JP2010271295A - Measuring reagent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To detect nephritis, prepare an animal model, and develop a nephritis therapeutic agent using peptide originating in type-IV collagen NC1 region and its antibody. <P>SOLUTION: Peptides and their antibodies (newly purified autoantibodies and antibodies of animal origin prepared by the conventional method) are used. Specifically, a measuring reagent of an EIA method (including an ELISA method) using these peptides as antigens is developed, and an anti-peptide antibody is found from a nephritis sample. The measuring method may be any method as long as it is immunoreaction, and can be speedily introduced when it is a method used for a reagent presently available in the market. Detection of a peptide antibody has extremely higher sensitivity than that of detection of an anti-NC1 antibody. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

The present invention relates to the use of peptides derived from type IV collagen and antibodies thereof. In particular, it is a measurement reagent using a peptide derived from the NC1 region of type IV collagen and its antibody.

Type IV collagen is divided into a triple-stranded helical structure 7S, a central helical region (hereinafter referred to as TH), and a non-helical NC1 region. Conventionally, the triple-stranded helical region of type IV collagen is the name of type IV collagen, and it is detected from nephritic urine, and its measuring reagent is used for clinical diagnostics. Further, it was found that the NC1 region of type IV collagen (hereinafter referred to as NC1) is useful for measuring the urinary concentration of NC1 in nephritis (Japanese Patent Application No. 2003-436150).

Type IV collagen has six chains from a1 chain to a6 chain, and one type IV collagen molecule is composed of three a chains (Reference: J Cell Biol 130 (5): 1219-1229, 1995). The antigen of Goodpascher's syndrome (a narrow sense anti-glomerular basement membrane (GBM) antibody nephritis), which is a rare nephritis, is considered to be a specific conformation site on the a3 chain of NC1 (Reference: J Biol Chem). 274 (16): 11267-11274, 1999.). In anti-glomerular basement membrane (GBM) antibody nephritis, anti-GBM antibody is measured in blood by a measurement method using a huge GBM other than NC1 of a3 chain as an antigen, and its reagent kit is used as a clinical diagnostic agent. ing.

Problems to be solved by the invention

These had the following problems. When using a type IV collagen triple chain region as a standard product with a measurement reagent kit or preparing an anti-type IV collagen triple chain region antibody, it is usually necessary to extract the triple chain from the placenta, kidney or eyeball of a human or animal. Yes, it takes a lot of time and special skills. NC1 requires much labor and technology for extraction and purification as well as triple strand. Since type IV collagen has a large molecular weight, including triple-stranded helical region (hereinafter referred to as triple-stranded) and NC1, the entire triple-stranded and NC1 molecules measured in urine are equally involved in the disease. Instead, some may be involved in the disease as a core.
In view of nephritis, efforts are being made to obtain proteins and peptides different from those of healthy individuals from the blood and urine of patients, not limited to type IV collagen. However, the substance which distinguishes a healthy person from a nephritis patient has not been specified yet.
In fact, separating proteins and peptides from patient samples (tissues, extracts, blood, urine, etc.) by electrophoresis is an attempt to find the core part related to the disease. Neither peptide nor peptide has been identified.
In anti-GBM antibody nephritis, the antigenic site is specified, but there is no report of detection of protein or peptide as an alternative to the antigen from patient's urine and serum samples.

Means for solving the problem

The inventor of the present application has accomplished this application as a result of many years of research on type IV collagen. It has already reported peptides (literature; medicine and pharmacy 57 (5): 605-613, 2007. yp5, 8, 10, 12, 13) and their antibodies (newly purified autoantibodies and conventional ones). Animal-derived antibody produced by the technique).

Specifically, a reagent for measuring the EIA method (including ELISA method) using these peptides (hereinafter referred to as peptides) as an antigen was developed, and anti-peptide antibodies were found from nephritis samples. As long as the measurement method is an immune reaction, any method may be used, and any method used for a commercially available reagent can be introduced quickly.
The detection of peptide antibodies is much more sensitive than the detection of anti-NC1 antibodies.
The high sensitivity makes distinction between a healthy person and nephritis clearer with a small amount of specimen. For example, if urine is used as a sample, the measurement of anti-NC1 antibody uses 100 ul at a dilution of 4-5 times, but the measurement of anti-peptide antibody only requires 50 ul at a dilution of 10-20 times. If a small amount of serum is a sample, it is desirable as a test that it can be used after sufficiently diluted. If the dilution ratio is not increased, the same immunization method enables a shorter measurement time.

