Method for determination of anti-lactoferrin activity in microorganisms

FIELD: medicine, medicinal microbiology.

SUBSTANCE: method involves growing microorganism culture to be studied in solid nutrient medium followed by preparing microbial suspension and its incubation in the presence of lactoferrin. Control sample is prepared in parallel series. Control and experimental samples are incubated, supernatant is removed from bacterial cells and lactoferrin concentration is determined in supernatant of experimental and control sample by immunoenzyme analysis. Then anti-lactoferrin activity is calculated by difference of concentrations of residual lactoferrin in experimental and control samples. This method provides enhancing the sensitivity and precision in carrying out the quantitative evaluation of anti-lactoferrin activity in broad spectrum of microorganisms that is urgent in diagnosis and prognosis of diseases with bacterial etiology. Invention can be used in determination of persistent indices of microorganisms for assay of their etiological significance in pathological processes.

EFFECT: improved assay method.

3 tbl, 3 ex

 

The invention relates to Microbiology and can be used in laboratories, research institutes, and also in the bacteriological laboratories of clinical agencies determine the persistent characteristics of microorganisms to establish their etiological significance in pathological processes.

Currently, the nature of the flow of most infections has changed in the direction of increasing the share of protracted and chronic forms [1]. Prolonged observation of bacteria in the body of the host (hosting), as well as the transition of infectious process in the chronic form provide mechanisms persistence of microorganisms in which inactivation/degradation of natural factors anti-infective resistance: lysozyme, complement, interferon, platelet-derived cationic protein, lactoferrin [2].

It is established that natural glycoprotein lactoferrin is one of the most important components of biochemical protection of human and animal possesses bacteriostatic and bactericidal action, takes part in the processes of local protection of the majority of the barrier epithelia of the respiratory, urinary, digestive tracts, in the regulation of humoral and cellular immunological responses, hematopoiesis, affects the complement system, adjustable lighting angle is t granoulozitopoez [3].

There is a method of determining antilactoferrin activity of microorganisms (Alf), which studied the culture of the microorganisms are grown in a liquid nutrient medium grown cultures treated with chloroform, separating the supernatant and mix it with lactoferrin, in parallel with an experienced cook two control samples, control 1 instead of the supernatant analyzed cultures of microorganisms add liquid nutrient medium, and the control 2 is prepared from a buffered saline solution and a liquid nutrient medium, the test and control samples incubated, after incubation add test-culture of Micrococcus luteus risk No. 211001 and conducting a first measurement of the optical density in the experience and controls immediately upon reaching mode temperature (37° (C) and the second measurement after 18-24 hours of incubation, then count alpha by the change in optical density in the experience and controls [4].

However, this method has some significant drawbacks. The known method is qualitative and does not allow to Express the level antilactoferrin activity in absolute terms, which is important in ecological and epidemiological studies, because the level of quantitative values of secreted factors persistence of microorganisms aimed at inactivation FA is tori natural resistance, strains of microorganisms isolated from different sources (external environment, patients, bakterionositeli, healthy people and. so on)differ significantly [2]. The quantitative determination of alpha values of different microorganisms will establish the etiological significance in pathological processes, to study the role of alpha in the mechanisms of persistence and forming bacteria carrier.

In addition, incubation of supernatants, not living cultures of microorganisms with lactoferrin significantly reduces the incidence of Alf bacteria, because in the first case can only be detected constitutive inhibitors lactoferrin. However, for the expression of many proteins of bacteria requires the cultivation of living microorganisms in a medium containing the substrate or inducer, which cannot be done by incubation of lactoferrin with supernatants mikroorganizmov.

Another significant disadvantage of this method is that the use of living indicator culture .luteus is not possible to determine antilactoferrin activity in strains of microorganisms, producing lysozyme [5], gestonorone proteins [6], hydrogen peroxide, other antagonistically active substance, which is typical for such microorganisms as bifidobacteria, lactobacilli, streptococci, Pseudomonas aeruginosa and others, because these biological the automatic active substances can exert an antimicrobial effect on microcock (bacteriolytic, bactericidal and others). However, supernatant the studied microorganisms can stimulate the growth indicator of culture, which leads to false-positive results [7]. Treatment of cultures of microorganisms chloroform leads to the release of some bacterial pigments, which distorts the results of determining antilactoferrin activity [8].

