JPH0792457B2 - High-sensitivity antibody measurement method - Google Patents

Description

TECHNICAL FIELD The present invention relates to a highly sensitive method for measuring an antigen-specific antibody.

[Conventional technology]

The measurement of antigen-specific antibodies in body fluids is very important for clinical examination. That is, it is widely used in antibody tests for infectious diseases, autoantibody tests, and the like. Conventional measurement methods in this field are largely 2
It can be divided into two technologies. The first technique is to react an antigen labeled with a radioisotope with a sample containing an antigen-specific antibody, precipitate the resulting immune complex with a precipitant, and measure the radioactivity in the precipitate. is there. In the second technique, an antigen is immobilized on a solid-phase carrier, a sample containing the antigen-specific antibody is reacted with the solid-phase carrier, and further an anti-immunoglobulin antibody labeled with a radioisotope or an enzyme is reacted with the antigen-specific antibody. Is a method of measuring. First
An example of this technique is the polyethylene glycol precipitation method using radioisotope-labeled insulin of Palmer and Asplin in the measurement of anti-insulin antibody [Science 2
Vol. 22, p. 1337 (1983)]. In addition, there is an ammonium sulfate precipitation method using radioisotope-labeled DNA of RS Farr et al. In the measurement of anti-DNA antibody [Science, Vol. 161, page 806 (1968)]. On the other hand, as an example of the second technique, LJ Nell et al.
abetes) Vol. 34, p. 60 (1985)], in which insulin was immobilized on a 96-well plate, a sample was added to the plate, and the bound human immunoglobulin was subjected to enzyme-labeled anti-human immunoglobulin antibody to be used for enzyme immunoassay. There is a method of quantifying.

[Problems to be solved by the invention]

However, the first technique is a method using a radioisotope, and there are problems in terms of safety, equipment, and shelf life of reagents. In the second technique, a large amount of antigen non-specific antibody is contained in the sample, which is non-specifically adsorbed on the solid phase, resulting in a high background, resulting in poor measurement sensitivity. Atsuta

In view of the above circumstances, an object of the present invention is to provide a simpler and more sensitive method for measuring an antigen-specific antibody than a conventional method.

[Means for solving problems]

Briefly describing the present invention, the present invention relates to a method for measuring an antibody, which comprises the following steps (A), (B), (C) and (D) (A) Step of adding antigen of antibody to form antigen-antibody complex (B) Step of separating the antigen-antibody complex from the test liquid (C) Step of dissociating antigen from the antigen-antibody complex (D ) A step of measuring the dissociated antigen by an immunoassay method.

The present inventor reacts a specimen containing an antibody with an antigen,
After forming the antibody complex, the antigen-antibody complex is separated, the antigen in the complex is dissociated by a dissociation agent, and the amount of this antigen is conventionally well-known immunological assay method, especially Sangertien enzyme. The inventors have found that the antigen-specific antibody in a sample can be measured with much higher sensitivity than the conventional method by measuring by an immunoassay, and completed the present invention.

Examples of test liquids that can be measured by the method of the present invention include body fluids such as serum, plasma, spinal fluid, saliva, and urine.

As the antigen used in the method of the present invention, an antigen that specifically binds to the antigen-specific antibody to be measured and whose antigenicity is not changed by the treatment with the dissociating agent during the step of the present invention is used. For example, in the measurement of anti-insulin antibody, human-, porcine-, bovine-derived and chemically modified insulin, as well as genetically engineered insulin, are used as the antigen.

Further, as a method for separating the antigen-antibody complex in the method of the present invention (step B), a precipitation method using polyethylene glycol, ammonium sulfate or the like is used. When the free antigen cannot be separated by the precipitation method, it is used as a separation method. The solid-phased anti-immunoglobulin antibody or the anti-immunoglobulin antibody itself can be used, and the antigen-antibody complex can be separated without changing the antigenicity.

In addition, it is preferable to remove free antigens present in the test liquid from the test liquid prior to the step of separating the antigen-antibody complex. At this time, for example, an adsorption removal method using dextran-Chacoal particles is used.

