SU1672365A1 - Method of exotoxin determination - Google Patents

Abstract

This invention relates to biotechnology and can be used to determine the amount of β exotoxin in entomopathogenic preparations. The aim of the invention is to simplify the method and increase the reliability of the results of the determination of β-exotoxin. Trilon B is added to the suspension of the β-exotoxin preparation or the culture fluid, then the supernatant obtained is centrifuged, streaked onto a Silufol UV ₂₅₄ plate, chromatographed over the propanol-ammonia-water system, β-Exotoxin is chromatographed, the eluate is filtered through a membrane filter to remove particles of Silufol, the optical density of the filtered eluate is determined at wavelengths of 260, 230 nm, the concentrations of β - exotoxin are calculated according to the formula C _at (wt.%) = 6 D ₂₆₀ - D ₂₃₀ / 5.75 ^. V ₀ ^. V ₂ / M ₀ ^. V ₁ ^. 16.6, where V ₀ is the volume of ml in which the sample of the preparation is suspended

M ₀ - the sample of the drug (mg), V ₁ - the volume of solution applied to the chromatogram

V ₂ is the volume of solution used for spectrophotometry. In the case of studying the culture liquid, V ₀ and M ₀ = 1, V ₁ is the volume of the culture liquid applied to the chromatogram. The deviation from the average value does not exceed 2 - 3%. 5 tab.

Description

This invention relates to biotechnology and can be used to determine the amount of exotoxin in entomopathogenic preparations.

The purpose of the invention is to simplify the method and increase the reliability of the results of exotoxin determination.

The method for determining / -exotoxin is as follows.

The entomopathogenic preparation used is dissolved in a saturated aqueous solution of Trilon B. The suspension is centrifuged, the undissolved substances are removed, the β-exotoxin contained in the supernatant is purified by thin layer chromatography, and the eluate obtained after chromatography is filtered through a 0.1-p membrane filter. 0.2 µm, the optical density of the filtrate is measured at wavelengths of 260 and 230 nm, and the amount of / 9-exotoxin is determined when the coefficient value is calculated by the formula

about

4 N3 CJ O SL

Set

 6 D260 - D230Vo V2

 - H.M. 01

5.75m0Vi 16.6 wt

where V0 is the volume in which the sample of the preparation is suspended, ml;

t0 - drug weight, mg;

Vi is the volume of the solution applied to the chromatogram, ml;

Va is the volume of the solution used for spectrophotometry, ml, to determine the EC (mg / ml) in the culture fluid in the formula, m0 V0 1 is assumed.

16.6 - the absorption of pure / 3-exotoxin at a concentration of 1 mg / ml;

D2GO, D230 - absorption of the eluate at the corresponding wavelengths.

The addition of Trilon B (ethylenediaminetetraacetic acid disodium salt) is extremely important for preparations obtained on nutrient media containing divalent metal ions (calcium chloride, magnesium chloride or sulfuric acid, etc.).

Exotoxin has the ability to form poorly soluble complexes with divalent metal ions and therefore, in the presence of bivalent ions in the nutrient medium, a part of the / -exotoxin is bound and is not separated when isolated by conventional methods. This leads to significantly underestimated results of the determination of β-exotoxin both in the preparation and in the culture fluid. For example, in preparations prepared on a multicomponent medium containing 0.2% CaCl2, 20-25% / 3-exotoxin in the bound state, and 60% to 70% bound on an acetate medium containing 3-4% calcium acetate. ft-exotoxin. Adding an excess of Trilon B, which binds divalent ions, results in the release and dissolution of β-exotoxin.

Table 1 shows the results of studies on the example of pure (purity 99.5%) / -exotoxin.

Example 1. To a 2 ml aqueous solution of P-exotoxin at a concentration of 6 ml / ml (0.6%), 20 µl of a 5% aqueous solution of CaClz is added. To speed up the precipitation process, a few drops of a 25% aqueous ammonia solution are added, bringing the pH to 8.0 (precipitation is judged by cloudiness of the solution). To 2 ml of the suspension containing the insoluble complex of / 3-exotoxin with calcium, as well as free exotoxin, add 6 ml of a saturated solution of trilon B or 6 ml of distilled water (control) and filter through a Vladipor Mg 1 filter or Nfe 2 (pore size 0 , 1 - 0.2 microns). The same is done in the absence of calcium chloride. In the filtrates, the value of optical density at a wavelength of 260 nm is determined.

