JPS59214767A - Kit for measuring specific protein in human pregnancy - Google Patents

Abstract

PURPOSE:To determine SP1 in a number of sample liquids quickly and accurately by a radio immunoassay by arranging an anti-human SP1 (pregnancy specific protein) antibody immobilizing vessel, a radioactive substance labeled human SP1 labeled human SP1 and a buffer liquid. CONSTITUTION:Labeled human SP1 is dissolved into a buffer liquid to prepare a standard calibration curve. Radioactive substance labeled human SP1 is preferably <125>I or <135>I labeled one and the labeling of human SP1 is done by the chloroamine T method or the like. A vessel is preferably a test tube-shaped on male of a synthetic resin. Anti-human SP1 antibody is obtained by emulsifying human SP1 with Freund's Adjuvant and sensitizing the emulsion to an animal such as rabbit and guinea pig. The anti-human SP1 antibody is diluted in the buffer liquid and after it is allowed to stand in the vessel, the dilusion liquid is removed. Then, the product is washed and dried to obtained anti-human SP1 antibody immobilization vessel. The radioactive substance-labeled SP1 and a buffer liquid are added to the vessel and after allowed to stand, it is washed to make an antibody immobilization vessel in which about 40-60% of the labeled SP1 is bonded. The buffer liquid herein used is, for example, a 0.1M phosphate- buffered physiological salt solution with pH7.

Description

[Detailed description of the invention] Unexploded rib Ji-1: Pregnancy-specific protein (S
P,) Rapid and positive by total radioimmunoassay? 7
1) SP, related to a measurement kit that is capable of quantifying K.

(A
rch, GynMk, , 210.440, 197
1), and its serum concentration is attracting attention as an indicator of placental function and fetal growth.

The purpose of the present invention is to quickly and accurately test a large number of subjects.
The purpose is to provide bottlings that measure SPi to F.

Wood seller] is anti-human si', antibody specific volume, radioactive material J
A human SPI titration kit consisting of 1-labeled human SP, 1-labeled cow/hig) SP, and a buffer solution is included.

SP, which is related to unexploded j, is 1-4, which will be described later.
It is used to create a calibration curve and is made from an undried product of Touyu Seihito SP1.

Such a standard human SPI can be obtained by the method described in ii-j. The standard h si'+ is used as a melody Y (used in a loose drawing), and this one extra analogy is 1 to 100n.
Divide into 4 to 5 concentration levels so that f/me is used as standard L1 (used once to create a calibration curve).

Radioactive substance labeling (SP), for example, IZI
, +351-labeled or preferred, per human urine 5P11μ2, e.g. +251-C
- If the specific radioactivity is around 105~1O'cpm 2@
That's true. Radioactive substance labeled human SP? i, h) SP,
using methods known per se, such as the talolamine 1 method (Reference 1).
), the lactoperoxidase method (see Reference 2), and the Porton-Notter reagent method (see Reference 3).

The container in the anti-human sP, antibody immobilization device is preferably a test tube-like container, which preferably has a diameter of 5 to 151,111 mm.
11. The length is approximately between 50 and loo. The material may be synthetic resin such as polystyrene, polyglopylene, polyethylene, or glass, and synthetic resin is preferred because it is easy to store and handle and is inexpensive.

It can be obtained by emulsifying, for example, human SP or iFloyd adjuvant to the human SP1 antibody to be immobilized on the container, and then sensitizing the animal such as a rabbit, guinea pig, rat, or goat. anti-hito from anti-saraura) SP
+Ra production of antibodies is carried out by treatments such as ammonium sulfate precipitation, ion exchange chromatography, molecular sieves, etc., either alone or in combination.

Examples of methods for immobilizing the anti-Human SP+ antibody on a container include the following methods. Anti-human sp.

Antibodies, for example, 100 times, 400 times, 1600 times, 6
400x, 25600x, 102400x, 40960x
Dilute to approximately 0 times. Here, the buffer solution is acetate,
Phosphate, tris-hydrochloride etc. are available, especially O
, OIM Sodium Acetate Relaxation Buffer i45.5 is preferred. An appropriate amount of the antibody dilution solution (for example, 0.5 to 1.5
-) ff: Put in a container, for example, 1 to 2 at 0 to 37°C
After allowing it to stand for 4 hours, completely remove the diluent. Washing is preferably carried out 1 to 3 times using excess volumes (for example 1 to 2 ml) of washing liquid. Cleaning liquids include distilled water, physiological saline, and 0.0
A solution containing 1 to 0.1% albumin or Tween-20 can be used. After washing and drying the container, each container for anti-Human SP+ antibody identification can be prepared. Then, the antibody immobilized container obtained in this way was labeled with a radioactive substance SP! .

For example, +25i mark at SP + about 10000c
Add pm buffer (e.g. p) 16.5 to 8.5
), the gold amount is preferably l-, for example 0 to 87
Let stand at °C for 2 to 24 hours. After standing, the sample is washed using the same method as above, and radioactivity is measured. Thus,
It is preferable to use a gold antibody-immobilized container to which about 40 to 60% of the used radioactive substance labeled SP binds for the kit of the present invention.

