SU1573427A1 - Method of determining theophylline in blood serum - Google Patents

Abstract

The invention relates to immunochemistry, specifically to a method for determining theophylline in biological fluids and can be used for drug monitoring in theophylline therapy: monitoring the concentration of the drug in patients, selecting individual doses to increase the effectiveness of therapy and to eliminate toxic effects. The aim of the invention is to increase the sensitivity and speed of the method. Measurement of theophylline in serum is based on the competition between the theophylline from the sample and the theophylline associated with the solid phase by azo-peroxidase-labeled antibodies to theophylline. For analysis, a few µl of serum or plasma is required (a drop of blood from a finger is enough), the analysis time of 30 samples is about 40 minutes, the sensitivity of the method is 0.01 µg / ml of theophylline.

Description

The invention relates to the field of immunochemistry, specifically to a method for determining theophylline in biological fluids, and can be used for drug monitoring in theophylline therapy: monitoring the concentration of the drug in patients, selecting individual doses to increase the effectiveness of therapy and eliminate toxic effects.

The purpose of the invention is to increase the sensitivity and acceleration of the method.

The method is carried out as follows.

For immunization, a theophylline conjugated antigen with a protein is used, which has the following structure: (theo-azo-BSA)

ABOUT

N

CHj-N-V

(

ate

(bovine serum albumin).

This antigen allows antibodies to be produced with binding constants.

10 l / mol. By specificity, these antibodies are similar to the antibodies described in the known method.

Immunoglobulin fractions were isolated from antithyophylline sera and peroxidase-labeled by a well-known method. For sorption on the solid phase, several congenerized anions were tested.

YU

31573427

conjugate of theophylline and a conjugate was cut for use of the theophylline determination method, which allows to achieve maximum sensitivity. ,

When immunized with a conjugated ntigen, three types of nitel are always formed: on the carrier, on the hapten and on the ground between the hapten and the protein. When orbiting on the solid phase of that hen-th ata, which was used for immunization, all three types of antibodies bind, a complete inhibition of the binding of antibodies to the antigen free drug does not succeed, due to which the sensitivity of the method is low. In the proposed method, it avn 0.5-1 μg / ml. To eliminate the effect of antibodies to the carrier, a conjugated antigen was synthesized, which is different from that used for immunization with a carrier protein; .

ABOUT

CHo-N VV ™ 30; kX N NU m

25

N N CH3

(human serum albumin).

When using such a conjugate for sorption on the solid phase, the sensitivity of the method increased to 0 μg / ml.

The next stage of work was the synthesis of cognate, which differs from that used for immunization not only as an antigen carrier, but also as a type of chemical bond between hapten and protein.

These conjugates had the following structures:

About SNS

zo

- 35

40

JS VNH (CH2) 5NH

About N N

N CH3

(human serum albumin)

ABOUT

SS-N,

h)

O1

OC-N

XX VNHCH CONH

45

50

55

(h

ko to but ga yes

va no no ki ne and shi sy in te a ne ne g s n n m m aa (in

in to p

at

with i

2 to 1

Sn (human serum albumin)

 N

.N

ABOUT

XXVCHiCH CONH

  h h „

N

15th and 20th

25

zo

35

40

45

50

55

sns

(human serum albumin).

The use of the second and third conjugated antigens does not lead to a significant increase in the sensitivity of theophylline determination, while the use of the first coyugat increases the sensitivity of the method to 0.01 µg / ml.

For analysis, the conjugated antigen theo-g / a-CSA is sorbed on the solid phase (polystyrene 96-well plates, tubes, beads) in concentrations sufficient to saturate the solid phase (they were selected experimentally). 1-10 μl aliquots of blood serum from patients who received theophylline, control donor sera or donor sera with a known amount of theophylline added to them are mixed with diluted peroxide-labeled antibodies to theophylline and incubated for 20 minutes with the antigen sorbed on the solid phase. The amount of antibodies bound to the antigen is estimated by the activity of the enzyme label in comparison with the control. Peroxidase activity can be measured using any available substrate that gives a colored product (orthophenylene diamine, 5-aminosalicylic acid, o-dianisidine, etc.) after reaction with the enzyme.

Dilution of antibodies is preselected in such a way that in a control experiment (donor sera without theophyllic), the absorbance values will be 1-1.3.

