DE2850503C2 - Methods for the detection and determination of antibiotics - Google Patents

Description

2. The method according to claim 1, characterized in that steps (A) and (B) are carried out simultaneously will.

3. The method according to claim 1 or 2, characterized in that in step (A) or microorganisms Parts thereof immobilized on a water-insoluble support are used and the separation (C) is done by removing the carrier from the liquid and washing.

4. The method according to any one of claims 1 to 3, characterized in that the amount of the solid Phase bound labeled substance is determined.

5. The method according to any one of claims 1 to 4, characterized in that C-labeled compounds, ^I25 J-labeled compounds or compounds labeled irit a peroxidase are used ^{as the labeled substance M.}

6. The method according to any one of claims 1 to 5, characterized in that in step (A) a strain of one Microorganism with a temperature of optimal growth above 50 ° C is used.

7. The method according to any one of claims 1 to 6, characterized in that in step (A) a microorganism of the genus Bacillus stearothermophilus is used.

8. The method according to any one of claims 1 to 7, characterized in that the labeled substance in Step (B) the antibiotic to be detected or determined is used in marked form.

The invention relates to a method for the rapid, sensitive detection and determination of Antibiotics in liquids such as milk, body fluids, meat extracts and fermentation broths, for example. The possibility of detecting small concentrations of antibiotics in liquids is numerous Areas of application of great importance, for example in the food industry, where the use from antibiotics to the treatment of animals that produce food to a need for a fast, precise test procedure, which is also used, for example, in food wholesaling and the like. applicable is

Since penicillins are used for the treatment of mastitis in dairy cattle, detection methods for rapid and accurate detection and elimination of penicillin-containing milk is of particular importance. That increasing medical interest in the problem of human ingestion of small amounts of antibiotics the diet accordingly requires in the range of 0.010 to 0.050 with the presence of penicillins in milk IU / ml or above simple screening methods for the detection of such small amounts of penicillin and other antibiotics.

Numerous methods for the detection of antibiotics are already known. Most of them are based on microbiological processes, the presence or absence of penicillin or other antibiotics by observing the inhibition of the growth of antibiotic sensitive microorganisms in the presence the test sample is determined. Previously, such procedures required incubation times of 4 hours or above; by using strains of microorganisms that are hypersensitive to antibiotics the time required to carry out the tests could be reduced to 2 to 2.5 hours.

Other methods for the detection of antibiotics make as a test basis of various other specific ones Properties of the antibiotic to be determined or the class of antibiotics to be determined Use. For example, in the literature (J. F. Rusling et al., Immobilized Enzyme-Based Flowing-Stream Analyzer for Measurement of Penicillin in Fermentation Broths, Analytical Chemistry, Vol.48 (1976) 1211) a test indicated that is based on the enzymal hydrolysis of penicillin with an immobilized // - lactamase derivative is based.

By Palmer (I.M.A. Palmer et al., Simple Ultrasensitive Test for Detecting Penicillin in Milk,). Dairy Science.

hS 50 (1967) 1390) a method for the detection of penicillin was also developed, which is based on the growth of Hacillus subtilis spores on culture medium-spore-dye paper discs. those in a small sample grow from air exposed milk. With evaporation the water content of the milk increases at the same time the concentration of any penicillin present and then leads to a color change of the dye,

which gives an indication of concentration

In US-PS 35 86 483 a method for the detection of tetracydine antibiotics in liquids is described, in which the liquid is absorbed on an absorbent strip, which is a complex-forming metal! which forms a fluorescent metal complex with the antibiotic, the fluorescence of the Metal complex is observed under UV light.

The above and other previously available methods for the detection of antibiotics differ very different from each other in terms of their sensitivity and speed. It is assumed, that none of the previously known tests for the detection of small concentrations of 0.01 IU penicillin / ml (6 ng / ml) is suitable in measurement times of less than 1 hour

In Em. J. Biochem. 72 (1977) 341-352, studies to determine the ability of various unlabeled / -lactam antibiotics to bind to certain cell membrane proteins on the basis of the so-called competitive assay are described. In these investigations, however, the concentrations of the labeled and unlabelled antibiotic were known. Furthermore, membranes from special bacteria (E. coif KN 126) were used can use very different classes of antibiotics, was accordingly not obvious to the person skilled in the art.
The principle of the competitive assay was also known from DE-AS 22 06 103

Such a test method would be a quick and at the same time reliable determination in an economical manner for example, enable milk samples from relatively small batches of antibiotics in over Contain the concentrations permitted in accordance with the relevant regulations.

