JP2007024661A - Quantifying method of sugar chain deficient iga molecule and quantifying kit therefor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a simple and rapid quantifying method of a sugar chain deficient IgA molecule, and a quantifying kit. <P>SOLUTION: A sample containing a sugar chain is brought into contact with a carrier to which streptococcal IgA bonding peptide is fixed to bring a substance specifically bonded to the sugar chain into contact with the carrier. Then, the quantity of the substance bonded to the carrier is measured. Further, the quantifying kit is constituted of the streptococcal IgA bonding peptide, the carrier for fixing the streptococcal IgA bonding peptide and the substance specifically bonded to the sugar chain. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for quantifying a sugar chain-deficient IgA molecule and a quantification kit. More specifically, using a carrier on which Streptococcal IgA-binding peptide (SAP) is immobilized, a substance that specifically binds to a sugar chain is detected. The present invention relates to a simple and rapid quantitative method and quantitative kit.

  IgA nephropathy is chronic glomerulonephritis characterized by the deposition of IgA immune complexes thought to be blood-derived within the glomerular mesangial region of the kidney. In Japan, it accounts for more than 30% of primary kidney disease, the most common single kidney disease, of which 15-30% have a poor prognosis and shift to renal failure. A number of studies including the present inventors have partially revealed the onset / progression mechanism of IgA nephropathy, but there are still many unclear points. In recent years, it has become clear that a sugar chain-deficient IgA molecule is present in the serum of patients with this disease, and that this sugar chain-deficient state is involved in the formation of pathological immune complexes and IgA deposition in the glomerular mesangial region. It is coming. In sugar chain-deficient IgA, it has been reported that the content of galactose and sialic acid in the O-linked sugar chain present in the hinge part of IgA is smaller than that in healthy individuals. In addition, IgA nephropathy patients have a decreased enzyme activity of β-1,3-galactosyltransferase responsible for binding of galactose to N-acetylgalactosamine in the production of O-linked sugar chains as compared with healthy individuals ( Non-patent document 1). However, details of why sugar chain failure occurs in sugar chain-deficient IgA molecules remain unclear. The main cause is that the method for detecting and quantifying sugar chain-deficient IgA is complicated and expensive, and therefore, analysis in many cases and change due to the course of treatment cannot be analyzed sufficiently.

  Known methods for quantification of IgA molecular sugar chain modification deficiency to date include separation and purification of IgA from serum, separation and purification of hinge region using proteolytic enzyme (Patent Document 1), and MALDI-TOF mass spectrometer. It requires extremely complicated steps such as analysis. For this reason, large-scale equipment, complicated processes, and advanced knowledge of glycan analysis are required, which is highly specialized and has been conducted in a limited number of facilities.

In the separation of IgA from serum, conventionally, a lectin, Jacalin, has often been used. However, since jacalin is a lectin that recognizes the sugar chain Galβ-1,3-GalNAc of IgA, there is a bias that IgA having galactose is selectively purified at the purification stage, and sugar chain-deficient IgA is detected. It was not suitable for the purpose.
Nichirenkai 2002, 44: 514-523 JP-A-8-228795

  As described above, it is clear that a sugar chain-deficient IgA molecule is involved in IgA nephropathy, and if this can be easily quantified over time and with quantitativeness, it can be used not only for pathological analysis but also for diagnostic treatment. Is also extremely useful. Therefore, an object of the present invention is to provide a simple and rapid quantification method and quantification kit for a sugar chain-deficient IgA molecule.

  As a result of intensive studies in view of the above problems, a method for quantifying a sugar chain-deficient IgA molecule using a streptococcal IgA-binding peptide (SAP) that specifically recognizes the Fc region of IgA was found, The present invention has been completed. That is, the present invention is as follows.

  The method for quantifying a sugar chain-deficient IgA molecule in a sample according to claim 1 comprises contacting a sample containing a sugar chain with a carrier on which a streptococcal IgA-binding peptide is immobilized, and contacting a substance that specifically binds to the sugar chain. A contact step and a measurement step of measuring the amount of the substance bound to the carrier.

  The quantitative method according to claim 2 is characterized in that, in claim 1, the substance that specifically binds to the sugar chain is a lectin.

  The quantitative method according to claim 3 is characterized in that, in claim 1 or 2, the substance that specifically binds to the sugar chain is labeled.

  The quantification kit for sugar chain-deficient IgA molecules in the sample of claim 4 comprises a streptococcal IgA-binding peptide, a carrier for immobilizing the streptococcal IgA-binding peptide, and a substance that specifically binds to the sugar chain. Features.

