JPS6338163A - Method for measuring antigen - Google Patents

Abstract

PURPOSE:To measure an antigen and hapten in the presence of an antibody by adding an acid to a specimen liquid contg. the antigen or hapten and the antibody thereof,then neutralizing the liquid. CONSTITUTION:The acid such as hydrochloric acid, nitric acid or sulfuric acid is added to the specimen liquid such as serum, plasma, saliva or urine contg. the antigen or hapten and the antibody thereof to deactivate only the antigen which affects the measurement of the antigen or hapten under an acidic condition. the specimen liquid is then neutralized by sodium hydroxide, potassium hydroxide, etc. The neutralized specimen liquid is immunologically measured by, which the measurement of the quantity of the antigen and hapten in the presence of the antigen is permitted.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a simple method for measuring an antigen in a sample in which antibodies against the antigen are present.

[Conventional technology]

Various antigens and haptens in body fluids such as serum? Measuring is important in clinical tests for diagnosing diseases. However, antibodies against the antigen or hapten are often present in body fluids such as serum, making measurement of the antigen or hapten difficult. As a method for measuring antigens in body fluids containing antibodies against the antigen, measurement of the total amount of insulin in body fluids containing anti-insulin antibodies has not been reported. Conventionally, there are two methods for this. One method is to extract and measure insulin from body fluids containing insulin antibodies using acidic alcohol [G., M., Grodsky (G.
, M. Grod-sky), P. H. Forsham, Journal of Clinical Investigation (J., C.
11n, Engineering Best, ), Volume 39, Page 1070 (
1960), L, C1, heading (L.

G, HecLing), Diabetologia (Di, a
betolo-gia), Volume 8, page 260 (19
72) 10 Another method is to neutralize anti-insulin antibodies and precipitate them with polyethylene glycol after acidifying the body fluid sample.Insulin? It is a method of measuring (
acid/polyethylene glycol treatment) CS, Nakagawa (S
, Nakagawa) et al., Diabetes (Dia
betes), Vol. 22, p. 590 (1973), H
, Kuzuya H. et al., Diabetes, Vol. 26, p. 22 (1977)].

[Problem that the invention seeks to solve]

However, these methods have drawbacks.

First, both of these two methods are complex to operate. Both extraction with acidic alcohols and precipitation with polyethylene glycol are complicated. Second, the types of antigens that can be extracted with acidic alcohol are limited, and the number of antigens that cannot be precipitated with polyethylene glycol is also limited.

In view of the above-mentioned circumstances, the purpose of the present invention is to be simpler and more efficient than conventional methods. The object of the present invention is to provide a complete method for measuring antigens and haptens in the presence of antibodies that can be applied to the measurement of type 1 antigens and butenes.

[Means for solving the eight points between 5' and 5'] To summarize the present invention, the present invention relates to a method for determining an antigen or hapten in the presence of an antibody, which comprises the following steps (A) and (B). ) and (C)CA) Acid is added to the test solution containing the antigen or hapten and its antibody, and the antibody that does not impair antigenicity but affects the immunoassay of the antigen or hapten is incubated under acidic conditions. (B) neutralizing the test solution; (C) neutralizing the test solution; and (C) neutralizing the test solution.

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

The acid used in step (A) of the present invention is not particularly limited, and examples include hydrochloric acid, nitric acid, sulfuric acid, perchloric acid, and the like.

In addition, although the antigenicity is impaired, the acidic conditions that cause total inactivation of the antibody, which affects the immunoassay of the antigen or JD hapten, are not constant depending on the type of antigen and hapten, but when the antigen is insulin, growth hormone, :/ In the case of thyroid stimulating hormone, alpha-fetoprotein, etc., the purpose can be achieved at pH 15 or lower, preferably at about pH 1, and at 4°C to 50°C for several tens of hours.

The base used in the neutralization in step (B) of the present invention is not particularly limited, and hydroxide, potassium hydroxide, ammonium hydroxide, etc. are used.

