RU2282192C1 - Method of preparing diagnosticum to reveal diphtheria toxin - Google Patents

Abstract

FIELD: medicinal microbiology.

SUBSTANCE: gist of invention resides in using polymer carrier prepared by a method of anionic polymerization of acryl aldehyde monomer in presence of sodium hydroxide as polymerization initiator, said polymer carrier being in the form of colored spherically-shaped equigranular particles 2±0.5 μm in diameter and having surface reactive aldehyde groups in quantity 1.3-1.6 μmol/g carrier, which carrier is washed with 0.1 M carbonate buffer solution having pH 9.0-9.2, centrifuged for 10 min at 3000 rpm, immobilized by suspending washed out carrier in 0.1 M carbonate buffer solution, pH 9.0-9.2, to which diphtheria antitoxic immunoglobuline is then added. Components are held in contact for 2 h at 20^°C, cooled to 7^°C, kept at this temperature for 16-18 h, and stabilized. Stabilization is effected by adding 1% gelatose solution in 0.9% sodium chloride solution, stirring for 2 h at 20^°C, and lyophilization of diagnosticum. Diphtheria toxin is revealed using space agglomeration reaction.

EFFECT: increased specificity of diagnosticum.

3 cl, 3 ex

Description

The present invention relates to medical microbiology and can be used in laboratory diagnostics for rapid analyzes in the detection of diphtheria toxin produced by toxigenic cultures of corynebacterium diphtheria.

At present, despite the outlined tendency towards a decrease in the incidence rate, the country's epidemic troubles persist, as evidenced by the high severity of the course of diphtheria and mortality from it.

Laboratory diagnosis of diphtheria to date is represented by traditional bacteriological methods - isolating the pathogen in a pure culture, identifying it by biochemical characteristics and determining the toxigenicity of the culture in the precipitation reaction in agar. The methods used do not meet the modern requirements of the analysis due to the complexity, low sensitivity (the inability to detect toxigenic strains at a low level of toxin formation) and the issuance of the final test result only for 4-5 days.

To reduce the time needed to diagnose diphtheria and increase the sensitivity of the method, it is necessary to develop drugs and research methods aimed at indicating a pathogenic agent - diphtheria toxin.

Currently, considerable attention is paid to the production of polymer latex carriers of antigens and antibodies, which differ from biological (erythrocyte) carriers by the absence of their own antigenicity, stability during long-term storage, constant physical parameters and chemical (covalent) interaction with ligands.

Known methods for detecting diphtheria toxin (see "Laboratory diagnosis of diphtheria infection" Guide. - M .: Information and Publishing Center of the State Committee for Sanitary and Epidemiological Surveillance of Russia, 1995. - p. 42-61), which consist in carrying out accelerated methods for diagnosing diphtheria infection using enzyme-linked immunosorbent assay ( ELISA) and indirect hemagglutination reactions to detect diphtheria toxin (RNGA).

However, despite the relatively high sensitivity, these methods have disadvantages, namely:

- for the conduct of ELISA requires expensive reagents and hardware;

- RNGA is characterized by instability and non-standard properties of the erythrocyte biological carrier, as well as the absence of a lyophilized erythrocyte diagnostic product, the shelf life of the produced liquid erythrocyte diagnosticum is one year.

For the prototype, a method was selected for producing a preparation for indicating the toxin of the diphtheria pathogen in a liquid nutrient medium (see US Pat. RU No. 2104714, 02.20.98), including treatment with a glycine-saline buffer solution with a molarity of 0.05, pH 8.2 gamma globulin immune to diphtheria toxin with its subsequent immobilization on a mixture of polystyrene latex with a dispersion of 1.1-1.2 microns, activated by zinc methacrylate with volume inclusion, and a carboxylated polystyrene latex with a dispersion of 0.2-0.25 microns, and to stabilize the diagnosticum in it doba 20 ml of a 0.1% solution of bovine serum albumin in a 0.05 M glycine-saline buffer solution with a pH of 8.2 containing 12% polyethylene glycol - 6000 are added.

However, the known method has several disadvantages:

- firstly, a mixture of latexes of different dispersion and requiring additional activation by zinc methacrylate and methacrylic acid to create reactive groups for interaction with protein molecules of diphtheria immunoglobulin is used as a carrier, which complicates the diagnosticum technology;

- secondly, the serological reaction is performed on glass, the results in the form of unpainted agglutinants (particles) indicate the absence of any demonstrative reaction, while the sensitivity (RLA) is 10 ^-3 -10 ^-4 Lf / ml.

