JPH081438B2 - Enzyme immunoassay - Google Patents

Description

TECHNICAL FIELD The present invention relates to an enzyme immunoassay method.

[Prior Art] Conventionally, as an enzyme immunoassay, a method in which an antigenic substance bound to a first antibody on an insoluble solid is reacted with a second antibody labeled with an enzyme and the amount of the enzyme is measured (a so-called sandwich method) ) [For example, Eiji Ishikawa, Tadashi Kawai, Kiyoshi Miyai, "Enzyme-linked immunosorbent assay", Medical Institute P45 (1982)].

[Problems to be Solved by the Invention] However, with this technique, when the antibody titer of the second antibody is low, sufficient measurement sensitivity cannot be obtained.

[Means for Solving the Problem] The present inventors have conducted extensive studies on enzyme immunoassays that provide sufficient measurement sensitivity even when a second antibody having a low antibody titer is used, and as a result, the present invention Arrived

That is, the present invention relates to carcinoembryonic antigen (CEA) and thyroid stimulating hormone (TSH).
An antibody on an insoluble solid that recognizes an antigenic substance selected from (first antibody), an antigenic substance selected from the CEA and TSH, and the antigenic substance derived from an animal species different from the first antibody An immune complex obtained by reacting an antibody (second antibody) at the same time and an immunoglobulin derived from the same animal species as the first antibody, which is labeled with peroxidase, are derived from the same animal species as the second antibody. An enzyme immunoassay method characterized in that the antigenic substance is quantified by reacting with a peroxidase-labeled antibody (third antibody) that recognizes, then reacting with the peroxidase-labeled antibody, and then measuring the enzyme amount. Is.

The present invention is a method for measuring an antigenic substance selected from carcinoembryonic antigen (CEA) and thyroid stimulating hormone (TSH). CEA and TSH are antigenic substances that require particularly sensitive measurement systems.

Examples of the insoluble solid include siliceous inorganic carriers [glass (porous glass, matte glass, etc.), silica gel, bentonite, etc.], magnetic materials, and organic carriers (plastic, dextran, mouth paper, etc.). Of these, preferred are glass (glass beads and glass test tubes) and plastic (plastic tubes and plastic trays), which are simple and stable to which antibodies can be bound and which are easy to handle.

The first antibody is a double-cloned antibody obtained by immunizing a mammal such as rabbit, goat, sheep, or guinea pig with the above antigenic substance by a conventionally known method.
Alternatively, there is a mouse-derived monoclonal antibody obtained by culturing or ascites the hybridoma cells obtained by cell fusion of antibody-producing cells (derived from mouse) against the above-mentioned antigenic substance and myeloma cells (derived from mouse). These antibodies are used after being purified by a conventionally known purification method such as ammonium sulfate, DEAE-cellulose chromatography, affinity chromatography and the like.

As a method of binding the first antibody on the insoluble solid,
A method of chemically bonding glass and an antibody (using a silane coupling agent and optionally a crosslinking agent) (for example, JP-A-54-108696) or a physical adsorption method (for example, US Pat. No. 4,280,992 and No. 3652761), or a method of physically adsorbing an antibody to a plastic [eg, E. Engbar, J. Johnson, P. Perlman; Biokim. Buy office.
Actor (E. Engvall, J. Johnson, P. Parlmann; Biochim. Bio
phys.Acta), 251 (1971) 427-434].

The second antibody is a monoclonal antibody that recognizes the above-mentioned antigenic substance derived from an animal species different from that of the first antibody (for example, when the first antibody is a rabbit, an antibody such as goat, sheep or guinea pig; or the first antibody is In the case of sheep, antibodies such as rabbits, goats, and guinea pigs); or, in the case where the first antibody is a monoclonal antibody that recognizes the above antigenic substance derived from an animal species other than mouse, antibody-producing cells (derived from mouse) against the above antigenic substance And a myeloma cell (derived from mouse), a monoclonal antibody obtained by culturing or ascites the hybridoma cell obtained by cell fusion. The form of the second antibody includes antiserum, hybridoma culture solution, ascites,
Alternatively, there are immunoglobulins obtained by purifying these.

