JP2004191382A - Test reagent kit - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method and a reagent which easily detect and measure autoimmune diseases and infectious diseases without causing pain in patients. <P>SOLUTION: The method and the reagent for using urine of patients of autoimmune diseases and infectious diseases as a sample and detecting an antibody or an antigen corresponding to a disease by immunoreaction are provided. Renal diseases or IgA nephropathy is specified as the autoimmune diseases and the infectious diseases. IgA is specified as the antibody, and NC1 is specified as the antigen. Ape urine can be used for a measuring reagent as a positive control in a detection method by immunoreaction. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

DETAILED DESCRIPTION OF THE INVENTION

Industrial applications

The present invention relates to a detection method and a measurement reagent used for detecting an autoimmune disease or an infectious disease.

Conventionally, serum has been used to detect autoimmune diseases and infectious diseases.
A method has been adopted in which blood is collected from a patient suffering from an autoimmune disease or an infectious disease, and an antibody or antigen corresponding to the disease in serum is detected by an immune reaction (for example, measurement of "rheumatoid factor", p. 726). "Clinical Examination Guide 2001-2002", Bunkodo Co., Ltd.).
Here, “patients suffering from autoimmune disease or infectious disease” include not only patients who have developed the disease but also immunogens and causative bacteria (including bacteria such as mold and tuberculosis bacteria and viruses) and their antibodies before the onset. This includes those who have it in the body. Viruses include hepatitis virus (types A, B, C, D, E, etc.), influenza virus, AIDS virus, varicella virus and SARS virus.
Serum specimens are painful at the time of blood collection, especially for infants. In addition, the injection needle must be replaced for each patient in order to prevent infection, and there is a problem that labor and cost are required. Therefore, the emergence of a clinically simpler diagnostic method and diagnostic reagent has been desired.

Problems the invention is trying to solve

The present inventor provides a method and a reagent which can easily and easily measure an autoimmune disease or an infectious disease without pain of a patient.