As a method for dissociating the antigen from the antigen-antibody complex of the present invention (step C), a known method, for example, treatment with acid, iodine and magnesium chloride is used. In the present invention, acid treatment is particularly preferable among these methods, and strong acid treatment around pH 1 at which the dissociated antibody is inactivated is desirable. That is, the other antibody dissociated in the dissociation step of the method of the present invention must be inactivated in the next step D, but the above dissociation treatment also inactivates the antibody at the same time. Therefore, in the method of the present invention, the dissociation and the inactivation of the antibody may be performed in separate steps, if necessary.

As a method for measuring the dissociated antigen used in the present invention, various well-known immunological assay methods are conventionally used. Among them, the Sangertien enzyme immunoassay method is suitable, and for example, K-h luan as an insulin assay method. (Kh Ruan) et al. [Clin. Chim. Acta. No. 147]
Volume 167 (1985)]. As an antibody to be used, an IgG fraction purified from an antiserum obtained by immunizing an animal with an antigen by a conventional method, or a monoclonal antibody prepared by a generally well-known method is suitable.

The method for measuring a highly sensitive antibody according to the present invention will be described in detail below.

First, an excess amount of the antigen is added to the test liquid and incubated at about 37 ° C. for several hours, preferably about 3 hours. After this,
Dextran-Chyacol is added as an adsorbent for free antigen and incubated for about 5 minutes with occasional mixing.
This mixture was centrifuged at 1500 g for 15 minutes, the supernatant was taken,
The same centrifugation operation is repeated again. This supernatant was mixed with bovine serum albumin (hereinafter referred to as BSA) -containing phosphate buffered saline, and a polyethylene glycol aqueous solution was added as a precipitant and mixed well, followed by centrifugation at 1500 g for 15 minutes.
Mix the precipitate well with the same precipitant and perform the same centrifugation.
This operation is repeated twice. Suspend the precipitate with cold acetone 1500
Centrifuge at g for 15 minutes. The precipitate is dried at room temperature, suspended in distilled water, and hydrochloric acid is added as a dissociator. This mixed solution is incubated at room temperature for 1 hour to dissociate the antigen from the antigen-antibody complex. After this, Tris-HCl buffer (p
H8.0) is added and neutralized, and then phosphate-buffered saline containing BSA and NaN ₃ is added. By the above operation, the antigen captured by the antigen-specific antibody is extracted. The amount of this antigen is measured by the following method. First, the antibody IgG or its F
_(ab ' _{) 2} in a microplate for enzyme immunoassay made of plastic, solid ball such as plastic ball, plastic tube and the like at 4 ° C in a solvent such as phosphate buffered saline.
The solid phase carrier and the antibody are physically bound by reacting for 12 hours or more.

Then, the antigen-containing solution dissociated from the antigen-specific antibody by the above-mentioned method is mixed with the antibody-bound solid phase at 4 ° C to 37 ° C for 4 hours.
Hours to 24 hours, preferably 4 hours at 37 ° C, and 12 at 4 ° C
After capturing the antigen by reacting for a period of time, after washing, an enzyme-labeled antibody or its F _(ab ' _{) 2} , preferably a solution of the Fab' such as phosphate buffered saline is added, By reacting at 4 ° C. to 37 ° C. for 4 hours to 12 hours, preferably at 37 ° C. for 4 hours, the antigen captured on the solid phase is bound to the enzyme-labeled antibody. Alkaline phosphatase, β-D-galactosidase, peroxidase and the like are available as the labeling enzyme, and peroxidase is suitable. After washing the solids, a coloring reagent, a fluorescent reagent or an issuing reagent is added and reacted, and the absorbance, fluorescence intensity, emission intensity, etc. are measured. As such a substrate, for example, in the case of peroxidase, a coloring reagent such as 5-aminosalicylic acid and o-phenylenediamine, a fluorescent reagent such as 3- (p-hydroxyphenyl) propionic acid, and luminol together with H ₂ O ₂ as a substrate. Luminescent reagents and the like are used, among which fluorescent reagents are most suitable.

In addition, a solid phase carrier to which an antibody is bound, an antigen, an enzyme-labeled antibody Fa
It is also possible to perform a one-step method in which b ′ is reacted at the same time and measurement is performed.