The objective of the proposed method of determining antilactoferrin activity of microorganisms is to increase the sensitivity of the method and accuracy of quantitative assessment of alpha.

To solve this problem in the present method of determining alpha investigated the culture of microorganisms grown on solid nutrient medium, grown agar culture prepared microbial suspension, mix it with lactoferrin, simultaneously prepare control, in control instead of the microbial suspension type liquid nutrient medium, an experimental sample and a control incubated separating the supernatant from the bacterial cells by centrifugation, determine the concentration of lactoferrin in the experimental and control samples by enzyme immunoassay and then expect alpha formula

Alpha=SC-Co,

where alpha - antilactoferrin activity, ng inactivated lactoferrin/ml supernatant;

SC - concentration of lactoferrin in control, ng/ml;

Co - Konz is ntrace lactoferrin in the experience, ng/ml

New features of the proposed method are:

- studied the culture of microorganisms grown on solid nutrient medium, grown agar culture prepared microbial suspension;

- incubated microbial suspension prepared from agar culture, lactoferrin; in parallel with an experienced breakdown incubated test sample prepared from a liquid nutrient medium and lactoferrin;

- the residual concentration of lactoferrin in the experimental and control samples determined by enzyme immunoassay;

- expect antilactoferrin activity by the formula

Alpha=SC-Co,

where alpha - antilactoferrin activity, ng inactivated lactoferrin/ml supernatant;

SC - concentration of lactoferrin in control, ng/ml;

The concentration of lactoferrin in the experience, ng/ml

A new set of features allows for the implementation of the claimed invention to obtain a new technical result, which is that the proposed method of determining antilactoferrin activity of microorganisms allows for increase sensitivity and improve the accuracy of quantitative estimates alpha microorganisms to determine quantitative values of this characteristic over a wide range of microorganisms. The method allows for the difference in the level number is i.i.d. values alpha microorganisms make the diagnosis and prediction of diseases of bacterial etiology, conduct ecological and epidemiological studies to evaluate the impact of drugs on this topic.

Know the use of reagents for quantitative immunoassay for determination of lactoferrin in serum [9].

The use of reagents for immunoassay determination of lactoferrin in order to detect alpha microorganisms in the sources of patent and scientific literature is not found.

The authors experimentally found that the incubation of lactoferrin with live cultures of microorganisms definition of alpha is more reliable compared to the same time, if the incubation lactoferrin significantly compared with the same time, if the incubation of lactoferrin to spend with their supernatants.

Was conducted the following experiments.

In the experimental series, the first batch cultures of microorganisms Escherichia coli, Staphylococcus epidermidis, Salmonella enteritidis was grown on mesopatamia agar at a temperature of 37°C for 24 h, were preparing their suspension in mesopatamia broth (109CFU/ml). Next, the first batch of suspension in equal volumes were mixed with lactoferrin in a concentration of 100 ng/ml in the Second game of the same crops at the same time was first grown in mesopatamia broth for 24 h at 37 ° °C, then the supernatant was separated from the bacterial cells by centrifugation in accordance with the s 20 min (8000 rpm) and mixed with lactoferrin in a concentration of 100 mg/ml In parallel to the first and second batches of cultures of microorganisms were prepared control sample from the nutrient broth and lactoferrin in the same concentration that was used in the experimental samples. The test and control samples of both parties of the studied cultures of microorganisms were incubated overnight at 37 ° °C and centrifuged for 20 min (8000 rpm), received the cell-free supernatant, in which enzyme-linked immunosorbent assay (ELISA) using reagents for the quantitative determination of lactoferrin (JSC “Vector-best”) by measuring optical density (OD492) photometer Labsystems Multiskan (Finland) and the subsequent recalculation via a calibration curve was determined by the concentration of lactoferrin in the experimental and control samples. According to the difference of concentrations of lactoferrin in the experiment and control were determined by inactivation of lactoferrin and judge antilactoferrin activity of microorganisms.

