CA1108516A - Antibiotic detection method - Google Patents
Antibiotic detection methodInfo
- Publication number
- CA1108516A CA1108516A CA316,133A CA316133A CA1108516A CA 1108516 A CA1108516 A CA 1108516A CA 316133 A CA316133 A CA 316133A CA 1108516 A CA1108516 A CA 1108516A
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- CA
- Canada
- Prior art keywords
- antibiotic
- tagged
- set forth
- sample
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
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Classifications
-
- F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
- F04—POSITIVE - DISPLACEMENT MACHINES FOR LIQUIDS; PUMPS FOR LIQUIDS OR ELASTIC FLUIDS
- F04B—POSITIVE-DISPLACEMENT MACHINES FOR LIQUIDS; PUMPS
- F04B39/00—Component parts, details, or accessories, of pumps or pumping systems specially adapted for elastic fluids, not otherwise provided for in, or of interest apart from, groups F04B25/00 - F04B37/00
- F04B39/12—Casings; Cylinders; Cylinder heads; Fluid connections
- F04B39/127—Mounting of a cylinder block in a casing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/18—Testing for antimicrobial activity of a material
Abstract
ABSTRACT OF THE DISCLOSURE
A process for rapidly detecting as little as .001 I.U.
of antibiotic in a sample of liquid such as milk. The process comprises the steps of incubating the sample together with a tagged antibiotic or antibiotic precursor and antibiotic sensitive cells under conditions which allow antibiotic molecules to attach to receptor sites in or on the cells, separating the cells with immobilized antibiotic from the remainder of the reaction mixture, and determining the quantity of tagged antibiotic on the cells. The amount of tagged antibiotic on the cells is a function of the quantity of antibiotic present in the sample.
A process for rapidly detecting as little as .001 I.U.
of antibiotic in a sample of liquid such as milk. The process comprises the steps of incubating the sample together with a tagged antibiotic or antibiotic precursor and antibiotic sensitive cells under conditions which allow antibiotic molecules to attach to receptor sites in or on the cells, separating the cells with immobilized antibiotic from the remainder of the reaction mixture, and determining the quantity of tagged antibiotic on the cells. The amount of tagged antibiotic on the cells is a function of the quantity of antibiotic present in the sample.
Description
1~851~i This invention relates to a rapid, sensitive method for detecting the presence of antibiotics in liquids such as milk, body fluids, meat extracts, and fermentation broths.
The ability to detect small concentrations of anti-biotics in liquids is important in various situations. One example is the food industry where the use of antibiotics in the treatment of animals whieh produce food stuffs has created a need for a rapid, accurate test method which can be used in the field by bulk food handlers and the like. Because peni-cillins are used to treat mastitis in dairy cattle, penicillin detection methods suitable for rapidly and accurately screening milk are partieularly important. Thus, the growing medical eoneern about ingestion of small amounts of antibioties by humans is directing attention to the incidence of penieillin in milk at levels in the range of 0.010 - 0.050 I.U./ml or greater and to simple screening methods for detecting such minute quantities of penicillin and other antibiotics.
At the present time, there are a variety of known antibiotie deteetion procedures. Most of these involve miero-biologieal teehniques wherein the presenee or absenee of peni-eillin or other antibiotie is determined by observing the inhibition of growth of antibiotie sensitive mieroorganisms in the presenee of the test sample. Formerly, sueh proeedures required ineubation times of four hours or more, but by employing mieroorganism strains "super-sensitive" to antibioties, the time required to perform the assays has been redueed to between 2 and 2 1/2 hours.
Other antibiotie detection techniques exploit various other unique properties of the antibiotic or class of antibiotics ~a~
1 to be detected as a test basis. Thus, in Immobilized Enzyme-Based Flowing-Stream Analyzer for Measurement of Penicillin in Fermentation Broths, J.F. Rusling et al., Analytical Chemistry, Vol. 48, p. 1211, (July, 1976), a test based on the enzymatic hydrolysis of penicillin with an immobilized ~ lactamase derivative is disclosed. Simple Ultrasensitive Test for Detecting Penicillin in Milk, J.M.A. Palmer et al., J. Dairy Science, Vol. 50, p. 1390, discloses a penicillin detection method based on the growth of Bacillus subtilis spores on nutrient-spore-dye paper discs residing in a small sample of milk exposed to the air. As the water content of the milk evaporates, penicillin concentration, if any, increases and induces a colour change in the dye which is indicative of concentration.
U.S. Patent No. 3,586,483 to J.G. Heider et al. discloses a method of detecting tetracycline antibiotics in fluids by absorbing the fluid on an absorbent strip containing a complexing metal which forms a fluorescent metal complex with the antibiotic, and by observing the fluorescence of the metal complex under ultraviolet light.
The foregoing and other available detection techniques vary widely with respect to their sensitivity and speed. It is believed that no presently available test is capable of detecting as little as 0.01 I.U. of penicillin per milliliter (6 ng/ml) in less than an hour. If such a test were available, it would become economical to rapidly and reliably determine, for example, whether milk sampled in the field from relatively small batches contained antibiotic concentrations in excess of Food and Drug Administration standards.
SUMMARY OF THE INVENTION
The instant invention provides a novel procedure for 11 .3 85~
1 detecting the presence of antibiotics in liquids. Certain embodiments of the invention are ideally suited for screening milk for penicillin type antibiotics, and the invention will be described in detail with reference to this application.
However, in view of the description which follows, it will be apparent that the invention may be used to assay body fluids, meat extracts, fermentation broths and the like for a variety of antibiotic drugs. The outstanding advantage of the process of the invention is that it is capable of detecting very small antibiotic concentrations, e.g., .001 I.U./ml of penicillin, and can do so rapidly, e.g., in less than about 10 minutes.
In its broadest aspects, the process of the invention comprises the steps of incubating the sample with cells, or in some cases, cellular subunits, of an antibiotic sensitive microorganism under conditions to allow antibiotic molecules that may be present in the sample to attach to receptor sites associated with the cells, incubating the cell-sample mixture with a tagged antibiotic or antibiotic precursor under the same conditions, separating the cells from the liquid portion of the reaction mixture, determining the amount of tagged antibiotic present either in association with the separated cells or the remaining liquid, and comparing the determination to a standard. If antibiotic molecules were present in the sample, then the tagged and untagged molecules both seek to attach themselves onto a finite number o receptor sites on the cells or cellular subunits, and the amount of tagged antibiotic which becomes immobilized is inversely proportional to the concentration of antibiotic in the test sample. Similarly, the amount of tagged antibiotic which remains in the liquid phase will be a function of original antibiotic concentration.
~r.5~8S~l6 1 The surprising sensitivity and speed of the test is believed to be a consequence of both the high binding constant characteristic of the attraction between antibiotic molecules and receptor sites on cell walls or other subcellular structures of antibiotic sensitive organisms, and the specificity of anti-biotic molecules for their sites of action on such organisms.
