immune reaction regardless of whether the detection target is an antigen or an antibody. For detection, a luminescence method is used, for example, isotope, chemiluminescence, or bioluminescence. Aggregation and light scattering may be used without using the luminescence method, or electrophoresis, sedimentation, and the like may be used. Antibody measurement by ELISA is shown as an example of human measurement. Add 50-100-fold diluted serum to the antigen-immobilized microplate and allow it to react. After washing, add enzyme-labeled anti-human (matched to the animal to be measured) antibody, react, wash, and wash the enzyme substrate solution. Is added, and the reaction stop solution is added in 5 to 30 minutes while observing the color density, and the absorbance is immediately measured at a wavelength of 450 nm.

Effect of the invention

The present invention is useful as a method for detecting tissue damage due to hypertension and for assisting treatment. In addition, the measurement in animals and humans is an index for grasping the degree of disease, antihypertensive agent, lifestyle-related disease improving agent (anticholesterol agent, antilipid agent, etc.), antidiabetic agent, and drug evaluation.

Measurement target;
Four rat strains (WKY, SHR, SHRsp, SHRcp) ♂ each of five 6-week-old and 21-week-old plasmas,
Measurement item: anti-type IV collagen NC1 antibody,
Measurement method: NC1 (derived from bovine kidney) on a 96-well microplate → Plasma (diluted 100 times) → Incubation and washing →
Addition of enzyme-labeled anti-rat IgG antibody → Incubation and washing →
Enzyme substrate solution added → 5-30 minutes later while observing color →
Reaction stop solution (1N sulfuric acid) added → Immediately measured absorbance at a wavelength of 450 nm (average absorbance, standard deviation);
6-week-old SHRcp (0.653, 0.127), SHRsp (0.412, 0.071), SHR (0.262, 0.036), WKY (0.243, 0.016)
21 weeks of age SHRcp (0.826, 0.110), SHRsp (0.629, 0.071), SHR (0.470, 0.036), WKY (0.520, 0.065)
Conclusion;
1) Rats with metabolic syndrome (SHRcp) have an increased anti-NC1 antibody from an early stage, which is an index of tissue damage associated with the metabolic state. It is an index of microvascular disorders including cerebrovascular

Measurement subjects: serum of healthy human subjects (86 cases) and hypertensive patients (11 cases),
Measurement item: anti-type IV collagen NC1 antibody,
Measurement method: Same as in Example 1, except that the enzyme-labeled antibody is an anti-human antibody (average absorbance is displayed as Ave., standard deviation is displayed as SD);
Healthy subjects (0.467, 0.186), hypertensive patients (0.632, 0.405)
The cut-off value was Ave + 2SD (0.839)
The conclusion that the cut-off value or more was 4 cases (36%) in 11 hypertensive patients and 8 cases in 86 healthy subjects was concluded;

Claims (4)

A method for detecting a disorder of a biological tissue basement membrane in which type IV collagen excluding kidneys and lungs is present. A blood sample collected from a human and / or an animal is used as a sample, and it is derived from a damaged biological tissue basement membrane. Analysis of the presence or absence of type IV collagen NC1 region or its peptide, or antibody thereof, and the measurement value of type IV collagen NC1 region or its peptide, or antibody thereof derived from a damaged biological tissue basement membrane The measured value of the type IV collagen NC1 region derived from the damaged biological tissue basement membrane in the sample or the peptide thereof or the antibody thereof is higher than the cutoff value calculated on the basis of the cut-off value. A method comprising showing that a basement membrane of a living tissue in which IV collagen is present is damaged. A method for detecting damage to a basement membrane of a living tissue in which type IV collagen is present is a method for finding a hypertension group in humans , and for finding a stroke group and metabolic syndrome in non-human animals. In the method, blood collected from each of humans and / or animals is used as a sample, and the detected value of the type IV collagen NC1 region derived from the damaged biological tissue basement membrane or the peptide thereof or the antibody thereof is When the cutoff value calculated based on the detected value of the type IV collagen NC1 region derived from the biological tissue basement membrane or its peptide or antibody thereof is damaged, the biological tissue basement membrane in which type IV collagen exists is caused. A method consisting of showing that A method for diagnosing a disorder in the type IV collagen NC1 region of a hypertension group, metabolic syndrome or pre-stroke group using anti-NC1 antibody measurement. A method for distinguishing the possibility of a pre-stroke group from a hypertensive group using anti-NC1 antibody measurement.