JPS59212435A - Protein related to basement membrane derived from human placenta - Google Patents

Abstract

PURPOSE:The titled novel protein recoverable from the human placenta, having high specificity to ovarian cancer, and vesicular mole, useful for diagnosing and remedying these diseases. CONSTITUTION:Novel protein having high specificity to ovarian cancer, and vesicular mole, obtainable from the human placenta, having about 100,000 molecular weight by gel filtration, 3.7pH of isoelectric point, mobility in beta-globulin range by electrophoresis, and 10wt% calculated as hexose saccharide content. The protein related to basement membrane derived from the human placenta (BMAP for short) is recoverable from the human placenta by a process such as alkali extraction, sodium dodecyl sulfate extraction, antigen.antibody complex extraction, etc. Human placenta from which blood is removed is preferable as the human placenta, and preferably fresh. BMAP shows positive only to ovarian cancer and vesicular mole, but negative to normal people and other malignant diseases, and has very high specificity.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel protein (human placenta-derived basement membrane-related protein) recovered from human placenta and highly specific to ovarian cancer and hydatidiform mole.

Proteins derived from human placenta are broadly divided into two groups: water-soluble antigens and insoluble antigens. There are 12 types of soluble antigens, and blood coagulation ratio factors, 7 erythins, etc. belong to this group.

By the way, the present inventors discovered that there is a water-soluble protein highly specific to ovarian cancer and hydatidiform mole in the human placenta, especially in its basal membrane, and wondered how to isolate this protein. Along with establishing
After investigating its properties, it was confirmed that the substance was a new substance, and the present invention was completed.

The invention can be recovered from human placenta, has a molecular weight of about 100,000 as determined by gel filtration, an isoelectric point of pH 3.7, a mobility of β-globulin by electrophoresis, and a sugar content of 10% (as a hexose). human placenta-derived basement membrane-related protein (hereinafter referred to as
Regarding BMAP.

The BMAP of the present invention is usually extracted from human fetus m by: ■ alkaline extraction, ■ SDS (sodium dodecyl sulfate) extraction, and ■
It is recovered by methods such as antigen-antibody complex extraction.

The human placenta to be used in the present invention is preferably one after blood components have been removed, preferably fresh, and preferably fresh or frozen.

Note that blood components are usually removed by washing with distilled water, physiological saline, or the like.

BMAP is collected from the placenta in the following manner.

(Method 1) To extract from the placenta, first, immerse the placenta and suspend it in distilled water, physiological saline, buffer, etc. Homogenize and collect the precipitate by centrifugation. Is this sediment an alkaline water bath solution with a pH of 8 to 10? Add, homogenate, and supernatant pH
4 to 5.5 and perform acid precipitation. This precipitate fraction? The mixture is again bath-dissolved in the alkaline water bath solution, homogenized, and the dried supernatant is subjected to salting-out fractionation using 25 to 35% ammonium sulfate saturation. The precipitate is collected, dissolved in a pH 6-8 buffer, and the salt removed, followed by column chromatography using a weak to medium basic anion exchanger. The column was contacted with a BMAP solution equilibrated with a low ionic strength buffer of pH 6 to 8, and HM
AP k is coupled. After washing the column, increase the ionic strength and elute and collect it in HMAP'. This collected product is subjected to affinity chromatography using an immobilized anti-N (normal human serum) antibody column, and then adsorbed onto the contaminated protein ligation column. Let me see, BMAP ke elution koma made tea.

(Second method) As the M"J method for different BMAPOs, placenta mince is suspended in physiological saline, etc., homogenized, centrifuged sediment is collected, and the placenta is dissolved in a low salt concentration buffer containing γbisDs with a pH of 7 to 9. There is a way to extract BMAP; b. BM
AP is 0.3-1.0% SDS 17J concentrated Jlj
It appears in the soluble fraction. 18-30 for both
% saturated ammonium sulfate, and the precipitated fraction? to recover. After removing the salt from the precipitated fraction, affinity chromatography was performed using immobilized anti-BMAP antibody to purify B.
Get MAP'f.

(Third method) Furthermore, in another method of BMAP manufactured by IWJ, after the above-mentioned 0.3-1% SDS extraction and supernatant were thoroughly dialyzed against physiological saline, insoluble in addition to anti-BMAP serum was added. Antigen-antibody complex? The T core is recovered as a precipitate. After adding a dissociating agent to this, gel filtration fractionation is performed to obtain purified BMAP. In addition,
For example, the carrier used for gel filtration has a molecular weight of 5.
Compounds ranging from oooo to 5ooooo, such as sodium thiocyanate, are widely available.

The characteristics of the HMAP thus obtained are as follows.

1. The protein BMAP has a molecular weight of approximately 100,002 as measured by the Kell filtration method, an isoelectric point of pH 3.7, a mobility of β-Clopuri in the 7 region of 4 by electrophoresis, and a sugar content of approximately 10% as hexose. Since it shows high specificity for ovarian cancer and hydatidiform mole, it is useful for diagnosis and treatment of ovarian cancer, search for hydatidiform mole, etc.

In order to explain the BMAP'i provided by the present invention in detail, experimental examples and manufacturing examples are shown below.

(1) Experimental Example 1: Measurement of Molecular Weight The molecular weight was measured by the method of Andrew B using the Kell filtration method. - That is, purified BMAP 10 tR9 was dissolved in a small amount of phosphate buffered saline (PBS) together with a molecular weight marker and applied to Sephadex G-150 (Pharmacia Co., Ltd. Tucolumn (2.5X 100tx)). Respectively eluted 17F, molecular weight marker and BM
Based on the location of AP, it was calculated that the molecular weight of BMAP was approximately 100,000.

