SU1429026A1 - Method of producing conjugates of ferment and antibodies for immunoferment analysis - Google Patents

Description

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i The invention relates to medicine, / a. specifically, bioorganic chemistry, and a method for preparing enzyme and antibody conjugates for enzyme immunoassay.

The aim of the invention is to shorten the production time of the target prod product.

 The method is carried out as follows.

I The conjugation of enzymes with antibodies according to the proposed method is carried out in water or in 0.15 M NaCl at pH A., 0

, 0, adding the salt transition-15 solution was repeated three times.

3. Adding to (antibodies, labeled gats were added to 20 shets. The initial p 20 was 1:25 s after cubing for 4. Washing the plate. 5. Adding su to the Substrate Mixture

go metal (). The reaction mixture is stirred for 5–20 minutes, after which it is dialyzed against 0.15 M NaCl. The reaction was followed by taking aliquots of the reaction mixture (2 µg of antibodies), and applied to the wells for immunoassay, pre-coated with antigen. After inc3. Introduction to the tablet conjugate (antibodies labeled with an enzyme). Conjugate was applied at 200 μl per well of the plate. The initial dilution of the conjugate 20 was 1:25, followed by a step 2. Incubate for 1 h at 37 C. 4. Washing the plate (see item 2). .five. Adding substrate mixture. Substrate mixture for alkaline phosphatase is

camp for 1 hour and rinsing with 0.01 M

phosphate buffer containing 1 m 0.15 M 25 10 mM solution of para-nitrophenyl phosphate

in 1 M diethylolamine, pH 9.8. Substrate mixture in a volume of 200 µl insertion

Nad and 0.05% Tween-20 directly determine the enzymatic activity of the sorbed conjugate.

Example 1. A. Synthesis of an alkaline phosphatase conjugate and antibodies with the same power as Cr.

To 250 µg of alkaline phosphatase (1DF) seals (specific activity of 1200 units per 1 µg of protein) dissolved in: 250 µl of 0.15 M NaCl containing 0.01 Msz: MgCl-, (pH 9.0) were added 21 µl antibodies against human IgG with a concentration of 6 mg / ml, which are in a solution of 0.15 M NaCl, and 19 µl of a 3% solution. The final concentration of Cg was 40 2 mg / ml. The reaction proceeded at room temperature for 20 minutes. The reaction mixture was dialyzed against a 0.15 M solution of NaCl containing 0.001 M MgCl2 for 2 hours at room temperature. The resulting conjugate was diluted 10-fold with 0.02 M Tris-HC buffer, 0.01 M MgCl2, 5% human serum albumin (USA) pH b, 0, containing 0.01X NaNj.

B, Statement of the reaction of enzyme-labeled antibodies (REMA) with the resulting conjugate.

1. Immobilization of antigen to halogen50

whether in each well of the tablet,

at room temperature for

45 min. . -

6. Stop the enzymatic reaction. To each well of the tablet with the substrate mixture was added 50 µl

 1 M NaOH.

7. Record the results of the reaction. The optical density of the substrate mixture in each well was measured on a microphotocell calorimeter at a wavelength of 405 nm. Optical density

in the wells with immobilized IgG was Cd, which corresponds to 190 ng, while in the wells with immobilized BSA OED4O5 0.15, which corresponds to 30 ng.

8. Selection of the optimal Cr concentration for conjugation of antibodies with alkaline phosphatase.

Selection was carried out in experiments with a small amount of proteins. Each well was filled with 20 µl of alkaline phosphatase with a concentration of 40 µl / ml, 20 µl of antibodies to IgG with a concentration of 20 µl / ml and 10 µl of a solution of Cg in such concentrations so that the final concentration of ions

blow phase In the microtitre chromium wells in the reaction mixture, 200 μl were diluted with a solution of 1, 2, 3, 4, and 5 mg / ml. antigen concentration of protein The reaction mixture was incubated

20 µg / ml in OB01 M carbonate buffer-40 min at room temperature.

re (pH 9.6} and incubated for then the contents of each well (50 μl)

0262

4 h at room temperature. After incubation, the plates are washed from the unbound antigen. Bovine serum albumin (BSA) was used as a control instead of antigen.