Various well-known immunoassay methods can be used to measure the antigen or hapten in step (C) of the present invention.

Hereinafter, the antigen measurement method according to the present invention will be explained in detail.

First, phosphate buffered saline containing bovine serum albumin (hereinafter abbreviated as BSA) is completely added to the test solution, hydrochloric acid is added to the solution to adjust the pH to approximately 1, and the solution is left at room temperature for about 60 minutes. Neutralize this by adding sodium hydroxide, and then add phosphate buffered saline containing BSA and NaN1.

The amount of antigen in the sample thus prepared is measured by the following method. That is, antibody IgG or F (
a b') t 5r: Insolubilized solid phase carrier such as a plastic microtiter plate, plastic cleft ball, plastic tube, etc. with the above sample at 4°C ~
After reacting at 37°C for 4 to 24 hours, preferably at 57C for 4 hours, and washing with phosphate buffered saline, this? After reacting with enzyme-labeled antibody engineered gG or its F(ab')1, preferably its Fab' at 4°C to 37°C for 4 to 12 hours, preferably at 20°C for 4 hours, phosphate buffered saline was added. After washing with water, the enzyme activity bound to the solid phase was measured. Labeling enzymes include alkaline phosphatase, β-
Examples include D-galactosidase and peroxidase, with peroxidase being suitable. In addition, as the enzyme's t1, the beam IJ,'<.

For example, in the case of peroxidase, H! of group 1a! 0! Chromogenic substrates such as 5-aminosalicylic acid and 0-phenylenediamine, fluorescent substrates such as 3-(p-hydroxyphenyl)propionic acid, and luminescent substrates such as luminol are used.
Among these, fluorescent substrates are suitable.

It is also possible to carry out a one-step method in which a solid phase carrier in which the antibody is insolubilized, an antigen, and an enzyme-labeled antibody Fab' are reacted simultaneously to carry out the measurement.

In the case of the hapten measurement method according to the present invention, it can be carried out in the same manner as above, but in the case of hapten, the method according to the present invention
C) In the step, a conventionally known immunoassay method,
For example, a competitive binding immunoassay may be used.

〔Example〕

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

Actual case 1: Measuring insulin! (A buffer solution (C) of α115d in human serum α005d
10 containing 1 mol/l NaC2 and 1r/zEsA
mmo1/l phosphorus [Na% pH 7,0) f added,
Add 3 mo1/l HOL of α01- to this and adjust the pH
After adjusting f to approximately 1, it was left to stand at room temperature for 60 minutes. This is V
Add 2.9-5 mol/1) JaOH of α01- to the mixture, add 2.9-5 mol/1) of α01- buffer solution (1.8 mol/7
NaC6 and 5r/l BEIA and 15y/1Na
11103 m01/l phosphorus p Na including N1 f,
pH 7.0) was added. Insulin in the thus prepared sample [C15-
-h%Ruan) et al., Cl1n, Chim, Acta, Vol. 147, p. 167 (1985)]. That is, ]sample α15−
Mixed with whole anti-insulin antibody-conjugated polystyrene bow, 37
The reaction was carried out at ℃ for 4 hours. Note that the anti-insulin antibody-bound polystyrene balls were prepared as follows.

The anti-insulin antibody was obtained by salting out guinea pig anti-insulin antiserum (Medical and Biological Research Institute, Nagoya) with Na1SO4 and purifying it by DBAM-cellulose column chromatography to obtain an anti-insulin antibody (K, Ishikawa (L)). Journal of Immunoassay, Vol. 4, p. 209 (1983)]. This anti-insulin antibody engineered gG polystyrene ball [diameter x 2 m (Precision Plastic Ball Co., Chicago). )] was physically adsorbed to make it insolubilized [ K, Ishikawa and Ni, Ka M
(K, KatO), Scandinavian Journal of Immunology (5cand, J, Engineering
), Volume 8 (Supplement 7), Page 43 (1978)].

