JP3309900B2 - Method for detecting anti-FKBP12 autoantibody - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for detecting anti-FKBP12 autoantibodies, and more particularly, to diagnosing various pathological conditions and elucidating the mechanism of their development, and before and after transplanting an organ into a subject. Anti-FKBP contributes to diagnosing whether a subject is suitable for organ transplantation, etc.
12 A method for detecting an autoantibody.

[0002]

[Prior Art] FK506 binding protein 12 (FKBP1
2) was discovered in 1990 as a cytoplasmic receptor protein of FK506, which is now widely used as an immunosuppressant after liver transplantation and kidney transplantation.

[0003] Today, it is known that FKBP12 and its analogous proteins are widely present not only in mammals such as humans but also in cells of bacteria, insects and plants.

[0004] When the properties of FKBP12 are examined immunologically, FKBP12 is a protein directly involved in the immune response, and binds to FK506 to suppress the production of cytokines such as IL-2. It has been reported to reduce rejection after transplantation and other transplants (Thomson
AW, Bonham CA, Zeevi A. Mode of cation of tacrolim
us (FK506): molecular and cellular mechanisms.Ther
Drug Monit 1995; 17: 584., And Fung JJ. Eliasziw
M, Todo S et al. The Pittsburgh randomizedtrial of
tacrolimus compared to cyclosporine for hepatic t
ransplantation. J Am Coll Surg 1996; 183: 177.). Physiologically, FKBP12 is TGF-β
It has been reported that it binds to the family type 1 receptor and suppresses the signal of this receptor, and that it controls the function of the cytoplasmic calcium channel (Wang. T, Li B, Danielson PD, et al. Th
eimmunophilin FKBP12 functions as a common inhibit
or of the TGF-β familytype I receptors. Cell 199
6; 86: 435; and Brillantes AB, Ondrias K, Scott A,
et al. Stabilization of calcium release channel (Ry
anodine receptor) function by FK506-binding protei
n. Cell 1994; 77: 513.), suggesting that the protein has an important role not only in the immune system but also in the physiology of the cell itself.

[0005] On the other hand, in conditions causing immunological abnormalities (for example, autoimmune diseases, conditions observed in patients who have received transplanted organs, etc.), the thyroid gland, erythrocytes,
Various antibodies that react with myelin, stomach, DNA, etc., that is, autoantibodies have been recognized, and many autoantibodies have been reported. Some of these autoantibodies have been applied clinically as markers in the diagnosis of disease and its activity, and others have been considered to be the direct cause of diseases that cause immune abnormalities such as autoimmune diseases. Have been.

[0006] Therefore, determining whether or not an autoantibody is present in a subject and quantifying the autoantibody in the subject are one of means for diagnosing whether or not the subject is in a disease state. And eventually to elucidate the pathogenesis of the disease. Therefore, detecting autoantibodies in a subject and quantifying the amount of the autoantibodies in the subject are considered to contribute to diagnosis and research of various disease states. From these facts, it is expected that the detection and quantification of autoantibodies against FKBP12 will contribute to the diagnosis of various diseases and the elucidation of the mechanism of the development of FKBP12.

However, there has been no report on the existence of an autoantibody against FKBP12 (hereinafter, referred to as "anti-FKBP12 autoantibody"), and accordingly, a method for detecting and quantifying an anti-FKBP12 autoantibody has not been developed. .

[0008]

DISCLOSURE OF THE INVENTION The present invention has been made to solve the above problems, and it is an object of the present invention to easily detect anti-FKBP12 autoantibodies present in human plasma and the like. An object of the present invention is to provide a method that contributes to the quantification of an anti-FKBP12 autoantibody.

[0009]

In order to achieve the above object, a method for detecting an anti-FKBP12 autoantibody according to the present invention comprises the following steps: (A) adding FKBP12 to a protein solid phase carrier; (B) adding a blocking solution to the protein solid phase carrier to block a region of the protein solid phase carrier to which FKBP12 is not bound; (C) anti-FKBP12 binding to the protein solid phase carrier; A test solution expected to contain an FKBP12 autoantibody was added to a protein solid phase carrier, and an anti-FKBP12
(D) adding an enzyme-labeled antibody capable of binding to the anti-FKBP12 autoantibody to the protein solid phase carrier,
An autoantibody and an enzyme-labeled antibody are bound to form an anti-FKBP12
Detecting autoantibodies.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION A method for detecting an anti-FKBP12 autoantibody, which includes the above steps (A) to (D), comprises an enzyme immunoassay (enzyme-li) using an enzyme-labeled secondary antibody.
nked immunosorbent assay, hereinafter referred to as "ELISA". ) Detection method. Hereinafter, each of the steps will be described. In the present invention, FKBP1
No. 2 utilizes the antigen-antibody reaction used as an antigen and the anti-FKBP12 autoantibody used as an antibody.

