WO2004042371A2

WO2004042371A2 - Method for quick diagnosis of kidney diseases

Info

Publication number: WO2004042371A2
Application number: PCT/JP2003/014187
Authority: WO
Inventors: Kazuo Onuma; Hisashi Narimatsu; Hiroko Iwasaki; Tomomi Kubota
Original assignee: Nat Inst Of Advanced Ind Scien; Amersham Biosciences Corp; Kazuo Onuma; Hisashi Narimatsu; Hiroko Iwasaki; Tomomi Kubota
Priority date: 2002-11-08
Filing date: 2003-11-07
Publication date: 2004-05-21
Also published as: JPWO2004042371A1; AU2003277600A1; AU2003277600A8

Description

Specification

Rapid diagnostic method for IgA nephropathy

The present invention evaluates the physical quantity of a IgA protein present in the blood of a nephropathy patient, particularly an IgA nephropathy patient, using a light scattering method, and compares the physical quantity with the IgA protein of a healthy subject. The present invention relates to a system capable of nondestructively swift, painless and precise diagnosis of IgA nephropathy specific phenomena. Landscape technology

IgA nephropathy is a kidney disease caused by the selective deposition of IgA in the glomerular mesangial region of the kidney. According to statistical data from 2000, it is estimated that about half of all dialysis patients nationwide have 100,000 chronic nephritis, and half have IgA nephropathy. The only method of definitive diagnosis of this disease was to confirm that IgA was deposited in the mesangium region by fluorescent antibody staining by observing glomeruli by renal biopsy. Thus, the only diagnostic method for this disease was tissue observation by renal biopsy, which placed a heavy burden on patients. Therefore, the actual pathology has not been clarified.

In addition, there are two examples of IgA light scattering measurement in which the shape of the IgA molecule itself is observed by neutron scattering and small-angle X-ray scattering or in which the size of the IgA monomer is measured (Non-patent Documents 1 and 2). However, even though the sample is not derived from humans, or from humans, it only deals with samples from healthy subjects, and has not studied the molecular interaction of IgA. Furthermore, there is no mention of the most important possibility of diagnosing IgA nephropathy by comparing various physical quantities of IgA molecules derived from blood of healthy subjects and patients. In addition, neutron scattering requires a large-scale facility and has very limited experimental opportunities, so general clinical diagnosis It cannot be easily applied to

As described above, a system capable of rapidly and painlessly diagnosing IgA nephropathy has not been known so far.

(Non-patent document 1)

Gilmour, S et al., Smal 1-angle neutron scattering studies of the conformation of myeloma protein MOPC315 and its Fab fragment, and the interaction with a monovalent dinitrophenyl hapten.Proceedings of the Royal Society of

London. Series B. Biological Sciences, Volume 211, Issue 1185, March 27, 1981, 433-453

(Non-patent document 2)

Boehm, MK et al., The Fab and Fc fragments of IgAl exhibit a different arra ngement from that in IgG: a study by X-ray and neutron solution scattering g and homology modeling, Journal of Molecular Biology, Volume 286, Issue.

5, March 12, 1999, pp. 1421-1447 Disclosure of the Invention

The present invention has been made in view of such a situation, and an object of the present invention is to provide a system that can rapidly and painlessly diagnose IgA nephropathy. More specifically, the physical quantity of IgA protein present in a test sample such as blood of a nephropathy patient is evaluated using a light scattering method, and is compared with that of a healthy subject to determine IgA nephropathy. It is an object of the present invention to provide a system that can make a diagnosis quickly and painlessly.