The invention is accordingly based on the object of providing a method to the detriment of a large number different antibiotic substances in different liquid media to indicate that so formed It can be that it is very fast with usable sensitivity or, on the other hand, a little slower at The procedure for the detection and determination of antibiotics works with an extraordinarily high sensitivity intended to detect very small amounts of antibiotics of around 1 ng / ml in liquid samples of Materials such as milk, body fluids, meat residues, fermentation broths and the like. be suitable and

^ enable operation without a centrifuge or scintillation counter; it is also intended to be in a convenient manner

ψ can also be carried out outside of laboratories.

|| The object is achieved according to the claims. Advantageous developments are the subject of the subclaims

woe

r _; . The method according to the invention for the detection and determination of antibiotics in liquid samples is

% characterized by the following steps:

(A) Incubation of the sample with a microorganism with a recipe capable of binding the antibiotic

; · Gate posts

^; : under conditions under which the molecules of an antibiotic present in the sample bind

can enter into with the receptor sites, the use of E. coli for detection or for Determination of>? - lactam antibiotics is excluded

(B) Incubation of the mixture from (A) with a labeled one capable of binding to the receptor sites Substance that is a labeled antibiotic, a labeled antibiotic derivative, or a labeled antibiotic precursor is,

(C) separation of the solid phase from the liquid after incubation,

; (D) Determination of the amount of the labeled substance or labeled substance bound to the solid phase

: Substance in the liquid and

'■■. (E) Comparison of the determination with a standard.

Certain embodiments of the method according to the invention are ideally suited for the detection and elimination of milk containing antibiotics of the penicillin type.
ί The invention is explained in more detail below with reference to this, but only exemplary application

explained. From the following it emerges that the concept of the invention for the investigation of body fluids (cn, Meat extracts, fermentation broths, etc. can be relied on to numerous antibiotic substances can.

The most important advantage of the procedure according to the invention is that it makes the detection very small Anlibiotic concentrations of, for example, 0.001 IU penicillin / ml are made possible in a very rapid manner, for example in less than about 10 minutes.

If there are molecules of an antibiotic in the sample, the labeled as well as the unlabeled

len molecules bound to a limited number of receptor sites on the cells, the amount of

'. thereby immobilized antibiotic reversed the concentration of the antibiotic in the test sample

is proportional. The amount of the labeled remaining in the liquid phase depends in an analogous manner Antibiotic from the original antibiotic concentration.

The surprisingly high sensitivity and speed of the method according to the invention on the one hand the high binding constant of the interaction between the antibiotic molecules and the receptor sites on the cell walls or other subcellular structures of organisms sensitive to antibiotics and on the other hand the specificity of antibiotic molecules for such interacting receptor sites attributed to such organisms. The binding constant for penicillins at receptor sites Gram-positive cells, for example, is orders of magnitude higher than the binding constant for the immunochemical Antigen-antibody reaction on which analog detection methods (so-called immunoassays) are based.

In a particularly important variant of the method according to the invention, the rapid test

for use in milk monitoring carried out a single incubation step in which the against Antibiotic-sensitive microorganism, the sample to be examined and the labeled antibiotic or the labeled antibiotic precursor can be used, followed by the separation step by centrifugation will. The speed as well as the sensitivity of this method is determined by the use of Cell strains increased against a detectable class of antibiotics or a specific one are hypersensitive to the antibiotic to be detected.

According to a further development of this concept, the antibiotic-sensitive microorganisms used are preferably strains which have a temperature of optimal growth above about 50 ^° C. In this case, the incubation can be carried out at relatively high temperatures, which in turn, for kinetic reasons, accelerates the reaction rate of the reaction between the antibiotic and the receptor site.

Preferred microorganisms for carrying out tests for /? - lactam antibiotics are Bacillus stearothermophilus (ATTC 10 149 or 15 952). According to the invention, however, cells of various other against Antibiotics of hypersensitive strains are used; in cases where a lower sensitive wedge is sufficient, only normally sensitive strains can be used.

Examples of cell strains suitable according to the invention are certain mutants of E. coli, Ps. Acruginosa, B. subtilis and S. aureus, but the invention is not limited to the use of the microorganisms mentioned limited

When, in appropriate tests, the speed achieved is less of a concern than the speed achieved Sensitivity, after a corresponding implementation of the method according to the invention, the Sample and cell culture are incubated for a period of time, after which the labeled antibiotic is later added is added. When using sensitive microorganisms with an optimally high growth rate it was found that the sensitivity and the incubation time do not increase linearly with time Should not exceed 15 minutes.