  The quantitative kit according to claim 5 is characterized in that, in claim 4, the substance that specifically binds to the sugar chain is a lectin.

  The quantitative kit according to claim 6 is characterized in that, in claim 4 or 5, the substance that specifically binds to the sugar chain is labeled.

  According to the method for quantifying sugar chain-deficient IgA molecules in the sample of claim 1, since the structure is simple and the number of components is small, the sugar chain-deficient IgA molecules can be quantified easily and rapidly. This quantitative method is very useful not only for pathological analysis but also for diagnostic treatment.

  According to the quantification method of claim 2, sugar chain-deficient IgA molecules can be detected more accurately by using a substance that specifically binds to a sugar chain as a lectin.

  According to the quantification method of claim 3, the sugar chain-deficient IgA molecule can be quantified easily and rapidly by labeling the substance that specifically binds to the sugar chain.

  According to the quantification kit for sugar chain-deficient IgA molecules in the sample of claim 4, the quantification kit for simple and rapid quantification of sugar chain-deficient IgA molecules is provided because the configuration is simple and the number of components is small. it can. Moreover, this quantitative kit is extremely useful not only for pathological analysis but also for diagnostic treatment.

  According to the quantification kit of claim 5, a quantification kit for detecting a sugar chain-deficient IgA molecule more accurately can be provided by using a lectin as a substance that specifically binds to a sugar chain.

  According to the quantification kit of claim 6, it is possible to provide a quantification kit for quantifying sugar chain-deficient IgA molecules simply and quickly by labeling a substance that specifically binds to a sugar chain.

The present invention will be described below.
1. Method for quantifying sugar chain-deficient IgA molecules The method for quantifying sugar chain-deficient IgA molecules in a sample according to the present invention comprises contacting a sample containing a sugar chain with a carrier on which a streptococcal IgA-binding peptide is immobilized, and A contact step of contacting a substance that binds to the carrier, and a measurement step of measuring the amount of the substance bound to the carrier. According to the present invention, a simple and rapid method for quantifying sugar chain-deficient IgA molecules has become possible.

  Sugar chain-deficient IgA is human IgA in which the content of galactose and sialic acid in the O-linked sugar chain present in the hinge part of IgA is less than that of healthy people. As mentioned above, sugar chain-deficient IgA molecules are known to be involved in IgA nephropathy.

  Streptococcal IgA-binding peptide (SAP) (hereinafter referred to as SAP) is a peptide derived from streptococci that specifically recognizes and binds to the Fc region of human IgA (The Journal of Immunology, 2002, 169: 1357-1364). It has been reported that 99% or more of human IgA is adsorbed on the SAP column, and SAP is a peptide having high affinity with human IgA.

(A) Carrier on which SAP is fixed Examples of the shape of the carrier include plates, tubes, beads, and gels. For example, a 96-well microplate may be used as a carrier, and a large number of samples may be quantified at once.

  As a method for fixing the carrier to the SAP, methods conventionally known in the art such as a physical adsorption method and a covalent bonding method can be used. For example, SAP is dissolved in a buffer solution of about pH 7-9 (for example, phosphate buffer, phosphate buffered saline (PBS), carbonate buffer, etc.), added to the solid phase, and allowed to stand at 4 ° C. overnight. It can be fixed by incubating.

  After fixing SAP to the carrier, it is preferable to add a blocking substance to the carrier to coat the portion where SAP is not fixed. Such blocking substances include serum albumin, casein, skim milk, gelatin and the like.

By the above method, a carrier on which SAP is immobilized can be produced.
(B) Contact process A contact process is described. The sample is not limited as long as it contains or may contain a sugar chain-deficient IgA molecule. Specifically, biological samples, such as serum, are mentioned, for example.

  First, the carrier on which the SAP is immobilized is brought into contact with the sample. Since SAP is a peptide having high affinity with IgA as described above, IgA in the sample specifically reacts with and binds to SAP by contacting the sample with a carrier on which SAP is immobilized. The IgA molecule is then bound to the carrier via SAP. At this time, the sugar chain-deficient IgA molecule bound to SAP exposes N-acetylgalactosamine (GalNAc) in the hinge region, mannose (Man), etc. in the Fc region. These are found when sugar chain-deficient IgA molecules are deficient in galactose or sialic acid.