Subsequently, the inventor of the present application detected the antigenic peptide from urine by the EIA method using an anti-peptide antibody prepared by administering the peptide to animals (rats, guinea pigs and rabbits).

As a result, anti-NC1 antibody, NC1, anti-peptide antibody and peptide are also detected in urine where urine protein is negative or positive or negative, and in urine where mild nephritis is considered. I found that they do not necessarily match.

When urine is used as a sample from a patient, it is appropriate to collect samples during the day except for morning urine, especially in the morning. Furthermore, morning collection is good for hypertensive diseases.
In the conventional urine protein test, early morning urine is used because the protein is most concentrated in the day. However, the inventor found that anti-NC1 antibody was highest during the day, especially in the morning, in the study of nephritis (chronic nephritis, hypertensive nephritis, diabetic nephritis, IgA nephropathy, etc.). It was. The early morning urine of nephritis had the lowest anti-NC1 antibody in the day, and in some cases it was within the normal range within an average plus 2 times the standard deviation (2SD) of healthy subjects, making it difficult to identify as nephritis.
In the measurement of healthy subjects, there was no significant fluctuation during the day even after exercise.
The use of morning test samples is especially effective for patients with hypertensive nephritis and nephritis who are taking antihypertensive drugs. In addition, kidneys with nephritis are considered to have a narrower tolerable range when loaded than healthy individuals. Therefore, it is considered effective to select a labor suitable for the patient's body by examining the kidney function test with a load as in the case of performing a load test with a treadmill in the examination of the heart. In that sense, it is best to determine whether the current living environment or working environment is appropriate by examining urine frequently in daily life as a renal function test without conducting a special stress test. Think. It should be noted that the heart function test by the treadmill is considered dangerous when the heart is clearly damaged.

The invention's effect

Since the measurement results of the anti-peptide antibody and the peptide were the same as those of the anti-NC1 antibody, the detection of the anti-peptide antibody and the peptide can be used in place of the measurement of the NC1 or anti-NC1 antibody, and the result is more sensitive. Bring.
Since the peptide to be used is a synthetic product, a certain quality can be obtained unlike NC1 of biological extraction. The peptide can be used as an antigen of an anti-peptide antibody or as a standard for peptide measurement. Anti-peptide antibodies can also be used for immunohistochemical staining, which is a type of immune reaction.
Furthermore, peptide and anti-peptide antibodies can be used as nephritis models and therapeutic agents for nephritis by administering them to animals.
Hereinafter, although an Example is shown, this application is not limited to this.

Search for peptide in urine / experiment 71219
Outline of experiment; Immobilization of sample by EIA method; Immobilization of diluted urine on microplate (Maxi soap NUNK) Healthy patient (n = 10) Nephritis patient (n = 9); 2-fold diluted urine 50ul / well
Blocking agent: 0.2% Tween-PBS 300ul / well
Detection antibody: anti-yp8 antibody (rabbit derived) X1,000 50 ul / well, anti-yp12 antibody (rabbit derived) X1,000 50 ul / well,
Enzyme-labeled anti-rabbit antibody; (goat-derived CAPPEL) X5,000 50ul / well
Chromogenic substrate; TMB (+) (DAKO) 50ul / well
Reaction stop solution (1N sulfuric acid) 50ul / well
Antibody dilution and sample dilution are 0.05% Tween-PBS
After the operation, the absorbance was measured at A450 nm and measured;
yp8 yp12
Healthy person no. 01 0.132 0.214
02 0.321 0.572
03 0.042 0.057
04 0.000 0.000
05 0.016 0.037
11 0.002 0.028
12 0.000 0.029
13 0.000 0.000
14 0.728 1.829
15 0.026 0.042
AVE. 0.128 0.281
SD. 0.233 0.571
AVE + 2SD / 0.594 1.423
Nephritis patient no. N02 0.216 0.245
N03 0.289 0.367
N04 0.781 0.834
N05 0.722 0.749
N11 0.249 0.890
N12 0.277 0.602
N13 0.000 0.019 After recovery from nephrotic syndrome
N14 0.086 0.222
N15 0.306 0.779
Positive standard 0.625 0.636
(All after blank deduction)
Blank value 0.082 0.144