According to a preferred embodiment of the method according to the invention, for the detection of ^ -Lactam antibiotics such as benzylpenicillin, cephalosporin, ampicillin, oxacillin, methicillin, cloxacillin, Cephaloridin and cephalotin as labeled antibiotic benzylpenicillin or the antibiotic precursor 6-aminopenicillanic acid is used. Other antibiotics that can be detected by the method according to the invention are for example erythromycin, lincomycin, actinomycin, vancomycin, bacitracin and aminoglycosides such as such as streptomycin, gentamycin, kanamycin and neomycin. However, the invention is not based on evidence or the determination of the antibiotics mentioned is limited.

The method according to the invention is well suited for the detection of such penicillins or penicillin-like ones Antibiotics that inhibit cell proliferation by sticking to the appropriate sites attach to the cell membrane, inhibiting cell wall synthesis.

Antibiotics with other sites of interaction such as rifamycin and tetracyclines can, however can also be detected according to the invention.

The antibiotic can be labeled with a radioactive atom, an enzyme, an enzyme inhibitor or about a coenzyme.

According to the invention, labeled substances that ^{contain 14 carbon atoms in the structure of the antibiotic molecule are preferred, as well as those that contain a 125} I atom linked by reaction with, for example, tyrosine or suitable derivatives thereof, as well as antibiotics that react with an enzyme such as a peroxidase are marked.

According to another important development of the invention, cell parts containing receptor sites are used used, which on an insoluble carrier such as cotton gauze and the like. are immobilized. Through this the separation step is facilitated, since the carrier after incubation in a simple manner from the Liquid can be removed and washed.

The use of a device for the detection of radioactive decay products can also be done by marking be avoided with an enzyme. In this case, the presence of the enzyme label is determined by incubating the support with a solution of the enzyme substrate which is under the catalytic influence of the enzyme undergoes a detectable chemical change. A preferred system comprises peroxidase as a marking enzyme and a substrate solution with H2O2 and 4-aminoantipyrine, which in the presence of Peroxidase shows a color change

Corresponding test sets for carrying out the method according to the invention for the detection of antibiotics in liquid samples contain a certain amount of concentration-stabilized, antibiotic-sensitive and preferably cells hypersensitive to antibiotics, a certain amount of a labeled antibiotic or antibiotic precursors that seek a high constant of binding to the receptor sites of cells is selected, as well as a standard with which the results of the reagents performed Tests can be compared.

According to a preferred embodiment, such test sets comprise freeze-dried Bacillus stearothermophilus, as a labeled antibiotic radioactive, for example ^{benzylpenicillin labeled with 14} C, and as ao standard either a standard curve or at least one sample that contains a known concentration of the antibiotic to be detected or of antibiotics of the antibiotic class to be detected.

According to another embodiment, the test set has a water-insoluble carrier on which the opposite

cells that are hypersensitive to penicillin are immobilized, and also enzyme-labeled 6-aminopenicillanic acid a solution of the enzyme substrate which, under the catalytic influence of the labeling enzyme, causes a color change The marking enzyme can be a peroxidase; the substrate solution contains H2O2, phenol and 4-aminoantipyrine.

The invention is explained below with reference to the drawing; it shows

Fig. 1 is a block diagram to explain the inventive method for detection or for

Determination of antibiotics and

F i g. 2 a calibration curve for benzyl penicillin.

The method of the invention comprises four main steps: incubation; at the ilas, if necessary, in tier Sample available antibiotic as well as a marked antibiotic at antibiotic unisplintlliche /.eilen ge are bound, the step of separation, in which the cells with an antibiotic bound to them from remaining system are separated, a determination step in which a note aiif the amount of either labeled antibiotic associated with the cells or present in the separated fluid is obtained, and a comparison step in which the obtained determination result with a standard is compared.

As the concentration of the antibiotic in the test sample increases, fewer appear on the cells bound labeled antibiotic molecules and correspondingly more labeled antibiotic molecules in the separated liquid.

By preparing a series of samples with known antibiotic concentration and treatment according to the method according to the invention can produce a calibration curve of the antibiotic concentration as a function of the count rate (counts per minute) in the case of radioactive labeling or other equivalent Variables are set up. Investigations of unknown materials lead to a quantitative value of the Anti-antibiotic concentration by comparison with such a calibration curve.

An indication of the presence of antibiotics in a sample can also be obtained by comparing the result of the test sample with the labeled antibiotic with the results of carried out in parallel Experiments can be obtained with samples that contain known amounts of antibiotics. If only that The presence of antibiotics in concentrations above a certain maximum value should be checked, the required comparison step can be carried out indirectly. This is done by determining the Result with the test sample and comparison with a previously determined result that corresponds to the critical Antibiotic concentration was correlated. This simple comparison is made by standardizing the reagents and procedure enables. When performing the test, there is inherently a comparison made performed when the test result is interpreted.

