JP2001519357A - Anti-BiP antibodies in rheumatoid arthritis and methods and test kits for their detection - Google Patents

Abstract

  (57) [Summary] The present invention relates to the determination of anti-heavy chain binding protein antibodies (anti-BiP antibodies) and anti-BiP antibodies in body fluids from patients with rheumatoid arthritis. In particular, it relates in vitro to the determination of said antibody as an autoantibody for the serodiagnosis of rheumatoid arthritis in the differential diagnosis of other rheumatic diseases.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to anti-heavy chain binding protein specific antibodies (anti-BiP antibodies) and methods and test kits for their evaluation in body fluids. It also relates to the evaluation of these antibodies as autoantibodies in the forensic diagnosis of rheumatoid arthritis in the differential diagnosis of other rheumatic diseases.

[0002] Rheumatoid arthritis (RA)
It is the most common inflammatory joint disease with a prevalence of 1-2% of the population. Autoantibodies, mainly rheumatoid factors, appear in patients with ongoing RA. Therefore, rheumatoid factor is the only serological marker in the diagnosis of RA. Nevertheless, they also frequently appear in other rheumatic diseases, such as systemic lupus erythematosus, systemic scleroderma, and mixed connective tissue disease, as well as in healthy populations. Rheumatoid factor specificity for RA is only 74
% And its sensitivity in RA patients is 68% [Blas St, Spe
ccker Ch, Lakomek HJ, Schneider EM, Schw
ochau M "A new 68k autoantigen detected by rheumatoid arthritis (RA) specific autoantibodies" Ann Rheum Dis 1995; 54: 355-
60]. RA is a heterogeneous (he) that is difficult to diagnose, as exemplified by seven ACR criteria, of which at least four criteria should be met.
terogenous disease (Arnett FC, Edwardy)
SM, Bloch DA, McShane DJ, Fries JF, Coop
erns, Healey LA, Kaplan S, Liang MH, Lu
thra HS, Medsger TA jr. , Mitchell DM, N
ustadt DH, Pinals RS, Schaller JG, Shar
p JT, Wilder RL, Hunder GG). The American Rheumatism Association revised the criteria for the classification of rheumatoid arthritis in 1987 (Arthr
itis Rheum 1988; 31: 315-24). It contains only a single serological marker, namely rheumatoid factor. Thus, improved diagnostics, including even more specific serological markers, may be acceptable for appropriate treatment.

[0003] Heavy chain binding protein (BiP) [glucose regulatory protein 78 (Grp78)
)] Is a so-called molecular chaperone (chap) located in the endoplasmic reticulum.
erone). Its function is to help the protein fold and complex the protein with other subunits that travel along the secretory pathway. BiP is a member of the Hsp70 family of stress proteins,
It is a protein that is ubiquitously expressed and essential and highly conserved in yeast. Therefore, BiP has never been taken out in the context of RA. The reason may be that anti-BiP antibodies are directed against glycoside epitopes that are not expressed in common expression systems.

[0004] Accordingly, the present invention provides means for isolating anti-BiP antibodies from RA patients, anti-BiP antibodies and the means for evaluating their titers and developing appropriate test kits. Anti-BiP antibodies were purified by natural (biochemically purified) or recombinant BiP bound to a solid phase. By this means, antibodies could be purified from body fluids. Antibody elution was performed by applying chaotropic material. According to the present invention, natural (biochemically purified) or recombinant BiP is bound to a solid phase by adsorption or covalent bonding or by electrotransfer and used to identify anti-BiP antibodies in immunoassays. Is done. The glycosylated form of the antigen is of particular interest and interest. The method of the present invention for determining an anti-BiP antibody in a body fluid is to bind BiP to a solid phase and perform an immunoassay. After chemical activation of the solid phase, if necessary, BiP is bound to the solid phase by adsorption or covalent bonding. The antigen in this assay is natural (biochemically purified) or recombinant BiP. The solid phase is any of polystyrene, polyvinylidene fluoride, or nitrocellulose in various geometries (eg, microtiter plates, vials, or spherical or flat supports). Anti-BiP antibodies are determined in an immunoassay in bound form. Antibody binding is mediated by a suitable buffer containing non-ionic or ionic detergents or proteins. Bound anti-BiP antibodies are determined by specific conjugates to particular immunoglobulin classes or some of them.

