JPS5856694A - Reagent for assay of antinuclear antibody - Google Patents

Abstract

PURPOSE:To obtain a reagent for the assay of the titled substance, by combining an insolubilized antigen obtained by insolubilizing an antigen capable of inducing the production of an antinuclear antibody, with a labeled reagent obtained by bonding an enzyme with the second antibody or protein A which is unreactive with the insolubilized antigen and reactive specifically with the antinuclear antibody. CONSTITUTION:In the enzyme immunoassay of an antinuclear antibody such as anti-poly-ADP-ribose antibody, anti-dsDNA antibody, etc., an insolubilized antigen obtained by bonding a plastic carrier such as polystyrene with an antigen such as poly-ADP-ribose, DNA, etc. capable of inducing the production of the antinuclear antibody, is used as a reagent to be reacted immunologically with the antinuclear antibody to be determined. The reagent is combined with another reagent obtained by the enzymatic labeling of the second antibody or protein A which does not cause the specific reaction with said antigen but cause specific reaction with the object to be determined.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a measuring reagent used in enzyme assay for antinuclear antibodies.

Anti-nuclear antibodies, especially anti-double-stranded DNA antibodies, are predicted to be closely related to the onset of systemic lupus erythematosus (hereinafter referred to as SLE), which is a typical autoimmune disease. Furthermore, in addition to anti-double-stranded DNA antibodies, the existence of anti-PoIJ ADP-IJ Bose antibodies was revealed, and it was demonstrated that they are frequently detected in SLE patients. Bo IJ ADP
-! J-bose regulates DNA synthesis, cell differentiation and DNA
It is known that it is closely related to the repair of A. PoIJ ADP - Natural antibodies against IJ Bose are S
The high frequency of appearance in LH suggests that anti-polyADP-ribose antibodies are closely related to the pathology of SLE and its modification. Therefore, methods for measuring these antinuclear antibodies have been developed.

Radioimmunoassay (hereinafter referred to as RIA) is often used as a method for measuring antinuclear antibodies due to its sensitivity and quantitative nature. However, measurements by RIA cannot be carried out outside of facilities that are legally permitted to handle radioactive materials, which has hindered its widespread use.

In place of RIA, which has such restrictions on use, enzyme immunoassays (hereinafter referred to as EIA), which use enzymes instead of radioisotopes as labeling agents, have been developed and are becoming popular in various fields. However, the measurement of antinuclear antibodies, especially the measurement of anti-IJ ADP-IJ Bose, is lacking in examples, and there is a desire to develop an analysis method using EIA.

RIA and EIA are based on the same principle except for using different labeling agents, and their methods can be roughly divided into competition method and sandwich method, and the latter method is suitable for analyzing antinuclear antibodies. However, in these methods, a solid phase method using a solid phase reagent in which an antigen or antibody is bound to a solid support as a reagent is used to facilitate the separation of the bound products produced by the antigen-antibody reaction from the reaction system. It is superior in terms of simplicity.

From this perspective, the present inventors conducted research on the measurement of antinuclear antibodies by the Sand-Deutsch method using a solid-phase probe, and arrived at the present invention.

The present invention is a reagent for measuring antinuclear antibodies by EIA, in which an antigen that induces the production of the antinuclear antibody to be measured is bonded to a plastic carrier, and an insolubilized antigen is bonded to the insoluble antigen, which does not react specifically with the antigen and does not react with the antigen to be measured. The present invention relates to a reagent for measuring an anti-nuclear antibody, characterized in that it is composed of a second antibody that specifically reacts with a certain anti-nuclear antibody, or a labeling reagent in which an enzyme is bound to protein A.

If you explain it in detail in T, it will be anti-Bo! j ADP −
! In the measurement of antinuclear antibodies such as j-bose antibody and anti-double-stranded DNA antibody by EIA, the antinuclear antibody that is the object of measurement is used as a partner reagent to cause the antinuclear antibody production. - The reagent is an insolubilized antigen made by bonding an antigen such as ribose or DNA to a plastic carrier such as polystyrene, and the antigen used in the usual sandwich method is replaced with a labeled antigen labeled with an enzyme. is a reagent for measuring an antinuclear antibody that is used in combination with a second antibody that does not react specifically with the target to be measured or a reagent labeled with protein A with an enzyme.