The present inventors have conducted intensive research to solve the above-mentioned problems. First, the present inventors collected blood from a healthy subject and a patient with IgA nephropathy, and crudely purified IgA using an affinity column. The obtained sample was analyzed using the light scattering method. We examined whether it was possible to determine whether or not IgA nephropathy could be detected by detecting differences in the size, distribution, and intermolecular interactions of IgA monomers and aggregates. A comparison of the decay rates in the second-order autocorrelation function showed that the decay time of the patient specimen was significantly faster. In addition, when a two-particle component analysis is performed on the assumption of exponential decay and a sample of a healthy subject is compared with a patient sample, the relationship between the relaxation time and the scattering angle tends to be dissipated in the sample of a healthy subject, and the linearity is poor. Very bad, the radius of the small particles was determined to be about 13-20 nm, but in the patient sample, the relationship between the relaxation time and the scattering angle showed a very good linear relationship for the small particles, and the particle radius converged to 7-8 nm The result was. Furthermore, using a solution with the same IgA concentration, a comparison was made between a healthy subject and a patient sample with respect to the relative scattered light intensity produced by an IgA monomer or an IgA complex. The difference ranged from 2 to 50 times. From these facts, the present inventors have found that it is possible to diagnose IgA nephropathy by analyzing IgA in a sample derived from a subject using a light scattering method, and completed the present invention. Was.

By using the diagnostic method developed by the present inventors, it is possible to easily and quickly diagnose IgA nephropathy simply by collecting blood without performing tissue biopsy of IgA nephropathy patients. Become. In addition, the diagnostic method of the present invention can also be used to examine the progress of IgA nephropathy and to determine the risk of developing IgA nephropathy in the future.

The present invention relates to a method for rapid diagnosis of IgA nephropathy, more specifically,

[1] A method for diagnosing nephropathy, comprising the following steps (A) to (C):

(A) Measure the absolute value, time change, and scattering angle dependence of the scattered light of the incident light irradiated on the aggregated particles containing IgA protein or IgA protein present in the test sample derived from the subject Process,

(B) a step of evaluating one of the following physical quantities (a) to (f) based on the value measured in the step (A);

(a) Scattered light intensity at any scattering angle

(b) Autocorrelation function at any scattering angle

(c) Molecular weight, radius of gyration, fractal dimension, or particle shape based on the analysis in (a) above (d) Diffusion coefficient and hydrodynamic radius based on the analysis of (b) above

(e) Relaxation time and scattering angle

(f) Dependence of diffusion coefficient and hydrodynamic radius calculated from (d) or (e) as a function of IgA protein concentration

(C) Step of comparing the physical quantity of step (B) with that of healthy human IgA protein

(2) The diagnostic method according to (1), wherein the incident light is polarized or unpolarized ordinary light, X-rays, synchrotron radiation, or neutrons.

(3) the diagnostic method according to (1), wherein the scattering is dynamic light scattering or static light scattering, (4) the test sample is a blood sample, (1) to (3), Diagnostic methods,

[5] The diagnostic method according to any one of [1] to [4], wherein the nephropathy is IgA nephropathy.

The present invention provides a method for diagnosing nephropathy using light scattering. In general, the light scattering method is a method for evaluating the diffusion coefficient, intermolecular interaction, molecular weight, structure, and the like of particles undergoing Brownian motion in a solution. Due to Brownian motion, the intensity of light scattered from particles changes with time. By measuring the time change of the scattered light intensity, the diffusion coefficient of the particles can be determined. Since the diffusion coefficient is inversely proportional to the particle size, the particle size is known, and the interparticle interaction can be determined by measuring the particle concentration dependence of the diffusion coefficient. More specifically, by measuring the absolute value, time change, and scattering angle dependence of the scattered light of the light (incident light) applied to the particles moving in Brownian motion in the solution, the molecular weight of the target particle, ( (Translation and rotation) A method to evaluate the diffusion coefficient, hydrodynamic radius, radius of inertia, fractal dimension, and physical quantity of particle shape. Furthermore, it is possible to evaluate the interaction between particles by measuring the time change of the scattered light intensity with the light scattering method as a function of the concentration of the particles.

The present invention uses a light scattering method for IgA protein present in the blood of IgA nephropathy patients. The present invention relates to a method for rapidly and painlessly diagnosing a phenomenon specific to IgA nephropathy by evaluating the above physical quantities using the above and comparing and examining the physical quantity with the case of a healthy subject's IgA protein. The present invention is based on the inventor's finding that it is possible to detect the size of IgA molecules between IgA patients and healthy subjects, or differences in interactions between IgA molecules, by using a light scattering method. The present invention relates to a method for diagnosing whether or not a subject has nephropathy based on the findings found by the present inventors.