The immunoglobulin concentration of the second antibody can take various values depending on the concentration of the antigenic substance and the concentration of the peroxidase-labeled antibody in the test sample, but is usually 0.1 to 1000 μg / ml, preferably 1 to 200 μg / ml. .

The immune complex can be obtained by a method of simultaneously reacting the first antibody on the insoluble solid, the antigenic substance, and the second antibody. For example, by incubating an insoluble solid bound with a first antibody, a test sample containing an antigenic substance, and a second antibody-containing buffer at the same time,
An immune complex can be obtained. Incubation is carried out under normal conditions (eg 5 to 50 ° C, preferably 30 to 40 ° C).
The temperature may be 5 ° C to 2 days, preferably 5 to 30 minutes).

After incubation, wash solution (eg distilled water,
Add physiological saline, phosphate buffer, etc.) and then aspirate the washing solution with an aspirator. By performing this operation several times (for example, 2 to 5 times), the immune complex can be separated from the unreacted substance.

As the third antibody, an antibody that recognizes an immunoglobulin derived from the same animal species as the second antibody [for example, when the second antibody is a rabbit, an anti-rabbit immunoglobulin antibody (guinea pig); when the second antibody is a goat antibody, Anti-goat immunoglobulin antibody (mouse); or if the second antibody is mouse,
Anti-mouse immunoglobulin antibody (rabbit) and the like].

As a combination of the first antibody, the second antibody, and the third antibody, nonspecific adsorption is small, and thus the first antibody and the third antibody are derived from the same animal species. For example, a first antibody rabbit, a second antibody mouse monoclonal, and a third antibody rabbit; a first antibody guinea pig, a second antibody sheep, and a third antibody guinea pig; a first antibody sheep, a second antibody rabbit, and a third antibody. Sheep; first antibody goat, second antibody mouse monoclonal, and third antibody goat. Particularly preferred combination is, because of its high specificity for an antigenic substance, a first antibody rabbit, a second antibody mouse monoclonal clone, and a third antibody rabbit; or a first antibody goat, a second antibody mouse monoclonal clone, And the third antibody goat.

The reason why peroxidase is used as the enzyme for labeling the third antibody is that antibody labeling is easier and higher sensitivity can be obtained than when alkaline phosphatase or β-galactosidase is used.

Labeling of the third antibody with peroxidase can be performed by a conventional method, for example, S. YOSHITAKE, M. IMAGAWA, E.ISHIKAWA, et. al., J. Bioche
m.,) 92 (1982) 1413-1424.

In the present invention, the reaction between the immune complex obtained by the simultaneous reaction and the peroxidase-labeled antibody can be performed by a usual method. For example, the immunocomplex is transferred to a peroxidase-labeled antibody solution and incubated. Incubation can be performed under usual conditions (for example, a temperature of 5 to 50 ° C, preferably 30 to 40 ° C, 5 minutes to 2 days, preferably 5 to 30 minutes).

The reaction product of the above immunocomplex and the peroxidase-labeled antibody is washed with a washing solution (for example, distilled water, physiological saline, phosphate buffer, etc.), and then the substrate [for example, 5-aminosalicylic acid, o-phenylenediamine, ABTS [2,2'-azinodi (3-ethylbenzthiazoline) -6'-sulfonic acid], etc., preferably o-phenylenediamine]
Incubate. Incubation can be performed under normal conditions (for example, a temperature of 5 to 50 ° C., preferably 30 to 40 ° C., 5 minutes to 1 hour, preferably 5 to 30 minutes). After incubation, a reaction stop solution (for example, sulfuric acid, hydrochloric acid, etc.) is added, and the enzyme activity of peroxidase is quantified by the absorbance.

[Examples] The present invention is further described below with reference to Examples and Comparative Examples, but the present invention is not limited thereto.