Since antigens such as polyADP-lipose are expensive, their effective use is preferred. Although plastic has the ability to bind antigens as a solid phase carrier without the use of a specific binding agent, it is desirable to have a more efficient binding method, and as a result of extensive research, the present inventors discovered that plastic surface After pre-treating the poIJ L-IJ, the surface of the plastic surface was contacted with an antigen solution and evaporated to dryness. The insolubilized antigen produced in this way produces good measurement results with less non-specific reactions that interfere with measurements.To prevent non-specific reactions, post-treatment is usually performed using bovine serum albumin, but the insolubilized antigen of the present invention compared to the normally used concentration of 1-2%.
Non-specific reactions can be prevented by treatment with bovine serum albumin at an extremely low concentration of about 0.005 to 0.1%.

As the solid phase carrier used for the insolubilized antigen of the present invention, various plastics such as polyvinyl, polyamide, polyolefin, polyether, polyester, polycarbonate, and polyurethane can be used.

The carrier may be in any shape such as beads, rods, and tubes. Recently, microtightness plates are often used for the purpose of miniaturizing measurements, and they are also suitable for the present invention. As shown in Example 1, a microtitration plate with a capacity of 300 μt was used, and only 100 nf (
50μt of 2μf/ml solution)
-Measurement is possible using IJ Bose.

In the normal Sand Inch method, a labeling reagent is used which is a labeling agent bound to the antigen used for the insolubilized antigen, but in the present invention, instead of an antigen such as BoIJ ADP-ribose, the antinuclear protein to be measured is used as a labeling reagent. A labeling reagent in which an enzyme is bound to a substance that reacts specifically with the antibody but not with the antigen is used. As such a substance, a second antibody such as an anti-immunoglobulin antibody, protein A, etc. can be used.

The labeling reagent of the present invention does not use an expensive antigen, so it is economical, and has the advantage that it has a large amount of bound enzyme, which is a labeling agent, in the reagent, exhibiting extremely high activity, and improving sensitivity.

The enzyme may be selected from those commonly used in EIA, but alkaline phosphatase obtained from calf small intestine has high activity and reacts quickly with the enzyme substrate, making it possible to shorten the enzyme reaction time to 10 minutes. Very suitable. Since the labeling reagent of the present invention uses a second antibody or protein A, it may also bind to antibodies other than the measurement target present in the sample. Therefore, non-specific reactions tend to occur with the insolubilized antigen, and if an unintended antibody binds, the measurement will be interfered with. However, as mentioned above, the insolubilized antigen of the present invention has less non-specific reactions, and by using them in combination, good measurements can be made.

SL by measuring antinuclear antibodies using the measuring reagent of the present invention.
In addition to being capable of quickly diagnosing H., for example, in the process of producing antibodies by culturing lymphoid cells, it is extremely useful because antibodies can be easily measured without concentrating the culture fluid. The present invention will be explained in more detail with reference to Examples and Test Examples.
Not limited to NA antibodies, but anti-histone J'+'+
This also includes other types of anti-cough antibodies such as anti-cough antibodies, anti-SmK antibodies, and anti-RNP antibodies.

Example 1 Insolubilized antigen) (1) Pretreatment of solid phase carrier A 96-sized plastic microtitration plate (Dyna
Polyvinylplate U type (manufactured by Tech Co., Ltd.) was used as a solid phase carrier. Strong positive charge Po IJ L -IJ
100 μt of a solution prepared by dissolving John Molecular Weight: 4×10', Sigma) in distilled water to a concentration of 50 μf/ml was added to each well, and left at room temperature for 1 hour, followed by Tris-salt buffer (2
5mM) Squirrel, 140mM NaCl.

The cells were washed four times using TBS (pH 7.4 or below).

(11) Binding of polyADP-lipose by the method of Kanai, Y-et al., J-Bioche et al.
m-.

88.917-920.1980), the concentration of which was adjusted to 2 μf/mt using TBS, was added in an amount of 50 μt to each hole of the microtitration plate obtained in (1). The mixture was evaporated to dryness at °C overnight and washed four times with TBS.

