JP3353133B2 - Antibodies and assays using them - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to an antibody and a measuring method using the same. More specifically, the present invention relates to human chromogranins A.
The present invention relates to a novel antibody (particularly, antiserum) having specific reactivity to CgA), and a method for measuring and detecting human chromogranin A using the novel antibody.

[0002]

2. Description of the Related Art Recently, several documents have been published by Konecki,
DS et al., J. Biol. Chem. 262, 17026-17030, 1987 and Mouland, AJ et al., J. Biol. Chem. 269, 6918-6926,
According to 1994, the 344-374 sequence on the amino acid sequence of human chromogranin A forms a sequence of amino acid residues represented by the following formula (I) EEEED NRDSS MKLSF RARAY GFRGP GPQLRR (I) as a result of structural analysis of the gene. It has been reported that the peptide is presumed to be a peptide.

[0003]

SUMMARY OF THE INVENTION Human chromogranin A is a soluble acidic glycoprotein present in chromaffin granules in human chromaffin cells. And today,
The relationship between this and human diseases (eg, diseases of the sympathetic nervous system) has been studied. The present invention has been made based on such a background, and an object of the present invention is to recognize human chromogranin A, in particular, by recognizing the above-mentioned 344-374 sequence, thereby to make specific reactivity to human chromogranin A. Antibodies, specifically antisera, monoclonal antibodies, polyclonal antibodies and I antibodies having such specificity.
gY. Another object of the present invention is to
It is an object of the present invention to provide a method for specifically measuring and detecting human chromogranin A and its related peptide using the above antibody.

[0004]

Means for Solving the Problems The present invention specifically provides a peptide having the amino acid residue sequence represented by the following formula (I): EEED NRDSS MKLSF RARAY GFRGP GPQLRR (I) as an antigen, and excluding humans. By immunizing blood animals,
The present invention relates to antibodies produced and obtained and having specific reactivity to human chromogranin A. Further, the present invention provides a method for immunizing a warm-blooded animal other than a human using a peptide having an amino acid residue sequence represented by the following formula (II): NRDSS MKLSF RARAY GFRGF GPQLRR (II) as an antigen.
The present invention relates to antibodies produced and obtained and having specific reactivity to human chromogranin A. Where:
Each symbol shown in the sequences of (I) and (II) represents the next amino acid residue, respectively. The configuration of these amino acid residues is D, L or DL, but is usually in the L-form. E: Glutamic acid (Glu) D: Aspartic acid (Asp) N: Asparagine (Asn) R: Arginine (Arg) S: Serine (Ser) M: Methionine (Met) K・ ・ ・ Lysine (Lys) L ・ ・ ・ Leucine (Leu) F ・ ・ ・ Phenylalanine (Phe) A ・ ・ ・ Alanine (Ala) Y ・ ・ ・ Tyrosine (Tyr) G ・ ・ ・ Glycine (Gly) P ・ ・-Proline (Pro) Q ... Glutamine (Gln) The immunity in the present invention is humoral immunity, and the obtained antibodies include antisera, monoclonal antibodies, polyclonal antibodies, and general antibodies such as IgY. .
These antibodies are used to measure and detect chromogranin A in human tissues, blood, urine, spinal fluid and saliva by various immunoassays.
Further, it can be an effective means for measuring and detecting a peptide related thereto, and is useful for elucidation and study of a stimulus-secretion mechanism. Therefore, the present invention also relates to a method for specifically measuring and detecting human chromogranin A and its related peptide, which is subjected to immunoassay using the above antibody.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION The antiserum according to the present invention comprises, for example, a peptide represented by the following formula (III) or (IV): H-EEEEEDNRDSSMKLSF RARAYGFRGGPGPQLRR-OH (III) Used as an antigen, produced by immunizing warm-blooded animals such as rabbits, sheep and guinea pigs, and obtained from the blood of the warm-blooded animal. The antiserum obtained has specific reactivity to the peptides of the above formulas (III) and (IV), and is characteristically characterized by a specific reactivity to human chromogranin A. Having.

