JP6757041B2 - Measurement method of anti-DNA antibody - Google Patents

Description

The present invention is a method for measuring an anti-DNA antibody by reacting a DNA fragment labeled with a non-radioactive compound with a sample and measuring the amount of the labeled DNA fragment. (a) Labeling with a non-radioactive compound The DNA fragment was reacted with the sample, (b) after the reaction, a precipitant was added to the reaction solution, the liquid phase and the precipitated phase were separated (B / F separation), and (c) after the B / F separation, The amount of the labeled DNA fragment in the liquid phase is measured, (d) the amount of anti-DNA antibody is calculated from the measurement result of the amount of DNA fragment, and (e) the concentration of the DNA fragment to be used is Y (μg / μg /). mL), when the chain length is X (bp), use the one within the range represented by the following [Equation 1].
[Number 1]
400 ≤ X ≤ 2000
Y ≧ 0.1
0.0006X-0.4595 ≤ Y ≤ 0.0005X + 0.3744
(F) When the obtained measurement result is compared with the result of measuring the same sample by a known Farr-RIA method, the correlation coefficient between the two measurement results is 0.93 or more. It relates to a method for measuring a DNA antibody.

Patients with systemic lupus erythematosus (SLE), one of the autoimmune diseases Anti-DNA antibody, which is an autoantibody that binds to DNA, is present in the serum, and this autoantibody is the main cause of glomerulonephritis that frequently occurs in patients with SLE. It is thought that it is a causative substance. Therefore, the measurement of anti-DNA antibody is regarded as an important index for the diagnosis and activity evaluation of SLE.
Conventionally, as a typical method for measuring an anti-DNA antibody, a DNA fragment is labeled with ¹²⁵ I to prepare a labeled DNA, and this labeled DNA is used as an antigen with the anti-DNA antibody in the sample. After the antigen-antibody reaction, the labeled DNA that is not bound to the antigen-antibody complex is separated by the ammonium sulfate precipitation method (B / F separation), and the radioactivity contained in the precipitate is measured in the sample. The Farr-RIA method (Patent Document 1) for measuring the amount of anti-DNA antibody is known.
In addition to the RIA method, many Non-RIA methods are also known, and the ELISA method and the FEIA method (Patent Document 2, Patent Document 3, Patent Document 4) using immobilized DNA in which DNA is immobilized on a carrier. , Non-Patent Document 1), CLIA method, CLEIA method (Non-Patent Document 2) and the like have been reported.
The Farr-RIA method detects high-affinity anti-DNA antibodies, but cannot distinguish between antibody classes. On the other hand, the Non-RIA method detects both low-affinity and high-affinity anti-DNA antibodies, but there is a difference that the antibody class can be distinguished. From this, it is known that the Farr-RIA method and the Non-RIA method have a low correlation, and they are used properly depending on the desired use (diagnosis / disease grasping) and the patient (Non-Patent Document 4).

Japanese Patent No. 2649100 Japanese Unexamined Patent Publication No. 9-33529 Special Fair 4-40662 Gazette Japanese Unexamined Patent Publication No. 60-253869

Ann Rheum Dis, 61: 1099-1102 (2002) Clinica Chimica Acta 424: 141-147 (2013) Arthritis Rheum. 26 (1): 52-62. (1983) Journal of the Japanese Society of Internal Medicine Vol. 94, No. 10, October 10, 2005 Yaksik No. 0120 No. 1 Approval criteria for in-vitro diagnostic drugs Attachment 1

Among the many measurement methods, clinically, the Farr-RIA method is recognized to more accurately reflect the disease activity of SLE (Non-Patent Document 1, Non-Patent Document 3), and Farr-RIA. By tracking the measured values of the method, it is used to judge the effectiveness of treatment for SLE patients and to determine the treatment policy and the like.
However, the Farr-RIA method requires a dedicated measuring device, requires strict control of radioisotopes, and has many problems such as high cost.
On the other hand, although the Non-RIA method can overcome the problems of the Farr-RIA method, it does not show a good correlation with the Farr-RIA method, and problems such as the inability to accurately grasp the pathological condition of SLE have been pointed out.
In general, not only in the diagnosis of SLE, but also in the field of in-vitro diagnostic drugs, etc., when considering replacement of an existing measurement method with a new measurement method, the existing measurement method and the new measurement method have a correlation. Is required to have a very high correlation that the correlation coefficient between the two is 0.90 or more when the prescribed statistical processing is performed (Non-Patent Document 5), and the correlation coefficient is 0 in practice. Without a very high correlation coefficient of .93 or higher, the user's request cannot be satisfied.
Therefore, it is an object of the present invention to provide a measurement method that shows a good correlation with the Farr-RIA method while being a Non-RIA method.

The present inventors have various points regarding the points for enhancing the correlation in order to develop a measurement method for measuring the anti-DNA antibody in the sample with good correlation with the Farr-RIA method, although it is a Non-RIA method. As a result of examination from the viewpoint, (1) the DNA fragment used as an antigen is represented by the above formula [Equation 1] when the concentration of the DNA fragment is Y (μg / mL) and the strand length is X (bp). It is essential to use (2) a B / F separation step that separates the liquid phase and the precipitated phase after the antigen-antibody reaction, and (3) a DNA fragment labeled with a non-radioactive compound was used. In the case, it was found that the correlation was improved by measuring the amount of labeled DNA fragment in the liquid phase instead of the precipitated phase after the B / F separation.
By further brushing up these findings, it is possible to measure in the range of anti-DNA antibody concentration equivalent to that of the Farr-RIA method, and it is possible to obtain a result showing a good correlation with the Farr-RIA method. It was confirmed. The present invention has been completed based on such novel findings, and provides the following inventions.