For example, the binding constant for penicillins with receptor sites on gram positive cells is orders of magnitude greater than the binding constant for the antigen-antibody immunochemical reaction on which analogous detection techniques (immunoassays) have been based.
In an important embodiment of the process of the invention, in the interest of providing a rapid test suitable for use in screening milk, a single incubation is conducted involving the antibiotic sensitive microorganism, the sample to be tested, and the tagged antibiotic or antibiotic precursor, and the separation step is effected by centrifugation. Both the speed and sensitivity of the assay are promoted by employing cell strains which are supersensitive to a class of antibiotics or specific antibiotic to be detected. Extending this concept one step further, the preferred antibiotic sensitive microorganisms are strains having a temperature of optimum growth above about 50C. In this situation, the incubation can be conducted at relatively high temperatures and the kinetics of the antibiotic-receptor site organic reaction are enhanced.
The preferred microorganism for conducting assays for ~ lactam antibiotics is Bacillus stearothermophilus (A.T.C.C. No. 10149 or 15952). However, various other antibiotic supersensitive cell strains can be used, and indeed, merely sensitive strains can be used if reduced sensitivity can be tolerated. Nonlimiting 5ll6 1 examples of suitable cell strains include certain mutants of E. coli, Ps. aeruginosa, B. subtilis, and S. aureus.
In embodiments of the process of the invention wherein speed is less important than sensitivity, the sample and cell culture are incubated for a time and the tagged antibiotic is added later. When employing sensitive microorganisms having the high optimum growth rate, it has been noted that sensitivity does not increase linearly with time and that incubations of greater than 15 minutes in duration should not be conducted.
In preferred embodiments of the invention, the tagged antibiotic is benzylpenicillin, or the antibiotic pre-cursor 6 amino penicillanic acid, and the antibiotic to be detected is a ~ lactam antibiotic such as benzylpenicillin, cephalosporin, ampicillin, oxacillin, methicillin, cloxacillin, cephaloridine, and cephalothin. Other nonlimiting antibiotics which can be detected include erythromycin, lincomycin, actino-mysin, vancomycin, bacitracin and aminoglycosides such as streptomycin, gentamycin, kanamycin, and neomycin. The process of the invention works exceptionally well with penicillin or penicillin-like antibiotics of the type which inhibit cell reproduction by attaching to sites on cell membranes to inhibit cell wall synthesis. However, antibiotics having other sites of action such as rifamycin and tetracyclines can also be detected. The tagged antibiotic can be tagged with a radio-active atom, an enzyme, enzyme inhibitor, or a conenzyme.
Presently preferred tags include a 14C incorporated in the structure of the antibiotic molecule, an 125I atom attached to the antibiotic via reaction with, for example, tyrosine or suitable derivative thereof, and an enzyme such as peroxidase.
In another important embodiment of the inventlon, cell ~85~6 1 parts containing receptor sites are immobilized on an insoluble support, e.g., a cotton swab. This approach facilitates the separation step since, after incubation, the support can simply be removed from the liquid and washed. The need for a device capable of detecting radioactive decay products can be eliminated by tagging with an enzyme. In this case, the determination of the presence-of the enzyme tag is made by incubating the support with an enzyme substrate solution capable of undergoing a detectable chemical change under the catalytic influence of the enzyme. A preferred system includes a peroxidase tag and a substrate solution comprising H202 and 4-amino antipyrine which changes colour in the presence of peroxidase.
In accordance with another aspect of the invention, a test set for detexmining the presence of antibiotics in liquid samples is provided. The set has a quantity of con-centration stabilized antibiotic sensitive (preferably super-sensitive) cells, a quantity of tagged antibiotic or antiblotic precursor selected to have a high binding constant with the sites on the cells, and a standard against which the results of tests made with the reagents can be compared. In preferred embodiments, the test set includes freeze dried Bacillus stearothermophilus, the tagged antibiotic is benzylpenicillin tagged with a radioactive atom, e.g., 14C, and the standard is either a standard curve or at least one sample containing a known concentration of the antibiotic or class of antibiotics to be detected.
Another embodiment of the test set comprises a water insoluble support having penicillin super sensitive cells or cell membranes immobilized thereon, enzyme-tagged 6 amino-penicillanic acid, and an enzyme substrate solution capable of ~ 85~.6 1 undergoing a colour change in res~nse to the catalyticinfluence of the enzyme tag. The enzyme tag can be peroxidase;
the substrate solution comprises H202, phenol, and 4 amino antipyrine.
Accordingly, objects of the invention include the pro-vision of an antibiotic detection technique which is capable of detecting a large number of different antibiotic drugs in a variety of liquid media, which can be adapted to be very rapid and acceptably sensitive, and which can be adapted to be slightly slower, but extraordinarily sensitive. Another object of the invention is to provide a fast antibiotic detection technique which can be conveniently conducted outside of the laboratory. Another object is to provide a test set suitable for determining the presence of as little as 1 ng/ml of antibiotic in a liquid sampled from materials such as milk, body fluids, meat residues, fermentation broths and the like.
Yet another object is to provide a test set which enables the process of the invention to be conducted without a centrifuge or scintillation counter.
These and other objects and features of the invention will be apparent from the following detailed description of some important embodiments. -;`
BRIEF DESCRIPTION OF THE DRAWINGS
r FIGURE 1 is a flow chart illustrating the antibiotic detection process of the invention; and FIGURE 2 shows a standard curve for benzylpenicillin.
DESCRIPTION OF THE PREFERRED EMBODIMENT
The process of the instant invention involves four basic steps: an incubation wherein antibiotic (if any) in the 85~6 1 sample and tagged antibiotic become bound to antibiotic sensitive cells or cell subunits; a separation wherein the cells with bound antibiotic are isolated from the remainder of the system;
a determination wherein an indication of the quantity of tagged antibiotic associated either with the cells or the separated ~7a-`'~
1 liquid is obtained; and a comparison wherein the determination is compared with a standard. As the concentration of antibiotic in the test sample increases, fewer tagged antibiotic molecules will report in association with the cells ~and more with the separated liquid).
By preparing a series of samples of known antibiotic concentration and treating them in accordance with the process of the invention, a standard curve of antibiotic concentration versus counts per minute (in case of radioactive tags) or the like can be produced. Assays of unknowns will give a quantitative indication of the antibiotic concentration if compared with such a curve. An indication of the presence of antibiotic in a sample may also be obtained by comparing the test sample read out of tagged antibiotic with the results of assays run in parallel with samples containing known amounts of antibiotic.
If only the presence of antibiotic in concentrations above a certain maximum is sought, the necessary comparison step may be conducted indirectly. This is done by noting the read out associated with the test sample and comparing with a previously determined read out which has been correlated with the critical antibiotic concentration This simple comparison is made possible by the standardization of reagents and procedure.
- Thus, the technician conducting the test inherently makes the comparison when he interprets the read out.
It is believed that the favourable sensitivity and speed characteristic of the assay are traceable to the typically high binding constants for the reactions between antibiotics and cells and to the specificity of the antibiotics for their sites of action. Thus, in the process of the invention, even extremely small quantities of antibiotic in a test sample rapidly 51~
1 become attached to receptor sites on the cell membrane or other location on the microorganism with which it is incubated.
