JPS587279B2 - How to detect antibiotics - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a rapid and sensitive method for the detection of antibiotics that may be present in liquids such as milk, body fluids, meat extracts and fermented meat juices.

The ability to detect antibiotics that may be present in low concentrations in fluids is important in a variety of situations.

One example is the food industry, where the use of antibiotics to treat animals that produce food materials requires rapid and accurate testing methods that can be used in the field by bulk food handlers and others. It has become.

Since penicillin is used to treat mastitis in dairy cows, a penicillin detection method suitable for rapid and accurate screening of milk is particularly important.

Thus, there has been growing medical interest in the small intake of antibiotics by humans, with concentrations ranging from 0.0 1 0 to 0.0 5 in milk.
0 I. U. Attention has been directed to the effects of penicillin amounts in the (International Units)/ml range or higher and to simple screening methods for detecting such minute amounts of penicillin and other antibiotics.

Various antibiotic detection methods are known today.

Most of them are thus based on microbiological techniques that determine the presence or absence of penicillin or other antibiotics by observing the inhibition of the growth of antibiotic-sensitive microorganisms in the presence of the test sample.

Such methods previously required 4 or more hours of incubation, but the use of microbial strains that are "hypersensitive" to antibiotics has reduced the assay time to 2 to 2.5 hours. Ta.

Other antibiotic detection methods also utilize other unique properties of the antibiotic being detected as a test basis.

Thus, J. F. r Immo of Rusling et al.
bilized Enzyme-BasedF low
ing-S stream Analyzer for
Measurement of Penicill
in inFermentation Broths
(Immobilized enzyme-based flow analyzer for measuring penicillin in fermented meat juices) JC A
Nutical Chemistry, Vol. 4
8, p]-2 1 1 (July 1976 issue)] discloses a test based on the enzymatic hydrolysis of penicillin using an immobilized β-lactamase derivative.

Also, J.M.A. Palmer et al.
Ultrasensitive Test for De
tecting Penicillin in Mil
(A simple ultra-sensitive test for the detection of penicillin in dairy cows)” (J. Dairy Science
, Vol. 50, p. 390) describes the determination of Bacillus subtilis on vegetative, spore, and dye paper discs in small samples of milk exposed to air.
A penicillin detection method based on the development of spores (S. ilis) has been disclosed.

In this case, as the water in the milk evaporates, the concentration of penicillin, if present, increases and induces a color change in the dye that is indicative of the concentration.

Furthermore, J. G. U.S. Patent No. 3 of Heider et al.
No. 586,483 discloses a method for detecting the antibiotic tetracycline in a fluid by absorbing the fluid into an absorbent strip containing an arsenic metal which together with the antibiotic forms a fluorescent metal complex. A method is then disclosed for detecting the antibiotic by observing the fluorescence of the metal complex under ultraviolet light.

However, these and other effective detection methods vary widely in sensitivity and speed.

Therefore, today, 0.01 I.I. per ml. U, (
6n. g/m. 1) It is believed that no test method is capable of detecting penicillin in as little as one hour.

If effective, such a test would, for example, quickly and reliably determine whether milk sampled from relatively small batches in the field has antibiotic concentrations higher than the Food and Drug Administration Standards. That would be economical.

In general, the present invention provides a novel method for detecting antibiotics that may be present in a liquid.

Since certain embodiments of the invention are ideally suited for screening milk for penicillin-type antibiotics, the invention will be detailed with respect to this application.

However, it will be apparent in view of the following description that the present invention may be used to assay body fluids, meat extracts, fermented meat juices, etc. for various antibiotic agents.

A significant advantage of the present invention is that it provides very low antibiotic concentrations, e.g. It is possible to detect penicillin concentrations of OO1I,U,/ml, and to do so very quickly, for example within about 10 minutes.

In its broadest aspect, the method of the invention comprises providing a sample with a cell or subcellular unit of an antibiotic-sensitive microorganism under conditions that cause antibiotic molecules that may be present in the sample to bind to receptor sites associated with said cells. incubating this cell-sample mixture with a labeled antibiotic or precursor under the same conditions, separating the cells from the liquid portion of the reaction mixture, and incubating the cell-sample mixture with a labeled antibiotic or its precursor under the same conditions; to measure the amount of labeled antibiotic present,
and comparing the measurements obtained with standards.

If antibiotic molecules are present in the sample, then both labeled and unlabeled molecules will attempt to bind to a limited number of receptor sites on the cell or cellular unit. The amount of labeled antibiotic immobilized is inversely proportional to the concentration of antibiotic in the test sample.