The buffer solution truly used for the Misfire J kit is for antigen-antibody reactions, with a pH of 6 to 9 and a salt concentration of 0. O
A thickness of about 1 to 0.2M is preferable. Specifically, for example, a 0.1 MIJ salt buffered physiological saline solution, pH 7, is used.

The method for defining SP using the kit of the present invention is as follows.

Anti-Human SP1, radioactively labeled human SP1 in antibody immobilized container
A buffer solution and a test solution (test urine) are added and incubated. In this way, the immobilized antibody, the radioactive substance-labeled human sp, and the SP1 in the test solution form conjugates (8 bodies) through an antigen-antibody reaction.

By the way, in the present invention, since the anti-Human SP and antibody are immobilized on the container, the free radioactive substance-labeled SF1 and the free SP in the test solution (that is, these correspond to the F form)
can be easily removed by removing the waste in the container. i.e. ij B/F min PIP in "misfire"
, is extremely easy.

After performing B/F separation, the radioactivity of the container is measured. At this time, the SP in the test solution, the larger the amount, the more radioactive substance f1
Since less labeled human urine SP1 binds, the radioactivity value becomes smaller.

On the other hand, standard calibration curves were prepared at various concentrations of the standard H)SP instead of the two test samples, and the SP I in the test solution was determined by comparing this with the radioactivity value of the test box. (goods) to do.

Create the line as follows.

NX1 (10
(However, BO is the radioactivity value when bound to the maximum amount of anti-human sp, antibody-immobilized container, and standard hsP at each dilution) SP, and radioactive substance-labeled human Sl ', the radioactivity value when combined,
The horizontal axis shows human SP Hiti (n W/”48:: l
By taking k and connecting the two at each concentration of standard human SP+, 'C' is created.

Therefore, the -5X100 value of each test solution should be adjusted to match the standard calibration edge, and the human SP contained in the test solution, the amount (
n2/volume (e)) can be measured.

Example 1 (Preparation of anti-human sP, antibody-immobilized test tube) Anti-human sPI antibody was purified from rabbit anti-blood grooves against SP by ammonium sulfate precipitation method and DEAE-cellulose chromatography, and then purified with 001M sodium acetate, It was diluted 6400 times using pH 5.5. 1me of this diluted solution was added to a polystyrene test tube (1, Ox
The mixture was placed in a 7.5 ml of water and allowed to stand at 87°C for 4 hours. After removing the solution in the test tube by suction, it was washed twice with 1,5- of physiological saline containing 0.05% bovine serum alguminum. Washed once with 1.5 mg of the solution.

It was dried by blowing air at 25°C.

Reference Example 1 (Measurement of SP using this kit) Anti-Human SP, antibody-immobilized phosphate-buffered physiological earth solution tD 0.8 rnt, +257-labeled SP, solution 0
．． 1 rtt (containing about 110,000 cp) and 0.1 + d t of the specimen, stirred, and allowed to stand at 37°C for 4 hours. 1
．． After washing twice with ., 5me, radioactivity was determined using a comma counter. On the other hand, in the reaction solution described above, instead of the analyte, O, Lm of the standard #-8PI dissolved
ll (as SPl, 1nf', 4n?, 16nf
, 64n5i', 128ny) was used to prepare a standard calibration curve. The amount of human SP contained in the broken bones was determined by comparing the reaction with the radioactivity calibration curve of the removed bones.

Reference Example 2 Human placenta extract 400j [Human placenta (average weight 600g)
420 pieces of minced meat suspended in physiological saline solution] was mixed with IN-H (adjusted to pH 4.8 with l, left to cure for 3 to 4 days, then unneeded, The mucoid fraction and heme fraction were removed by centrifugation (3000 rpm
Hi. Furthermore, by attaching the ammonium sulfate side to the brim (at p 147.0) and saturating it with 5 strips, the resulting sedimentation centrifugation fraction IWI: was removed by 1 minute, and the obtained upper layer was added to the skin by adding ammonium sulfate to a length. The resulting sediment was dissolved in 0.02 M phosphate buffer (CpH 6,8) K and dialyzed against the same buffer. Using a molecular weight marker, we separated both fractions with a molecular weight of 80,000 to 120,000 Daltons.
- It was applied to a Sepharose column to dissolve SP. The resulting unadsorbed fraction was atomized by 40% saturation with ammonium sulfate, then dialyzed against the same buffer solution, and then lyophilized to obtain dry powders of SP and BQf. This SP, if necessary, can be further processed by high performance liquid chromatography or
It can be highly purified by an electrophoretic method such as MAD77j'-j)'/ng and used as an antigen for immunization against hmJ.

Reference 1) Greenwood, F. C., et al.
+Biochem.

J, 89.114 (1968) 2) Miyachi Y, et al * J,
Cl1n.

Endocrinol, Metab, 84, 2
8 (1972) 8) Bolton, A.h.
and Hunter, W.M.

Claims (1)

[Claims] (1) A human pregnancy-specific protein quantification kit consisting of an anti-human pregnancy-specific protein antibody immobilization container, a radioactive substance-labeled human pregnancy-specific protein, a standard human pregnancy-specific protein, and a buffer solution.