JP3227690B2 - Anti-neutrophil cytoplasmic antibody measurement method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for measuring anti-neutrophil cytoplasmic antibodies.

[0002]

2. Description of the Related Art Anti-neutrophil cytoplasmic antibodies (hereinafter, referred to as ANCA) are examined by a fluorescent antibody indirect method (hereinafter, referred to as IIF method), and cytoplasm is stained based on the staining pattern (Cytoplasmic) -ANCA (hereinafter, referred to as ANCA). , C-ANCA), the periphery of the nucleus is stained (Perinuclear) -ANCA
(Hereinafter, referred to as P-ANCA). In recent years, CA has been diagnosed with Wegener's granulomatosis, an intractable renal disease.
Since the measurement of NCA has become very effective for early diagnosis, the measurement of C-ANCA has attracted attention. However, the measurement of C-ANCA by the IIF method required considerable experience and skill in the interpretation of the technique and the fluorescence pattern and was not suitable for routine clinical examination.

[0003] Then, C-ANC was converted to α-granules of human neutrophils.
Since the corresponding antigen of A was identified, a reagent by enzyme immunoassay (EIA method) utilizing an immunological action was commercially available, and C-ANCA was measured simply and in a short time. In this method, a surface of a synthetic resin plate or the like is coated with a C-ANCA antigen purified from human neutrophils, a specimen is put in a dried state, a color former is added, and the absorbance is measured. .

[0004]

However, CA
Since the NCA antigen is purified from α-granules of human neutrophils, it is possible that other substances may be mixed. The α-granules of human neutrophils include, in addition to the C-ANCA antigen, myeloperoxidase (hereinafter referred to as MPO), elastase, lactoferrin, cathepsin G and the like.
Coexists with A antigen. The aforementioned coexisting antigen is an anti-myeloperoxidase antibody (hereinafter referred to as MPO-A) present in the sample.
However, there is a drawback that an antigen-antibody reaction occurs with an antibody other than C-ANCA such as NCA, and a substance other than C-ANCA is measured. For example, C-ANCA and MPO
-Since ANCA has different clinical significance, MPO-A
Measuring non-specific reactions by NCA as C-ANCA was confusing and problematic in diagnosis. The present invention is to solve this problem.

[0005]

SUMMARY OF THE INVENTION The present invention provides an anti-neutrophil cytoplasmic antibody characterized by suppressing a non-specific reaction caused by a myeloperoxidase antigen coexisting with an antigen of the anti-neutrophil cytoplasmic antibody in an antigen-antibody reaction. It is a measuring method. Departure
In the literature, the antigen-antibody reaction is defined as the anti-neutrophil cytoplasm in the sample.
Antibody (C-ANCA) and reaction of C-ANCA antigen in reagent
Say hello. In the present invention, the anti-neutrophil cytoplasmic antibody
As a measurement method, a sandwich method using the above antigen-antibody reaction was used.
Such as the switch immunoassay.

The present invention also provides an anti-neutrophil cytoplasmic antibody characterized in that myeloperoxidase antigen coexisting with the antigen of the anti-neutrophil cytoplasmic antibody in the antigen-antibody reaction is specifically suppressed with a buffer solution adjusted to pH. It is a measuring method.

Further, the present invention provides an anti-neutrophil, wherein the myeloperoxidase antigen coexisting with the antigen of the anti-neutrophil cytoplasmic antibody in the antigen-antibody reaction is specifically inhibited by a buffer adjusted to pH 2 to 5. This is a method for measuring sphere cytoplasmic antibodies.

[0008] Buffers include sodium citrate-sodium dihydrogen phosphate buffer, sodium citrate-hydrochloric acid buffer, acetic acid-sodium hydroxide buffer, succinic acid-sodium hydroxide buffer, ββ-dimethylglutaric acid -PH range of sodium hydroxide buffer, glycine-hydrochloric acid buffer, potassium phthalate-hydrochloric acid buffer, citric acid-sodium citrate buffer, acetic acid-sodium acetate buffer, potassium chloride-hydrochloric acid buffer, etc. Anything can be maintained as long as it can be maintained.

Although the effective pH range is 2 to 5, it should be noted that if the pH is too acidic, the activity of the target C-ANCA antigen is suppressed even if it is effective for non-specific reaction by MPO antigen. is there.

[0010] The C-ANCA antigen may be immobilized directly on an insoluble carrier or may be added separately. The insoluble carrier is glass,
Agarose, dextran, cellulose, polyacrylamide, polystyrene, homopolypeptide, latex, polycarbonate, polypropylene, aminoalkyl silica glass, silicon rubber, etc. are used, and the surface is covalently bonded, ion bonded, physically adsorbed, etc. By C-
After attaching the ANCA antigen, it is dried and immobilized.

[0011]

As described above, p-CCA is present on the C-ANCA antigen in the reagent.
H by adding the prepared buffer to C-ANCA antigen
Since non-specific reactions due to coexisting MPO antigens are suppressed , MPO-ANCA in the sample is reduced to C-
Accurate C-ANCA without measuring as ANCA
The value can be measured.