In this regard, it is well known that many antibiotics operate by attaching themselves onto certain cell locations, thereby preventing normal reproductive metabolism. For example, vancomycin, bacitracin, penicillins, and cephalosporins are known to inhibit cell reproduction by attaching to receptor sites on the cell membranes to thereby prevent cell wall synthesis. See, for example, Correlation between Growth Inhibition and the Binding of Various Penicillins and Cephalosporins to Staphylococcus aureus, J.R. Edwards et al., Journal of Bacteriology, August 1969, p. 459-462; and The Actions of Penicillin and Other Antibiotics on Bacterial Cell Wall Synthsis, J.L. Strominger, Hopkins Med. J., V. 133, p. 63 (1973). Other antibiotics have sites of action somewhat less accessible than the cell membrane, but nevertheless operate by attaching themselves to specific cell locations such as ribosomes or nucleic acids. Accordingly, in addition to the ~ lactam antibiotics and other antibiotics set forth above, the process of the invention can detect erythromycin, lincomycin, actinomycin, and tetracyclines, as well as aminoglycosides such as streptomycin, neomycin, etc. These latter substances have ' various sites of action associated with the cells such as ribosomes and the like.
If tagged antibiotics are included in the incubation with the sample, then tagged and untagged molecules compete for the available receptor sites. If the tagged molecules are added after the sample has been incubated with the cells for a time, then tagged antibiotic molecules attach to remaining receptor sites. In either case, since by detecting the presence 5~
1 of tagged molecules immobilized on the cells one can obtain a measure of the extent of tagged antibiotic binding, an indirect measure of untagged molecule binding results since the amount of tagged antibiotic on the cells is a function of the quantity of antibiotic present in the sample.
Stated differently, the process of the invention depends on tagged antibiotic molecules and antibiotic which may be present in the sample seeking to react with a finite number of receptor sites on the cells either simultaneously or sequentially. Thus, a successful test may be conducted where the antibiotic to be detected and the tagged substance are the same antibiotic. However, any suitably tagged member of the class of antibiotics or related molecules such as antibiotic precursors or derivatives which attach to the same group of receptor sites as the antibiotic to be detected can be used in .
the incubation. For example, any specific penicillin or cephalo- -sporin antibiotic, suitably tagged, can be used to detect the same, any other, or a mixture of penicillins or cephalosporins.
However, preferred antibiotics used for tagging are those members of a given class which have a high binding constant such as benzylpenicillin (penicillin G),ampicillin, or cephalosporidine in the class of ~ lactam antibiotics. An example of an antibiotic precursor in this class is 6 amino penicillanic acid; an example of a derivative is the reaction product of 6 amino penicillanic acid and tyrosine. As used below, the term "tagged antibiotic" or "tagged substance"
includes tagged antibiotics, antibiotic precursors and deri-vatives, and other related substances which have an affinity for the sites of action of their parent antibiotic.
Regarding the nature of the tag, the state of the art ~3S5~
1 is such that there are several types of tags available. Thus, the tagged antibiotic can contain a 14C, or I, or other radioactive atom detectable by a counter or the like, or any enzyme, enzyme inhibitor, (e.g. methatrexate) or coenzyme (e.g. NAD) which are detectable by subjecting the tagged anti-biotic to a substrate solution which undergoes a detectable chemical reaction under the catalytic influence of the tag, or in which a reaction is inhibited. Excellent results have been obtained using radioactive tagged antibiotics, for example, C tagged benzylpenicillin (penicillin G), which is now commercially available. Those skilled in the art will appreciate that in most cases a derivative to the antibiotic molecule must be synthesized in order to furnish a site of attachment for a radioactive iodine atom. Enzyme tags have also been used with success,and coenzymes such as dehydrogenase coenzymes can be employed. The presence of the enzyme tag is determined in a ! known manner using an enzyme substrate solution which, for example, changes colour on exposure to the enzyme. The quanti-tative determination of the coenzyme is accomplished by sub-jecting the separated cells (or remaining liquid) to a solution containing a substrate which changes colour when acted upon by an enzyme system, a critical component of which is the coenzyme.
The other enzymes in the system are included in the solution.
Broadly, the cells which can be used in the process of the invention comprise any cell which is sensitive to the antibiotic to be detected. Thus, for example, any microorganism that is inhibited by penicillin could be used to detect peni-cillin type antibiotics. However, to improve sensitivity, it is much preferred to employ a microorganism which is "super-sensitive" to the antibiotic to be detected. In recent years, 35~L6 1 many strains of cells supersensitive to either specific anti-biotics or classes of antibiotics have become available.
Examples of ~ lactam sensitive microorganisms include B. subtilis, B. megaterium, S. aureus, Ps. aeuginosa, and certain mutants of _. coli. Most favourable results in terms of speed and sensitivity have been achieved using supersensitive microorganism strains which have an optimum temperature of growth generally in excess of about 50C. The preferred microorganism in this regard is Bacillus stearothermophilus (A.T.C.C. No. 10149).
B. stearothermophilus A.T.C.C. No. 15952 may also be used.
Because the incubations can be conducted at high temperature with this type of microorganism, the rate of antibiotic binding is increased.
It should be noted that whole cells need not necessarily be used in the process of the invention. Thus, the cellular - subunits on which the receptor sites are located, e.g., cell walls or membranes, ribosomes, bound enzymes, etc., may be used in place of the intact cells. For example, penicillin G is known to bind to and inhibit the action of D-alanine carboxy-20 peptidase which is normally immobilized on cell membranes.
However, methods are now available for the isolation of such enzymes, and these may be used in place of whole cells ~see, Purification and Characterization of Thermophilic D-Alanine Carboxypeptidase from Membranes of B. stearothermophilus, R.
Rogers et al., J. Biol. Chem. V. 249 p. t4863-4876). While separation of such subunits from the remainder of the reaction mixture may be difficult to accomplish quickly unless, as taught herein, the subunits are immobilized on an insoluble solid or the like, those skilled in the art will appreciate that the use of such materials is the equivalent of using whole cells.
1 As used herein~ the term "cell parts" accordingly refers to whole cells as well as cellular subunits such as cell membranes or other structures containing receptor sites.
The process of the invention can also be practiced using cell parts containing receptor sites immobilized on a suitable support such as a cotton swab, collagen matrix, poly-styrene well or bead, polyacrylamide gel, or a small quantity of a shape retaining nutrient agar attached to a dip stick. In this situation, separation of the cells from the liquid com-ponents of the reaction mixture is greatly simplified andspeeded up. When whole cells are used a preferred method of separation is by centrifugation. However, other methods of separating the cells from the remainder of the reaction mixture, e.g., ultrafiltration, can be employed.
