JPH0440662B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to enzyme immunoassays for antinuclear antibodies. Antinuclear 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), a typical autoimmune disease. In addition to anti-double-stranded DNA antibodies, the existence of anti-poly ADP-ribose antibodies was also revealed.
It has been demonstrated that it is frequently detected in SLE patients (Y. Kanai et al: Nature, 269 , 175-177,
1977). PolyADP-ribose regulates DNA synthesis,
It is known to be closely related to cell differentiation and DNA repair. The frequent appearance of natural antibodies against polyADP-ribose in SLE 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 performed outside of legally permitted facilities that 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 EIAs), which use enzymes instead of radioisotopes as labeling agents, have been developed and are becoming popular in various fields. However, there are no examples of measuring anti-nuclear antibodies, especially anti-poly ADP-ribose antibodies, and the development of an analysis method using EIA is desired. RIA and EIA are based on the same principle except for the use of different labeling agents, and their methods can be roughly divided into the competition method and the Sand-Deutsch method, and the latter method is suitable for analyzing antinuclear antibodies. In addition, in these methods, the solid phase method uses a solid phase reagent in which the antigen or antibody is bound to a solid support as a reagent in order to easily separate the bound product generated by the antinuclear antibody reaction from the reaction system. It is superior in terms of simplicity. From this point of view, the present inventors studied the measurement of antinuclear antibodies by the Sand-Deutsch method using a solid phase reagent, and arrived at the present invention. That is, the present invention provides a method for measuring antinuclear antibodies in human serum by enzyme immunoassay (EIA), which involves bringing an antigen that induces the production of antinuclear antibodies to be measured into contact with a plastic carrier, and then evaporating to dryness. The antinuclear antibody to be measured is reacted with the insolubilized antigen bound by the method, and then an enzyme is added to a second antibody or protein A that does not specifically react with the antigen but specifically reacts with the antinuclear antibody to be measured. The present invention relates to a method of reacting bound labeled reagents and measuring antinuclear antibodies based on the amount of reacted labeled reagents. More specifically, in the present invention, in the measurement of antinuclear antibodies such as anti-poly ADP-ribose antibodies and anti-double-stranded DNA antibodies by EIA, it is used as a counterpart reagent to immunoreact with the antinuclear antibody to be measured. The reagent is an insolubilized antigen obtained by contacting an antigen such as polyADP-ribose or DNA that induces antinuclear antibody production with a plastic carrier such as polystyrene treated with poly-L-lysine and evaporating to dryness, Furthermore, instead of the antigen employed in the normal Sand-Deutsch method, a labeled antigen labeled with an enzyme is used, and a second antibody or protein A that does not have a specific reaction with the antigen but has a specific reaction with the antinuclear antibody to be measured is used. A reagent labeled with an enzyme is used as a labeling reagent. Note that protein A is a protein with a molecular weight of 42,000 derived from Staphylococcus aureus cells. The effective use of antigens such as polyADP-ribose is preferred due to their high valence. Plastic has the ability to bind antigens without using a specific binding agent as a solid phase carrier, and the present inventor has already published a method for quantifying polyADP-ribose in various animal organs in Biochemistry, Vol. 53. , No. 8, August 25, 1982
Published in Japan, p. 658, when developing the EIA method that can directly quantify polyADP-ribose below ng, 0.1-1 ng of polyADP-ribose was used to create a calibration curve.
ADP-ribose was attached to the microplate via poly-L-lysine. However, for the determination of anti-nuclear antibodies, in particular anti-poly ADP-ribose antibodies according to the invention, a larger amount of poly ADP-ribose, i.e. 5-1000 ng, preferably 20-500 ng, in as sparing an amount as possible, is required. An efficient method for binding to microplates is desired. Therefore, as a result of intensive research, the present inventor pretreated the plastic surface with poly-L-lysine as detailed in Example 1, brought it into contact with an antigen solution, and then evaporated it to dryness, which had never been done before. Using a method of binding antigens to plastic surfaces, it was possible to achieve the goal of stronger antigen binding. The insolubilized antigen produced by this method has few nonspecific reactions that interfere with measurement, and good measurement results can be obtained. In order to prevent non-specific reactions, post-treatment is usually performed using bovine serum albumin, but in the insolubilized antigen of the present invention, the concentration is extremely low, about 0.005 to 0.1%, compared to the normally used concentration of 1 to 2%. Non-specific reactions can be prevented by treatment with bovine serum albumin. As the solid phase carrier used for the insolubilized antigen of the present invention, various plastics such as polyvinyl resins such as polystyrene, polyamides, polyolefins, polyethers, polyesters, polycarbonates, and polyurethanes can be used. The carrier may be in any shape such as beads, rods, or 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 volume of 300 μl/well was used, and a small amount per measurement was used.