The technical task of the invention consisted in the development of a diagnosticum for the detection of diphtheria toxin with high sensitivity, stability of properties and high demonstrativity.

This object is achieved in that in the known method for producing a diagnosticum for detecting diphtheria toxin, including washing the polymer carrier with a buffer solution, immobilizing the immunoglobulin to diphtheria toxin on the particles of the carrier and then stabilizing, the carrier is a polymer carrier obtained by the anionic polymerization of monomer-acrylic aldehyde in the presence of a polymerization initiator - sodium hydroxide and representing colored monodisperse particles of sph of a trimeric form with a diameter of 2 ± 0.5 μm with reactive aldehyde groups on the particle surface in an amount of 1.3-1.6 μmol / g of support, the support is washed with a 0.1 M carbonate buffer solution with a pH of 9.0-9.2, centrifuged at 3000 rpm for 10 minutes and immobilization is carried out by suspending the washed carrier in 0.1 M carbonate buffer, pH 9.0-9.2 and adding diphtheria antitoxic immunoglobulin to it, left for contact for 2 hours at 20 ° C, cooled to 7 ° C and incubated for 16-18 hours, stabilization is carried out by introducing into the suspension 1% ra creates zhelatozy 0.9% sodium chloride solution, left under stirring at 20 ° C for 2 hours, conduct lyophilization diagnosticum, diphtheria toxin and detection reaction is carried out in bulk agglomeration.

In addition, lyophilization is carried out by introducing into the precipitate a diagnosticum up to (20-22) ml of 3% gelatin-sucrose medium, then pouring into ampoules and then drying at a final temperature of 22 ° C for a day.

The volumetric agglomeration reaction is carried out in tablets by adding 50 μl of 1.0% normal rabbit serum, 50 μl of suspension of the test culture and 25 μl of diagnosticum to the wells, followed by stirring for 1 minute and contacting at a temperature of (20-22) ° С for 1 5-2 hours.

The method is as follows.

To carry out the method, a polymer carrier is required, which, in accordance with the production schedule (RP) No. 979-00 of June 20, 2000, is obtained by the method of anionic polymerization of a monomer — acrylic aldehyde (acroline) in the presence of a polymerization initiator — sodium hydroxide. As a result of synthesis, monodisperse particles of a spherical shape of the same diameter ranging from (2 ± 0.5) μm with reaction aldehyde groups on the surface of spherical particles (microspheres) are obtained. The number of aldehyde groups is 1.3-1.6 μmol / g of the carrier and they are determined by a special method using a photoelectric colorimeter by changing the optical density. At the same time, at the polymerization stage, dye is added - safranin, which stains the microspheres in a lilac-pink color.

An example of obtaining a diagnosticum.

Initially, 100 mg of the carrier was placed in a centrifuge cup and washed twice with 0.1 M 10 ml carbonate buffer solution, pH 9.0-9.2 at (3000 ± 100) rpm for 10 minutes. The washed carrier is suspended in 1.5 ml of 0.1 M carbonate buffer solution. Then, 1.5 ml of a buffer solution and 450 μg / ml of diphtheria antitoxic immunoglobulin are added to the suspension of microspheres for immobilization and, while stirring, they are left in contact for 2 hours at a temperature of 20 ° С, as a result, the aldehyde reaction groups on the particle surface interact specifically amino groups of protein molecules of diphtheria immunoglobulin.

After that, the suspension is cooled to 7 ° C and incubated for 16-18 hours.

The next step in obtaining a diagnosticum is to stabilize the suspension obtained. To do this, add 2 ml of a 1.0% gelatose solution to a 0.9% sodium chloride solution and leave it under constant stirring for 2 hours at a temperature of 20 ° C.

As a result of stabilization of gelatose, it allows blocking reaction groups on particles that did not bind to a solution of a specific antitoxic immunoglobulin during immobilization.

Then the suspension of the diagnosticum is centrifuged and washed three times with 0.9% sodium chloride solution at 3000 rpm for 5 minutes, obtaining a precipitate.

