DE2850503A1 - PROCEDURE AND TEST SET FOR DETECTION AND DETERMINATION OF ANTIBIOTICS - Google Patents

Description

Stanley Ely CHARM, Newton, Massachusetts, _ V.St.A.

Procedure and test set for verification and determination of antibiotics

The invention relates to a method and a test set for rapid, sensitive detection and determination of the Presence of antibiotics in liquids such as milk, body fluids, meat extracts and fermentation broths.

The ability to detect small concentrations of antibiotics in liquids has numerous uses of great importance, for example in the food industry, where the use of antibiotics to treat animals that produce food, to a need for a quick, accurate

See also test procedures that can be used, for example, / in ^{mid-life 1} ! - wholesale and the like.

65- (CHM-OOI 0IP (2)) / SF-nu

21/077

Since penicillins are used to treat mastitis in dairy cattle, detection methods are needed quickly and accurate detection and elimination of penicillin-containing milk is of particular importance. The rising medical Accordingly, interest in the problem of human ingestion of small amounts of antibiotics via driving requires if penicillins in milk are present in the range from 0.010 to 0.050 IU / ml or above, simple Screening method 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 microbiological based Operations wherein the presence or absence of penicillin or other antibiotics by observation the inhibition of the growth of antibiotic-sensitive microorganisms in the presence of the test sample was determined will. Previously, such procedures required incubation times of 4 hours or more; by using strains of microorganisms that were hypersensitive to antibiotics were able to carry out the experiments required time can be reduced to 2 to 2.5 hours.

Other methods for the detection of antibiotics make use of various other specific properties of the antibiotic to be determined or of the class of antibiotics to be determined as a test basis. Thus, in the literature (JF Rusling et al., Immobilized Enzyme-Based Flowing Stream Analyzer for Measurement of penicillin in fermentation Broths, Analytical Chemistry, Vol. 48 (1976) 1211) given a test that matic on the enzj hydrolysis of Penicillin with a

903821/0774

immobilized β- lactamase derivative is based.

Palmer (JMA Palmer et al., Simple Ultrasensitive Test for Detecting Penicillin in Milk, J. Dairy Science, ^ Q , 1390) also developed a method for the detection of penicillin, which is based on the growth of spores of Bacillus subtilis on nutrient medium Spore-dye-paper disks, which are removed from the air in a small sample.

is based.

Set milk grow, / With the evaporation of the water content In the milk, the concentration of any penicillin that may be present increases at the same time and then leads to a change in color of the dye, which gives an indication of the concentration.

In U.S. Patent 3,586,483 there is a method for detecting Tetracycline antibiotics in liquids are described in which the liquid is absorbed onto an absorbent strip that contains a complex-forming metal that forms a fluorescent metal complex with the antibiotic forms, whereby the fluorescence of the metal complex is observed under UV light.

The above and other previously available methods for the detection of antibiotics differ with respect to one another their sensitivity and their speed are very different from each other. It can be assumed that none of the previously known tests for the detection of small concentrations of 0.01 IU penicillin / ml (6 ng / ml) in measurement times of less than 1 h suitable is.

Such a test procedure would be more economical Way enable a quick and at the same time reliable determination, for example, whether milk samples from

9-8 21/0774

- -ίο -

relatively small batches of antibiotics in concentrations above the permitted levels in accordance with the relevant regulations contain lying quantities.

The invention is accordingly based on the object Procedure as well as a test set for the detection of a large number indicate various antibiotic substances in various liquid media that can be formed in this way, that they are very fast with usable sensitivity or, on the other hand, somewhat slower with extraordinarily high sensitivity work. The method for the detection and determination of antibiotics should also be convenient Way can also be carried out outside of laboratories; the test set according to the invention should at the same time prove very low levels of antibiotics of around 1 ng / ml in liquid samples of materials such as milk, body fluids, Meat residues, fermentation broths and the like are suitable and an implementation of the method according to the invention without a centrifuge or scintillation counter.

The invention is a method and a test set for Evidence of the presence of antibiotics in liquids as well as their determination. Certain embodiments of the invention are ideally suited for the detection and elimination of milk, the antibiotics from Contains penicillin type.

The invention is explained in more detail below with reference to this, but only exemplary, application. From the following it emerges that the invention for the investigation of body fluids, meat extracts, Fermentation broths and the like can be used on numerous antibiotic substances.

909821/0774

The most important advantage of the procedure according to the invention lies in the fact that they detect very small concentrations of antibiotics, for example 0.001 IU Penicillin / ml made possible in a very rapid manner at the same time, for example in less than about 10 minutes.