Measurement is possible using 100 ng (50 μl of 2 μg/ml solution) of polyADP-ribose. In the normal Sand-Deutsche method, a labeling reagent is used that is a labeling agent bound to the antigen used for the insolubilized antigen, but in the present invention, instead of using an antigen such as polyADP-ribose, a labeling reagent that is specific to the antinuclear antibody to be measured is used. The labeling reagent used is an enzyme bound to a substance that reacts with the antigen but does not show a specific reaction with the antigen. 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 can 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, reducing the enzyme reaction time to 10 minutes. This is extremely 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, if there is a non-specific reaction to the insolubilized antigen and an unintended antibody binds to it, the measurement will be interfered with. However, as mentioned above, the insolubilized antigen of the present invention has little non-specific reaction, and good measurements can be made by using them in combination. The measurement of antinuclear antibodies using the measurement reagent of the present invention not only enables rapid diagnosis of SLE, etc., but also facilitates the production of antibodies by culturing lymphoid cells without concentrating the culture medium. It is also possible and extremely useful to measure antibodies. As mentioned above, the present inventor has previously disclosed ELISA, a method for measuring trace amounts of polyADP-ribose in various animal organs. However, with this method, it is impossible to directly measure the polyADP-ribose concentration in the various animal organs to be measured, that is, to directly apply the organ extract to the EIA system, due to interfering substances. Therefore, by performing various treatments and isolating the polyADP-ribose fraction, it is possible to extract the polyADP contained in various organs.
-It is possible to measure the amount of ribose, but there is no mention of this. Furthermore, because polyADP-ribose in human serum forms a complex with natural antibodies, etc., it is not possible to directly measure the polyADP-ribose content in human serum. However, in the present invention, polyADP
-Unlike when measuring ribose antigen, animals,
Especially antinuclear antibodies in human serum, especially anti-poly ADP-
This invention differs not only in the difference in the measurement target but also from the previous description by the present inventors in that it discloses a method for directly measuring ribose antibodies without any processing steps. The above description is common to the present invention in that a solid phase method is used when measuring an antigen, but the reaction system includes a reaction system prepared experimentally in advance, for example, a highly specific
Generally, an Ig-type monoclonal antibody that does not react with any antigen other than the relevant antigen, or a polyclonal antibody that does not non-specifically adhere to a plastic carrier is generally used. However, in the system for measuring anti-nuclear antibodies in human serum, especially in patient serum, especially in the serum of patients with collagen diseases, etc., the serum of patients with collagen diseases etc. interferes with the measurement of the target antibodies. Anti-nuclear antibody-specific measurements are generally interfered with because they contain large amounts of other immunoglobulins and immune complexes that cause nonspecific adsorption to the solid phase plate. However, in the present invention, despite these interferences, in order to use a second antibody that reacts specifically with only the specific antibody for the antigen to be measured, or a labeling reagent in which protein A and enzyme are bonded, , it was achieved to specifically measure the antinuclear antibody to be measured. In addition, for the quantitative determination of antigens such as polyADP-ribose by the present inventor, which has already been announced, an experimentally produced antibody with sufficient specificity is used, so protein A
- It is not necessary to use an enzyme-linked labeling reagent, and sufficient results can be obtained simply by using an anti-IgG-enzyme-linked labeling reagent. On the other hand, in the present invention, a second antibody or a labeling reagent in which protein A and an enzyme are bonded is used, which does not react specifically with the antigen that induces the production of antinuclear antibodies, but reacts specifically with the antinuclear antibody that is the measurement target. . When using a labeling reagent in which protein A and an enzyme are bonded, it is generally necessary to use expensive or highly concentrated bovine serum albumin such as fetal bovine serum to block nonspecific adsorption of antibodies to the solid support. While such blockers are normally used, the present invention is characterized in that it is sufficient to use an inexpensive blocker such as bovine serum albumin at a low concentration or skim milk powder as described above. The present invention will be explained in more detail with reference to Examples and Test Examples.