Example 1 [Measurement of Carcinoembryonic Antigen (CEA)] (a) Preparation of Human CEA Standard Solution High CEA obtained by perchloric acid extraction method from colorectal cancer liver metastases was used as WHO CEA International Reference.
CE manufactured by Dynabot, using the standard (63/701)
The concentration was assayed with the A measurement kit "CEA-EIA", and the concentration was adjusted to 5, 10, 30 and 60 ng / ml with 0.02M phosphate buffer.

(B) Production of anti-CEA monoclonal antibody Mice (Balb / c) were immunized with a high concentration of human CEA. After 6 weeks, a cell suspension was prepared from the spleen and then
About 1 × 10 ⁸ cells of spleen and about 2 × 10 ⁷ mouse myeloma (myeloma) cells were fused by PEG treatment. The fused cells were cultured in HAT medium, and the target antibody-producing cells (hybridomas) were selected by screening.
This hybridoma was grown as a single cell group (single clone) by cloning, and then the single clone was subjected to ascites in mice. By purifying the obtained ascites, highly specific anti-CEA monoclonal immunoglobulin was prepared.

(C) Preparation of peroxidase-labeled anti-mouse immunoglobulin antibody An anti-mouse immunoglobulin antibody (manufactured by Dako) was bound to peroxidase by the method described in the above-mentioned document [J. Biochem, 92 (1982) 1413-1424] to give peroxidase. Labeled anti-mouse immunoglobulin antibody was obtained. This reagent was usually diluted with a buffer containing 1% bovine serum albumin 10 to 5000 times before use.

(D) Preparation of Anti-CEA Double Clone Antibody-Bound Glass Beads According to the method described in US Pat. No. 3652761, the surface of glass beads was coated with an anti-CEA double clone (rabbit) antibody (manufactured by Dako).

(E) Quantification of human CEA In vitro, anti-CEA double clone (rabbit) antibody-bonded glass beads (1) and human CEA standard solution (60 ng / ml, 50 μl)
After incubation in 300 μl of 0.02 M phosphate buffer containing 10 to 100 μg / ml of CEA monoclonal antibody (37 ° C., 15 minutes), the beads were washed with distilled water. Next, the beads were transferred to 300 μl of peroxidase-labeled anti-mouse immunoglobulin antibody solution and incubated (37 ° C, 15 minutes).
did. After washing the beads again with distilled water, the beads were incubated (37 ° C., 15 minutes) in 400 μl of the substrate solution (hydrogen peroxide-containing ortho-phenylenediamine solution). Next, the reaction was stopped by adding 3 ml of 1.5 N sulfuric acid. The absorbance of this solution at 492 nm was measured to quantify the enzyme activity of peroxidase bound to the beads.

Similarly, the enzyme activity was measured when human CEA standard solutions 30, 10, 5 and 0 ng / ml were used to prepare a calibration curve.

FIG. 1 is a calibration curve for human CEA measurement by this measurement method.

The measurement sensitivity, measurement area, and measurement accuracy were as follows.

Measurement sensitivity 1.0ng / ml Measurement area 0-60ng / ml Measurement accuracy 5.0 ± 0.4ng / ml (Coefficient of variation 8.0%) 38.2 ± 2.2ng / ml (Coefficient of variation 5.8%) Comparative Example 1 [Human CEA by conventional sandwich method Measurement] For comparison with Example 1, human CEA was measured by the conventional side-itch method.

(A) Preparation of human CEA standard solution Same as in Example 1 (a).

(B) Production of Anti-CEA Monoclonal Immunoglobulin Same as in Example 1 (b).

(C) Preparation of Peroxidase-Labeled Anti-CEA Monoclonal Immunoglobulin According to Example 1 (c), the anti-CEA monoclonal immunoglobulin was bound to peroxidase to prepare a peroxidase-labeled anti-CEA monoclonal immunoglobulin.
This reagent is usually a 10% buffer containing 1% bovine serum albumin.
It was diluted 5000 times before use.

(D) Preparation of anti-CEA double-cloned antibody-bound glass beads Same as in Example 1 (d).