Similarly, the amount of labeled antibiotic remaining in the liquid phase is a function of the original antibiotic concentration.

The extraordinary sensitivity and speed of the method of the present invention are due to the high binding constants that characterize the attractive forces between the antibiotic molecule and the receptor site on the cell wall or other cellular part of the antibiotic-sensitive organism, and the Organism's second site of action (receptor site)
It is believed that this is due to the specificity of

For example, in the case of penicillin, the binding constant with receptor sites on Gram-positive cells is an order of magnitude higher than that in the antigen-antibody immunochemical reaction that is the basis of conventional similar detection methods (immunoassays). It is a value.

In an important embodiment of the method of the invention, a single test involving an antibiotic-sensitive microorganism, a test sample and a labeled antibiotic or antibiotic precursor is used to provide a rapid test suitable for use when screening milk. Incubation is carried out and a separation step is carried out by centrifugation.

Both the speed and sensitivity of detection or assays can be enhanced by using cell lines that are hypersensitive to a class of multiple antibiotics or a particular single antibiotic to be detected.

Taking this concept one step further, preferred antibiotic-sensitive microorganisms are those strains or strains that have optimal growth temperatures greater than about 50°C.

In this case, incubation can be carried out at relatively high temperatures, thus increasing the kinetics of the reaction between antibiotic and receptor.

The preferred microorganism for assaying β-lactam antibiotics is Bacillus stearothermophilus.
s stearothermophilus ) /
(ATCC Ya 1049 or 15952), however, various other antibiotic hypersensitive cell lines can also be used.

In fact, only sensitive strains can be used when lower sensitivity is tolerated.

Non-limiting examples of suitable cell lines include E. Coli
, Ps. aeruginosa,
B. subtilis and S. subtilis. Included are certain mutants of P. aureus.

In embodiments of the method of the invention, where sensitivity takes precedence over speed, the labeled antibiotic is added after the sample and cultured cells have been incubated for a period of time.

When using sensitive microorganisms with high optimal growth rates, it was noted that the sensitivity did not increase linearly with time, and that the incubation time increased by 1.
This means that it should not last longer than 5 minutes.

In a preferred embodiment of the invention, the labeled antibiotic is benzylpenicillin or the antibiotic precursor 6-aminobenicillanic acid, and the antibiotic to be detected is benzylpenicillin, cephalosporin, ampicillin,
Beta-lactam antibiotics such as oxacillin, methicillin, cloxacillin, cephaloridine and cephalothin.

Other non-limiting antibiotics that can be detected include erythromycin, lincomycin, actinomycin, pancomycin, bacitracin and aminoglycosides such as streptomycin, dientamicin, kanamycin and neomycin.

The method of the invention works particularly well with penicillin or penicillin-like antibiotics that bind to multiple sites on the cell membrane and inhibit cell wall synthesis, thereby inhibiting cell regeneration.

However, antibiotics such as rifamycins and tetracyclines with other sites of action can also be detected.

Labeled antibiotics can be labeled with radioactive atoms, enzymes, enzyme inhibitors, or coenzymes.

Currently preferred labels include 14C incorporated into the molecular structure of the antibiotic, such as 125 conjugated to the antibiotic through reaction with tyrosine or a suitable derivative thereof.
atoms, and enzymes such as peroxidases.

In another important embodiment of the invention, the cell part containing the receptor site is immobilized on an insoluble support, such as a cotton swab.

This strategy facilitates the separation process. This is because after incubation the support can be easily removed from the liquid and washed.

The need for equipment that can detect radioactive decay products is
can be eliminated by using enzyme labels.

In this case, the presence of the enzyme label is determined by incubating the support with an enzyme substrate solution that can exhibit a detectable chemical change under the catalytic influence of the enzyme.

A preferred system includes a peroxidase label and a substrate solution containing 4-aminoantipyrine and H2S2 that changes color in the presence of the peroxidase.

According to another aspect of the invention, a test set is provided for determining the presence or absence of antibiotics in a liquid sample.

This set includes concentration-stabilized antibiotic sensitive (preferably hypersensitive) cells, labeled antibiotics or antibiotic precursors selected to have high binding constants for receptor sites on the cells, and Contains standards for comparing the results of tests performed with the reagent.

In a preferred embodiment, the test set comprises lyophilized Bacillus stearothermophilus, and the labeled antibiotic is benzylpenicillin labeled with a radioactive atom, such as C, and the standard is a standard curve or
At least one sample containing known concentrations of multiple antibiotics or a single antibiotic of the class to be detected.

