JPS5830667A - Marked immunologic active substance - 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 provides a labeled immunologically active reagent, a method for producing this reagent, a method for detecting or measuring a substance with this reagent, and the use of this reagent in an immunological detection method. Regarding.

Many immunologically active substances, such as antibodies,
For example, it is known that 1g51makin cannot be labeled using peroxidase without losing its immunological activity.

This means that a%Xδhler and Milstein (Na
True.

This is particularly true for monoclonal antibodies obtained according to the known method of 256.495f, 1975).

Therefore, there is a need to find a universal method by which immunologically active substances can be labeled without loss of immunological activity.

Within the scope of the present invention, it has now been discovered that biotin can be covalently bound to an immunologically active substance, and subsequently avidin can be covalently bound to a #substance.

Therefore, the present invention relates to a complex consisting of an immunologically active substance to which biotin is covalently bound and avidin to which an enzyme is covalently bound.

Antibodies or antigens are particularly suitable as immunologically active substances. Monoclonal murine antibodies are particularly suitable.

In the case of monoclonal mouse antibodies, B [directed against hepatitis virus surface antigen or carcinoembryonic antigen (CEA)]
It has been discovered that nine are particularly selected for use against.

Within the scope of the invention all enzymes used in immunological methods, ie for example alkaline phosphatase! Leic acid dehydronase, glucose-
6-phosphate dehydrobenase, glucose oxidase, melf amylase, galactosidase, acetylcholinesterase, and peroxidase are considered. In this connection, peroxidases have been found to be of particular choice. Peroxidase from horseradish is particularly suitable.

The method for producing the complex according to the invention consists in covalently binding biotin to the immunologically active substance, covalently binding avidin to the enzyme, and mixing the two obtained products with each other. The complex obtained is then lyophilized in a manner known per se.

The covalent bonding of biotin with an immunologically active substance is carried out in a manner known per se, ie the immunologically active substance in a strongly basic buffer solution is combined with biotin succinimate in dimethyl sulfoxide. By processing with midoester solution. The product is then phosphoricated with V phosphate and purified by dialysis against a sodium cyclide solution. The phosphate buffered sodium chloride solution [can contain a disinfectant such as temerosal J (Fluka) or melfen.

Since the ratio between the immunologically active substance and the biotin succinido ester is critical for the success of the reaction, this ratio is determined individually for each immunologically active substance. Must.

Conjugation of enzymes, especially horseradish peroxidase, to avidin by known methods, such as Wilao in "Immunofluorescence and Related Staining Techniques J 1978, p. 215.
This can be carried out by the method of N. and Nakane.

For the production of the conjugate according to the invention, a solution of the immunologically active substance to which biotin is covalently bound can be treated with a solution of avidin to which the enzyme is covalently bound. This treatment can be carried out at room temperature.

The complex formed can be stabilized by freeze-drying in a manner known per se.

The complex according to the invention can be used in any immunological detection method. The so-called Sand Inch method has been found to be a particularly suitable method.

The invention therefore also relates to the Sandwich method for the detection or determination of certain substances, which method comprises an immunologically active reaction that is bound or becomes bound to a water-insoluble carrier. The substance to be measured together with a partner is inked into a complex consisting of an immunologically active substance to which biotin is covalently bound and avidin to which an enzyme is covalently bound, and the solid and liquid phases are separated. and then measuring the enzyme activity in either the solid or liquid phase and concluding from the result the presence or amount of the substance to be measured.

Antigens are particularly considered as substances to be measured with this sandwich method. It has been discovered that hepatitis B virus surface antigen and carcinoembryonic antigen (OKA) are antigens of particular choice.

In this method, two different antibodies directed against the same antigen as immunologically active reaction partners, but directed against different engineered topes of this antigen, are preferred. used. In this case, two different single mouse antibodies come into particular consideration.

The following examples illustrate the invention.

The single anti-hepatitis B virus surface antigen antibody (anti-HBm) used in the examples was 1. roundal o
f-engineering mmunological Method 35
Mice are immunized with the Bil hepatitis virus surface antigen obtained by the method of IFazaka et al. as described in 1-21 (1980). The myeloma line IFO described by 'Il&Mek& (cited above) is used for fusion. The obtained hyartaoma is injected intraperitoneally into BALB/O mice in a manner known per se and the ascites fluid formed is used to obtain antibodies. As an example we use two antibodies directed against different epitopes of the hepatitis B virus surface antigen, designated anti-HBa-1 and anti-HBa-2, respectively. The Ig() fraction of this antibody is obtained by precipitation of ascites fluid against ammonium sulfate and chromatography on diethylamine ethyl cellulose.