[0006] The antibody according to the present invention including such an antiserum can be obtained, for example, by the following procedure. (1) First, the peptides represented by the above formulas (I) and (II) are synthesized by a solid phase method or a liquid phase method, and then the obtained crude product is subjected to 95% by reverse phase liquid chromatography.
Purify to the above purity. According to the solid phase method, a method in which amino acids are introduced one by one from the amino acid residue at the carboxyl terminus of the peptide to be synthesized and the peptide chain is successively extended is suitably employed. In this case, the use of a peptide synthesizer is very convenient. In the above synthesis,
Trityl chloride, tert.-butyl ester, etc. are used as the protecting group for the carboxyl group not involved in the reaction,
Further, as a protecting group for an amino group not involved in the reaction, ter
A t.-butyloxycarbonyl group or the like is used. (2) Next, the purified peptide is adjusted to an antigen for immunization by, for example, the following methods (i) to (iii). (i) water-soluble carbodiimide, for example, 1-ethyl-3-
(3-dimethylaminopropyl) carbodiimide hydrochloride (ECDI) or dimethylsuberimidate (DM
A method in which a peptide is bound to a carrier such as a serum protein by the action of a condensing agent such as S), and this is used as an antigen. (ii) glutaraldehyde, bis-diazonated benzidine,
Alternatively, a method for preparing a high molecular antigen by forming a bridge between the peptide and the carrier using a bifunctional reagent such as p-diazonium phenylacetic acid. (iii) A method in which a peptide is adsorbed on fine particles of carbon or an inert polymer such as polyvinylpyrrolidone, and this is used as an antigen. (3) Next, by immunizing a warm-blooded animal with the antigen thus obtained, an antibody is produced in a body fluid such as blood of the warm-blooded animal or in an egg laid by the warm-blooded animal, and get. For example, in the case of warm-blooded animals such as rabbits, sheep, and guinea pigs, the above-mentioned antigen is usually immunized to the warm-blooded animal multiple times with Freund's complete adjuvant to produce an antiserum, and the blood And collect antiserum according to a conventional method. In the case of a chicken, the chicken is immunized a plurality of times with the above antigen to produce IgY in the egg laid by the chicken, and crude IgY is collected from the egg yolk of the chicken according to a conventional method. To obtain IgY. Alternatively, a mouse is immunized multiple times with the above antigen together with Freund's complete adjuvant to produce an antibody, and the antibody-producing cells are fused with myeloma cells according to a cell fusion method, and cloning is performed. Monoclonal antibodies are obtained by isolating cells of a single clone producing the desired antibody.

[0007] Thus, the antiserum, IgY and monoclonal antibody of the present invention obtained by the above-described procedure can be obtained by combining all or all of the amino acid residue sequences represented by the above formulas (I) and / or (II). A peptide containing a portion thereof and a property specifically reacting with human chromogranin A, and therefore, a human chromogranin A and a human chromogranin A-related peptide containing the sequences of the above formulas (I) and (II) Targeted immunoassays (eg, radioimmunoassay (RIA), radioimmunometric assay (IRMA), enzyme immunoassay (EIA), enzyme immunometric assay (ELISA), fluorescence immunoassay (FIA), laser-based immunoassays, etc.) Highly sensitive measurement / detection becomes possible.

[0008]

The present invention will be described in more detail with reference to the following examples. However, these examples are non-limiting and the scope of the invention should not be construed as being limited by these examples. In the following examples, unless otherwise specified, the configuration of the amino acid residue shows an L-configuration. The following abbreviations are used: Fmoc: 9-fluorenylmethoxycarbonyl group tBoc: tert.-butoxycarbonyl group DMF: N, N-dimethylformamide Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl group OtBu: tert -Butyl ether group tBu: tert.-butyl group Trt: trityl group HATU: 7-azabenzotriazol-1-yl-
1,1,3,3-tetramethyluronium hexafluorophosphate