Means for solving the problem

The present inventors have confirmed by quantitative measurement that a small amount of protein is also present in urine of a healthy person who is determined to be negative for protein in a commonly used urine qualitative test. He further questioned that trace amounts of proteins were treated as if they were one, and continued to analyze urine proteins and found that there were countless unexplained various proteins (eg, healthy Fact that NC1 is excreted in the urine of the elderly). In addition, it was confirmed that various globulins including IgA as well as IgG were present in this trace protein as well as antibodies, and it was clarified that an antibody corresponding to a specific antigen appearing in serum at the time of disease was also present in urine. Was completed. Of course, urine and serum vary in the appearance time and amount of antigens and antibodies depending on the stage at the time of disease.
In addition, in patients with autoimmune diseases and infectious diseases, it has been found that urine has an antigen as well as serum, and thus, in the same manner as antibody measurement, an antigen measurement method and reagent have been completed.
Furthermore, we have determined that it is possible to use the monkey biological sample (serum, urine, etc.) of the experimental model as a positive standard at the time of measurement. Ease of availability
Autoimmune diseases include nephritis, rheumatoid arthritis, insulin-dependent diabetes mellitus, collagen disease and the like.
For example, nephritis is understood as a type of allergic reaction that develops through an antigen-antibody complex. As far as nephritis is limited to glomerular diseases, acute progressive glomerulonephritis including anti-glomerular basement membrane (GBM) antibody nephritis, IgA nephropathy, membranous nephropathy, Glomerulonephritis, acute post-streptococcal glomerulonephritis, minimal change nephrotic syndrome, focal glomerulosclerosis. Examples of the secondary nephropathy include diabetic nephropathy.
An infectious disease refers to a case where a pathogenic bacterium has invaded the body. Examples of the infectious disease include visceral mycosis, hepatitis B infection, HIV infection, chlamydia infection and the like. It is also possible to determine by urine antibody measurement.
In nephritis, a representative of autoimmune diseases, antibodies are deposited on glomeruli and others, but the corresponding antigens are largely unknown. Elucidation of what this antigen is is awaited.
In an anti-GBM antibody nephritis as an example in which the antigen has been elucidated, it is known that the antigen is NC1. However, since NC1 is difficult to purify, there is currently no kit as an in vitro diagnostic agent as a reagent for detecting an antibody against purified NC1.
At present, in the world, an antibody detection kit for anti-GBM antibody nephritis uses a GBM extract containing NC1 as an antigen, and therefore, a detection reagent having low sensitivity and low detection accuracy is only commercially available.
Here, the inventor of the present application thought that sensitivity would be increased if a kit was prepared using a purified NC1 product, and as a result of diligent research, completed the “anti-NC1 antibody detection kit”.
By the way, according to a general classification, the glomeruli of the kidney consist of cell subdivisions and extracellular matrix.
The cell subdivision is mesangial cells, epithelial cells, endothelial cells and Bowman's capsule epithelial cells, and the extracellular matrix is constituted by the mesangial matrix, glomerular basement membrane and Bowman's capsule basement membrane. The major components of the extracellular matrix of the glomerulus are glycoproteins such as collagen, mainly laminin, proteoglycan, and fibronectin, mainly of type 4 collagen, and the proportions of these components differ between the mesangial substrate and the glomerular basement membrane. (P. 413, "Extracellular Matrix" published by Medical Review, 1996).
Therefore, the present inventor has proposed that the NC1 and type 4 collagen triple-chain regions constituting type 4 collagen can be all antigens of nephritis regardless of primary or secondary, and only the glomerular basement membrane is used as an antigen. It is presumed that it is widely scattered, including the mesangial region. In other words, it was presumed that NC1 and the type 4 collagen triple-chain region were antigens even in unidentified nephritis other than anti-GBM antibody nephritis, for example, in IgA nephropathy. In other words, if IgA is deposited in the mesangial region in IgA nephropathy whose antigen has not been determined, use of an anti-GBM antibody measurement reagent using NC1 (or type 4 collagen triple-chain region) as an antigen, rather than IgG antibody measurement, It was determined that the measurement should be performed by an immune reaction using antibody measurement, and the "anti-NC1IgA antibody measurement kit" was found to be useful for serum measurement and also applicable to urine measurement.
Here, the diagnostic criteria for IgA nephropathy will be described.
Conventionally, the only diagnostic method for IgA nephropathy is a renal biopsy as a definitive diagnosis. Specifically, "diffuse IgA granule deposition mainly in the mesangial region" is determined by fluorescent antibody or enzyme antibody staining. (1071 "Clinical tests 2001-2002", published by Bunkodo).
Therefore, the “anti-NC1 IgA antibody measurement kit” of the present invention can measure serum and urine of IgA nephropathy, and thus does not require a conventional renal biopsy. It frees you from time and remarkable skill acquisition.
Further, the "anti-NC1 antibody measurement kit (NC1 may be replaced with type 4 collagen)" of the present invention is useful for detecting primary and secondary nephritis such as diabetic nephritis.
Especially for "anti-GBM antibody nephritis", "anti-NC1 antibody measurement kit" is extremely sensitive because it uses NC1 which is abundant in GBM as antigen, and IgA antibody in urine as well as serum can be used. IgG antibodies can also be measured sharply, and particularly sensitive to IgA in urine.
In any of the antibody measurement kits of the present invention, urine measurement of an antibody can serve as a substitute for serum measurement, and particularly, IgA measurement in urine is excellent in distinguishing between a healthy group and a disease group.
By the way, as described below, the inventor considers that almost all nephritis is the same even though the disease name is different.
In other words, globulin binds to complement or globulin for some reason and grows larger or becomes more sticky, so it happens to be caught in an unspecified fine area of the kidney, and because it is the most widespread, it has more opportunities to contact It is considered that NC1 constituting type 4 collagen and the triple chain region of type 4 collagen were recognized as antigens with the passage of time and inflammation occurred.
The reason is that the "anti-NC1 antibody measurement kit (NC1 may be replaced with type 4 collagen)" and the "NC1 measurement kit (NC1 may be replaced with type 4 collagen)" of the present invention are primary and diabetic nephritis. This is because secondary nephritis can be detected irrespective of secondary nephritis and the like, and it is more useful in urine samples, especially in early samples.
The present inventor has established the following specific means for detecting an autoimmune disease or an infectious disease. The present invention is not limited to the described measuring methods and reagents.
That is, a method for detecting an anti-NC1 antibody generated in an anti-GBM antibody nephritis from urine of a patient or the like and a measurement reagent will be illustrated and described in the case of serum.

