JPS63273496A - Monoclonal antibody, hybridoma and measurement of human growth hormone - Google Patents

Abstract

PURPOSE:To obtain a hybridoma to produce a monoclonal antibody specifically reacting with a specific human growth hormone, by cell fusion and screening. CONSTITUTION:Splenocyte of an animal immunized against human growth hormone (hGH) extracted from pituitary and a myeloma cell system of another animal are subjected to cell fusion. The fused cell thus obtained is cloned by limiting dilution and screening is carried out by enzymatic immunoassay method using hGH extracted from the pituitary and hGH having 20,000mol.wt. to give a strain to produce a monoclonal antibody which specifically reacts only with hGH having 22,000mol.wt. but not substantially with 20KhGH, namely hybridoma CL.B1. Only 20KhGH among human growth hormone of patient can specifically determined by using a monoclonal antibody produced by the hybridoma.

Description

[Detailed Description of the Invention] [Field of Industrial Application] The present invention provides human growth hormone (hereinafter abbreviated as hGH) useful for diagnosis of growth disorder diseases such as acromegaly and pituitary dwarfism, and for pituitary function tests. ), a hybridoma that produces the same, and a method for measuring human growth hormone using the monoclonal antibody.

[Prior art]

Conventional hG)I is a molecule Wi consisting of 191 amino acid residues.
22,000 peptide hormone (hereinafter abbreviated as 22KhGH) It was thought that there was only one type of hGH, but recently it was discovered that hGH (abbreviated as 20KhGH), a molecule of 1120,000 molecules consisting of 176 amino acids, is present in the anterior pituitary gland, blood, and urine of humans. ) was found to exist. Regarding its physiological activity, it was found that its growth promoting effect is equivalent to that of 22Khco, but it lacks the insulin-like activity, lipolytic effect and diabetic pathogenicity that 22KhGH possesses. Therefore, it is thought that the various growth hormone actions are solely due to 22K hGH, and therefore, it is desirable to measure the in vivo concentration of 22K hGH in diagnosing various diseases caused by abnormal secretion of hGH.

On the other hand, radioimmunoassay is currently used to measure hGH, but the measurement sensitivity is 0.5 to 1. Ong
Since the basal blood level cannot be measured due to the relatively low level of hGH, diagnosis is made by measuring hGH after a challenge test with insulin, arginine, etc.

However, there are quite a number of cases in which the physiological secretion dynamics have failed (hGH measurement using natural secretion dynamics has been desired).

Therefore, a highly sensitive enzyme immunoassay method capable of measuring the basal level of GH in blood and urine was developed, and has already been reported [Kitanta, Ishikawa et al., Clinica Chimica Acta (CIln
lca, Chlmlca, ^cta)-l 工, (1
9B? ) pp. 229-235].

[Problem that the invention seeks to solve]

In view of this current situation, diagnosis by measuring the dynamics of natural secretion of hGH is more sensitive, and the ability to develop substances or measurement methods that can quantify only 22K hGH or separately from 20K hGH (a long-awaited goal). It is a place where
Therefore, an object of the present invention is to provide such a substance, a hybridoma that produces the same, and a method for measuring human growth hormone using the substance.

[Means for solving problems]

This object is solved by a method for measuring human growth hormone using a monoclonal antibody, a hybridoma, and a monoclonal antibody according to the present invention.The present invention is as follows.

■A monoclonal antibody that specifically reacts with 22K hGH and does not substantially react with 20K hGH. ■A hybridoma that produces the monoclonal antibody.

(2) A method for measuring human growth hormone using the monoclonal antibody.

The monoclonal antibody of the present invention is hybridoma CL,
B1. It can be produced by culturing the hybridoma or from the ascites of a mouse inoculated with the hybridoma.

Hybridoma CL, B1. is produced by cloning hybridomas obtained by so-called cell fusion and selecting clones having specificity for 22K hGH.

Specifically, the following means are exemplified.