-Peptide synthesis- Example 1: H-EEEEEDNRDSSMKLSFFRARAY
Synthesis of GFRGPGPQLRR-OH (I) The peptide synthesis was carried out as follows by an automatic operation according to a solid phase method using a peptide synthesizer. First, 0.77 g of Fmoc-L-Arg (Pbf) -polyethylene glycol-resin was placed in a reactor of a peptide synthesizer (9050 Plus Pepsynthesizer, USA), and amino acids were sequentially bonded to each residue one by one to obtain a target. Was synthesized. One cycle of the synthesis includes the following operating procedures. 1) Washing with DMF (1 minute) 2) Removal of protecting group (6 minutes) 3) Washing with DMF (7 minutes) 4) Activation of Fmoc-amino acid by HATU 5) Coupling (condensation) of Fmoc-amino acid ) Reaction (60 minutes) 6) Washing with DMF (4 minutes) During the synthesis, the inside of the reactor was kept under a nitrogen atmosphere by purging with nitrogen to minimize oxidation by air. . In addition, procedure 2), that is, elimination of the protecting group was carried out by treating in a 20% piperidine solution in DMF for 6 minutes. For procedures 4) and 5), successive coupling of each Fmoc-amino acid (0.4 mmol) was performed in DMF in the presence of HATU (0.4 mmol) for 60 minutes. At that time, Fmoc-
The following protected amino acids were used as amino acids. F
moc-Ala, Fmoc-Arg (Pbf), Fmo
c-Met, Fmoc-Asn (Trt), Fmoc-
Asp (OtBu), Fmoc-Gln (Trt), F
moc-Glu (OtBu), Fmoc-Leu, Fm
oc-Lys (tBoc), Fmoc-Gly, Fmo
c-Phe, Fmoc-Pro, Fmoc-Ser (t
Bu), Fmoc-Tyr (tBu). Thus, 1.41 g of the desired protected peptide-resin was obtained. After completion of the synthesis, protected peptide-resin (1.01 g) was added to m-cresol (0.6 ml), ethanedithiol (1.8 m).
l), treated in the presence of a mixture of thioanisole (3.6 ml) and trifluoroacetic acid (24.0 ml) at room temperature for 2 hours, after which the resin is removed by filtration and the trifluoroacetic acid is then reduced in vacuo. Distilled off under. Upon addition of dry diethyl ether to the residue, the peptide precipitates.
This was further centrifuged at 0 ° C., and the precipitated peptide was washed with diethyl ether. By repeating this series of operations, a crude peptide (201 mg) was obtained. Thereafter, the obtained crude peptide was purified by reverse-phase high performance liquid chromatography (HPLC). The column used was YMC-Pack-D-ODS-5 (20 ×
250 mm). The elution was performed using 0.01 N HCl /
The measurement was performed under the condition of a linear concentration gradient of CH ₃ CN from 83/17 to 75/25 (flow rate: 7 ml / min, detection: 230 nm). Then, the purity of the obtained purified peptide was tested by analytical reverse-phase HPLC. The implementation conditions are as follows. Column: YMC-Pack-R-ODS-5 (4.6 ×
Solvent: 0.01 N HCl / CH ₃ CN DC concentration gradient: gradient from 95/5 to 40/60 Flow rate: 1.0 ml / min Detection: 210 nm The purified peptide of interest is a sharp single at 13.12 minutes Eluted as a peak. In addition, the amino acid residue sequence was assayed using a sequencer (Type 477A, Applied Biosystems, USA). As a result, it was confirmed that the target product was a peptide having the amino acid residue sequence of the title (I). Was done.

Example 2: H-NRDSSMKLSFFRARA
Synthesis of YGFRGFGPQLRR-OH (II) This peptide was synthesized by the peptide synthesis apparatus used in Example 1 in the same manner as in Example 1 by an automatic operation according to the solid phase method. That is, the same amount of Fmo was added to the reactor of the synthesis apparatus.
c-L-Arg (Pbf) -polyethylene glycol-
The resin was added, and amino acids were sequentially bonded to the resin one residue at a time to synthesize a target peptide chain. As the Fmoc-amino acid, the same protected amino acid as in Example 1 was used except for the protected amino acid of Fmoc-Glu (OtBu). The operating procedure of the synthesis cycle and other conditions for each synthesis are the same as in Example 1. Thus, 1.30 g of the desired protected peptide-resin was obtained. After completion of the synthesis, the protected peptide-resin (1.00 g) was added to m-cresol (0.6 ml) and ethanedithiol (1.8 m).
l), treated in the presence of a mixture of thioanisole (3.6 ml) and trifluoroacetic acid (24.0 ml) at room temperature for 2 hours, after which the resin is removed by filtration and the trifluoroacetic acid is then reduced in vacuo. Distilled off under. Upon addition of dry diethyl ether to the residue, the peptide precipitates.
This was further centrifuged at 0 ° C., and the precipitated peptide was washed with diethyl ether. By repeating this series of operations, a crude peptide (190 mg) was obtained. Thereafter, the obtained crude peptide was purified by reverse-phase high-performance liquid chromatography (HPLC) according to the same procedure and conditions as in Example 1. Then, the purity of the obtained purified peptide was tested by analytical reverse-phase HPLC. The implementation conditions are the same as in Example 1. The target purified peptide eluted as a sharp single peak at 14.60 minutes. In addition, the amino acid residue sequence was tested in the same manner as in Example 1 by using a sequencer (type 477A
Applied Biosystems, USA)
As a result, it was confirmed that the target product was a peptide having the amino acid residue sequence of the title (II).