  In the next step, a substance that specifically binds to the exposed sugar chain is brought into contact with the carrier on which the SAP bound with IgA is immobilized. Thereby, the substance that specifically binds to the sugar chain specifically reacts with and binds to the sugar chain-deficient IgA molecule. A substance that specifically binds to a sugar chain is bound to a carrier via a sugar chain-deficient IgA molecule and SAP. As a substance that specifically binds to a sugar chain, a substance that specifically binds to these GalNAc, Man and the like is preferable. Examples thereof include lectins such as SBA (soybean agglutinin) and ConA (concanavalin A). SBA is a lectin that specifically binds to GalNAc. ConA is a lectin that specifically binds to Man.

  It is preferable to incubate at about 0 ° C. to about 45 ° C., for example, about 37 ° C. in order to sufficiently react when these are brought into contact. The incubation time is not particularly limited as long as it can sufficiently react.

  Moreover, it is preferable to wash | clean the surface of a support | carrier with a washing | cleaning liquid after reaction of a contact process. This washing is performed to remove the molecules that did not specifically bind as described above, and is performed under conditions that do not release substances that specifically bind to sugar chains, IgA molecules, SAPs, and the like. The washing solution is not particularly limited, and for example, a buffer solution (for example, phosphate buffer solution, PBS, Tris-HCl buffer solution, etc.) to which a nonionic surfactant such as a Tween surfactant is added can be used.

(C) Measurement process After passing through the above contact process, it transfers to a fixed_quantity | quantitative_assay process. The quantification step is a step of quantifying a substance that specifically binds to the sugar chain bound to the carrier. In addition, it is preferable that the substance that specifically binds to the sugar chain is labeled with a labeling substance because measurement is easy in the measurement step. The substance that specifically binds to the sugar chain can be labeled conventionally known in the art, such as an enzyme label, a radioactive label, or a fluorescent label. For example, when a substance that specifically binds to a sugar chain is labeled with biotin, an enzyme to which streptavidin or the like is bound (for example, peroxidase) is added to bind biotin and streptavidin, and then streptavidin or the like is bound. By adding an enzyme substrate, a chromogenic substrate, and the like, the degree of color development of the product due to the enzyme reaction can be measured for a change in absorbance. In addition, when a substance that specifically binds to a sugar chain is labeled with a fluorescent substance or a chemiluminescent substance, fluorescence or luminescence can be measured.

  The quantification method of the present invention is based on the ability to correlate the measurement result obtained from a sample with the measurement result obtained from a known standard substance. That is, the sugar chain in the sample can be quantified by preparing a calibration curve from the above measurement results with a sugar chain standard solution (for example, a sugar chain containing GalNAc) at a known concentration in advance. As described above, the sugar chain-deficient IgA molecule bound to SAP exposes GalNAc in the hinge region, Man and the like in the Fc region. Therefore, by quantifying these, the sugar chain-deficient IgA molecule can be quantified.

2. Glucose-deficient IgA molecule quantification kit The glycan-deficient IgA molecule quantification kit in the sample of the present invention specifically binds to a sugar chain with a streptococcal IgA-binding peptide, a carrier for immobilizing the streptococcal IgA-binding peptide. And a substance. Similarly to the method for quantifying sugar chain-deficient IgA molecules of the present invention, the Streptococcus IgA-binding peptide is hereinafter referred to as SAP. The description of SAP, the carrier that immobilizes SAP, and the substance that specifically binds to a sugar chain is the same as the method for quantifying a sugar chain-deficient IgA molecule of the present invention.

  The quantification kit of the present invention is not particularly limited as long as it contains at least the SAP, a carrier for immobilizing SAP, and a substance that specifically binds to a sugar chain. Constituent components such as a substance that specifically binds to a sugar, a reagent that labels a substance that specifically binds to a sugar chain, a reagent that detects a substance that specifically binds to a sugar chain, or a reagent that detects a labeled sugar chain Can be added as. In addition, SAP can be immobilized on a carrier in advance, or a substance that specifically binds to a sugar chain can be labeled in advance and used as a component of a quantitative kit. In addition to these components, blocking substances, washing solutions, specimen dilution solutions, enzyme reaction stop solutions, instruments (microtiter plates, pipettes, etc.) and the like may be included.

  These components can be stored in separate containers and stored as kits that can be used according to the quantification method of the present invention at the time of use.

  Hereinafter, specific examples will be described, but the present invention is not limited to the following examples.