As follows from Table 1, the addition of Trilon B does not affect the absorption of / -exotoxin. When CaC12 is added, about 33% / 3-exotoxin precipitates, resulting in a decrease in optical density. When Trilon B is added, the result almost coincides with the values obtained by dissolving pure / J-exotoxin

in water.

In chromatography, 100 µl of the sample was applied in a strip to a 20 x 20 ml Silufol UV 254 plate. The amount of / -exotoxin applied to the plate,

should be such that, after elution, to ensure absorption above the sensitivity threshold of the spectrophotometer used.

Filtration of the eluate is carried out with

the purpose of removing silica gel particles that cause light scattering of samples, reflecting on the accuracy of optical density determination. The concentration of / J-exotoxin in the preparation is determined by the formula, where the first factor (in parentheses) gives the true absorption of ft-exotoxin, subtracting the contribution of impurities present in the preparations:

25

g 6 D260 - D230 V1 V2 et5,75 mo V 16.6

 0.84 / 6D260-D230, May. %

where V0 is the volume of the solution of Trilon B in which the sample of the preparation is suspended, ml; gl0 - drug weight, mg; Vi is the volume of the solution applied to the chromatogram, ml;

V2 - volume of the spectrophotometric solution, ml;

16.6 - the absorption of pure / 3-exotoxin at a concentration of 1 mg / ml at 260 nm.

To determine the SET. mg / ml, in the culture fluid m0 - V0 1. The determination of the accuracy of the method is carried out by control experiments using highly purified / 3-exotoxin (purity 96 - 98%). Then samples are prepared with the previously known content of / -exotoxin (in particular, the preparation lepidocide, which does not contain / -exotoxin, is used as a filler.

The results are presented in table 2. Statistical analysis of these dependencies showed that the accuracy of the proposed method is higher than ± 3%.

PRI mme R 2. The test preparation (0.5 g) is suspended in 8 ml of a saturated aqueous solution of trylon B. The suspension is centrifuged at 8000 rpm for 10 minutes. The supernatant (100 µl) is applied by microspray to a UV 254 force plate. x 20. on the edge of the witness is applied - pure / 9-exotoxin. The plate is chromatographed in the propanol 25% - ammonia - water system (10: 2: 5). The position of the separated substance is determined in UV light. The adsorbent is transferred to a tube for elution with 5 ml of 0.01 M ammonia solution. The eluate is filtered through a Vladipor membrane filter (0.1 - 0.2 µm) to remove the particles of Silufol, and the optical density is measured at wavelengths of 260 and 230 nm. The concentration of β-exotoxin is determined by the indicated formula.

The results are presented in table 3 and 4.

Example 3. In the culture fluid (5 ml) containing ft-exotoxin, 20 mg of Trilon B is added, centrifuged at 8000 rpm for 5 minutes, and 10 μm of the supernatant are applied in a strip on a plate (20 x 20) of UV 254 forcefol. Further, according to Example 2. The concentration of β-exotoxin is determined by the formula given in Example 1, assuming m0 V0 1.

The results are presented in table.5.

The proposed method is simple, has a sensitivity that allows determining the concentration of ft-exotoxin in all industrial preparations and in the culture fluid at different stages of production of the preparations, as well as during their storage. The method does not require scarce equipment and reagents, the analysis can be carried out within 30 minutes, which makes it possible to attribute it to express methods. The method can be useful in research and production laboratories as an alternative to long-term bio-testing and semi-quantitative methods used for determining ft-exotoxin.

Claims (1)

Invention Formula The method of determining / -exotoxin, which involves removing insoluble substances from the sample under investigation, followed by purifying ft-exotoxin by thin layer chromatography, measuring the optical density of the resulting eluate and determining the amount of exotoxin by calculation, in order to simplify the method and increase the reliability, before removing insoluble substances, a three / yun B sample is introduced into the sample under investigation; before measuring the optical density, the eluate is filtered on membrane ultrafilters with a size of rum then 0.1 - 0.2 microns. and the number Seth exotoxin calculated by the formula Set -  6 D260 - D230Vo 2 about.-3. vu vz mchg ° L 5.75m0Vi 16.6 wt where D260 and D230 - absorption of the eluate at the respective wavelengths; V0 is the volume, D of which the suspension of the preparation is suspended, ml, then - the sample of the drug, mg; Vi is the volume of the solution applied to the chromatogram, ml; (2) the volume of the solution used for spectrophotometry, ml, to determine the EC, mg / ml, in the culture fluid in the formula, m0 V0 1 is assumed. Table 1 table 2 Table 3 Table 4 Table 5