In the above method, when using serum as the test liquid,
A calibration curve can be prepared by performing a reaction using an antigen-specific antibody having a known concentration, which is purified using an affinity column such as antigen-binding-Sepharose 4B from patient serum instead of serum. The amount of antigen-specific antibody in the test serum can be determined from a calibration curve.

〔Example〕

Hereinafter, the present invention will be described in more detail with reference to Examples.
The present invention is not limited to these examples.

Example 1 1.0 g of a sample of an anti-insulin antibody-positive patient serum diluted to various concentrations with normal human serum was added with 1.0 g of 16 μU of insulin [Actrapid MC, Novo Industrials, Copenhagen]. 0.44 ml of a solution prepared by dissolving 0.44 ml of 10 mM sodium phosphate buffer pH 7.0 (buffer A) containing / l BSA and 0.1 M NaCl was added and incubated at 37 ° C for 3 hours.
This was followed by the addition of 0.055 ml dextran-Chyacol suspension and agitated for 5 minutes with occasional agitation. Dextran-Chyacol is Dixon [Clinical
Chemistry (Clin. Chem.) Vol. 20, p. 1275 (1974)]
It was created by the method of. However, BSA [Fraction V, Armor Pharmaceutical Co., Kankakee]
And Norit A (Hanui Chemical Co., Kyoto) were used in place of human serum albumin and Norito NK (or Norito GSX). Dextran-Chyacol was prepared as a suspension in dry weight containing 60 mg of Chajacol in 1 ml.

1500g of the reaction solution treated with dextran-Chacol
The mixture was centrifuged for 15 minutes, and this supernatant was further centrifuged again at 1500 g for 15 minutes. To 0.55 ml of this supernatant, 0.45 ml of buffer A and 1 ml of a polyethylene glycol (# 6000 Hanii Chemical Co., Ltd., Kyoto) solution (250 g / l) were added, mixed well, and centrifuged at 1500 g for 15 minutes. This precipitate was washed twice with 1 ml of a polyethylene glycol (# 6000 Hanai Chemical Co., Kyoto) solution (125 g / l). To this precipitate, 1 ml of cold acetone was added and suspended, and the suspension was centrifuged at 1500 g for 15 minutes. The precipitate was dried at room temperature, suspended in 0.07 ml of distilled water, added with 0.01 ml of 0.8M HCl and mixed well. After left at room temperature for 1 hour, it was neutralized with 0.02 ml of 1M Tris-HCl buffer pH 8.0, and further 0.05 ml of 3 g / l BSA, 3 g / l NaN ₃ and 1.2M Na.
30 mM sodium phosphate buffer containing Cl was added and mixed well. 0.15 ml of the thus prepared sample was mixed with polystyrene balls conjugated with anti-insulin antibody and reacted at 37 ° C. for 4 hours and subsequently at 4 ° C. for 12 hours. The anti-insulin antibody-bonded polystyrene balls were prepared as follows. As the anti-insulin antibody, guinea pig-derived anti-insulin antiserum (Medical and Biological Research Institute, Nagoya) was salted out with Na ₂ SO ₄ and purified by DEAE-cellulose column chromatography according to a conventional method to obtain an IgG fraction. This anti-insulin antibody IgG solution (0.
1g / l) Polystyrene balls (diameter 3.2mm, Precision Plastic Ball Company (Precision
Plastic Ball Co.) Chicago] and soaked Ishikawa et al. [Scandinavian Journal of Immunology (Scand.J.Im
munol.) Vol. 8, p. 43 (1978)]. The polystyrene ball bound with the anti-insulin antibody was reacted with the above sample, and was adjusted to 10 mM containing 0.1 M NaCl.
It was washed twice with sodium phosphate buffer pH 7.0 (buffer B), and then reacted with 5 ng of peroxidase-labeled anti-insulin antibody Fab 'solution 0.15 ml at 20 ° C for 4 hours.