In the examples used in the
cal Methods 32 (1980), 297-3
25 May 1979, but as the starting cell line for fusion.
Precipitated under storm CRL 8006 along with TCC1
It is obtained using the myeloma line 8p210.l-Ag, which became publicly available on November 24, 19981. Fusion expansion was performed using mouse pancreatic cells immunized with CBA. Immunizations of mice were carried out in a manner analogous to Table 1 of the aforementioned publication, with the first two immunizations being carried out using 50 μ9 (JA) in each case, and immunizations 3 and 4 Omitted, immunization 5 was performed with 50 μg of CEA, and immunizations 6 to 8 were each performed with 200 μg of CEA. Two different immunizations directed against different epitopes of the CEA antigen Appropriate monoclonal antibodies are used, designated anti-CBA (C-3) and anti-CBA (c'-19), respectively.

Example 1 The IgG fraction of anti-HBa-1 or anti-CEA (C-5) antibody was added to a 0.1 mol/J sodium bicarbonate solution (pH 8,2-8,5) overnight for 2-8 days. ℃
The protein concentration was adjusted to 11ui/1117. Additionally, 120 trout/- of completely freshly prepared biotin succinimide ester S solution (1 w97d in dimethyl sulfate) are mixed per 1-1 of the protein solution and the mixture is incubated for 4 hours at room temperature. The biotin-antibody conjugate was then added at 0.66H9/! of melfen against phosphate buffered saline at 2-8°C. 10/- of bovine serum albumin is mixed as a protein stabilizer.

To bind peroxidase to acadin, dissolve 5 Da of avidin in 2.5-2 of Q, i mo-4ij/J of sodium bicarbonate solution (p?19.5) and incubate in the same solution overnight. Dialyze at -8°C. i from wasabi
owg peroxidase was dissolved in 3.0-heat bath water and 3i! Leave it at a warm temperature for 1 hour, then use the freshly prepared 60.1
mcJ / , 0 of J persodium oxate aqueous solution
．． 5- and incubate for exactly 20 minutes at room temperature. The peroxidase is then liberated from the excess sodium periodate by ultrafiltration and concentrated to the original volume with a falcon. After ultrafiltration, the peroxidase solution is mixed with the Navidin solution, after which the mixture is allowed to incubate for 2 hours at room temperature. In order to reduce the Schiff base and at the same time stabilize the covalent bond, the newly prepared Q;1mcJ/
0.5- of the sodium borohydride aqueous solution of 4
Mixed with peroxidase solution 1. Add the mixture to 2-
Incubate at 8°C for at least 2 hours. The Acdin-peroxidase conjugate is then dialyzed against phosphate buffered saline containing 0166Q/1 melfen and stabilized with 101119/- comb serum albumin.

To form a complex, 554 anti-HBa-1-antibody-biotene conjugates were combined with >55 anti-CEA (c
-3) Mixing the antibody-biotin conjugate with the 484 7vidin-peroxidase conjugate.

The mixture is allowed to incubate for 15 minutes at room temperature. The formation of the complex carried out can be checked by incubating a portion of this solution, after appropriate dilution with phosphate buffered saline, with anti-Max IgG antibody bound to an insoluble carrier. If complex formation has been carried out, then after separation of the liquid phase the enzyme activity can be detected on the solid phase in known manner.

This complex can be lyophilized using conventional methods.

For use according to the invention, anti-HBs-1-
For antibodies, the conjugate is 0 = 25 moA/J sodium hydrogen phosphate, 101//J bovine serum aldemin, 0.6
6 da/Kan no Melfuen and 0.04.9/Yu no 4-
Dilute with a solution containing amino-antipyrine and adjust to -7.0, and in the case of anti-CBA (C-3) antibody to 0.
20 moJ/J sodium hydrogen phosphate, 211/
J bovine serum albumin, 20% Yai serum, 0.51
The solution was diluted with a solution containing 17 ml of "Chime 4S" and 0.1 g/J of 4-amino-antiquiline, and the pH was adjusted to 6.5.