(Step A) In the present invention, first, FKBP12 is added to a protein solid phase carrier,
KBP12 is bound to the protein solid phase carrier.

The protein solid phase carrier is not particularly limited as long as it can adsorb and carry the protein. For example, polypropylene or polystyrene test tubes, beads, nitrocellulose membrane, polyvinyl difluoride (PVDF) membrane And a microtiter plate. From the viewpoint of efficiently binding proteins to a protein solid phase carrier, simplifying the steps, and reducing the burden of operation in each step, a microtiter plate is preferable, and a microtiter plate chemically treated for ELISA is preferably used. More preferred.

[0013] In order to bind more to the protein solid phase carrier, FKBP12 is preferably added in the form of an aqueous solution, and more preferably as a buffer from the viewpoint of pH adjustment and the like. As a buffer to be used, PBS is preferable.

After FKBP12 is bound to the protein solid phase carrier, FKBP12 that has not bound to the protein solid phase carrier is
In order to remove KBP12, it is preferable to wash the protein solid phase carrier. As the washing solution, an aqueous solution containing a surfactant (more preferably, a buffer solution) is preferable.
In the following steps (B) to (D), it is similarly preferable to appropriately wash the protein solid phase carrier, and those skilled in the art in view of the present specification appropriately consider the necessity of such washing. It is possible to recognize and appropriately select the cleaning solution and the number of times of cleaning in each cleaning.

As a control, the same aqueous solution (preferably, a buffer solution) as described above is prepared except that FKBP12 is not contained. Further, when the anti-FKBP12 autoantibody is quantified using the detection method according to the present invention,
A standard curve for each immunoplate was obtained and the anti-FKBP12
To calculate the concentration of the autoantibody, an aqueous solution (preferably a buffer) containing various concentrations of immunoglobulin (hereinafter, referred to as “Ig”) is prepared.

The buffer containing FKBP12, the buffer containing various concentrations of Ig, and the buffer containing no protein are added to a protein solid phase carrier.
A protein solid phase carrier to which FKBP12 or various concentrations of Ig are bound and a carrier to which no protein is bound are prepared.

(Step B) Next, a blocking solution is added to the protein solid phase carrier to block a region of the protein solid phase carrier to which FKBP12 or Ig is not bound (that is, non-specific). An adsorption prevention operation is performed) to prevent Igs other than the anti-FKBP12 autoantibody contained in the subject from binding to the protein solid phase carrier.

[0018] The blocking solution is a solution capable of coating a portion of the protein solid phase carrier other than the region to which FKBP12 is bound, and preventing excess protein from binding to the protein solid phase carrier at such a site. Point to. Examples of such a blocking solution include a solution obtained by diluting skim milk with a PBS solution, a solution obtained by diluting a protein that does not interact with a labeling substance of another secondary antibody, a solution containing a surfactant, and the like. Is preferably diluted with a PBS solution, and a surfactant is further added to make the skim milk PB.
Solutions diluted with S solution are preferred. Such blocking solutions may be used alone or in combination of two or more. In addition, as a surfactant, Tween (trade name) or the like can be used.

In this way, FKBP12 was bound by a protein which did not participate in the antigen-antibody reaction in the next step (C) and did not interact with the enzyme-labeled antibody used in the subsequent step (D). Since the portion other than the region is coated, the anti-FK is applied after the following step (C).
Even when a solution containing Ig other than the BP12 autoantibody is added to the protein solid phase carrier, the Ig does not bind to the protein solid phase carrier, so that the anti-FKBP12
There is no adverse effect on detection, quantification, etc., and the accuracy is improved.

(Regarding step C) Then, anti-FKBP1
(2) A test solution expected to contain the autoantibody is added to the protein solid phase carrier described above to bind the anti-FKBP12 autoantibody to FKBP12.