Antibody producing cells are tile cells, lymph node cells, B-lymphocytes from the immunized animal. Examples of the immunogen include pituitary-extracted hGH (eg, trade name Thalescormon), and examples of the animals to be immunized include mice, rats, and the like.

The immunogen is administered in a conventional manner, for example in combination with an adjuvant. Freund's complete or incomplete adjuvant is preferred as adjuvant. Immunization is performed by administering at least two times at intervals of 2 to 6 weeks.

For example, tile cells are obtained from the thus immunized animal,
Fuse with myeloma cell line. This fusion is based on Köhler (KB)
hler) and Milstein
Known method [Nature Ni, 49
5-497, 1975], for example, mice, rats, etc. are used as myeloma cell lines.

The resulting fused cells are cloned by a limited dilution method or the like. Furthermore, among them, pituitary extracted hGH (Cresscormon) and 20K hGH (U, J.

Enzyme immunoassay method (kindly provided by Robert Lewis)
22Kh by screening by ELISA method)
Monoclonal antibody-producing strains that specifically react only with GH and do not substantially react with 20KhGH, namely hybridomas CL, B1. can be obtained.

The hybridoma is a nearly spherical floating cell and is stored at 37°C.
When cultured at 5% CO□, 100% humidity, and pH 7.2 to 7.4, the number of cells will double in about 9 to 24 hours. Moreover, the number of chromosomes is 109-121.

Hybridoma CL, B1. is freezing method (below -80℃)
It can be stored in For example, the freezing conditions are 10
'/ld/vial, RP M I 164070
%, fetal bovine serum 20%, dimethyl sulfoxide 10%
The temperature is lowered at a rate of 1°C/min in a solution of

The hybridoma CL, B1. has applied for deposit to the Institute of Microbial Technology, Agency of Industrial Science and Technology, and has already been informed by the institute that the deposit has been rejected as it is outside the range of microorganisms that can be deposited. It has already been shipped from the place.

The monoclonal antibody of the present invention can be recovered from the culture supernatant of the above hybridoma, or from mouse ascites, serum, etc.

The medium for culturing the hybridoma contains a carbon source,
Contains a nitrogen source, vitamins and inorganic salts as necessary,
An example of such a medium is RPMI-1640 supplemented with fetal calf serum (for example, about 10%).

The culture temperature is 36 to 37.5°C (preferably 37°C), the culture period is 1 to 4 (preferably 2 to 3 days), and the pH is 6.5 to 7.
．． 5 (preferably 7.2-7.4), 5% Cot 95
%air %Usually 1-2 X 1 under 100% humidity conditions
Recovery from mouse ascites, serum, etc., which is transplanted at a cell concentration of 0'/@l, is performed, for example, as follows. 6i
! ! 0.5- pristane (2,6+ 10+ 14-
tetramethylpentadecane, A
ldrich) is administered intraperitoneally, and 2 weeks to 1 month later, CL and B1.5X10' are administered intraperitoneally. 10-14
Collect ascitic fluid from the mice after 1 day.

Although this monoclonal antibody can be used without purification, it is preferably purified before use.

For example, it is purified by operations such as ammonium sulfate salting out and DEAE cellulose chromatography to obtain an IgG fraction.

The monoclonal antibody of the present invention has a high affinity for 22K hGH (9,6xLO"z1 molar affinity constant). Furthermore, the cross-reactivity for 20K hGH is 15%.
The proportion of 20KhGH in blood and urine is as low as 1.
0% or less [Baumann et al., Journal of Clinical Endocrinology and Metapolide (Journal of C11ni)]
cal Endocrinology and Met
a-bolite) 56. pp. 305-311, 946
952, 1983], there is no substantial effect on the 22KhGH measurement system.

The monoclonal antibody has a 1g class (subclass) of IgG, and has 4 polypeptides (2 heavy chains, 2 heavy chains at molecules 147 to 50, molecular weight 25 to 29k).
It is a glycoprotein consisting of p+ of 6.1 to 7.0.