1. A method and a reagent for detecting an anti-NC1 antibody in serum and / or urine.
And a method and reagent for detecting anti-type 4 collagen antibody from serum and / or urine.
As reagents, 1) a plate coated with NC1 or type 4 collagen (here referred to as a triple-stranded region), 2) an enzyme-labeled anti-human IgG (or IgA) antibody, 3) a chromogenic substrate (TMB), 4) a reaction stop Measure using liquid (sulfuric acid).
At this time, the positive standard may be obtained from a human patient, but is better obtained from an experimental model monkey, as found by the inventors. An experimental model of a monkey that is managed and raised and produced can be a more stable standard.

The immune reaction is typically an enzyme immune reaction, but is not limited thereto, and includes an AB method, an RIA method, an immunoluminescence method, a precipitation reaction, an agglutination reaction and the like. The enzyme-labeled antibody in the enzyme immunoreaction may be a polyclonal or monoclonal antibody. It may be a radioactive substance (RIA method), a substance labeled with a luminescent substance (immunoluminescence method), or a non-labeled substance (sedimentation method, aggregation method).
The reaction method is not limited to the sandwich method, and may be a competitive method or the like, but the sandwich method is particularly preferable. As a configuration of the measurement reagent, the plate coated with NC1 or type 4 collagen may be made of glass or a magnetic substance, or may be absent without using the solid phase method.
When the plate is coated with NC1 or type 4 collagen (hereinafter referred to as antigen), the plate may be indirectly coated and the coating substance may be avidin, biotin, or a component in which these are combined.
The antigen may be not only a biological extract or a recombinant but also a constituent peptide (including a specific fraction and a synthetic product).
The animal species of the antigen used for the measurement reagent is desirably human, and may be monkeys, cows, pigs, chickens, sheep, goats, rabbits, rats, or other animals, and is not limited thereto. Further, the antigen may be a mixture of a plurality of animal species.
The organ from which the antigen is derived is preferably, but not limited to, the kidney.
Further, as the second antibody, an anti-human IgA antibody is particularly desirable, and an anti-human IgG antibody is also desirable, but it is not limited thereto, and may be an anti-human IgM antibody, another anti-human immunoglobulin antibody or a mixture thereof. When the measurement target is an animal other than a human such as a rat or a mouse, the aforementioned human reagent component can be measured according to the target animal. However, in the case of monkeys, the human one can be used as it is.

The invention's effect

The present invention makes it possible to easily detect an antibody corresponding to an antigen of a disease by using urine as a sample in an immune abnormality or an infectious disease. In addition, antigens can be detected in urine samples.
Furthermore, in the case of a nephritis patient detected according to the present invention, during blood purification, an antibody corresponding to the antigen "NC1 and / or type 4 collagen" and / or an antigen corresponding to the anti- "NC1 and / or type 4 collagen" antibody is adsorbed and removed. By doing so, the effect of purification can be further enhanced.