It is believed that the extremely favorable sensitivity and high sensitivity achieved in the present invention Speed of the test for the typically high binding constants of the conversions between antibiotics and cells and the specificity of antibiotics with respect to those that can interact with them Receptor sites are based. In the procedure according to the invention, extremely small amounts are also used of antibiotics in a test sample very quickly at the receptor sites on the cell membranes or at others Places on the microorganism with which the incubation takes place.

In this regard, it is already well known that many antibiotics work by acting on certain Cell sites are bound, whereby the normal proliferation metabolism is prevented. So is for example Vancomycin, Bacitracin, Penicillins and Cephalosporins are known to promote cell reproduction by being bound to receptor sites on cell membranes, thereby inhibiting cell wall synthesis is blocked (see, for example, J. R. Edwards et al., Correlation between Growth Inhibition and the Binding of Various Penicillins and Cephalosporins to Staphylococcus aureus, Journal of Bacteriology, August 1969, pp. 459-462, and J. L Strominger, The Actions of Penicillin and Other Antibiotics on Bacterial Cell Wall Synthesis, Hopkins Med. J. 133 (1973) 63).

For other antibiotics, the corresponding points of interaction are somewhat less accessible than the cell membrane; however, antibiotics work by binding to specific cell sites such as ribosomes or nucleic acids. With the procedure according to the invention it is also possible to correspondingly y? -lactam antibiotics and other antibiotics of the type mentioned above, including erythromycin, lincomycin, actinomycin and tetracyclines as well as aminoglycosides such as streptomycin, neomycin and the like, and determine quantitatively. In the case of the latter substances, there are various points of interaction at the Cells, for example on the ribosomes and the like.

In the incubation according to the invention of the labeled antibiotics together with the antibiotics to be examined In the probe, labeled and unlabeled molecules compete for the accessible receptor sites. If the labeled molecules after incubating the sample with the cells for a certain time are added, the labeled antibiotic molecules are correspondingly attached to the remaining receptor parts bound. Since by detection and determination of the presence of labeled, bound on the cells Molecules in both cases a measured value for the amount of bound labeled antibiotic molecules is obtained, this indirectly results in a measured value for bound unlabeled molecules, since the amount of labeled antibiotic on the cells is a function of the amount of antibiotic in the sample

In other words, the procedure according to the invention is based on the fact that labeled antibiotic molecules and non-labeled molecules of the antibiotic present in the test sample react with a limited number of receptor sites on the cells either simultaneously or one after the other. A successful test can therefore already be carried out if the antibiotic to be detected and the labeled antibiotic are the same. For incubation in the procedure according to the invention, however, other suitably labeled compounds from the class of antibiotics or related molecules such as antibiptic precursors or derivatives that react with the same group of receptor sites as the antibiotic to be determined can also be used in the same way. For example, certain penicillins or cephalosporins can be used after suitable labeling to determine the same substances, other substances or mixtures of penicillins or cephalosporins. Antibiotics preferred for labeling are, however, those compounds of a given compound class which have a high binding constant, for example benzylpenicillin (penicillin G) ₁ ampicillin or cephalosporidin from the class of the ^ -lactam antibiotics.

An example of an antibiotic precursor from this class of substances is 6-aminopenicillanic acid; a An example of a corresponding derivative is the reaction product of 6-aminopenicillanic acid with tyrosine.

As stated above, be under a labeled antibiotic or under a labeled Substance-labeled antibiotics, antibiotic precursors and their derivatives, as well as other related ones

s understood substances that have an affinity for interacting sites of their corresponding Have stem antibiotics.

With regard to the type of marking, various types of marking are currently available. The labeled antibiotic can accordingly contain a ¹⁴ C-AtOm, a ¹²⁵ J-AtOm or other radioactive atoms which z. B. can be detected with a counter, furthermore enzymes, enzyme inhibitors, for example methotrexate, or coenzymes, for example NAD, which are detectable in that the labeled antibiotic is reacted with a substrate solution which results in a detectable chemical reaction under the catalytic influence of the label, or in which a reaction is inhibited. Excellent results have been obtained using radiolabelled antibiotics such as ¹⁴ C-labeled ben? Vlpenicillin (Penicillin G), which is commercially available.