The method for measuring human growth hormone using the monoclonal antibody of the present invention is not particularly limited as long as the monoclonal antibody can be used and human growth hormone can be measured. For example, enzyme immunoassay, radioimmunoassay, fluorescence immunoassay, etc. are exemplified, and enzyme immunoassay is particularly preferred.

For example, in enzyme immunoassay, the monoclonal antibody is bound to an insoluble support to obtain an antibody-bound insoluble support, and the so-called sandwich enzyme enzyme immunoassay using this insoluble support is particularly preferred.

The measurement method of the present invention will be explained below based on the sandwich enzyme immunoassay method.

Examples of the insoluble support include polystyrene spheres, polystyrene tubes, silicone pieces, microplates, and polystyrene spheres are particularly preferred.

The monoclonal antibody TgG fraction may be bound to these insoluble supports by a known chemical binding method, but a physical adsorption method is sufficient. That is, the above 1 gG fraction is dissolved in a phosphate buffer, etc., the above-mentioned insoluble support is added, and the mixture is heated at 0°C to
After being left at room temperature for 1 hour to 48 hours, preferably at 4°C to 10°C for 10 hours to 24 hours, bovine serum albumin,
Wash with a phosphate buffer solution containing a stabilizer such as sodium azide, and store at 2 to 8°C.

In the present invention, first, a test solution containing hGH is reacted with a monoclonal antibody 1gG fraction-bound insoluble support, and 22Kh
Bind GH. The reaction is carried out at 20°C to 40°C, preferably 25°C to 37°C, for 1 hour to overnight, preferably for 3 hours to
The test is carried out for 8 hours, and the hGH-containing test solution may be one containing human growth hormone, such as serum, plasma, or urine.

Next, the anti-22K hGH monoclonal antibody bound to 22K hGH was reacted with a labeled substance (hereinafter referred to as the label) obtained by labeling the anti-hGH antibody with an enzyme to the 1gG fraction-bound insoluble support, and the anti-22K hGH monoclonal antibody A sandwich-like complex consisting of the 1gG fraction-22KhGH-labeled substance is formed.

The anti-hGH antibody used here as a label may be an antibody that specifically reacts with hGH, and may be an antibody that reacts only with 22K hGH or with 20K hGH, and may be either a monoclonal antibody or a polyclonal antibody, and is particularly preferably is an affinity-purified anti-hGH rabbit polyclonal antibody, and in order to reduce non-specific adsorption, an F (ab')x+ Fab' fragment or the like is preferable.

Furthermore, examples of enzymes include peroxidase, β-galactosidase, alkaline phosphatase, glucose oxidase, etc., but peroxidase with less nonspecific adsorption is preferred.

This enzyme is then used with anti-hGH antibody or anti-hGH antibody F.
(ab')z, Fab' fragments can be labeled by known methods. For example, enzyme 1! A method in which the chain is oxidized with periodic acid and an amino group such as an anti-hGH antibody is bonded to the generated aldehyde group. For example, a maleimide group or a pyridyl sulfide group is introduced into the enzyme, and the thiol present in the anti-hGHF ab' fragment is Examples include a method of bonding with a group.

In the present invention, the activity of the enzyme used for labeling in the obtained sandwich-like complex is measured. Since the amount of activity of this enzyme depends on the amount of 22KhGH initially reacted, 22KhGH in the test solution can be measured.

The activity of an enzyme is measured by adding a substance that serves as a substrate for the enzyme, but for peroxidase, a substrate that produces a fluorescent substance such as hydrogen peroxide and p-hydroxyphenylpropionic acid is desirable as a highly sensitive measurement method.

In the enzyme immunoassay method of the present invention, 22K hGH
can be specifically measured, but the standard hGH used for preparing the standard curve is 22K hGI purified from pituitary hGH.
However, the content of 20K hGH in pituitary hGH is said to be below 10% [Lewis (
J. Lewis) et al., Journal of Biological Chemistry〇, of Biolog
ical Che+wistry) 253 (197
8) pp. 2679-2687], pituitary hGH has a constant reaction rate with the 22K hGH-specific monoclonal antibody, and WHO measurement 1st 1. If hGH of a certain quality can be used by correcting it using R and P, the standard product is 20KhG).
H can be used. Therefore, the displayed value is the pituitary gland extracted hGH conversion value.