Determined that incubated with lactoferrin live culture of E. coli iactiveaware 4,18±0,77 ng/ml lactoferrin, and during the incubation with lactoferrin supernatant of E. coli culture lactoferrin not inaktivirovanie. Accordingly a living culture S.enteritidis iactiveaware 7,92±1,06 ng/ml of lactoferrin and its supernatant - 3,06±0.11 ng/ml lactoferrin. Live culture S.epidermidis iactiveaware 2,40±0.09 ng/ml of lactoferrin and its with pernament not inaktiverad lactoferrin.

This confirms that the determination of the ability of microorganisms to inactivate lactoferrin during the incubation of lactoferrin with supernatants microorganisms significantly less compared to the same time, if the incubation of lactoferrin to spend with live cultures of microorganisms.

There are various methods for the determination of lactoferrin in whey of blood of man, of which the most commonly used ELISA [9] and immunodiffusion methods, in particular the method of radial immunodiffusion (RID) by Mancini [10] using, for example, monospecific antisera to the lactoferrin manufactured 000 “Microflora” in mniam them. Hingamisega (Moscow). However, the sensitivity of the method of REED significantly inferior ELISA (respectively 3 μg/ml and 1 ng/ml) [10].

Authors of studies were conducted to compare the ability to inactivate lactoferrin 11 microbial culture collection of the Institute of cellular and intracellular symbiosis, Ural branch of the Russian Academy of Sciences (Orenburg) using ELISA method and REED to determine the residual concentration of lactoferrin and alpha calculation. The research results are summarized in table 1.

As can be seen from table 1, when using ELISA ability to inactivate lactoferrin was detected in 10 of the 11 cultures of microorganisms (which amounted to 90.9 per cent) and a range of quantitative changes inactivated the CSOs lactoferrin was 0-17,3 ng/ml When using REED in all the investigated cultures failed to detect alpha due to low compared to ELISA, the sensitivity of this method.

This confirms that the authors use in the claimed method ELISA to determine the residual concentration of lactoferrin and alpha calculation of microorganisms due to the unique specificity of immunochemical analysis in combination with high sensitivity detection of enzyme labels [11] allows high accuracy to quantify antilactoferrin the activity of microorganisms.

Table 1.

Antilactoferrin the activity of microorganisms in various methods of detecting the number of inactivated lactoferrin
Type of microorganismThe values of alpha, ng/ml
REIDELISA
Staphylococcus epidermidis02,40
Staphylococcus hominis01,97
Staphylococcus xylosus05,23
Shigella flexneri0to 6.58
Providencia rettgeri013,50
Escherichia coli04,18
Klebsiella oxytoca0/td> 5,31
Klebsiella pneumoniae02,22
Candida albicans07,45
Salmonella enteritidis0the 17.3
Salmonella typhimurium00

Authors for incubation of the microbial suspension with lactoferrin was used, the concentration of lactoferrin 100 ng/ml Choosing this concentration was determined by the fact that the most reproducible results in ELISA can be obtained by using the average concentrations of lactoferrin, which is determined with the ELISA reagents [11]. Given that the range of detectable concentrations of lactoferrin with the used reagents ELISA (JSC “Vector-best”, Russia) was 0-120 ng/ml, the average concentrations of lactoferrin are 60-100 ng/ml In later authors experimentally was selected concentration of lactoferrin 100 ng/ml, which does not inhibit the growth of test organism (table 2).

As can be seen from table 2, the concentration of lactoferrin higher than 100 ng/ml had a bacteriostatic action, and lactoferrin in a concentration of 100 ng/ml and lower concentrations had no effect on bacterial growth. This confirms the validity of the choice of a given concentration of lactoferrin to study alpha microorganisms enzyme, analisa is.

