Ammonium Sulfate Precipitation Procedure
Serum (20 ml) was diluted 5 times with phosphate buffer (NaH2PO4¡¦2H2O pH7.2 ), and solution of 100 ml was made. To make the 30% saturated ammonium sulfate fraction, 17.6 g ammonium sulfate was added to this solution, and it was stirred for 15 min and incubated for 30 min at 4¡î. After centrifugation (20,000¡ßg, 30 min, 4¡î) the pellet was dissolved with the phosphate-buffered saline (PBS(-)) .To make the 30-40% saturated ammonium sulfate fraction, 6.2 g of ammonium sulfate was added to this supernatant. The same process was repeated 6.3 g ammonium sulfate for 40-50% saturated ammonium sulfate fraction, 6.6 g ammonium sulfate for 50-60% saturated ammonium sulfate fraction, 6.9 g ammonium sulfate for 60-70% saturated ammonium sulfate fraction, and 7.2 g ammonium sulfate for 70-80% saturated ammonium sulfate fraction. All fractions were dialyzed with the purpose of desalting.
The concentration of protein in all fractions was unified at 30 mg/ml. Fractions with protein concentrations under 30 mg/ml were concentrated by centrifugation (10000 rpm, 4¡î) with Microcon YM-10 (MILLIPORE). Fractions with protein concentrations of more than 30 mg/ml were diluted with PBS (-).

Indirect ELISA
We used two types of 96-well microtiter plate. SUMILON Multi Well plate (Sumitomo Bakelite, cat.No.MS-8096) was used as a 96-well tissue culture plate, and Nunc-Immuno Plate PolySorp Surface (Nalge Nunc International) was used as a 96-well ELISA plate. Fractions and the samples which were diluted with PBS(-) 1 to 2048 times were added at 100 ¦Ìl to each well, and incubated overnight at 4¡î. After 2 washes with PBS(-), 96-well microtiter plates were incubated with 200 ¦Ìl of BSA (5% in PBS(-)) for 30 min at room temperature. After 4 washes with PBS(-), the plates were incubated with 100¦Ìl of anti-MUC1 mouse IgG (HMPV) (10¦Ìg/ml ) (PharMingen International) for 1 h at room temperature. Following 4 washes with Wash Solution Concentrate (KPL) diluted 20 times with Mili-Q water, in the plates were incubated with 100 ¦Ìl of HRP-conjugated rabbit anti-mouse IgG(DAKO) diluted 1 : 500 in PBS(-) for 1 h at room temperature. Then 100 ¦Ìl of ABTS-Component Microwell Peroxidase Substrate (KPL) was incubated in these plates for 30 min at room temperature. The substrate reaction was determined at 405 nm in Spectra and Rainbow Readers (STL-Libinsruments).

Western blotting
Each fraction (10 ¦Ìl) was mixed with Laemmli buffer (10 ¦Ìl) and boiled for 2.5 min. The samples were electrophoresed for 80 min at 20 mA, 200 V with 10% polyacrylamide gel and then electrotransferred for 2 h at 200 mA, 60 V onto Immobilon Transfer Membranes (PVDF membrane) (MILLIPORE) in a chamber. Briefly, the PVDF membrane was preincubated with 5% (w/v) skim milk in PBS (-) for 30 min and then incubated with HMPV (1 ¦Ìg/ml) and 5% (w/v) skim milk overnight at 4¡î. After 4 washes with 0.1% Tween/PBS, the PVDF membrane was incubated with HRP conjugated anti mouse goat IgG (1¦Ìg/ml) and 5% (w/v) skim milk for 2 h at room temperature. This membrane was washed 3 times with DDW. Washing with 0.1% Tween/PBS for 20min was repeated 4 times, and then washing with PBS (-) for 10 min. Finally, Konica Immunostain HRP-1000 (Konica) was used to stain the membrane.

RESULTS
To detect MUC1 mucin in serum, indirect ELISA was performed with 96-well tissue culture plate and HMPV. When serum was added directly to the wells of a 96-well tissue culture plate, no coloring of the solution was observed with indirect ELISA. However, coloring of solution was found with indirect ELISA when the serum was diluted with PBS (-) 16 to 256 times. The highest level of absorbance was observed with a serum dilution of 128 times. There was no coloring of solution with indirect ELISA performed to detect MUC1 mucin in the serum with a 96-well ELISA plate and HMPV (Fig. 1). After concentration of protein of all fractions, the 30% saturation, 30-40% saturation, 40-50% saturation, 50-60% saturation, 60-70% saturation, and 70-80% saturation were unified into 30 mg/ml, serial dilutions of each fraction, and indirect ELISA was performed with 96-well tissue culture plates and HMPV.
No coloring was seen in the serial dilution of the fractions of 30% saturation or 70-80% saturation, though there was coloring in the serial dilution of the 40-50% saturation to 60-70% saturation ammonium sulfate fraction. In the serial dilution of the 60-70% saturation ammonium sulfate fraction, a rise in the level of absorbance was observed at a low dilution magnification. In the serial dilution of the 30-40% saturation ammonium sulfate fraction, a rise in the level of absorbance was observed from 1¡ß to 128¡ß dilution magnifications. In the serial dilution of 40-50% saturation ammonium sulfate fraction, a rise in the level of absorbance was observed from 1¡ß to 1024¡ß dilution magnifications, and the level of absorbance of 1¡ß, 2¡ß, and 4¡ßmagnifications were lower than 8¡ßmagnification. In the serial dilution of the 50-60% saturation ammonium sulfate fraction, a rise in the level of absorbance was not observed from 1¡ß to 4¡ß dilution magnifications, and the absorbance rose with the increase of dilution magnification until a 32¡ß dilution magnification, and then the absorbance decreased up to a dilution magnification of 128¡ß (Fig. 2). To confirm the existence of MUC1 mucin, Western blotting was performed for all fractions. The concentration of protein of each fraction was unified to 30 mg/ml, as is the indirect ELISA. (Fig.3)
The band on the lane of 30-40% saturation, 40-50% saturation, 50-60% saturation ammonium sulfate fraction suggests the existence MUC1 mucin in each fraction.
These data shows that:
1. MUC1 mucin can adsorb to a 96-well tissue culture plate, but not ELISA plate.
2.MUC1 mucin molecules is detected in the 30-40% saturation, 40-50% saturation, and 50-60% saturation ammonium sulfate fractions, and most abundant in the fraction between 40 and 50% saturation.
3.Plenty of MUC1 mucin molecules also exist in the fraction between 50 and 60% saturation of ammonium sulfate, and an inhibitor that prevents the adsorption between MUC1 mucin and the tissue culture plate may also exist.
To investigate the existence of the inhibitor in the fraction between the 50 and 60% saturation of ammonium sulfate, the solution (100¦Ìl) which was made from the same volume of 40-50% and 50-60% saturation ammonium sulfate fractions was examined by indirect ELISA with the 96-well tissue culture plate and HMPV (Fig. 4). The level of absorbance of the 40-50% saturation ammonium sulfate fraction diluted 2 times with PBS(-) was higher than that of the 40-50% saturation ammonium sulfate fraction. The level of absorbance of the solution containing both the 40-50% saturation and 50-60% saturation ammonium sulfate fractions was extremely suppressed, more so than the 40-50% saturation ammonium sulfate fraction diluted 2 times with PBS(-).
To investigate the existence of the inhibitor and MUC1 mucin in each fraction, indirect ELISA was performed for the serial dilution of the 40-50% saturation ammonium sulfate fraction, the mixed solution with 40-50% saturation and 50-60% saturation ammonium sulfate fractions, and the 50-60% saturation ammonium sulfate fraction with the 96-well tissue culture plate and HMPV (Fig. 5). Without dilution the rise of absorbance of the mixed solution with both the 40-50% saturation and 50-60% saturation ammonium sulfate fractions was not observed, but a rise in absorbance did occur when it was diluted. The rise of absorbance started at lower dilution magnifications. The peak value was higher than in the 50-60% saturation ammonium sulfate fraction and lower than in the 40-50% saturation ammonium sulfate fraction.
We did the same experiments to the sera of two volunteers, and obtained the results which were the same as this time. (data was not shown)

DISCUSSION
Although the existence of MUC1 mucin was confirmed by Western blotting, the level of absorbance of the 40-50% and 50-60% saturation ammonium sulfate fractions was suppressed or disappeared at low dilution magnification, and rose as the dilution magnification increased. This phenomenon may suggest the existence of an inhibitor which can effectively prevent the binding between MUC1 mucin and 96-well tissue culture plate, and that the concentration of this inhibitor is less than that of MUC1 mucin. A large amount of the inhibitor exists in the 50-60% saturation ammonium sulfate fraction, and a small amount in the 40-50% saturation ammonium sulfate fraction. Due to the presence of the inhibitor with a high binding capacity to 96-well tissue culture plates, MUC1 mucin can not bind with the plate at low dilution magnifications. At the 32¡ß to 128¡ß magnifications of dilution, the concentration of inhibitor is too low to sufficiently prevent the binding of MUC1 mucin and the plate. When the concentration of MUC1 mucin becomes higher than that of the inhibitor, MUC1 mucin is able to adsorb to the plate. At high magnifications of dilution, the concentration of MUC1 mucin is insufficient to color the substrate solution.
The result of indirect ELISA showing that the level of absorbance of the 40-50% saturation ammonium sulfate fraction diluted 2 times with PBS(-) was higher than the 40-50% saturation ammonium sulfate fraction, and that the level of absorbance of the mixed solution of 40-50% and 50-60% saturation ammonium sulfate fractions was suppressed much more than the 40-50% saturation ammonium sulfate fraction diluted 2 times with PBS(-), dose not contradict our hypothesis.
Monoclonal antibody HMPV recognizes the peptide epitope (APDTR) of the core protein of MUC1 mucin (6). There are many other anti-MUC1 core protein antibodies, and a protein with a molecular weight of 70 kDa was detected in serum by Western blotting with these antibodies. The molecular weight of 70 kDa of this protein is too small for high molecular weight mucins such as MUC1 mucin even if it is the secreted form. This protein was thought to be a degradation product of MUC1 mucin resulting from protease cleavage, or thought to be another protein that contains the same peptide epitope that is recognized by anti-MUC1 core protein antibodies. If this protein is a degradation product, the cleavage must occur at specific sites in the core protein of MUC1 mucin, because of the consistent size of the 70kDa between different individual (7). In our experiment, MUC1 mucin was detected as a 70kDa molecule in the 30-40% saturation, 40-50% saturation, and 50-60% saturation ammonium sulfate fractions.
In many studies, sandwich ELISA was performed to detect MUC1 mucin because MUC1 mucin can not adsorb to the 96-well ELISA plate which is made from polystyrene, making it very difficult to detect this mucin by direct or indirect ELISA with this plate. Recently, a 96-well ELISA plate for glycoprotein made of vinyl chloride has come on the market, and electrochemiluminescence immunoassay (ECLIA) has been used to detect MUC1 mucin being detected in serum clinically, but there is no report of MUC1 mucin being detected in serum by indirect ELISA with 96-well tissue culture plate. The details on how the surface of the tissue culture plate is treated have not been revealed. So far as we know, the 96-well tissue culture plate was also made of polystyrene, but when it was treated by electric discharge, hydroxyl groups appeared on the surface of the plate, thereby increasing the hydrophilicity of the plate. This treatment makes it so that MUC1 mucin is able to adhere to the 96-well tissue culture plate.
To detect MUC1 mucin, indirect ELISA was performed with tissue culture plates from several companies, and with these plates MUC1 mucin could be detected just as with the products of SUMILON. (data was not shown) In this study, other mucins were not examined as to weather they could be determined by indirect ELISA with a tissue culture plate and their core protein antibody. We consider that the present method is one of the easiest methods to detect MUC1 mucin in solution, and perhaps a substitution for other assays.
The fact that the binding capacity between MUC1 mucin and polystyrene plates with surfaces treated to be hydrophilic, and the existence of an inhibitor that prevents the binding of MUC1 mucin to this hydrophilic polystyrene plate, may be good clues to undestanding the bioactivity of MUC1 mucin in vivo. Today, ICAM-1 (8), Selectins (9), Sialoadhesin (10), Grb2 (11), beta-catenin (12), and GSK-3 beta (13) are the known as the ligands of MUC1 mucin. A molecule which has hydrophilic sites similar to that of the tissue culture plate can be a ligand of MUC1 mucin. The fact that MUC1 mucin can be adsorbed onto tissue culture plates can be a good clue to finding new ligands.
MUC1 mucin is overexpressed on cell surfaces of many kinds of carcinoma, and the level of MUC1 mucin in serum rises in cancer patients. It was reported that MUC1 mucin in serum suppresses the activation and proliferation of T cells (14)(15), that MUC1 mucin expressed on cell surfaces inhibits E-cadherin mediated cell-cell interactions (16), and that MUC1 mucin may accelerate the invasion and metastasis (17). If MUC1 mucin in serum influences the immune system and mechanism of metastasis of cancer cells, an inhibitor which prevents adsorption between MUC1 mucin and tissue culture plate may also influence them. In the future, the present findings would contribute to the search for new ligands of MUC1 mucin, to identify these inhibitor molecules, and to elucidate the interactions between MUC1 mucin and these inhibitors in vivo.
In conclusion, we found that MUC1 mucin in serum can be adsorbed to 96-well tissue culture plate, and that there is an inhibitor in serum that prevents the adsorption between MUC1 mucin and 96-well tissue culture plate.

ACKNOWLEDGEMENTS
The authors thank Dr. Kiyohide Kojima, Dr. Hiroyasu Akatsu and Mr. Yasushige Ishikawa for helpful advice and Mr. Takeshi Kanesaka for technical assistance.

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