(E) CEA quantification In a test tube, anti-CEA double clone (rabbit) antibody-bound glass beads (1) and human CEA standard solution 60 ng / ml, 50 μl 0.02
Incubation in 200 μl of M phosphate buffer (37 ℃, 1
After 5 minutes), the beads were washed with distilled water. Next, the beads were transferred to 300 μl of peroxidase-labeled anti-CEA monoclonal immunoglobulin solution and incubated (37 ° C., 15 minutes). After washing the beads again with distilled water, the beads were washed with a substrate solution (hydrogen peroxide-containing ortho-
Incubation (37 ° C., 15 minutes) in 400 μl of phenylenediamine solution). Next, the reaction was stopped by adding 3 ml of 1.5 N sulfuric acid. The absorbance of this solution at 492 nm was measured to quantify the enzyme activity of the enzyme bound to the beads. Similarly, the enzyme activity was measured when human CEA standard solutions 30, 10, 5 and 0 ng / ml were used to prepare a calibration curve.

FIG. 2 is a calibration curve for human CEA measurement according to Comparative Example 1.

The measurement sensitivity, measurement area, and measurement accuracy were as follows.

Measurement sensitivity 4.5 ng / ml Measurement area 0-60 ng / ml Measurement accuracy 5.3 ± 0.9 ng / ml (variation coefficient 17.0%) 37.5 ± 5.3 ng / ml (variation coefficient 14.1%) Comparative Example 2 [Three antibody sequential reaction method Measurement of Human CEA] For comparison with Example 1, the reaction of the first antibody and the antigenic substance, the antigenic substance and the second antibody, and the reaction of the second antibody and the third antibody are successively performed to measure human CEA. went.

(A) Preparation of human CEA standard solution Same as in Example 1 (a).

(B) Production of Anti-CEA Monoclonal Immunoglobulin Same as in Example 1 (b).

(C) Preparation of peroxidase-labeled anti-mouse immunoglobulin antibody This was carried out according to Example 1 (c).

(D) Preparation of anti-CEA double-cloned antibody-bound glass beads Same as in Example 1 (d).

(E) CEA quantification In a test tube, anti-CEA double clone (rabbit) antibody-bound glass beads (1) and human CEA standard solution (60 ng / ml, 50 μl) were added 0.02
Incubation in 300 μl of M phosphate buffer (37 ℃, 1
After 5 minutes), the beads were washed with distilled water. Next, the beads were transferred to 300 μl of 0.02 M phosphate buffer containing 10 to 100 μg / ml of anti-CEA monoclonal antibody, incubated (37 ° C., 15 minutes), and then washed with distilled water. Further, the beads were transferred into 300 μl of peroxidase-labeled anti-mouse immunoglobulin antibody solution and incubated (37 ° C., 15 minutes). After washing the beads again with distilled water, the beads were incubated (37 ° C., 15 minutes) in 400 μl of the substrate solution (hydrogen peroxide-containing ortho-phenylenediamine solution). Next, 1.5 normal sulfuric acid 3m
The reaction was stopped by adding l. The absorbance of this solution at 492 nm was measured to quantify the enzyme activity of the enzyme bound to the beads.

Similarly, the enzyme activity was measured when human CEA standard solutions 30, 10, 5 and 0 ng / ml were used to prepare a calibration curve.

FIG. 3 is a calibration curve for human CEA measurement according to Comparative Example 2.

The measurement sensitivity, measurement area, and measurement accuracy were as follows.

Measurement sensitivity 3.9 ng / ml Measurement area 0-60 ng / ml Measurement accuracy 6.6 ± 0.9 ng / ml (variation coefficient 13.6%) 40.2 ± 5.8 ng / ml (variation coefficient 14.4%) Example 2 [of thyroid stimulating hormone (TSH)] Measurement] (a) Preparation of human TSH standard solution Human TSH obtained from UCB Bioproducts (50 μg /
Vial) diluted with 0.02M phosphate buffer, 50, 25, 5,
2.5 and 0 IU / ml human TSH standard solutions were prepared.

(B) Production of anti-TSH monoclonal antibody Using the above human TSH, an anti-human TSH monoclonal antibody was obtained according to the method of Example 1 (b).