Another embodiment of the test set is an insoluble support with penicillin hypersensitive cells or a cell membrane immobilized on the support,
It consists of 6-aminopenicillanic acid labeled with an enzyme and an enzyme substrate solution that can exhibit a color change in response to the catalytic influence of the enzyme label.

Thus, the enzyme label may be peroxidase and the substrate solution consists of H202, phenol and 4-aminoantipyrine.

Accordingly, one object of the present invention is to provide an antibiotic detection method capable of detecting various antibiotic agents in various liquid media, which is adaptable to very high speed and with acceptable sensitivity. , and to provide a Vine detection method adapted to provide very high sensitivity even at slightly reduced speeds.

Another object of the invention is to provide a rapid antibiotic detection method that can be conveniently performed outside the laboratory.

Another object of the present invention is to provide a test set suitable for detecting antibiotics present in amounts as low as 1 to 1/ml in liquids sampled from substances such as milk, body fluids, meat extracts, fermented meat juices, etc. It is to provide.

Yet another object is to provide a vine test set in which the method of the invention can be carried out without centrifugation or scintillation counters.

The above and other objects and features of the invention will become apparent from the following description, which shows some important embodiments.

The invention will now be described in detail with respect to its preferred embodiments.

The method of the present invention involves four basic steps: an incubation step in which the labeled antibiotic and the antibiotic, if present in the sample, bind to antibiotic-sensitive cells or subcellular units; a separation step to isolate the labeled antibiotic from the rest of the system; a measurement step to indicate the amount of labeled antibiotic associated with either the cells or the separated fluid; and a comparison step to compare the measurement results to a standard.

Thus, the higher the antibiotic concentration in the test sample, the fewer labeled antibiotic molecules will be associated with the cells and the more labeled antibiotic molecules will be associated with the separation liquid.

By preparing a series of antibiotic samples of known concentrations and processing them according to the method of the present invention, a standard curve of antibiotic concentration plotted against counts per minute (
(in the case of radioactive labels) or analogs can be created.

Calibration of unknown samples will yield a quantitative indication of antibiotic concentration when compared to such a curve.

The presence or absence of an antibiotic in a sample can also be indicated by comparing readings of a labeled antibiotic test sample to the results of a parallel assay on a known amount of antibiotic-containing sample.

If one only wants to determine whether an antibiotic is present at a concentration above a certain maximum value, the necessary comparison step can be carried out indirectly.

This is accomplished by noting the reading associated with the test sample and comparing this value to a predetermined reading that correlates to the critical concentration of antibiotic.

This easy comparison is made possible by standardization of reagents and procedures.

Thus, the comparison is something that the technician performing the test can do with his or her in-house ability when interpreting the readings.

We believe that the advantageous sensitivity and kinetic properties of the assay method can be attributed to the typically high binding constants in antibiotic-cell reactions and the specificity of the antibiotic for cellular receptor sites. It will be done.

Thus, in the method of the invention, even minute amounts of antibiotic in the test sample are rapidly bound to receptor sites on the cell membrane or elsewhere of the microorganism with which the antibiotic is incubated.

In this regard, it is well known that many antibiotics bind themselves to specific cellular locations and thereby prevent normal regenerative metabolism.

For example, vancomycin, bacitracin, penicillin, and cephalosporins are known to bind to receptor sites in cell membranes and prevent cell wall synthesis, thereby inhibiting cell regeneration.

[For example, J. R. Edwards et al.
rre lation between Growth
Inhibition and the B indi
ng of Various Penicilli
ns and Cephalosporins to S
"Correlation between binding of various penicillins and cephalosporins to Taphylococcus aureus and growth inhibition", Jo
Urnal of Bacteriology, p45
9-462 (August 1969); and J, L,
S trom inger's r The Actio
ns of Penicillin and Other
Antibiotics on Bacterial
CellWall Synthesis (The Effects of Penicillin and Other Antibiotics on Bacterial Cell Wall Synthesis)'', Hopkins Med. J. , Vol.
, 133, p. 63 (1.973)].

Other antibiotics have sites of action with somewhat less accessibility than the cell membrane, but nevertheless act by binding themselves to specific cellular locations such as sobosomes or nucleic acids.

Thus, in addition to the β-lactam antibiotics and other antibiotics already mentioned, the method of the invention can detect erythromycin, lincomycin, actinomycin and tetracyclines as well as aminoglycosides such as streptomycin, neomycin, and the like.

This latter substance has various sites of action associated with cells such as ribosomes and the like.

When a labeled antibiotic is included in the sample and cell incubation, the labeled and unlabeled molecules compete for binding to available receptor sites.