〔Effect of the invention〕

The hybridoma of the present invention produces a monoclonal antibody that specifically reacts with 22KhGH and does not substantially react with 20KhGf, and in a method for measuring human growth hormone using the monoclonal antibody, for example, Concentration 1 pg/lll7 or more, human blood concentration 5
22KhG (22KhG) at a level of pg/@Z or higher can be quantified with high sensitivity by enzyme immunoassay.

Furthermore, by combining the conventional sandwich enzyme immunoassay method using an insoluble support bound with a polyclonal or monoclonal antibody that reacts only with 22KhG1 (and 20KhGH), 22KhGH
and 20K hGH can be quantified with high sensitivity.

〔Example〕

The present invention will be explained below with reference to Examples, but the present invention is not defined by these Examples.

Example 1 Preparation of anti-22K hGH monoclonal antibody fi+ Immuno Freund's complete adjuvant (Freund'sco
complete adjuvantHFCA; Difc
o Lab, Detroit.

2 2 K h G mixed with Michigan)
H (Crescor so on; Kabi Vitrum
AB, + Stockho1m + Sweden)
100/Ig was intraperitoneally administered to BALB/C female 5-week-old mice (Nippon Talea, Tokyo). Thereafter, Freund's incomplete adjuvant (Freun) was administered three times at 3-week intervals.
d' 5incos+plete adjuvant;
22KhGH100q mixed with F r A) was administered intraperitoneally. Three weeks later, they were boosted by intravenous administration of 22KhGH100q dissolved in physiological buffer.

(2) Preparation of immunized lung cells Four days after the final booster immunization, the lungs were removed and Eagle's MEM
Lung cells were obtained by suspending them in a medium (Osaka University Research Institute, Osaka).

(3) Cell fusion mouse myeloma cell p3X63-Ag8・Ul (P2O3
; ATCC CRL, 1597: Curt, To
p, Microbiol.

Immuno+, 81.1-7.1978) at 10%
Fetal bovine serum (Fe2 HGibco+ Chagri
The cells were cultured in RPMI-1640 medium (Osaka University Institute of Technology) supplemented with Ni Falls, Ohio), and the cells were collected in the logarithmic growth phase and used for cell fusion. The fusion method is Kijhler.
) et al. (Nature) 256°495
-497.1975). That is, lung cells and myeloma cells were suspended in serum-free Eagle's MEM medium at a ratio of 10:1 and centrifuged for 7 minutes at 1.00 Orpm (Tomy Seiko CD-10OR). 3 in the sediment
45% (w/w) polyethylene glycol (P[!G) 6000 (Sigma Chem
ical Coot st.

Louis+ MC) dissolved 'tfX 1 sr was added over 1 minute and incubated for 8 minutes at room temperature (23±3°C).Next, serum-incompatible Eagle MEM medium 16d was added at a rate of 2 m/min and suspended. It got cloudy. Furthermore, 10 tf of Eagle's MEM medium without serum was gradually added.
diluted.

Centrifuge at 1*OOOrpm for 7 min and then add 10% FC3-RPMI-,16 to obtain a cell concentration of 6X10'/s7 as mouse bone [111 cells].
The mixture was resuspended in 40 medium and dispensed into 966 microplates (Jumin Bakelite MS-3096F) at 0.2 m/hole. The fused cells were cultured at 37°C in 5% Co. One day after Ii cell fusion, half the medium was added to fresh H
AT medium (10-'M pipoxanthine (Sigma C
hea+1cal Go,), 4X10-'M aminopterin (Sigma Chemical Co,),
1.6 Xl0-'M Thymidine (Sigma Chem
ical Co,) containing RPMI-1640 medium]. Thereafter, half-volume replacement with HAT medium was performed three times every two days.

