JPH0792454B2 - Antigen measurement method - Google Patents

Description

TECHNICAL FIELD The present invention relates to a simple method for measuring an antigen in a sample in which an antibody against the antigen is present.

[Conventional technology]

Measuring various antigens and haptens in body fluids such as serum is important in clinical examinations for diagnosing diseases. However, antibodies against the antigen or hapten are often present in body fluids such as serum, which makes it difficult to measure the antigen and the hapten. As a method for measuring an antigen in a body fluid in which an antibody against the antigen is present, measurement of the total amount of insulin in a body fluid in which an anti-insulin antibody is present has been reported. There are two conventional methods for this. One of them is to measure insulin from body fluid containing anti-insulin antibody by extracting it with acidic alcohol [GM Grodsky (GMGr
odsky), PH Forsham, Journal of Clinical Investigation (J.
Clin.Invest.), Vol. 39, p. 1070 (1960), LG Heding, LG Diabetologi.
a), Vol. 8, p. 260 (1972)]. The other is a method of measuring insulin after acid treatment of a body fluid sample, neutralization of anti-insulin antibody and precipitation removal with polyethylene glycol (acid / polyethylene glycol treatment) [S. Nakagawa (S. Nakagawa). ) Et al, Deiabetes (Di
abetes), Vol. 22, p. 590 (1973), H. Kuzuy
a) et al., De Abetes, Vol. 26, p. 22 (1977)].

[Problems to be solved by the invention]

However, these methods have drawbacks. First, both of these two methods are complex to operate. Extraction with acidic alcohol and precipitation with polyethylene glycol are complicated. Second, the types of antigens that can be extracted with acidic alcohol are limited, and the types of antigens that do not precipitate with polyethylene glycol are also limited.

In view of the above circumstances, an object of the present invention is to provide a method for measuring an antigen and a hapten in the presence of an antibody, which is simpler than conventional methods and can be applied to the measurement of many types of antigens and haptens. It is in.

[Means for solving problems]

Briefly describing the present invention, the present invention relates to a method for measuring an antigen or a hapten in the presence of an antibody, which comprises the following steps (A), (B) and (C) (A) antigen or hapten and its antibody: A step of adding an acid to a test liquid containing the antibody to inactivate an antibody which does not impair the antigenicity but affects the immunological measurement of the antigen or the hapten under acidic conditions of pH 3.5 or lower (B) It is characterized by including the step of neutralizing the test solution (C) immunologically measuring the amount of the antigen or hapten in the test solution.

Serum as an example of the test liquid that can be measured by the method of the present invention,
Body fluids such as plasma, cerebrospinal fluid, saliva and urine can be mentioned.

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

In addition, the acidic condition that inactivates the antibody that does not impair the antigenicity but affects the immunological measurement of the antigen or hapten is not constant depending on the type of the antigen and hapten, but the antigen is insulin, growth hormone, or thyroid stimulation. In the case of hormones, alpha-fetoprotein, etc., the purpose can be achieved under the conditions of pH 3.5 or less, preferably about pH 1, several minutes to several tens hours, and 4 ° C to 50 ° C.

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

As the method for measuring the antigen or hapten in the step (C) of the present invention, various well-known immunological measurement methods are used.

Hereinafter, the antigen measuring method according to the present invention will be described in detail.

First, bovine serum albumin (hereinafter abbreviated as BSA) -containing phosphate buffered saline is added to the test solution, and hydrochloric acid is added to adjust the pH to about 1, and then the solution is left to stand at room temperature for about 60 minutes.
Sodium hydroxide was added to this to neutralize, and BSA and NaN
Phosphate buffered saline containing ₃ is added.

The amount of antigen in the sample thus prepared is measured by the following method. That is, the antibody IgG or F (ab ′) ₂
A solid phase carrier such as a plastic microtiter plate, plastic ball, plastic tube, etc. insolubilized with the above sample is reacted with the above sample at 4 ° C to 37 ° C for 4 hours to 24 hours, preferably 37 ° C for 4 hours to obtain a phosphate buffer. After washing with physiological saline, this was labeled with enzyme-labeled antibody IgG or its F
(ab ′) ₂ , preferably with Fab ′ at 4 ° C. to 37 ° C. for 4 hours
After reacting for 12 hours, preferably at 20 ° C. for 4 hours, after washing with phosphate buffered saline, the enzyme activity bound to the solid phase was measured. Alkaline phosphatase as a labeling enzyme,
There are β-D-galactosidase, peroxidase and the like, and peroxidase is suitable. As the enzyme substrate, for example, in the case of peroxidase, a coloring substrate such as 5-aminosalicylic acid and O-phenylenediamine, a fluorescent substrate such as 3- (P-hydroxyphenyl) propionic acid, and H ₂ O ₂ as a substrate, A luminescent substrate such as luminol is used, among which a fluorescent substrate is suitable.