(C) Preparation of anti-TSH double-cloned antibody-bound glass beads Anti-TSH double-cloned (rabbit) antibody (manufactured by Dako) was used to obtain anti-TSH double-cloned antibody-bound glass beads by the method of Example 1 (d). It was

(D) Quantification of human TSH In a test tube, 1 glass of anti-TSH double-cloned (rabbit) antibody-bound glass and 100 μl of human TSH standard 50 IU / ml solution were added to anti-TSH monoclonal antibody in an amount of 10 to 100 μg / ml and 0.02 M phosphoric acid After incubation in 200 μl of buffer (37 ° C., 30 to 60 minutes), the beads were washed with distilled water. Example 1
The beads were transferred to 300 μl of the peroxidase-labeled anti-mouse immunoglobulin antibody solution prepared in (c) and incubated (37 ° C., 30 to 60 minutes). After washing the beads again with distilled water, the beads were incubated (10 to 30 ° C., 30 minutes) in 400 μl of a substrate solution (ortho-phenylenediamine solution containing hydrogen peroxide). next,
The reaction was stopped by adding 3 ml of 1.5N sulfuric acid. 492n of this liquid
The absorbance of m was measured to quantify the enzyme activity of the enzyme bound to the beads.

Similarly, the enzyme activity was measured when human TSH standard solutions 25, 5, 2.5 and 0 IU / ml were used as test samples, and a calibration curve was prepared.

FIG. 4 is a calibration curve for human TSH measurement according to Example 2.

The measurement sensitivity, measurement area, and measurement accuracy were as follows.

Measurement sensitivity 2.0 IU / ml Measurement area 0-50 IU / ml Measurement accuracy 2.8 ± 0.2 IU / ml (variation coefficient 7.1%) 26.0 ± 1.4 ng / ml (variation coefficient 5.4%) Comparative Example 3 [Human TSH by conventional sandwich method Measurement] For comparison with Example 2, human TSH was measured by the conventional sandwich method.

(A) Preparation of human TSH standard solution Same as in Example 2 (a).

b) Production of Anti-TSH Monoclonal Immunoglobulin Same as in Example 2 (b).

(C) Preparation of Peroxidase-Labeled Anti-TSH Monoclonal Immunoglobulin According to Example 1 (c), the above anti-TSH monoclonal immunoglobulin was combined with peroxidase to prepare a peroxidase-labeled anti-TSH monoclonal immunoglobulin.
This reagent is usually 10% buffer containing 1% bovine serum albumin.
It was used after diluting ~ 5000 times.

(D) Preparation of anti-TSH double-cloned antibody-bound glass beads Same as in Example 2 (c).

(E) Quantification of TSH In a test tube, 1 glass of anti-TSH double-cloned antibody-bonded glass beads and 100 μl of human TSH standard 50 IU / ml solution in 200 μl of 0.02 M phosphate buffer containing 10-100 μg / ml of anti-TSH double-cloned antibody. After incubation (37 ° C., 60 minutes), the beads were washed with distilled water. Then peroxidase-labeled
The beads were transferred into 300 μl of TSH monoclonal immunoglobulin solution and incubated (37 ° C., 60 minutes). After washing the beads again with distilled water, the beads are used as a substrate solution (hydrogen peroxide-containing ortho-phenylenediamine solution).
Incubation (37 ° C., 30 minutes) in 400 μl.
Next, the reaction was stopped by adding 3 ml of 1.5 N sulfuric acid. The absorbance of this solution at 492 nm was measured to quantify the enzyme activity of the enzyme bound to the beads.

Similarly, the enzyme activity was measured using human TSH standard solutions 25, 5, 2.5 and 0 IU / ml, and a calibration curve was prepared.

FIG. 5 is a calibration curve for human TSH measurement according to Comparative Example 3.

The measurement sensitivity, measurement area, and measurement accuracy were as follows.

Measurement sensitivity 6.5 IU / ml Measurement area 0-50 IU / ml Measurement accuracy 2.6 ± 0.7 IU / ml (Variation coefficient 26.9%) 25.5 ± 4.3 IU / ml (Variation coefficient 16.9%) Comparative example 4 [Three antibody sequential reaction method Measurement of Human TSH] For comparison with Example 2, human TSH was measured by the conventional three antibody sandwich sequential reaction method.