After 10 days, cell proliferation was observed in approximately 100% of the microplate holes.

(4) Antibody assay Eleven days after cell fusion, the presence of antibodies against 22K hGH in the culture supernatant of the hybridoma cells was screened by enzyme immunoassay, so-called ELISA. That is, 100 μl of a 22K hGH solution dissolved in phosphate buffered saline (P B 5) pH 7.2 was added to a polyvinyl chloride 966 microplate (Falcon Microtest).
m flexible assembly plate;
The incubage solution was removed at 37° C. and 27° C. for 2 hours, and then the PvC assay plate was dried. In order to prevent non-specific binding, bovine serum albumin (BSA) was adsorbed using the same procedure.
It was used as an antigen plate. 1% BSA, 0.05% (
v/v) Tween 20@(Polyoxyethylene・
100μ of culture supernatant per well diluted appropriately with phosphate buffered saline pH 7.2 (hereinafter abbreviated as PBST) containing sorbitan monolaurate (Gyudon Kagaku). In addition, the cells were incubated for 2 hours at 37°C. After that, it was washed three times with PBST, and then calcium- and magnesium-free P
alkalinephos diluted 103 times in BS
pha tase label anti-mouse immu
noglobulin-antibody (New
England Nuclear) 1 per well
00 μl was added and cultured at 37° C. for 2 hours. PBST
After washing 5 times with
phosphate (PNPP)) as a substrate, color is developed by incubating at 37°C for 15 minutes,
Multiscan (Titerteck Multiscan)
405n at an MCC+ Flow l,ab,)
The absorbance at m was measured.

As a result of the assay, 170 wells were positive (approximately 35% of cell proliferation wells).

None of the culture supernatants from wells that showed binding to 22K hGH bound BSA. This indicates high specificity.

(5) Cloning of hybridomas Hybridomas producing antibodies showing high binding ability to 22K hGH were placed in 966 microplates using BAL8/C mouse thymocytes as feeder layer (IXIO'' cells/well). The cells were seeded at 0.5 cells/well.Culturing was continued at 37°C in the presence of 5% CO while half of the medium was replaced with fresh medium every 3 to 4 days, to allow the cells to sufficiently proliferate.

1. The antibody titer of each well was assayed as in (4), and wells showing good proliferation and high antibody titer were selected.

(6) Hybridoma culture Hybridoma 5 grown in culture after cloning
BAL treated with Bristane 2 weeks prior to X10' cells
B/C mice were inoculated intraperitoneally.

After 10 to 14 days, ascitic fluid was collected from the mouse's abdominal cavity.

(7) Antibody titer in ascites The antibody titer in ascites was examined by radioimmunoassay (RIA). That is, 50μ of ascites appropriately diluted with PBS was added to the wells of a 96-well microplate. / well, and further diluted with PBS.'l-22KhG
H (50 μCi/Pg) was dispensed at 50 μl/well and incubated at 37° C. for 2 hours and at 4° C. overnight. The next day, anti-mouse immunoglobulin antibody (Miles Labor
atorles+ Inc,) 50u7! /well, and left at 37°C for 1 hour.

After further centrifugation, the wells were counted using a T-counter (Be
ckman Instruments, Inc.
Fullerton, (CA)).

As a result of the assay, clones CL and Bl exhibited high antibody titers with a dilution of 2.8XlOs, which represents 50% of the maximum antigen binding amount.

(8) Identification of immunoglobulin class of antibodies 75 μl of hybridoma culture supernatant was applied to Ophthalony's monoclonal antibody typing kit based on double immunodiffusion method (Seikagaku Corporation) at room temperature (23 ± 3°C) for 24 hours. After incubation, the antigen-antibody precipitate was observed and the subclass was identified. As an antiserum, mouse immunoglobulin γ1
, sheep polyclonal antibodies against T2, rtb, γ5, α and μ (Mtles Laboratory
S, Inc.) was used.