２）結論
１）本発明の「抗ＮＣ１抗体測定キット」は、既存の体外診断用医薬品と同様に、ヒト検体を測定できる。加えてより高感度である。
−ＥＵＲ社キットではカットオフ値（１０Ｕ）でのＯＤが、陰性（０Ｕ）の１６倍であるのに、本発明キットでは２９倍である。
２）両測定試薬は、サル血清もヒトと同様に測定できる。従って、サルの血清はヒトの抗ＧＢＭ抗体腎炎検体の測定時に標準として使用し得る。
３）ヒト検体を指標として、サル血清を測定する時、発明の「抗ＮＣ１抗体測定キット」は、既存の体外診断用医薬品（６Ｗ）より早く陽性を認める（２Ｗ）。よって、発明キットはヒトの抗ＧＢＭ抗体腎炎の早期検出に有用である。
３ヒトの抗ＧＢＭ抗体腎炎では血清にＩｇＡ抗体も存在するかを検討した。
１）前述「抗ＮＣ１抗体測定キット」で「ＨＲＰ標識抗ヒトＩｇＧウサギ抗体」を「ＨＲＰ標識抗ヒトＩｇＡウサギ抗体」に置き換えて測定した。
２）測定結果

３）結論
１）ヒトの抗ＧＢＭ抗体腎炎にはＩｇＡ抗体も存在する。よって、「抗ＮＣ１抗体測定キット」によるＩｇＡ抗体の測定はヒト抗ＧＢＭ抗体腎炎の検出に利用できる。ＩｇＡのカットオフ値と健常値のＯＤがほぼ等しいことは、ＩｇＧのそれが離れている時に、カットオフ値を示す検体が健常群であることを明瞭にする。
４「抗ＮＣ１抗体測定キット」は尿中の抗体を測定できるか検討した。
１）測定結果

２）結論
１）「抗ＮＣ１抗体測定キット」は尿中のＩｇＡ抗体もＩｇＧ抗体も測定できる。従って、尿の測定は血清測定の代用となり得る。
２）尿測定では、血清と異なり、ＩｇＡが優位となるので、これを測定するのが望ましい。
５「ＮＣ１測定キット」の作製（サンドイッチＥＬＩＳＡ法で実施）
１）キットの構成
抗ＮＣ１抗体結合マイクロプレート；抗血清１２０ｕｌ／ｗｅｌｌ
前出ＮＣ１で作製したラット由来抗血清を１０００倍希釈
検体希釈液；ＰＢＳ（含ＢＳＡ，Ｔｗｅｅｅｎ２０）
抗ＮＣ１抗体
前出ＮＣ１で作製したウサギ由来抗血清を５０００倍希釈
ＨＲＰ標識抗ウサギＩｇＧ抗体（ヤギ由来）
発色基質液；ＴＭＢ
反応停止液；１Ｎ硫酸
洗浄液；ＰＢＳ（含Ｔｗｅｅｎ２０）
２）操作方法
抗体結合マイクロプレートを１回洗浄→検体添加（ヒト尿はそのまま使用、サル血清・サル尿は希釈）→２時間後３回洗浄→抗ＮＣ１抗体添加→２時間後３回洗浄→ＨＲＰ標識抗ウサギＩｇＧ抗体添加→１時間後３回洗浄→発色基質液添加→５分後に反応停止液添加→吸光度測定（４５０ｎｍ）
３）ヒト検体；ヒト男健常者（４４才、１２才、１０才）の早朝一番尿市販のテルモ尿試験紙でいずれも陰性
４）サル検体；「準備」で作製した抗ＧＢＭ抗体腎炎モデル
５）測定結果１