Example 3 Biotinylated GG-EEEEEDNRD
SSMKLSFRARAYGFRGPGPQLRR-O
Synthesis of H... (V) The protected peptide-resin [having the amino acid residue sequence of (I)] (0.30 g) synthesized in Example 1 was used as a starting material, and was automatically operated using a peptide synthesizer. , Example 1
According to the methods and conditions described in
The group was removed, followed by two successive couplings of Fmoc-Gly (0.15 mmol) followed by biotin-N
Biotinylation was performed using -hydroxysuccinimide ester. As a result, 0.31 g of the target biotinylated protected peptide-resin was obtained. After completion of the synthesis, biotinylated protected peptide-resin (0.31 g)
-Cresol (0.3 ml), ethanedithiol (0.
9 ml), thioanisole (1.8 ml) and trifluoroacetic acid (12.0 ml) in the presence of a mixture at room temperature for 2 hours, after which the resin is removed by filtration and the trifluoroacetic acid is then removed under reduced pressure. Distilled off under. Thereafter, a series of operations similar to those in Example 1 were performed to obtain a crude peptide (39 mg). Thereafter, the obtained crude peptide was subjected to reverse phase high performance liquid chromatography (HPL).
Purified by C). The column is YMC-Pack-D-
ODS-5 (20 × 250 mm) was used, and elution was performed using a linear concentration gradient of 0.01 N HCl / CH ₃ CN from 82/18 to 74/26 (flow rate: 7 ml / min, detection: 230 nm). Was performed under the following conditions. Then, the purity of the obtained purified peptide was tested by analytical reverse-phase HPLC. The implementation conditions are as follows. Column: YMC-Pack-D-ODS-5 (4.6 ×
Solvent: 0.01 N HCl / CH ₃ CN DC concentration gradient: gradient from 95/5 to 40/60 Flow rate: 1.0 ml / min Detection: 210 nm The purified peptide of interest is a sharp single at 14.33 minutes Eluted as a peak. At the same time, the amino acid residue sequence was tested in the same manner as in Example 1, and as a result,
It was confirmed to be a biotinylated peptide having the amino acid residue sequence of (V).

-Preparation of antigen for immunization, immunization, and acquisition of antibody- Example 4: Acquisition of antiserum
The solution was dissolved in 1.5 ml of a 50% aqueous solution of polyvinylpyrrolidone (PVP) of No. 00, followed by addition of 1.6 ml of Freund's complete adjuvant, and the mixture was stirred with a homogenizer under ice-cooling for 30 minutes. 1.5 ml of the obtained emulsion is used as a rabbit (body weight: 2.0 to 2.2 kg).
Was injected intradermally. Booster immunizations were performed by repeating the bi-weekly dose at half the initial injection dose.
Thereafter, whole blood of the rabbit was collected according to a conventional method, and then the blood was left at 37 ° C. for 1 hour, then at 4 ° C. overnight, and then centrifuged at 3,000 rpm to remove the serum. This was frozen and stored. Using peptide (II) obtained in Example 2, an antigen for immunization was prepared according to the same procedure as described above, and the rabbit was immunized with the antigen. Thereafter, an antiserum was obtained by an ordinary method.

Example 5: Obtaining antiserum 9.0 mg of peptide (I) obtained in Example 1 was added to 10 ml of a carrier keyhole limpet hemocyanin (KLH) in a 0.1 M sodium bicarbonate solution, and the condensing agent dimethylsuberimi was added. Binding was performed in the presence of 5 mg of date (DMS.2HCl). By dialysis of the reaction mixture, the peptide
(I) [Human CgA (344-374) -KLH complex 1
8 mg were obtained. Thus, 3.6 mg of this composite was 50%
It was dissolved in 3.0 ml of an aqueous solution of polyvinylpyrrolidone, and subsequently 3.6 ml of Freund's complete adjuvant was added thereto, followed by mixing with a homogenizer. The obtained emulsion was divided into two equal parts, and the two equal parts were injected and immunized into two house rabbits by the same procedure and under the same conditions as in Example 4 above. Thereafter, whole blood of the rabbit was collected in accordance with a conventional method, and then serum was obtained according to the same procedure as in Example 4 described above, and the serum was stored frozen. Further, using the peptide (II) obtained in Example 2, an antigen for immunization was prepared according to the same procedure as above, and the rabbit was immunized with the antigen.
Thereafter, an antiserum was obtained by an ordinary method.