The monoclonal antibodies CL, B1 class and Safrilus were IgG.

(9) Determination of antibody affinity The antibody affinity was determined by tA. That is, in the method (7), a certain amount of "'?-22KhGH (50μCi
/ pg, 10,000-20,000 cps) as well as non-radioactive standards of various concentrations! Add 1i22KhGH, determine the 22KhGHft that binds to the antibody, and
tchard method (Ann, N, Y, ^cad,
Sci., 51.660-672°1949).

The binding constant of monoclonal antibodies CL and B1 to 22KhGH is 9.6X10M! It was calculated to be 1 mol.

Determination of binding specificity The binding specificity of monoclonal antibodies CL and B1 was determined using EL4S.
^Determined by law. That is, in method (4), 22
Instead of KhGH, 20KhGH (hGH lacking amino acids 32-46; Dr, U, J.

Lewis, 5cripps Memorial H
o5pital+ La Jolla.

CA), human prolactin hPRL (Calb
iochem-Behring Corp, La
Jolla, CA), methionyl 22KhGH (γ-
hGH, recombinant DNA preparation; Kabi Vitrum
AB, Stockholm, Sweden
), 32 (hGH lacking amino acids 135-140).

Dr. U. J. Lewis + 5cripps
Memorial Ho5pital.

La Jolla+ CA) and human chorionic somatomamotolobin (hcsHPharmacia F
ine Chemicals AB, Uppsala,
Microplates were prepared and assayed.

Assay results showed that monoclonal antibodies CL and Bl were γ
It bound to -hGH,32 to the same extent as 22KhGH, but only showed a low binding of less than 15% to 20Kh(1,H).Also, almost no cross-reactivity was observed for hPRL and hC3. (less than 1%).

Purification of αD monoclonal antibody Ammonium sulfate fractionation of each ascites obtained in (6) above (40% saturation)
After that, add 205M phosphate buffer (p) 1 containing 20mM NaCl.
DEAE cellulose (DH-52) equilibrated with 7.8)
Gradient elution was performed using a 4° 11M phosphate buffer (pH 7,8) containing 400s+M of sodium chloride, and the percentage of IgG was fractionated, followed by 0.1%
0.1M phosphate buffer (pH 7) containing sodium azide
, 0) overnight at 4°C and stored at 4°C.

Example 2 Preparation of antiserum and anti-hGH rabbit polyclonal antibody fil Immunization A rabbit is first immunized with hGH extracted from the pituitary gland (Thales Cormon) together with Freund's complete adjuvant. 1100p
hGH in 0.1 M phosphate buffer (pH 7,0) 1-
was mixed with Freund's complete adjuvant 1-, and the emulsion was subcutaneously administered to dozens of places on the back of rabbits, and then 2-
Booster immunizations were given weekly using the same administration method. After each booster immunization, serum was collected and the binding activity with ``I-hGH was examined.The antiserum showed a binding rate of 30% at a dilution rate of 1/25600.

(2) Purification of antiserum After the rabbit antiserum obtained in step 11 above was fractionated with sodium sulfate (18% saturation), it was added to 17.5d phosphate buffer (pH 6).
, 3) equilibrated with DEAE-cellulose (DE-5
2. The non-adsorbed fraction of the What+wan Co.) column was collected to obtain a 1gG fraction of anti-hGH polyclonal antibody.

Example 3 <22K hGH enzyme immunoassay based on the Sandermansch method (1) Anti-22K hGH mouse monoclonal antibody TgG
Preparation of fraction-bound insoluble support 0.1% sodium azide containing 0.1% sodium azide-containing anti-22KhGH mouse monoclonal antibody IgG fraction obtained in Example 1.
The concentration of 1 M phosphate buffer (pH 7,0) solution was adjusted to 0.1.
Adjust to 5 dawn/ll17.