The peroxidase-labeled anti-insulin antibody Fab 'was prepared as follows. That is, the guinea pig-derived anti-insulin antibody IgG prepared as described above was digested with pepsin by a conventional method to obtain an F _(ab ' _{) 2} fraction, which was reduced with 2-mercaptoethylamine to obtain Fab'. This was analyzed by the method of Hashida et al. [Journal of Applied Biochemistry (J. Appl. Biochem.) Vol. 6, p. 56 (1984)] and horseradish-derived peroxidase [grada I, Peeringer Mannheim ( Boehringe
r Mannheim), Mannheim] using N-succinimidyl-6-maleimido hexanoate (Dojindo Laboratories, Kumamoto).

Furthermore, 1.6 mg of a mercaptosuccinylated insulin prepared from the reaction product by the method described by Kh Luang et al. [Clinica Kimika Actor Vol. 147, p. 167 (1985)].
Was purified by insulin-Sepharose 4B affinity column chromatography, which was coupled to activated thiol Sepharose 4B (Pharmacid Fine Chemicals AB, Ubusala), and further purified by Ultrogel AcA44 (LKB, Stockstock).
Purified with peroxidase-labeled anti-insulin antibody
I got Fab '. The insulated police balls were washed twice with buffer B and the peroxidase activity adsorbed on the polystyrene balls was measured. The activity of peroxidase is H ₂ O ₂ and 3- (P-hydroxyphenyl) propionic acid [Aldrich Chemical Company Inc.
O. Inc.), Milwaukee], and Imagawa et al. [Analytical Letters (Anal Lett) 16: 1509 (198).
3)] was used for measurement at 30 ° C. The fluorescence intensity was expressed as a relative intensity with respect to a solution in which 1 mg quinine was dissolved in 1 0.05 MH ₂ SO ₄ , and was measured at an excitation wavelength of 320 nm and a fluorescence wavelength of 405 nm using a Shimadzu spectrofluorometer (RF-510 Shimadzu Corporation, Kyoto). The measurement results of the five types of samples are shown by the solid line in FIG. That is, FIG. 1 is a graph showing a comparison of the sensitivities of the method of the present invention (solid line) and the conventional method (dotted line) in the relationship between the serum dilution rate (horizontal axis) and the relative fluorescence intensity (vertical axis).

Comparative Example 1 The same sample as in Example 1 was measured by the method described above by LJ Nell et al. [Dia Beaches, Vol. 34, page 60 (1985)] as a conventional method. 0.1 ml of patient serum sample was brought to pH 6.0 with 0.125 ml of 32 mM HCl, to which was added 0.05 ml of the dextran-Chyacol solution used in Example 1, the mixture was stirred occasionally for 5 minutes, and 0.125 ml of 8 mM NaO was added.
Add H to neutralize. This mixed solution was centrifuged at 1500 g for 10 minutes, and the supernatant was further centrifuged at 1500 g for 10 minutes. This supernatant was further centrifuged at 1500 g for 10 minutes. The supernatant was diluted 7500 times with the buffer A used in Example 1, and 0.15 ml of this was reacted with polystyrene balls having insulin bound thereto at 37 ° C. for 4 hours and then at 4 ° C. for 12 hours. Insulin-bonded polystyrene balls were previously prepared with normal rabbit IgG (2 mg / m
l) was physically adsorbed on polystyrene balls in the same manner as in Example 1 and treated with insulin and glutaraldehyde [T. Kohno], Journal
Of Biochemistry (J. Biochem.) Vol. 98, 379
Page (1985)]. The reaction product of the serum sample and the insulin-bound polystyrene balls was washed twice with buffer B of Example 1 and then 5 ng of peroxidase-labeled anti-human IgG.
[Γ-Chain] The above polystyrene balls are immersed in a 0.15 ml solution of antibody Fab ′ and reacted at 20 ° C. for 4 hours. As the peroxidase-labeled anti-human IgG (γ-chain) antibody Fab ′, rabbit-derived anti-human IgG (γ-chain) antiserum (Medical and Biological Research Institute, Nagoya) was used, and the same procedure as in Example 1 was performed. It was prepared by the method of Hashida et al. [Journal of Applied Biochemistry, Vol. 6, page 56 (1984)]. The reaction product was washed twice with buffer B of Example 1 and the peroxidase activity adsorbed on polystyrene balls was measured by the same method as in Example 1.
The results are shown by the dotted line in FIG. From this result, the measurement method according to the present invention performed in Example 1 is 1000 to 30 as compared with the conventional method.
It is shown that the sensitivity is much higher at 00 times and the background is low.

