JPS6283664A - Reagent for immunoassay - Google Patents

Abstract

PURPOSE:To make it possible to shorten the time required in immunoassay and to measure PTH with high accuracy while performing antigen-antibody reaction within a shorter time, by constituting a reagent for immunoassay of hPTH[(39-43)-68] labelled with a marker. CONSTITUTION:As anti-hPTH antibody used in immunoassay using a tracer, one having specificity to hPT and capable of confirming the center region of hPTH, especially, a region from 39-th alanine to 68-th glycine as an antigenic determinant is used. In a solid phase method, the antibody converted to a solid phase on an insoluble carrier is used and, in a liquid phase method, a BF separating agent is used after antigen-antibody reaction to perform BF separation. The quantity of a marker is measured, for example, by measuring the radioactivity of an radioactive element and the activity of enzyme can be measured by a spectroscopic or fluorescent technique.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel reagent used in hPTH (human parathyroid hormone) immunoassay.

[Prior art]

PTH is a peptide consisting of 84 constituent amino acids, and measurement of its blood concentration plays an important role in the diagnosis and pathological analysis of various parathyroid diseases and metabolic bone diseases related to abnormalities in calcium and phosphorus metabolism. .

Since radioimmunoassay was developed by Berson et al. in 1963 as a method for measuring PTH (prO
c, fiJatl, , 4Cad, 3Ci, 11.
3A, VOl, 49, p613 (1963))
, various improvements have been made.

Initially, bovine PTH (1-84) labeled with a radioactive isotope was used as a tracer, but bovine PTH (1-84) has a high non-specific binding property and can be used for example on test tube walls. There were problems with measurement sensitivity and accuracy, such as adsorption to other materials.

In recent years, a homogeneous RIA method using synthetic human PTH fragments as tracers and standards has been published, and Marx et al.
68) By using fragments, there is less adsorption to the glass wall, storage stability, and BF by DCC.
We report an RIA method with excellent separation performance (JCli
n, EndOCrinol, 1vfetab, .

Vol, 58, pp76-84 (1981)). In addition, 5harp mule, N of hPTH as a tracer.
A tyrosine residue was introduced at the end (Tyr 43) h
By using F'TH(4a-68), hPT
15-5096 than when using H(4,4-68)
Sensitivity improvement Surukotowo was reported [(:1inica,
(:himica.

Acta Vol, 145. pp 59-68, (19
85) ).

Radioimmunoassays using other PTH fragments include hPTH(1-84) (e.g. 7ttx
as Reports f3i0. fi7flld,,
VOl, 83, p457 (1975)), hPTH
(44-64) (Ligand Review, Vo
l, 2.1)? (1980) ), hPTH (65
-84) (e.g. Hormones and Clinical Studies, Vol. 30
%I) p885-888, pI) 1309-1313,
(1982) is known.

[Problem that the invention seeks to solve]

In the conventional hPTH radioimmunoassay,
Although problems such as measurement sensitivity were resolved, there were 3 to 3 problems with the assay.
The assay system required a long time of 4 days, and there was a desire to develop an assay system that could quickly and accurately perform measurements in a shorter period of time.

[Means for solving problems]

As a result of intensive research to solve the above problems, the present inventors discovered hPTH [(39
~48) - By using the labeled product of -68), hP
The present invention was completed based on the finding that the TH' immunoassay operation can be completed within 24 hours.

That is, the present invention provides hPTH [
(89-48)-68). Furthermore, the present invention provides a competitive hPTH immunoassay reagent kit, which contains hPTHE(39-43)-68) labeled with a labeling substance as a tracer as a constituent reagent.

In the present invention, hPT used as a tracer)
[((39-43)-68) has the following N-terminus at 39
to the 43rd amino acid sequence.