Example 6: Acquisition of IgY White hens (22-24 weeks old) are bred with free access to food and water under normal daily fluctuations. The peptide (I) obtained in Example 1 and the peptide (II) obtained in Example 2 were used as antigens. 300 μg of antigen was dissolved in 0.50 ml of 0.01 M phosphate buffer (pH 7.2) and then mixed with an equal volume of complete Freund's adjuvant. The obtained emulsion was injected into several subcutaneous sites in the abdomen of chickens. As a boost, half of the dose of the first immunization was injected every 2-3 weeks. The chicken eggs laid by the chickens were collected every day from the day three weeks after the start of immunization, the egg laying date was entered for each egg, and the entered egg was stored at 4 ° C. -Extraction of IgY-Ten preserved eggs were divided, the yolk was separated, and washed with water. Remove the yolk film and leave the remaining suspension 200ml
, A 0.01 M potassium phosphate buffer solution containing 0.1 M NaCl (buffer A) is added to make 300 ml. Add an equal amount of 7% (W / V) polyethylene glycol (PEG) 60
00 solution (dissolved in buffer A) was added. The resulting mixture was centrifuged (14,000) at 4 ° C. for 10 minutes.
Xg), the precipitate separates. The supernatant was filtered with a cotton cloth, and then solid PEG was added to a concentration of 12% (W / V) and stirred. Thereafter, when all PEG was dissolved, it was centrifuged again for 5 minutes (14,000 ×
g). Thereafter, the obtained precipitate containing IgY was washed with 2
Suspended in 00 ml of buffer A, add an equal volume of 24%
A (W / V) PEG solution (dissolved in buffer A) was added, and the resulting mixture was similarly centrifuged to regenerate the precipitate. Finally, the resulting precipitate is
Crude IgY was obtained by suspending in 0 ml of buffer A and dialysis against the same buffer followed by centrifugation. -Purification of IgY- Crude IgY was purified by the following chromatography. i) Anion exchange chromatography: The supernatant was washed with 0.015 M potassium phosphate buffer (pH 8.0).
DEAE cellulose equilibrated with (DE50, whatma
n) Add to 50 ml of column. Next, elution is performed stepwise using 0.2 M potassium phosphate buffer (pH 8.0). The fraction containing IgY was added again to the same column, and then 0.015 M to 0.2 M potassium phosphate buffer (pH
8.0) with a linear gradient. ii) Cation exchange chromatography: 100 ml of a CM cellulose (CM52, whatman) column [equilibrated with 0.01 M phosphate buffer (pH 5.2)] was added with a fraction containing IgY for peptide (I). Then, stepwise elution is performed using 0.2 M phosphate buffer (pH 5.0). iii) Affinity chromatography: The fraction containing IgY was further added to a Sepharose 4B (Pharmacia) column to which an antigen (peptide (I)) was bound, followed by washing with a phosphate buffer, followed by 0.5 M acetic acid. (PH
3.0). Each fraction obtained was assayed for titer by ELISA and 280 nm
The IgY eluted fraction was determined by the absorption of.

Example 7: Preparation of Monoclonal Antibody -Preparation of Monoclonal Antibody- (Immunization to Animals) Antigen (peptide (I)) was applied to 5 healthy mice (BALB / C) every two weeks according to a conventional method. Mice were given three intraperitoneal doses and then the antibody titers were examined, and the mouse with the highest antibody titer was selected and used for the next cell fusion. (Cell fusion) Under aseptic conditions, the spleen was removed from the body of the selected mouse, and lymphocytes were collected from the spleen as antibody-producing cells according to a conventional method. Then, the obtained lymphocytes are mixed with myeloma (myeloma) cells, and then 45% polyethylene glycol (PEG) is used as a cell fusion promoting substance.
Are added to cause the cells to fuse. As a medium at this time, an RPMI medium 1640 (Gibco) was used. Lysolecithin, glycerol oleate, and the like can also be used as the accelerating substance. (Screening and cloning) The fused cells were cultured in a HAT selection medium, and the cultured cells were screened according to an ELISA method to select antibody-producing hybridomas capable of producing a peptide (I) -specific antibody. This was cloned according to the limiting dilution method. When the clones have grown to some extent, the same screening is performed again.
Screening and cloning were repeated until a single clone was obtained. (Preparation of ascites) Single clone (monoclonal antibody-producing hybridoma) obtained by the above screening
Was cultured and grown in large amounts, and it was intraperitoneally administered to a pre-blistered nude mouse (BALB / C), and then the ascites produced was collected from the administered mouse. -Purification of monoclonal antibody- The monoclonal antibody in the obtained ascites was purified from protein A.
Purify by (Bio-Rad) affinity chromatography. At that time, the content of immunoglobulin IgG was calculated by absorption at 280 nm. (Test for antibody titer) Purified monoclonal antibody
Culture screening according to the ELISA method,
The titer of the monoclonal antibody against peptide (I) was assayed. The test method is as described in the following procedures (i) to (iv). (i) Immobilization reaction plate 96-well microplate (MaxisorpNunc Immuno Pl
ate (Nunc 442404)), and inject 0.1 μg / 0.1 ml of the antigen peptide (I) solution into each well.
The 96-well microplate was left at 4 ° C. overnight. (ii) First reaction 0.1 ml of a monoclonal antibody solution (different concentration of each solution) of the culture supernatant was added to each well, and then the 96-well microplate was left at room temperature for 3 hours. And anti-human CgA (34
0.1 ml of the monoclonal antibody solution of 4-374) was added to each well, and this was left at room temperature for 3 hours. (iii) Second reaction Thereafter, 0.1 ml of a 5000-fold diluted solution of horseradish peroxidase (HRP) -labeled anti-mouse IgG (Bio-Rad) was added to each well, and this was added at room temperature for 2 hours. Reacted. (iv) Third reaction Then, 0.1 ml of an o-phenylenediamine solution of a substrate solution was added to each well, followed by reacting at room temperature for 10 minutes to develop a color.
Stopped by the addition of H ₂ SO ₄ and immediately
Photometric measurements were performed on each well using a microplate photometer. Thus, good linearity was observed in the obtained graphs of antibody concentration and light intensity, and peptide (I)
(Ie, monoclonal antibody against human CgA (344-374)) showed very high binding activity in a dose-dependent manner.