2, Washing tablets. The contents of the wells were removed and the plates were thoroughly washed of non-aligned Q protein with a tap of running water. The wells were then filled with washing fluid (tap water containing 0.05% Tween 20) and rinsed again with tap water. Laundering procedure

repeat thrice.

3. Introduction to the tablet conjugate (antibodies labeled with an enzyme). Conjugate was applied at 200 μl per well of the plate. The initial dilution of the conjugate was 1:25, followed by Step 2. In a cubic vial for 1 h at 37 C. 4. Washing the plate (see item 2). .five. Adding substrate mixture. Substrate mixture for alkaline phosphatase is

Ikubatsi

whether in each well of the tablet,

at room temperature for

45 min. . -

6. Stop the enzymatic reaction. To each well of the tablet with the substrate mixture was added 50 µl

 1 M NaOH.

7. Record the results of the reaction. The optical density of the substrate mixture in each well was measured on a microphotocell calorimeter at a wavelength of 405 nm. Optical density

in the wells with immobilized IgG was Cd, which corresponds to 190 ng, while in the wells with immobilized BSA OED4O5 0.15, which corresponds to 30 ng.

8. Selection of the optimal Cr concentration for conjugation of antibodies with alkaline phosphatase.

Selection was carried out in experiments with a small amount of proteins. Each well was filled with 20 µl of alkaline phosphatase with a concentration of 40 µl / ml, 20 µl of antibodies to IgG with a concentration of 20 µl / ml and 10 µl of a solution of Cg in such concentrations so that the final concentration of ions

transferred to the well with pre-immobilized antigen. Incubated for 1 h at 37 C. The plate was washed and the substrate mixture was added.

The optimal concentration of salt Cg corresponded to the highest optical density and was 2 mg / ml.

Example 2. A. Preparation of glucose oxidase conjugate and Q antibodies with Cr.

To 500 µl of purified glucose oxidase with a concentration of 4 mg / ml, located in 0.15 M Nad (pH 7.0), was added

tech auto Ehsareader at a wavelength of 492 nm. As a result of the reaction, it was found that the optical density in the well with immobilized IgG was D 49 1.0, which corresponds to 180 ng of enzyme delivered to the plate, while in the wells with immobilized BSA 0.08, which corresponds to 5 ng of enzyme .

B. Determination of the degree of enzyme binding to antibodies in the preparation of the conjugate. .

On a column of IgG inflicted horse

250 µl of antibodies to IgG with concentration. - 15 g at glucose oxidase antibodies to

20

it is 4 mg / ml in 0.15 M NaCl and CrCl3 solution to a final concentration of 5 mg / ml.

The reaction proceeded at room temperature for 20 minutes, after which the resulting conjugate was removed against 0.15 M Nad for 2 hours at room temperature.

B. Setting REM with the resulting conjugate.

1. Immobilization of the antigen to the solid phase. 0.15 ml of antigen with a protein concentration of 14 µg / ml in carbonate buffer solution (pH 9.6) Q was added to the wells of a microtiter tablet and incubated overnight at 4 ° C. After incubation, the tablet was washed of unbound antigens with tap water containing 0.05% twin-20. Two rows of tablet sensitized human IgG and two rows of BSA,

2. Introduction to the tablet conjugate. Conjugate was made in 150 μl per well of the plate. 1.5 μg of protein was added to each well. Incubation 1 hour with

about

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37 C, and then washing the tablet from unwashed protein.

3. The introduction of the substrate mixture. Substrate mixture for glucose oxidase was prepared as follows. 2 ml of orthophenylene diamine, 200 mg of glucose and

1 mg of peroxidase was dissolved in 20 ml of phosphate-buffered saline (pH 7.4). A substrate mixture in a volume of 150 μl was added to each well of the plate. Incubated at room temperature for 7 minutes.

4. Stop the enzymatic reaction. 59 mp of 50% sulfuric acid was added to each well of the plate to the substrate mixture.

5. Recording of the reaction results The optical density of the substrate mixture in each well was measured on DynaIgG with glucose oxidase activity corresponding to 30 µg of purified enzyme. In the solution passed through the column, the activity of glucose oxidase was determined using a substrate mixture containing orthophenylene diamine and peroxidase. The enzyme activity at the column corresponded to 12 μg of glu :: ozooxidase. Consequently, when preparing the enzyme-antibody complex conjugate, it is bound to 60% of the enzyme.