The nephropathy diagnosed by the present invention usually refers to IgA nephropathy, but is not necessarily limited thereto. For example, it is also possible to diagnose diabetic nephropathy.

The light scattering method generally includes a dynamic light scattering method, a static light scattering method, a small-angle X-ray scattering method, a neutron scattering method, and the like. In the present invention, the diagnostic method of the present invention can be carried out using any of these scattering methods.

The measurement principle of the most commonly used dynamic light scattering method (Dynamic Light Scattering, also known as Quasi-elastic Light Scattering) is described below.

Assuming that the scattered light intensity is I, the following autocorrelation function is defined for the time change of the scattered light intensity.

g ² (a, t)-l = <I (q, t) I (Q, 0)> / <I (Q)> ² (Equation 1)

In Equation 1, gdt) is a second-order autocorrelation function, I (Q, t) is the time, and the scattering angle (scatter vector) is the scattered light intensity at Q, and I (Q, 0) is the measurement start time. I (Q) means the average value of the scattered light intensity during the entire measurement time. <> Indicates ensemble average. The quadratic autocorrelation function is related to the relaxation time of a particle, and Equation 2.

g ² (Q, t) — 1 = [∑a „xexp (1 / te _n xt) ⁰ )] ² (n = l, 2, 3, · · ·) (Equation 2)

The simplest system is a monodisperse (one average particle size) system with weak interaction between particles and no anisotropy of particle morphology, where n = 1 and / 3 = 1. In a system such as a polymer in which the interaction between particles becomes remarkable, 3 is not always 1 and can take a value of 1 or less. one On the other hand, when particles with different average sizes exist in the solution, the quadratic autocorrelation function is the sum of the relaxation times of each particle. That is, n = l, 2,. If the particles are large and move slowly, the relaxation time will be long. Conversely, if the particles are violently Browning with small particles, the relaxation time will be short. Figure 1 schematically shows examples of both.

In the actual measurement, we assume what kind of particles exist in the solution and analyze the quadratic self-correlation function to find the relaxation time of each particle at the scattering angle Q. Equation 3 has a relationship between the relaxation time and the diffusion coefficient and scattering angle of the particles.

1 / T = Q ² XD (Equation 3)

Here, the scattering angle is changed, determine the relaxation time at each of the Q value, 1 / Tet Q ^'2 when the plotted as both axes, if approximated by a straight line passing through the origin, the movement of being measured particles Can be concluded to be translational diffusion. On the other hand, the translational diffusion coefficient obtained by the above process includes factors such as structuring of the hydration region around the particles due to the interaction between the particles. Therefore, if you want to find the true diffusion coefficient, perform the above measurement as a function of particle concentration to find the translational diffusion coefficient at infinite dilution. The following relationship holds between the translational diffusion coefficient and particle size (hydrodynamic radius: r) determined by such a procedure (Stokes-Einstein equation).

r = k T / 6 π 7? D (Equation 4)

k is the Boltzman constant, T is the absolute temperature of the solution, and /? is the viscosity of the solvent. The diagnostic method of the present invention comprises the following steps: first, the absolute value, the time change, and the scattered light of the incident light applied to the IgA protein or the aggregated particles containing the IgA protein present in the test sample derived from the subject. The scattering angle dependence is measured (step (A)). As the temperature at the time of measurement in this step, for example, 25 can be suitably indicated, but it is not particularly limited to this temperature.

In a preferred embodiment of the above step (A), scattering of incident light applied to an aggregated particle containing IgA protein or IgA protein present in the test sample derived from the subject For light, its absolute value, the autocorrelation function based on temporal change, the scattering angle dependence of the autocorrelation function, in the diagnostic methods of the _c the present invention for performing IgA concentration dependence of the measurement of diffusion coefficients calculated from the autocorrelation function For example, when the subject's nephropathy is significantly advanced, it is possible to diagnose nephropathy by measuring only the scattered light intensity in the above step (A). In general, by measuring all of the “absolute value”, “time change”, and “scattering angle dependency” of the scattered light in the above step (A), the accuracy of diagnosis of nephropathy is improved. In the method, it is preferable to measure all of them.