Also, when a labeled molecule is added after the sample has been incubated with cells for a period of time, the labeled molecule binds to the remaining receptor sites.

Thus, in both cases, binding measures of labeled antibiotics can be obtained by detecting the presence of labeled molecules immobilized on cells, so that binding of unlabeled molecules is also measured indirectly. .

This is because the amount of labeled antibiotic on the cells is a function of the amount of antibiotic present in the sample.

Stated differently, the method of the present invention attempts to simultaneously react molecules of a labeled antibiotic and antibiotics that may be present in the sample to a limited number of receptor sites on a cell; depends on whether the reactions are to be performed sequentially.

Thus, the test can be successfully performed when the antibiotic to be detected and the labeled substance are the same antibiotic.

However, any appropriately labeled class of antibiotics or related molecules such as antibiotic precursors or derivatives that bind to the same group of receptor sites as the antibiotic to be detected can be used in the incubation. Can be used.

For example, any appropriately labeled antibiotic penicillin or cephalosporin can be used to detect the same antibiotic, any other or mixed penicillin or mixed cephalosporin.

However, preferred antibiotics used for labeling are certain antibiotics with high binding constants, such as cephalosporidins, benzylpenicillin (penicillin G), or ampicillin, which belong to the beta-lactam antibiotic family.

An example of an antibiotic precursor of this β-lactam is 6-aminopenicillanic acid, and an example of a derivative is the reaction product of 6-aminopenicillanic acid and tyrosine.

When the term "labeled antibiotic" or "labeled substance" is used below, it refers to labeled antibiotics, their precursors and derivatives, and other related substances that have affinity for the receptor site of the parent antibiotic. Includes those with.

Regarding the types of signs, the current state of the industry is such that several types of signs are available.

Thus, the labeled antibiotic may contain 14C, 125 or other radioactive atoms that can be detected by a counter, etc.
or enzymes, enzyme inhibitors (e.g. methotrexate) or coenzymes (e.g. NAD), and the labeled antibiotic is subjected to a substrate solution in which the reaction is inhibited or exhibits a detectable chemical reaction under the catalytic influence of the label. It can include things that can be detected by

Excellent results have been demonstrated by the use of radiolabeled antibiotics, such as the commercially available “c” benzylpenicillin (penicillin G).
) is obtained by using

As will be appreciated by those skilled in the art, derivatives to antibiotic molecules must often be synthesized to provide binding sites for the radioactive iodine atoms.

Enzyme labels have also been used successfully, and coenzymes such as dehydrokenase can also be used.

The presence of an enzyme label is determined in a known manner, for example using an enzyme substrate solution that changes color upon exposure to the enzyme.

Quantitative measurement of coenzymes is performed using separated cells (or residual fluid).
is carried out by subjecting it to a substrate-containing solution that changes color under the influence of an enzyme system.

Therefore, the critical components of enzyme systems are coenzymes.

Other enzymes of this system are also included in the substrate solution.

Generally, cells that can be used in the method of the invention include any cell that is sensitive to the antibiotic to be detected.

Thus, for example, any microorganism inhibited by penicillin could be used to detect penicillin-type antibiotics.

However, to increase sensitivity it is much more preferable to use microorganisms that are "hypersensitive" to the antibiotic being detected.

In recent years, multilineage cells that are hypersensitive to specific antibiotics or classes of antibiotics have become available.

An example of a β-lactam sensitive microorganism is B. Subtilis, B. Megatherium, S. Aureus, Ps. aeruginosa and E. Some mutants of E. coli are included.

The most beneficial effect in terms of both speed and sensitivity is generally about 5
This was achieved using a hypersensitive microbial strain with an optimal growth temperature above 0°C.

A preferred microorganism in this regard is Bacillus stearothermophilus (ATCC No. 10149).

B. Stearothermophilus ATCC No. 1 5 9
5 2 is also used.

When using this type of microorganism, incubation can be carried out at high temperatures, thereby increasing the rate of antibiotic binding.

It should be noted that the method of the present invention does not necessarily require the use of whole cells.

Thus, instead of complete cells, subcellular units in which receptor sites or sites of action are located, such as cell walls or cell membranes, ribosomes, conjugated enzymes, etc., can be used.

For example, penicillin G is normally immobilized on the cell membrane.
- Known to bind to alanine carboxypeptidase and inhibit its action.

