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  Отзывы специалистов. Часть 1

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The Acta of REKITSEN-RD antitoxic activity and sorptive of capacity studies

версия для печати

The ministry of a defense of Russian Federation Research institute of microbiology

According to the program of structural reorganization of economy and external communications of the Kirov territory, subroutine "Biotechnology", project of "Bioforage", sub-project "REKITSEN-RD" in the period from 1.09.94 till 25.01.95 on the basis of Research Institute of microbiology Russian Federation Ministry of Defense the analysis of REKITSEN-RD antitoxic (toxins neutralizing) activity and of sorptive capacity was carried out with the substance produced by TOO "ЭкоБиоТехноЦентр" (Moscow). Three series of REKITSEN-RD were subjected to tests under conditional designations C-1, C-2, C-3.

For testing of antitoxic activity specially prepared standard templates of the following toxins were used:

  • type A botulinic toxin with specific activity 2x107 DL50/mg of protein in working delution 100 DL50/ml;
  • shigelligenous toxin with specific activity 1,3x104 LD50/ml in working delution 1000 DL50/ml;
  • thermolabile escherichia coli produced enterotoxin (similar to choleraic toxin on the mechanism of action) with specific activity 1,8x103 DL50/mg of protein in working delution 1000 DL50/ml.

DL50 values for botulinic and shigelligenous toxins were carried out in intraperitoneal infection of white mice with a step of delution equal 2.

For thermolabile escherichia coli produced enterotoxin ED50 values were determined on white mice in the test of an edema of a back extremity (step of delution equal 5). The calculations of LD50 and ED50 values were carried out using Kerber method in Ashmarin's modification. The working delutions of toxins prepared just before the experiments. For definition of REKITSEN-RD sorptive capacity a stain Congo red of "pure" qualification was used in concentration previously experimentally found as optimal.

Definition of REKITSEN-RD antitoxic activity
Portions of REKITSEN-RD weighting 1,0±0,001 g were brought into test-glass with 10 ml of the appropriate toxin in working delution and carefully mixed. In control tests 1,0+0,1 ml of normal saline solution were added instead. The samples were initiated in a water ultra thermostat within 1 hour at 37,0±0,1С and centrifuged on a refrigerator centrifuge at 5000 rpm and 10±1С within 30 minutes. Supernatant was taken and tested for a titre of toxins in experimental and control samples using white mice bioassays.

The samples containing botulinic and shigelligenous toxins were diluted with normal saline solution by a step equal 2 and injected intraperitoneously to white mice in a volume of 0,5 ml. The animals were observed within 7 days with registration of death rate.

In thermolabile escherichia coli produced enterotoxin tests the samples were diluted with a step 5 and injected into animals' plantar surface of one back extremity in volume 0,05 ml. The same amount of a normal saline solution was injected into an opposite extremity (control). In 48 hours the animals were deadened by a Aether and amputated the extremities on a knee joint. The amputated extremities weighed using torsion balance to within 1 mg. The difference of weight in experimental and control extremities more than 65 mg was treated as an evidence of presence of thermolabile escherichia coli produced enterotoxin. All works were carried out in aseptic conditions.

The results of titration were used for calculation of relative toxicity index under the following formula:

RT - relative toxicity;
Co - concentration of a toxin in the experimental sample;
Ск - concentration of a toxin in the control sample.
The results of researches are represented in tab. 1.

Thus, under the conditions of the study 1 g of REKITSEN-RD is able to neutralize about 900 mice DL50 of type A botulinic toxin, 9000 DL50 of schigellogenous toxin and 8600 ED50 of a thermolabile escherichia coli produced enterotoxin.

Table 1. Results of the analysis toxin containing samples after processing by REKITSEN-RD

REKITSEN-RD antitoxic activity concerning toxins
Thermolabile enterotoxin
RT, %
RT, %
RT, %
The control

The notes. 1. Means were obtained from results of three experiments with 95% reliability Ki = 1/RT - factor of an inactivation of a toxin.

Definition of REKITSEN-RD sorptive capacity

REKITSEN-RD sorptive capacity was investigated in relation of a Congo red stain. Concentration of a stain was determined photometrically on a spectrophotometer SF-26. For this purposes calibration diagram was previously built ranging 10...136 meg/ml stain concentrations. Previously working concentration of a stain was experimentally determined as 90 meg/ml.

Portions of REKITSEN-RD weighting 11,0±0,001g admixed with 10 ml of working delution of a stain and maintained in a ultra thermostat within 1 hour at 37,0+0,1С, periodically mixing. Then samples were centrifuged at 6000 rpm within 30 minutes. Supernatant was poured in others test glasses and determined stain concentrations of on a spectrophotometer. The difference of concentrations in a control and experimental samples was used for calculating of a sorption factor (Fs), which measured in units of absorption (UA). One UA corresponds to one microgram of the stain absorbed. The results of the analysis are submitted in tab. 2.

Table 2. Results of REKITSEN-RD sorptive capacity definition

Initial preparation Concentration of a stain, meg/ml Factor of a sorption, UA
Initial After interaction with REKITSEN-RD
REKITSEN-RD C-1 90±1 28±3 62±3
REKITSEN-RD C-2 90±1 32±2 58±2
REKITSEN-RD C-3 90±1 31±3 59±3

The notes

  1. The means of concentration of a stain and Fs were determined with 95% probability by results of four independent experiments.
  2. Fs is designed per gram of REKITSEN-RD.

Thus, as a result of the researches the commission has made the following conclusions:

  1. REKITSEN-RD has expressed nonspecific toxin neutralizing activity concerning to type A botulinic, shigellogenous and thermolabile escherichia coli produced enterotoxin.
  2. The antitoxic properties of REKITSEN-RD are obviously caused by its ability to bind albuminous toxins, that proves to be true by results of the analysis of REKITSEN-RD sorptive activity
    concerning Congo red (Fs = 59,6+4,35).
  3. The results received allow to assume REKITSEN-RD to have antitoxic activity concerning other albuminous toxins of biological origin.
  4. In view of that the thermolabile escherichia coli produced enterotoxin is similar on a structure and properties to a choleragen, salmonellas enterotoxins and some other intestinal bacteria, REKITSEN-RD can appear an effective agent of prophylaxis and treatment of alimentary poisonings with various albuminous toxins. For confirmation of this assumption it is expedient to carry out additional experiments on laboratory animals receiving a forage, containing the additives of toxins and REKITSEN-RD.


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