10 m containing anti-insulin antibody-conjugated polystyrene balls reacted with the above sample in an amount of C1 mol/1 NaC2f.
mol/l Phosphorus 6xqag (pHy, o) 2
- After washing with 5nf of affinity f# anti-insulin Fab'-horseradish peroxidase complex solution (115m), the reaction was carried out at 20°C for 4 hours. The peroxyguse-labeled anti-insulin Fab' was prepared as follows.

That is, the guinea pig-derived anti-insulin antibody engineered gG to F (a b5k, then F
ab' 5r: E0 Ishikawa et al., Journal op.
-Maleimidohexanoate (Isotope Chemistry Institute, Kumamoto)
After binding using [S, Hashida (S, I(ashid
a) et al., Journal of Applied Biochemistry (J.

Appl, Biochem, ), Volume 6, Page 56 (
1984), was prepared by insulin-Sepharose 4B affinity column chromatography in which mercaptosuccinylated insulin was coupled to activated thiol Sepharose 4B (Pharmacia Fine Chemicals AB, Uppsala), and further Ultrogel A (!A44(LKB.

A peroxidase-labeled anti-insulin tab' was obtained (see Luan et al., supra). The buffer used for incubating the enzyme-labeled antibody was CL 1 mol.
/A 10 mmol containing NaC6 and α1%B8Ai
/j Sodium phosphate solution (pH 7.0).

After washing the polystyrene balls incubated as described above with the washing buffer 2-, the peroxidase activity adsorbed on the polystyrene balls was measured.

Peroxidase activity is activated by H2C and 3-(p) as substrates.
-hydroxyphenyl)propionic acid (Aldrich
Chemical Company Incorporated (Milwaukee) at 30°C
Vol. 1509 (1983)]. Fluorescence intensity? '11 It is expressed as the relative intensity to the solution containing 1 ton of IIO5mol/l H2BO3K of Misaki Kinin, and the excitation wavelength is 320n.
The amount of insulin in the sample was determined from the ffk line measured using standard insulin. Table 1 shows the measurement results for 15 types of serum samples. In other words, Table 1 shows a comparison of the measurement results between the method of the present invention and the conventional method (acid/polyethylene glycol treatment) and without pretreatment, using serum with and without anti-insulin antibodies. be.

Table 1 l-) -90155129 2+ 49 109 100 5 + 117 206 200 5+ 97 185 185 6 + 168 236 221 7 + 113 240 221 9+65167151 10 + 47 63 60 11 No Maru 7.5 7.0
6,112 Normal 13
15 113 Normal 12
13 1314 Normal
13 15 1315 Normal
9.1 9.1 a2 Comparison Row 1 Insulin was measured using a method combining conventional acid treatment and polyethylene glycol method. That is, various human serum (1 mo1/1 Ho of 0.01 d of 105-
After mixing with t and leaving at room temperature for 60 minutes, ao 7+
d's 250? /l polyethylene glycol mixed with α
After adding 1 mol/1 NaOI of 01-, 150
Centrifuged at 0'AP for 45 minutes (S. Nakagawa et al.
(see Diabetes).

This supernatant α01@/ (114- buffer solution (α42mo
l/1NaC4,1,I P/l BSA, 1
．． Insulin was measured as described above after mixing with 10 mmol/l phosphate Na pH 7,0) containing 1 r/z NaN1. The results are shown in the acid/polyethylene glycol treatment column of Table 1.

As is clear from Table 1, the measurement method according to the present invention carried out in Example 1 can produce similar results to the conventional method, much more easily than the conventional method.

ratio! Example 2 Insulin was measured by the enzyme immunoassay method described in Example 11 simply without pretreatment of the serum sample. The results are shown in the column without pretreatment in Table 1. From this result, if there is no anti-insulin antibody in the serum sample, Example 1
, the same results as Comparative Example 1 are obtained, but in the presence of anti-insulin antibodies, all samples show clearly lower values than in Example 1 and Comparison Row 1, and in the presence of antibodies, the antigen amount can be accurately measured. It shows that it is not possible.