However, a variety of methods are available to isolate such enzymes, and they can be used in place of whole cells (see R. Rogers et al., "Purific
ation and Characterization
n of The her mophi 1 ic D -
A lanine C arboxypeptidas
efromMembranes of B. steam
"Purification and characterization of thermophilic D-alanine carboxypeptidase from B. stearothermophilus membranes", J. Biol.
Chem,, Vol. 249 p. 4863-4876].

As will be appreciated by those skilled in the art, if such subunits are immobilized on an insoluble solid or the like as taught herein, they can be separated more rapidly from other components of the reaction mixture; Use of such substances is equivalent to using whole cells.

Accordingly, when the term "cellular portion" is used herein, it is intended to mean the entire cell as well as subcellular dimensions such as the cell membrane or other structural parts containing receptor sites.

The method of the invention also includes cotton swabs, collagen matrices,
It can also be carried out using receptor site-containing cell parts immobilized on a suitable support such as polystyrene walls or beads, polyacrylamide gels, or a small amount of shape-retentive agar that is attached to a dipstick. .

In this case, separating the cells from the liquid component of the reaction mixture becomes much easier and is also speeded up.

When whole cells are used, a preferred separation method is by centrifugation.

However, other methods of separating cells from the remaining components of the reaction mixture can also be used, such as ultrafiltration.

In view of the above description, it will be appreciated that the tests of the present invention may be adapted to detect various antibiotics or mixtures thereof that may be present in different liquid media.

In addition, various labeled antibiotics and cells or subcellular units can be used, and both qualitative and quantitative tests can be designed.

Here, the present invention is first applied to penicillin at 0.001 I.D.
U. A specific non-limiting embodiment is described that is suitable for rapid screening of milk present in amounts as small as 1/ml.

Example 1 Bacillus stearothermophilus was grown as follows.

Culture solution: Solution A - Difco yeast 2.5
8 kg extract deifco bactotrip 2.58 kg ton dibasic sodium phosphate 1.54 kg mu basic potassium phosphate 515 g ammonium sulfate 515 g distilled water 515 l Solution B monoanhydrous magnesium sulfate 103 g distilled water
1. ．． 2 9l Solution C Calcium monochloride 2H20 12.9g Magnesium chloride 0.52g Distilled water
1.29l solution D-glucose
2.58 kg distilled water 12.9 l Conditions: 60°C, aeration, 10% Inoculum growth time: 2.5 hours Growth method: Solution A is sterilized in a fermenter.

Separately, sterilize solution B and add 2.
Add to solution A in a 5 ml volume.

The final magnesium sulfate concentration in this culture solution was adjusted to o. 2
g/l.

Separately, solution C was sterilized, and 2. Add to solution A in a 5 ml volume.

The final compound concentration in this culture solution is set to 0.001 g/l of calcium chloride/2H20 and 0.0025 g/l of magnesium chloride.

Separately, solution D is sterilized and added to solution A in an amount of 25 ml per All solution.

The final glucose concentration in this culture solution is set to 5 g/l.

At that time, first, the temperature of solution A placed in the fermenter was set to 60-65
Should be in the °C range.

Then, while stirring vigorously, add solution B to this fermenter.
Add solution C and solution D.

Vigorous stirring and slow addition of Solution C were required to prevent the formation of a precipitate.

The complete culture medium has a pH of 6.9 without pH adjustment.

Antifoam agent Dow Corning
Add B at a concentration of 0.1%.

Requires very intense aeration to develop the organism.

At the start of fermentation, 11 carboys containing 6 liters of complete culture
Incubate with the contents of two Erlenmeyer flasks.

Each flask contains 150 ml of overnight growth medium.
shall be.

The optical density reading is O. When 1.50 (600 mμ) is reached, use the entire contents of the carboy to 60. Incubate the e-fermenter.

When the solids content is 0.03%, this 60 liter fermenter is used to incubate a 530 liter fermenter.

The obtained cells were 0. 2 M NH4Cl (pH
7. 0) Wash twice with 5 volumes, then 0. Wash twice with 10 volumes of OIM magnesium acetate, 0.01 M mercaptoethanol and 0.02 M Tris-HCI buffer (pH 7.8).

Next, the cells are centrifuged, the supernatant is tecanted, and then resuspended in a mixture of solutions A, B, and C, and aliquoted into vials.

The contents of each vial can be lyophilized by known methods and stored at about 4°C for extended periods of time.

Freeze-drying in growth medium (-glucose solution) preserves maximum antibiotic binding activity of the cells.

Such lyophilized cells constitute an ideal concentration-stabilizing cell source for use in test sets.

The labeled antibiotic used was benzylpenicillin (50 μCurie/mg) containing 14C atoms, Amers
It was purchased from Ham Searle.