The procedure according to the invention comprises in principle following steps:

(A) Incubation of the sample with cells or in some cases with Zeil subunits of a microorganism sensitive to antibiotics under conditions under which the molecules of the antibiotic possibly present in the sample are bound to the receptor sites of the cells can be

(B) Incubation of the cell-sample Gemisclis with a labeled Antibiotic or antibiotic precursor under the same conditions,

(C) separation of the cells from the liquid part of the reaction mixture,

(S) determining the amount of labeled antibiotic bound to either the separated cells or in the \ ^r erb biological liquid

and

(E) Comparison of the determination result with a standard.

If there are molecules of an antibiotic in the sample, the labeled as well as the unlabeled molecules at a limited number of receptor sites on the Cells or cellular units bound, with the amount of the antibiotic immobilized in this way is inversely proportional to the concentration of the antibiotic in the test sample is. In an analogous manner, the amount of the labeled antibiotic remaining in the liquid phase depends on the original antibiotic concentration.

The surprisingly high sensitivity and speed of the test method according to the invention is on the one hand the high binding constant of the interaction between the antibiotic molecules and the recipe or st eilen to the Cell walls or other subcellular structures organisms sensitive to antibiotics and, on the other hand, the specificity of antibiotic molecules for those that are capable of interacting Receptor sites on such organisms

90982 1/0774

attributed to. The binding constant for Penici.3 line The prescription on gram-positive cells is for example orders of magnitude higher than the binding constant for the: i mmunhemical antigeri-antibody reaction on which analogous detection methods (so-called immunoassays) are based .

In a particularly important embodiment variant of the invention The procedure is a single incubation step as a rapid test for use in lii Ic awakening made in which the antibiotic-sensitive microorganism, the sample to be examined and the labeled antibiotic or the labeled antibiotic precursor are used, followed by the separation step is done by centrifugation. The speed as well as the sensitivity of this method is increased by using it of cell strains raised against a class of antibiotics to be detected or a specific one are hypersensitive to the antibiotic to be detected.

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

Preferred microorganisms for carrying out Testa for β- lactam antibiotics is Bacillus stearothermophilus (ATTG No. 1014-9 or 15 952). According to the invention can

0 9 8 21/0774

however, various others hypersensitive to antibiotics as well Cell strains are used, in cases where there may be reduced sensitivity, atich only normally sensitive strains can be used.

Examples of cell strains suitable according to the invention are certain mutants of E. c-pli , Fs. Ae.ruj ^ inpsa, B. subt i lis and {3 .. aureus $, however, the invention is not limited to the use of the microorganisms mentioned.

If, in appropriate tests, the speed achieved is less important than the sensitivity achieved, the sample and the cell culture can be incubated for a period of time, after which the method according to the invention is carried out accordingly, after which the labeled antibiotic is added later. When using sensitive microorganisms with optimal high growth rate was found that the sensitivity does not increase linearly with time ", and the incubation period I5 ^m i ⁿ should not exceed.

According to a preferred embodiment of the invention Procedure is called the labeled antibiotic benzylpenicillin or the antibiotic precursor 6-aminopenicillanic acid used, whereby the antibiotic to be detected is a / 3-lactam antibiotic such as benzylpenicillin, Is cephalosporin, ampicillin, oxacillin, methicillin, cloxacillin, cephaloridin and cephalotin. Other Antibiotics detectable 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.

909821/0774

However, the invention is not limited to the detection or determination of the antibiotics mentioned.

The method according to the invention is well suited for detection of penicillin or penicillin-like antibiotics of the type that thereby inhibits cell proliferation, that the antibiotic to the appropriate places on the cell membrane with inhibition of the cell wall synthesis attaches.

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

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

According to the invention, labeled substances are preferred which

O atoms contained in the structure of the antibiotic molecule, and also those which a linked by reaction with, for example, tyrosine, or suitable derivatives thereof - containing ⁷ J-atom, further having antibiotic, an enzyme such as peroxidase as a label.

According to another important development of the invention, the cell parts containing the receptor sites are exposed an insoluble carrier such as cotton gauze and the like. Immobilized. This facilitates the separation step as the carrier after incubation can be easily removed from the liquid and washed.

The use of a device for the detection of radioactive decay products can also be avoided by labeling with an enzyme. In this case, the presence of the enzyme label is determined by incubating the carrier with a solution of the enzyme substrate which is subject to a detectable chemical change under the catalytic influence of the enzyme. A preferred system comprises peroxidase as a labeling enzyme and a substrate solution with Έ. ^ 2 VE-ü ^ ~ aminoantipyrine, which shows a color change in the presence of peroxidase.

The invention also provides a test set for performing the method according to the invention for the detection of antibiotics in liquid samples; the test set has a specific Amount of concentration-stabilized, antibiotic-sensitive and preferably hypersensitive to antibiotics Cells, a certain amount of a labeled antibiotic or antibiotic precursor that is after a high Binding constant towards the receptor sites of the cells is selected, as well as a standard with which the results of the tests carried out on the reagents can be compared.

