JPS6117065A - Measuring reagent for human chorionic gonadotropin - Google Patents

Abstract

PURPOSE:To enable a high sensitive measurement free from cross reaction with other hormons, by combining a monoclonal antibody highly specific to the whole molecular structure of human chorionic gonadotropin and a monoclonal antibody highly specific to the beta subunit of HCG. CONSTITUTION:After the fusion of a spleen cell obtained by immunizing a mouse with human chorionic gonadotropin (HCG) and a marrow seed cell derived from a mouse, a cell for producing an antibody is selected against HCG by cloning them. A monoclonal antibody highly specific to the whole molecular structure of HCG and a monoclonal antibody highly specific to the beta subunit thereof are obtained from the cell. For example, a sample to be measured is added to an insoluble solid supporting an atibody highly specific to the beta subunit to be incubated and then, an antibody highly specific to the whole molecular structure of HCG labelled with enzyme, radioactive element or the like to be incubated. Thereafter, activity or the like of the label of the labelled antibody bonded to a solid phase is measured. Thus, HCG can be measured accurately not affected by luteal hormon or others.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention is an invention of a reagent used for highly specific immunochemical measurement of human chorionic gonadotropin (hereinafter abbreviated as HOG).

HOG is a protein hormone secreted by the villous tissue of the human placenta, and detection of HOG in urine is used for early diagnosis of pregnancy.

In recent years, it has become clear that some malignant tumors produce a large amount of HOG, and measurement of HOG in body fluids is also used for diagnosis and follow-up of malignant tumors.

Conventional HOG measurement methods include radioimmunoassay (aadio
-immunoassay (hereinafter abbreviated as RIA), enzyme immunoassay (Bnlyme+rrmunoassay)
Immunochemical methods such as the latex method (hereinafter abbreviated as EIA) and the latex method are often used.

Problems to be solved by the invention HOG has a molecular weight of 37, consisting of α and β subunits.
000 glycoprotein hormones. The α subunit has an amino acid sequence similar to that of α subunits such as luteinizing hormone (hereinafter abbreviated as LH), follicle stimulating hormone (hereinafter abbreviated as FSH), and thyroid stimulating hormone (hereinafter abbreviated as TSH). On the other hand, the β subunit of HOG,
It has a different amino acid sequence from the β subunits of FSH and TSH, but is similar to the β subunit of LH. Specifically, the β subunit of HOG has a peptide chain that is 30 longer at the carboxyl group end, but 92 of the 115 amino acid sequences from the ami7 end are common to the LH β subunit.

For the above reasons, in the immunochemical assay of HOG, LH
Cross-reactions are a major problem.

It is known that polyclonal antibodies obtained through normal immunoassays exhibit some degree of cross-reactivity with LH, and immunochemical assays using such antibodies have resulted in samples in which it is difficult to distinguish between HOG and LH. It was not a highly specific measurement method.

Many reports have also been made regarding HOG monoclonal antibodies. wada et a+, (01in,
0hen.

Volume 28, page 1862 (19B2) Tsukima, HO that has been suppressed
reported the Sand-Germany EIA method of HOG using a monoclonal antibody against the α subunit of G and an enzyme-labeled monoclonal antibody against the β subunit.

However, according to their report, the cross-reaction with LH is relatively high at 8.3% (there seems to be a problem with samples showing high levels of LH).

Means for Solving the Problems In view of the above circumstances, the inventor has conducted intensive research and has found that
In the HOG sandwich method using two types of monoclonal antibodies, LH,
The present invention has been completed by devising a highly specific measurement method for HOG that shows almost no cross-reactivity with , FSH, etc.

The measurement method using the reagent of the present invention will be described in more detail.

The monoclonal antibodies used in the present invention can be obtained using cell fusion technology (Natural Vol. 256, p. 495 (1).
975)). The manufacturing method will be briefly outlined below.

First, mice are immunized with HOG as an antigen, and the resulting tile cells are fused with mouse-derived myeloma cells, followed by cloning to select cells that produce antibodies against HOG. The monoclonal antibodies produced by the obtained cells are HO
It is a specific antibody that corresponds to a specific part of the G molecule.

Next, the specificity of the monoclonal antibody obtained in this way was examined by RIA, and HO, which has low cross-reactivity with LH,
A monoclonal antibody that is highly specific for the entire molecular structure of G and a monoclonal antibody that is highly specific for the β subunit of HOG are selected. Furthermore, a monoclonal antibody highly specific to the HOG β subunit can also be obtained by immunizing a mouse with the HOG β subunit and cloning the antibody after cell fusion.

These monoclonal antibodies are, for example, 001-1 Misaki/
ml aqueous solution or immobilized on an insoluble carrier,
Furthermore, the labeled antibody can be used as the measurement reagent of the present invention.

To measure HOG using the reagent of the present invention, one type of monoclonal antibody immobilized on an insoluble carrier is reacted with HQG as an antigen, and then another type of monoclonal antibody is labeled with a radioactive substance or an enzyme. A method of reacting a labeled antibody, the so-called Sandermansch method, is suitable.

Insoluble carriers used for immobilization include polystyrene spheres,
Glass beads, latex, silicone pieces, etc. are used.

The labeled antibodies employed in this case include radioactive isotopes (e.g., 1311, -, etc.), enzymes (e.g., peroxidase, β-galactosidase, alkaline phosphetase, etc.), etc. bound to monoclonal antibodies. It can be manufactured by

As a method for binding radioisotopes and antibodies, the chloramine T method (Nature, Vol. 194, p. 495 (L962
There are methods such as the periodic acid method (J, H
Istochem, Oytochem, Volume 22 10
84 (1974)), mixed acid anhydride method, carbodiimide method, etc.

A method for measuring HOG using the Sanderch method will be explained in more detail.