Table 2.

The sensitivity of microorganisms to lactoferrin
Type of microorganismThe growth of bacteria at different concentrations of lactoferrin (ng/ml)
025501002001000
Staphylococcus aureus++++--
Staphylococcus epidermidis++++--
Pseudomonas aeruginosa++++--
Escherichia coli++++--
Klebsiella pneumoniae++++--
Salmonella enteritidis++++--

For implementing the method used by human lactoferrin, crystalline, by Fluka (Switzerland), the degree of purity of the drug (according to gel electrophorese) more than 90%, the solubility of 1 mg/is l H 2Oh, catalog No. 61326 [12].

The method is as follows.

1. The investigated microorganism cultures grown on solid nutrient medium at 37°C for 18-24 hours

2. From grown agar culture are preparing a suspension of microorganisms (10 units optical turbidity standard of gisk named after. Lautareasca) in a liquid nutrient medium (for each type of microorganisms used corresponding to the type of culture media).

3. Prepare a solution of lactoferrin in a concentration of 100 ng/ml in buffered saline solution (Sigma P4417. Composition: 0.01 M phosphate buffer, 0.0027 M KCl, 0.137 M NaCl, pH to 7.4 at 25°).

4. A suspension of the investigated microbial culture volume of 50 µl contribute to the wells of a sterile polystyrene tablet and mixed with 50 μl of the prepared solution of lactoferrin.

5. In parallel with the experienced prepare a control sample in the wells of polystyrene tablet make 50 μl of a solution of lactoferrin and 50 ál of sterile liquid nutrient medium.

6. Samples incubated at 37°C for 18-24 hours

7. After incubation the contents of the wells are transferred into plastic tubes, centrifuged at 8000 rpm for 20 min, down to 50 μl of the supernatant in the test and control samples.

8. Working solution of buffer experimental and control samples diluted in 5 times (up to a volume of 250 μl).

9. Spend the definition of a mod who offered the concentration of lactoferrin in the supernatant experimental and control samples enzyme-linked immunosorbent assay using reagents JSC “VECTOR-BEST” (Russia) in accordance with instructions for use reagents. Measurement of the optical density of the test and control samples is carried out on the Multiskan photometer Labsystems, Finland) at a wavelength of 492 nm, recalculate the concentration of lactoferrin is carried out by constructing the calibration graphs. Found on the schedule of values of the concentration of lactoferrin is multiplied by a factor of dilution, i.e. at 5, and get results in ng/ml.

10. Expect antilactoferrin activity by the formula

Alpha=SC-Co,

where alpha - antilactoferrin activity, ng inactivated lactoferrin/ml supernatant;

SC - concentration of lactoferrin in control, ng/ml;

The concentration of lactoferrin in the experience, ng/ml

Antilactoferrin activity expressed in ng inactivated lactoferrin per ml of supernatant (ng/ml).

Example 1. The strain of Providencia rettgeri, isolated from a patient with a paraproctitis, we studied the ability to inactivate lactoferrin. To do this, from the daily agar culture was prepared suspension 10 units of optical turbidity standard of gisk named after. Laurasia in a liquid nutrient medium, 50 μl of suspension culture were made in well sterile polystyrene tablet, next to the hole was made to 50 μl of a solution of lactoferrin in a concentration of 100 ng/ml In the control sample instead of a culture of the microorganism in the hole made 50 μl of liquid culture media. After 18 h of incubation, the ri 37° With the contents of each well was transferred into plastic tubes, the supernatant was separated from the bacterial cells by centrifugation for 20 min (8000 rpm). Next, from each sample were selected by 50 μl of the supernatant was determined by its content of lactoferrin enzyme linked immunosorbent assay according to the instructions attached to the kit of reagents JSC “VECTOR-BEST” (Russia). The registration results were photometrically on the Multiskan photometer Labsystems, Finland) at a wavelength of 492 nm. The values of optical density of the experimental samples -0,144 and control -0,350. The concentration of lactoferrin in the experimental and in the control samples was calculated by a calibration curve based on the values of optical density of the samples. To build it was used in the form attached to the set: the x - axis the concentration of LF in the calibration samples (120 ng/ml, 60 ng/ml, 30 ng/ml, 15 ng/ml to 7.5 ng/ml 0 ng/ml); y-axis is the corresponding value of optical density (OD492). The values of concentration of lactoferrin in the experimental sample -15,12 ng/ml and in the control sample - 36.75 per ng/ml Antilactoferrin activity (alpha) was calculated by the formula