According to a preferred embodiment, the test set according to the invention comprises freeze-dried Bacillus stearothermophilus , the radioactive labeled antibiotic used, for example, benzylpenicillin labeled with C, and either a standard curve or at least a sample containing a known concentration of the antibiotic to be detected or the class of antibiotic to be detected is used as the standard.

909821/0774

According to another embodiment, the inventive Test kit on a water-insoluble carrier on which cells or cell membranes are hypersensitive to penicillin are immobilized, furthermore enzyme-labeled 6-aminopenicillanic acid and a solution of the enzyme substrate, which can provide a color change under the catalytic influence of the labeling enzyme. The labeling enzyme can be a peroxidase; the substrate solution contains 1 ^ 0.g, phenol and 4-amino-antipyrine.

The method according to the invention comprises four main steps: the incubation, in which this may be in the sample present antibiotic as well as a labeled antibiotic to antibiotic-sensitive cells or cell subunits

909821 / 077i

-yf-

tied, also the step of a separation, in which the cells with attached antibiotic from remaining system will be separated, a determination step in which an indication of the amount of either nait labeled antibiotic associated with the cells or the separated fluid is obtained, and a step of comparison, in which the determination result obtained is compared with a standard.

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

By preparing a series of samples with a known antibiotic concentration and treating them according to the method according to the invention, a standard curve of the antibiotic concentration as a function of the counts per minute in the case of radioactive labeling or a corresponding other criterion can be generated. Investigations of unknown materials lead to a quantitative value of the 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 marked antibiotic with the results of tests carried out in parallel with samples containing known amounts of antibiotics. If only the presence of antibiotics is in If concentrations above a certain maximum value are to be checked, the required comparison step can be performed

909821/0 774

be carried out indirectly. This is done through determination "" of the result with the test sample and comparison with a previously determined result which was correlated with the critical ariüibiotic concentration. This icfold comparison, is made by standardizing the Reagent ier> tmc5 procedure enables. During execution of the test, a comparison is inherently made when the test result is interpreted.

It is believed that the extremely favorable sensitivity and high speed achieved in the present invention of the test for the typically high binding constants the conversions between antibiotics and cells as well as the specificity of antibiotics compared to those with they are based on interacting receptor sites. So are extreme in the procedure according to the invention small amounts of antibiotics in a test sample very quickly at the receptor sites on the cell membranes or bound in other places on the microorganism, with which the incubation takes place.

In this regard, it is already well known that many antibiotics work by targeting specific sites be bound, whereby the normal proliferation metabolism is prevented. For example, from Vancomycin, bacitracin, penicillins and cephalosporins are well known to inhibit cell proliferation by binding to receptor sites on cell membranes , whereby the cell wall synthesis is blocked, (see, for example, the publications by J.R. Edwards et al., Correlation between Growth Inhibition and the Binding of Various Penicillins and Cephalosporins to Staphylococcus aureus, Journal of Bacteriology, August

909821/077 Λ

1969, pp. 4-59-4-62, and J. I /. Stromingei ·, The Actions of Penicillin and Other Antibiotics on Bacterial Cell Wall Synthesis, Hopkins Med. J., IJl (1973) 63)).

For other antibiotics, the corresponding points of interaction are somewhat less accessible than the cell membrane; yet Corresponding antibiotics work by binding to specific cell sites such as ribosomes or nucleic acids. With the procedure according to the invention can be in addition to ^ -lactam antibiotics and other antibiotics of the above kind also include erythromycin, lincomycin, actinomycin and tetracyclines as well Aminoglycosides such as streptomycin, neomycin and the like. prove and quantitatively determine. The latter substances have various points of interaction in the cells before, for example on the ribosomes u. like

In the incubation according to the invention, the labeled antibiotics compete with the sample to be examined labeled and unlabeled molecules around the accessible receptor sites. If the labeled molecules after are added to the incubation of the sample with the cells carried out for a certain time, the labeled Antibiotic molecules bound accordingly to the remaining receptor sites. Because by proof and determination the presence of labeled molecules immobilized on the cells in both cases a measured value for the Amount of bound labeled antibiotic molecules is obtained, this results indirectly in a measured value for bound unlabeled molecules as the amount of labeled antibiotic on the cells is a function of the

909821 / om

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 antibiotic 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 class of compounds which have a high binding constant, for example benzylpenicillin (penicillin G), ampicillin or cephalosporidin from the class of β- lactam antibiotics.

An example of an antibiotic precursor from this one The substance class represents 6-amino-penicillanic acid; a An example of a corresponding derivative is the reaction product of 6-aminopenicillanic acid with tyrosine.