(III) In order to block the surface of the plate obtained in post-treatment (11) that is not filled with antigen, 300 μt of TBS supplemented with 0.01% Ω bovine serum albumin was added to each hole and left for 1 hour. Back T
The plate was washed three times with BS to obtain insolubilized poIJ ADP-IJ Bose (insolubilized antigen). This product is stable for one month at 4°C under humidification.

Example 2 Insolubilized double-stranded DNA (insolubilized antigen) Commercially available calf thymus DNA was purified by the Mar-mur method.
J-Mo1. Biol・, 3 + 208.196
After protein removal purification according to 1), the protein was purified using the Stoller-Papalian method (5tollar, B, D, and Pa
palian, M., J-CIin, In-ve.
st., 66.210-219.1980)
Treat with l-nuclease to completely cleave the single-stranded DNA. This DNA was then extracted using the method of Kanai et al.
, Y-et al., J.Biochem,, 8
8.917-92 (L1980) and applied to a hydroxyapatite column in 0.2 M phosphate buffer (p
Only the double-stranded DNA eluted with H6,8) was collected.

This double-stranded DNA was further treated with KSI-nuclease,
It was confirmed by ultraviolet absorption at 260 nm that 81-nuclease was no longer cleavable at this stage.

Thereafter, the double-stranded DNA eluted using the above-mentioned method was collected from the hydroxyapatite column again. Following the same method as
The double-stranded DNA was bound to a micro titre rayon plate to obtain an infusible double-stranded DNA (insolubilized antigen).

Example 3 Alkaline phosphatase/Protein A conjugate (labeling reagent) 1.9 rl of alkaline phosphatase derived from calf small intestine (Sigma product, Type 41) and 0.75 mf of protein A (Pharmacia product) were added to a saline phosphate buffer (
Phosphate IQmM, salt 140mM, pH 7.4, below P
After dialyzing a solution containing 1mzK (abbreviated as BS) against PBS, glutaraldehyde (manufactured by Sigma; concentration 25%) was added to a final concentration of 02%, and the mixture was stirred at room temperature for 2 hours to perform binding. Gel sifting device (Uetrog)
Separate the combined product of alkaline phosphatase and protein A using el AcA 34 ml [1, IX 54 cm) and convert it into a protein using labeling reagent 10 at 220 trf/mt.
m1 was obtained.

The enzyme specific activity of this conjugate is 200U per mf of protein.
(Substrate 25mM paranitrophenyl phosphate, p
(under measurement conditions of H9, 5, 37°C), and the amount of the enzyme alone is 900 to 1100 U, making it an extremely highly active labeling reagent. This stuff has bovine serum albumin 0
It was stable for more than 4 months at 4° C. in the presence of 5% and 0.02% sodium nitride.

Example 4 Alkaline phosphatase/anti-mouse immunoglobulin antibody conjugate (labeling reagent) Anti-mouse immunoglobulin (IfG+IfM) antibody (Tufts University, Sto.
A conjugate of alkaline forsterase and anti-mouse immunoglobulin antibody (labeled reagent) was obtained in the same manner as in Example 3, using instead of protein A. (Yield tomt, protein “C140nf
i'ml).

Reference Example 1 Control Grade Example 1 (11) POIJADP-I
PoIJADP was prepared in the same manner as in Example 1 except that only TBS was used instead of the TBS solution of JBose
- A microtitreion plate without binding IJ Bose (antigen) was obtained and used as a control plate.

Test Example 1 Measurement of anti-PoIJ ADP-IJ Bose antibody in serum of SLE, addicts and normal subjects
ADP-IJ Bose antibody was measured.

(1) First reaction (reaction between insolubilized antigen and antibody) Example 1
Inactivated serum 100 diluted 20 times with TBS containing 001% bovine serum albumin was added to each well of the insolubilized poIJ ADP-ribose (insolubilized antigen) prepared by the method of
μL was added and incubated for 60 minutes at room temperature with slight shaking.

After removing the reaction solution, the plate was washed four times with TBS.

(11) Second reaction (reaction with labeling reagent) The alkaline foster protein A conjugate (labeling reagent) obtained by the method of Example 3 was diluted 100 times with TBS for the first reaction. Add 100 pl to each well of the finished plate,
Incubated for 60 minutes at room temperature. Remove the reaction solution,
Plates were washed 4 times with TBS.