In view of the foregoing it will be appreciated that the test of the invention may be adapted to detect the presence - of a variety of antibiotics or mixtures thereof in a number of different liquid media. A variety of tagged antibiotics and cells or cell subunits may be used, and either a qualitative or quantitative test may be designed. The invention will now be described with reference to specific, nonlimiting embodiments, the first of which is suitable for rapidly screening milk samples or the presence of as little as 0.001 I.UJ of penicillin.
Bacillus stearothermophilus was grown in accordance with the following.
Medium:
Solution A - Difco yeast extract 2.58 kg Difco bactotryptone 2.58 kg Sodium phosphate, dibasic 1.54 kg B5~6 Potassium phosphate, monobasic 515 g Ammonium sulfate 515 g Distilled water 515 liters Solution B - Magnesium sulfate, anhydrous 103 g Distilled water 1.29 liters Solution C - Calcium chloride 2 H2O 12.9 g Manganese chloride 0.52 g Distilled water 1.29 liters .-Solution D - Glucose Z.58 kg Distilled water 12.9 liters Conditions: 60C, aeration, 10% inoculum Growing time: 2.5 hours Procedure:
Solution A is sterilized in the fermentor. Solution B
is sterilized separately and 2.5 ml added to each liter of Solution A. The final concentration of magnesium sulfate in the medium is 0.2 g/liter. Solution C is sterilized separately and 2.5 ml added to each liter of Solution A. The final con-centration in the medium is:20 calcium chloride 2 H2O 0.025 g/liter manganese chloride 0.001 g/liter Solution D is sterilized separately and 25 ml added to each liter of Solution A. The final concentration of glucose in the medium is 5 g/liter.
The temperature of Solution A in the fermentor should be between 60-65C. Solutions B, C, and D are added with vigorous stirring of the fermentor. Slow addition of Solution C and vigorous stirring were necessary to prevent the formation of a precipitate. The complete medium has a pH of 6.9 without adjustment. Dow Corning B antifoam is added to give a concen-1 tration of 0.1%. Very vigorous aeration is required for growth of the organism.
When the fermentation is initiated, a carboy containing six liters of complete medium is inoculated with the contents of two one-liter Ehrlenmeyer flasks, each of which contains 150 ml of a culture grown overnight. When a reading of O.D.
0.150 (600 m,u)is attained, the entire contents of the carboy are used to inoculate the 60 liter fermentor. When the solids are 0.03%, the 60 liter fermentor is used to inoculate the 530 liter fermentor.
The cells are washed twice with five volumes of 0.2M
NH4Cl, pH 7.0, then twice with 10 volumes of a buffer containing O.OlM magnesium acetate, O.OlM mercaptoethanol, and 0.02 Tris-HCl buffer, pH 7.8.
The cells were then centrifuged and after decanting the supernatant, were resuspended in a mixed solution of A, B, and C (above) and dispensed into vials. The contents of each vial can be freeze dried by known methods and stored at about 4C
~ for extended periods of time. Freeze drying in the growth medium ; 20 (minus glucose solution) preserves the maximum antibiotic binding activity of the cells. Such freeze dried cells con-stitute an ideal concentration stabilized source of cells for use in a test set.
The tagged antibiotic employed was benzylpenicillin ~' 14 ~S~
: containing a C atom (~,u Curie/mg) and was purchased from Amersham Searle Co. This reagent can also be freeze dried for stability.
To obtain quantitative results and to act as a control, two or more liquid samples, one of which is known to be anti-biotic free and the other of which contains a known quantity of ., .
516 ~ ~
1 the antibiotic to be detected, e.g., 0.01 unit'of penicillin, can be tested in parallel. These samples can also be freeze dried for stability. Any of the foregoing reagents can be reconstituted with distilled water or phosphate buffer having a slightly acidic pH. For penicillins, binding occurs optimally between p~ 6.0-7Ø The stabilized cells, tagged antibiotic, and controls (or standard curve) constitute a test set suitable for conducting assays in accordance with the invention.
The reagents set forth above were used to prepare a standard curve and to test unknowns for the presence of penicillin.
One standard curve was prepared by subjecting 12 1.0 ml milk samples containing known concentrations of benzylpenicillin (penicillin G) to an incubation with a ~1) 100 ml sample of a B. stearothermophilus suspension (reconstituted from freeze dried material by adding 2.5 ml distilled water to a vial produced as set forth above) and (2) 14C tagged benzylpenicillin (penicillin G) in sufficient quantity to give about 10,000 counts/minute. The incubation was conducted for three minutes in a dry bath incubator set at 90C. The cells were then separated by centrifugation at 3,000 xg for two minutes. The supernatant was discarded, and the tube was rinsed with phos-phate bufer without disturbing the cells. The cells were then transferred from the tube to a scintillation vial using scintil-lation fluid as a wash. This latter step may be eliminated and the tagged molecules directly detected if a 125I tag is used. Results are set forth in Table I below:
11~8516 Standard Curve for Detection of Benzylpenicillin Background Average **
Sample [Benzylpen] Corrected Counts/10 C/CO
No.ng/ml Counts/10 min. Counts/10 min. min.
1 * 331 84 102 .007
The ability to detect small concentrations of anti-biotics in liquids is important in various situations. One example is the food industry where the use of antibiotics in the treatment of animals whieh produce food stuffs has created a need for a rapid, accurate test method which can be used in the field by bulk food handlers and the like. Because peni-cillins are used to treat mastitis in dairy cattle, penicillin detection methods suitable for rapidly and accurately screening milk are partieularly important. Thus, the growing medical eoneern about ingestion of small amounts of antibioties by humans is directing attention to the incidence of penieillin in milk at levels in the range of 0.010 - 0.050 I.U./ml or greater and to simple screening methods for detecting such minute quantities of penicillin and other antibiotics.
At the present time, there are a variety of known antibiotie deteetion procedures. Most of these involve miero-biologieal teehniques wherein the presenee or absenee of peni-eillin or other antibiotie is determined by observing the inhibition of growth of antibiotie sensitive mieroorganisms in the presenee of the test sample. Formerly, sueh proeedures required ineubation times of four hours or more, but by employing mieroorganism strains "super-sensitive" to antibioties, the time required to perform the assays has been redueed to between 2 and 2 1/2 hours.
Other antibiotie detection techniques exploit various other unique properties of the antibiotic or class of antibiotics ~a~
1 to be detected as a test basis. Thus, in Immobilized Enzyme-Based Flowing-Stream Analyzer for Measurement of Penicillin in Fermentation Broths, J.F. Rusling et al., Analytical Chemistry, Vol. 48, p. 1211, (July, 1976), a test based on the enzymatic hydrolysis of penicillin with an immobilized ~ lactamase derivative is disclosed. Simple Ultrasensitive Test for Detecting Penicillin in Milk, J.M.A. Palmer et al., J. Dairy Science, Vol. 50, p. 1390, discloses a penicillin detection method based on the growth of Bacillus subtilis spores on nutrient-spore-dye paper discs residing in a small sample of milk exposed to the air. As the water content of the milk evaporates, penicillin concentration, if any, increases and induces a colour change in the dye which is indicative of concentration.