In the present invention, the “incident light” for irradiating the IgA protein is not particularly limited in its kind and the like, but includes, for example, polarized (including quenched) and unpolarized ordinary light (eg, ultraviolet, visible, Infrared), X-rays, synchrotron radiation, neutrons and the like. In the present invention, ordinary light can be suitably used.

The “scattering” in the present invention means that the scattering method based on Rayleigh scattering or Mie scattering includes dynamic static scattering, small angle scattering, single particle scattering, and scattering using evanescent waves. Considering the simplicity of use in the method and the speed of measurement, dynamic scattering or static scattering is preferred, and dynamic scattering is more preferred. Examples of the test sample used in the diagnostic method of the present invention include a sample prepared from a body fluid, and it is usually a sample derived from blood or saliva, preferably from a blood of a subject. This is a prepared sample. In the present invention, it is desirable to prepare a sample derived from a healthy person as a control. At that time, preferably, the preparation is performed so that the IgA concentrations contained in the samples of the healthy subject and the subject are the same.

Preparation of a test sample from blood can be performed by any method as long as IgA can be purified. For example, the preparation can be performed by column chromatography, electrophoresis, or the like, which is a method generally performed by those skilled in the art. Preferably, the preparation is performed using a jackalin agarose column or an anti-IgA column in addition to the column. I do. Hereinafter, an example of a method for preparing a test sample of the present invention will be described. It is not limited to.

Apply 3-5 mL of serum (or plasma) to a Jackalin agarose column (column volume (CV) = 2fflL) equilibrated with 100 m Tris-HCl, \% NaN ₃ (J-buffer). After eluting the flow-through fraction with 10 CV J-buffer, elute with J-buffer containing 0.8 M galactose. The eluted fraction is designated as jackalin-purified IgA. The obtained fraction is concentrated and desalted with an ultrafiltration apparatus with a molecular weight cut of 10k, and equilibrated with 50 mM Tris-HCl buffer (pH 3) (three times of desalting). The protein concentration of the sample is determined by the Lowry method <The reagents and instruments used in the preparation of the test sample are not particularly limited to those described above. Any buffer can be used as long as it has a concentration of up to several hundred mM.

When preparing a test sample, it is preferable to perform filtering, as necessary, to remove dust.

The measurement in the above step (A) using the light scattering method in the present invention can be easily performed by those skilled in the art, usually using commercially available equipment. For example, DLS-700 (Otsuka Electronics Co., Ltd.) or the like can be used.

In this method, then, based on the value measured in step (A),

The physical quantity of any of (a) to (f) is evaluated (step (B)), and the physical quantity of step (B) is compared with the case of a healthy subject's IgA protein (step (C)).

(a) Scattered light intensity at any scattering angle

(b) Autocorrelation function at any scattering angle

(c) Molecular weight, radius of gyration, fractal dimension, or particle shape based on the analysis in (a) above

(d) Diffusion coefficient and hydrodynamic radius based on the analysis of (b) above

(e) Relaxation time and scattering angle

(f) Dependence of diffusion coefficient and hydrodynamic radius calculated from (d) or (e) as a function of IgA protein concentration The comparison with the case of a healthy subject's IgA protein in the above step (C) is performed by measuring a test sample of the subject by the light scattering method and also measuring a sample derived from a healthy subject as a control. The physical quantity of any of the above (a) to (f) can be compared with the case of a healthy person. Alternatively, the physical quantity may be compared with any of the physical quantities (a) to (f) described above for a healthy person calculated in advance.

In general, a smaller scattering angle is more susceptible to the influence of dust in the sample and stray light from the measurement container, and larger particles are more likely to be observed. Conversely, the higher the angle, the less the dust is affected, and the smaller the particles to be measured this time are more likely to be detected. As for the scattering angle of the present invention, those skilled in the art can appropriately select an optimum scattering angle based on the above findings. Normally, a scattering angle of 90 degrees is considered to have the least effect of dust and stray light. The scattering angle in the present invention is usually in the range of 10 to 150 degrees, and is actually 20 to 90 degrees, and furthermore, the influence of dust in the sample is eliminated as much as possible, and the measurement is simple. For example, 90 degrees is preferable.