(iii) Third reaction (enzyme substrate reaction) and measurement 2,
5mM halanitrophenyl phosphate to 2mM
MG Ct! The substrate was a solution dissolved in 10mt of 50mM carbonate buffer (pH 9,5),
Add 100 pl to each hole of the grate where the second reaction has finished,
After reacting at 37°C for 10 minutes, 100μt of this solution was taken out and 0.00μt was taken out. IN aqueous solution of caustic soda 400 μt
In addition, the enzymatic reaction was stopped.

410 nm of 500 μt of the reaction solution in each well obtained.
The ultraviolet absorption at K was measured.

In order to correct enzyme activity based on non-specific reaction, reference example 1
The first, second and third reactions were carried out in the same manner as above except that the control plate prepared by the above method was used in place of the insolubilized antigen, and the ultraviolet absorption was measured.

For each serum, the ultraviolet absorption when using the insolubilized antigen and the ultraviolet absorption when using the control grade were determined to be the true antibody activity, which is shown in Table 1.

A t-test showed that there was a difference of 0 between the measured values of SLE patients and normal people.
．． A significant difference of 001<P<0.01 was observed.

Table 2 shows the UV absorption when using the control plate. S
It is clear from the comparison with the measured values of normal subjects in Table 1 that there is no difference between LE patients and normal subjects, and non-specific reactions are extremely rare.

Test Example 2 Measurement of anti-double-stranded DNA antibodies Anti-double-stranded DNA antibodies in the serum of SLE patients and normal individuals were measured.

Insolubilized double-stranded DNA produced by the method of Example 2 in place of the insolubilized POIjADP-ribose of Test Example 1
The anti-double-stranded DNA antibody in the serum was measured using the same method as in Test Example 1 except that the anti-double-stranded DNA antibody was used as the insolubilized antigen.

The measurement results are shown in Table 3.

``A t-test revealed a significant difference of 0.001<P<0.01 between the measured values of SLE patients and normal subjects.

Test Example 3 Measurement of anti-poly ADP-ribose antibody produced by hybridoma Mouse myeloma cells 5P210-AP-14 and poly ADP
-C3H/He mouse tile cells immunized with ribose were prepared using the method of Andrzejervsky et al.
ki.

C, Jr. etat, J-Immunol-, 12
Cell fusion was carried out by the following method (4, 1499-1502, 1980). Regarding the obtained 11 hybridoma strains, the culture supernatant was not concentrated and the labeling reagent of Example 4 was used as the anti-vo IJADP-ribose antibody as a labeling reagent, but otherwise the method was the same as in Test Example 1. I measured it.

The measurement results are shown in Table 4. As shown in Table 4, polyJADP-ribose produced by hybridomas could be directly measured without concentrating the culture supernatant.

Table 1 Antibodies in serum! JADP-ribose antibodies, Table 2
Ultraviolet absorbance (A 41015 μt) on control plate Table 3
Anti-double-stranded DNA antibodies in serum, Table 4 Anti-poly ADP-lipose antibody B produced by hybridoma 1001 0.045C901
0,840D 601 0.250D
901 0.885D 902
0.050E 902
0.155F 503 0.86
0F 702 0.130F
801 0.115F 100
3 0.055G 1001
0.12, Patent applicant Mitsui Pharmaceutical Industries, Ltd. Procedural amendment (method) % formula % 1. Indication of the case 1982 Patent Application No. 150668 2 Name of the invention Reagent for measuring antinuclear antibodies 3 Case made by the person making the amendment Relationship with Patent application ← Person 4, Date of amendment order January 5, 1980 5, Notification of attachment of specification subject to amendment

Claims (1)

[Claims] A reagent for measuring anti-M antibodies by enzyme immunoassay includes: an insolubilized antigen in which an antigen to be measured that induces the production of a certain antinuclear antibody is bound to a plastic carrier;
An anti-nuclear antibody characterized in that the antigen does not have a specific reaction pot, but instead is combined with a second antibody that reacts specifically with the anti-nuclear antibody to be measured, or with a labeling reagent in which protein A is bound to an enzyme. Measurement reagent