U.S. Patent No. 3,586,483 to J.G. Heider et al. discloses a method of detecting tetracycline antibiotics in fluids by absorbing the fluid on an absorbent strip containing a complexing metal which forms a fluorescent metal complex with the antibiotic, and by observing the fluorescence of the metal complex under ultraviolet light.
The foregoing and other available detection techniques vary widely with respect to their sensitivity and speed. It is believed that no presently available test is capable of detecting as little as 0.01 I.U. of penicillin per milliliter (6 ng/ml) in less than an hour. If such a test were available, it would become economical to rapidly and reliably determine, for example, whether milk sampled in the field from relatively small batches contained antibiotic concentrations in excess of Food and Drug Administration standards.
SUMMARY OF THE INVENTION
The instant invention provides a novel procedure for 11 .3 85~
1 detecting the presence of antibiotics in liquids. Certain embodiments of the invention are ideally suited for screening milk for penicillin type antibiotics, and the invention will be described in detail with reference to this application.
However, in view of the description which follows, it will be apparent that the invention may be used to assay body fluids, meat extracts, fermentation broths and the like for a variety of antibiotic drugs. The outstanding advantage of the process of the invention is that it is capable of detecting very small antibiotic concentrations, e.g., .001 I.U./ml of penicillin, and can do so rapidly, e.g., in less than about 10 minutes.
In its broadest aspects, the process of the invention comprises the steps of incubating the sample with cells, or in some cases, cellular subunits, of an antibiotic sensitive microorganism under conditions to allow antibiotic molecules that may be present in the sample to attach to receptor sites associated with the cells, incubating the cell-sample mixture with a tagged antibiotic or antibiotic precursor under the same conditions, separating the cells from the liquid portion of the reaction mixture, determining the amount of tagged antibiotic present either in association with the separated cells or the remaining liquid, and comparing the determination to a standard. If antibiotic molecules were present in the sample, then the tagged and untagged molecules both seek to attach themselves onto a finite number o receptor sites on the cells or cellular subunits, and the amount of tagged antibiotic which becomes immobilized is inversely proportional to the concentration of antibiotic in the test sample. Similarly, the amount of tagged antibiotic which remains in the liquid phase will be a function of original antibiotic concentration.
~r.5~8S~l6 1 The surprising sensitivity and speed of the test is believed to be a consequence of both the high binding constant characteristic of the attraction between antibiotic molecules and receptor sites on cell walls or other subcellular structures of antibiotic sensitive organisms, and the specificity of anti-biotic molecules for their sites of action on such organisms.
For example, the binding constant for penicillins with receptor sites on gram positive cells is orders of magnitude greater than the binding constant for the antigen-antibody immunochemical reaction on which analogous detection techniques (immunoassays) have been based.
In an important embodiment of the process of the invention, in the interest of providing a rapid test suitable for use in screening milk, a single incubation is conducted involving the antibiotic sensitive microorganism, the sample to be tested, and the tagged antibiotic or antibiotic precursor, and the separation step is effected by centrifugation. Both the speed and sensitivity of the assay are promoted by employing cell strains which are supersensitive to a class of antibiotics or specific antibiotic to be detected. Extending this concept one step further, the preferred antibiotic sensitive microorganisms are strains having a temperature of optimum growth above about 50C. In this situation, the incubation can be conducted at relatively high temperatures and the kinetics of the antibiotic-receptor site organic reaction are enhanced.
The preferred microorganism for conducting assays for ~ lactam antibiotics is Bacillus stearothermophilus (A.T.C.C. No. 10149 or 15952). However, various other antibiotic supersensitive cell strains can be used, and indeed, merely sensitive strains can be used if reduced sensitivity can be tolerated. Nonlimiting 5ll6 1 examples of suitable cell strains include certain mutants of E. coli, Ps. aeruginosa, B. subtilis, and S. aureus.
In embodiments of the process of the invention wherein speed is less important than sensitivity, the sample and cell culture are incubated for a time and the tagged antibiotic is added later. When employing sensitive microorganisms having the high optimum growth rate, it has been noted that sensitivity does not increase linearly with time and that incubations of greater than 15 minutes in duration should not be conducted.
In preferred embodiments of the invention, the tagged antibiotic is benzylpenicillin, or the antibiotic pre-cursor 6 amino penicillanic acid, and the antibiotic to be detected is a ~ lactam antibiotic such as benzylpenicillin, cephalosporin, ampicillin, oxacillin, methicillin, cloxacillin, cephaloridine, and cephalothin. Other nonlimiting antibiotics which can be detected include erythromycin, lincomycin, actino-mysin, vancomycin, bacitracin and aminoglycosides such as streptomycin, gentamycin, kanamycin, and neomycin. The process of the invention works exceptionally well with penicillin or penicillin-like antibiotics of the type which inhibit cell reproduction by attaching to sites on cell membranes to inhibit cell wall synthesis. However, antibiotics having other sites of action such as rifamycin and tetracyclines can also be detected. The tagged antibiotic can be tagged with a radio-active atom, an enzyme, enzyme inhibitor, or a conenzyme.
Presently preferred tags include a 14C incorporated in the structure of the antibiotic molecule, an 125I atom attached to the antibiotic via reaction with, for example, tyrosine or suitable derivative thereof, and an enzyme such as peroxidase.
In another important embodiment of the inventlon, cell ~85~6 1 parts containing receptor sites are immobilized on an insoluble support, e.g., a cotton swab. This approach facilitates the separation step since, after incubation, the support can simply be removed from the liquid and washed. The need for a device capable of detecting radioactive decay products can be eliminated by tagging with an enzyme. In this case, the determination of the presence-of the enzyme tag is made by incubating the support with an enzyme substrate solution capable of undergoing a detectable chemical change under the catalytic influence of the enzyme. A preferred system includes a peroxidase tag and a substrate solution comprising H202 and 4-amino antipyrine which changes colour in the presence of peroxidase.
In accordance with another aspect of the invention, a test set for detexmining the presence of antibiotics in liquid samples is provided. The set has a quantity of con-centration stabilized antibiotic sensitive (preferably super-sensitive) cells, a quantity of tagged antibiotic or antiblotic precursor selected to have a high binding constant with the sites on the cells, and a standard against which the results of tests made with the reagents can be compared. In preferred embodiments, the test set includes freeze dried Bacillus stearothermophilus, the tagged antibiotic is benzylpenicillin tagged with a radioactive atom, e.g., 14C, and the standard is either a standard curve or at least one sample containing a known concentration of the antibiotic or class of antibiotics to be detected.
Another embodiment of the test set comprises a water insoluble support having penicillin super sensitive cells or cell membranes immobilized thereon, enzyme-tagged 6 amino-penicillanic acid, and an enzyme substrate solution capable of ~ 85~.6 1 undergoing a colour change in res~nse to the catalyticinfluence of the enzyme tag. The enzyme tag can be peroxidase;
the substrate solution comprises H202, phenol, and 4 amino antipyrine.