[1]
A method for measuring an anti-DNA antibody by reacting a DNA fragment labeled with a non-radioactive compound with a sample and measuring the amount of the labeled DNA fragment.
(A) The DNA fragment labeled with the non-radioactive compound is reacted with the sample,
(B) After the reaction, a precipitant is added to the reaction solution to separate the liquid phase and the precipitate phase (B / F separation).
(C) After B / F separation, the amount of labeled DNA fragment in the liquid phase was measured.
(D) Calculate the amount of anti-DNA antibody from the measurement result of the amount of DNA fragment,
When the concentration of the DNA fragment to be used is Y (μg / mL) and the chain length is X (bp), the DNA fragment used is within the range represented by the following [Equation 1]. ,
[Number 1]
400 ≤ X ≤ 2000
Y ≧ 0.1
0.0006X-0.4595 ≤ Y ≤ 0.0005X + 0.3744
(F) When the obtained measurement results and the results of measuring the same sample by the known Farr-RIA method are compared, the correlation coefficient between the two measurement results shows 0.93 or more.
A method for measuring an anti-DNA antibody.
[2]
A method for measuring an anti-DNA antibody by reacting a DNA fragment labeled with a non-radioactive compound with a sample and measuring the amount of the labeled DNA fragment.
(A) The DNA fragment labeled with the non-radioactive compound is reacted with the sample,
(B) After the reaction, a precipitant is added to the reaction solution to separate the liquid phase and the precipitate phase (B / F separation).
(C) After B / F separation, the amount of labeled DNA fragment in the liquid phase was measured.
(D) Calculate the amount of anti-DNA antibody from the measurement result of the amount of DNA fragment,
When the concentration of the DNA fragment to be used is Y (μg / mL) and the chain length is X (bp), which includes the step and (e') is used, the one within the range represented by the following [Equation 2] is used. And
[Number 2]
700 ≤ X ≤ 2000
Y ≧ 0.1
0.0006X-0.3595 ≤ Y ≤ 0.0005X + 0.3744
(F') When the obtained measurement result and the result of measuring the same sample by the known Farr-RIA method are compared, the correlation coefficient of both measurement results shows 0.95 or more.
A method for measuring an anti-DNA antibody.
[3]
The non-radioactive compounds used to label DNA fragments are biotin, 2,4-Dinitrophenol (DNP), digoxigenin (DIG), Fluorescein, HiBiT tags, and theirs. The measuring method according to [1] or [2], which is selected from the derivatives of.
[4]
The measuring method according to any one of [1] to [3], wherein the precipitant used is ammonium sulfate.
[5]
(G) When the concentration of the DNA fragment is Y (μg / mL) and the chain length is X (bp), the DNA labeled with a non-radioactive compound within the range represented by [Equation 1] below. fragment,
[Number 1]
400 ≤ X ≤ 2000
Y ≧ 0.1
0.0006X-0.4595 ≤ Y ≤ 0.0005X + 0.3744
It consists of (h) a precipitant for separating the liquid phase and the precipitated phase (B / F separation), and (i) a reagent for measuring the amount of labeled DNA fragment in the liquid phase after B / F separation. A reagent kit for measuring the amount of anti-DNA antibody in a sample by the measuring method according to any one of [1] to [4].

The measurement method of the present invention is described in the above formula [Equation 1] when (1) the concentration of the DNA fragment labeled with a non-radioactive compound as an antigen is Y (μg / mL) and the chain length is X (bp). Using the DNA fragment represented by, (2) after the antigen-antibody reaction, B / F separation is performed to separate the liquid phase and the precipitated phase, and (3) after the B / F separation, labeling in the liquid phase instead of the precipitated phase The three elements of measuring the amount of DNA fragment are simultaneously executed. By executing the above elements at the same time, it has become possible to obtain a measured value having a high correlation with the Farr-RIA method for the first time in the Non-RIA method.
For this reason, the present invention is different from the conventional Non-RIA method in terms of performance, and more accurately reflects the disease activity of SLE in clinical diagnosis that can replace the Farr-RIA method. A non-RIA-like method for measuring a DNA antibody can be provided.
Further, (1) DNA represented by the above formula [Equation 2] when the concentration of the DNA fragment labeled with a non-radioactive compound as an antigen is Y (μg / mL) and the strand length is X (bp). Using the fragments, (2) after the antigen-antibody reaction, perform B / F separation to separate the liquid phase and the precipitated phase, and (3) after the B / F separation, determine the amount of labeled DNA fragment in the liquid phase instead of the precipitated phase. By simultaneously executing the three elements of measurement, it is possible to detect with high sensitivity even if the anti-DNA antibody concentration in the sample is low, and to measure with high sensitivity even if the anti-DNA antibody concentration is high. It is possible to provide a Non-RIA-like method for measuring an anti-DNA antibody, which shows a better correlation with the Farr-RIA method.

FIG. 1 is a diagram showing the measurement sensitivity of Biotin / DNP-labeled DNA by an immunological measurement method. The horizontal axis of the graph represents the labeled DNA concentration (ng / mL), and the vertical axis represents RLU. FIG. 2 is a diagram showing the effect of the labeled DNA concentration to react with the sample at B / B ₀ % in the measurement of the anti-DNA antibody by the measurement of the labeled DNA (418 bp) in the B / F separation supernatant. The concentrations of labeled DNA to be examined were 0.1 μg / mL, 0.2 μg / mL, 0.4 μg / mL, 0.8 μg / mL, 1.6 μg / mL, and 3.2 μg / mL. The horizontal axis of the graph represents the anti-DNA antibody concentration (IU / mL), and the vertical axis represents the signal measurement value (B / B ₀ %). FIG. 3 is a diagram showing a range of labeled DNA concentration and chain length suitable for the measurement system of the present invention. In the graph, the horizontal axis represents the DNA strand length (bp) and the vertical axis represents the DNA concentration (μg / mL). The points plotted in the graph show that the measurement system at each point is (i) B / B ₀ % of 13.7 IU / mL, where a concentration-dependent signal decrease is observed from _{0 to} 200 IU / mL. There is 90% or less, rated at (iii) 218.2IU / mL of B / _{B 0%} is anti-DNA antibodies 131.8IU / mL of B / _{B 0%} more than twice, three criteria In the case of, "●: All the criteria of (i) to (iii) were satisfied" and "▲: (i) was satisfied, but either (ii) or (iii) (or both) was satisfied. It indicates whether it was "not" or "x: did not satisfy (i)". The colored part in the graph shows an evaluation of ▲ or more and a correlation coefficient of 0.93 or more, that is, a range of labeled DNA concentration and chain length suitable for the measurement system of the present invention. FIG. 4 is a correlation diagram between the Farr-RIA method for SLE patient serum and the labeled DNA measurement method in B / F separation supernatant (the present invention). The horizontal axis plots the measured value of the sample when measured with the commercially available Farr-RIA method measuring kit, and the vertical axis plots the measured value of the present invention (labeled DNA length 1159 bp, concentration 0.8 μg / mL), and the phases of both. The number of relationships is calculated. FIG. 5 is a diagram showing a range of labeled DNA concentration and chain length that is particularly suitable for the measurement system of the present invention. In the graph, the horizontal axis represents the DNA strand length (bp) and the vertical axis represents the DNA concentration (μg / mL). The points plotted in the graph show that the measurement system at each point is (i) B / B ₀ % of 13.7 IU / mL, where a concentration-dependent signal decrease is observed from _{0 to} 200 IU / mL. There is 90% or less, rated at (iii) 218.2IU / mL of B / _{B 0%} is anti-DNA antibodies 131.8IU / mL of B / _{B 0%} more than twice, three criteria In the case of, "●: All the criteria of (i) to (iii) were satisfied" and "▲: (i) was satisfied, but either (ii) or (iii) (or both) was satisfied. It indicates whether it was "not" or "x: did not satisfy (i)". The colored part in the graph shows ● or more as an evaluation and a correlation coefficient of 0.95 or more, that is, it indicates that it is in the range of the labeled DNA concentration and the chain length which are particularly suitable in the measurement system of the present invention. .. FIG. 6 is a correlation diagram between the Farr-RIA method and the FEIA method of SLE patient serum. The horizontal axis plots the measured value of the sample measured by the commercially available Farr-RIA method measuring kit, and the vertical axis plots the measured value of the sample measured by the commercially available FEIA method measuring kit, and the correlation coefficient between the two is calculated. ing. FIG. 7 shows the reactivity of Biotin / DNP-labeled DNA and Biotin / DIG-labeled DNA in the measurement of anti-DNA antibody by the measurement of labeled DNA in the B / F separation supernatant with respect to B / B ₀ %. It is a figure shown by. The horizontal axis of the graph represents the anti-DNA antibody concentration (IU / mL), and the vertical axis represents the signal measurement value (B / B ₀ %). FIG. 8 is a correlation diagram between the Farr-RIA method of SLE patient serum and the labeled DNA measurement method during B / F separation and precipitation (Reference Example 2). The horizontal axis shows the measured values of the sample measured with the commercially available Farr-RIA method measurement kit, and the vertical axis shows the labeled DNA measurement method during B / F separation and precipitation (labeled DNA chain lengths 1159 bp and 1274 bp, concentration 0.8 μg / mL) is plotted and the correlation coefficient between the two is calculated. FIG. 9 is a diagram showing the measurement sensitivity of HiBiT-labeled DNA by the HiBiT system. The horizontal axis of the graph represents the labeled DNA concentration (ng / mL), and the vertical axis represents RLU. FIG. 10 is a diagram showing the effect of the labeled DNA concentration to react with the sample at B / B ₀ % in the measurement of the anti-DNA antibody by the measurement of HiBiT-labeled DNA in the B / F separation supernatant. 0.2 μg / mL, 0.4 μg / mL, and 0.8 μg / mL were used as the concentrations of the labeled DNA to be examined. The horizontal axis of the graph represents the anti-DNA antibody concentration (IU / mL), and the vertical axis represents the signal measurement value (B / B ₀ %). FIG. 11 is a correlation diagram between the Farr-RIA method of SLE patient serum and the HiBiT-labeled DNA measurement method in the B / F separation supernatant. The horizontal axis plots the measured value of the sample when measured with the commercially available Farr-RIA method measurement kit, and the vertical axis plots the measured value of the present invention (HiBiT-labeled DNA length 1159 bp, concentration 0.8 μg / mL). The correlation coefficient is calculated.

