JPH07280806A - Measuring method for antineutrophilic cytoplasmic antibody - Google Patents

Abstract

PURPOSE:To measure a chromatic antineutrophilic cytoplasmic antibody (C- ANCA) accurately by retarding the nonspecific reaction due to myeloperoxidase (MPO) antigen coexisting with the antibody. CONSTITUTION:When the IgG fraction of an animal derived anti-human MPO antiserum is added to a C-ANCA antigen, it has no effect on the measurement of C-ANCA but only the measurement of MPO-ANCA lowers because the IgG fraction of anti-human neutrophilic MPO animal antiserum reacts previously on the MPO antigen to retard reaction between the MPO-ANCA and the MPO antigen. Since the nonspecific reaction of MPO antigen is retarded, the MPO- ANCA is prevented from being mismeasured as C-ANCA due to the MPO antigen coexisting with the C-ANCA antigen resulting in the accurate measurement of C-ANCA.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for measuring anti-neutrophil cytoplasmic antibody.

[0002]

2. Description of the Related Art Anti-neutrophil cytoplasmic antibody (hereinafter referred to as ANCA) is examined by a fluorescent antibody indirect method (hereinafter referred to as IIF method), and the cytoplasm is stained from its staining pattern (Cytoplasmic) -ANCA (hereinafter referred to as ANCA). , C-ANCA) and stain around the nucleus (Perinuclear) -ANCA
(Hereinafter, referred to as P-ANCA). In recent years, CA has been reported in Wegener's granulomatosis, which is a refractory renal disease.
Since the measurement of NCA has become extremely effective for early diagnosis, the measurement of C-ANCA has come to the forefront. However, the measurement of C-ANCA by the IIF method requires a considerable amount of experience and skill in the procedure and interpretation of the fluorescence pattern, and is not suitable for routine clinical examinations.

Then, C-ANC was formed in α granules of human neutrophils.
Since the corresponding antigen of A was identified, a reagent by an enzyme immunoassay method (EIA method) utilizing an immunological action was marketed, and C-ANCA came to be easily and quickly measured. This is a method in which the surface of a synthetic resin plate or the like is coated with C-ANCA antigen purified from human neutrophils, a sample is put in a dried state, a coloring agent is added, and the absorbance is measured. .

[0004]

However, C-A
Since the NCA antigen is purified from α granules of human neutrophils, other substances may possibly be mixed. In the α granules of human neutrophils, there are myeloperoxidase (hereinafter referred to as MPO), elastase, lactoferrin, cathepsin G, etc. in addition to C-ANCA antigen, and these are C-ANC.
It coexists with the A antigen. The aforementioned coexisting antigen is an anti-myeloperoxidase antibody (hereinafter referred to as MPO-A) present in the sample.
There is a drawback that it causes an antigen-antibody reaction with an antibody other than C-ANCA (such as NCA) to measure a substance other than C-ANCA. For example, C-ANCA and MPO
-As ANCA has different clinical significance, MPO-A
Measuring non-specific reactions by NCA as C-ANCA has been a diagnostic confusion and a problem. The present invention is to solve this problem.

[0005]

The present invention provides an anti-neutrophil cytoplasmic antibody characterized by suppressing a non-specific reaction caused by a myeloperoxidase antigen coexisting with the antigen of the anti-neutrophil cytoplasmic antibody in the antigen-antibody reaction. It is a measuring method.

Further, the present invention is characterized in that the myeloperoxidase antigen coexisting with the antigen of the antineutrophil cytoplasmic antibody in the antigen-antibody reaction is specifically suppressed by the anti-human neutrophil myeloperoxidase antiserum derived from a non-human animal. And a method for measuring anti-neutrophil cytoplasmic antibody.

The animal-derived anti-human neutrophil MPO antiserum immunizes non-human immunized animals with MPO antigen, and purifies this antiserum to obtain an IgG fraction. As the immunized animal, any animal other than human can be used, but mouse, rabbit, goat, sheep, pig, horse and the like are preferable. That is, the antiserum (Ig
G) does not cross-react with the secondary antibody, anti-human IgG antibody, and it is desirable to use those derived from the same immunized animal. Although unpurified antiserum itself may be used,
At this time, if there is a substance that may cause a non-specific reaction in the antiserum, it needs to be removed.

This animal-derived anti-human neutrophil MPO antiserum is added before or during the reaction between the specimen and the C-ANCA antigen, and the suppression effect is greater when added before the reaction.

The C-ANCA antigen may be directly immobilized on an insoluble carrier or 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 thereof is covalently bonded, ionicly bonded, physically adsorbed, etc. By C-
After the ANCA antigen is attached, it is dried and solid-phased.

[0010]

As described above, by adding the IgG fraction of the animal-derived anti-human MPO antiserum to the C-ANCA antigen, the non-specific reaction due to the MPO antigen is suppressed in advance.
MPO-with the MPO antigen coexisting with C-ANCA antigen
An accurate C-ANCA value can be measured without measuring ANCA as C-ANCA.