(Labeled antigen) Peptide (I) obtained in Example 1
Hunter and Chloramine T from Greenwood
According to the law (Hunter and Greenwood, Nature, 194, 495).
-496 pp. (1962), made into ¹²⁵ I. That is, the peptide (I) synthesized in Example 1 (that is, human CgA (344-37)
4)) 2.5 μg was dissolved in 2.5 μl of 0.01 N HCl solution and then added to 1 M phosphate buffer (pH 7.
3) A solution of 40 μl, 1 μl of ¹²⁵ I-Na solution (200 μCi, Amersham, UK) and 10 μg of chloramine T dissolved in 20 μl of purified water is added successively, followed by shaking the mixture for 20 seconds and then adding And sodium metabisulfite 20 dissolved in 20 μl of purified water.
The reaction was stopped by the addition of a solution of 10 μl of 10 μl of 10% potassium iodide. Thereafter, the mixture was added to 0.1
Purification was performed on a prepacked column (NAP-10, Pharmacia Biotech, Sweden) using 1 M acetic acid containing 10% bovine serum albumin as an eluent.

(Radioimmunoassay) As a diluent, bovine serum albumin (0.5% W / V), 0.025M
Ethylenediaminetetraacetate (EDTA) and
0.01 M phosphate buffer containing 0.14 M NaCl (pH
7.4) was used. Synthetic human CgA (344-374) was used as a standard product. Diluent 0.4
0.1 ml of the standard or specimen, 0.1 ml of the antiserum of this example (diluted (8500-fold final)) and 0.1 ml of labeled antigen (5000 to 10000 cpm) were mixed in an incubation tube. Boron siliconized glass tubes (Chase Instrument, USA) were used as the incubation tubes. The resulting mixture was incubated at 4 ° C. for 48 hours, after which it was added to diluted normal rabbit serum (1: 5).
0, Daiichi Radio Isotope Laboratory, Tokyo) 0.05m
l, 0.05 ml of diluted goat anti-rabbit IgG serum (1:10, Daiichi Radioisotope Laboratory, Tokyo) and 10
% (W / V) polyethylene glycol 6000 (molecular weight 7
500) 0.5 ml each. The mixture obtained was then incubated at 4 ° C. for a further 3 hours, after which it was centrifuged at 4 ° C. for 30 minutes (300 rpm).
0 rpm) and then remove the supernatant from this,
Subsequently, this was counted using a gamma counter (gamma counter). When the cross-reactivity of the antiserum of this example was examined by the above immunoassay, rat C
gA (359-389) clearly showed a cross-reactivity of 33.5%, but both rat CgA (375-389) and human CgA (360-374) showed substantially cross-reactivity. Did not. FIG. 1 shows a dose-response curve of CgA immunoreactivity in human plasma and human urine in the above-mentioned human chromogranin A (344-374) radioimmunoassay system, and FIG. 2 shows human adrenal gland in the same assay system. And C in human pancreatic extract
Figure 4 shows a dose response curve of gA immunoreactivity. It is particularly important here that the dose-response curves of human serum, urine and saliva, and adrenal gland, pituitary gland, and duodenum extracts were all based on the synthetic human CgA (344-
374), which point to the fact that they have no immunochemical differences. This point is clearly shown in the graphs of FIGS. Therefore, based on these results, the measurement system of this example shows that human-derived human serum, urine and saliva, and human CgA and / or human CgA (34
4-374) It has been proved that it can be used for measuring the immunological activity of human CgA-related peptide containing the sequence.
In addition, regarding the measurement of CgA immunoreactivity in human plasma in the above radioimmunoassay system, the reproducibility within and between measurements is summarized in the following table.