One aspect of the present invention is a Non-RIA-like method for measuring an anti-DNA antibody.

The samples to be measured in the present invention are human serum and plasma. In particular, specimens of patients with autoimmune diseases such as SLE are the targets of measurement.

The DNA fragment labeled with the non-radioactive compound used in the present invention refers to a DNA fragment to which a non-radioactive label (compound) is added.
The DNA fragment used in the present invention is double-stranded.
Further, only one type of DNA fragment may be used, or a mixture of two or more types of DNA fragments having different lengths may be used.
For such a DNA fragment, a DNA fragment having a desired length can be prepared according to a known method such as a PCR method or a restriction enzyme treatment from a plasmid. Moreover, there is no limitation on the sequence of the DNA fragment.

The type of the non-radioactive labeling compound used in the present invention can be appropriately selected depending on the measurement principle of the measurement method used. In addition, the number of labeling types is preferably single labeling or double labeling. Here, the single label refers to a state labeled with one kind of non-radiolabeled compound, and the double label means a state labeled with two kinds of non-radiolabeled compounds.
The number of labels may be introduced at one or more locations in one DNA fragment, and may be introduced at a plurality of locations.
When double-labeled DNA is used, it is preferable that each DNA end is labeled with a kind. In addition, both ends may be labeled with the same type of label.

Labels used in the present invention include biotin, 2,4-Dinitrophenol (DNP), digoxigenin (DIG), fluorescein, 5-bromodeoxyuridine (Bromodeoxyuridine). It can be selected from low molecular weight compounds such as His-tag, FLAG-tag, HA-tag, Myc-tag, peptide tags such as HiBiT tag, enzymes such as peroxidase and alkaline phosphatase, and derivatives thereof.
Among the above compounds, biotin, 2,4-Dinitrophenol (DNP), digoxigenin (DIG), and fluorescein (Digoxigenin: DIG) are difficult to precipitate in the B / F separation step. Fluorescein), HiBiT tags, and derivatives thereof are preferred.
As a method for labeling DNA using the label, it can be prepared by a conventional method usually used for each labeled compound. For example, taking the biotin-DNP double-labeled DNA exemplified in the examples as an example, a DNA fragment is amplified by PCR using a 5'-biotin-labeled F primer and a 5'-DNP-labeled R primer. An example of how to obtain it. However, the preparation method is not limited to the above method.

For the reaction between the labeled DNA and the sample, known conditions capable of carrying out a normal antigen-antibody reaction may be used. The concentration of the labeled DNA to be brought into contact with the sample to be measured can be appropriately selected according to the length of the DNA fragment used.

More specifically, the relationship between the chain length and the concentration of the labeled DNA fragment will be described. The labeled DNA used in the present invention preferably has an amount and a chain length within a specific range. The specific range is the range defined by the following [Equation 1] when the concentration of the labeled DNA is Y (μg / mL) and the strand length is X (bp).
[Number 1]
400 ≤ X ≤ 2000
Y ≧ 0.1
0.0006X-0.4595 ≤ Y ≤ 0.0005X + 0.3744
When a mixture of two or more types of DNA fragments having different lengths is used, the above-mentioned strand length refers to the average strand length. Further, even when a mixture of two or more kinds of DNA fragments having different lengths is used, the length of each DNA fragment is preferably within the above range.

When the concentration and chain length of the labeled DNA fragment are within the above ranges, it can be detected with high sensitivity even when the anti-DNA antibody concentration is low, and when the anti-DNA antibody concentration is high. Even if there is, it can be measured with high sensitivity. When the labeled DNA is used, the present invention shows a high correlation with the existing Farr-RIA method, specifically, a correlation coefficient of 0.93 or more.

The concentration and strand length of the labeled DNA fragment are more preferably in the range specified in [Equation 2] below.
[Number 2]
700 ≤ X ≤ 2000
Y ≧ 0.1
0.0006X-0.3595 ≤ Y ≤ 0.0005X + 0.3744
When the concentration and chain length of the labeled DNA fragment used are within the above ranges, the present invention can detect with high sensitivity even if the anti-DNA antibody concentration in the sample is low, and the anti-DNA antibody concentration is high. However, it can be measured with high sensitivity, and further, it shows a higher correlation with the existing Farr-RIA method, specifically, a correlation coefficient of 0.95 or more.

The clear reason for the fact that measurement can be performed with good sensitivity within the range specified in [Equation 1] to [Equation 2] above is unclear, but the amount of substance of the labeled DNA in the measurement system is important. It is presumed that there is. However, as can be seen from the results of the examples described later, the sensitivity of the measurement system and the like vary depending on the chain length even if the amount of substance is the same. It is presumed that this is because the behavior in the measurement system changes as the labeled DNA length increases, such as binding of two antibodies to one fragment.
For the above reasons, the suitable labeled DNA conditions used in the present invention cannot be defined by the final molar concentration in the measurement system or the like, and can only be described in a complicated format as in [Equation 1].
When a mixture of two or more types of DNA fragments having different lengths is used, the above-mentioned strand length refers to the average strand length.