This reagent can also be lyophilized for further stabilization.

Two or more fluid samples can be tested in parallel to obtain quantitative results and to serve as controls.

Thus, one of the liquid samples is known to be free of antibiotics, and the other sample contains a known amount of the antibiotic to be assayed, such as 0.01 unit of penicillin.

Such samples can also be lyophilized for further stabilization.

For all of the above reagents, use distilled water or slightly acidic pH.
can be reconstituted in a phosphate buffer with

In the case of penicillin, binding optimally occurs at a pH in the 6.0-7.0 range.

Stabilized cells, labeled antibiotics and controls (
or standard curve) constitute a suitable test set for carrying out the assay according to the present invention.

A standard curve was constructed using the above reagent combinations and unknown samples were tested for the presence or absence of penicillin.

First, a known concentration of benzylpenicillin (penicillin G)
Milk sample containing 1 2 1. 0 ml, (1)
B. 100 ml of a stearothermophilus suspension sample (reconstituted from the lyophilizate, added to a vial prepared as described above with 2.5 ml of distilled water) and (2) "Ci-identified benzylpenicillin ( Penicillin G
) (sufficient amount to give approximately 10,000 counts/min) to generate a standard curve.

Incubations were carried out for 3 minutes in a dry bath incubator set at 90°C.

Cells were then separated by centrifugation at 3000G for 2 minutes.

After draining the supernatant, the centrifuge tube was rinsed with phosphate buffer without disturbing the cells.

The cells were then transferred from the tube to a scintillation vial.

At this time, scintillation fluid was used as a washing liquid.

Thus, the latter step can be eliminated when using a 125 label, allowing the labeled molecule to be detected directly.

The results are shown in Table I below.

As can be seen from the table above, the amount of benzylpenicillin in the samples ranged from 0 (sample numbers 3 and 4) to 1. Ong
/ml (sample nos. 9 and 10).
A significant and rapid drop in counts occurred.

The materials in Table I are shown in Figure 2. This figure shows the sample count/
Concentration of penicillin (ng/ml) relative to zero count
This is a graph plotting.

Using the above table as a basis of comparison, 20 milk samples prepared by a commercial milk manufacturer were tested.

The method used was the same as the method used to create the standard curve above, except that as an additional step, the number of counts/min obtained from the test sample was compared with the standard curve, and the H
g/mi value as ■, U. /ml value (0.0 1 1
．． U. :6 ng).

These results are shown in Table 3 below: What is noteworthy is that in the first test above, 0.01I. U. The presence of moderately small amounts of penicillin was consistently detected.

It should also be noted that in the above method, the sample and labeled penicillin are incubated for only 3 minutes, with only 2 minutes of concurrent centrifugation.

This test was specifically designed to be as rapid as possible and with acceptable sensitivity.

B. Another test was carried out to demonstrate the highest possible sensitivity using stearothermophilus, labeled benzylpenicillin (1500 counts/min) and a 3 minute incubation in a dry bath incubator set at 90°C.

The results of this first test are shown in Table 1 below.

The data listed in Table ■ indicates the antibiotic concentration of the sample from 0 to 0.
0 0 1 I. U,, 0.002I,U, and 0.
005I. U. It is illustrated that the number of counts/min associated with the separated cell portion decreased significantly when increasing to .

This test can be performed within 10 minutes including a counting step.

Example 1 ■ Bacillus stearothermophilus was grown according to the method described in Example 1.

The resulting cell suspension was sonicated to lyse the cells.

Preferably, the test piece was immersed in a water bath during that time.

Unlysed cells were removed by centrifuging the sonicated suspension at 2500 G for 5 minutes.

In addition, cell membranes containing penicillin binding sites were isolated by centrifugation at 5000 G for 10 minutes.

Then adjust the pH to 11.0 with 5M NaOH.30g/l CNBr solution 100
100 cotton swabs (6.5 g of cotton) were added to each ml.

After 10 minutes, the cotton swab was removed, rinsed with NaHCO3 solution, and then treated with NaHCO3 solution (pH = 8.1.
) into a suspension of cell membranes prepared as above.

After stirring for 18 hours at 4°C, the cell membranes were covalently bound to the cotton swab using cyanogen bromide.

The cotton swab was then rinsed with distilled water and soaked in ethanolamine for 2 hours.
After soaking for an hour, it was rinsed again and dried.

Conjugate of penicillanic acid and peroxyguse (con
jugate) was prepared as follows.