Example 1: Preparation of theophylline conjugate bovine theophylline antigen

serum albumin (T-azo-BSA). i

To 20 mg of 8-aminotheophillin in 10 μl

of formic acid and 100 µl of 1N HH; 6 mg of NaNOj are added. at 0 ° C. After 20 minutes, the resulting diazonium salt of 8-aminotheophylline is added dropwise to a cooled solution of 100 mg of bovine serum albumin (BSA) in 1 ml of 0.15 M phosphate buffer, pH 8.2. The pH value is maintained equal to 8-9 by adding 1 n. NaOH. After stirring for 2 hours at 0-10 ° C, the mixture

divided on a column with Sephadex C-25 or dialysed against water until absorption in the dialysate at 290 nm becomes less than 0.05. The protein fraction is lyophilized. The content of theophylline in the resulting conjugated antigen is determined by the difference between the UV spectra of the conjugate and BSA, starting from E ° / о 87 for 8-aminotheophylline, then at E k - absorption of the conjugate at a concentration of 1 mg / ml and absorption of the original protein at the same concentration of 0.3, and mol. m of protein and 8-aminotheophylline, equal to 69000 and 200, respectively, receive a substitution in theophylline mole per mole of protein:

69000

Substitution

EC-0.3

The resulting conjugate contains 13 theophylline molecules for each protein molecule.

Take p2. Preparation of theophylline conjugated antigen with human serum albumin (Tg / a-CSA).

To a solution of 40 mg of 8-aminotheophilus and 90 mg of human serum albumin (HSA) and 2 ml of 0.01 n. NaOH, cooled to 10 ° C, is added over 30 minutes to 120 µl of 25% glutaraldehyde. The reaction mixture is stirred for 1 h and added dropwise with NaBH in 100 µl of water. After incubation for 1 hour at room temperature, the protein is separated from the low molecular weight reaction products according to Example 1. After incubation for 1 hour at room temperature, the protein is separated from the low molecular weight reaction products according to Example 1. The resulting conjugate lyophilized dried. According to the UV spectra, 15 theophylline molecules are bound in it with one protein molecule.

PRI me R 3. Immunization of animals and the production of serums.

Outbred gray rabbits are immunized with the conjugated antigen T-azo-BSA, 0.5 mg per animal. Conjugate is dissolved in 0.25 ml of physiological saline and mixed with 0.5 ml of Freund's adjuvant to form a stable emulsion. The mixture is injected subcutaneously in six places on the back of a rabbit. Immunization is repeated through

6 weeks and further with an interval of g-4 weeks with Freund's incomplete adjuvant. Blood is taken after 7 days. after immunization from the rabbit auricular vein, incubated for 30 minutes in a thermostat at 37 ° C, centrifuged at 3000 rpm for 30 minutes. The resulting whey is stored frozen at -20 ° C. The specificity of antibodies to theophylline is determined by the radioimmunopogic method for binding to H-theophylline and the enzyme-linked immunosorbent assay. The specificity of antibodies to theophylline, the cross-reaction in percent to theophylline is given in the table.

PRI me R 4 „Obtaining conjugate of immunoglobulin fraction of serum with peroxidase (1g-PO).

The immunoglobulin fraction is obtained from antithéophylline serum by salting out ammonium sulfate at 50% saturation. Horseradish peroxidase, 4 mg, is dissolved in 1 ml of distilled water, 0.2 ml of freshly prepared 100 mM NaJ04 s is added for 20 minutes at room temperature, the solution is nightized at 4 ° C against 1 mM a-acetate buffer, pH 4.4. 20 µl of 200 mM carbonate buffer, pH 9, 6.8 mg of immunoglobulins (dissolved in 1.0 ml of 10 mM carbonate buffer, pH 9.6) are added and stirred for 1 hour at room temperature. 0.1 ml of freshly prepared NaBH4 (4 mg / ml water) is added and incubated for 2 hours at 4 ° C. An equal volume of saturated ammonium sulfate is added, centrifuged, the precipitate is dissolved in water, dialysed against 0.15 M NaCl, BSA is added to 2% concentration, diluted twice with glycerol and stored at -20 ° C.

Example 5: Preparation of Theophylline Standard Solutions for Calibration Curves.

Exact samples of theophylline are dissolved in water at a concentration of 1 mg / l, then appropriate aliquots of this solution are added to a pool of 10 donor sera so as to obtain Standards containing 1,5,10,20 and 30 µg of the drug in 1 ml of serum .

Measurement of theophylline concentration in blood sera by the solid-phase immunoassay method.