Example 2 To detect Bm hepatitis virus surface antigen in human serum or blood cells, 0.05 to 0.15 of the sample was combined with the IgG fraction of anti-HBs-2 antibody as described in Example 1. Incubate for 1 hour at 45°C in a round-bottom microtitration chamber with 3.2 mm diameter (D) polystyrene beads coated with 0.05-D polystyrene beads. After incubation, the polystyrene beads were washed with hot water in a titration dish and then the peroxidase activity was determined.
4 Listerene C-s was incubated with 0.1-00-phenylenediamine (4*/d) in Q21 mcJ/J of potassium citrate buffer solution PH5=0 and 6 mJ/J of hydrogen peroxide. By doing this, it is measured in the solid state. After 30 minutes of enzymatic reaction, add 4N sulfuric acid 0.1
The reaction is stopped by adding 11 Il and 2.5 mo/J of sodium chloride, and the color reaction is read visually or with a photometer suitable for a microtitrator. Comparison with positive and negative samples confirms whether the sample under consideration contains hepatitis B virus surface antigen.

Example 3 In order to detect CgA in human serum or plasma, in each case 0.2- of the test solution (containing 2 g/J of bovine serum aldimine 0.2 mol/1 sodium phosphate buffer pH 6.5, 20% normal Yai serum, 0.1 g/l 4-amino-
Antipyrine, 0.5974 of "temerosal" and 480 ng/14 of single-loan mouse anti-sugar (C
-S)-biotin/avidin-peroxidase)-
Sansol or CEA standard solution (Ong/Ml CHA, 2.5 n
g/IIJ's (JA.

5. Qng/ILl cgA, 10 thll/- amA, 20n19/- CEA) and CRA control serum (5,0 nJ/- CEA).
) and in each case polystyrene beads (φ = 6.51 m) sensitized with a single mouse anti-CEA-(C-19) were added and the mixture was incubated at 37 °C. Incubate for 16 hours. after that.

The polystyrene beads were washed three times with 2 to 5111 g of hot water each time, in each case 0.0 to 511 l, in order to immunologically bind to the beads and measure the peroxidase activity.
5117 substrate buffer (0 = 1 m
oJ/J) and incubate for 30 minutes at 18-26°C. To stop the peroxidation activity and enhance the color development, 210-1N hydrochloric acid is mixed and the absorbance at a wavelength of 492 nm is measured photometrically within 30 minutes. Table 1, shown below, summarizes the cmAall constant values,
A comparison of these values with those obtained by ROCHE radioimmunoassay is shown.

Claims (9)

[Claims] (1) A complex consisting of an immunologically active substance to which piotin is covalently bound and avidin to which an enzyme is covalently bound. (2) The complex according to item (1) above, wherein the immunologically active substance is an antibody or an antigen. (3) The antibody mentioned above is a monoclonal mouse antibody.
2) Complex of terms. (4) The complex according to item (3) above, wherein the monoclonal mouse antibody is an antibody against BW hepatitis virus true surface antigen. (5) Item (1) above, where the enzyme is peroxidase.
A complex of any one of the items from item (4) to item (4). (6) The complex according to any one of the above items (1) to (5), wherein the peroxidase is horseradish peroxidase. (7) The complex according to any one of paragraphs (1) to (6) above, wherein the complex is present in lyophilized form. (8) above, consisting of covalently binding pyotin to an immunologically active substance, covalently binding avidin to an enzyme, and mixing the two resulting products with each other.
A method for producing a composite according to item (1). (9) The method of item (8) above, in which the obtained complex is freeze-dried. αI Sand-Deutsch method for the detection or amplification of certain substances, which are determined in conjunction with an immunologically active reaction partner that is bound to, or becomes bound to, a water-insoluble carrier. A complex consisting of the target substance, an immunologically active substance to which -otine is covalently bound, and avidin to which an enzyme is covalently bound is incubated, and after separating the same phase and liquid phase, the same phase or liquid expansion is performed. A method consisting of measuring the enzyme activity in any of the phases and concluding from the results the presence or abundance of the substance to be quantified. The method described in the above paragraph, in which the substance to be quantified is an antigen. az The method of paragraph αυ above, wherein the antigen is a Bm hepatitis virus surface antigen. 0S The immunologically active reaction partners are different antibodies directed against the same antigen, but different antibodies of this antigen.
Any one method from Section 03 to Section 03. Q4) The method of item 04 above, wherein the antibodies are two different monoclonal mouse antibodies. (151 The method of paragraph α above, wherein the monoclonal mouse antibody is an anti-BWi hepatitis virus surface antigen antibody. liG The method of any one of paragraphs 4 to 09 above, wherein the enzyme is peroxidase. aη The method of paragraph ae above, wherein the peroxidase is horseradish peroxidase. αυ Use of the complex according to paragraph (1) above in the Sanderutsch method.