The person skilled in the art is also familiar with the fact that to generate a linkage point for a radioactive iodine atom in most cases a derivative of the antibiotic moieküis has to be synthesized. Enzymes as reducers were also used with success according to the invention, and can also be used within the scope of the invention Coenzymes such as dehydrogenase coenzymes are used. The presence of the labeling enzyme is determined in a manner known per se using a substrate solution for the enzyme, which in the Reaction with the enzyme, for example, results in a color change. The quantitative determination of the coenzyme takes place in that the separated cells or the remaining liquid is reacted with a solution which contains a substrate that, when exposed to an enzyme system, is its crucial component The other enzymes in the system are contained in the solution.

In the method according to the invention, in general, all cells which are to be detected against them can be used Are antibiotic sensitive. For example, any microorganism that is affected by penicillin is inhibited, can be used for the detection of antibiotics of the penicillin type. To increase the Sensitivity, however, it is very preferred to use microorganisms that are against the to be detected Are over-sensitive to antibiotics. Recently there are numerous strains of microorganisms become available who are hypersensitive to either certain antibiotics or to classes of antibiotics are. Examples of microorganisms sensitive to /? - lactam type antibiotics are B. subtilis, B. megatherium, S. aureus and Ps. aeruginosa.

The best results in terms of speed and sensitivity have been obtained using hypersensitive strains of microorganisms achieved, which have a temperature of optimal growth, the is generally above about 50 ° C. A preferred microorganism in this regard is Bacillus stearothermophilus ATCC 10 149. B. stearothermophilus ATCC 15 952 can also be used. Since the Incubations with this type of microorganism can be carried out at high temperature, can this increases the speed at which the antibiotic is bound.

Penicillin G is known to bind to D-alanine carboxypeptidase and inhibit this enzyme, which is normally bound to cell membranes, e.g. B. on the membranes of B. stearothermophilus.

The method according to the invention can also be carried out using cell parts with receptor sites immobilized on a suitable support, for example cotton gauze, a Collagen matrix, polystyrene beads or flat or differently shaped polystyrene, polyacrylamide gel or a small amount of a dimensionally stable agar culture medium attached to a stick for dipping is provided. This enables the cells to be separated from the liquid components of the reaction mixture considerably simplified and accelerated.

If whole cells are used, the separation is preferably carried out by centrifugation. According to the invention, however, other methods for separating the cells from the remaining reaction mixture, for example ultrafiltration, can also be used.
It can be seen from the above that the method according to the invention can be used for the detection or determination of a large number of antibiotics or mixtures of antibiotics in a large number of different liquid media. Furthermore, a large number of labeled antibiotics and cells or cell subunits can be used; the method according to the invention can also be carried out as a qualitative as well as a quantitative test.

The invention is explained in more detail below with the aid of specific exemplary embodiments

The first example relates to the verification of the suitability of the method according to the invention for Rapid examination of milk samples for the presence of small amounts of 0.001 IU / ml penicillin.

example 1
Bacillus stearothermophilus was cultivated under the following conditions:

Medium: 248 kg Solution A - Difco yeast extract 248 kg Difco-Bactotrypton 144 kg Disodium phosphate 515 g Monopotassium phosphate 515 g Ammonium sulfate 5151 distilled water

28 50 503 Solution B - anhydrous magnesium sulfate 103 g distilled water 1,291 Solution C - Calcium chloride dihydrate UV> g Manganese chloride 0.52 g distilled water 1.291 Solution D - glucose 2.58 kg distilled water 12.91

Conditions: 60 ⁰ C, aeration, 10% inoculum.

Cultivation time: 2.5 hours.
/ ■; Procedure

Tl The solution A is sterilized in the fermenter. The solution B is sterilized independently and the solution

^ A was added in an amount of 2.5 ml per liter of solution A. The final concentration of magnesium sulfate im

Medium is 0.2 g / l. Solution C is sterilized separately and then solution A in an amount ■ of 2.5 ml per liter of solution A was added.

The final concentration in the medium is:

\ Calcium chloride dihydrate 0.025 g / l

; Manganese chloride 0.001 g / l.

':' ;! Solution D is sterilized separately and added to solution A in an amount of 25 ml per liter of solution A.

The final concentration of glucose in the medium is 5 g / l.

The temperature of solution A in the fermenter should be between 60 and 65 ° C. Solutions B, C and D

are added to the fermenter with vigorous stirring. To avoid the formation of a precipitate

; solution C must be added slowly and with vigorous stirring. Owns the full medium

without adjustment a pH value of 6.9. An antifoam agent is also added to the medium in a concentration of 0.1% added Very vigorous aeration is necessary for the cultivation of the microorganisms.