(AIa-pro 4eu-AIa-Pro) -
Arg −AS+) −Ala −Gly −5er
-Qlu -Arg-Pro -Arg -1ys-t
, ys -Qlu -Asp -Asn -Val -
Leu-Val-Glu-8er-)(is-
(, lu −1,ys −5er −Leu −Qly
Examples include H(43-68). When radioactive iodine (125, 1311) is used as a labeling substance, these fragments with a tyrosine residue introduced at the N-terminus are used. You only need to select one that matches mglfiWm-lum, and generally radioactive isotopes (for example, 1251.1311.3H, 14 (etc.), enzymes (
For example, β-galactosidase, beluoxinase,
Alkaline phosphatase, acetylcholinesterase, etc.), fluorescent substances (e.g., umbelliferone, fluorescein, rhodamine, fluorescein isocyanate, fluorescein isothiocyanate, phycoerythrin, etc.), chemiluminescent substances (e.g., luminol, etc.), and the like are applicable. Labeling methods for these labeling substances are all known, and may be carried out by any known method. For details of these general technical means, reference can be made to reviews, books, etc. (for example, "Radioimmunoassay" edited by Minoru Irie; "Radioimmunoassay" edited by Minoru Irie; (Kodansha Co., Ltd., published May 1, 1980), edited by Eiji Ishikawa et al., "Enzyme Immunoassay" 2nd edition (Igaku Shoin Co., Ltd., December 1980)
Published on May 15th), Nippon Rinrin, Volume 42, Spring Special Issue (
1984) rClinical Immunology Handbook J (Nippon Rinrinsha Co., Ltd., published March 20, 1984), No. 11
b8-1227 pages, H0■, Bounakis et al., “Meth
ods in Enzymology Vow.

70, [,)mmunochemical Techn.
iques part AJ (Academic Press,
1980), J. J., Lambon et al., j”Method
s in EnzymologyJ VOl, 78
, “Immunochemical Technique
es Part BJ, Ibid., VOI.

74 [mmunochemical technology
ues part (: J, (Academic Press,
(1981), etc.).

For example, when labeling with radioactive iodine such as 125 and 1811, chlora, min-T method, lactoperoxidase, etc. are applied, and when labeling with enzyme, acid anhydride method, carbodiimide method, glutaraldehyde crosslinking method, periodic acid method, etc. Cross-linking methods, maleimide cross-linking methods, etc. are applied (
(See Japanese Patent Application Laid-Open No. 55-26477).

The immunoassay to which the tracer of the present invention is applied involves performing a competitive binding reaction between the analyte and the tracer against a receptor such as an antibody, and measuring changes in the amount of bound or unbound label depending on their abundance ratio. Any method is acceptable as long as it is based on the measurement principle of quantifying the analyte using a method. Such an immunoassay system is based on the following: A liquid phase method in which an antigen-antibody reaction is carried out, followed by BF separation using a BP separation agent, and the amount of labeled B (bound tracer) or F (free tod-cer) is measured; The substance in the sample and 1-racer are reacted sequentially or simultaneously to cause an antigen-antibody reaction, and then separated into a liquid phase and a solid phase, F in the liquid phase or B in the solid phase.
There is a solid-phase method that measures the amount of labeling.

The anti-hPTH antibody used in the immunoassay using the tracer of the present invention has specificity for hPTHl, and has specificity for hPTHl in the mid region of hPTH.
In particular, any antigen that can recognize the region from alanine at position 39 to glycino at position 68 as an antigenic determinant may be used. Antibodies are prepared by conventional methods. For example, a method of immunizing a foreign animal such as a rabbit, rat, sheep, horse, cow, or chicken with bovine or human PTH or a central region PTH fragment and collecting antiserum from the immunized animal; Obtain antibody-producing cells such as lung cells, thymocytes, or peripheral lymph node cells, fuse these with thick myeloma cells, and culture the resulting hybridoma by cell culture or mouse ascites formation method,
It can be obtained by a method for obtaining produced antibodies.

In the solid phase method, an antibody immobilized on an insoluble carrier is used. Examples of insoluble carriers include silicone, glass, ceramic, polystyrene, and styrene.
Divinylbenzene copolymer, polyethylene, polypropylene, crosslinked polyacrylamide, cyanogen halide activated cellulose, 0M cellulose, DEAE cellulose,
Materials that can be used in general immunoassays/assays, such as cross-linked dextran, may be used. The shape of the carrier can be arbitrary, such as latex, disk, particulate, etc.
It may be in any shape such as a tube. Known methods are also applied to immobilize the antibody onto a solid carrier.
For example, physical adsorption methods, glutaraldehyde, succinic acid, 2,2-dipyridyl sulfide, performed within 24 hours. In the reaction, predetermined amounts of antibodies and tracers are used, and a competitive reaction is performed with PTH in the standard solution and sample solution. As the buffer, those commonly used in immunoassays are used, such as Tris-HCl buffer,
Phosphate buffer, acetate buffer, citrate buffer, etc. can be used.