Not limited to ADP-ribose antibodies and anti-double-stranded DNA antibodies, but also pore histone antibodies, anti-Sm antibodies,
This also includes other types of antinuclear antibodies such as anti-RNP antibodies. Production example 1 Production of insolubilized polyADP-ribose (insolubilized antigen) (i) Pretreatment of solid phase carrier A 96-well plastic microtitration plate (Dynatech, Polyvinylplate U type) with a volume of 300 μl per hole was prepared. It was used as a solid phase support. Poly-L-lysine (molecular weight: 4 x 10 ⁵ , Sigma), which has a strong positive charge, was dissolved in distilled water to a concentration of 50 μg/ml, and 100 μl was added to each hole.
Add Tris-salt buffer (25mM tri, 140mM NaCl, PH 7.4 or lower) and leave at room temperature for 1 hour.
The cells were washed four times using TBS (abbreviated as TBS). (ii) PolyADP-ribose binding Kanai et al. method (Kanai, Y. et al, J.
PolyADP-ribose (Biochem., 88 , 917-920, 1980) was added at 2μg/ribose using TBS.
Add 50 μl of the solution to each well of the microtitration plate obtained in (i).
After adding it in portions and evaporating it to dryness at 37°C,
Washed 4 times with TBS. (iii) Post-treatment To block the surface of the plate obtained in (ii) that is not filled with antigen, add 300 μl of TBS supplemented with 0.01% bovine serum albumin to each well and leave for 1 hour, then add TBS three times. It was washed to obtain insolubilized polyADP-ribose (insolubilized antigen). This product is stable for one month at 4°C under humidification. Production Example 2 Production of insolubilized double-stranded DNA (insolubilized antigen) Commercially available calf thymus DNA was deproteinized and purified according to Marmur's method (Marmur, J.Mol.Biol., 3 , 208, 1961). - Papalian method (Stollar, BD and Papalian, M., J. Clin.
Invest., 66 , 210-219, 1980) to completely cleave the single-stranded DNA. This DN is then processed using Kanai et al.'s method (Kanai,
0.2M on a hydroxyapatite column according to Y. et al.
Double strands eluted with phosphate buffer (PH6.8)
Only DNA was collected. This double-stranded DNA is further
S1-nuclease treatment, at this stage no longer
S1 - 260nm to ensure that it will not be cleaved by nuclease
This was confirmed by ultraviolet absorption. Thereafter, the double-stranded DNA was collected using the hydroxyapatite column and eluted using the above method. The double-stranded DNA thus obtained was converted into polyADP-
In place of ribose, double-stranded DNA was bound to a microtitration plate and insolubilized by following the same method as in Production Example 1.
(Insolubilized antigen) was obtained. Production example 3 Production of alkaline phosphatase/protein A conjugate (labeling reagent) 1.9 mg of alkaline phosphatase derived from calf small intestine (Sigma product, Type) and 0.75 mg of protein A (Pharmacia product) were mixed with salt and phosphate. Buffer solution (phosphoric acid 10mM, sodium chloride 140mM, PH7.4, hereinafter PBS)
After dialyzing the solution dissolved in 1 ml against PBS, glutaraldehyde (manufactured by Sigma; concentration 25%) was added to a final concentration of 0.2%, and the mixture was stirred at room temperature for 2 hours to perform binding. The combined product of alkaline phosphatase and protein A was separated using a gel filtration device (Uetrogel AcA 34 filled 1.1 x 54 cm), and the labeled reagent was added as a protein at 220 μg/ml.
Obtained 10ml. The enzyme specific activity of this conjugate is per mg of protein color.
200U (substrate 2.5mM paranitrophenyl phosphate, pH 9.5, under measurement conditions of 37°C),
Since the enzyme alone has a concentration of 900 to 1100 U, it is an extremely active labeling reagent. This product was stable for more than 4 months at 4°C in the presence of 0.5% bovine serum albumin and 0.02% sodium nitride. Production example 4 Production of alkaline phosphatase/anti-mouse immunoglobulin antibody conjugate (labeling reagent) Production example using anti-mouse immunoglobulin (IgG + IgM) antibody (donated by Professor Stollar, Tufts University) instead of protein A. According to the same method as in 3, an alkaline phosphatase/anti-mouse immunoglobulin antibody conjugate (labeling reagent) was obtained.