First, a certain type of monoclonal antibody (for example, a monoclonal antibody with high specificity for the β subunit of HOG) is insolubilized, the analyte to be measured is added to the solid phase, and after reaction at a certain temperature for a certain period of time, the reaction solution is washed. Remove. Next, another type of labeled monoclonal antibody (for example, a monoclonal antibody highly specific to the entire molecular structure of HOG) is added, the reaction is further allowed to proceed for a certain period of time, and the reaction solution is washed away.

If a radioactive isotope is used as the labeled antibody, directly measure the radioactivity of the reactant.

When an enzyme is used as the labeled antibody, a substrate for the enzyme is added to carry out an enzyme reaction, and the enzyme activity is measured.

The amount of radioactivity and enzyme activity obtained above increase in accordance with the amount of HOG in the sample to be measured, so
By drawing a calibration curve using a standard solution with a known OG concentration, the amount of HOG in a sample can be measured.

The first method mentioned above is a so-called two-step method, but measurement can also be performed using a one-step method. That is, an unknown specimen and a labeled antibody are simultaneously added to the insolubilized monoclonal antibody and allowed to react for a certain period of time. After washing and removing the reaction solution, H in the sample is determined by measuring the radioactivity or enzyme activity of the reaction product.
You can know the amount of OG.

The present invention will be further explained below with reference to Examples, but the present invention is not limited thereto.

Example 1 Measurement of HOG by Sand-Deutsch method (1) Preparation of monoclonal antibody 150 μg of HOG was mixed with complete Freund's adjuvant and administered intraperitoneally to BALB10 mice.

1 = After 3 months, 50 μg of HOG was administered intraperitoneally, and 6
The spleens of the mice were removed on the following day. Pile cells and myeloma cells (
P3-NSI/1-A74-1) were mixed at a ratio of 5=1, and cell fusion was performed in the presence of polyethylene glycol 4ooo.

Fused cells were selected by culturing in a hypoxanthine-aminopterin-thymidine medium, and the amount of HOG antibody in the culture supernatant was measured by RIA.

The active fused cells were converted into single clone-producing cells by limiting dilution, and then inoculated into mice that had been previously administered pristane to obtain ascites. Ascites was salted out with 50% saturated ammonium sulfate, and I and G fractions were purified using a DEAE cellulose column.

Regarding the specificity of the obtained monoclonal antibody, HOG
This study was conducted using the 几IA.

If the cross-reactivity with HOG is 1.00%, one monoclonal antibody shows less than 1 cross-reactivity with LI (2%), H (α subunit of 3G, β subunit of HOG, and FsH). , a monoclonal antibody with high specificity for the entire molecular structure of HOG.

Another monoclonal antibody is a crossantibody of HOG, L
HlFSI (shows less than 1 cross reaction with HO
G β-subunit) [It was a highly specific monoclonal antibody.

(2) Preparation of polystyrene spheres with insolubilized monoclonal antibodies Add the monoclonal antibody with high specificity to the β subunit of HOG obtained in (1) to a concentration of 0.01M/ml.
Polystyrene spheres (64mm φ
] and left at 30°C for 18 hours. After separating the polystyrene sphere from the liquid and washing, it was stored at 4°C in PBS containing 01% bovine serum albumin and used.

(6) Preparation of peroxidase-labeled antibody (1) H
Monoclonal antibody 5 highly specific for the entire molecular structure of OG
and horseradish-derived peroxidase (manufactured by Sigma) by a known method (Nakane P, K, an
d Kawai A: J, Histocher.
n cytochem.

22, p. 1084 (1974)) and purified with Sephadex G-200, and the highly active fraction was diluted 1000 times with PBS and subjected to measurement.

(4) Take the HOG standard solution or test sample in a sandwich test tube and add PBS for 20 minutes.
Set the volume to 0 μl. Next, one polystyrene sphere in which the monoclonal antibody obtained in (2) was insolubilized was added and reacted at 37°C for 2 hours, followed by washing twice with PBS.

Further, add 200 μl of the Tabell oxidase-labeled antibody obtained in (3).
1 and reacted at 37°C for 2 hours.

After washing twice with PBS, add 1 ml of a substrate solution containing 02% 0-7 22 diamine and 001 hydrogen peroxide, react for 1 hour in the dark, and then add 3 ml of 1M H2SO4 solution!
In addition, the reaction was stopped. Is the reaction solution 4927L? The absorbance of FL was measured using a spectrophotometer, a calibration curve was drawn using the absorbance of standard HOG, and the absorbance of the sample was applied.

The HOG concentration was calculated.

The measurement results are shown in Table 1.

Table 1 Example 2 of measuring HOG using the Sand-Deutsch method
LI (, HOG as a sample for cross-reaction measurement with FSH,
A sandwich method test was conducted in the same manner as in Example 1 using standard solutions of LH and FSH.

The measurement results are shown in Figure 1.

LH,'FSH showed no reaction at all even at a concentration of IQ 007Lg/rnl.

Effects of the Invention The method for measuring HOG using the reagent of the present invention
This is an extremely specific method for HOG, which exhibits a complete cross-reaction with HOG, and is an industrially useful method.

[Brief explanation of drawings]

The drawing shows the cross-reaction of HOG with LH and FSH in the Sand Inch method using the reagent of the present invention. Patent applicant Mitsui Toatsu Chemical Co., Ltd. ℃ MA: . . 6 = - (mu Otsu 6shi) Men γ □ Jun 6 Procedural amendment August 7, 1982

Claims (1)

[Claims] A reagent for measuring human chorionic gonadotropin, which comprises as essential components a monoclonal antibody highly specific to the entire molecular structure of human chorionic gonadotropin and a monoclonal antibody highly specific to the β subunit of human chorionic gonadotropin.