Example 2 It was confirmed by the following example that the assay method according to the present invention is not affected by free insulin coexisting in serum samples. 1.6 μU to 1600 μU of insulin (Actrapid MC, Novo Industrials, Copenhagen as described above) was added to normal human serum, and the assay method according to the present invention shown in Example 1 was performed. In addition, as a target, the measurement method excluding the adsorption removal operation of free insulin by dextran-chaycor in the measurement method of Example 1 was performed. Results are shown in FIG. That is, FIG. 2 is a graph showing the effect of free insulin in the assay method of the present invention in the relationship between the amount of added insulin (μU / tube, horizontal axis) and the relative fluorescence intensity (vertical axis). The white squares are the target experiments,
Open circles represent the results of the measuring method according to the present invention in Example 1.

From FIG. 2, in the coexistence of 1.6 μU to 160 μU of insulin, there is no effect on the measured value, and the amount of free insulin in the serum sample has no effect on the assay method of the present invention. It is clear.

Example 3 A calibration curve for quantifying anti-insulin antibody according to the present invention is shown in the following example.

Using the assay method according to the present invention in Example 1, instead of the patient serum sample, a calibration curve was obtained using samples obtained by variously diluting anti-insulin antibody IgG derived from patient serum of known concentration with normal human serum. In addition, as a subject, normal human Ig
G was used instead of patient serum-derived anti-insulin antibody IgG,
The same measurement was performed. The patient serum-derived anti-insulin antibody IgG was prepared as follows. 3 ml of patient serum containing anti-insulin antibody was added to 20 mM sodium phosphate buffer pH 7.
Diluted 2-fold with 0 and using insulin-bound Sepharose 4B, column chromatography as described in Example 1, 1.
Collect the fractions eluted with 3 mM HCl pH 2.5 and immediately
Neutralization was performed with 1 M sodium phosphate buffer (pH 7.0) to obtain patient serum-derived anti-insulin antibody IgG. The results of this example are shown in FIG. That is, FIG. 3 is a graph showing a calibration curve for the quantification of anti-insulin antibody according to the present invention in the relationship between IgG amount (mol / tube, horizontal axis) and relative fluorescence intensity (vertical axis). White circle indicates anti-insulin antibody derived from patient serum
In the case of IgG, open squares are those of normal human IgG.

From this result, it is clear that the assay method according to the present invention does not react with normal human IgG and that the peroxidase activity is increased depending on the concentration of the anti-insulin antibody, and that it is possible to quantify the anti-insulin antibody. Is. Furthermore, the measurement sensitivity according to the present invention was found to be 45 pg / tube or 450 ng / l. The measurement sensitivity is t-test (n-5,
p <0.001). This measurement sensitivity is
About 10,000 compared to the measurement sensitivity (4 mg / l) reported by LJ Nell et al. [Dia Beaches, Vol. 34, p. 60 (1985)].
It turned out to be a very sensitive measurement method of double.

[Effects of the Invention] As described in detail above, the present invention enables simple and highly sensitive measurement of an antibody, and as a result, enables measurement of an ultratrace amount of antibody that could not be conventionally measured.

[Brief description of drawings]

FIG. 1 is a graph showing a comparison of sensitivities when an anti-insulin antibody-containing serum sample was diluted to various concentrations and measured by the method according to the present invention (solid line) and the conventional method (dotted line).
FIG. 3 is a graph showing the effect of free insulin in the assay method of the present invention, and FIG. 3 is a graph showing a calibration curve for quantifying the anti-insulin antibody of the present invention.

Claims (1)

[Claims] 1. The following steps (A), (B), (C) and (D): A step of adding an antigen of the antibody to a test solution containing the antibody to form an antigen-antibody complex. (B) separating the antigen-antibody complex from the test liquid (C) dissociating the antigen from the antigen-antibody complex (D) measuring the dissociated antigen by an immunoassay A method for measuring an antibody, which comprises: