JPS6070358A - Reagent for immunity method - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to reagents for carrying out immunization methods comprising a solid support coated with an immunologically active material.

Various immunological methods, especially enzyme immunological methods, for the quantitative and qualitative determination of diagnostically and therapeutically important substances are based on the solid phase principle using water-insoluble carriers coated with immunologically active materials.

Water-insoluble carriers suitable for this purpose include organic and inorganic polymers such as amylases, dextran, natural or modified cellulose, polyacrylamides, agarose, magnetite, porous glass powders, polyvinylidene fluoride P (Kynar), test vessels (test tubes, titration plates or cupettes of glass or synthetic material), as well as solid bodies (glass rods and rods with a rod r1 end wing or membrane-like portion with a rod r1 end thick part of synthetic material). can be used. Particularly suitable carriers for immunological reagents generally have a diameter of about 6.65 mm.
~6.5 mm surface treated polystyrene beads.

Conventionally, these carriers coated (sensitized) with immunological reagents have to be constantly stored in solution because their immunological binding ability decreases when dried.

However, storing these carriers in whole solution causes various disadvantages when conducting actual tests using these carriers.

German Patent No. 2,910.707 proposes a method in which the sensitized carrier is washed with a buffer, treated with a sugar solution and then dried in air for about 30 minutes. The sensitized carrier coated with sugar shows no loss of immunological activity even after long-term storage. On the other hand, these carriers, especially when using monosaccharides, become sticky when they come into contact with moisture in the air;
Manual work, especially machine operation, using these carrier sounds has the disadvantage of being difficult and cumbersome.

The present invention was completed based on the surprising discovery that the above-mentioned drawbacks can be overcome by coating the sensitized beads not with sugar but with a sugar acid salt. The sensitized beads coated with sugar acids by this method do not exhibit the above-mentioned drawbacks, but can be stored exactly like the carrier according to German Patent No. 2,910,707 mentioned above.

The invention therefore relates to a reagent for carrying out an immunological method comprising a solid support coated with an immunologically active material, the coated support being coated with a salt of a sugar acid.

Possible salts of sugar acids include salts of glucuronic acid, manonic acid, galacturonic acid and gluconic acid. Suitable salts include alkali salts or ammonium salts for convenience. Particularly preferred are the sodium salts. Preferred sugar acid salts are sodium glucuronate or sodium glucuronate.

As an immunization method, enzyme immunotherapy can be considered.

Immunological materials that coat carriers include antigens,
Antibodies or haptens may be mentioned. Preferred antibodies are cancerous embryonic antigen (CF!A), HC().

Antibodies against interferon, hepatitis B surface antigen, human IgE, etc. A particularly preferred antibody is K6hle
This is a monoclonal mouse antibody prepared by a method known per se according to the cell fusion method of r and Milstein. In the case of antibodies against interferon, antibodies against recombinant interferon αA can be considered.

The carrier to be coated is preferably one having a diameter of about 6 mm and a diameter of 5 to 5 mm.
Mention may be made of 5.5 mm surface-treated polystyrene beads.

To prepare the coating, the carrier coated with the immunologically active material in a manner known per se is removed from the total buffer solution, drained and then treated with Pl (range 5-9, especially 6-6).
Immerse in Z5 saccharide-containing aqueous solution. If necessary, the pi of the aqueous solution can be adjusted with a buffer, but a small amount of buffer should be used so that the coated support is coated with the sugar acid and not with the buffer.

The aqueous solution contains 5 to 200 g/13 of a sugar acid salt. 20 to 100 g, especially 507 i/l is preferred.

If desired, stabilizers such as thimerosal can also be added to this solution.

After the carrier is removed from the buffer solution, it is dried, but there are no particular restrictions on the drying method in this case. Drying in air, 0~
It is preferable to perform this at 56°C, and immediately raise the temperature from room temperature to 45°C.
It is preferable to dry within this range. The optimum temperature is 57°C. However, drying can also be carried out in vacuum at 37° C. on a rotary evaporator or using a fluidized bed granulator. In the case of large batches, drying may be carried out by a fluidized bed method.

Similarly, there is no restriction on drying time, but drying time is 37°C in air.
Very good results have been obtained when drying for 24 hours. When drying in vacuum using a rotary evaporator, 1 to 2 hours is usually sufficient. When drying in a fluid bed granulator, about 10 minutes of drying is usually sufficient.

The invention will now be illustrated by the following examples.

Example 1 Polystyrene beads (diameter 6.55 mm) are sensitized with a monoclonal mouse antibody against recombinant interferon αA in a conventional manner in a buffer.

Remove the beads from the buffer and pass through a coarse sieve into 50 ml of water.
Suspend in an aqueous solution containing g/l sodium glucuronate for 10 minutes at room temperature. The beads are then sieved from this solution, drained and dried for 24 hours at 37°C.

A portion of the beads is stored at room temperature for one month, and another portion is stored at 67° C. for one month.

Both beads show only a very small, non-significant decrease in immunological activity after storage. Beads are dry even when exposed to moisture in the air.

Example 1a Polystyrene beads (diameter 6.65 mm) are sensitized with a monoclonal mouse antibody against recombinant interferon alpha A in standard f buffer.

Remove the beads from the buffer and drain them through a coarse sieve.
Suspend in an aqueous solution containing g/l sodium lacuronate for 10 minutes at room temperature. The beads are then removed from this solution with a sieve, drained of water, and dried at 37°C for 24 hours.

Example 1b Polystyrene beads (6.55 mm in diameter) are sensitized with a monoclonal mouse antibody against recombinant interferon alpha A in a buffer solution according to conventional methods.

Remove the beads from the buffer and drain through a coarse sieve for 50 min.
! ! Suspend in an aqueous solution containing sodium manonate/l for 10 minutes at room temperature. The beads are then removed from this solution with a sieve, drained and dried at 67°C for 24 hours.

Example 2 a) Polystyrene beads (diameter 6.65 mm) f
Sensitize with a monoclonal mouse antibody against CB8 in a buffer solution according to a conventional method. Remove the beads from the buffer and
Suspend in an aqueous solution containing sodium glucuronate at 100 ji/11 for 210 minutes at room temperature. The beads were then removed from this solution with a sieve, dried and evaporated in a rotary evaporator under vacuum at 676C.
Dry for an hour.

A portion of the beads was stored at room temperature for one month, and another portion was stored at '57°C for one month.

Both beads show only a very slight, non-significant decrease in immunological activity after storage. The beads remain dry even when exposed to moisture in the air.

For example, polystyrene beads (diameter 6.5 mm) are sensitized with a monoclonal mouse antibody against HC'G in a buffer solution according to a conventional method. The beads are removed from the buffer, drained through a coarse sieve, and suspended in an aqueous solution containing 10011/l sodium glucuronate for 10 minutes at room temperature. The beads are then sieved from this solution, drained and dried in a rotary evaporator under vacuum at 57° C. for 1 hour.

A portion of the beads is stored at room temperature for one month, and another portion is stored at 57° C. for one month.

Both beads show only a very slight, non-significant decrease in immunological activity after storage. The beads remain dry even when exposed to moisture in the air.

Example 4 a) Polystyrene beads (diameter 665 mm) are sensitized with a monoclonal mouse antibody against hepatitis B surface antigen in f buffer according to conventional methods. The beads were removed from the buffer and filtered through a coarse sieve into an aqueous solution containing 9.1001/l sodium glucuronate for 10 min at room temperature.
become cloudy The beads are then sieved from this solution and dried in a rotary evaporator under vacuum at 67° C. for 1 hour.

A portion of the beads was stored at room temperature for 1 month, and another portion was stored at 37° C. for 1 month).

Both beads show only a very slight, non-significant decrease in immunological activity after storage. Are the beads dry even when exposed to moisture in the air? maintain.

Even after storing a portion of the beads at room temperature for 7 months, 70% of the immunological activity remained.

b) Polystyrene beads (6.65 mm diameter)'
sensitize with a monoclonal mouse antibody against hepatitis B surface antigen in C buffer according to a conventional method. The beads are removed from the buffer, drained through a coarse sieve and suspended in an aqueous solution containing 100 g/l sodium gluconate for 1 D at room temperature. Then remove this giz from this solution with a sieve,
Filter in a fluidized bed granulator and dry at 7°C for 10 minutes.

A portion of the beads is stored at room temperature for one month, and another portion of the shredded beads is stored at 37° C. for one month.

Both beads show only a very slight, non-significant decrease in immunological activity after storage. The beads remain dry even when exposed to moisture in the air.

Example 5 Polystyrene gear (diameter 6.55 mm) is sensitized with a monoclonal mouse antibody against a human IgE antigen in f buffer according to a conventional method. The beads are removed from the buffer, drained through a coarse sieve and suspended in an aqueous solution containing 50 g/l sodium glucuronate for 10 minutes at room temperature. The beads are then removed from the solution with a sieve, drained of water, and dried at 37°C for 24 hours.

A portion of the beads is stored at 2-8°C for 1 hour, another portion at room temperature, and another portion at -1:37°C.

Both beads show only a very slight, non-significant decrease in immunological activity after storage. ♂－
remains dry even when exposed to moisture in the air.

Example 6 Quantification of interferon in patient serum (enzyme immunoassay) Test solution (0
, 1 mol/l sodium phosphate pi (6,5,10,
9/l BSA, 0.5 p9/mltviap
-7,1n-free human serum monoclonal mouse anti-interferon-peroxidase conjugate)
Add with a 0.05 mJt pipette to the patient plasma or interferon standard solution to be quantitated (0 units/rnl).

125 units/ml, 25 units/1nl! and 50
units/ml) and interferon standard serum (7t
For example, interferon 30 units/d) 0. ? 7' were mixed, each coated with sodium glucuronate-coated polystyrene sensitized with a monoclonal mouse antibody against recombinant interferon αA according to the method of Example 1.
(φ-=6.5mm), and heated to room temperature (18~26°O
) for 16 hours. The polystyrene beads were then washed three times with 2-5M distilled water each time, and each time was washed with a substrate buffer for gold peroxidase activity determination (PH
5. Add H to the 0.1 mol/l potassium citrate buffer of Q.
2O26 mmol/l, 0-phenylenediamine 2
0 mmol/l added) and transferred to room temperature (1
Incubation is performed for 30 minutes at 8-26°C.

Add 0.5 ml of 4N-HCf to completely stop the activity of peroxidase and simultaneously increase the absorbance of gold.
The absorbance is measured within 50 minutes at a wavelength of 492 nm. The ΔF value is the average of two measurements. Table 1 shows the measured values for interferon in comparison with the values obtained for interferon standard solutions.

Table 1 Interferon standard solution 0 units/mlo 0.0675 12.5 units/mlo, 24 25 units/m 10.427 50 units/mA 0.782 Control serum filtration O units/m 10.497 Patient plasma Interferon rot 8327 0. 060 0 units/WLII68328
0.060 0 tt, (683360,2059〃 A8!+38 0.062 0 tt /16B5ro9 0.145 5 # /168365 0.058 0 tt, vg8541
0.103 2 n /I68ro44 0. Example 7 Determination of CEA in patient plasma Into the required number of test tubes (10 x 75 mm), add the test solution (0.2 mol/lONaH2PO4/Na2HP).
O4 pH 6.5 and 2 g/l bovine serum albumin, 2
0% normal goat serum and 0.2 g/ml goat anti-CE
A-peroxidase conjugate) with a 0.2 ml't pipette and add the patient's plasma to be quantified or the CFA standard solution (CE
A Ong/ml, 2.5 n,! 7/ml, 1
0 ng/ml and 20 n9/ml) and CEA
Control serum (CFA 5. OnJ/mA'Shiage On,!
i'/rnl) 0.050 ml 2, each with monoclonal mouse anti-CEA according to the method of Example 2.
Polystyrene beads (φ = 6.5 mm) sensitized with
Perform incubation for 6 hours. The polystyrene beads were then washed 6 times with 2-5 d of distilled water each time and each time added to a substrate buffer for quantifying total peroxidase activity (26 mmol/l H2O in 0.1 mol/l potassium citrate buffer at pH 50, []- Phenyl diamine 20 mmol/
l addition) o, transferred to 5 mJ and incubated at room temperature (22 °C) for 60 min.
Incubate for 1 minute.

In order to stop the activity of peroxidase and increase the color intensity at the same time, I N -H (J 2.0 + d' plus 6
Measure the absorbance at 492 nm within 0 minutes. The ΔE value is the average of two measurements. Table 2 shows the measured values of CEA in comparison with the measured values by Roche's radioimmunoassay method.

To CE8, 5 ng/ml or less is normal range, 2.5 n,
Exceeding lil/ml is in the pathological range.

Second table OngAnlCk! A 0105 2.5n! ! /mAICEA Olro3010.0nJ
'/mAICEA O, 97820, On, 9. Δmc
mA 1.8595, Qng/m1CEA O, 540 718B 2.2 0.185 1.07220 1.
2 0.227 1.47218 1,. 2 0.21
5 1.5720ro 2. Rosen 2 kama 6.1B'-2
,072233,00,ro99 3.15 7247 3.1 0. Ro55 2.87258 4.
6 0.510 4.57215 4.9 0.5B5
5.57219 5.0 0.610 5.9818
0 8.6 0.850 8.57248 11.2
1.045 10.77262 14,. 2 1.36
0 14.57201 15.6 1.445 153
Example 8 Quantification of HCG in serum Inject the test solution (0.1 mol/j IJaH2PO4/Ni12HP) into the required number of test tubes (10 x 75 mm).
O4pH7O, 2. ! il/l of bovine serum albumin and 1,0/1. ! iI/ml monoclonal mouse anti-HC'G-peroxidase conjugate) 0.2d
Pipette the patient's plasma or HCG standard solution (0.25°50.100 and 2001U/1O
HcG containing) 0.050 mJ' was added to each Example 3.
Add polystyrene beads (φ-6, 5 mm) sensitized with monoclonal mouse anti-HCG and coated with sodium glucuronate according to the method described in 2007, and incubate at 37°C for 2 hours. The polystyrene beads were then washed three times with 2 to 5 drops of distilled water each time, and each was mixed with a substrate buffer for measuring peroxidase activity (26 mmol/l H2O in 0.1 mol/l potassium citrate buffer at pH 5.0).
X o-phenylenediamine 20 mmol/It'
fl:, added), transferred to 0.57 d and kept at room temperature (18-26
Incubate for 60 minutes at 10°C.

To stop peroxidase activity and increase color intensity at the same time, add 2N-H (Ji, Oml) and measure the absorbance at 492 nm within 50 minutes. ΔE is the average of two measurements. Quantitative HCG values in serum and patient serum are shown in Tables 3 and 4.

HCG ΔE492nm30G/! F 0 0, Oro 8 0.054 0.158 0.182
5 0.226-- - 50,0,4750,5440,5570,71610
00,9050,9550,9701,40 The HCG values in patient serum shown below were determined from standard curve 1). Standard curves 2), 5) and 4) were prepared with fresh HCG standards on separate days.

Table 4 (Patient serum) Serum Δ"492/3 Curse temperature IU/l HC() curve 1) Sol091080 0.041 0 A 2801 0.025 D Waste Ro G81 0.450 47 /162673 1.1+ 127 .76 1167 2.12 (G2) 470 flat 489
1 0975 (G50) 5,450 rot 3240 1.
0<S (1:20) 2,280/I64NB 1.4
75 (1:1000) 167.000 Example 9 a) Samples to be quantified, positive or negative, respectively, into the required number of test tubes (10 x 60 mm, coated with 1:200 μl of beads in this test tube). (HB
sA, pi' corresponds to about 20 ± 5 n, jil/ml) control o, ii pipette to each test solution (01 mo/L//1 Tris-acetate pH 7,
6, Thimerosal 0.59/l, 4-aminoantipyrine 0.4 g/l, Tween 20 0.29/13. Heparin Sodium Salt 40.00 D units USP/lz
Fetal bovine serum 20% v/v, goat anti-HBS-peroxidase conjugate) was mixed with 0,1 ml of polystyrene beads coated with sodium glucuronate and sensitized with monoclonal mouse anti-HBs according to the method of Example 4a.
φ-6.35 mm) f and incubation for 2 hours at 45°C in a water bath.

Next, the polystyrene beads were washed with 3 to 5 washes each time (N
a2HPO4 approx. 5.0 mmorph 6 N KH2PO
41.5 mmol/tJ, about 130 mmol/e of NaC, 1 and 0.029/M of Tween 20)
Wash twice and use substrate buffer for peroxidase activity measurement (p
H2O2 to 0.1 mol/l of potassium citrate of H5.0
6 mmol/l, 0-phenylenediamine 20 mmol/l added) in addition to 0.25 ml at room temperature (22°
Incubate in C) for 30 minutes. IN to stop peroxidase activity and increase color intensity
- Add H2sO41,0 and filtrate within 0 minutes to 490~4
Measure the absorbance at a wavelength of 92 nm.

Table 5 shows the ΔE values (average of two measurements) in a series of HBsA7 quantifications.

1 >1.500 16 0.111 2 >1.500 17 0.099! l >1.500 18 0. [)244 >1.5
00 19 0.028 5 0.869 20 0.024 6 0.499 21 0.0ro2 7 0.138 22 0.025 8 0.374 Control 9 0.198 Positive 0.826 10 0.118 0. 879 11 0371 0.849 12 0.398 Negative 0.040 1 0.370 0.0 6 14 0.226 0.031 15 0.246 b) Required number of test tubes (iQ x 60 mm, in this test tube Beads are coated with 200 μl) of each sample to be quantified or positive or negative (HBsA,
ji' corresponding to approximately 20±sng/mz) control o, 1 m/
! Sample solution (0.1 mo#/l Tris-acetate PH7.3, thimerosal 0.5 g/l, 4-aminoantipyrine 0.4 g/l)
, 0.24 j/l of Tween 20, heparin sodium salt 40.00 [1 unit usp/l, fetal bovine blood seedlings 20%, now goat HBs-peroxidase conjugate]
0. I Tnl and sensitized with monoclonal mouse anti-HBs gluconic acid according to the method of Example 4b).
FC polystyrene beads (φ-6.6
5 mm ) and incubation for 2 h at 45 °C in a water bath.

Next, the polystyrene beads were washed with a washing solution (Na
2HPO4 approx. 5.0 mmol/l, KH2PO4 approx. 1.5 mmol/1. , NaCf approximately 15 mmol/1
1 Tween 2000.02 g/l) 5 times, and substrate buffer for peroxidase activity measurement (pi (5,0 potassium citrate 0.1 mol/l and H2O 26 mmol/l).

0-phenylene cyamine 2U mmol/l addition) 0,
Add 25 ml and incubate for '50 minutes at room temperature (228C). 1N-H2SO to stop peroxidase activity and increase color intensity.

Add 1.0 mA' and the wavelength changes from 490 to 49 within 50 minutes.
Measure the absorbance at 2 nm.