After the fermentation has started, a container of 61% of the complete medium is inoculated with the contents of two [ II Erlenmeyer flasks, each containing 150 ml of an overnight culture. When an optical density of 0.150 (600 nm) is reached, the entire contents of the container are used to inoculate the 60-liter fermenter. If the solids content is 0.03%, the contents of the 60-liter fermenter are used for inoculation

a 530-liter fermenter was used.

The cells are twice with five times the volume of 0.2 M NHUCl, pH 7.0, and then twice Washed with ten times the volume of a buffer solution of pH 7.8, the 0.01 M magnesium acetate 0.01 M Contains mercaptoethanol and 0.02 M Tris-HCl buffer.

The cells are then centrifuged off and, after the supernatant has been decanted off, in a mixed solution of A, B and C resuspended as above and then divided into bottles. The content Each bottle can be freeze-dried in a known manner and stored at about 4 ° C for a long time will. Freeze-drying the cultured medium (without glucose solution) maintains the maximum antibiotic binding effect of cells received. Such freeze-dried cells represent an ideal concentration tion-stabilized cell source for use in the method according to the invention.

¹⁴ C-labeled benzylpenicillin (50 \ iC \ / mg) was used as the labeled antibiotic. This reagent ί can also be freeze-dried for reasons of stability.

'To obtain quantitative results and for control purposes, two or more liquid samples can be used

are examined in parallel, one of which is known to be antibiotic-free and the other of which is known to be a known amount of ^ 'contains antibiotic to be detected, for example 0.01 IU / ml penicillin. These samples can also be made from

& Safety reasons are freeze-dried. The above reagents can be mixed with distilled water

or a slightly acidic phosphate buffer. In the case of penicillins, the binding takes place on most favorable between pH 6.0 and 7.0.

Set the stabilized cells, the labeled antibiotic and the control material (or the calibration curve) represents a corresponding test set which is suitable for carrying out the method according to the invention

The reagents given above were used to set up a calibration curve and to examine unknown substances for the presence of penicillin. A calibration curve was obtained by mixing 12 milk samples of 1.0 ml containing known concentrations of benzylpenicillin (penicillin G) with (1) a 100 ml sample of a suspension of B. stearothermophilus (made from freeze-dried material by adding 2 £ ml of distilled water reconstituted to a bottle obtained as above) and (2) '^ -labeled benzylpenicillin (Penicillin G) were incubated in sufficient quantities to give a count rate of about 10,000 counts / min. The incubation was carried out for 3 min at 90 ° C. in a dry bath incubator. The cells were then centrifuged for 2 min at 3000 g. The supernatant was discarded and centrifuge tubes were rinsed with phosphate buffer without stirring the cells. using scintillation fluid as washing fluid.

If ¹²⁵ J is used as the marking substance, this last process step can be omitted, since the marked molecules can be detected directly.
The results obtained are shown in Table I:

Table I.

Calibration curve for the determination of benzylpenicillin

1 • \ 331 84 2 ·) 367 120 3 0 15 457 15 210 4th 0 15 582 15 335 5 1.0 13165 12 918 6th 1.0 13 368 13121 7th 5.0 9 859 9 612 8th 5.0 10639 10392 9 10 7 615 7368 10 10 8 277 8 030 11th 50 4175 3,920 12th 50 4,461 4 214

5 sample benzylpenicillin counts / 10 min background corrected mean - C / C ,, **)

No. (ng / ml) counts / 10 min counts / 10 min

102

Z. 'J JO / ΙΛ) J

10 3 0 15 457 15 210 \ 1 ₅₂ 725

13 0193

15 8 5.0 10 639 luJitt J ^

α in 7 Αίς 7 · Μ «ι

7 699

\ 4 071

20th

*) Addition of 20,000 IU benzylpenicillin (Peniciiiin G) to the sample. **) Count rate of the sample / count rate of the blank sample (without penicillin G in the sample).

As can be seen from the above results, there is an extraordinary and significant decrease in the counting rate 25 when the amount of benzylpenicillin in the sample is increased from zero (3.4) to 10 n ^ / ml (9.10).

The data in Table I are shown in FIG. 2, the ratio of the count rate of the sample to the Counting rate of the blank sample is plotted in ng / ml as a function of the penicillin concentration Using this table or the associated curve as a standard for comparison purposes

Tests carried out on 20 milk samples from a milk processing plant. It was

30 Proceed exactly as above to generate the calibration curve, with the addition of the one obtained with the test samples Counting rate compared with the calibration curve and the dimension ng / ml of the concentration of penicillin in IU / ml

(0.01 IU w 6 ng) were converted.