In addition, a solid phase carrier in which an antibody is insolubilized, an antigen, an enzyme-labeled antibody
It is also possible to carry out a one-step method in which Fab ′ is reacted at the same time to perform the measurement.

The hapten assay method according to the present invention can be performed in the same manner as above, but in the case of the hapten, in the step (C) according to the present invention, a conventionally known immunological assay method, for example, competitive binding immunoassay is performed. You can use the method.

〔Example〕

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

Example 1 Measurement of Insulin 0.15 ml of a buffer solution (0.1 mol / l N) was added to 0.005 ml of various human sera.
10 mmol / l Na phosphate, pH 7.0) containing aCl and 1 g / l BSA was added, and 0.01 ml of 3 mol / l HCl was added to this to adjust the pH to about 1
And then left for 60 minutes at room temperature. 0.01 ml of this 2.95
Add mol / l NaOH to neutralize, and add 0.01 ml of buffer solution (1.8 m
0.03 mol / l Na phosphate, pH 7.0) containing ol / l NaCl, 3 g / l BSA and 15 g / l NaN ₃ was added. Insulin in 0.15 ml of the sample prepared in this way was measured by the known Sangertian enzyme immunoassay [K-h, luan (K-h,
Ruan) et al., Clin.Chim.Act
a) Vol. 147, p. 167 (1985)]. In other words, sample 0.15m
Mix l with anti-insulin antibody conjugated polystyrene poles, 3
The reaction was carried out at 7 ° C for 4 hours. The anti-insulin antibody-bonded polystyrene balls were prepared as follows.

The anti-insulin antibody was DEAE-deposited by salting out guinea pig-derived anti-insulin antiserum (National Institute of Medical Biology, Nagoya) with Na ₂ SO _4.
Purification by cellulose column chromatography gave anti-insulin antibody IgG [E. Ishikawa et al., Journal of Immunoassay, Vol. 4, page 209 (1983)]. This anti-insulin antibody IgG was physically adsorbed and insolubilized on polystyrene balls [diameter 3.2 mm (Precision Plastic Ball, Chicago)] [E. Ishikawa and K. Kato, Scandinavian Journal]・ Immunology (Scand.J.Immu
nol.), Vol. 8 (Appendix 7), p. 43 (1978)]. Anti-insulin antibody-bonded polystyrene balls reacted with the above sample were added to 10 mmol / l Na phosphate solution containing 0.1 mol / l NaCl (pH 7.
0) After washing with 2 ml, 5 ng of affinity purified anti-insulin Fab'-horseradish peroxidase complex solution 0.15 m
The reaction was carried out for 4 hours at 20 ° C. Peroxidase-labeled anti-insulin Fab 'was prepared as follows. That is, F (ab ') ₂ and then Fab' were prepared from the guinea pig-derived anti-insulin antibody IgG prepared as described above (see E.
Ishikawa et al., Zynar of Immunoassay), using horseradish-derived peroxidase (grade I, Peeringer Mannheim, Mannheim) and N-succinimidyl-6-maleimidohexanoate (Dojindo Laboratories, Kumamoto). After joining, [S.Hashda (S.Hash
ida), Journal of Applied Biochemistry (J. Appl. Biochem.), Vol. 6, page 56 (198).
4)], Insulin-Sepharose 4B affinity-column chromatography, which was obtained by coupling mercaptosuccinylated insulin to activated thiol Sepharose 4B (Pharmacia Huaine Chemicals AB, Uppsala), and further purified by Ultrogel AcA44 (LKB, Stoskform). Purification was performed to obtain a peroxidase-labeled anti-insulin Fab '(see Khluan et al., Clinica Kimika Actor, supra). In addition, the buffer used in the ink labeling of the enzyme-labeled antibody was 0.1 mol / l NaCl and 0.1% BS.
It is a 10 mmol / l sodium phosphate solution (pH 7.0) containing A.