909821/0774

As already stated above, there are marked substances under a marked antibiotic or under a marked substance Antibiotics, antibiotic precursors and equivalent Derivatives as well as other related substances understood that have an affinity for interactable Make their appropriate parent antibiotics exhibit.

There are currently various types of marking Mark types available. The labeled antibiotic can accordingly be a carbon atom, a - ^ J atom or contain other radioactive atoms that can be detected with a counter or the like, also enzymes, enzyme inhibitors, for example methotrexate, or coenzymes (for example NAD), which can be detected by the fact that the labeled Antibiotic is reacted with a substrate solution that a detectable chemical reaction under the catalytic. Influence of the marking results or in which a reaction is inhibited. Excellent results were made using radiolabelled antibiotics such as

14-for example, with C-marked benzylpenicillin (penicillin G-) achieved, which is now commercially available is.

The person skilled in the art is also familiar that to generate a linkage point for a radioactive iodine atom in the most cases a derivative of the antibiotic molecule is synthesized must become. According to the invention, enzymes as marking substances have also been used successfully, according to the invention, coenzymes such as dehydrogenase coenzymes can also be used can be used. The presence of the enzyme marking substance is shown in a manner known per se

909821/077 *

Use of a substrate solution intended for the enzyme, which, for example, results in a color change when reacted with the enzyme. The quantitative determination of the coenzyme takes place in that the separated cells (or the remaining liquid) reacted with a solution that contains a substrate that will act upon exposure of an enzyme system, the decisive component of which is the Go enzyme, results in a color change. The other enzymes of the system are included in the solution.

Generally all cells which are sensitive to the antibiotic to be detected can be used in the method according to the invention. Sb, for example, any microorganism that is inhibited by penicillin can be used to detect penicillin-type antibiotics. In order to increase the sensitivity, however, it is very preferred to use microorganisms that are hypersensitive to the antibiotic to be detected. Recently, numerous strains of cells have become available that are hypersensitive to either certain antibiotics or to classes of antibiotics. Examples of microorganisms which are sensitive to antibiotics of the / 3-lactam type are B_. subtilis , B. megatherium , S. aureus , Ps . aeruginosa and certain mutants of E. coli . The most favorable results in terms of speed and sensitivity have been obtained using hypersensitive strains of organisms which have a temperature of optimum growth which is generally above about 50.degree. A preferred microorganism in this regard is Bacillus stearothermophilus (ATCG No. 10149). B. stearothermophilus ATCG No. 15952 can also be used

9098 21/077

Find. Since the incubations with this microorganism merfcyp can be carried out at high temperature, the rate of binding of the antibiotic can thereby be increased.

Whole cells do not necessarily have to be used to carry out the method according to the invention; accordingly, cellular subunits on which there are receptor parts, for example cell walls or membranes, ribosomes, bound enzymes and the like, can also be used instead of intact cells. For example, penicillin G is known to bind to D-alanine carboxypeptidase and inhibit this enzyme, which is normally immobilized on cell membranes. However, methods for isolating such enzymes are currently available which can be used in place of whole cells (cf. R. Rogers et al., Purification and Characterization of Thermophilic D-Alanine Garboxypeptidase from Membranes of B. stearothermophilus, J. Biol . Ohem. 24-9 ,

Although the rapid separation of such subunits from the remainder of the reaction mixture can be difficult if the subunits are not immobilized according to the invention on an insoluble solid or the like, is for the Nonetheless, it is apparent to those skilled in the art that the use of such materials means that the use of whole cells according to the invention is equivalent.

Cell parts or parts of cells are both whole cells and cellular subunits such as cell membranes or other structures understood that

909821/0774

Have receptor sites.

The method according to the invention can also be used of cell parts with receptor sites immobilized on a suitable support, for example on cotton gauze of a collagen matrix, polystyrene beads or flat or otherwise shaped Polystyrene, polyacrylamide gel or a small amount a dimensionally stable agar nutrient medium, which is provided on a stick for immersion. This will make the separation of the cells from the liquid components of the reaction mixture is considerably simplified and accelerated.

If whole cells are used, the separation is preferably carried out by centrifugation. According to the invention however, other methods of separating the cells from the remainder of the reaction mixture can also be used, for example Ultrafiltration, can be applied.

From the above it can be seen that the inventive The method and the test set according to the invention for the detection or determination of a large number of antibiotics or have antibiotic mixtures applied in a large number of different liquid media. Furthermore can a large number of labeled antibiotics and cells or cell subunits can also be used; Procedures can be carried out as a qualitative as well as a quantitative test.

The invention is illustrated below with the aid of specific exemplary embodiments explained in more detail, without being restricted to this.

A general process scheme is shown in the block diagram of FIG.