Measurement of urinary anti-peptide antibody / Experiment No. 80501
Outline of experiment;
Peptide-immobilized microplate (immobilized 10 ug / ml, 100 ul / well; NC1, (yp5, yp8, yp12)), blocking agent (PBS-0.2% Tween), sample urine; sample diluent (PBS- 0.05% Tween) 10-fold diluted 100 ul / well, room temperature x 1 hour, detection antibody (enzyme-labeled anti-human IgG / CAPPEL diluted 5,000 times) room temperature x 30 minutes, substrate solution (TMB (+) DAKO ) 100 ul / well Room temperature x 15 minutes, stop. Liquid (1N sulfuric acid) 100ul / wellA 450nm
Healthy people (n = 21)
NO. NC1 yp5 yp8 yp12
6 0.056 0.012 0.706 0.025
7 0.033 0.000 0.573 0.003
8 0.044 0.037 1.314 0.004
9 0.026 0.000 0.905 0.004
10 0.021 0.000 0.513 0.011
17 0.003 0.000 1.296 0.034
18 0.352 0.023 0.078 0.029
19 0.101 0.036 0.946 0.008
20 0.069 0.013 0.424 0.000
21 0.010 0.001 0.000 0.005
22 0.282 0.070 1.346 0.082
24 0.030 0.000 1.228 0.018
25 0.065 0.007 0.152 0.019
26 0.019 0.000 0.251 0.000
27 0.013 0.017 0.456 0.000
28 0.022 0.000 1.148 0.009
29 0.090 0.000 0.488 0.015
30 0.015 0.000 1.048 0.009
32 0.166 0.080 1.748 0.057
33 0.003 0.005 0.178 0.000
34 0.232 0.035 0.732 0.013
Ave. 0.080 0.016 0.739 0.016
SD. 0.097 0.023 0.490 0.021
Ave. + 2SD 0.274 0.062 1.719 0.058
Nephritis patients (n = 9)
6 0.449 0.512 2.754 0.301
7 0.661 0.630 2.754 0.300
8 0.304 0.474 2.754 0.196
9 0.113 0.050 1.420 0.022
10 0.306 0.233 2.752 0.094
16 0.225 0.412 2.754 0.125
17 0.792 0.998 2.754 0.502
18 0.122 0.223 2.354 0.088
19 0.209 0.068 2.053 0.071
Blank 0.089 0.172 0.746 0.075