Examples of the precipitant added after the reaction include ammonium sulfate, polyethylene glycol, polyamino acid, trichloroacetic acid, acetone, ethanol and the like. Among the above-mentioned precipitants, ammonium sulfate and polyethylene glycol are preferable, and ammonium sulfate is particularly preferable. The amount of the precipitant added can be appropriately set according to the precipitant to be selected. For example, when ammonium sulfate is selected as the precipitating agent, the method of ammonium sulfate precipitation is known by itself, and it is preferable to add ammonium sulfate so that the concentration in the reaction solution is 40 to 60% saturated, particularly 45 to 55% saturated. If it is 40% saturated or more, precipitation occurs, and if it is 60% or less, it has a good correlation with the Farr-RIA method.
B / F separation can be performed by a known method such as standing, decantation, filtration, or centrifugation. For example, in the case of centrifugation, it can be carried out under the conditions of 1800 × g, 4 ° C. and 30 minutes, and the conditions can be appropriately set.

Next, the amount of labeled DNA fragment in the liquid phase (supernatant) after B / F separation is measured. The supernatant may be subjected to measurement as it is, or may be appropriately diluted, concentrated, pH adjusted or the like as necessary. As will be clear from the examples described later, by measuring the amount of labeled DNA fragment in the liquid phase (supernatant) after B / F separation, the correlation coefficient of 0.93 or more required by the present invention can be achieved. On the other hand, the correlation coefficient of 0.93 or more cannot be achieved in the precipitation after B / F separation.

The method for measuring the amount of the labeled DNA fragment may be any method known per se that corresponds to the labeled compound, and is not particularly limited. In particular, as a method that is easy to operate and low in cost, a method that utilizes the specific affinity between molecules is preferable. Specifically, EIA, ELISA, CLEIA, FEIA, CLIA, ECLIA, FIA, and latex agglutination. Examples thereof include a method, an immunoturbidimetric method, an immunoreduction wax method, a surface plasmon resonance method, and a HiBiT system.
Among the above measurement methods, EIA, ELISA, CLEIA, FEIA, which utilize a detection system corresponding to a labeling agent, specifically, a biotin-streptavidin system, a DNP-anti-DNP antibody system, a DIG-anti-DIG antibody system, etc. CLIA, ELISA, FIA, and the HiBiT system, which is a homogenius luminescence measurement system using a luciferase complementary to a high affinity peptide (HiBiT peptide tag: amino acid sequence VSGWRLFKKIS), are exemplified as preferable, but are not limited thereto.

The amount of anti-DNA antibody in the sample is calculated from the measured amount of labeled DNA fragment in the liquid phase. That is, by reacting an anti-DNA antibody of an arbitrary concentration with a labeled DNA fragment and measuring the amount of the labeled DNA fragment that did not bind to the anti-DNA antibody by B / F separation, it was inversely proportional to the anti-DNA antibody concentration. A calibration curve can be drawn by utilizing the fact that a signal is obtained, and the anti-DNA antibody concentration can be calculated using this calibration curve.
Further, as a secondary advantage of the present invention for calculating the amount of anti-DNA antibody in a sample from the measured amount of labeled DNA fragment in the liquid phase, the concentration is higher than that of the existing Farr-RIA method, for example, 200 IU / mL or more. It is possible to measure with high sensitivity under the condition of high concentration of anti-DNA antibody. This is because the existing Farr-RIA method, which measures the signal increase of radiolabeled DNA and calculates the amount of anti-DNA antibody, causes the signal to reach a plateau under high anti-DNA antibody concentration conditions and cannot be measured with high sensitivity. On the other hand, in the present invention, since the amount of anti-DNA antibody is calculated from the decrease in signal, it is easier to measure under the condition of high concentration of anti-DNA antibody than the Farr-RIA method.

The correlation coefficient is obtained by plotting the measured values (X, Y) on the X-axis and the measurement result of the present invention on the X-axis and statistically processing the measurement result of the existing Farr-RIA method for the same sample. Can be done. The correlation coefficient of the present invention is required to be 0.93 or more.

One aspect of the present invention is a reagent kit for measuring the amount of anti-DNA antibody.

The reagent kit of the present invention
(G) When the concentration of the DNA fragment labeled with a non-radioactive compound is Y (μg / mL) and the strand length is X (bp), the DNA fragment within the range represented by the following [Equation 1] ,
[Number 1]
400 ≤ X ≤ 2000
Y ≧ 0.1
0.0006X-0.4595 ≤ Y ≤ 0.0005X + 0.3744
It consists of (h) a precipitant for separating the liquid phase and the precipitated phase (B / F separation), and (i) a reagent for measuring the amount of labeled DNA fragment in the liquid phase after B / F separation. This is a reagent kit for measuring the amount of anti-DNA antibody in a sample by the measurement method of the present invention described above.
In such a kit, as a reagent for measuring the amount of labeled DNA fragment, a reagent commonly used for detecting the label used can be used, and the best combination thereof can be appropriately selected. Good. As an example, a kit configuration such as a standard solution, a labeled DNA solution, a B / F separator, a diluent, an ALP-labeled DNP antibody solution, a streptavidin magnetic particle solution, a luminescent reagent, and a cleaning solution can be considered.

The method for measuring a Non-RIA anti-DNA antibody of the present invention is as follows: (1) The concentration of a DNA fragment labeled with a non-radioactive compound as an antigen is Y (μg / mL), and the chain length is X (bp). Sometimes, using the DNA fragment represented by the above formula [Equation 1], (2) after the antigen-antibody reaction, B / F separation is performed to separate the liquid phase and the precipitation phase, and (3) B / F separation and then precipitation. The biggest feature is to measure the amount of labeled DNA fragment in the liquid phase instead of the phase. By simultaneously executing these three elements, the anti-DNA antibody concentration in the sample is highly correlated with the existing Farr-RIA method, that is, the correlation coefficient is 0.93 or more, as demonstrated in the examples described later. Therefore, the measurement can be preferably performed with a correlation coefficient of 0.95 or more, more preferably a correlation coefficient of 0.97 or more. Since this high correlation can be obtained, the present invention can be used to diagnose whether a subject has SLE instead of the existing diagnostic method for SLE based on the Farr-RIA method, and to evaluate disease activity. ..