909821/0774

The first example relates to proof of suitability of the method according to the invention for the rapid examination of milk samples for the presence of small amounts of 0.001 IU penicillin.

example 1

Bacillus stearothermophilus was cultivated under the following conditions:

Medium:

Solution A - Dxfco yeast extract 2.58 kg Difco-Bactotrypton 2.58 kg Dinat riumpho sphat 1.54- kg Monopotassium sphat 515 S. Ammonium sulfate 515 S. distilled water 515 1 solution B - anhydrous magnesium sulfate 103 S. distilled water 1.29 1 solution C - calcium chloride dihydrate 12.9 S. Manganese chloride 0.52 S. distilled water 1.29 1 solution D - glucose 2.58 kg distilled water 12.9 1

Conditions: 60 ⁰ C, aeration, inoculum 10%

Cultivation time: 2.5 hours

21/0774

-, 20 -.

Procedure:

Solution A is sterilized in the fermenter. Solution B is sterilized independently and added to solution A in an amount of 2.5 ml per liter of solution A. The final concentration of magnesium sulfate in the medium is 0.2 g / l. Solution C is sterilized separately and then added to solution A in an amount of 2.5 ml per liter of solution A.
The final concentrations in the medium are:

Calcium Chloride Dihydrate 0.025 g / l Manganese chloride 0.001 g / l

Solution D is sterilized separately and solution A in an amount of 25 ml per liter of solution A was added. the The final concentration of glucose in the medium is 5

The temperature of solution A in the fermenter should be between 60 and 65 ^° C. Solutions B, 0 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. The complete medium has a pH of 6.9 without adjustment. An antifoam agent (Dow Corning B antifoam) is also added to the medium at a concentration of 0.1 % . Very vigorous ventilation is required to cultivate the microorganisms.

Compartment start of fermentation is a container with 6 1 des complete medium with the contents of two 1-1 Erlenmeyer flasks vaccinated, the {each I50 ml one overnight

909821/0774

grown culture included. When the optical density reaches 0.150 (600 nyu), the entire contents of the container are used to inoculate the 60-1 fermenter. If the solids content is 0.0% , the contents of the 60-1 fermenter are used to inoculate a 530-1 fermenter.

The cells are made twice with five times the volume 0.2 M NH ^ Ol, pH 7.0, and then twice with the washed ten times the volume of a buffer solution of pH 7 »8, which contains 0.01 M magnesium acetate, 0.01 M mercaptoethanol and 0.02 M Tris-HCl buffer.

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

Ali.

C-labeled benzylpenicillin was used as the labeled antibiotic (5OyUCViUg) used (source: Amersham Searle Co.). This reagent can also be freeze-dried for stability reasons.

For quantitative results and for control purposes, two or more liquid samples can be run in parallel

909824/0774

Unfei addiction, one of which is known to be antibiotic free and the other contains a known amount of the antibiotic to be detected, for example 0.01 IU Penicillin. These samples can also be freeze-dried for stability reasons »The reagents mentioned above can be reconstituted with distilled water or a slightly acidic phosphate buffer. at Penicillins are best bonded between pH 6.0 and 7.0.

Provide the stabilized cells, the labeled antibiotic and the control material (or the standard curve) represents the test set according to the invention which is suitable for carrying out the method according to the invention.

The reagents given above were used to generate a standard curve and to examine unknown substances for the presence of penicillin. A standard curve was obtained by mixing 12 1.0 ml milk samples containing known concentrations of benzylpenicillin (penicillin G) with (1) a 100 ml sample of a suspension of B_. stearothermophilus (reconstituted from freeze-dried material by adding 2.5 ml of distilled water to a bottle obtained as above) and (2) C-labeled benzylpenicillin (penicillin G) in an amount sufficient to give 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 the centrifuge tube was rinsed with phosphate buffer without agitating the cells. The cells were then placed in a centrifuge tube in a sintillation bottle

909821/077

ίο

transferred, using scintillation fluid as a wash

sigkeit was used.

12S

When using ^ J as a marking substance, this can the last step in the process is omitted because the labeled molecules can be detected directly.

The results obtained are shown in Table I. :

Table I.
Standard curve for the determination of benzylpenicillin

underground medium

Benzyl corrected value

Sample penicillin counts / counts / _{ρ / ρ} No. (ηκ / ml) p; en / 10min / 10 min / 10 min ⁷

0 0

1.0 1.0 5.0 5.0

10

10

50

50

331 84 367 120 15457 15210 15582 15335 13165 12918 13368 13121 9859 9612 10639 10392 7615 7368 8277 8030 4175 3920 4461 4214

102 , 5 0.007 15272 , 5 1,000 13019 , 0 0.853 10002 0.655 7699 0.504 4071 0.267

909 8 21/0774

* '' Addition of 20,000 ItI benzylpenicillin (penicillin G) for trial

** Counts of the sample / counts of the control experiment (without penicillin G in the sample)

As can be seen from the above results, occurs extraordinary and significant decrease in the count rate when the amount of benzylpenicillin in the samples of Zero (3, 4) is increased to 10 ng / ml (9, 10).