Test liquids expected to contain anti-FKBP12 autoantibodies include plasma, serum, synovial fluid, cerebrospinal fluid, urine, tissue extract, and cells derived from a subject (eg, mammals such as humans). Culture supernatant). When plasma is used, it is preferable to dilute it with an aqueous solution (preferably a buffer) in order to efficiently perform the antigen-antibody reaction described below. Note that such a diluted solution containing plasma may be referred to as a “plasma sample”.

As described above, when the test liquid as described above is added to the blocked protein solid phase carrier,
The anti-FKBP12 autoantibody in the test solution causes an antigen-antibody reaction with FKBP12 bound to the protein solid phase carrier,
The anti-FKBP12 autoantibody and FKBP12 specifically bind. Components (eg, proteins) other than the anti-FKBP12 autoantibody in the test solution do not bind to the protein solid phase carrier due to the blocking performed in the above step (B). FKB
Specific binding between the P12 autoantibody and FKBP12 is selectively performed.

Even if the blocking in the step (B) is performed, it is impossible to completely inhibit the Ig contained in the test solution from non-specifically binding to the protein solid phase carrier. Therefore, the test solution is also added to the carrier to which FKBP12 produced in the step (A) is not bound, and the non-specifically bound Ig is corrected as described in detail in the step (D). When the anti-FKBP12 autoantibody is quantified, a diluent used for preparing a plasma sample from plasma is added to a carrier to which Ig is bound.

(Step D) Subsequently, anti-FKBP1
(2) adding an enzyme-labeled antibody capable of binding to the autoantibody to the protein solid phase carrier to cause a secondary antibody reaction,
The P12 autoantibody is combined with the enzyme-labeled antibody to form an anti-FKB
Detect P12 autoantibodies.

[0025] The enzyme-labeled antibody used in this step may be any antibody that binds to the anti-FKBP12 autoantibody and has a label that can be detected by an appropriate means. For example, alkaline phosphatase (ALP) -labeled anti-human Ig
G antibody, alkaline phosphatase (ALP) -labeled anti-human IgM antibody, peroxidase-labeled anti-human IgM antibody,
And peroxidase-labeled anti-human IgG antibodies. When an ALP-labeled anti-human Ig antibody is used, a solution containing an enzyme substrate such as a fluorescent reactant is added,
Cause an enzymatic reaction. Such enzyme substrates include:
4-Methylumbelliferyl phosphate
feryl phosphate).

That is, when an enzyme-labeled antibody bound to an anti-FKBP12 autoantibody reacts with a fluorescent reactant, fluorescence is generated. By detecting this using a fluorescence intensity meter or the like, the anti-FKBP12 autoantibody is detected. You. On the other hand, when the anti-FKBP12 autoantibody is not present in the test solution, the anti-FKBP12 autoantibody does not bind to the enzyme-labeled antibody, and the enzyme-labeled antibody is removed from the solid phase carrier by washing or the like. Fluorescence does not occur and is not detected by a fluorescence intensity meter or the like. When a fluorescence intensity meter is used, the measured fluorescence is detected as a predetermined fluorescence intensity.

As means for detecting the anti-FKBP12 autoantibody reacted with the enzyme-labeled antibody as described above, not only the indirect means by detecting fluorescence as described above, but also other appropriate means ( For example, p-
Means for measuring absorbance at a specific wavelength using nitrophenylphosphoric acid, o-phenylene diamine, etc.) can also be used.

As a control, a carrier to which the protein prepared in step (A) was not bound was used, and the non-specifically bound I
g can be corrected. That is, by subtracting the fluorescence intensity of non-specific Ig bound to the carrier to which no protein is bound from the fluorescence intensity obtained using the fluorescence intensity meter as described above, the specificity of FKBP12 immobilized on solid phase is reduced. It is possible to detect specifically bound Ig, that is, an anti-FKBP12 autoantibody.

When the anti-FKBP12 autoantibody is quantified, the fluorescence intensity obtained by reacting a diluent instead of a plasma sample with a protein solid phase carrier to which various concentrations of Ig are bound is determined from each fluorescence intensity. Generate an Ig standard curve for each immunoplate. And anti-FKB present in the test solution
I emitting a fluorescence intensity equal to that of the P12 autoantibody
The concentration of g is calculated from this standard curve and defined as the concentration of anti-FKBP12 autoantibody (unit / ml). This allows comparison, ie quantification, of the results for different immunoplates.