A kit according to the invention for determining anti-BiP antibodies comprises: a natural (purified) or recombinant form of BiP, which is bound to a solid phase by adsorption or covalent bonding, Buffers containing non-ionic or ionic detergents for diluting biological fluids, antibodies against human immunoglobulin IgG, IgM, IgA, or a plurality of these immunoglobulin classes, and enzymes, fluorescence A specific conjugate consisting of a dye or radioactively labeled component; a buffer containing a non-ionic or ionic detergent or protein for diluting said conjugate; an unbound immunoreaction A buffer containing a nonionic or ionic detergent or protein for washing (removing) the body, a group for determining the enzymatic reaction Quality solution,-characterized by a stop solution for stopping the enzymatic reaction.

Due to the high prevalence of the antibodies of the present invention, they
Is a new diagnostic marker. Since BiP has essential cell functions, a relationship between anti-BiP antibodies and pathogenesis can be elicited. The antibodies of the present invention are also important diagnostic markers for the differential diagnosis of other rheumatic diseases where these antibodies are absent or have a fairly low incidence.

[0007] Immunologically established methods for determining anti-BiP antibodies, such as ELI
SA (enzyme linked immunosorbent assay)
), Sandwich-ELISA, and immunoblot are used. ELIS
Analysis of antibody reactivity by the A technique is superior because of its low cost and turnaround time.
Thus, ELISA is a screening tool, while immunoblotting is used to specify antibody reactivity in detail.

[0008] Depending on the results of serological markers (anti-BiP antibodies) in RA patients, 66
% Were found to be positive for ELISA and immunoblot for the anti-BiP autoantibodies of the invention. This screening of patient sera was performed on purified (native) BiP.

According to the present invention, it is mentioned that anti-BiP antibodies appear with high incidence in RA patients. These antibodies respond primarily to carbohydrate epitopes, a major determinant that is N-acetylglucosamine. The molecular and biochemical properties of BiP were determined (relative molecular mass, isoelectric point, amino acid sequence). Hereinafter, examples will be described to explain the present invention, but these examples do not limit the present invention.

[0010]

【Example】

Patients with various rheumatic diseases and 100
Human healthy donors were analyzed. Of the patients, 214 were RA patients, 47 were SLE patients, 23 were psoriatic arthritis patients, 24 were systemic sclerosis patients, and 6 were mixed connective tissue disease patients. , 13 are osteoarthritis patients,
13 patients with ankylosing spondylitis, 4 with reactive arthritis and 1
0 were patients with localized scleroderma. BiP is purified according to standardized purification steps
Purified from HeLa cells.

[0011]

Example 1 << Purification of anti-BiP antibody from body fluid of RA patient >> Binding of BiP to solid phase. Blocking of excess binding sites. Washing (removal) of unbound immunoreactants. Elution of specifically bound anti-BiP antibodies by treatment with a chaotropic salt (eg, guanidinium isothiocyanate 3M). Wash and concentrate antibodies.

[0012]

Example 2 << Preparation of ELISA for Determining Anti-BiP Antibody >> Binding of BiP to ELISA Solid Phase A 96-well polystyrene plate was used as the solid phase. Bi per well
Coating was performed using 10 to 15 ng of P. Carbonate buffer 50 mM (p
H9.5) and incubated overnight at 4 ° C. Subsequently, the plates were used as they were or stored frozen at -20 ° C.

Prior to coating the material, the sample buffer [nonidet (nonidet)
) The plate was pre-blocked with 100 μl of phosphate buffered saline containing P40 (PBS-NP40) (0.1%, pH 7.0)]. Samples (serum) were added at a dilution of 1:20, 50 μl per well and incubated at room temperature for more than 3 hours (application of RA or other rheumatic disease patient or healthy donor serum). For the standard curve, RA serum with outstanding reactivity was used as a positive control (standard) and given in 6 serial dilutions starting from 1:10. Following incubation, it was washed three times with wash buffer.

Incubation with alkaline phosphatase (AP) -conjugated anti-human IgG was performed at room temperature for 3 hours at a dilution of 1: 3000 in sample buffer (50 μl / well). Subsequently, it was cleaned in a washing buffer.

Substrate reaction Antigen-antibody reactivity staining was performed using BCIP / NBT (5-bromo-3-chloro-indolyl phosphate / nitro-blue tetrazolium) in alkaline buffer.
This was performed using

The antibody reactivity of a 1:20 dilution of the standard serum was defined as 10,000 relative units (RU). Therefore, each dilution 1:10, 1:30, 1:50, 1: 100, 1:
500 are 20,000 RU, 6,667 RU, 4,000 RU, 1
2,000RU and 200RU. Standard curves were determined by triplicate values. If the serum reached a value higher than the standard value, further dilutions were repeated. The resulting RU was then multiplied by a dilution factor higher than 1: 100. According to ELISA, anti-BiP antibodies were detected in 66% of RA patients and in 0.5% of other patients diagnosed with rheumatism, but not in healthy individuals.