Example 7. The analysis is carried out as in Example 6 with the difference that the theo-, azo-BSA antigen is absorbed on the solid phase, and the measured volume of serum is 10 µl (1:10 dilution).

Try on The analysis is carried out as in Example 7, with the difference that the conjugated-tezo-azo-CSA conjugated antigen was sorbed on the solid phase.

days 0.98). Thus, the duration of the analysis is reduced by 20 minutes compared with the known method with a higher sensitivity.

Claims (1)

Claim Method The determination of theophylline in The coefficient of variability of the method does not exceed 12%. The measurement results coincide with the data obtained. EXAMPLE 6 Conjugated with an-15 by liquid chromatography, high te gene-g / a-CSA is dissolved in 0.05 M CO 2 pressure (the correlating coefficient is sodium carbonate-bicarbonate buffer (pH 9.6) at a concentration of 1 μg / ml and adsorb onto polystyrene 96-well plates 100 μl / well, at 20 A ° C overnight, then the solid phase is washed with 0.05% Triton X-100 solution three times and three times with water. 1 µl aliquots of blood serum from patients treated with theophylline, or 25 serum, including obtaining donor sera containing antibodies by immunizing animals a known amount of the drug, developed with theophylline antigen up to 100 µl of buffer A: 0.01 M nat-protein, in which theophylline is a phosphate-addition buffer containing 0.15 M H-to protein through the O8 position of mo-NaCl, 0.05% tween-20 and 0.2% BSA, and mixtures of 30 and immunochemical peas were added with 100 µl of 1 g-PO in the same solvents on the solid phase, added to the ferment in concentration and 50 μg / ml. The mixture is shaken thoroughly and transferred to the solid phase with the adsorbed antigen, incubated for 20 minutes, the solid phase is washed and the substrate solution is poured (4 μl of 30% HaOi and 4 mg of ortho-phenylenediamine per 100 ml of 0.1 M sodium carbonate). citrate buffer, pH 5.0) detection at 492 nm. The concentration of the theophilic 40 protein is azo-bonded, and peroxidase and up to 100 μl are used as relien per dilution of aliquots of agents. From the obtained value of conjugate m, in which theophylline absorption absorption, equal, for example, 0.6, to the tapemethylenediamine protein, the concentration of the theophilic bond can be determined, the protein in the conjugate, which is introduced into sorption is different; mi, it is equal to 0.16 μg / mp, and, respectively, on antigenicity of protein, using, in the initial sample, 16 μg / ml. Immunization. immobilized reagents of the obtained antibodies and the serum under study, followed by measuring the optical intensity of the mixture, characterized in that, in order to increase the sensitivity of the method and its acceleration, a conjugate is used in which theophylline is attached to Example 7. The analysis is carried out as in Example 6 with the difference that the teloazo-BSA antigen is absorbed on the solid phase, and the measured volume of serum is 10 µl (1:10 dilution). Try on The analysis is carried out as in Example 7, with the difference that the conjugated theo-azo-HSA was sorbed on the solid phase. days 0.98). Thus, the duration of the analysis is reduced by 20 minutes compared with the known method with a higher sensitivity. Claim Method The determination of theophylline in The coefficient of variability of the method does not exceed 12%. The measurement results coincide with the data obtained by liquid chromatography with a high COG pressure (coefficient of correl - serum, which includes the production of antibodies by immunizing animals with a kyogenic antigen, theophylline protein, in which theophylline is attached to the protein through O8, the position of the molecule, sorbn and immunochemical pea genes on the solid phase, is added by azo bond, and reagents use peroxidase and conjugate t, in which theophylline is attached to a tapemethylenediamine bond protein, wherein the protein in the conjugate used for sorption is different; By antigenicity of the protein used for immunization. serum, which includes the production of antibodies by immunizing animals with a kyogenic antigen, theophylline protein, in which theophylline is attached to the protein through O8, the position of the molecule, sorbn and immunochemical pea genes on the solid phase, is added by azo bond, and reagents use peroxidase and conjugate t, in which theophylline is attached to a tapemethylenediamine bond protein, wherein the protein in the conjugate used for sorption is different; By antigenicity of the protein used for immunization. immobilized reagents of the obtained antibodies and the serum under study, followed by measuring the optical rate of the mixture, characterized in that, in order to increase the sensitivity of the method and its acceleration, conjugate in which theophylline is attached to