Accordingly, objects of the invention include the pro-vision of an antibiotic detection technique which is capable of detecting a large number of different antibiotic drugs in a variety of liquid media, which can be adapted to be very rapid and acceptably sensitive, and which can be adapted to be slightly slower, but extraordinarily sensitive. Another object of the invention is to provide a fast antibiotic detection technique which can be conveniently conducted outside of the laboratory. Another object is to provide a test set suitable for determining the presence of as little as 1 ng/ml of antibiotic in a liquid sampled from materials such as milk, body fluids, meat residues, fermentation broths and the like.
Yet another object is to provide a test set which enables the process of the invention to be conducted without a centrifuge or scintillation counter.
These and other objects and features of the invention will be apparent from the following detailed description of some important embodiments. -;`
BRIEF DESCRIPTION OF THE DRAWINGS
r FIGURE 1 is a flow chart illustrating the antibiotic detection process of the invention; and FIGURE 2 shows a standard curve for benzylpenicillin.
DESCRIPTION OF THE PREFERRED EMBODIMENT
The process of the instant invention involves four basic steps: an incubation wherein antibiotic (if any) in the 85~6 1 sample and tagged antibiotic become bound to antibiotic sensitive cells or cell subunits; a separation wherein the cells with bound antibiotic are isolated from the remainder of the system;
a determination wherein an indication of the quantity of tagged antibiotic associated either with the cells or the separated ~7a-`'~
1 liquid is obtained; and a comparison wherein the determination is compared with a standard. As the concentration of antibiotic in the test sample increases, fewer tagged antibiotic molecules will report in association with the cells ~and more with the separated liquid).
By preparing a series of samples of known antibiotic concentration and treating them in accordance with the process of the invention, a standard curve of antibiotic concentration versus counts per minute (in case of radioactive tags) or the like can be produced. Assays of unknowns will give a quantitative indication of the antibiotic concentration if compared with such a curve. An indication of the presence of antibiotic in a sample may also be obtained by comparing the test sample read out of tagged antibiotic with the results of assays run in parallel with samples containing known amounts of antibiotic.
If only the presence of antibiotic in concentrations above a certain maximum is sought, the necessary comparison step may be conducted indirectly. This is done by noting the read out associated with the test sample and comparing with a previously determined read out which has been correlated with the critical antibiotic concentration This simple comparison is made possible by the standardization of reagents and procedure.
- Thus, the technician conducting the test inherently makes the comparison when he interprets the read out.
It is believed that the favourable sensitivity and speed characteristic of the assay are traceable to the typically high binding constants for the reactions between antibiotics and cells and to the specificity of the antibiotics for their sites of action. Thus, in the process of the invention, even extremely small quantities of antibiotic in a test sample rapidly 51~
1 become attached to receptor sites on the cell membrane or other location on the microorganism with which it is incubated.
In this regard, it is well known that many antibiotics operate by attaching themselves onto certain cell locations, thereby preventing normal reproductive metabolism. For example, vancomycin, bacitracin, penicillins, and cephalosporins are known to inhibit cell reproduction by attaching to receptor sites on the cell membranes to thereby prevent cell wall synthesis. See, for example, Correlation between Growth Inhibition and the Binding of Various Penicillins and Cephalosporins to Staphylococcus aureus, J.R. Edwards et al., Journal of Bacteriology, August 1969, p. 459-462; and The Actions of Penicillin and Other Antibiotics on Bacterial Cell Wall Synthsis, J.L. Strominger, Hopkins Med. J., V. 133, p. 63 (1973). Other antibiotics have sites of action somewhat less accessible than the cell membrane, but nevertheless operate by attaching themselves to specific cell locations such as ribosomes or nucleic acids. Accordingly, in addition to the ~ lactam antibiotics and other antibiotics set forth above, the process of the invention can detect erythromycin, lincomycin, actinomycin, and tetracyclines, as well as aminoglycosides such as streptomycin, neomycin, etc. These latter substances have ' various sites of action associated with the cells such as ribosomes and the like.
If tagged antibiotics are included in the incubation with the sample, then tagged and untagged molecules compete for the available receptor sites. If the tagged molecules are added after the sample has been incubated with the cells for a time, then tagged antibiotic molecules attach to remaining receptor sites. In either case, since by detecting the presence 5~
1 of tagged molecules immobilized on the cells one can obtain a measure of the extent of tagged antibiotic binding, an indirect measure of untagged molecule binding results since the amount of tagged antibiotic on the cells is a function of the quantity of antibiotic present in the sample.
Stated differently, the process of the invention depends on tagged antibiotic molecules and antibiotic which may be present in the sample seeking to react with a finite number of receptor sites on the cells either simultaneously or sequentially. Thus, a successful test may be conducted where the antibiotic to be detected and the tagged substance are the same antibiotic. However, any suitably tagged member of the class of antibiotics or related molecules such as antibiotic precursors or derivatives which attach to the same group of receptor sites as the antibiotic to be detected can be used in .
the incubation. For example, any specific penicillin or cephalo- -sporin antibiotic, suitably tagged, can be used to detect the same, any other, or a mixture of penicillins or cephalosporins.
However, preferred antibiotics used for tagging are those members of a given class which have a high binding constant such as benzylpenicillin (penicillin G),ampicillin, or cephalosporidine in the class of ~ lactam antibiotics. An example of an antibiotic precursor in this class is 6 amino penicillanic acid; an example of a derivative is the reaction product of 6 amino penicillanic acid and tyrosine. As used below, the term "tagged antibiotic" or "tagged substance"
includes tagged antibiotics, antibiotic precursors and deri-vatives, and other related substances which have an affinity for the sites of action of their parent antibiotic.
Regarding the nature of the tag, the state of the art ~3S5~
1 is such that there are several types of tags available. Thus, the tagged antibiotic can contain a 14C, or I, or other radioactive atom detectable by a counter or the like, or any enzyme, enzyme inhibitor, (e.g. methatrexate) or coenzyme (e.g. NAD) which are detectable by subjecting the tagged anti-biotic to a substrate solution which undergoes a detectable chemical reaction under the catalytic influence of the tag, or in which a reaction is inhibited. Excellent results have been obtained using radioactive tagged antibiotics, for example, C tagged benzylpenicillin (penicillin G), which is now commercially available. Those skilled in the art will appreciate that in most cases a derivative to the antibiotic molecule must be synthesized in order to furnish a site of attachment for a radioactive iodine atom. Enzyme tags have also been used with success,and coenzymes such as dehydrogenase coenzymes can be employed. The presence of the enzyme tag is determined in a ! known manner using an enzyme substrate solution which, for example, changes colour on exposure to the enzyme. The quanti-tative determination of the coenzyme is accomplished by sub-jecting the separated cells (or remaining liquid) to a solution containing a substrate which changes colour when acted upon by an enzyme system, a critical component of which is the coenzyme.
The other enzymes in the system are included in the solution.