That is, 6 aminopenicillanic acid 2 8. An alkaline solution (pH=9.0) containing 1 mg of peroxidase and an aqueous phosphate buffered peroxidase solution containing 8.8 mg of peroxidase and 8.27 mg of carbodiimide were mixed.

The reaction mixture was kept under stirring at 4° C. for 24 hours.

As a result, peroxidase and 6-aminopenicillanic acid were covalently bonded.

The conjugate solution thus obtained was dialyzed against 0.05M phosphate buffered saline (PBS).

After the first change in solution, penicillin activity disappeared from the PBS.

After four changes of solution, the penicillin-peroxidase conjugate was ready for use.

Assays using reagents prepared as described above involve applying a cotton swab to a solution of conjugate. Each test sample (or control) along with 0 ml
This is done by adding

The reaction mixture is incubated for 3 minutes at 50° C. in a dry bath incubator.

During this time, peroxidase-labeled penicillanic acid and penicillin, if present in the sample, compete with each other and try to bind to the active site on the cell membrane that is attached to the cotton swab.

The sample is then poured from the tube, the swab is washed with water to remove unbound conjugate, and 1 ml of substrate solution is added to the tube.
Add.

The substrate solution consisted of 1.0 ml of 0.3% H202, 25 mg of 4-aminoatipyrine, and 20 ml of phenol dissolved in 50 ml of distilled water.

The swab substrate was then incubated at 25°C.

If less than 0.05 units of penicillin are present in the sample, enough conjugate is immobilized on the swab to cause a visually detectable pink-to-red color change in about 15 minutes through the action of peroxidase.

If no color appears after 15 minutes, the sample is 0.05 units/m
It has a penicillin content of more than 1.

For more accurate results, use a spectrophotometer at 510 m.
The optical density of the substrate at μ can be read.

The results of a representative test are shown below: Example III Another test was conducted to illustrate the speed and sensitivity with which streptomycin could be detected. Streptomycin was added to a 65 ml milk sample at a concentration of Ong/ml.
, 1 ng/ml, 10 ng/ml, 100 ng/ml
ml, 1000ng/ml and 106
The mixture was mixed to a concentration of ng/ml.

Each sample was given a B. Stearothermophilus lyophilized product was reconstituted by diluting 0.2 ml with 8 ml of water and tritium-labeled dihydrostreptomycin (228,000 counts/min) was added.
) (for example, commercially available from Amersham. Searle) were mixed.

(In addition, the cells to be centrifuged as described in Example
Prior to lyophilization, a suitable concentration of B. A stearothermophilus suspension is obtained.

) Heating this mixture facilitates the binding of tritium-labeled dihydrostreptomycin at the binding site within the cell.

Therefore, in this example, the sample, which was initially at 10-20°C, was heated to 90°C in a dry block heater so that the sample temperature was 60-65°C.

The samples were then centrifuged at 3000 G for 3 minutes, the supernatant liquid was decanted, and each precipitate was washed twice with distilled water.

The precipitate (cells) was then transferred to a scintillation vial with scintillation fluid and the counts were read for 1 minute.

These test results are shown in the table below: As can be seen from the table above, the amount of streptomycin in the sample was varied from 0 (sample number 1) to 1, 10 and 100.
As the number of counts increases to ng/ml and above, the number of counts decreases significantly.

Therefore, by processing a sample according to the above method and comparing the counts with the data in the table, 1 ng in the sample can be determined.
It is possible to detect streptomycin present in small amounts as low as /ml.

Such a determination can be accomplished within about 8 minutes.

As recognized by those skilled in the art in view of the above description,
For example, even lower concentrations of antibiotics can be detected by incubating the sample with antibiotic-sensitive cells for a sufficient period of time to allow all of the antibiotic present in the sample to bind to receptors.

That is, by adding labeled antibiotic after incubation, it binds to the remaining receptors.

Apart from such modifications, the detection method is substantially the same as described above.

In that case, the speed will be slightly lower, but the sensitivity will be much higher.

The invention may be embodied in other specific forms without departing from its spirit or essential characteristics.

The embodiments described above should therefore be considered in all respects as illustrative rather than restrictive.

The scope of the invention is indicated by the claims rather than by the foregoing description.

Therefore, all modifications and changes that come within the meaning and range of equivalency of the claims are intended to be included in the present invention.

[Brief explanation of the drawing]

FIG. 1 is a flow sheet illustrating the antibiotic detection method of the present invention, and FIG. 2 is a graph showing a standard curve for implementing the present invention in detecting benzylpenicillin.