・測定結果２

６）結論
１）健常血液及び健常尿中には抗原ＮＣ１が存在する。
２）血清よりも尿の方が、健常時と疾患時の開きが大きく、指標とし易い。
６「抗ＨＢｃ抗体測定キット」の作製（ＥＬＩＳＡ法で実施）
１）キットの構成
ＨＢｃ結合マイクロプレート；
検体希釈液；ＰＢＳ（含ＢＳＡ，Ｔｗｅｅｅｎ２０）
ＨＲＰ標識抗ヒトＩｇＧ抗体
発色基質液；ＭＢ
反応停止液；１Ｎ硫酸
洗浄液；ＰＢＳ（含Ｔｗｅｅｎ２０）
２）操作方法
ＨＢｃ抗原結合マイクロプレートを１回洗浄→検体添加（ヒト尿を２倍希釈し使用）→室温で２時間反応後３回洗浄→ＨＲＰ標識抗ヒトＩｇＧ抗体添加→室温で１時間反応後３回洗浄→発色基質液添加→１５分後に反応停止液添加→直ちに吸光度測定（４５０ｎｍ）
３）ヒト検体
血清測定でＨＢｃ抗体陽性を呈した男子３名と陰性男子３名の随時尿を用いた。
４）測定結果
尿でも血清陽性者は全て陽性を、陰性者は全て陰性を示した。
５）結論
Ｂ型肝炎の検査で、従来血清に用いられている抗ＨＢｃ抗体の測定は、随時の尿測定でも有用な判定を示す。
７「ＨＢｓ測定キット」の作製（サンドイッチＥＬＩＳＡ法で実施）
１）キットの構成
抗ＨＢｓ抗体結合マイクロプレート；
ポリクローナル抗体（カルテット社、ウサギ由来）を緩衝液（ｐＨ９．６）で１０００倍希釈してコート
検体希釈液；ＰＢＳ（含ＢＳＡ，Ｔｗｅｅｅｎ２０）
抗ＨＢｓ抗体
モノクローナル抗体（カルテット社、マウス由来）を緩衝液（ｐＨ７．４）で１０００倍希釈
ＨＲＰ標識抗マウスＩｇＧ抗体（ウサギ由来）
発色基質液；ＴＭＢ
反応停市液；１Ｎ硫酸
洗浄液；ＰＢＳ（含Ｔｗｅｅｎ２０）
２）操作方法
抗体結合マイクロプレートを１回洗浄→検体添加（ヒト尿をそのまま使用）→２時間後３回洗浄→抗ＨＢｓ抗体添加→２時間後３回洗浄→ＨＲＰ標識抗マウスＩｇＧ抗体添加→１時間後３回洗浄→発色基質液添加→１５分後に反応停止液添加→直ちに吸光度測定（４５０ｎｍ）
３）ヒト検体
血清測定でＨＢｓ抗原陽性を呈した男女各１名と陰性男子３名の随時尿を用いた。
４）測定結果
尿でも血清陽性者は全て陽性を、陰性者は全て陰性を示した。
５）結論
Ｂ型肝炎の検査で、従来血清に用いられているＨＢｓ抗原の測定は、随時の尿測定でも有用な判定を示す。Preparation: For use as a positive standard, a monkey anti-glomerular basement membrane (GBM) antibody nephritis model was prepared under the following conditions.
Antigen; NC1
Bovine kidney glomerular basement membrane-derived type 4 collagen NC1 region purified product administration; 13 mg of NC together with the same amount of FCA administered intradermally to the back skin of cynomolgus monkeys Serum was collected at 1, 2, and 6 weeks after NC1 administration.
Urine was collected at 1, 2, and 6 weeks after NC1 administration.
Examples; All experiments were performed at room temperature unless otherwise noted.
1. Preparation of "anti-NC1 antibody measurement kit" (performed by ELISA method)
1) Kit composition NC1 binding microplate; NC1: 0.05 to 0.20 ug / well
Sample diluent: PBS (including BSA, Tween 20)
HRP-labeled anti-human IgG (or IgA) rabbit antibody coloring substrate solution; TMB
Reaction stop solution; 1N sulfuric acid washing solution; PBS (including Tween 20)
2) Operation method Wash the antigen-binding microplate once → Add sample (use human sample as it is, dilute monkey serum and monkey urine) → Wash 3 times after 2 hours → Add HRP-labeled anti-human IgG (or IgA) rabbit antibody → Washing 3 times after 1 hour → Addition of chromogenic substrate solution → Addition of reaction stop solution after 5 minutes → Absorbance measurement (450 nm)
3) Human specimen; anti-glomerular basement membrane (GBM) antibody nephritis serum Commercially available standard "anti-glomerular basement membrane (GBM) antibody nephritis measurement reagent" (in vitro diagnostic drug) (Euro-Diagnostica, imported) Sales / Nipro)
4) Monkey specimen: It was examined whether the anti-GBM antibody nephritis model 2 “anti-NC1 antibody measurement kit” prepared in “Preparation” can measure human anti-GBM antibody nephritis.
1) Measurement results