As mentioned above, from the results of Example 1, Comparison Column 1, and Comparison Column 2, the method of the present invention performed in Practical Column 1 can be used more easily than the conventional method.
antibody? It is shown that the antigen can be accurately measured even in samples containing V.

Example 2 Measurement of Insulin 12.5 μt of α5 in 50 μt of various human serum samples
After adding molAHOt to pH 6xs, 25μt
Dextran - Charcoal! ! It was added to the solution to adsorb free insulin. This mixture clJrc was reacted for 5 minutes with stirring, and the Q of j2.5tiL,
Add 38 mol/1 NaOHf and neutralize 15008r
The mixture was centrifuged for 15 minutes, and the supernatant was centrifuged again in the same manner. A 70 μt portion of this supernatant contains 35 at 1 f/L NaN1.1 containing 2 μg U insulin.
．． Or/L BSA, a, 1mo1/z Na
(Contains 10 mmol/l Na phosphate (pH 7,0
) was added and reacted at 4°C for 24 hours.

A portion of this reaction solution is 15 μt and 105 μt is 12/l.
BSA, α1 mol/1Na (containing 10 mm
ol/l Na phosphate (pH 7,0) was added. Example 1 Insulin in the sample prepared by K.
It was measured by the method of the present invention in the same manner as in 1. Table 2 shows the measurement results of 15 types of serum samples and the recovery rate of added insulin.
(method of the present invention).

The results of measuring various samples that were shredded and not subjected to acid treatment as in the method of the present invention are shown in the column without pretreatment in Table 2.

As is clear from Table 2, when anti-insulin antibodies were not present in the serum sample, the method of the present invention and the method without pretreatment yielded similar recovery rates. However, in samples containing anti-insulin antibodies, the method without pretreatment has a lower recovery rate than the method of the present invention.

That is, when measured by the method of the present invention, it is 94-1
This shows a good recovery rate of 0.02%, but when measured without pretreatment, the recovery rate is between 39 and 104%, indicating that it is not possible to accurately measure the amount of antigen in the presence of antibodies.

The above results indicate that the method of the present invention allows accurate measurement of antigens in samples containing antibodies.

〔Effect of the invention〕

As explained in detail above, according to the method of the present invention, the amounts of many types of antigens and habten can be measured in the presence of antibodies with much greater ease than with conventional methods.

Proceedings Amendment (Section 9.) September 5, 1986 Director-General of the Patent Office Akio Kuro IB 1, Indication of the case 1985 Patent Distribution No. 181955 λ Name of the invention
Relation to the Case of Person Making Amendments to Antigen Fermentation 1. Address of Patent Licensee: 1-24, 3-24 Otsukadainishi, Miyazaki City, Miyazaki Prefecture Name: Sakae Ishikawa, 302, Chisai Shinbashi Chuo Building, Telephone: (437)-3467 Name Patent Attorney (7850) Nakamoto
Hiroshi (and 2 others) i Date of correction order Voluntary correction
i & correction target par, -
'(1) Detailed explanation of the invention in the specification.
', -, t□ - 2 Contents of amendment The detailed description of the invention column in the specification will be amended as follows.

+11 Change “several tens of hours” to “several minutes to several minutes” on page 5, line 4 of the specification.
"Dozens of hours," he corrected.

Claims (1)

[Claims] 1. The following steps (A), (B) and (C) (A) Adding an acid to a test solution containing an antigen or hapten and its antibody, but not impairing antigenicity. (B) neutralizing the test solution; (C) reducing the amount of the antigen or hapten in the test solution by immunization. 1. A method for measuring an antigen or hapten, comprising a step of chemically measuring it.