The results of these tests are shown in Table II below:

Table II

Comparison of the sensitivity of the method according to the invention with a microbiological method

₄₀ sample no. Known penicillin concentration test results according to the invention

(IU / ml) microbiological procedures obtained

(IU / ml) test results c

(IU / ml)

45 1 0.005 - 0.007

0.009 0.008 0.003 0.002

50 6 0.007 - 0.006

- 0.001 0.1 0.005

- 0.004 0.06 0.07

55 11 U ₁ U ^ u 0.04 0.05

0.03 0.04

0.02 0.03

0.05 0.06

- 0.01 0.01 0.02 0.09 0.10 0.08 0.09 0.07 0.08

- <0.001

65

Among the above results, it is found that the tests were consistent with the presence of a small Amount of 0.01 IU / ml penicillin was detected after about 6 min test duration (without the counting time).

The procedure was such that the sample and the marked penicillin were incubated simultaneously for only 3 minutes

0.007
1,000 Ψ
I. 0.853 I. 0.655 1 0.504 0.267

1 0.005 2 0.005 3 0.003 4th 0.002 5 0.000 6th 0.007 7th 0.000 8th 0.042 9 0.003 10 0.030 11th 0.020 12th 0.020 13th 0.025 14th 0.037 15th 0.007 16 0.017 17th 0.043 18th 0.022 19th 0.030 20th 0.000

were centrifuged for 2 min. The test conditions are selected so that a useful Sensitivity as well as the highest possible speed is achieved

To explain the sensitivity achievable using "B. stearothermophilus and marked benzylpenicillin (counting rate 1500 min- '), a further series of tests was carried out, each of which was ^{incubated for 3 min at 90 °} C. in a dry bath incubator. The test results obtained in this way are Table III shows that there is a significant decrease in the count rate of the separated cell fractions when the antibiotic concentration of the sample is increased from 0 to 0.001 and 0.002 to 0.005 IU / ml be performed.

Table III

Sample No. Amount of penicillin G in the sample

; Count rate of cells } Mean value of the counting ».? (min- ') (min- ') 365 I. 311 338 207
227 217 191
176 183.5 97
103 100 89
112 100.5 27 Example 2

10 365 1 " _o

2 0

3 0.001

4 0.001

5 0.002

6 0.002 176 / "" ¹ ^

7 0.005

8 0.005

9 0.010

10 0.010

11-27

Following the procedure of Example 1, Bacillus stearothermophilus was cultivated. The resultant Cell suspension was then sonicated to lyse the cells with ultrasound, with the sample being placed in an ice bath was immersed. Unlysed cells were spun at the sonicated suspension by centrifuging for 5 min 2500 g separated. The binding sites for cell membranes containing penicillin were centrifuged for 10 min isolated at a relative centrifugal acceleration of 5000.

100 pieces of cotton gauze (6.5 g sections) were then added to 100 ml (CNBr solution at a concentration of 30 g / l, which had been adjusted to pH 11.0 with 5 M NaOH. After 10 min, the pieces of gauze were removed with NaHCCh solution washed and added to the cell membranes, which were prepared as above and suspended in a NaHCCh solution of pH 8.1. After 18 h stirring at 4 ⁰ C, the cell membranes were covalently linked by the cyanogen bromide bound to the Mullstücken. the Mullstücke were then washed with distilled water, soaked in ethanolamine for 2 hours, washed again and then dried. ⁴ "

A conjugate of 6-aminopenicillanic acid and peroxidase was prepared as follows:

An alkaline solution (pH 9.0) with 28.1 mg of 6-aminopenicillanic acid was mixed with a peroxidase solution, buffered with phosphate buffer, with 8.8 mg of peroxidase and 8.2 mg of carbodiimide in water. The reaction mixture was kept at 4 ° C. with stirring for 24 hours. In this way the peroxidase and 6-aminopenicillanic acid were covalently linked. The conjugate solution was then dialyzed against a PBS saline solution buffered with 0.05 M phosphate buffer; penicillin activity disappeared from the solution after the first switch. After changing the solution four times, the penicillin-peroxidase conjugate was ready for use. The test using the reagents prepared as above was carried out in such a way that a piece of cotton wool was added to the corresponding test samples or control samples together with 100 μl of the conjugal solution. The reaction mixture was then ^{incubated for 3 min at 50 °} C. in a dry incubator. During this reaction time, any penicillin present in the sample and the 6-aminopenicillanic acid labeled with peroxidase competed for the attachment points on the cell membranes attached to the cotton pieces. The sample was then poured from the tube and the cheesecloth washed with water to remove unbound conjugate; then 1 ml of substrate solution was added to each tube. The substrate solution consisted of 1.0 ml of 0.3% H 2-2.25 mg of 4-aminoantipyrine and 20 ml of phenol, dissolved in 50 ml of distilled water.