Alpha=36.75 per-15,12=21,63 ng/ml,

where 21,63 - antilactoferrin activity, ng inactivated lactoferrin/ml supernatant;

of 36.75 - is the concentration of lactoferrin in ng/ml, in the context of the role;

15,12 - is the concentration of lactoferrin in ng/ml in the experience. Thus, antilactoferrin activity of the strain of Providencia rettgeri is 21,63 ng/ml.

Example 2. 63 strains of microorganisms from the collection of the Institute of cellular and intracellular symbiosis, Ural branch of the Russian Academy of Sciences (Orenburg) was determined antilactoferrin activity using the proposed method and the prototype method.

When using the prototype method the investigated microorganism cultures were grown in liquid nutrient medium grown cultures were treated with chloroform, separating the supernatant and mixed it with lactoferrin, in parallel with experienced prepared two control samples, control 1 instead of the supernatant analyzed cultures of microorganisms were added to a liquid nutrient medium, and control 2 was prepared from buffered saline and liquid nutrient medium, the test and control samples were incubated, after incubation were added to the test culture of Micrococcus luteus risk No. 211001 and carried out the first measurement of the optical density in the experience and the controls directly on the achievement of the regime temperature (37°) And the second measurement after 24 h incubation, then counted antilactoferrin activity by the change in optical density in the experience and the controls. When using the proposed method the study Kul is URS microorganisms were grown on solid nutrient medium, from grown agar cultures were prepared microbial suspension, mixed it with lactoferrin prepared in parallel control, in control instead of the microbial suspension was added to a liquid nutrient medium, an experimental sample and a control were incubated, separating the supernatant from the bacterial cells by centrifugation, was determined by the concentration of lactoferrin in the experimental and control samples by enzyme immunoassay and then counted antilactoferrin activity according to the difference of the residual concentration of lactoferrin in the experience and control. Comparative analysis of results of determination of alpha microorganisms these methods are presented in table 3.

As can be seen from table 3, the use of the proposed method allowed to detect alpha at 100% of the strains studied microorganisms, and in the method-prototype - only 68.5% of the studied strains.

In addition, in the inventive method, the level of detected alpha microorganisms was higher than the method of the prototype: for strains of S. aureus, not producing lysozyme - 35.6% in the claimed method against 24.3% in the method-prototype; for E. coli, respectively, 59,65% compared to 44.7%; for K. pneumoniae, respectively 47,77% against 29.3%; for S. typhimurium, respectively 75,0% compared to 48.5%.

Table 3.

Comparative analysis of the sensitivity of the determination of the ALPHA micro the of organisms using the proposed method and the prototype method
Type of microorganismThe number of strainsAverage values antilactoferrin activity (M±m)
prototype methodthe inventive method
%%ng/ml
Strains of Staphylococcus aureus, not producing lysozyme524,3±3,4835,6±5,213,7±3,65
Listingproducer Staphylococcus aureus10it is impossible to determine in 100% of cases23,61±2,479,1±0,88
Pseudomonas aeruginosa10it is impossible to determine in 100% of cases26,28±3,599,01±0,98
Escherichia coli1044,7±to 4.4159,65±ceiling of 5.60*22,9±2,90
Klebsiella pneumoniae829,3±2,9647,77±6,20*18,4±1,25
Salmonella enteritidis1239,1±3,32with 58.33±6,41*5,98±0,86
Salmonella typhimurium848,5±7,6275,0±8,75*of 6.29±0,91
Note: * - significance of difference between the proposed method and the method of the prototype at p<0,05.