2) Conclusion 1) The "anti-NC1 antibody measurement kit" of the present invention can measure a human sample in the same manner as an existing in vitro diagnostic drug. In addition, it has higher sensitivity.
-The OD at the cut-off value (10 U) is 16 times that of the negative (0 U) in the kit of EUR, but 29 times in the kit of the present invention.
2) Both measurement reagents can measure monkey serum in the same manner as humans. Thus, monkey serum can be used as a standard when measuring human anti-GBM antibody nephritis samples.
3) When monkey serum is measured using a human sample as an index, the "anti-NC1 antibody measurement kit" of the present invention recognizes positivity earlier (2W) than the existing in vitro diagnostic drug (6W). Therefore, the kit of the invention is useful for early detection of human anti-GBM antibody nephritis.
In three human anti-GBM antibody nephritis, it was examined whether the serum also contained an IgA antibody.
1) The measurement was performed by replacing the “HRP-labeled anti-human IgG rabbit antibody” with the “HRP-labeled anti-human IgA rabbit antibody” using the “anti-NC1 antibody measurement kit” described above.
2) Measurement results

3) Conclusion 1) IgA antibodies are also present in human anti-GBM antibody nephritis. Therefore, measurement of the IgA antibody using the “anti-NC1 antibody measurement kit” can be used for detecting human anti-GBM antibody nephritis. The fact that the cut-off value of IgA and the OD of the healthy value are almost equal makes it clear that the sample showing the cut-off value is a healthy group when it is separated from that of IgG.
4 It was examined whether the “anti-NC1 antibody measurement kit” can measure antibodies in urine.
1) Measurement results

2) Conclusion 1) The “anti-NC1 antibody measurement kit” can measure both IgA antibodies and IgG antibodies in urine. Therefore, measuring urine can be a substitute for measuring serum.
2) In urine measurement, unlike serum, IgA is dominant, and it is desirable to measure it.
5 Preparation of “NC1 measurement kit” (performed by sandwich ELISA method)
1) Composition of kit Anti-NC1 antibody binding microplate; antiserum 120 ul / well
The antiserum derived from the rat prepared in NC1 described above is diluted 1000-fold with a sample; PBS (containing BSA, Tween 20)
Anti-NC1 Antibody Rabbit-derived antiserum prepared by the above-mentioned NC1 was diluted 5000-fold HRP-labeled anti-rabbit IgG antibody (from goat)
Chromogenic substrate solution; TMB
Reaction stop solution; 1N sulfuric acid washing solution; PBS (including Tween 20)
2) Operation method Wash the antibody-bound microplate once → Add sample (use human urine as it is, dilute monkey serum and monkey urine) → Wash 3 times after 2 hours → Add anti-NC1 antibody → Wash 3 times after 2 hours → Addition of HRP-labeled anti-rabbit IgG antibody → Wash 3 times after 1 hour → Addition of chromogenic substrate solution → Addition of reaction stop solution after 5 minutes → Absorbance measurement (450 nm)
3) Human specimen: Human male healthy person (44 years, 12 years, 10 years) Early in the morning, all negative on commercial urtera test paper 4) Monkey specimen; anti-GBM antibody nephritis model prepared in "Preparation" 5) Measurement result 1