[0011]

Example A C-ANCA antigen derived from normal human neutrophils was immobilized on a Nunc microplate (96 wells). Using a phosphate buffer (pH 7.3), the concentrations of anti-human neutrophil MPO rabbit antiserum IgG fractions (manufactured by Athens Research Co.) were adjusted to the concentrations shown in Table 1. Dispense 200 μl / well of this into a microplate,
Incubated at 60 ° C for 60 minutes. After that, the reaction solution was removed, and a phosphate buffer solution (pH 7.
It was washed in 2). Next, C-ANC diluted 75 times with a phosphate buffer (pH 7.3) as a sample in each well
200 μl / well of A-containing human serum or MPO-ANCA-containing human serum was added, and after incubation at 25 ° C. for 60 minutes, a phosphate buffer containing Tween 20 (p
It was washed with H7.2). Furthermore, phosphate buffer (pH
Orion alkaline phosphatase-labeled anti-human IgG antibody prepared in 7.3) was dispensed at 200 μl / well and incubated at 25 ° C. for 60 minutes. After that, the reaction solution is removed and washed with a phosphate buffer solution (pH 7.2) containing Tween 20, and then diethanolamine-HCL
200 μm of Sigma's disodium p-nitrophenyl phosphate adjusted with a buffer solution (pH 9.7) was used as a color developing agent.
l / well was added to each well, and the difference in absorbance between 0 minutes and 60 minutes was measured. Absorbance is measured by Corona, type MTP120
It measured at the wavelength of 405 nm using the microplate reader of. The results are shown in Table 1. Here, the measured value OD (Optical Density) in the table represents the absorbance.

Anti-human neutrophil MPO rabbit antiserum Ig
The concentration of G fraction is defined by the antibody titer (μg antigen / ml).
The antibody titer is an antibody activity value, and indicates the amount of antigen that reacts in 1 ml of antiserum. The antiserum used this time is
The immunized animal was immunized with MPO antigen purified from human neutrophils. Perform an antigen-antibody reaction with this antiserum and MPO antigen (molecular weight: 150,000) purified from human neutrophils,
The amount of antigen precipitated by the reaction was determined and used as the antibody titer.

[0013]

[Table 1]

From this, the addition of the anti-human neutrophil MPO rabbit antiserum IgG fraction did not affect the measured value of C-ANCA at all, and lowered only the measured value of MPO-ANCA. This is because the anti-human neutrophil MPO rabbit antiserum IgG fraction reacted beforehand with MPO antigen,
This is because the reaction between O-ANCA and MPO antigen is suppressed. Also, the effective concentration of this anti-human neutrophil MPO rabbit antiserum IgG fraction is, as is clear from the above table, the effect at an antibody titer of 0.49 μg / antigen / ml of rabbit antiserum. Shows.

[0015]

Comparative Example 1 C-ANCA-containing human serum was used as a conventional C-ANCA assay reagent except for the dispensing of the IgG fraction of anti-human neutrophil MPO rabbit antiserum added as a non-specific reaction inhibitor in Example. , MPO-ANCA-containing human serum and normal human serum were used as samples and measured in the same manner as in the examples. The results are shown in Table 2.

[0016]

[Table 2]

From this result, when the average value + 2 × SD (standard deviation) from the measurement value of the healthy human serum is defined as a normal value, the normal value is (0.009 + 2 × 0.00028 = 0.00956), so that C−A
The NCA-containing human serum and the MPO-ANCA-containing human serum are clearly different from the normal value. This is CA
MPO-A was added to wells on which NCA antigen was immobilized.
Since NCA is reacting, it can be said that a reaction is occurring with the MPO antigen that coexists with the C-ANCA antigen.

[0018]

Comparative Example 2 Bovine serum albumin (BSA) manufactured by Organon was used as a non-specific reaction inhibitor generally used in place of the IgG fraction of the anti-human neutrophil MPO rabbit antiserum added in the example. It was added to the C-ANCA measurement reagent. Then, the measurement was performed using C-ANCA-containing human serum and MPO-ANCA-containing human serum as specimens in the same manner as in the examples. The results are shown in Table 3.

[0019]

[Table 3]

The experimental results using BSA, which is a general non-specific reaction inhibitor, shows that MPO-ANCA was used regardless of the amount added.
Since the measured values of contained human serum are almost the same, BS
It can be seen that A has no effect of specifically suppressing the MPO antigen.

[0021]

When the measuring method of the present invention is adopted, C-A
A non-specific reaction in the C-ANCA antigen can be suppressed without deactivating the activity of the NCA antigen. That is, C-ANCA
Only the MPO antigen that coexists with the antigen can be specifically suppressed, and the target C-ANCA can be accurately measured.

Claims (2)

[Claims] 1. A method for measuring an anti-neutrophil cytoplasmic antibody, which comprises suppressing a non-specific reaction due to a myeloperoxidase antigen coexisting with an antigen of an anti-neutrophil cytoplasmic antibody in an antigen-antibody reaction. 2. An anti-human neutrophil myeloperoxidase antiserum derived from a non-human animal which specifically inhibits a myeloperoxidase antigen that coexists with an antigen of an anti-neutrophil cytoplasmic antibody in an antigen-antibody reaction. Method for measuring neutrophil cytoplasmic antibody.