The final step in obtaining a diagnosticum is lyophilization, in which the sediment is adjusted to 20 ml with a 3% gelatin-sucrose drying medium and poured into 1 ml ampoules with a syringe. Lyophilization is carried out for 23 hours at a final temperature of (22 ± 2) ° C.

The contents of the ampoules are frozen by alcohol cooling at a temperature of (-45) ° C for 10 minutes. Frozen diagnosticum is placed in a low-temperature cabinet at a temperature of (-50) ° C and maintained for 17 hours, after which ampoules with frozen diagnosticum are placed in the chamber of a vacuum-drying apparatus.

Reaction Procedure

Example 1

The study of material with a dense nutrient medium in the reaction of bulk agglomeration (RAW). For the reaction, the test material is preliminarily prepared; for this, pathological material from the oropharynx, tonsils, and nose, taken with a dry swab, is seeded on a plate with blood-tellurite agar, incubated in a thermostat at 37 ° C for 24 hours, a suspension is prepared from suspicious colonies in vitro by adding 1-2 ml of 0.9% sodium chloride solution.

In parallel, microbial suspension of the control toxigenic strain of corynebacteria diphtheria with a concentration of 10 ⁹ μ / ml is examined. To conduct RAO in the wells of the tablet for immunological reactions (see table 1) in rows A, B, C, D make 50 μl of 1% solution of normal rabbit serum (NCC). Then, 50 μl of a commercial diphtheria toxoid (diluted 1: 1000 or 0.3 Lf per 1 ml) is pipetted into the first two wells of row A and titrated, i.e. make serial two-fold dilutions of 12 wells. Then transferred to row B and titrated to 11 wells. In series with the first two wells of making .mu.l of 50 C. diphtheria mitis 1092 (tox ⁺⁾ bacterial suspension control toxigenic strain (September ¹⁰ mg / ml) and titrate with twofold increments of 12 ^th well.

Then, in the first two wells of row A Pipette 50 ul of suspension culture under study and titrated twice to 12 ^th wells.

After that, in each well (rows A, B, C, D), 1 drop (25 μl) of diagnosticum is pipetted.

The contents of the wells are mixed by rocking the tablet for 1 minute and left at a temperature of (20-22) ° C for 1.5-2 hours. To control the diagnosticum for the absence of spontaneous agglomeration, only NKS and the diagnostic preparation are added to the B wells 11-12 in a row.

For a positive result (+) take a color bright pink agglomerate, lining the entire bottom of the hole evenly. In negative (-) cases and in the control, a compact ring or “point” is formed in the center of the well.

Thus, from table 1 shows:

- row A - indicates the presence of diphtheria toxin in the sample (+) (pink agglomerate);

- row B - up to 7 holes the reaction is positive, and from 7 to 10 holes indicate a negative result (pink rings);

- row C - 6 holes are positive with the control strain (toxigenic) (+), and with 7 holes (-), i.e. the toxin content in the control toxigenic strain corresponds to 1.9 × 10 ^-4 Lf / ml;

- row D - the test culture with diphtheria toxin is confirmed up to 6 holes (+), and from 6 holes (-).

The sensitivity of the reaction with a commercial diphtheria toxoid is 3 × 10 ^-6 Lf / ml (row B) - the minimum detectable concentration of toxoid. The sensitivity with the control toxigenic strain C. diphtheria mitis 1092 was 1.9 × 10 ^-4 Lf / ml (row C), i.e. the toxin content in the control toxigenic strain corresponds to 1.9 × 10 ^-4 Lf / ml. A positive reaction (RAO) with the studied culture (row D) indicates that the culture is toxigenic, and the production of toxin in the culture quantitatively corresponds to 3.8 × 10 ^-4 Lf / ml (5 holes).

Example 2

It differs from Example 1 in that the pathological material under investigation is seeded in a liquid nutrient medium (Hottinger broth, potassium tellurite, cattle) and after 18 hours of incubation in a thermostat at 37 ° C, the supernatant of the culture medium is examined (see table 2, row C). Rows A and B - positive control of radioactive waste with a commercial diphtheria toxoid, as in example 1. Row C - broth culture of the studied pathogen. Row D - broth nutrient medium without a pathogen - control of the culture medium.