[Table 1] Table 1 ──────────────────────────────────── Human CgA (344-374) (fmol / tube) Mean standard deviation (SD) CV (%) ──────────────────────────────────再現 Reproducibility within measurement (n = 10) Human plasma 1 226.8 12.9 5.7 Human plasma 2 286.6 18.4 6.4 Human plasma 3 128.9 5.2 4.0 Human plasma 4 255.5 8.8 3.4 Human plasma 5 199.2 4.8 2.4 Reproducibility between measurements (n = 5) Human plasma 6 255.1 35.5 13.9 Human plasma 7 310.7 42.5 13.7 Human plasma 8 163.0 21.2 13.0 Human plasma 9 264.7 17.3 6.5 Human plasma 10 214.0 17.4 8.1 ────────────────────抗体 Further, an antibody against the peptide (II) obtained in Example 2 was prepared in the same manner as in Example 1, and the antibody was subjected to immunoassay. Similar cross-reactivity and above were observed.

(Enzyme immunoassay) Goat anti-rabbit Ig
A 96-well microplate on which G was immobilized was used. First, 0.9% NaCl and 0.0
Wash well enough with wash solution containing 5% Tween 20, then inject 300 μl of phosphate buffer (pH 7.4) containing 0.1% bovine serum albumin into each well, then seal the plate and Was stored at room temperature for 2 hours. Thereafter, the liquid in each well is drained off, and the same washing operation is performed using the above washing solution for 3 times.
Repeated times. Next, 50 μl of phosphate buffer containing 5% normal goat serum and 0.1% bovine serum albumin was added to each well.
μl was injected, followed by the addition of 50 μl of the analyte to be measured or 50 μl of a standard human CgA (344-374) solution to each well. Furthermore, biotinylated GlyGly-human CgA (344-374) synthesized in Example 3.
Of a solution of anti-human CgA (344-37)
4) 100 μl of serum solution was added to each well. Thereafter, the microplate was sealed and reacted at room temperature for 2 hours. During the reaction, the plate was shaken slowly using a microplate shaker. Thereafter, the liquid in each well was drained off, and the same washing operation was repeated three times using the above washing solution. Thereafter, each well was added to a phosphate buffer containing 0.1% bovine serum albumin (p.
100 μl of a solution of streptozotocin-horseradish peroxidase (HRP) dissolved in H7.4) was injected,
The microplate was then sealed and shaken at room temperature for 2 hours, after which the liquid in each well was drained off, and
The same cleaning was performed using the same cleaning liquid as above. Then, in each well, a solution of enzyme substrate in 0.1 M sodium phosphate-citrate buffer containing 0.015% hydrogen peroxide was added.
μl was added. And seal the microplate,
The mixture was slowly shaken at room temperature for 15 minutes, and then 100 μl of 2N H ₂ SO ₄ was added as an enzyme reaction stop solution. Then, using a microplate absorptiometer, wavelength 490
The absorbance of the liquid in each well was measured in nm.
The absorbance was measured for each of the seven different concentrations of the human CgA (344-374) standard solution, and a calibration curve was prepared from the measured values, and the human CgA concentration in each sample was measured based on the calibration curve. did.