・ Measurement result 2

6) Conclusion 1) The antigen NC1 is present in healthy blood and healthy urine.
2) Urine has a larger gap between normal and disease states than serum and is easier to use as an index than serum.
6 Preparation of “anti-HBc antibody measurement kit” (performed by ELISA method)
1) Composition of kit HBc-binding microplate;
Sample diluent: PBS (including BSA, Tween 20)
HRP-labeled anti-human IgG antibody coloring substrate solution; MB
Reaction stop solution; 1N sulfuric acid washing solution; PBS (including Tween 20)
2) Operation method Wash HBc antigen-binding microplate once → Add sample (use human urine diluted 2-fold) → Wash at room temperature for 2 hours and wash 3 times → Add HRP-labeled anti-human IgG antibody → Reaction at room temperature for 1 hour After washing three times → addition of a chromogenic substrate solution → addition of a reaction stop solution after 15 minutes → immediate measurement of absorbance (450 nm)
3) Urinary urine of three boys and three negative boys who showed HBc antibody positive in the serum measurement of human samples was used.
4) Measurement results In urine, all seropositive persons were positive, and all negative persons were negative.
5) Conclusion In the test for hepatitis B, measurement of anti-HBc antibody conventionally used in serum shows a useful judgment even in occasional urine measurement.
7 Preparation of “HBs measurement kit” (performed by sandwich ELISA method)
1) Components of the kit: anti-HBs antibody binding microplate;
Polyclonal antibody (quartet, derived from rabbit) diluted 1000-fold with buffer solution (pH 9.6) to dilute a coated sample; PBS (containing BSA, Tween 20)
Anti-HBs antibody monoclonal antibody (quartet, mouse) diluted 1000-fold with buffer (pH 7.4) HRP-labeled anti-mouse IgG antibody (rabbit)
Chromogenic substrate solution; TMB
Reaction stop solution; 1N sulfuric acid washing solution; PBS (including Tween 20)
2) Operation method Wash the antibody-bound microplate once → Add sample (use human urine as it is) → Wash 3 times after 2 hours → Add anti-HBs antibody → Wash 3 times after 2 hours → Add HRP-labeled anti-mouse IgG antibody → Washing 3 times after 1 hour → Addition of chromogenic substrate solution → Addition of reaction stop solution after 15 minutes → Immediate absorbance measurement (450 nm)
3) Urinary urine of one male and three male and three negative males presenting HBs antigen positive in the serum measurement of the human sample was used.
4) Measurement results In urine, all seropositive persons were positive, and all negative persons were negative.
5) Conclusion In the test for hepatitis B, the measurement of HBs antigen conventionally used in serum shows useful judgment even in urine measurement at any time.

Claims (7)

A method and a reagent for detecting an antibody or antigen corresponding to a disease by using urine as a sample to detect an autoimmune disease or an infectious disease in humans by using an immune reaction. The method and reagent according to claim 1, wherein "antibody" is specified as "antibody or antigen" in claim 1. 2. The method and reagent according to claim 1, wherein "autoimmune disease or infectious disease" is specified as "renal disease". The method and the measuring reagent according to claim 1, wherein monkey urine is used as a positive standard in the "method and method for detecting by immunological reaction" in claim 1. The method and the measuring reagent according to claim 1, wherein the "antibody" according to claim 1 is specified as "IgA". Claim 1 wherein "autoimmune disease or infectious disease" is specified as "IgA nephropathy", "urine" is replaced with "serum", and "antibody or antigen" is specified as "IgA or NC1". Methods and reagents. A method and a reagent for detecting an antibody or antigen corresponding to a disease or the like by an immunoreaction using urine as a sample to detect an autoimmune disease or an infectious disease in an animal other than a human.