The data in Table I are shown graphically in Figure 2, The ratio of the count rate of the sample to the count rate of the blank sample as a function of the penicillin concentration is plotted in ng / ml.

Using this table or the associated curve as a standard for comparison purposes, tests were carried out on 20 milk samples obtained from a commercially available milk processing facility. The procedure was exactly as above to generate the standard curve, with the difference that an additional step of comparing the counting rate (counts / min) obtained with the test samples with the curve was carried out, the dimension ng / ml of the penicillin in IU / ml ( 0.01 IU - ^ 6 ng) was converted.

The results of these experiments appear from the following Table II shows:

909821A0774

285Q503

Table II

Comparison of the sensitivity of the invention Procedure with a microbiological

procedure

Test results OF INVENTION & ungsgebekannte after the measure received ■ penicillin microbiological test resulting concentration see method nisse P robe Ήτ. (IU / ml) (Ül / ml) (IU / ml)

0.005 - 0.007 0.005 - 0.009 0.003 - 0.008 0.002 - 0.003 0.000 - 0.002 0.007 - 0.006 0.000 - 0.001 0.042 0.1 0.005 0.003 - 0.004 0.030 0.06 0.07 0.020 0.04 0.05 0.020 0.03 0.04 0.025 0.02 0.03 0.037 o _r o5 0, first floor 0.007 - 0.01 0.017 0.01 0.02 0.043 a, 09 0.10

909821/0774

Table II (continued)

known penicillin concentration sample no. (IU / ml)

Test results
after this
microbiological process
(IU / ml)

test results obtained according to the invention
(IU / ml)

0.022 0.030 0.000

0.08
0.0?

0.09

0.08

, .0.001

From the above results, it is found that the presence of a small amount is consistent with the tests of 0.01 IU 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 only incubated for 3 minutes at the same time, followed by centrifugation 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 stearothermophilus using B. and labeled benzylpenicillin (I5OO counts / min) achievable sensitivity was carried out a further series of tests using each incubated for 3 min at 90 ⁰ G in a dry bath incubator. The test results obtained are shown in Table III. It can be seen from this that there is a significant decrease in the counts / min 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. This test can count in less than including

909821/07 74

2H

285G503

10 rain can be carried out.

Tabel 111 Average
(Counts /
/ min) Sample Wr. Amount of
Penicillin G.
in the rehearsal
(IU) Count rate of
Cells (tough
lungs / min) 338 1
2 0
0 365
311 217 3
4- 0.001
0.001 o- o-
o cu
CU CU 183.5 5
6th 0.002
0.002 191
176 100 O- 00 0.005
0.005 97
103 100.5 9
10 0.010
0.010 89
112 11 27

Example 2

Following the procedure of Example 1, Bacillus stearothermophilus was cultivated. The resulting cell suspension is then sonicated to lyse the cells with ultrasound, the sample preferably in a

909821/0774

Ice bath being immersed. Spruce become lysed ropes separated by centrifuging the sonicated suspension at 25OO g for 5 min. The binding sites for penicillin Cell membranes are isolated by centrifugation at 5000 g for 10 min.

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

A conjugate of penicillanic acid and peroxidase was made like is made as follows:

An alkaline solution (pH 9.0) with 28.1 mg of 6-aminopenicillanic acid is mixed with a peroxidase solution with 8.8 mg of peroxidase and 8.2 mg of carbodiimide in water, which is buffered with a phosphate buffer. The reaction mixture is ^{kept at 4 °} C. for 24 h with stirring. In this way, the peroxidase and the 6-amino penicillanic acid are covalently linked. The conjugate solution is then dialyzed against a saline solution (PBS) buffered with 0.05 M phosphate buffer; penicillin activity disappears from the PBS after the first change of solution. After four changes of solution, the penicillin peroxidase conjugate is ready for use. The test using the as above

90982t / 0? 74

The reagents prepared are made in such a way that a piece of cotton wool is added to the corresponding test samples or control samples together with 100 μl of the conjugate solution. The reaction mixture is then ^{incubated for 3 at 50 °} C. in a dry incubator. During this reaction time, any penicillin present in the sample and the penicillanic acid labeled with peroxidase compete for the attachment points on the cell membranes bound to the pieces of cotton wool. The sample is then poured out of the tube, after which the piece of gauze is washed with water to remove unbound conjugate; then 1 ml of substrate solution is added to each tube. The substrate solution consists of 1.0 ml 0.3 % H ₂ O ₂ * 25 mg 4-aminoantipyrine and 20 ml phenol, dissolved in 50 ml distilled water.