In (a) above, the scattered light intensity measured by the light scattering method on the test sample derived from the subject is compared with the scattered light intensity of a healthy person. Although it is possible to make a diagnosis of nephropathy based on the absolute value of the scattered light intensity in the subject, it is usually preferable to use a sample judged to be healthy as a control. That is, the scattered light intensity is compared with the scattered light intensity in a healthy person (comparison of the relative scattered light intensity). For example, a diagnosis of nephropathy can be made based on the value of the scattered light intensity in the subject when the scattered light intensity in a healthy person is set to “1”.

As an example, when the scattering angle at which the effect of dust is considered to be the least is 90 degrees, as shown in FIG. There is a slight difference in relative scattered light intensity. When the relative scattered light intensity in the subject is usually 0.25 or less, preferably 0.2 or less, more preferably 0.15 or less, the subject is determined to have nephropathy. . In the present invention, "the subject has nephropathy" usually means that "the subject has nephropathy" from a physiological point of view. In the above comparison, more preferably, in order to determine the value, a statistically averaged scattered light intensity is calculated for a sample group that can be definitely determined to be a healthy person from both physiological and main light scattering measurements. It is advisable to measure the scattered light intensities of samples from a sufficient number of patients and compare them.

Even nephropathy varies in severity from mild to severe. Usually, it is determined that the lower the value of the scattered light intensity, the more advanced the nephropathy (the higher the degree of the nephropathy) or the higher the risk of developing nephropathy in the future.

In addition, even if they are physiologically healthy, they may be a reserve arm for nephropathy. In one embodiment of the present invention, the diagnostic method of the present invention can be performed using a healthy subject who does not exhibit nephropathy as a subject. That is, when the method of the present invention is performed on such a subject and it is determined that the subject has nephropathy, the subject is likely to have nephropathy in the future (high). Conceivable.

In the above (b), the autocorrelation function can be calculated by the above equation 1 based on the scattered light intensity and the time change measured by the light scattering method for the test sample derived from the subject. This autocorrelation function is compared with the autocorrelation function of a healthy person.

At any scattering angle, a graph can be drawn around time (log) and autocorrelation function. In the present invention, nephropathy can be determined based on the difference between the shapes of the graphs drawn for the healthy subject and the subject. As an example, when the scattering angle is 90 degrees, a graph as shown in FIG. 2 described later is drawn for a healthy person and an IgA patient. As shown in Fig. 2, the autocorrelation function at a scattering angle of 90 degrees has a clear difference in the graph drawn with the time (logarithm) and the autocorrelation function as axes. Therefore, when the graph drawn in the subject shows the shape of the graph in the IgA patient shown in FIG. 2, that is, as described later, when the autocorrelation function rapidly attenuates with time, Is judged to be nephropathy. Quantitatively, it is possible to determine nephropathy by focusing on the decay rate of the autocorrelation function. For example, in FIG. 2, a healthy person shows a slow decay. In contrast, the decay time is significantly faster in patients than in healthy individuals. Therefore, if the subject shows a faster decay rate as compared to a healthy subject, it can be determined that the subject has nephropathy or is likely to develop nephropathy.

It is also possible to determine nephropathy by comparing the values of the autocorrelation function at a specific “time”. In the present invention, usually, the IgA concentration in the sample derived from the subject and the healthy subject is made the same as described above, and artifacts such as stray light from the container are removed as much as possible during the measurement. Thus, the value of the autocorrelation function at time zero is the same for both the healthy subject and the subject.