Claims (1)

[Scope of Claims] 1. A method for detecting an antibiotic that may be present in a liquid sample, comprising: A. incubating said sample with a microorganism or part of a microorganism having an acceptor site capable of binding said antibiotic;
The incubation is carried out under conditions that bind the antibiotic molecule to the receptor site when present in the sample, and B. incubating the mixture of step A with a labeling substance capable of binding to the receptor site;
C. separating the solid phase and the liquid phase; D. measuring the amount of labeling substance associated with the solid phase or the liquid phase; E. A method comprising the steps of comparing the measured value with a standard to determine the presence or absence of ciliobiotic material in the sample. 2. The method according to claim 1, wherein the amount of labeling substance associated with the solid phase is measured. 3. The method according to claim 1 or 2, wherein steps A and B are carried out simultaneously. 4. The method according to any one of claims 1 to 3, wherein the labeling substance is a labeled antibiotic, or a derivative or precursor thereof. 5. The method according to any one of claims 1 to 4, wherein the label of the labeling substance is a radioactive atom, an enzyme, or a coenzyme. 6. The method according to any one of claims 1 to 5, wherein the microorganism is sensitive to ultrasensitive to the antibiotic to be detected. 7. The method of claim 6, wherein the microorganism sensitive to antibiotics is of a strain or strain with an optimal growth temperature higher than about 50°C, and the incubation temperature is higher than 50°C. . 8. Any one of claims 1 to 7, wherein the antibiotic to be detected is a β-lactam antibiotic, the labeling substance includes a β-lactam moiety, and the microorganism is a β-lactam antibiotic sensitive microorganism. or the method described. 9 Microorganism Cavacillus stearothermophilus (B
acillus stearothermophilu
s). The method according to any one of claims 1 to 8. 10. The method according to claim 4, wherein the antibiotic to be detected and the labeled antibiotic are the same. 11 The antibiotic to be detected is selected from the group consisting of benzylpenicillin, cephalosporin, ampicillin, oxacillin, methicillin, cloxacillin, cephalosporin, and cephalothin, and the labeled antibiotic is a 6-aminopenic acid labeled with a radioactive element. The method according to claim 4, wherein the radiolabeled substance is selected from the group consisting of silanic acid and benzylpenicillin. 12. The method according to any one of claims 4 to 7, wherein the antibiotic to be detected is streptomycin, and the labeling substance is tritium-labeled di- or tetrahydrostreptomycin. 13. The method according to any one of claims 1 to 12, wherein the separation step is performed by centrifugation. 14. Any of claims 1 to 13, wherein the microorganism or part of the microorganism is immobilized on a water-insoluble support, and the separation step is carried out by removing the support from the liquid and washing it. or the method described. 15. The method according to any one of claims 1 to 14, wherein the liquid sample is selected from the group consisting of milk, body fluid, fermented meat juice, and liquid extractable from meat. 16. The method of any one of claims 1 to 15, wherein the standard used in step E is a standard curve of antibiotic concentration plotted against a quantitative representation of the amount of labeled substance associated with the solid phase. . 17 An enzyme label is used and an indication of the amount of enzyme present is obtained on incubation with an enzyme substrate solution capable of showing a color change in response to the catalytic influence of the enzyme label and also used in step E. Claim 5, or any claim dependent thereon, wherein the standard consists of optical density values measured under standard conditions, which values correspond to the concentration of the antibiotic to be selected. Range of items 6 to 1
The method described in any of Section 5. 18 A test set for the assay of antibiotics that may be present in a liquid sample. Detection 1 - Concentration-stabilized microorganism or microorganism moiety having an acceptor site that reacts with the intended antibiotic; B. a labeling substance capable of binding to the receptor site when incubated with the microorganism or microbial moiety; and C. A test set comprising a combination of standards by which the results of a test performed on a liquid sample with said microorganism and said labeling substance are compared to provide a qualitative or quantitative determination of said antibiotic in the sample. 19. The test set according to claim 18, wherein reagent B is labeled with tritium, 125 or 14C. 20 Reagent A consists of a water-insoluble support on which cell parts of antibiotic-hypersensitive microorganisms are immobilized, and Reagent B consists of an enzyme-labeled antibiotic; 19. The test set of claim 18, which also includes an enzyme substrate solution capable of exhibiting a responsive color change. 21 Claims 18 to 21, wherein the microorganism or microorganism part is produced from Bacillus stearothermophilus.
Test set as described in any of Item 20. 22. Claims in which the standard is a standard curve, at least one sample containing a known concentration of the antibiotic to be detected, and a standard reading corresponding to a selected antibiotic concentration, measured under standard test conditions. The test set according to any one of the ranges 18 to 21.