For details of the measurement method utilizing the specific affinity between molecules such as EIA, ELISA, CLEIA, FEIA, CLIA, ECLIA, and FIA that can be adopted in the present invention, for example, the following documents may be referred to. ..
(b) "Enzyme Immunoassay" edited by Hideharu Ishikawa et al. (2nd edition) (Igaku-Shoin Co., Ltd., published on December 15, 1982)
(c) Extra edition of clinical pathology Special issue No. 53 "Immunoassay for clinical examination-technology and application-" (Clinical Pathology Publishing Association, published in 1983)
(d) "Biotechnology Encyclopedia" (CMC Co., Ltd., published on October 9, 1986)
(e) "Methods in ENZYMOLOGY Vol.70" (Immunochemical techniques (Part A))
(f) "Methods in ENZYMOLOGY Vol.73" (Immunochemical techniques (Part B))
(g) "Methods in ENZYMOLOGY Vol.74" (Immunochemical techniques (Part C))
(h) "Methods in ENZYMOLOGY Vol.84" (Immunochemical techniques (Part D: Selected Immunoassay))
(i) "Methods in ENZYMOLOGY Vol.92" (Immunochemical techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods))
[(E)-(i) are published by Academic Press]
(j) Biochemical Measurement Study Group (Norihiro Kobayashi et al.), "Immunometry" (Kodansha Co., Ltd., published on December 20, 2014)

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

(Example 1)
(1-1) Construction of Non-RIA measurement system using non-radioactive labeled DNA fragment
[Reagent preparation]
5'-Biotin-labeled Forward primer (sequence 1: 5'-GCGCCATTCGCCCATTCAG-3'primer with Biotin-labeled at the 5'end) and 5'-Ditintrophenol (DNP) -labeled Reverse primer (sequence with DNP-labeled at the 5'end) A primer for 2: 5'-ATTTTTGTGATGCTCGTCAGGGG-3') was ordered, pUC19 to 418 bp were amplified by PCR, and about 100 μg of Biotin / DNP-labeled DNA was obtained. Biotin / DNP-labeled DNA was prepared at 0.0001 to 100,000 ng / mL with 50 mM Tris (pH 8) and 1 mM EDTA. Goat anti-DNP Affinity Purified (BETHYL, A150-117A) was labeled with an ALP labeling kit (Alkaline phosphatase Labeling Kit-SH, Dojin Kagaku, LK13) to obtain an ALP-labeled anti-DNP antibody.

[Measurement of labeled DNA]
Sufficient reaction of 30 μL of the first reagent (50 mM HEPES (pH 7.2)) containing various concentrations of Biotin / DNP-labeled DNA and 30 μL of the second reagent (ALP-labeled anti-DNP antibody) instead of the sample at 37 ° C. After that, 30 μL of the third reagent (streptavidin magnetic particles) was added and mixed, and the reaction was further carried out at 37 ° C. After the reaction, the cells were washed, 100 μL of a luminescent substrate was added, and the luminescence intensity was measured to detect Biotin / DNP-labeled DNA.

The results are shown in FIG. As shown in FIG. 1, since an RLU difference was observed between DNA 0 ng / mL and 0.32 ng / mL, it was possible to detect if the amount of Biotin / DNP-labeled DNA was 0.32 ng / mL or more. It was judged. Further, up to about 200 ng / mL, the RLU value showed good linearity with respect to the amount of DNA. From the above results, it was shown that measurement is possible if the concentration of Biotin / DNP-labeled DNA in the reaction solution is within the range of 0.32 to 200 ng / mL.

(1-2) Measurement of labeled DNA in supernatant after B / F separation SLE patient serum appropriately diluted with bovine serum, which has been priced with a commercially available kit (Seti) based on the measurement principle of the Farr-RIA method. As a standard solution, 25 μL of this was added to a test tube, 200 μL of a 0.2 to 3.2 μg / mL Biotin / DNP-labeled DNA solution (418 bp) was added, mixed, and incubated at 37 ° C. for 2 hours.
After the reaction, 1000 μL of cold ammonium sulfate (61.25% saturated) was added so that the concentration of ammonium sulfate in the reaction solution was 50%, and the mixture was mixed. Centrifuge at 2000 xg, 4 ° C. for 15 minutes.

10 μL of the obtained supernatant after centrifugation, 30 μL of the first reagent (50 mM HEPES (pH 7.2)) and 30 μL of the second reagent (ALP-labeled anti-DNP antibody) were mixed, and the mixture was sufficiently reacted at 37 ° C. 30 μL of 3 reagents (streptavidin magnetic particles) were added and mixed, and the mixture was sufficiently reacted at 37 ° C. After the reaction, the cells were washed, 100 μL of a luminescent substrate was added, and the luminescence intensity was measured to detect labeled DNA in the supernatant.

The results are shown in FIG. 2 and Table 1.
In addition, "evaluation" in Table 1 below means that (i) concentration-dependent signal decrease is observed from _{0 to} 200 IU / mL, and (ii) B / B ₀ % of 13.7 IU / mL is 90%. hereinafter become, when evaluated in (iii) 218.2IU / mL of B / _{B 0%} is anti-DNA antibodies 131.8IU / mL of B / _{B 0%} more than twice, three criteria , "○: All the criteria of (i) to (iii) were satisfied", "Δ: (i) was satisfied, but either (ii) or (iii) (or both) was not satisfied" , "X: (i) was not satisfied", the evaluation is performed.
"○" in Table 1 corresponds to "●" in Fig. 3, which will be described later, "△" in Table 1 corresponds to "▲" in Fig. 3, and "×" in Table 1 corresponds to "×" in Fig. 3. ing.
△ (or ▲) is a symbol selected to differentiate from ○ (or ●) in the graph, and actually means that it meets a practical level. Therefore, it should be clearly stated that it has no negative meaning other than being an intermediate evaluation between ○ evaluation and × evaluation.

When the biotin / DNP-labeled DNA concentration was 0.1-0.4 μg / mL, a concentration-dependent signal decrease of the anti-DNA antibody was observed in the whole range of 0 to 200 IU / mL. From this, it was clarified that the anti-DNA antibody can be measured by the present invention.
If labeled DNA concentration is more than _{0.2μg / mL, B / B 0} % of anti-DNA antibodies 218.2IU / mL become anti-DNA antibody 131.8IU / mL of B / _{B 0%} of 2 times or more, anti It was clarified that it can be measured even in the high concentration range of DNA antibody.
When the Biotin / DNP-labeled DNA concentration is 0.2 μg / mL or less, the B / B ₀ % of the anti-DNA antibody 13.7 IU / mL is 90% or less, and even if the anti-DNA antibody concentration is in the low concentration range, It became clear that it can be measured with good sensitivity.
Taken together, the anti-DNA antibody can be measured when the labeled DNA length is 418 bp and the biotin / DNP-labeled DNA concentration is 0.1-0.4 μg / mL, with 0.2 μg / mL being preferable. It became clear that.

(Example 1-3) Examination of relationship between DNA fragment concentration and strand length 5'-Biotin-labeled Forward primer (primer of sequence 1: 5'-GCGCCATTTCGCCATTCAG-3' having Biotin-labeled at the 5'end) and 5' -Diintrophenol (DNP) -labeled Reverse primer (primer of sequence 3: 5'-GCACCCATCTCAGCGATCTGTC-3' with DNP-labeled at the 5'end) was used to amplify pUC19 to 1159bp by PCR to obtain Biotin / DNP-labeled DNA. About 100 μg was obtained. Using a SLE patient serum appropriately diluted with bovine serum, which has been priced with a commercial kit (Seti) based on the measurement principle of the Farr-RIA method, as a standard solution, add 25 μL to a test tube, and 0.2 to 0.8 μg. 200 μL of / mL Biotin / DNP-labeled DNA solution (1159 bp) was added, mixed, and incubated at 37 ° C. for 2 hours.
After the reaction, 1000 μL of cold ammonium sulfate (61.25% saturated) was added so that the concentration of ammonium sulfate in the reaction solution was 50%, and the mixture was mixed. Centrifuge at 2000 xg, 4 ° C. for 15 minutes.