Add a polystyrene ball (Precisio) to this antibody solution.
nPlastic Ba11) was soaked at 4°C for 24 hours to adsorb the antibody onto the ball. Next, the antibody soaking solution was collected, and the polystyrene ball was soaked in buffer AC0.1% BSA.

Contains 0.1% salt and 0.1% sodium azide 10
Wash 5 times with mM phosphate buffer (pl+ 7.0) and use.

(2) Peroxidase labeling of rabbit anti-hGHFab' Rabbit anti-hGHIg obtained in Example 2 (2) above
The G fraction was dialyzed against 0.1 M acetate buffer (pH 4, 5), 2% (w/v) pepsin was added to the anti-hGHI gG fraction, and the mixture was digested at 37°C for 20 hours.

The reaction was stopped with IN sodium hydroxide, and Ultrogel was equilibrated with 0.1 MM phosphate buffer (pH 8.0).
Gel filtration was performed using an A44 (LKB) column, and F (ab
Next, the F (ab')g fraction was added to 0.1 M phosphate buffer containing 0.2 M NaCl (pl+7.
After dialysis at 0) at 4°C overnight, affinity purification is performed. In other words, after equilibrating the pituitary gland extraction hGH-binding Sepharose 4B column with the above buffer, the F(ab') fraction is applied to the column and adsorbed. let After further washing the column with the same buffer, 0.1M glycine-hydrochloric acid buffer (pH 2,
5), the specific antibody F(ab') was fractionated at 0 and 5 M.
Elute into a container containing Tris-HCl buffer (pH 8,0).

Next, this specific antibody F(ab')g fraction was dialyzed against 0.1M phosphate buffer (pH 6.0), and 0.5-5μ/45
Prepare 0 μl solution. This contains 5mMEDTA.
, 1 M + J #Rffl buffer (pH 6,0) 0, 1 M mercaptoethylamine 5
0μ! and incubate at 37°C for 90 minutes.

Next, 0.1M phosphate buffer (pH 6) containing 5mM EDTA was added.
Gel filtration is performed using a Sephadex G-25 (Pharmacia) column equilibrated with .

Separately from the above operation, a pyridyl sulfide group is bonded to horseradish-derived peroxidase (hereinafter abbreviated as POD). That is, 6 μm of POD O,IM was dissolved in phosphate buffer (pH 7.0), and 50 times the molar amount of N
-Add an ethanol solution of succinimidyl-3-(2-pyridyldithio)propionate and incubate for 30 minutes at 30°C. This was mixed with 0.1M phosphate buffer (pH 6).
, 0), and 1 minute fraction of pyridyldithio group-bound PODii is collected.

Next, equimolar amounts of pyridyldithio group-bonded POD are added to the Fab' fraction, and the mixture is allowed to stand at 4°C for 20 hours. This mixture is gel-filtered through an Ultrogel AcA44 (LKB Co.) column equilibrated with 0.1M phosphate buffer (pH 6.5), and a rabbit anti-hG HFab'-POD fraction is collected.

BSA and thimerosal are added as protective stabilizers at 0.1% and o,oos%, respectively, and stored at 4°C until use.

(3) 10 test tubes for measurement method calibration curve (inner diameter 10 mm, length 75 mm)
Prepare two sets, each containing pituitary gland extracted hGH standard product (0,0
,1,0,3,1,3,10,30,100,300,
11000p/150. Add 150 pl of ul).

Add 20 μl of human serum to the sample test tube, and add calibration curve buffer (0.1% BSA, 0.4M NaCl, and 0.1% BSA, 0.4M NaCl, and 0.1% NaCl).
10mM phosphate buffer containing 1% sodium azide pH 7
, 0) Add 130 μl and mix well.

In the case of a urine sample, mix 100 ul of urine and 50 μl of the buffer solution. 6. Next, lightly sandwich the antibody-binding ball prepared in step +11 above with a bottle set, and absorb the adhering liquid with filter paper.
Place one in each test tube. After shaking and heating each test tube at 37°C for 6 hours (first reaction), the reaction solution in each test tube was removed by suction, 2 d of physiological saline was added, washed twice, the washing solution was removed by suction, and the reaction solution was separated in advance. Example 2 (l) and (
The enzyme-labeled antibody prepared in 2) was added to 0.1% BSA and O.
, diluted with 10mM phosphate buffer containing 1M sodium chloride, 10n
g/150μl of each solution adjusted to 150μl! and transfer the washed polystyrene balls thereto. After shaking and warming each test tube at 20° C. for 6 hours (second reaction), the unreacted enzyme-labeled antibody solution in each test tube is removed by suction, and the same washing operation as above is performed.

Next, add 0.1 M phosphate buffer (p11) to another test tube in advance.
Add 100 μ2 of a 0.6% p-hydroxypropionic acid (HPPA) solution of POD substrate dissolved in 7.0) and transfer the washed polystyrene balls thereto.

Also, from this reaction step, POD can be used as a reagent blank.
Prepare two test tubes containing only the base fHPPA and perform the subsequent operations. Add 50 μl of 0.015% hydrogen peroxide to each test tube containing the above substrate, and shake and heat at 30°C for 90 minutes (enzyme reaction). After that, the reaction was stopped with 0.05 M glycine-sodium hydroxide buffer (pH 10,3) 2.5-, and quinine sulfate (1Pg/d, 0.1N sulfuric acid) was used as a control, and the reaction was measured using a fluorophotometer. , subtract the reagent blank value from the relative fluorescence intensity and plot the standard ha)((
pg), take the relative fluorescence intensity on the vertical axis, plot each measurement value and draw a calibration curve. Read the amount of hGtI (pg) corresponding to the relative intensity of 20 μl of the sample from the calibration curve, and multiply that value by 50 times. The amount obtained is defined as the amount of hGH per sample.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing a calibration curve for enzyme immunoassay.

Claims (12)

[Claims] (1) Human growth hormone with a molecular weight of 22,000 (hereinafter referred to as 2
2K hGH) and has a molecular weight of 20,000.
A monoclonal antibody that does not substantially react with 20KhGH human growth hormone (hereinafter abbreviated as 20KhGH). (2) The monoclonal antibody according to claim (1), which is a mouse monoclonal antibody that has an affinity constant for 22K hGH of 10^8 l/mol or more and does not substantially react with 20K hGH. (3) Hybridoma CL. B1. The monoclonal antibody according to claim (1), which is produced by: (4) A hybridoma that produces the monoclonal antibody according to claim (1). (5) Hybridoma CL. B1. The hybridoma according to claim (4). (6) A method for measuring human growth hormone, which comprises using the monoclonal antibody according to claim (1). (7) The method according to claim (6), wherein the method for measuring human growth hormone is an enzyme immunoassay. (8) Antibody-bonded insoluble support and human growth hormone (
A test solution containing hGH) was reacted with 22K
After only hGH is specifically bound to the antibody-bound insoluble support, an antibody that specifically reacts with hGH is reacted with an enzyme-labeled label to form an antibody-22KhGH-labeled complex on the antibody-bound insoluble support. Claim (7) is characterized in that 22KhGH is specifically measured by forming the antibody and then measuring the enzyme activity amount of the label bound to the antibody-bound insoluble support. How to measure human growth hormone. (9) The method according to claim (8), characterized in that an enzyme-labeled anti-hGH polyclonal antibody is used as the label, which is an enzyme-labeled antibody that specifically reacts with hGH. (10) The method according to claim (9), characterized in that an affinity-purified Fab' fragment of rabbit anti-hGH antibody is used as the anti-hGH polyclonal antibody. (11) Claim (7) characterized in that a peroxidase-labeled substance is used as the enzyme-labeled substance.
The method according to any one of Items to Items (10). (12) Any one of claims (7) to (10) characterized in that when measuring the amount of enzyme activity, a compound that is converted into a fluorescent substance by an enzymatic reaction is used as an enzyme substrate. Method described.