(Enzyme immunometric assay) As a diluent, a phosphate buffer (pH 7.2) containing 0.1% bovine serum albumin and 50% normal pig serum was used. As a buffer, 0.1% bovine serum albumin, 2.
A phosphate buffer (pH 7.2) containing 5% normal goat serum and 0.05% Tween 20 was used. The washing solution is 0.9N NaC containing 0.025% Tween20.
1 solution was used. A 96-well microplate on which a human CgA (344-374) monoclonal antibody (500-fold dilution) was immobilized was used. First, each well was sufficiently washed with 300 μl of the above-mentioned washing solution, and then
Inject 300 μl of the above buffer into each well,
Subsequently, the plate was sealed and it was stored at room temperature for 2 hours. Thereafter, the liquid in each well was drained off, and then the same washing operation using these washing solution and buffer was repeated three times. Next, 50 μl of the above buffer solution was injected into each well, and then the sample 1 to be measured was added.
00 μl or 100 μl of human CgA (344-374) standard solution (0-800 pg / ml) was added to each well. Thereafter, the microplate was sealed, and this was shaken at room temperature for 2 hours using a microplate shaker. Thereafter, the liquid in each well was drained off, and then the same washing operation was performed using the above washing solution. Repeated three times. Thereafter, 100 μl of an anti-human CgA (344-374) serum (diluted 5000-fold) solution was added to each well. Then, the microplate was sealed, and this was shaken at room temperature for 2 hours. Thereafter, the liquid in each well was removed, and then the same washing operation was repeated three times using the above washing solution. Then, each well was filled with horseradish peroxidase (H
RP) 100 μl of standard goat anti-rabbit IgG solution was added. Then, the microplate was sealed, and this was shaken at room temperature for 2 hours. Thereafter, the liquid in each well was drained off, and then the same washing operation as described above was performed. Thereafter, 100 μl of a solution of the enzyme substrate in 0.1 M sodium phosphate-citrate buffer (pH 5.0) containing 0.015% hydrogen peroxide was added to each well. Then, the microplate was sealed, and slowly shaken at room temperature for 15 minutes. Then, 100 µl of 2N H ₂ SO ₄ was used as an enzyme reaction stop solution.
1 was added. Next, the absorbance of the liquid in each well was measured at a wavelength of 490 nm using a microplate absorbance meter. The absorbance was measured for each of the seven different concentrations of the human CgA (344-374) standard solution, and a calibration curve was prepared from the measured values, and the human CgA concentration in each sample was measured based on the calibration curve. did. Thus, also in the enzyme immunoassay and the enzyme immunometric assay described above, the antiserum of this example exhibited a clear cross-reactivity with human CgA (344-374). Therefore, based on these results, the measurement system of this example is suitable for human CgA,
Alternatively, it has been revealed that the peptide can be effectively used for measuring the immune activity of a human / CgA-related peptide containing a human / CgA (344-374) sequence.

[0020]

As described above, according to the present invention, an antiserum, a monoclonal antibody, or the like, which shows specific reactivity to human chromogranin A by recognizing human chromogranin A, in particular, the 344-374 sequence. Antibodies such as polyclonal antibodies and IgY are provided. Also,
According to the method of the present invention, human chromogranin A and a human chromogranin comprising the amino acid residue sequence of formula (I) or (II)
In an immunoassay targeting the chromogranin A-related peptide, the effect of enabling high-sensitivity measurement / detection of the peptide is obtained.
Chromogranin A and its related peptides in urine, bone marrow fluid, saliva and the like can be measured and detected effectively, and for example, they have the advantage of being useful for elucidation and study of the stimulus-secretion mechanism.

[Brief description of the drawings]

FIG. 1. This is human chromogranin A (344-3).
74 is a graph showing a dose response curve of CgA immunoreactivity in human plasma and human urine in a radioimmunoassay system.

FIG. 2. This is human chromogranin A (344-3).
74) A graph showing a dose response curve of CgA immunoreactivity in human adrenal gland and human pancreas extract in a radioimmunoassay system.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI // C07K 14/47 C07K 14/47 C12N 15/02 C12R 1:91 (C12P 21/08 C12N 15/00 C C12R 1:91 (56) References Kazuo Suzuki et al., Clinical Study of Blood Chromogranin-A Measurement by Elisa Method Using Synthetic Peptide, Dokkyo Medical Association Magazine, Japan, 1992, Vol. 8, No. 1, 83- 91 Andrew J. et al. M et. al, Human Chromogranin A Gene, J. Am. Biol. Chem, 1994, Vol. 269, no. 9, 6918-6926 (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 33/50-33/98 JICST file (JOIS) GenBank / EMBL / DDBJ / GeneSeq BIOSIS / WPI (DIALOG)

Claims (3)

(57) [Claims] Claims 1. A peptide having an amino acid sequence represented by the following formula (I): EEEED NRDSS MKLSF RARAY GFRGP GPQLRR (I) is used as an antigen, and is produced and obtained by immunizing a warm-blooded animal other than humans. An antibody having specific reactivity to human chromogranin A. 2. A peptide having an amino acid sequence represented by the following formula (II): NRDSS MKLSF RARAY GFRGF GPQLRR (II) is used as an antigen, and is produced and obtained by immunizing a warm-blooded animal other than a human. Antibodies having specific reactivity to human chromogranin A. 3. A method for specifically measuring and detecting human chromogranin A and its related peptide, comprising performing an immunoassay using the antibody according to claim 1 or 2.