The use of the proposed method allowed us to identify and range of quantitative values lactoferrin, inactivated studied microorganisms, and formed 5,98-to 22.9 ng/ml (table 3), it is impossible to determine the method of the prototype.

Example 3.

12 strains of S.enteritidis isolated from patients with salmonellosis and 8 strains of S.enteritidis isolated from reconvalescent Salmonella bakterionositelej was determined Alfa by the claimed method. It was found that the average number of inactivating lactoferrin in strains isolated from reconvalescent Salmonella bakterionositelej amounted 9,52±0,98 ng/ml, and in strains isolated from patients with salmonellosis -5,98±0.61 ng/ml, This indicates that Salmonella bakterionositelej are strains with a higher alpha than from patients with salmonellosis.

Thus, the inventive method allows to differentiate the studied cultures of microorganisms on the quantitative values of the alpha level, which is important for establishing the etiological significance of this property microorganisms in pathological processes.

Thus, the use of the proposed method of determining antilactoferrin activity of microorganisms allows for increasing the sensitivity of the method and improve the accuracy of quantitative assessment to determine it from a broader range of microorganisms and with greater accuracy, which is important in diagnosing the prediction of microbial diseases etiology, the execution of the eco-epidemiological studies.

Sources of information

1. Bukharin O.V., Usvyatsov BJ Hosting (medico-ecological aspect). - Ekaterinburg: Ural branch of the Russian Academy of Sciences, 1996. - S.

2. Bukharin O.V. Usvyatsov BJ Kartashova O.L. Biology of pathogenic cocci. - M.: Medicine, 2002. - Ñ.38-48.

3. Brock J.H. The discrimination oflactoferrin //Biochem. Cell Biol. - 2002. - V.80, no. 1. - P.1-6.

4. RF patent №2156807, IPC 7 12 Q 1/02, 1/04, publ (prototype).

5. Bukharin O.V. Vasilyev N.V., Usvyatsov BJ Lysozyme microorganisms. - Tomsk: Publishing house of Tomsk University, 1984. - P.17.

6. Application No. 2001133150/13 from 06.12.01, IPC 7 12 Q 1/02, a positive decision from 30.09.02.

7. RF patent No. 2175673, IPC 7 C 12 Q 1/04, 1/14, publ. 10.11.01.

8. Smirnov V.V., Kiprianova E.A. Bacteria of the genus Pseudomonas. -Kiev: Naukova Dumka, 1990. - P.106-108.

9. Nemtseva ER and other Immunoassay method for the detection of human lactoferrin and its use for the diagnosis of purulent-septic complications //Questions of medical chemistry. 1995. V.41. No. 3. - P.58-61.

10. Laboratory methods in the clinic. The Handbook, edited by V.V. Menshikov. - M.: Medicine, 1987. - 365 S.

11. Egorov A. M., Osipov, A.P., Dzantiev B.B., Gavrilova E.M. Theory and practice of immunoassay. - M.: Higher. HQ., 1991. - P.3.

12. The company's catalog "Fluka"(chemical company): Laboratory chemicals. - 2001/2002. - S.

The method of determining antilactoferrin activity of microorganisms, distinguish the different topics that studied the culture of microorganisms grown on solid nutrient medium, of the grown culture is prepared by experienced test microbial suspension, simultaneously prepare a control sample representing a liquid nutrient medium, then each sample is mixed with solution of lactoferrin and incubated, then separate the supernatant from the microbial cells by centrifugation, and then determine the residual concentration of lactoferrin in the experimental and control samples by enzyme immunoassay and determine antilactoferrin activity of the studied microorganisms by the formula

Alpha=SC-Co,

where alpha - antilactoferrin activity, ng inactivated lactoferrin/ml supernatant;

SC - concentration of lactoferrin in control, ng/ml;

The concentration of lactoferrin in the experience, ng/ml



 

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