The Mullstücke are incubated together with the substrate then at 25 ⁰ C. If less than 0.05 units of penicillin are present in the sample, sufficient conjugate is immobilized on the piece of cheesecloth so that under the action of the peroxidase a visually detectable coloration from pink to red occurs in about 15 minutes. If no coloration occurs after 15 rain, the sample contained more than 0.05 IU penicillin / ml.

To achieve more precise results, the optical density of the substrate can be measured spectrophotometrically at 5 ^ 0 nm will. Results of exemplary tests:

21/0774

Penicillin concentration Optic density (IU) (50 ma)

1. 0.05 0.75

0.78

0.00 1.10

1.05

2. 0.05 0.70

0.77 ο, οο 1.05

1.00

mean optical density

0.05 IU ^ penicillin / ml: 0.75

mean optical density

0.00 IU penicillin / ml: 1.05

Example g '.

To explain the speed and sensitivity achievable in the detection and determination of streptomycin a further series of tests was carried out as follows:

6 5- ^ffi l milk samples were mixed with streptomycin, so that samples with 0, 1, 10, 100, 1000 and 10 ⁶ ng / ml resulted. Each sample was treated with a tritiated dihydrostreptomycin (228,000 counts / min; commercially available, source: Amersham Searle Co.) and with a suspension of B_ obtained as in Example 1 above and reconstituted from 0.2 ml of freeze-dried material by dilution with 8 ml of water. ste ar othe rmophxlu s mixed. (An appropriate concentration of the material prior to freeze-drying is obtained by resuspending the through

90982 1/077 k

-yt-

Centrifuge separated cells as in Example 1 in at such a concentration that a 1: 500 dilution at 600 nm gives an optical density of 0.60).

The mixtures were used to facilitate the binding of the tritiated hydroxydrostreptomycin to. the corresponding binding sites of the cells. In this example, the sample originally located on 10 to 20 ⁰ C were heated according min in a heat block of 90 ⁰ C 3, so that the temperature of the samples reached 60 to 65 ⁰ C. The samples were then centrifuged for 3 min at 3000 g, after which the supernatant was decanted off and the centrifugate was washed twice with distilled water. The centrifugate consisting of the cells was then transferred to scintillation flasks with scintillation fluid, whereupon the counts / / min were determined. The test results obtained are shown in Table IV:

Table IV Count rate
(Counts /
/ min) ratio of
Count rate of the sample
to count rate of
Blank sample (%) sample
No. Streptomycin
conc entrat ion
(ng / ml) 30,441 100 1 0 25,849 84.9 2 1 22,644 74.5 3 10 18.123 59.6 4th 100 14,819 48.7 VJl 1000 8,707 28.6 6th 10 ⁶

909821/0774

2850-5

As can be seen from the above results, when from
O (sample 1) a significant increase in the amount of streptomycin in the sample above 1 \ 10 and 100 ng / ml
Decrease in the count rate.

The presence of streptomycin in samples at the low level of 1 ng / ml £ ann accordingly by treating the sample according to the above procedure and comparing the
Counting result can be verified with the table values or the calibration curve. Such determinations can be carried out in about 8 minutes.

As can be seen from the above, it is also possible according to the invention to detect even smaller concentrations of antibiotics, for example by the fact that the sample is exposed to cells sensitive to the antibiotic during
is incubated for a time which is sufficient for all of the antibiotic present in the sample to be able to bind to the corresponding receptor parts. In this case, the labeled antibiotic is added afterwards
and reacts with receptor sites that have still remained free.
Apart from this modification, the procedure is essentially identical to that given above and
represents a somewhat slower, but even more sensitive test.

In summary, the invention relates to a method and a test set for the rapid determination of small concentrations of, for example, 0.001 ITJ antibiotics in liquid samples, e.g. B. in milk. According to the procedure according to the invention, the sample is marked together with a
Antibiotic or an antibiotic precursor and

909821/0774

antibiotic sensitive cells incubated under conditions among which the antibiotic molecules bound to appropriate receptor sites in or on the cells whereupon the cells with the immobilized antibiotic are separated from the remaining reaction mixture and the Henge of the labeled antibiotic on the cells is determined. The amount of labeled bound on the cells Antibiotic is a function of the amount of antibiotic present in the sample used.

The invention is not restricted to the embodiments explained above by way of example; also analogous embodiments and further developments are included in the concept of the invention.