[0017]

Example 3 HeLa cells were dissolved in a buffer containing gardol, urea, and 2-mercaptoethanol and incubated at 70 ° C for 15 minutes. Lysed cells were supplemented with saturated cesium chloride until a density of ∂ = 1.6 g / cm ³ was reached and centrifuged at 10,000 g for 2 hours. The resulting protein suspension is
Homogenized in lysis buffer containing the non-ionic detergent NP40 instead of Gardol, followed by dialysis against 25 mM Tris buffer. After the material was precipitated with ethanol, sodium dodecyl sulfate gel electrophoresis (SD
S-PAGE) (acrylamide gradient = 10-20%). The gel was blotted onto polyvinylidene fluoride (PVDF).

[0018] The electrotransfer was blocked with PBS-NP40 and BSA and cut into small pieces. Each piece was incubated with serum diluted 1:20 at 4 ° C. overnight.
Subsequently, the small pieces were washed with a washing buffer (PBS-NP40), and then incubated with an alkaline phosphatase-conjugated secondary antibody diluted 1: 5000 for 1 hour. Following washing in PBS-NP40, the strips were incubated in AP buffer (pH 8.7) and substrate was added (BCIP / NBT). When properly stained, the immune response was stopped by neutral or basic buffer.

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] March 31, 2000 (2000.3.31)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

Claims (15)

[Claims] 1. A human anti-heavy chain binding protein antibody obtained by immunoadsorption to natural (biochemically purified) or recombinant human heavy chain binding protein (BiP) and elution by chaotropic salt treatment (Anti-BiP antibody). 2. The method for determining an anti-BiP antibody in a body fluid according to claim 1, wherein BiP is bound to a solid phase and applied to an immunoassay. 3. The method according to claim 2, wherein the BiP antigen is bound to the solid phase by adsorption or covalent bonding. 4. The method according to claim 2, wherein the BiP antigen is bound by adsorption or covalent bonding after chemically activating the solid phase in advance. 5. A natural BiP, a biochemically purified BiP, or a recombinant BiP.
5. The method according to claim 2, wherein P is applied as an antigen. 6. The method according to claim 2, wherein the BiP is bound by a specific anti-BiP monoclonal antibody. 7. The method according to claim 2, wherein the solid phase comprises polystyrene, polyvinylidene fluoride or nitrocellulose. 8. The method according to claim 2, wherein the solid phase consists of various geometries. 9. The solid phase-containing carrier is a microtiter plate or a tube, or a spherical or flat support.
9. The method according to any one of claims 1 to 8. 10. The method according to claim 2, wherein the anti-BiP antibody is determined as the binding in an immunoassay. 11. The anti-BiP antibody is bound in a buffer containing a nonionic or ionic surfactant or protein.
The method according to 0. 12. The method according to claim 2, wherein the bound anti-BiP antibody is determined by specific conjugates for a particular immunoglobulin class or several classes. 13. The specific conjugate is a human immunoglobulin (Ig) G
, IgM, or IgA, or an antibody against any of these immunoglobulins, and an enzyme, a fluorescent dye, or a radiolabeled component.
The method according to claim 2. 14. BiP bound to a solid phase.-A buffer for diluting the biological fluid to be analyzed.-A specific conjugate.-A buffer for dilution of said conjugate.-An unbound immunoreaction. The test kit for determining an anti-BiP antibody according to claim 1, comprising a buffer for washing the body, a substrate solution, and a stop solution. 15. A buffer for diluting the biological fluid to be analyzed, which contains BiP, which is bound to the solid phase by adsorption or covalent bonding, or a nonionic or ionic detergent or protein. Fluids-specific conjugates of human immunoglobulin IgG, IgM, or IgA, or antibodies to some of these immunoglobulins, and enzymes, fluorochromes, or radiolabeled components-non-ionic or ionic Conjugate Dilution Buffer Containing a Surfactant or Protein for Washing (Rinsing / Removing) Unbound Immunoreactants Containing Nonionic or Ionic Surfactants, Proteins, or Buffers 15. A buffer solution-a substrate solution for detecting the enzymatic reaction-a stop solution for stopping the enzymatic reaction. The test kit described.