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

Peroxidase activity was measured at 30 ° C. using H ₂ O ₂ and 3- (p-hydroxyphenyl) propionic acid (Aldrich Chemical Company, Inc., Milwaukee) as substrates [Imakawa et al., Analytical Letters (Anal. Lett.)]. 16: 1509 (198
3)]. The fluorescence intensity is 1mg quinine 1 0.05mol / l H ₂ SO ₄
It is expressed as the relative intensity for the liquid dissolved in
The amount of insulin in the sample was determined from a calibration curve using standard insulin, which was measured using a Shimadzu spectrofluorometer (RF-510 Shimadzu Corporation, Kyoto) at 0 nm and a fluorescence wavelength of 405 nm. Table 1 shows the measurement results of 15 serum samples. That is, Table 1 shows a comparison of the measurement results between the method of the present invention and the conventional method (treatment with acid / polyethylene glycol) without pretreatment, using serum with and without anti-insulin antibody.

Comparative Example 1 Insulin was measured by a method combining a conventional acid treatment and a polyethylene glycol method. That is, 0.05 ml of various human sera was mixed with 0.01 ml of 1 mol / l HCl, and the mixture was allowed to stand at room temperature.
After standing for 60 minutes, mix with 0.07 ml of 250 g / l polyethylene glycol and add 0.01 ml of 1 mol / l NaOH, then 1500xg
Centrifugation at 45 minutes (see S. Nakagawa et al., Deiapetes, supra). 0.01 ml of this supernatant was added to 0.14 ml of buffer solution (0.42 mol / l
10 mmol / l N phosphate containing NaCl, 1.1 g / l BSA, 1.1 g / l NaN ₃
a, pH 7.0) and insulin was measured as above after mixing. The results are shown in the column of acid / polyethylene glycol treatment in Table 1.

As is apparent from Table 1, the measuring method according to the present invention performed in Example 1 can obtain the same result as the conventional method very easily as compared with the conventional method.

Comparative Example 2 Insulin was measured simply by the enzyme immunoassay described in Example 1 without pretreatment of serum samples. The results are shown in Table 1.
Column without pretreatment. From this result, in the case where there is no anti-insulin antibody in the serum sample,
The same results as in Comparative Example 1 are obtained, but in the presence of the anti-insulin antibody, all samples show clearly lower values than those in Example 1 and Comparative Example 1, and the amount of antigen cannot be accurately measured in the presence of the antibody. Is shown.

As described above, from the results of Example 1, Comparative Example 1 and Comparative Example 2, the method of the present invention performed in Example 1 is simpler than the conventional method.
It is shown that the antigen can be accurately measured even in samples containing antibodies.

Example 2 Insulin determination 12.5 μl of 0.5 mol / l HC in 50 μl of various human serum samples
After adding 1 to adjust the pH to 3.5, 25 μl of the dextran-chaycor suspension was added to adsorb free insulin.
The mixture is allowed to react for 5 minutes with stirring and 12.5
The mixture was neutralized by adding μl of 0.38 mol / l NaOH, centrifuged at 1500 × g for 15 minutes, and the supernatant was centrifuged again in the same manner. 70 μl of this supernatant contains 2 μIU insulin 35
10 μl containing μl 1 g / l NaN ₃ , 1.0 g / l BSA, 0.1 mol / l NaCl
Ol / l sodium phosphate (pH 7.0) was added and reacted at 4 ° C for 24 hours. 15 μl of this reaction solution was added to 105 μl of 1 g / l BSA, 0.1
10 mmol / l Na phosphate (pH 7.0) containing mol / l NaCl was added.
Insulin in the sample thus prepared was measured by the method of the present invention as in Example 1. Table 2 shows the measurement results of 15 serum samples and the recovery rate of added insulin.
(The method of the present invention).

In addition, the results obtained by measuring various samples not subjected to the 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 the anti-insulin antibody was not present in the serum sample, similar recoveries were obtained by the method of the present invention and the method without pretreatment. However, in the sample in which the anti-insulin antibody is present, the recovery rate of the method without pretreatment is lower than that of the method of the present invention. That is, when measured by the method of the present invention 94 ~
It shows a good recovery rate of 102%, but when measured without pretreatment, the recovery rate is 39 to 104%, indicating that an accurate amount of antigen cannot be measured in the presence of the antibody.

From the above results, it is shown that the method of the present invention can accurately measure the antigen in the sample containing the antibody.

〔The invention's effect〕

As described above in detail, according to the method of the present invention, the amounts of many kinds of antigens and haptens can be measured in the presence of an antibody much more easily than the conventional methods.

Claims (1)

[Claims] 1. The following steps (A), (B) and (C) (A) An acid is added to a test solution containing an antigen or a hapten and its antibody so that the antigenicity is not impaired, but the antigen or the hapten. Of inactivating an antibody that affects the immunological measurement of E. coli under acidic conditions of pH 3.5 or lower (B) Neutralizing the test solution (C) Amount of antigen or hapten in the test solution A method for measuring an antigen or a hapten, which comprises the step of immunologically measuring