Broadly, the cells which can be used in the process of the invention comprise any cell which is sensitive to the antibiotic to be detected. Thus, for example, any microorganism that is inhibited by penicillin could be used to detect peni-cillin type antibiotics. However, to improve sensitivity, it is much preferred to employ a microorganism which is "super-sensitive" to the antibiotic to be detected. In recent years, 35~L6 1 many strains of cells supersensitive to either specific anti-biotics or classes of antibiotics have become available.
Examples of ~ lactam sensitive microorganisms include B. subtilis, B. megaterium, S. aureus, Ps. aeuginosa, and certain mutants of _. coli. Most favourable results in terms of speed and sensitivity have been achieved using supersensitive microorganism strains which have an optimum temperature of growth generally in excess of about 50C. The preferred microorganism in this regard is Bacillus stearothermophilus (A.T.C.C. No. 10149).
B. stearothermophilus A.T.C.C. No. 15952 may also be used.
Because the incubations can be conducted at high temperature with this type of microorganism, the rate of antibiotic binding is increased.
It should be noted that whole cells need not necessarily be used in the process of the invention. Thus, the cellular - subunits on which the receptor sites are located, e.g., cell walls or membranes, ribosomes, bound enzymes, etc., may be used in place of the intact cells. For example, penicillin G is known to bind to and inhibit the action of D-alanine carboxy-20 peptidase which is normally immobilized on cell membranes.
However, methods are now available for the isolation of such enzymes, and these may be used in place of whole cells ~see, Purification and Characterization of Thermophilic D-Alanine Carboxypeptidase from Membranes of B. stearothermophilus, R.
Rogers et al., J. Biol. Chem. V. 249 p. t4863-4876). While separation of such subunits from the remainder of the reaction mixture may be difficult to accomplish quickly unless, as taught herein, the subunits are immobilized on an insoluble solid or the like, those skilled in the art will appreciate that the use of such materials is the equivalent of using whole cells.
1 As used herein~ the term "cell parts" accordingly refers to whole cells as well as cellular subunits such as cell membranes or other structures containing receptor sites.
The process of the invention can also be practiced using cell parts containing receptor sites immobilized on a suitable support such as a cotton swab, collagen matrix, poly-styrene well or bead, polyacrylamide gel, or a small quantity of a shape retaining nutrient agar attached to a dip stick. In this situation, separation of the cells from the liquid com-ponents of the reaction mixture is greatly simplified andspeeded up. When whole cells are used a preferred method of separation is by centrifugation. However, other methods of separating the cells from the remainder of the reaction mixture, e.g., ultrafiltration, can be employed.
In view of the foregoing it will be appreciated that the test of the invention may be adapted to detect the presence - of a variety of antibiotics or mixtures thereof in a number of different liquid media. A variety of tagged antibiotics and cells or cell subunits may be used, and either a qualitative or quantitative test may be designed. The invention will now be described with reference to specific, nonlimiting embodiments, the first of which is suitable for rapidly screening milk samples or the presence of as little as 0.001 I.UJ of penicillin.
Bacillus stearothermophilus was grown in accordance with the following.
Medium:
Solution A - Difco yeast extract 2.58 kg Difco bactotryptone 2.58 kg Sodium phosphate, dibasic 1.54 kg B5~6 Potassium phosphate, monobasic 515 g Ammonium sulfate 515 g Distilled water 515 liters Solution B - Magnesium sulfate, anhydrous 103 g Distilled water 1.29 liters Solution C - Calcium chloride 2 H2O 12.9 g Manganese chloride 0.52 g Distilled water 1.29 liters .-Solution D - Glucose Z.58 kg Distilled water 12.9 liters Conditions: 60C, aeration, 10% inoculum Growing time: 2.5 hours Procedure:
Solution A is sterilized in the fermentor. Solution B
is sterilized separately and 2.5 ml added to each liter of Solution A. The final concentration of magnesium sulfate in the medium is 0.2 g/liter. Solution C is sterilized separately and 2.5 ml added to each liter of Solution A. The final con-centration in the medium is:20 calcium chloride 2 H2O 0.025 g/liter manganese chloride 0.001 g/liter Solution D is sterilized separately and 25 ml added to each liter of Solution A. The final concentration of glucose in the medium is 5 g/liter.
The temperature of Solution A in the fermentor should be between 60-65C. Solutions B, C, and D are added with vigorous stirring of the fermentor. Slow addition of Solution C and vigorous stirring were necessary to prevent the formation of a precipitate. The complete medium has a pH of 6.9 without adjustment. Dow Corning B antifoam is added to give a concen-1 tration of 0.1%. Very vigorous aeration is required for growth of the organism.
When the fermentation is initiated, a carboy containing six liters of complete medium is inoculated with the contents of two one-liter Ehrlenmeyer flasks, each of which contains 150 ml of a culture grown overnight. When a reading of O.D.
0.150 (600 m,u)is attained, the entire contents of the carboy are used to inoculate the 60 liter fermentor. When the solids are 0.03%, the 60 liter fermentor is used to inoculate the 530 liter fermentor.
The cells are washed twice with five volumes of 0.2M
NH4Cl, pH 7.0, then twice with 10 volumes of a buffer containing O.OlM magnesium acetate, O.OlM mercaptoethanol, and 0.02 Tris-HCl buffer, pH 7.8.
The cells were then centrifuged and after decanting the supernatant, were resuspended in a mixed solution of A, B, and C (above) and dispensed into vials. The contents of each vial can be freeze dried by known methods and stored at about 4C
~ for extended periods of time. Freeze drying in the growth medium ; 20 (minus glucose solution) preserves the maximum antibiotic binding activity of the cells. Such freeze dried cells con-stitute an ideal concentration stabilized source of cells for use in a test set.
The tagged antibiotic employed was benzylpenicillin ~' 14 ~S~
: containing a C atom (~,u Curie/mg) and was purchased from Amersham Searle Co. This reagent can also be freeze dried for stability.
To obtain quantitative results and to act as a control, two or more liquid samples, one of which is known to be anti-biotic free and the other of which contains a known quantity of ., .
516 ~ ~
1 the antibiotic to be detected, e.g., 0.01 unit'of penicillin, can be tested in parallel. These samples can also be freeze dried for stability. Any of the foregoing reagents can be reconstituted with distilled water or phosphate buffer having a slightly acidic pH. For penicillins, binding occurs optimally between p~ 6.0-7Ø The stabilized cells, tagged antibiotic, and controls (or standard curve) constitute a test set suitable for conducting assays in accordance with the invention.
The reagents set forth above were used to prepare a standard curve and to test unknowns for the presence of penicillin.