10 μL of the obtained supernatant after centrifugation, 30 μL of the first reagent (50 mM HEPES (pH 7.2)) and 30 μL of the second reagent (ALP-labeled anti-DNP antibody) were mixed, and the mixture was sufficiently reacted at 37 ° C. 30 μL of 3 reagents (streptavidin magnetic particles) were added and mixed, and the mixture was sufficiently reacted at 37 ° C. After the reaction, the cells were washed, 100 μL of a luminescent substrate was added, and the luminescence intensity was measured to detect labeled DNA in the supernatant.

The above 0.2 to 0.8 μg / mL Biotin / DNP labeled DNA solution (1159 bp) was replaced with a 0.2 to 1.2 μg / mL Biotin / DNP labeled DNA solution (1862 bp), and the same study was conducted. It was. For PCR amplification, pUC19 was used as a template, and 5'-Biotin-labeled Forward primer (primer of sequence 1: 5'-GCGCCATTCGCCATTCAGG-3'with Biotin-labeled at the 5'end) and 5'-Ditintrophenol (DNP) -labeled Reverse. Primers (primers of sequence 4: 5'-AACTGGATCTCAACAGCGGTAAG-3' having a DNP label at the 5'end) were used.

As a result of this example, the results shown in Table 2 below were obtained. As for the "evaluation" in Table 2 below, as in Table 1, (i) a concentration-dependent signal decrease is observed from 0 to 200 IU / mL, and (ii) 13.7 IU / mL B / B. Three evaluation criteria: ₀ % is 90% or less, and (iii) 218.2 IU / mL B / B ₀ % is more than twice the anti-DNA antibody 131.8 IU / mL B / B ₀ %. When evaluated in, "○: Satisfied all the criteria of (i) to (iii)" and "Δ: (i) was satisfied, but either (ii) or (iii) (or both). The evaluation was made according to the criteria of "does not meet" and "x: did not meet (i)".

In the case of labeled DNA strand length 1159 bp, a signal decrease dependent on anti-DNA antibody concentration was observed at all labeled DNA concentrations, and B / B ₀ % of anti-DNA antibody 13.7 IU / mL was 90% or less. It showed good sensitivity. Above all, if the labeled DNA concentrations greater than 0.4 [mu] g / mL, anti-DNA antibody 218.2IU / mL of B / _{B 0%} becomes anti-DNA antibody 131.8IU / mL of B / _{B 0%} of 2 times or more , It was possible to measure well even in the high concentration range of anti-DNA antibody.

When the labeled DNA length is 1862 bp, the anti-DNA antibody 131.8 IU / mL B / B ₀ % and the anti-DNA antibody 218.2 IU / mL B / B ₀ % are present at a labeled DNA concentration of 0.4 μg / mL or less. Indistinguishable, no concentration-dependent signal decrease could be observed in the high concentration range of anti-DNA antibody. On the other hand, at a labeled DNA concentration of 0.8 μg / mL or higher, an anti-DNA antibody concentration-dependent signal decrease was observed, and the B / B ₀ % of the anti-DNA antibody 13.7 IU / mL was 90% or less, and the anti-DNA antibody 218. .2IU / mL of B / _{B 0%} becomes anti-DNA antibody 131.8IU / mL of B / _{B 0%} more than twice, it was possible to divide measure with good sensitivity.
Comparing the results of the labeled DNA length of 1159 bp and 0.2 μg / mL and the results of the labeled DNA length of 1159 bp and 0.4 μg / mL with the results of the labeled DNA length of 1862 bp and 0.4 μg / mL, 1159 bp. In 1862bp, the sensitivity is poor, whereas even if the amount of the labeled DNA is the same, the chain length of the labeled DNA is high. It was clarified that when they are different, there is a difference in terms of sensitivity and the like.

From the results of Examples 1-2 and 1-3, the relationship between the concentration and the chain length of the labeled DNA used in the present invention was analyzed.
Results at concentration and strand length of each labeled DNA show that (i) concentration-dependent signal reduction is observed at 0-200 IU / mL, (ii) 90% B / B ₀ % at 13.7 IU / mL. hereinafter become, evaluated by (iii) 218.2IU / mL of B / _{B 0%} is anti-DNA antibodies 131.8IU / mL of B / _{B 0%} more than twice, three criteria, " ●: All the criteria of (i) to (iii) were met "," ▲: (i) was met, but either (or both) of (ii) or (iii) was not met "," X: did not satisfy (i) ”, and the results were shown as the shape of the plot points in the table of FIG.
From this result, in the present invention, when the concentration of the labeled DNA is Y (μg / mL) and the chain length is X (bp), the range is defined by the following [Equation 1]. , It was clarified that the anti-DNA antibody 0 to 200 IU / mL can be measured with high sensitivity in a concentration-dependent manner.
[Number 1]
400 ≤ X ≤ 2000
Y ≧ 0.1
0.0006X-0.4595 ≤ Y ≤ 0.0005X + 0.3744
The range defined by the above [Equation 1] is shown in color in the table of FIG.

(1-4) Correlation test between the method of the present invention and the Farr-RIA method A standard solution of SLE patient serum appropriately diluted with bovine serum, which has been priced with a commercially available kit (Seti) based on the measurement principle of the Farr-RIA method. In addition, 25 μL of SLE patient serum (prescribed by Farr-RIA method and FEIA method) was added, and Biotin / DNP-labeled DNA solution (418 bp was 0.2 μg / mL, 1159 bp was 0.8 μg / mL, and 1862 bp was 1.2 μg / mL) was added and mixed, and the mixture was incubated at 37 ° C. for 2 hours. After the reaction, 1000 μL of cold ammonium sulfate (61.25% saturated) was added so that the concentration of ammonium sulfate in the reaction solution was 50%, and the mixture was mixed. Centrifuge at 2000 xg, 4 ° C. for 15 minutes.

After centrifugation, 10 μL of the obtained supernatant, 30 μL of the first reagent (50 mM HEPES (pH 7.2)) and 30 μL of the second reagent (ALP-labeled anti-DNP antibody) were mixed, and the mixture was sufficiently reacted at 37 ° C. 30 μL of 3 reagents (streptavidin magnetic particles) were added and mixed, and the mixture was sufficiently reacted at 37 ° C.
After the reaction, the cells were washed, 100 μL of a luminescent substrate was added, and the luminescence intensity was measured to detect labeled DNA.
The correlation between the present invention and the Farr-RIA method was calculated by comparing the amount of the anti-DNA antibody of the present invention calculated from the amount of labeled DNA with the amount of the anti-DNA antibody measured by the Farr-RIA method by statistical processing. As an example of the calculation method, the result of 1159 bp is shown in FIG. 4, and the result of the correlation coefficient at each labeled DNA length is summarized in Table 3.