[0012]

[ Example 1] A C-ANCA antigen derived from normal human neutrophils immobilized on a Nunc microplate (96 wells) was prepared. The pH of the sodium citrate-sodium dihydrogen phosphate buffer is adjusted to pH 3 to 6, and the pH of the potassium chloride-hydrochloric acid buffer is adjusted to p.
Adjusted to H1-2. Buffer solution adjusted to each pH is 200
Dispense μl / well into microplates at 25 ° C.
Incubated for 0 minutes. Thereafter, the buffer was removed, and a phosphate buffer containing Tween 20 (pH 7.2) was used.
And washed. Next, C-ANCA-containing human serum or MPO-ANCA-containing human serum diluted 75-fold with a phosphate buffer (pH 7.3) was added to each well as a sample.
After adding 0 μl / well and incubating at 25 ° C. for 60 minutes, a phosphate buffer containing Tween 20 (pH
Washing was performed in 7.2). In addition, phosphate buffer (pH
The alkaline phosphatase-labeled anti-human IgG antibody manufactured by Orion prepared in 7.3) was dispensed at 200 μl / well and incubated at 25 ° C. for 60 minutes. afterwards
The buffer was removed, washed with a phosphate buffer containing Tween 20 (pH 7.2), and then diethanolamine-HCl.
200 μl of Sigma disodium p-nitrophenyl phosphate adjusted with a buffer (pH 9.7)
1 / well was added, and the absorbance difference between 0 minute and 60 minutes was measured. The measurement of the absorbance difference is performed by Corona, type MTP12
The measurement was performed at a wavelength of 405 nm using a microplate reader of No. 0. The measured values OD (Optica) in the table
1 Density) represents the absorbance difference . Table 1 shows the results.

[0013]

[Table 1]

The value of MPO-ANCA-containing human serum in the C-ANCA measuring reagent not adjusted to pH was 0.848.
(Comparative Example 1) , the buffering solution has an inhibitory effect from pH 5 or less . On the other hand, when the buffer is adjusted to a strong acid having a pH of 1, the nonspecific reaction of MPO-ANCA is remarkably suppressed, but the specific reaction of C-ANCA is also suppressed, which does not satisfy the object of the present invention. Than this,
The effective pH range of the buffer is pH 2-5. The buffer in this range without affecting the measurement of C-ANCA containing human serum, MPO-ANCA containing human
It has the effect of lowering only serum measurements.

[ Example 2] A C-ANCA antigen derived from normal human neutrophils immobilized on a microplate (96 wells) was prepared. PH 3 sodium citrate-sodium dihydrogen phosphate buffer
In ~ 6, the pH of the potassium chloride-hydrochloric acid buffer was adjusted to 2. These were dispensed at 200 μl / well into a microplate and incubated at 25 ° C. for 0 hour, 0.5 hour, 1 hour, and 3 hours. Hereinafter, the measurement was performed in the same procedure as in Example 1. Table 2 shows the results when the reaction time is 0 hour.
Table 3 shows the results of 0.5 hour, and Table 4 shows the results of 1 hour.
Table 5 shows the time results.

[0016]

[Table 2]

[0017]

[Table 3]

[0018]

[Table 4]

[0019]

[Table 5]

As is clear from Tables 1 to 5 , the pH was adjusted to 2
The buffer prepared in 5 had a reaction time of 0 hour (immediately after the reaction).
Has an effect of suppressing MPO antigen coexisting with C-ANCA, and has a higher inhibitory effect as the reaction time elapses. Therefore, when a buffer is used to suppress the non-specific reaction due to the MPO antigen, it is necessary to consider not only the pH but also the reaction time depending on the degree of the non-specific reaction due to the MPO antigen.

Comparative Example 1 A conventional C-AN was prepared in the same manner as in Example 1 except that a sodium citrate-sodium dihydrogen phosphate buffer or a potassium chloride-hydrochloric acid buffer prepared as a nonspecific reaction inhibitor was dispensed.
CA measurement reagent, C-ANCA-containing human serum, MPO-
ANCA-containing human serum and healthy human serum were used as samples and measured in the same manner as in Example 1. Table 6 shows the results.
Shown in

[0022]

[Table 6]

From these results, assuming that the average value + 2 × SD (standard deviation) is the normal value from the measured value of the serum of a healthy person, the normal value is (0.009 + 2 × 0.00028 = 0.00956).
There is a clear difference between NCA-containing human serum and MPO-ANCA-containing human serum from normal values. This is CA
The NCA antigen to the wells was immobilized, M in the serum
Since PO-ANCA is reacting, C-ANCA
It is thought that the reaction with the MPO antigen coexisting with the antigen has occurred.
Can be obtained .

[0024]

According to the measurement method of the present invention, the reagent
MPO without inactivating the activity of C-ANCA antigen
Nonspecific reaction of antigen can be suppressed. That is, C-ANCA
Only the MPO antigen coexisting with the antigen is specifically suppressed, and the desired C-ANCA can be accurately measured.

Claims (1)

(57) [Claims] In the antigen-antibody reaction, a myeloperoxidase antigen coexisting with an antigen of an anti-neutrophil cytoplasmic antibody is subjected to pH
A method for measuring an anti-neutrophil cytoplasmic antibody, wherein the antibody is specifically inhibited by a buffer prepared in any one of 2 to 5.