(2) Experimental Example 2: Measurement of isoelectric point An isoelectric point analysis method was used. That is, pH 3 to 6 manufactured by LKB
The column was packed into a 7-piece amphorite column for isoelectric point analysis, and after 10 days of purified BMAP was applied, electricity was applied for 48 hours at a voltage of 400 volts. Thereafter, each fraction was separated, and the BMAPQ fraction along with the pH value was detected by an immunochemical method (1ζ detection method using antiserum) to determine the isoelectric point. A value of pI = pH 3.7 was obtained.

(3) Experimental Example 3: Normal human serum equilibrated by sufficient dialysis against 0, that is, 0.05M barbital buffer, was subjected to electrophoresis using a moderate mobility immunoelectrophoresis method.
NL BMAP k dissolved in the same buffer and anti-BMAP
After applying the serum and leaving it overnight, the BM was determined from the sedimentation line at the formation site.
The mobility of AP is determined to be 0 (4) Experimental example 4: Measurement of sugar content The determination of hexose is performed using phenol, which is one of the colorimetric methods.
By the sulfuric acid method. That is, for every 1 d of purified BMAP solution containing 10 to 70 sugars, 5% phenol is added! /
Mix vigorously, then add 5 d of concentrated sulfuric acid, mix well, and leave at room temperature for 30 minutes.

Then, the color is yellow-orange and measured at a wavelength of f 490 nm. Glucose as a control? The hexose content of BΔtori was determined from the calibration curve f′ obtained using the same method.
It was calculated as 0%.

(5) Experimental example s: 6 cell specificity/b experiment BMAP
Using a specific antibody against BMAP and open ML enzyme immunoassay (detection sensitivity of 100/- or more), serum samples from normal and various patients were examined. As a result, BMA
P was detected in normal subjects (10 cases) and pregnant women (8 cases).
Furthermore, uterine cancer, a malignant disease in the gynecological field (10 cases)
However, among the four ovarian cancer cases, 2 cases (50%
) were detected at high concentrations of 300 nE and 450 nE, respectively. At the same time, the content fluid of 6 cases of hydatidiform mole, which is a question of transition to malignant chorionic epithelium, was positive in all cases in the range of 120 to 600 days.

In addition, a total of 39 cases of 12 other malignant diseases other than gynecological diseases, including esophageal cancer and liver cancer, were examined, but all were negative.

From the above, BMAP is a protein that is specifically detected in ovarian cancer, and is considered to be an effective substance for diagnosis and treatment of ovarian cancer.

Similarly, Ishikawa is considered in the search for hydatidiform mole.

Production Example 1 Centrifugal sedimentation of physiological saline/suspension of human placenta (400 pieces) (centrifugal sedimentation) Add 0.1 M charcoal salt solution and homogenize, and per supernatant, 30
Perform polysaturated ammonium sulfate fractionation. The ammonium sulfate precipitate was mixed with ammonium sulfate by dialyzing it against 0.05M phosphorus nitrogen (pH, 7.0). After removal, it is subjected to DEAE-cellulose column chromatography.

After filling the resin with a sample liquid that had been equilibrated with 0.05M phosphate buffer (pH 7.0),
Wash thoroughly with 1M phosphate buffer (PH7,
After removing contaminant proteins, elute the target product 2 with 0.15 MIJ acid buffer (pH 7.0), then add anti-NH
8 (Normal human serum) Perform affinity chromatography using an antibody-SE7 allose column, and then absorb the mixed protein into a decolumn to determine the purpose and D-'b BM.
APk elution/purification procedure. The N products of the obtained BMAP are:
4.5. @'t's one.

Production Example 2 Centrifugal sediment from Production Example 1 2. Add 2 volumes of SDS dissolved in 0.05 M IJ and Pan7er hydrochloric acid (pH 8,0) to extract BMAP. BMAP is 0.
Between 3% SDS and 1.0% SDS appears in the soluble fraction. The supernatant is precipitated with 20% monobasic ammonium sulfate.

In the same manner as the first method, after dialysis against phosphate buffer, affinity chromatography was performed using anti-BMAP antibody-Sepharose (3.5 M
Sodium thiocyanate (pH 8,0,) k)
BMAP Bonjona. The obtained purified BMAP was 2.7 g.
Met.

Production example 3 1% SDS extraction of production flJ2, upper'lfr 500
d 2-, anti-Br4aP antiserum (rabbit) 25 ml! In addition, insoluble antigen-antibody conjugates are available. 3.5
After dissociation of this complex with sodium thiocyanate (pH 8,0), J3MAP as antigen and antibody were combined with Sephadex G-150 cologne (pH 8,0) using a preparative plate/:IO purified BMAP U 1. 5 da, private anti-B antibody was obtained by 43 da.

The purified BMAP obtained here, like the purified products obtained in Production Examples 1 and 2, was used as a sensitizing antigen in a certain H French assay. BMAP antibody is R
It can be used as a sensitizing antibody for PHA reagents.

To the patent release shell Mi Tori Juji Co., Ltd. 224-

Claims (1)

[Claims] Can be recovered from human placenta and has a molecular weight of approximately 1 by gel filtration method.
00,000, the isoelectric point is pH 3.7, and the mobility due to electrophoresis is
β-globulin region, sugar content is 10 as hexose
% protein and is a human placenta-derived basement membrane-related protein that is highly specific to ovarian cancer and hydatidiform mole.