For example, if the value of the autocorrelation function at time zero is 0.45 to 0.5, the value of the autocorrelation function at time 0.1 ms for the patient is about 1/1. 5 to 1/30. Therefore, to give an example, an autocorrelation function for the subject at a time of 0.1 millisecond is calculated, and the value is usually 0 to 0.1, preferably 0 to 0.05, more preferably 0 to 0.05. When で 0.01, the subject is determined to have nephropathy. Those skilled in the art can appropriately select “time” that shows the largest difference in the value of the autocorrelation function, and compare the values of the autocorrelation function for healthy subjects and subjects. Also, the lower the value of the autocorrelation function at a particular time (eg, 0.1 ms), the more advanced the nephropathy (the more advanced), or the risk of developing nephropathy in the future Is determined to be high. For example, if the value of the autocorrelation function at a time of 0.1 milliseconds falls below 0.2, it can be determined that there is a risk of onset in the future. In the above (d) and (e), Based on the autocorrelation function of the examiner, the relaxation time (T) is calculated by Equation 2 above, and the relationship between the relaxation time and the scattering angle is obtained. At that time, the linearity and deviation of the relaxation time and scattering angle, and the calculated diffusion coefficient and hydrodynamic radius are used as the reference. For example, the relaxation time and scattering angle When a graph is drawn in this way, it usually shows a linear relationship as shown in Figure 3. From this relationship, the (translational) diffusion coefficient and hydrodynamic radius are calculated. As an example, if the relaxation time and the scattering angle show the relationship as depicted in Fig. 3, the diffusion coefficient of IgA in healthy subjects is 12-19 x lO- ^l2 mVs, and the corresponding hydrodynamic radius is It is determined to be about 13-20 nm. The diffusion coefficient of IgA in patients is 30.5 to 35 x iO- ¹² mVs, and the corresponding hydrodynamic radius is determined to be about 7 to 8 MI. Thus, a diagnosis of nephropathy can be made for a subject based on the difference between the diffusion coefficient of IgA and the hydrodynamic radius between a healthy person and a patient. For example, each of the diffusion coefficient及beauty hydrodynamic radius of IgA in a subject, usually ^{^{28. 8~35 10- |? 2 111 2}} /5 and ~ 8. 5 nm, rather preferably is from 30.5 to 35 X 10- ^l2 m ² / s and? ~ 8 nm, more preferably in the case of ^{^{32. 6~35 x 10- 12 m 2 /}} s and 7 to 7. 5 nm, the subject is a nephropathy Is determined.

In the above (f), the relationship between the above diffusion coefficient and the hydrodynamic radius in the subject and the IgA protein concentration is determined. That is, the particle-particle interaction can be evaluated by determining the dependence of the diffusion coefficient on the IgA protein (particle) concentration as a function. For example, in a graph in which the particle concentration is plotted on the horizontal axis and the diffusion coefficient is plotted on the vertical axis, if the shape of the drawn line is “upward to the right”, it is determined that there is a repulsive interaction. On the other hand, if the shape of the line is “downward to the right”, it is determined that the line has attractive interaction (Fig. 5). The present inventors have revealed that the interaction between IgA molecules in healthy subjects and patients is different. Therefore, a diagnosis of nephropathy can be made based on the difference in the interaction between IgA molecules. For example, nephropathy can be determined based on the difference between the shapes of the graphs drawn as described above for a healthy person and a subject. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a schematic diagram of the correlation function. The vertical axis represents the autocorrelation function gd, t), and the horizontal axis represents time (log). Figure 2 shows a comparison of autocorrelation functions between healthy subjects and IgA patients (5 donors each). A is a healthy person and B is an IgA patient.

Figure 3 shows the relationship between the relaxation time and the scattering angle for the representative donations of healthy subjects and patients. A is a healthy person and B is a patient.

FIG. 4 is a comparison diagram of the scattered light intensity of all samples at a scattering angle of 90 degrees. The vertical axis represents the relative scattered light intensity, and the horizontal axis represents the sample number.

FIG. 5 is a diagram schematically showing the relationship between the particle concentration and the diffusion coefficient when an attractive or repulsive interaction is shown. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to these Examples.

[Example 1]

Physical quantities were evaluated using light scattering method for healthy subjects and IgA nephropathy patients, and blood samples from each of the five samples were compared and compared. All samples were adjusted so that the concentration of IgA protein in the measurement solution was equal.

Figure 2A shows the second-order autocorrelation function of five healthy subjects at a scattering angle of 90 degrees. The point compared is the decay rate. In healthy subjects, all five samples showed a slow decay and converged to 0 in about 1 ms. Four of the five specimens were almost identical, but the remaining one showed a slightly faster decay than the other four. In contrast, the decay time of the patient sample was significantly faster than that of the healthy subject. In the fastest one, the correlation function converged to 0 in about 0.1 ms. Even at the slowest, for example, the value of the correlation function at a time of 0.1 millisecond was 50% smaller than that of a healthy person.