Example 3. A. Synthesis of peroxidase conjugate with antibodies using Cr.

300 μl of purified horseradish peroxidase (with a concentration of 5 mg / ml in 0.15 M NaCl, pH 4.0) were infused with 150 μl of antibodies to IgG with a concentration of 5 mg / ml in 0.15 M Nad ( pH 4.0) and solution.-CrCl 3 up to a concentration of 4 mg / ml, of course. The reaction was carried out for 10 min at room temperature, after which the resulting conjugate was ∆dialized against 0.15 M NaCl for 2 h at Room temperature.

B. Setting REM with the resulting conjugate. The formulation of the REM was performed as described in examples 1 and 2. The substrate mixture for

Proxides are orthophenylenediami dissolved in methyl alcohol. For 20 ml of citrate buffer solution (pH 4.7), 200 µl of a mixture of ortho-phenylenediamine + methanol was taken and 2 µl of 30% was added. Substrate mix in a volume of 100 μl was added to the wells of the plate. Incubated at room temperature for 20 min. To stop the reaction, 25 µl of 50% sulfuric acid was added to the substrate mixture. The results were recorded by measuring the optical density

40

45

50

55

Q

tech auto Ehsareader at a wavelength of 492 nm. As a result of the reaction, it was obtained that the optical density in the wells with immobilized IgG was D 49 1.0, which corresponds to 180 ng of enzyme delivered to the plate, while in the wells with immobilized BSA 0.08, which corresponds to 5 ng of enzyme.

B. Determination of the degree of enzyme binding to antibodies in the preparation of the conjugate. .

An IgG conjugate of glucose oxidase was applied to an IgG column with antibodies to

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Q

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IgG with glucose oxidase activity corresponding to 30 μg of purified enzyme. In the solution passed through the column using a substrate mixture containing orthophenylene diamine. and peroxidase, the glucose oxidase activity was determined. The enzyme activity associated with the column corresponded to 12 μg of glu :: ozooxidase. Therefore, upon receipt of the conjugate in the enzyme-antibody complex, 60% of the enzyme is bound.

Example 3. A. Synthesis of peroxidase conjugate with antibodies using Cr.

300 μl of purified horseradish peroxidase (with a concentration of 5 mg / ml, in 0.15 M NaCl, pH 4.0) were infused with 150 μl of antibodies to IgG with a concentration of 5 mg / ml in 0.15 M Nad (pH 4.0) and solution. -CrCl3 to a final concentration of 4 mg / ml. The reaction proceeded for 10 min at room temperature, after which the resulting conjugate was ∆dialized against 0.15 M NaCl for 2 h at Room temperature.

B. Setting REM with the resulting conjugate. The formulation of REMA was carried out as described in Examples 1 and 2. The substrate mixture for the peroxidase is orthophenylenediamine, dissolved in methyl alcohol. For 20 ml of citrate buffer solution (pH 4.7), 200 µl of a mixture of ortho-phenylenediamine + methanol was taken and 2 µl of 30% was added. The substrate mixture in a volume of 100 μl was applied to the wells. Incubated at room temperature for 20 min. To stop the reaction, 25 µl of 50% sulfuric acid was added to the substrate mixture. The results were recorded by measuring the optical density

0

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0

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substrate mixture on Dynatech auto Ebjsareader at a wavelength of 492 nm. I I

As a result of the reaction, it was found that the op-density in the wells with the immobilized IgG was D432, which corresponds to 220 ng of sufficient) enzyme per plate, while in the wells with immobilized BSA

92, which corresponds to 10 ng ffmenta.

The time for c-onyugates to remove a cross-linking agent is 5 to 20 minutes, while in 14

The known method is 2 hours.

Claims (1)

Invention Formula ten The method of obtaining enzyme conjugate and antibodies for enzyme immunoassay by incubating enzymes with antibodies in the presence of a cross-linking agent, characterized in that chlorine chromium is used as a cross-linking agent to shorten the time required to obtain the desired product. - by titels to IgG at pH 4.0-9.0.