After the antigen-antibody reaction, in the case of a liquid phase method, B
F separation takes place. As the BF separation agent, DCC (dextran-coated activated carbon), polyethylene 7 glycol, ammonium sulfate, a second antibody against an anti-PTH antibody, etc. are used. The second antibody can be used as a soluble antibody, but it can also be used as an immobilized antibody on an insoluble carrier as described above. Either BF is precipitated or adsorbed by such a separating agent, and the two are separated. In the case of solid phase method, lBF is used for suction, tilt-1 filtration, etc.
Separation can be performed.

Measurement of label 1 is performed by employing known means depending on the type of each label substance. For example, the radioactivity of a radioisotope can be measured using a device capable of measuring the amount of radioactivity of a radioisotope, such as a liquid scintillation counter or a gamma counter. The enzymatic activity of an enzyme can be measured using an activity measurement method depending on the type of enzyme, by selecting a substrate and measuring the amount or rate of substance consumption or production as the enzymatic reaction progresses using electrochemical, spectroscopic, or fluorescent methods. This can be done by measuring using standard techniques.

The PTH content in the test sample is determined by comparing the measured value of the labeled mold for the sample with the measured value of the label for the standard solution.

The composition of reagents other than the tracer of the present invention in a reagent kit for PTH immunoassay using the tracer of the present invention is selected and determined depending on the immunoassay system. For example, the basic reagent composition of a kit for liquid phase method is as follows.

■ Tracer 0 of the present invention Anti-PTH antibody reagent ■ PTH standard solution ■ BF separation agent The basic reagent composition of the solid phase method kit is as follows.

■ Tracer of the present invention ■ Immobilized anti-PTH antibody reagent ■ PTH standard solution For example, an immunoassay kit using an enzyme as a label further combines these basic reagent compositions with a substrate solution, a reaction stopper, etc. changes to;
Addition is optional.

〔Effect of the invention〕

According to the present invention, labeled hpTH [(
By using 39-43)-683, labeled hPTH (44-6
8), the binding reaction rate to the anti-PTH antibody was improved, the antigen-antibody reaction could be carried out in a shorter time, and the time required for the assay could be significantly shortened. Furthermore, it is possible to measure PTH with higher accuracy in a lower concentration region.

Examples Example 1 0.05M+) phosphoric acid buffer (pH 7,4) 208g
After adding 12J 1mC1 to a small test tube,
r42-h PTH(43-68) (Calbi
Add 112.5 μg of chloramine T (111 g/ml of 005M phosphate buffer (pH 7.4) solution), stir, and stir. Add 200μe of .05M phosphate buffer (pH 7,48,2) and add this to a Sephadex G-25 column (diameter 1G x 30α
) and gel filtration. 0.1 M) Lis-HCl buffer (0.1 M)
, 1% BSA, 10mM EDTA, I)H8,2), the radioactivity of each fraction 5] was measured, the fractions of the first radioactivity peak were collected, and (125■)-Tyr42-hPTH (
48-68) content classification was obtained.

Example 2 A radioimmunoassay kit for measuring PTH was prepared consisting of the following constituent reagents.

1. PTH standard solution hPTH (1-84) Opg/ml 5 ml 1 vial 10
0 pg/wt Lmt 1 vial 20
0 pg/xt 1 ml 1 vial 4
00 +) g/gl1M11 vial 800 pg/sapon 1 ml 1 vial 1600 pg/m
e 1 shoulder l 1 vial 3200 [) g/
Tan/1 mt1 vial 2, PTH antiserum solution anti-PTH chicken serum 10.5m/1 vial 1
('25I)PTH solution (125I)-Tyr42-hPTH(43-68)1
, 171C4/11 shoulder t 1 vial 4, second antibody solution anti-chicken IgG goat serum 1.0.5 ml
1 Vial Reference Example I PTH standard solution is hPTH (1-84) mixed with 0.1 M ToIJS hydrochloric acid buffer (10 m
The samples were diluted to various concentrations using MEDTA (pH 8, 2).