As a result of measurement using SLE patient sera, as shown in Table 3, the measurement system of the present invention showed a high correlation of 0.93 or more regardless of the labeled DNA length. In particular, when the labeled DNA length was 1159 bp, the correlation coefficient was 0.9758, which was the best correlation. Further, from the results of 418 bp and 1159 bp above, it was found that the labeled DNA length to be used must be 672 bp or more in order for the correlation coefficient to be 0.95 or more in the present invention.
In other words, from the results of this example, when the labeled DNA has a concentration of the labeled DNA of Y (μg / mL) and a chain length of X (bp), the case where the relational expression of [Equation 1] is satisfied. If the evaluation of the present invention is Δ or more, the correlation coefficient between the present invention and the Farr-RIA method is 0.93 or more, and the relational expression of the following [Equation 2] is satisfied, the present invention It was clarified that the evaluation of the invention was ◯ or higher and the correlation coefficient was 0.95 or higher.
[Number 2]
700 ≤ X ≤ 2000
Y ≧ 0.1
0.0006X-0.3595 ≤ Y ≤ 0.0005X + 0.3744
The range satisfying the relational expression of the above [Equation 2] is shown in FIG.

(Reference Example 1) Correlation test between existing Non-RIA method and Farr-RIA method SLE patient serum appropriately diluted with bovine serum, which has been priced with a commercially available kit (Seti) based on the measurement principle of Farr-RIA method, is used. As a standard solution, the anti-DNA antibody concentration was measured using a commercially available kit (Area dsDNA, Fadia) based on the measurement principle of the FEIA method. The measured values of both were compared by statistical processing, and the correlation between the FEIA method and the Farr-RIA method was calculated. The result is shown in FIG.

As shown in FIG. 6, the correlation coefficient between the FEIA method and the Farr-RIA method was 0.67, and it could not be said that a good correlation was shown.

(Example 2) Comparison of reactivity by type of label A 1159 bp Biotin / DIG-labeled DNA fragment was amplified by PCR by the method described in Example 1-3. In addition, a 5'-Biotin-labeled Forward primer (a primer for a sequence having a Biotin-labeled at the 5'end, a primer of 5: 5'-CTCACTGATTAAGCATTGGTAACTGTC-3') and a 5'-Digoxigenin (DIG) -labeled Reverse primer (DIG-labeled at the 5'end). Biotin / DIG-labeled DNA fragments from pUC19 to 1274 bp were amplified by PCR using a primer of sequence 6: 5'-CTGATGCGGTATTTTTCCTTACG-3').

Using a SLE patient serum appropriately diluted with bovine serum, which has been priced with a commercial kit (Seti) based on the measurement principle of the Farr-RIA method, as a standard solution, add 25 μL to a test tube, and 0.2 to 0.8 μg. 200 μL of / mL Biotin / DIG-labeled DNA solution (a mixture of equal amounts of 1159 bp and 1274 bp) was added, mixed, and incubated at 37 ° C. for 2 hours.
After the reaction, 1000 μL of cold ammonium sulfate (61.25% saturated) was added so that the concentration of ammonium sulfate in the reaction solution was 50%, and the mixture was mixed. Centrifuge at 2000 xg, 4 ° C. for 15 minutes.

10 μL of the obtained supernatant after centrifugation, 30 μL of the first reagent (50 mM HEPES (pH 7.2)) and 30 μL of the second reagent (ALP-labeled anti-DIG antibody) were mixed, and the mixture was sufficiently reacted at 37 ° C. 30 μL of 3 reagents (streptavidin magnetic particles) were added and mixed, and the mixture was sufficiently reacted at 37 ° C. After the reaction, the cells were washed, 100 μL of a luminescent substrate was added, and the luminescence intensity was measured to detect labeled DNA in the supernatant.

As a result of this example, 0.4 μg / mL Biotin / DIG-labeled DNA showed the same reactivity as 0.8 μg / mL Biotin / DNP-labeled DNA (Fig. 7).
From this, it was clarified that the present invention can be measured regardless of the type of label used.

(Reference Example 2) Correlation test when labeled DNA in the precipitate is measured instead of the supernatant after B / F separation. Priced with a commercially available kit (Seti) based on the measurement principle of the Farr-RIA method. SLE patient serum diluted appropriately with bovine serum was used as a standard solution, and 25 μL of SLE patient serum (valued by Farr-RIA method and FEIA method) was added to Biotin / DNP-labeled DNA solution (1159 bp and 1274 bp, 0. 8 μg / mL) was added and mixed, and the mixture was incubated at 37 ° C. for 2 hours. After the reaction, 1000 μL of cold ammonium sulfate (61.25% saturated) was added so that the concentration of ammonium sulfate in the reaction solution was 50%, and the mixture was mixed. Centrifuge at 2000 xg, 4 ° C. for 15 minutes.

The obtained precipitate after centrifugation was redissolved with the first reagent (50 mM HEPES (pH 7.2)), 10 μL of the lysate, 30 μL of the first reagent (50 mM HEPES (pH 7.2)), and the second reagent (ALP-labeled antibody). 30 μL of DNP antibody) was mixed and reacted sufficiently at 37 ° C., then 30 μL of a third reagent (streptavidin magnetic particles) was added and mixed, and the reaction was sufficiently reacted at 37 ° C.

The result is shown in FIG.
As shown in FIG. 8, the correlation coefficient with the Farr-RIA method when measuring the precipitation after centrifugation was 0.89, which was not a satisfactory result in terms of correlation.
(Example 3)
(3-1) Construction of Non-RIA measurement system using HiBiT-labeled DNA fragment
[Reagent preparation]
A DNA fragment into which an alkyne was introduced (from pUC19, DNA fragments of 1159 bp and 1274 bp were prepared by PCR using an alkyne-labeled primer having the same sequence as in Examples 1-3 and 2) and a terminal azide-labeled HiBiT. The tag was bound by a click reaction to prepare a HiBiT-labeled DNA fragment.

[Measurement of labeled DNA]
Twenty-five μL of HiBiT-labeled DNA (mixture of equal amount of 1159 bp and 1274 bp) of various concentrations was dispensed into 96-well half area white plate (Conring). 25 μL of the detection reagent prepared according to the manual of Nano Glo HiBiT Lytic Detection System (Promega) was added, and the mixture was stirred at 600 rpm for 3 minutes and then allowed to stand in the dark for 10 minutes. The emission intensity was detected with a luminometer (GloMax).

The results are shown in FIG. As shown in FIG. 9, since an RLU difference was observed between DNA 0 ng / mL and 1 ng / mL, it was judged that detection was possible if the amount of HiBiT-labeled DNA was 1 ng / mL or more. In addition, the RLU value showed good linearity with respect to the amount of DNA up to about 1000 ng / mL.
From the above results, it was shown that measurement is possible if the concentration of HiBiT-labeled DNA in the reaction solution is in the range of 1 to 1000 ng / mL.