1. Material Luminunk chemiluminescence plate: (Luminunk F96-polysorb white # 436111).
Washing solution: Tween-PBS (0.05% Tween20-PBS): 1 ml of 10% Tween20 was mixed with 200 ml of PBS.
Blocking solution and serum dilution buffer (1% BSA / Tween-PBS): 0.5 g of BSA was dissolved in 50 ml of Tween-PBS and stored frozen. It could be stored for several months.
Biotinylated lectin solution (5 μg / ml in blocking solution): 10 μl of biotinylated lectin (1 mg / ml) was dissolved in 2 ml of 1% BSA / Tween-PBS.
HRP-labeled streptavidin (5 μg / ml in blocking solution): 4 μl of HRP-labeled streptavidin was dissolved in 2 ml of 1% BSA / Tween-PBS.
Luminescent substrate for peroxidase: ECL Plus Western Blotting Detection Reagents (RPN2132) (Amersham Bioscience)

2. Immobilization of SAP peptide on ELISA plate 5 mg of SAP peptide was dissolved (100 μg / ml) in 50 ml of PBS (pH 7.5). After dispensing 1 ml each, it was stored frozen at -80 ° C. 100 μl of the SAP solution per well was added to a luminung chemiluminescence plate and sealed with a plate sealer. And it incubated at 4 degreeC overnight. Thereafter, the SAP solution was removed after the reaction, and the plate was washed 3 times with a Tween-PBS washing solution. 200 μl of blocking solution (1% BSA-Tween-PBS) was added to each well of the plate and allowed to stand at room temperature for 1 hour, after which the blocking solution was removed.

3. 100 μl of serum diluted 500 times with an adsorption blocking solution of IgA molecule and biotinylated lectin was added to each well. After incubating at room temperature for 1 hour, the diluted serum was removed, and the plate was washed 5 times with a washing solution (the 5th time was 1% BSA-Tween-PBS). As a biotinylated lectin solution, 100 μl of B-1235 (biotinylated lectin: 1% BSA-PBS) was added. B-1235 is Vicilla villosa lectin from vector laboratories and binds to N-acetylgalactosamine to which galactose is not bound. For B-1235, the stock solution of 2 mg / ml was diluted 400 times and adjusted to 5 μg / ml. Wells with control HRP anti-IgA contained blocking solution. Incubated for 1 hour at room temperature and washed 5 times with Tween-PBS.

4). Measurement by chemiluminescence Add 100 μl of HRP-labeled streptavidin solution (VEC SA-5004 Streptavidin, HRP binding, 1 mg / ml, 14000) (1% BSA-PBS-Tween diluted to 2 μg / ml (500 times)) at room temperature Incubated for 1 hour. Wells with control HRP anti-IgA received a 1: 10000 dilution of HRP anti-IgA. Thereafter, the plate was washed 5 times with PBS Tween. 100 μl of a detection solution (ECL plus detection solution) was added to each well of the plate, and chemiluminescence was detected with Mitras LB940 from 5 minutes after the start of the reaction.

5. Results The above steps 1 to 4 were carried out on 11 IgA nephropathy patients with incomplete IgA sugar chains and 11 healthy subjects as controls. The results are shown in FIGS. FIG. 1 and FIG. 2 revealed that the amount of N-acetylgalactosamine in IgA nephropathy patients was significantly higher than that in healthy subjects. This is because the sugar chain insufficiency of the present invention is characterized by detecting a substance that specifically binds to a sugar chain using a carrier on which Streptococcal IgA-binding peptide (SAP) is immobilized. It shows the effectiveness of the quantitative method for IgA molecules.

FIG. 1 is a diagram showing a box-whisker diagram obtained from a result of chemiluminescence by an IgA nephropathy patient having a defective IgA sugar chain and a healthy person. IgAN represents an IgA nephropathy patient having a defective IgA sugar chain, and control represents a healthy person. FIG. 2 is a diagram showing the results of a t-test on chemiluminescence by an IgA nephropathy patient having a deficient IgA sugar chain and a healthy person.

Claims (6)

A contact step in which a sample containing a sugar chain is brought into contact with a carrier on which Streptococcus IgA-binding peptide is immobilized, and a substance that specifically binds to the sugar chain is brought into contact; and a measurement for measuring the amount of the substance bound to the carrier And a method for quantifying a sugar chain-deficient IgA molecule in a sample. The method according to claim 1, wherein the substance that specifically binds to the sugar chain is a lectin. The method according to claim 1 or 2, wherein the substance that specifically binds to the sugar chain is labeled. A kit for quantifying sugar chain-deficient IgA molecules in a sample, comprising a streptococcal IgA-binding peptide, a carrier for immobilizing the streptococcal IgA-binding peptide, and a substance that specifically binds to a sugar chain. The quantitative kit according to claim 4, wherein the substance that specifically binds to the sugar chain is a lectin. 6. The quantification kit according to claim 4 or 5, wherein the substance that specifically binds to the sugar chain is labeled.

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