(Yield: 10ml, protein: 140μg/ml). Reference production example Control plate PolyADP-ribose in (ii) of Production Example 1
A microtitration plate that did not bind polyADP-ribose (antigen) was obtained by the same method as in Production Example 1 except that only TBS was used instead of the TBS solution, and used as a control plate. Example 1 Measurement of anti-poly ADP-ribose antibodies Anti-poly ADP-ribose antibodies in the serum of SLE patients and normal individuals were measured. (i) First reaction (reaction between insolubilized antigen and antibody)
0.01 in each well of ADP-ribose (insolubilized antigen)
100 μl of inactivated serum diluted 20 times with TBS supplemented with % bovine blood albumin, ie, serum whose complement activity had been destroyed by heating, was added and incubated at room temperature for 60 minutes with slight shaking. After removing the reaction solution, the plate was washed four times with TBS. (ii) Second reaction (reaction with labeling reagent) The alkaline phosphtase protein A conjugate (labeling reagent) obtained by the method of Production Example 3 was added to TBS.
100 μl of the 100-fold diluted solution was added to each well of the plate where the first reaction had been completed, and incubated at room temperature for 60 minutes. Remove the reaction solution and remove the plate.
Washed 4 times with TBS. (iii) Third reaction (enzyme substrate reaction) and measurement A solution of 2.5mM paranitrophenyl phosphate dissolved in 10ml of 50mM carbonate buffer (PH9.5) containing 2mM _MgCl2 was used as the substrate. 2 Into each well of the plate after the reaction
After adding 100 μl and reacting at 37° C. for 10 minutes, 100 μl of this solution was taken out and added to 400 μl of a 0.1N caustic soda aqueous solution to stop the enzyme reaction. Of the 500 μl of reaction solution obtained in each well, 5 μl of the 410 nm
The ultraviolet absorption was measured. To correct enzyme activity based on non-specific reactions,
The first, second and third reactions were carried out in the same manner as above except that a control plate prepared by the method of the reference production example was used instead of the insolubilized antigen, and the ultraviolet absorption was measured. For each serum, the true antibody activity was determined by subtracting the ultraviolet absorption when using the control plate from the ultraviolet absorption when using the insolubilized antigen, and this is shown in Table 1. The t-test (Student's t-test) is a significant difference test between the measured values of SLE patients and normal people.
A significant difference of 0.001<P<0.01 was observed. Table 2 shows the UV absorption when using the control plate. It is clear from the comparison with the measured values of normal people in Table 1 that there is no difference between SLE patients and normal people, and there are very few nonspecific reactions. From these facts, according to the method of the present invention,
It is possible to measure ADP-ribose antibodies,
Therefore, it can be seen that diagnosis of SLE patients, etc. is possible. Example 2 Measurement of anti-double-stranded DNA antibodies Anti-double-stranded DNA antibodies in serum of SLE patients and normal subjects
DNA antibodies were measured. Insolubilized double-stranded polyADP-ribose produced by the method of Production Example 2 in place of the insolubilized polyADP-ribose of Example 1
Anti-double-stranded DNA antibodies in serum were measured using the same measuring method as in Example 1 except that DNA was used as an 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 people, indicating that it was possible to measure anti-double-stranded DNA antibodies. Reference example Measurement of anti-poly ADP-ribose produced by hybridoma Mouse myeloma cells SP2/O-Ag-14 and poly
C3H/He mouse splenocytes immunized with ADP-ribose were prepared using the method of Andrzejervski et al. (Andrzejervski, C. Jr. et al, J. Immunol., 124 ,
1499-1502, 1980).
For the 11 obtained hybridoma strains, the culture supernatant was taken and the anti-polyADP-ribose antibody was measured without concentration using the labeling reagent of Production Example 4 as the labeling reagent, but otherwise in the same manner as in Example 1. . The measurement results are shown in Table 4. As shown in Table 4, anti-polyADP-ribose antibodies produced by hybridomas could be directly measured without concentrating the culture supernatant.

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Claims (1)

[Claims] 1. In a method for measuring antinuclear antibodies in human serum by enzyme immunoassay, the antigen that induces the production of antinuclear antibodies to be measured is poly-L-
The anti-nuclear antibody is reacted with the bound insolubilized antigen by contacting with a lysine-treated plastic carrier and then evaporated to dryness, and then reacts specifically with the anti-nuclear antibody without reacting specifically with the antigen. A method in which a second antibody or protein A is reacted with a labeling reagent in which an enzyme is bound, and the amount of the antinuclear antibody is measured based on the amount of the reacted labeling reagent.