(A) Preparation of human TSH standard solution Same as in Example 2 (a).

(B) Production of anti-TSH monoclonal antibody This was carried out according to Example 2 (b).

(C) Preparation of anti-TSH double-cloned antibody-bound glass beads Same as in Example 2 (c).

(D) Quantification of human TSH 1 tube of anti-TSH double clone (rabbit) antibody-bound glass beads and 100 μl of human TSH standard 50 IU / ml solution were placed in a test tube.
Incubation in 200 μl of 0.02 M phosphate buffer (37
Then, the beads were washed with distilled water.
Next, the beads were transferred to 300 μl of 0.02 M phosphate buffer containing 10-100 μg / ml of anti-TSH monoclonal antibody, incubated (37 ° C., 30 minutes), and then washed with distilled water. Furthermore, the beads were transferred to 300 μl of the peroxidase-labeled anti-mouse immunoglobulin antibody solution prepared in Example 1 (c) and incubated (37 ° C., 30 minutes). After washing the beads again with distilled water, the beads were incubated in 400 μl of the substrate solution (hydrogen peroxide-containing ortho-phenylenediamine solution) (37 ° C, 30 minutes).
did. Next, the reaction was stopped by adding 3 ml of 1.5 N sulfuric acid.
The absorbance of this solution at 492 nm was measured to quantify the enzyme activity of the enzyme bound to the beads.

Similarly, the enzyme activity was measured when human TSH standard solutions 25, 5, 2.5 and 0 IU / ml were used as test samples, and a calibration curve was prepared.

FIG. 6 is a calibration curve for human TSH measurement according to Comparative Example 4.

The measurement sensitivity, measurement area, and measurement accuracy were as follows.

Measurement sensitivity 5.7 IU / ml Measurement area 0-50 IU / ml Measurement accuracy 3.0 ± 0.6 IU / ml (Variation coefficient 20.0%) 28.2 ± 4.7 IU / ml (Variation coefficient 16.6%) [Effect of the invention] In the conventional side-itch method When the antibody titer of the enzyme-labeled secondary antibody was low, both the measurement sensitivity and accuracy were low, and normal measurement was impossible. Therefore, an attempt was made to measure by the three-antibody sequential method, but although the sensitivity was slightly increased, it could not be an effective measuring method.

According to the assay method of the present invention, sufficient assay sensitivity can be obtained even when the antibody titer of the secondary antibody is low. For example, high sensitivity can be obtained even when the second antibody is an unpurified antibody such as antiserum, culture solution or ascites fluid. Further, when a monoclonal antibody is used as the second antibody, it is possible to perform highly specific measurement with respect to the target antigenic substance.

From the above points, the present invention can be applied to diagnostic reagents for CEA and TSH, which are antigenic substances for which particularly sensitive measurement systems are required.

[Brief description of drawings]

Figures 1, 2 and 3 show the calibration curve for human CEA measurement,
FIG. 4, FIG. 5 and FIG. 6 are calibration curves of human TSH.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP 60-187861 (JP, A) JP 59-163565 (JP, A) JP 61-40568 (JP, A)

Claims (3)

[Claims] 1. An antibody (first antibody) on an insoluble solid that recognizes an antigenic substance selected from carcinoembryonic antigen (CEA) and thyroid stimulating hormone (TSH), the antigenic substance selected from CEA and TSH, And an immune complex obtained by simultaneously reacting an antibody (second antibody) that recognizes the antigenic substance derived from an animal species different from the first antibody, and the same animal as the first antibody labeled with peroxidase The antigenic substance is quantified by reacting with a peroxidase-labeled antibody (a third antibody) that is a species-derived antibody that recognizes immunoglobulin derived from the same animal species as the second antibody, and then measures the amount of peroxidase. An enzyme immunoassay method characterized by: 2. The assay method according to claim 1, wherein the second antibody is a monoclonal antibody. 3. The measuring method according to claim 1 or 2, wherein the insoluble solid is glass or plastic.