Based on table 2:

- row A - indicates the presence of diphtheria toxin sample (+) - single agglomerates;

- row B - from 7-10 holes indicate a (-) negative result (pink “rings”);

- row C - indicate the presence of toxin in 6 wells at a dilution (1:64), the sensitivity of the RAW with the studied culture was 1.9 × 10 ^-4 Lf / MA;

- row D - negative results, because with a culture medium without a pathogen, the diagnosticum does not respond.

Example 3

It differs from Example 1 in that diphtheroids that do not produce toxin are used as the test culture: C. xerosis 127 (row C) (see table 3), C. hofmanii Aleshin (row D). Rows A and B positive RAW control with commercial diphtheria toxoid.

Analysis of the reaction showed that with diphtheroids, namely C. xerosis 127 (row C) and C. hofmanii "Aleshin" (row D), a clear negative reaction of RAO is observed, since diphtheroids do not produce diphtheria toxin.

Based on the above examples, we can conclude that the diagnosticum developed by the Rostov NIPCH specialists is used to study cultures grown on both solid and liquid nutrient media, which provides a wider range of its use in laboratory diagnosis of diphtheria.

In addition, the examples show the specificity of the diagnosticum, since there are no cross-reactions with atoxigenic strains of corynebacteria closely related diphtheroids (C. xerosis, C. hofmanii).

The proposed method for producing a diagnosticum allows covalent interaction of the reaction aldehyde groups located on the spherical surface of the carrier particles with the amino groups of the diphtheria immunoglobulin protein molecules, which provides the diagnosticum with high sensitivity (10 ^-5 -10 ^-6 Lf / ml) and specificity.

The lyophilized diagnosticum has stable properties, since it retains primary activity for 4 years.

In addition, the diagnosticum obtained is economical, since 1 ampoule is enough for 50 tests, it is simple to set up, it does not require special training of medical personnel, and it is highly reproducible and demonstrative of the reaction results due to its bright color.

Table 1 Ranks Holes one 2 3 four 5 6 7 8 9 10 eleven 12 BUT + + + + + + + + + + + + AT + + + + + + ± - - - TO
- TO
- FROM + + + + + + - - - - - - D + + + + + - - - - - - -

table 2 Ranks Holes one 2 3 four 5 6 7 8 9 10 eleven 12 BUT + + + + + + + + + + + + AT + + + + + + - - - - TO
- TO
- FROM + + + + + + D - - - - - - -

Table 3 Ranks Holes one 2 3 four 5 6 7 8 9 10 eleven 12 BUT + + + + + + + + + + + + AT + + + + + + - - - - TO
- TO
- FROM - - - - - - - - - - D - - - - - - - - - -

Claims (3)

1. A method of obtaining a diagnosticum for detecting diphtheria toxin, comprising washing the polymer carrier with a buffer solution, immobilizing an immunoglobulin to diphtheria toxin on particles of the carrier and then stabilizing, characterized in that the carrier is a polymer carrier obtained by anionic polymerization of an acrylic aldehyde monomer in the presence of a polymerization initiator - sodium hydroxide and representing colored monodisperse particles of a spherical shape with a diameter of 2 ± 0.5 μm with reaction aldehyde groups on the surface of the particles in an amount of 1.3-1.6 μmol / g of carrier, wash the carrier with 0.1 M carbonate buffer solution with a pH of 9.0-9.2, centrifuge at 3000 rpm for 10 min and immobilization is carried out by suspending the washed carrier in 0.1 M carbonate buffer, pH 9.0-9.2, and adding diphtheria antitoxic immunoglobulin to it, left for contact for 2 hours at 20 ° C, cooled to 7 ° C and incubated for 16-18 hours, stabilization is carried out by introducing into suspension a 1% solution of gelatose on a 0.9% solution of chl sodium chloride, is left under stirring at 20 ° C for 2 hours, lyophilized diagnosticum, diphtheria toxin and detection reaction is carried out in bulk agglomeration. 2. The method according to claim 1, characterized in that the lyophilization is carried out by introducing into the precipitate diagnosticum up to (20-22) ml of 3% gelatin-sucrose medium, then pouring into ampoules and then drying at a final temperature of 22 ° C for days. 3. The method according to claim 1, characterized in that the volumetric agglomeration reaction is carried out in tablets by adding to the wells 50 μl of 1% normal rabbit serum, 50 μl of suspension of the test culture and 25 μl of diagnosticum, followed by stirring for 1 min and contacting at a temperature (20-22) ° C for 1.5-2 hours