909821/0774

Blank page

Claims (21)

Expectations 1. Procedure for the detection or determination of antibiotics in liquid samples, characterized by the following steps: (A) Incubation of the sample with a microorganism or parts thereof to bind the antibiotic, capable receptor sites under conditions under which the molecules of the A sample of the antibiotic present can bind to the receptor sites, (B) Incubation of the mixture from (A) with a labeled substance that is an antibiotic or a derivative or contains a precursor thereof capable of binding to the receptor sites are, 65- (CHM-OOI CIP (2)) / SF-nu 909821/0774 AL WSPECTEO (C) separation of the solid phase from the liquid, (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. 2. The method according to claim 1, characterized in that the amount of the labeled substance associated with the solid phase is determined. 3. Method according to claim 1 or 2, characterized in that steps (A) and (B) are carried out simultaneously will. 4. The method according to any one of claims 1 to 3 »thereby characterized in that the labeled substance is one with a radioactive atom, an enzyme or a coenzyme labeled substance is used. 5. The method according to any one of claims 1 to 4-, characterized in that the antibiotic to be detected or determined is a ß- lactam antibiotic, and a substance with a labeled substance
/ 3 -lactam part and a microorganism sensitive to β- lactam antibiotics can be used as the microorganism. 6. The method according to any one of claims 1 to 5, characterized in that the microorganism Bacillus 909821/0 7 f 4 ORIGINAL INSPECTED stearothermophilus is used. 7. The method according to any one of claims 1 to 6, characterized characterized as being more sensitive to antibiotics Microorganism a microorganism that is hypersensitive to the antibiotic to be detected or determined is used. 8. The method according to any one of claims 1 to 7, characterized in that as more sensitive to antibiotics Microorganism a strain with a temperature of optimal growth above 50 ° C is used. 9. The method according to any one of claims 1 to 8, characterized in that the separation (C) by centrifugation is made. 10. The method according to any one of claims 1 to 9, characterized characterized in that microorganisms or parts thereof are used as microorganisms or parts thereof, which are immobilized on a water-insoluble support, and the separation (C) by removing the support from the liquid and washing is made. 11. The method according to any one of claims 1 to 10, characterized characterized in that the liquid sample is milk, body fluids, fluids extracted from meat or fermentation broths can be used. 12. The method according to any one of claims 1 to 11, characterized in that the same as the labeled antibiotic Antibiotic like that to be detected or to "909821/0774 28 & 0503 determinant is used. 13 «The method according to any one of claims 1 to 12, characterized marked that as to be detected or determined Antibiotic benzylpenicillin, cephalosporin, ampicillin, oxacillin, methicillin, cloxacillin, Cephalosporidin or Cephaloün and, as a labeled antibiotic, radioactively labeled 6-aminopeniciü1 acidic or radiolabelled benzylpenicillin can be used. 14. The method according to any one of claims 1 to 12, characterized characterized in that streptomycin as the antibiotic to be detected or to be determined and as marked Substance tritiated tetrahydrostreptomycin used will. 15 · The method according to any one of claims 1 to 14, characterized characterized, dsß as a standard in step (E) a calibration curve of the dependence of the amount of the solid Phase bound labeled substance is used by the concentration of the antibiotic. 16. The method according to any one of claims 1 to 12 or 14 to 15, characterized in that the labeled substance an enzyme-labeled substance is used and the amount of enzyme present is incubated with a substrate solution is determined for this enzyme, which shows a change in color under the catalytic influence of the labeling enzyme, the standard in (E) the optical density measured under standard conditions is used, which is a certain 9 0 9 8 ^ 1/0 7 f 4 ORlGiNAL INSPECTED Antibiotic concentration. 17. Test set ζτχγ implementation of the method according to one of the Claims 1 to 16 for the technical specification or for the determination of antibiotics in liquid samples, marked by (A) Concentration-stabilized cells or cell parts with receptor sites capable of reacting with the antibiotic, (B) a labeled antibiotic, a labeled antibiotic precursor, or a derivative thereof, the can bind to the receptor sites during incubation with the cells or cell parts , and (C) a standard by which the results of the Cells and the labeled material tests performed can be compared. 18. Test set according to claim I7, characterized in that the reagent (A) has a water-insoluble carrier on which cell parts of a hypersensitive to antibiotics Microorganism are immobilized and reagent (B) contains an enzyme-labeled antibiotic, wherein the set further contains a substrate solution for the corresponding enzyme, which under the catalytic Influence of the marking enzyme a color change shows. 19. Test set according to claim 17 or 18, characterized 909821/0774 indicates that the reagent (A) is made from Bacillus stearothermophilus . 20. Test set according to claim 17 or 19, characterized in that that reagent (B) with tritium, ^ J or C is highlighted. 21. Test set according to one of claims 17 to 20, characterized characterized in that there is a calibration curve and / or at least one sample with a known standard (E) Concentration of the antibiotic to be detected or determined and / or one below standardized Results obtained under test conditions and corresponding to a specific antibiotic concentration füx * includes the marker substance. 909821/0 774