Measurement of Anti-Peptide Antibody by EIA Method 1 Sample and Method 1) Sample Urine from nephritis patients (19 cases) was used as a sample. The controls were urine test paper and urine protein negative healthy urine (34 cases) (Table 1).
2) Method (1) yp5 (amino acid sequence at positions 61-80 of a5 (IV) NC1, STMPFMFCCNNNVCNFASRN), yp8 (amino acid sequence at positions 106-125, PFISRCAVCEAPAVVIAVHS), yp12 (amino acid sequence at positions 166-185) SCLEEFRSAPFIECHGRRGTC) was applied to different 96-well microplates at 0.5 μg / hole and allowed to stand overnight at 4-8 ° C. to be solid-phased.
(2) PBS (containing 0.2% -Tween 20) as a blocking agent was added to the solid-phased microplate and left for 2 hours.
(3) Next, the plate was washed with PBS (containing 0.05% -Tween 20), urine diluted 10-fold with PBS was added at 50 μl / well, and left at room temperature for 2 hours.
(4) Subsequently, the plate was washed, 50 μl / well of enzyme-labeled anti-human IgG antibody (from rabbit, DAKO) was added, and left at room temperature for 1 hour.
(5) Further washing, 50 μl / well of enzyme substrate solution (TMB (+), DAKO) was added, 10 minutes later, 50 μl of reaction stop solution (1N-sulfuric acid) was added, and the absorbance immediately at a wavelength of 450 nm (A450 nm) Was measured using a Bio-Rad Laboratories Model 680.
2 Measurement Results The absorbance of each anti-peptide antibody at A450 nm was as follows.
(1) Anti-yp5 antibody: 34 healthy subject urine samples had an average (0.347) and standard deviation (SD, 0.108), and 19 urine patients with nephritis had an average (0.989) and standard deviation (0.400) )Met. Assuming that the average + 2SD of healthy subjects is the cut-off point (0.563), there were 2 positive cases (positive rate 6%) in healthy subjects and 18 positive cases (positive rate 95%) in nephritis patients.
(2) Anti-yp8 antibody: 34 healthy subject urine samples with mean (0.369) and standard deviation (SD, 0.121), 19 nephritis patient urine samples with mean (1.820) and standard deviation (0.746) )Met. Assuming that the average + 2SD of healthy subjects is the cut-off point (0.611), there were no positives in healthy subjects (positive rate 0%), and 18 positive cases (positive rate 95%) in nephritis patients.
(3) Anti-yp12 antibody: 34 healthy subject urine samples had a mean (0.067) and standard deviation (SD, 0.016), and 25 urine patients with nephritis had a mean (0.300) and standard deviation (0.194). )Met. Assuming that the average of healthy subjects + 2SD is a cut-off point (0.099), there were 2 positive cases (positive rate 6%) in healthy subjects and 16 positive cases (positive rate 84%) in nephritis patients.
The measured values are as follows.
H4NA5-5 H4N5-8 H4N5-12
0.460 0.508 0.065
0.574 0.298 0.089
0.322 0.275 0.058
0.394 0.546 0.063
0.295 0.285 0.060
0.344 0.434 0.071
0.216 0.217 0.055
0.386 0.469 0.065
0.395 0.404 0.059
0.310 0.265 0.053
0.421 0.567 0.067
0.232 0.269 0.056
0.330 0.285 0.061
0.682 0.457 0.120
0.294 0.297 0.057
0.236 0.390 0.063
0.314 0.257 0.087
0.416 0.388 0.057
0.250 0.486 0.061
0.248 0.242 0.057
0.409 0.569 0.113
0.360 0.490 0.088
0.215 0.335 0.054
0.307 0.237 0.054
0.288 0.251 0.054
0.328 0.448 0.059
0.303 0.574 0.055
0.383 0.502 0.075
0.184 0.330 0.065
0.289 0.228 0.059
0.531 0.321 0.070
0.401 0.170 0.065
Below nephritis Below nephritis Below nephritis 1.251 2.247 0.442
0.813 1.363 0.189
0.842 1.881 0.259
1.187 2.536 0.322
1.093 2.299 0.625
1.739 2.704 0.523
1.414 2.362 0.456
0.796 2.108 0.227
0.596 0.882 0.085
0.664 1.489 0.228
0.660 1.284 0.121
1.498 2.467 0.508
0.245 0.241 0.060
0.655 0.624 0.117
1.328 2.659 0.504
0.723 1.182 0.092
1.606 2.728 0.685
0.721 1.493 0.201
0.957 2.041 0.239
Each case was examined to see if the measured values differed during the sampling time. This example (female, 52 years old) has just been diagnosed with chronic nephritis at a university hospital outpatient clinic that had been used as hypertension. Urine was collected from time to time during two consecutive days of work, and the measurement was performed simultaneously with the measurement of the title. The results were as follows.
Anti-yp5 antibody (about 6 pm on the first day; 1.264 → about 11 pm on the first day; 0.713 → about 6 am on the 2nd; 0.486 → about 10 am on the 2nd; 0.957 → 3 pm on the 2nd 0.612), anti-yp8 antibody (around 6 pm on the first day; 2.374 → 11 pm on the first day; 1.468 → 6 am on the 2nd; 1.015 → 10 am on the 2nd; 2.041-> 2 days around 3:00 pm; 1.562), anti-yp12 antibody (first day around 6:00 pm; 0.345-> first day around 11 pm; 0.158-> 2 days early morning urine / around 6:00 am; 0.129-> 2 days around 10 am; 0.239-> 2 days around 3:00 pm; 0.195).
The cut-off point at the time of simultaneous measurement is 0.563 for anti-yp5 antibody, 0.611 for anti-yp8 antibody, and 0.099 for anti-yp12 antibody, so any early morning urine of chronic nephritis measured (around 6 am) The antibody is also within the cut-off point and is in the normal range. However, the urine collected at other times was positive for all antibodies. From this result, in this example, the importance of resting (normalization of early morning urine on the second day) and the burden of work on the kidneys (high value at 6 pm on the first day) were shown. In healthy subjects (2 cases), the urinary antibody remained in the healthy range within the cut-off point even after playing sports. It seems that collection is appropriate as a sample reflecting kidney function.
3 Conclusion As a result of measurement, there are a wide range of antibodies having a peptide on a5 (IV) NC1 as an antigen in nephritis urine, and these antibody measurements are useful for detecting nephritis.

Claims (4)

Yp5 (amino acid sequence at positions 61 to 80 of a5 (IV) NC1, STMPFMFCCNNNVCNFASRN), yp8 (amino acid sequence at positions 106 to 125, PFISRCAVCEAPAVVIAVHS), yp12 (amino acid sequence at positions 166 to 185, SCLEEFRSSAPFIEGGRGC)
A method for detecting nephritis by immune reaction from a sample using a corresponding antibody. A method for detecting nephritis by immunoreaction of a corresponding peptide from a sample, using an anti-peptide antibody produced by the peptide of claim 1. A nephritis model prepared using the peptide of claim 1 and / or the anti-peptide antibody of claim 2. A therapeutic agent for nephritis using the peptide of claim 1 and / or the anti-peptide antibody of claim 2.