(3-2) Measurement of HiBiT-labeled DNA in supernatant after B / F separation SLE patients appropriately diluted with bovine serum, priced with a commercially available kit (Seti) based on the measurement principle of the Farr-RIA method. Using serum as a standard solution, 25 μL of this was added to a test tube, 200 μL of 0.2 to 0.8 μg / mL HiBiT-labeled DNA solution was added, mixed, and incubated at 37 ° C. for 2 hours.
After the reaction, 1000 μL of cold ammonium sulfate (61.25% saturated) was added so that the concentration of ammonium sulfate in the reaction solution was 50%, and the mixture was mixed. Centrifuge at 2000 xg, 4 ° C. for 15 minutes.

After centrifugation, 25 μL of the obtained supernatant and 25 μL of the detection reagent prepared according to the manual of Nano Glo HiBiT Lytic Detection System (Promega) were added, and the mixture was stirred at 600 rpm for 3 minutes and then allowed to stand in the dark for 10 minutes. The emission intensity was detected with a luminometer (GloMax).

The results are shown in FIGS. 10 and 4. As for the "evaluation" in Table 4, similarly to Tables 1 and 2, (i) a concentration-dependent signal decrease is observed at 0 to 200 IU / mL, and (ii) 13.7 IU / mL B / B. Three evaluation criteria: ₀ % is 90% or less, and (iii) 218.2 IU / mL B / B ₀ % is more than twice the anti-DNA antibody 131.8 IU / mL B / B ₀ %. When evaluated in, "○: Satisfied all the criteria of (i) to (iii)" and "Δ: (i) was satisfied, but either (ii) or (iii) (or both). The evaluation was made according to the criteria of "does not meet" and "x: did not meet (i)".

A concentration-dependent signal reduction of anti-DNA antibody was observed in the range of 0.2 to 0.8 μg / mL of HiBiT-labeled DNA concentration. From this, it was clarified that the anti-DNA antibody can also be measured by HiBiT-labeled DNA.
Above all, when the labeled DNA concentration was 0.4 and 0.8 μg / mL, the B / B ₀ % of the anti-DNA antibody 13.7 IU / mL was 90% or less, and the measurement in the low-level anti-DNA antibody region was good. , and the and, B / _{B 0%} of anti-DNA antibodies 218.2IU / mL become anti-DNA antibody 131.8IU / mL of B / _{B 0%} of 2 times or more, good sensitivity even in the anti-DNA antibody altitude zone It became clear that it can be measured with.

(3-3) Correlation test between the method of the present invention and the Farr-RIA method using HiBiT-labeled DNA SLE appropriately diluted with bovine serum, which has been priced with a commercially available kit (Seti) based on the measurement principle of the Farr-RIA method. Use patient serum as a standard solution, add 25 μL of SLE patient serum (valued by Farr-RIA method and FEIA method), add 200 μL of HiBiT-labeled DNA solution (0.8 μg / mL), mix, and mix at 37 ° C. Incubated for 2 hours. After the reaction, 1000 μL of cold ammonium sulfate (61.25% saturated) was added so that the concentration of ammonium sulfate in the reaction solution was 50%, and the mixture was mixed. Centrifuge at 2000 xg, 4 ° C. for 15 minutes.

After centrifugation, 25 μL of the obtained supernatant and 25 μL of the detection reagent prepared according to the manual of Nano Glo HiBiT Lytic Detection System (Promega) were added, and the mixture was stirred at 600 rpm for 3 minutes and then allowed to stand in the dark for 10 minutes. The emission intensity was detected with a luminometer (GloMax).
The correlation between the present invention and the Farr-RIA method was calculated by comparing the amount of the anti-DNA antibody of the present invention calculated from the amount of labeled DNA with the amount of the anti-DNA antibody measured by the Farr-RIA method by statistical processing. The results are shown in FIG.

As a result of the correlation test, it was clarified that even if HiBiT-labeled DNA is used, a high correlation of 0.98 is shown when the relational expression between the concentration of the DNA fragment and the strand length specified in the present invention is satisfied. It was.

Claims (5)

A method for measuring an anti-DNA antibody by reacting a DNA fragment labeled with a non-radioactive compound with a sample and measuring the amount of the labeled DNA fragment.
(A) The DNA fragment labeled with the non-radioactive compound is reacted with the sample,
(B) After the reaction, a precipitant is added to the reaction solution to separate the liquid phase and the precipitate phase (B / F separation).
(C) After B / F separation, the amount of labeled DNA fragment in the liquid phase was measured.
(D) Calculate the amount of anti-DNA antibody from the measurement result of the amount of DNA fragment,
When the concentration of the DNA fragment to be used is Y (μg / mL) and the chain length is X (bp), the DNA fragment used is within the range represented by the following [Equation 1]. ,
[Number 1]
400 ≤ X ≤ 2000
Y ≧ 0.1
0.0006X-0.4595 ≤ Y ≤ 0.0005X + 0.3744
(F) When the obtained measurement results and the results of measuring the same sample by the known Farr-RIA method are compared, the correlation coefficient between the two measurement results shows 0.93 or more.
A method for measuring an anti-DNA antibody. A method for measuring an anti-DNA antibody by reacting a DNA fragment labeled with a non-radioactive compound with a sample and measuring the amount of the labeled DNA fragment.
(A) The DNA fragment labeled with the non-radioactive compound is reacted with the sample,
(B) After the reaction, a precipitant is added to the reaction solution to separate the liquid phase and the precipitate phase (B / F separation).
(C) After B / F separation, the amount of labeled DNA fragment in the liquid phase was measured.
(D) Calculate the amount of anti-DNA antibody from the measurement result of the amount of DNA fragment,
When the concentration of the DNA fragment to be used is Y (μg / mL) and the chain length is X (bp), which includes the step and (e') is used, the one within the range represented by the following [Equation 2] is used. And
[Number 2]
700 ≤ X ≤ 2000
Y ≧ 0.1
0.0006X-0.3595 ≤ Y ≤ 0.0005X + 0.3744
(F') When the obtained measurement result and the result of measuring the same sample by the known Farr-RIA method are compared, the correlation coefficient of both measurement results shows 0.95 or more.
A method for measuring an anti-DNA antibody. The non-radioactive compounds used to label DNA fragments are biotin, 2,4-Dinitrophenol (DNP), digoxigenin (DIG), Fluorescein, HiBiT tags, and theirs. The measuring method according to claim 1 or 2, which is selected from the derivatives of the above. The measuring method according to any one of claims 1 to 3, wherein the precipitant used is ammonium sulfate. (G) When the concentration of the DNA fragment is Y (μg / mL) and the chain length is X (bp), the DNA labeled with a non-radioactive compound within the range represented by [Equation 1] below. fragment,
[Number 1]
400 ≤ X ≤ 2000
Y ≧ 0.1
0.0006X-0.4595 ≤ Y ≤ 0.0005X + 0.3744
It consists of (h) a precipitant for separating the liquid phase and the precipitated phase (B / F separation), and (i) a reagent for measuring the amount of labeled DNA fragment in the liquid phase after B / F separation. A reagent kit for measuring the amount of anti-DNA antibody in a sample by the measuring method according to any one of claims 1 to 4.

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