Table 6 shows the ΔE values (average of two measurements) in a series of HBsAjl quantitative tests. These values are based on freshly prepared beads (AA), heated at 24-26°C for 1 month or at 3°C.
This was obtained using beads stored at 5°C.

1 0.190 0.15ro 0.1122 0.09
4 0.109 0.0845 0.100 0.08
5 0.08ro4 0.074 0.070 0.05
55 0.789 0.790 0.8816 0.9
27 0.896 1.0137 0.949 0.9
02 1.09B8 1.066 1.072 1.2
039 0.577 0.696 0.663 Example 10 Into the required number of test tubes (10
25.Heat treatment (56℃/filtration 0 minutes) Fetal bovine serum 200
ml/11 Thimerosal 0.25 g/l and monoclonal mouse anti-human) rgFj-peroxidase conjugate 0.2 ml/l) Pipette into 0.2 ml' of serum or plasma sample to be tested or human) IgE
Standard solution (IgF 400 units/ml, 200 units/rL
l, 100 Units/ml, 50 Units/ml and 0 Units/ml) and 0.05 ml of beads each sensitized with monoclonal (mouse) anti-human IgE and coated with sodium glucuronate according to the method of Example 5 ( 6,
65 mm) and incubate at 18-26°C for 2 hours. Then add 3 to 5 beads of formula styrene each time.
Washed 5 times with mJ of deionized water and prepared with 0-phenylenediamine substrate buffer (pH 5,
0 potassium citrate 0.1 mol/l, H2O 26 mmol/l and O-phenylenediamine 20 mmol/l
'fr, added) 0.5 ml, 18-26°C
Incubate for 30 minutes. Kb K 0.5 to stop peroxidase activity and increase color intensity
Mo/'/l -H2SO4-1g/l -Na2
Add S2052, 01nl and measure the absorbance at a wavelength of 492 nm. ΔE is the average of two measurements. H)
The IgE quantitative results are shown in Table 7.

Claims (1)

[Scope of Claims] (1) A reagent for carrying out an immunological method ( 2) The reagent according to claim 1, in which the immunological method is an enzyme immunotherapy; (3) the immunologically active material is an antigen; any one of claims 1 and 2; (4) The immunologically active material is an antibody.The reagent (5) according to any one of claims 1 and 2.The immunologically active material is a hapten. The reagent (6) antibody according to any one of claims 1 and 2 is an antibody against the hapten 8 surface antigen.The reagent (7) antibody according to claim 4 is (8) The reagent according to claim 4, in which the antibody is a CEA antibody (the reagent according to claim 4, in which the 91C'FA antibody is a monoclonal mouse antibody) The reagent according to claim 10, in which the antibody is an HCG antibody. The reagent according to claim 10, in which the antibody is a monoclonal mouse antibody (121 antibody is an interferon antibody. The reagent according to claim 4, which is an antibody (13) The reagent according to claim 12, wherein the antibody is an antibody against recombinant interferon αA (14)
(15) The reagent according to claim 14, wherein the antibody is a monoclonal mouse antibody. (15) The reagent according to claim 14, wherein the antibody is an antibody against human IgE. (16) The antibody is a monoclonal mouse antibody. A reagent according to any one of claims 1 to 16, wherein the carrier is a surface-treated polystyrene bead, and a salt of a sugar acid is The reagent cod according to any one of claims 1 to 17, which is a glucuronic acid salt; The reagent cod according to any one of claims 1 to 17, wherein the sugar acid salt is a manonate The reagent Cυ according to any one of claims 1 to 17, wherein the reagent (a) salt of sugar acid is a lacturonic acid salt. The reagent salt according to any one of claims 1 to 17, which is a gluconate salt, is an alkali salt or an ammonium salt, according to claims 18 to 21. The reagent (c) according to claim 23, wherein the salt is a sodium salt. The reagent (24) according to claim 22, wherein the salt is sodium glucuronate. 29. The reagent according to claim 23, wherein the salt is sodium gluconate.