Reference Example 2 A PTH antiserum solution was prepared by immunizing with bovine PTH as an antigen by the method of Hruska et al.
Cl1n■nvest, Vol, 56, pp3
9-45, (1975).

Reference example 3 (125■)-Tyr 4 2-h PTH
(43-68) was obtained in Example 1 by 0.1
M l-IJs hydrochloric acid buffer (0,196B SA, t
It was prepared by diluting it using OmMEDTA% pH 8,2).

Reference example 4 Anti-chicken IgG goat serum is commercially available chicken Ig
Cq↑ was sensitized by subcutaneous injection, blood was collected, and centrifuged to obtain 1f[L supernatant. Polyethylene glycol was added to this to prepare a second antibody solution.

Prepare a small test tube with the application example number (1 to 18) written on it, and
．． 2 was used for total count, Nα3.4 was used for non-specific binding value (NSB), and Nα5-18 was used for standard curve.

PTH standard HOpV/me 800ue Na 8.
4 and each standard solution (0,1'O'0,200.40
0.800.1600,8200p9/rin/)200μ
1 was dispensed into Nα5-18.

200 μe of the serum coated with the serum was dispensed into separately prepared sample test tubes (2 tubes).

100 μl of PTH antiserum was added to all test tubes and mixed using a portex mixer.

It was left at room temperature for 4 hours.

(12J) PTH solution 1001ml (D5-Eufuni was added and mixed using a portic mixer.

It was left in ice water for 18 hours.

One tie of the second antibody solution was dispensed into all test tubes containing Nα1.2 and mixed using a portex mixer.

It was left in ice water for 1 hour.

All test tubes except Nα1.2 were cooled and centrifuged (300
01m, 2-8°0120 minutes).

The upper yWH was removed by suction using an aspirator, and the radioactivity in all test tubes was measured using a gamma counter.

The binding rate was calculated from the radioactivity measurement results using the following formula.

(Bo) - (NSB) fBl Average measured value of the test sample or each standard solution (B
L) NSB average measured value (Bo) Average measured value of standard solution Opg/Go Semi-logarithm The horizontal axis of the graph paper shows each concentration of PTH standard solution, and the vertical axis shows the B/Bo (%) corresponding to each standard solution. 9 was plotted to create a standard curve.

The B/Bo Kl of the test sample was similarly calculated, and the degree of PTHa was read from the standard curve.

As a measurement control, Bo/T (%l) is calculated to determine the binding titer between the tracer and the antibody.

Comparative example Tyr 43 h PT'H (44-68) (Be
(125I)-Tyr48hPTH (44-68) in the same manner as in Example 1 and Reference Example 3 using
was prepared. This was used as a control, and the operation method described in the application example was used to observe the difference in the binding rate between the two. The results are shown in the table below.

The standard curves prepared using both were shown in FIG. 1.

From these results, it is clear that a sufficiently high binding rate can be obtained with the tracer of the present invention, whereas with the control tracer, the reaction is insufficient within the assay time of the application example, and a high binding rate cannot be obtained.

When a clinical specimen (patient serum) was measured using both tracers according to the procedure described in the application example, a good positive correlation was obtained as shown in FIG.

Correlation coefficient γ=0°9687 N=17 Regression formula:
Y=-0,0164+1.0838X

[Brief explanation of drawings]

FIG. 1 shows a standard curve of PTH created by RIA method using the tracer of the present invention and the conventional tracer,
FIG. 2 shows a correlation diagram of the results of measuring clinical specimens using these methods. Patent applicant (677) Yamasa Soy Sauce Co., Ltd.'/T%
$1 figure

Claims (1)

[Scope of Claims] 1) An immunoassay reagent comprising hPTH [(39-43)-68] labeled with a labeling substance. 2) The immunoassay reagent according to claim 1, wherein the labeling substance is a radioactive isotope. 3) In the hPTH competitive immunoassay kit, hPTH [(
39-43)-68] as constituent reagents.