In reality, there is a reserve arm of IgA nephropathy even if it is physiologically determined to be healthy. On the other hand, the disease progression of IgA nephropathy varies from mild to severe. This look Looking at Fig. 2 in this regard, it is thought that among healthy subjects, a sample whose correlation function decreases rapidly may be in the future. Differences in patient samples can be interpreted as corresponding to the stage of IgA nephropathy. [Example 2] Comparison of relaxation time and scattering angle

The relationship between the relaxation time and the scattering angle was determined for a representative sample of healthy subjects and patients (Fig. 3). As described above, the (translational) diffusion coefficient and particle radius were calculated from this relationship.

First of all, it was judged that it was appropriate to analyze the correlation function on the assumption that there are two types of large particles and small particles in the solution for healthy subjects. However, for both particles, the relationship between the relaxation time and the scattering angle tended to dissipate, and did not show a very clear linear relationship. This tendency was common to all healthy subjects. Here, since the possibility of impurities other than IgA cannot be denied for the large particles, detailed investigation was not performed hereafter. The radius of the small particles (written as fast mode in FIG. 3) is Although it is about 13ηm, the distribution is considerably wide depending on the sample, and it was determined to be about 13-20nm in this measurement result.

On the other hand, the patient sample was clearly different. The correlation function could be analyzed for patient samples assuming that there are large and small particles in the solution, but the percentage of large particles was very small. As a result, for large particles, the relationship between the relaxation time and the scattering angle largely dissipated from the straight line. On the other hand, small particles (fast mode) dominating in patient samples show very clear linearity in relation to relaxation time and scattering angle, with particle radii in the range of 7-8ηιη for all samples. Converged. From these results, the following significant differences were observed between the healthy sample and the patient sample.

1) When two types of particles are present in the solution and the correlation function is deduced as exhibiting exponential decay, the relationship between relaxation time and scattering angle tends to dissipate in healthy subjects, The linearity was very poor. In this case, the radius of the small particles was determined to be about 13-20 nm, while in the patient sample, the relationship between the relaxation time and the scattering angle showed a very good linear relationship for the small particles, and the particle radius converged to 7-8 nm. This value seems to correspond to the monomer of IgA protein.

2) From the difference in particle size corresponding to the first mode, the difference in the scattered light intensity at the same scattering angle between the healthy subject and the patient sample clearly appeared.

[Example 3] Comparison of relative scattered light intensity

Fig. 4 shows the relative scattered light intensities of all healthy subjects and patients at a scattering angle of 90 degrees. FIG. 4 is a diagram in which the scattered light intensity of the first sample of a healthy person is standardized as 1. Obviously, the scattered light intensity was low in the patient sample. The difference from a healthy person was 2 to 50 times. Industrial potential

The present invention provides a method for diagnosing nephropathy using the light scattering method. According to this method, nephropathy can be precisely diagnosed simply and quickly and nondestructively by merely collecting blood without performing a tissue biopsy of a nephropathy patient. In addition, the diagnostic method of the present invention also enables the examination of the degree of nephropathy and the determination of the risk of developing nephropathy in the future.

Claims

The scope of the claims

1. A method for diagnosing nephropathy, comprising the following steps (A) to (C).

(B) a step of evaluating any of the following (a) to (O) based on the value measured in the step (A),

(a) Scattered light intensity at any scattering angle

(b) Autocorrelation function at any scattering angle

(e) Relaxation time and scattering angle

(f) Dependency of diffusion coefficient and hydrodynamic radius calculated from (d) or (e) as a function of IgA protein concentration

(C) Step of comparing the physical quantity of step (B) with the case of healthy human IgA protein

2. The diagnostic method according to claim 1, wherein the incident light is polarized or unpolarized ordinary light, X-rays, synchrotron radiation, or neutrons.

3. The diagnostic method according to claim 1, wherein the scattering is dynamic light scattering or static light scattering.

4. The diagnostic method according to claim 1, wherein the test sample is a blood sample.

5. The diagnostic method according to any one of claims 1 to 4, wherein the nephropathy is IgA nephropathy.