Thus, by detecting anti-FKBP12 autoantibodies that may be present in a subject mainly suffering from an autoimmune disease, a subject before and after transplantation of an organ, or how much anti-FKBP12 autoantibodies are present By measuring the presence or absence, it becomes possible to diagnose various pathological conditions and elucidate the mechanism of their occurrence, to diagnose whether or not the subject is suitable for organ transplantation before and after transplanting the organ to the subject, etc. . Such a diagnosis is described in detail in the following examples.

[0031]

The present invention will be described in more detail with reference to the following examples, which are used for illustrative purposes only and should not be used for limiting purposes.

Example 1 Plasma was obtained from a total of 126 unspecified patients suffering from various autoimmune diseases, shown in Table 1 below, and 33 healthy subjects as controls:

[0033]

[Table 1]

Prior to use, all plasma was -20%.
C. In addition, two wells were used for all the measurement items (duplicate), and the average value was used as the measurement value of the measurement item.

Each plasma obtained from the above 159 persons was
A plasma sample was prepared by diluting 40-fold with an aqueous solution containing% Block Ace / 0.1% Tween 20, and then using this plasma sample, anti-FKBP12 autoantibodies were modified with a few modifications as follows. Indirect ELIS added
It was quantified by Method A.

(Regarding process A)
B, two microtiter plates (trade name "Nunc-Imm
unoplate Maxisorp F96 "(manufactured by Inter Med, hereinafter simply referred to as" plate ") was used to measure IgG and IgM anti-FKBP12 autoantibodies. Plate A
(For IgG) and plate B (for IgM)
The following contents were added to the three portions and incubated overnight at 4 ° C., and the wells were coated with the respective contents: Plate A (Al) human recombinant FKBP12 (Sigma Chem)
ical) (concentration 5 μg / ml, 50
(A2) PBS (50 μl); (A3) dilution series of human IgG in PBS (1000
ng / ml, 500 ng / ml, 250 ng / ml, 1
25 ng / ml, 62.5 ng / ml, 31.25 ng
/ Ml, 15.625 ng / ml, and 7.8125
(8 series of ng / ml, 50 μl each) Plate B (B1) Same as (Al); (B2) Same as (A2); (B3) Dilution series of human IgM in PBS solution (50 μl each as above). The wells were then washed three times, 0.05
% Tween 20 in PBS (Tween PB
Washing was performed three times in S) to remove unreacted components that did not coat the wells.

(Regarding Step B) 250 μl of an aqueous solution of 4% block ace powder (manufactured by Dainippon Pharmaceutical Co., Ltd.) was added to each of the wells (A1) to (B3), incubated at room temperature for 2 hours, and subjected to human genetic modification. FKBP1
Areas of the wells not coated with 2 or human Ig were blocked with the above powder. 1 with Tween PBS
After washing twice to remove unreacted block ace powder, 0.4% EDTA · 2Na, 4% block ace,
And 50 μl of an aqueous solution containing 0.1% Tween 20 was added to each well.

(Regarding Step C) The plasma samples (50 μl) obtained from the above 159 persons were added to the wells (A1) and (A2), and (B1) and (B2).
And incubate for 2 hours at room temperature with shaking to generate an antigen-antibody reaction of anti-FKBP12 autoantibodies and FKBP12 in wells (A1) and (B1) that may be contained in the plasma sample. In the wells of (A2) and (B2), non-specific binding of Ig to the plate occurred. Then 7 times, Tw
After washing with eenPBS, unreacted anti-FKBP12 autoantibody was removed from the wells. (A3) and (B)
In the well 3), 4% block ace / 0.1% Tw
An aqueous solution (50 μl) containing een20 was added.

(Regarding Step D) In each well of (A1), (A2) and (A3), 1% block ace / 0.
100 μl of an alkaline phosphatase-labeled anti-human IgG antibody F (ab) ₂ fraction diluted 5000-fold with an aqueous solution containing 1% Tween 20 (Kirkegaard & Perry Labo
ratories, hereinafter simply referred to as “labeled antibody”) and incubated at room temperature for 2 hours to bind the anti-FKBP12 autoantibody and the labeled antibody. The wells were washed seven times and unreacted labeled antibody was removed from the wells. Similarly, in each of the wells (B1), (B2) and (B3), 100 μl of alkaline phosphatase-labeled anti-human IgM diluted 5000-fold with an aqueous solution containing 1% block ace / 0.1% Tween 20 was added. Antibody F (a
b ') _Two fractions (Kirkegaard & Perry Laboratories) were added, incubated at room temperature for 2 hours, and the wells were
Washed twice.