The pieces of cheesecloth were then incubated together with the substrate at 25 ° C. If less than 0.05 Units of penicillin were present in the sample, sufficient conjugate was immobilized on the piece of gauze so that then under the action of peroxidase a visually detectable color from pink to red in about 15 minutes occurred. If no coloration occurred after 15 minutes, the sample contained more than 0.05 IU penicillin / ml.

For more accurate results, the optical density of the substrate at 510 nm can be spectrophotometrically be measured. Results of exemplary tests:

15th 20th 25th 30th

40 45 50 55 60

28 50 503 Optical density
(510 nm) Penicillin concentration
(IU / ml) 0.75
0.78
1.10
1.05 1. 0.05
0.00 0.70
0.77
1.05
1.00 2. 0.05
0.00 0.75
1.05 medium optical density
at 0.05 IU penicillin / ml:
medium optical density
without peniciliin: Example 3

To explain the speed that can be achieved in the detection and determination of streptomycin and Sensitivity, another series of tests was carried out as follows:

5 of 6 5 ml milk samples were mixed with streptomycin, so that samples with 0, 1, 10, 100, 1000 and 10 ⁶ ng / ml resultiertea Each sample was measured with a tritium-labeled dihydrostreptomycin (count 228,000 / min- ^1; commercially available ) and mixed with a suspension of B. stearothermophilux obtained as in Example 1 above and reconstituted from 0.2 ml of freeze-dried material by dilution with 8 ml of water in Example 1, obtained at such a concentration that a 1: 500 dilution at 600 nm gave an optical density of 0.60).

The mixtures were used to facilitate the binding of the tritiated dihydrostreptoniycin to the corresponding binding sites of the cells heated. In this example, the originally 10 to 20 ° C located samples heated accordingly in a heating block of 90 ° C for 3 min, so that the temperature of the Samples reached 60 to 65 ° C. The samples were then subjected to relative centrifugal acceleration for 3 minutes centrifuged by 3000, after which the supernatant is decanted and the centrifugate twice with washed with distilled water. The centrifugate consisting of the cells was then with Transfer scintillation fluid to scintillation bottles and determine the count rate. The received Experimental results are shown in Table IV:

Table IV

Sample No. Streptomycin Concentration Count Rate

(ng / ml) (min- ¹ )

Ratio of sample count rate to blank sample count rate (%)

1 0 2 1 3 10 4th 100 5 1000 6th 10 «

30 441 25 849 22 644 18 123 14 819 8 707

100
84.9 74.5 59.6 48.7 28.6

As can be seen from the above results, it occurs from 0 (sample 1) through 1.10 and 100 ng / ml and above As the amount of streptomycin in the sample increases, there is a significant decrease in the count rate.

The presence of streptomycin in samples as low as 1 ng / ml can accordingly be through Treatment of the sample according to the above procedure and comparison of the counting result with the table values or the calibration curve can be detected. Such determinations can be carried out in about 8 minutes will.

As can be seen from the above, it is also possible according to the invention to also use smaller concentrations To prove antibiotics, for example, that the sample is sensitive to the antibiotic Cells are incubated for a time sufficient to keep all in the sample present antibiotic can be bound to appropriate receptor sites. The labeled antibiotic is then added in this case and reacts with the receptor sites that have remained free. Apart from that of this modification, the procedure is essentially identical to that given above and provides a slightly slower but more sensitive test.

For this purpose 2 sheets of drawings

Claims (1)

Patent claims: 1. A method for the detection and determination of antibiotics in liquid samples, characterized by following steps: (A) Incubation of the sample with a microorganism capable of binding the antibiotic Receptor sites under conditions under which the molecules of an antibiotic present in the sample have a Binding with the receptor sites can enter into, with the use of E. coli for detection or for the determination of ^ -lactam antibiotics is excluded, (B) Incubation of the mixture from (A) with a labeled one capable of binding to the receptor sites Substance that is a labeled antibiotic, a labeled antibiotic derivative, or a labeled Is an antibiotic precursor, (C) separation of the solid phase from the liquid after incubation, (D) Determination of the amount of the labeled substance or labeled substance bound to the solid phase Substance in the liquid and
(E) Comparison of the determination with a standard.