10% diethanolamine and 0.5 m
Containing MMgCl ₂ 0.5mM4- methylumbelliferyl Philippines Le phosphate solution (Sigma Co., pH 10,
(100 μl each) was added to each of the wells (A1) to (B3) and incubated for 30 minutes to allow the labeled antibody to bind to the enzyme substrate.

Next, the fluorescence intensity of each well was measured using a fluorescence intensity meter NJ-5000 (manufactured by Inter Med) at an excitation wavelength of 3 nm.
The measurement was performed under the conditions of 60 nm and emission wavelength of 460 nm. At this time, a well in which the operations from the steps (A) to (D) are not performed at all is set as a blank, and the value of the well is adjusted to zero so that the sample having the maximum value in the whole well is obtained. The sensitivity was adjusted so that the fluorescence intensity was in the range of 300 to 400.

Further, the value obtained by subtracting the fluorescence intensity in the well (A2) from the fluorescence intensity in the well (A1) was defined as the value of IgG specifically bound to FKBP12. Similarly, from (B1) and (B2), FKBP12
The value of IgM specifically bound to was determined. Using the fluorescence intensity in the wells of (A3) and (B3), IgG
Alternatively, a standard curve of IgM was obtained, the concentration of IgG or IgM specifically bound to FKBP12 was calculated, and this value was used as the anti-FKBP12 antibody titer in each sample. Ig
For both G and IgM, the antibody titer of the average value of normal persons + standard deviation x 2 or more (16.9 units / ml or more for IgG, 17.5 units / ml or more for IgM) is positive, and the value less than this is negative. And The results are shown in Table 2 below.

[0043]

[Table 2]

From the above Table 2, it is understood that the proportion of the presence of anti-FKBP12 autoantibodies is higher in patients suffering from autoimmune diseases such as systemic erythematosus than in healthy persons. Therefore, the anti-FKBP12 of the present invention
The method for detecting autoantibodies is useful for diagnosis of various diseases (particularly autoimmune diseases).

(Example 2) Plasma was used for living donor liver transplantation
The experiment was performed in substantially the same manner as described above, except that it was obtained from the name. The results are shown in Table 3 below. In addition, living donor liver transplantation patient 4
Of the 7 patients, 22 had rejection after transplantation of the liver (R (+)), and 25 had no rejection (R (-)).

[0046]

[Table 3]

From Table 3 above, it can be seen that anti-FKBP12
It is understood that subjects positive for autoantibodies have a high probability of exhibiting rejection, and subjects positive for anti-FKBP12 autoantibodies after transplantation have a high mortality. Therefore,
By the method according to the present invention before and after transplantation, the risk of a patient can be predicted in advance by detecting the presence of an anti-FKBP12 autoantibody in a subject. For high-risk patients, more careful monitoring of the clinical course and increasing or changing the type of immunosuppressant can prevent rejection and reduce mortality from complications.

[0048]

Industrial Applicability According to the present invention, anti-FKBP12 autoantibodies which contribute to diagnosis of various disease states and elucidation of the mechanism of their occurrence, prediction of the risk of the subject before and after transplanting an organ to the subject, etc. Are provided.

Claims (1)

(57) [Claims] 1. A method for detecting an anti-FKBP12 autoantibody comprising the following steps: (A) a step of adding FKBP12 to a protein solid phase carrier and binding the FKBP12 to the protein solid phase carrier; (B) blocking A solution is added to the protein solid phase carrier, and the FKBP12
(C) adding a test solution expected to contain an anti-FKBP12 autoantibody to the protein solid phase carrier,
A step of binding a P12 autoantibody to FKBP12; (D) adding an enzyme-labeled antibody capable of binding to the anti-FKBP12 autoantibody to the protein solid phase carrier;
A P12 autoantibody is bound to the enzyme-labeled antibody to form an anti-FK
Detecting the BP12 autoantibody.