One standard curve was prepared by subjecting 12 1.0 ml milk samples containing known concentrations of benzylpenicillin (penicillin G) to an incubation with a ~1) 100 ml sample of a B. stearothermophilus suspension (reconstituted from freeze dried material by adding 2.5 ml distilled water to a vial produced as set forth above) and (2) 14C tagged benzylpenicillin (penicillin G) in sufficient quantity to give about 10,000 counts/minute. The incubation was conducted for three minutes in a dry bath incubator set at 90C. The cells were then separated by centrifugation at 3,000 xg for two minutes. The supernatant was discarded, and the tube was rinsed with phos-phate bufer without disturbing the cells. The cells were then transferred from the tube to a scintillation vial using scintil-lation fluid as a wash. This latter step may be eliminated and the tagged molecules directly detected if a 125I tag is used. Results are set forth in Table I below:
11~8516 Standard Curve for Detection of Benzylpenicillin Background Average **
Sample [Benzylpen] Corrected Counts/10 C/CO
No.ng/ml Counts/10 min. Counts/10 min. min.
1 * 331 84 102 .007
2 * 367 120
3 0 15457 15210 15272.5 1.000
4 0 15582 15335 1.0 13165 12918 13019.5 .853 6 1.0 13368 13121 7 5.0 9859 9~12 10002. .655 8 5.0 10639 10392 7699 .504 11 50 4175 3920 4071 .267 * 20,000 units benzylpenicillin (penicillin G) added to sample.
** Counts of sample/counts of control ~zero penicillin G
~ - present in sample).
As can be appreciated from the foregoing table, a significant and dramatic decrease in the count occurs as the amount of benzylpenicillin in the samples is increased from zero (3,4) to 10 ng/ml (9, 10). The data in Table I is graphically pre-sented in Fig. 2, wherein penicillin concentration in ng/ml is plotted vs. sampie count/zero count.
Using the foregoing table as a standard of comparison, tests were conducted on 20 milk samples prepared by a commercial milk processor. The procedure employed was identical to the procedure for preparing the standard curve set forth above except ~185~6 1 for the additional step of comparing the counts/minute obtained with the test samples with the curve and converting ng/ml of penicillin to I.U./ml (0.01 I.U.~Y6 ng). The results of these r tests are set forth in Table II below.
TABLE II
Comparison of Sensitivity of Microbiological Technique and Technique of the Invention Assay Results Known Penicillin Using Test Results Sample No. Concentration Microbiological (per ml) IU/ml Technique (IU/ml) IU
~ 1 .005 - .007 ; 2 .005 - ~009 3 ,003 - .008 4 .002 - ,003 ' ~ .002 6 .007 - .006 7 ~ - .001 8 .042 .1 .005 9 .003 - .004 .030 .06 .07 11 .020 .04 .05 12 .020 .03 .04 13 .025 .02 ,03 14 .037 .05 .06 .007 - .01 16 .017 .01 .02 17 .043 .09 .10 18 .022 .08 .09 19 .030 .07 .08 .000 - <.001
** Counts of sample/counts of control ~zero penicillin G
~ - present in sample).
As can be appreciated from the foregoing table, a significant and dramatic decrease in the count occurs as the amount of benzylpenicillin in the samples is increased from zero (3,4) to 10 ng/ml (9, 10). The data in Table I is graphically pre-sented in Fig. 2, wherein penicillin concentration in ng/ml is plotted vs. sampie count/zero count.
Using the foregoing table as a standard of comparison, tests were conducted on 20 milk samples prepared by a commercial milk processor. The procedure employed was identical to the procedure for preparing the standard curve set forth above except ~185~6 1 for the additional step of comparing the counts/minute obtained with the test samples with the curve and converting ng/ml of penicillin to I.U./ml (0.01 I.U.~Y6 ng). The results of these r tests are set forth in Table II below.
TABLE II
Comparison of Sensitivity of Microbiological Technique and Technique of the Invention Assay Results Known Penicillin Using Test Results Sample No. Concentration Microbiological (per ml) IU/ml Technique (IU/ml) IU
~ 1 .005 - .007 ; 2 .005 - ~009 3 ,003 - .008 4 .002 - ,003 ' ~ .002 6 .007 - .006 7 ~ - .001 8 .042 .1 .005 9 .003 - .004 .030 .06 .07 11 .020 .04 .05 12 .020 .03 .04 13 .025 .02 ,03 14 .037 .05 .06 .007 - .01 16 .017 .01 .02 17 .043 .09 .10 18 .022 .08 .09 19 .030 .07 .08 .000 - <.001
5~6 1 It should be noted that the foregoing tests consistently detected the presence of as little as 0.01 I.U./ml of penicillin in about 6 minutes-of testing time (not including counting time).
It should also be noted that the foregoing procedure involved the simultaneous incubation of both the sample and tagged penicillin for only three minutes and a two minute cen-trifugation. This test was specifically designed to be acceptably sensitive and as rapid as possible.
Another group of tests were run to demonstrate the sensitivity possible using B. stearothermophilus, tagged benzylpenicillin (1500 CPM), and the three minute incubation in a dry bath incubator set at 90C. The results of this series of tests are set forth in Table III below. The data set forth in Table III illustrate that a significant depression in the counts/minute associated with the separated cell fraction occurs when the antibiotic concentration of the sample is raised from zero up through 0.001 I.U./ml, 0.002 I.U./ml, and 0.005 I.U./ml. This test may be conducted, including the count-ing step, in less than 10 minutes.
TABLE III
I.U./ml Pen. G Counts/Min. Average Sample No. present in sample of cells Counts/Min.
3 .001 207 4 .001 227 .002 191 183.5
It should also be noted that the foregoing procedure involved the simultaneous incubation of both the sample and tagged penicillin for only three minutes and a two minute cen-trifugation. This test was specifically designed to be acceptably sensitive and as rapid as possible.
Another group of tests were run to demonstrate the sensitivity possible using B. stearothermophilus, tagged benzylpenicillin (1500 CPM), and the three minute incubation in a dry bath incubator set at 90C. The results of this series of tests are set forth in Table III below. The data set forth in Table III illustrate that a significant depression in the counts/minute associated with the separated cell fraction occurs when the antibiotic concentration of the sample is raised from zero up through 0.001 I.U./ml, 0.002 I.U./ml, and 0.005 I.U./ml. This test may be conducted, including the count-ing step, in less than 10 minutes.
TABLE III
I.U./ml Pen. G Counts/Min. Average Sample No. present in sample of cells Counts/Min.
3 .001 207 4 .001 227 .002 191 183.5
6 .002 176
7 .005 97 100
8 .005 103 ,,~` .
1 TABLE III -continued
1 TABLE III -continued
9 .010 89 100.5 .010112 EXAMPLE II
.
- Bacillus stearothermophilus was grown in accordance with the procedure set forth in Example I. The resulting cell suspension is then sonicated to lyse the cells, preferably while the sample is immersed in an ice bath. Unlysed cells are removed by centrifuging the sonicated suspension at 2500xg for five minutes. The cell membranes containing penicillin binding sites are isolated by centrifugation for 10 minutes at 5000 xg.
One hundred cotton swabs (cotton sections 6.5 g) were then added to 100 ml of a 30 g/l CNBr solution adjusted to pH=11.0 with 5 M NaOH. After 10 minutes, the swabs are removed, rinsed with Na~CO3 solution, and added to the cell membranes produced as set forth above contained in a suspension of NaHCO3 solution, pH=8.1. After stirring for 18 hr. at 4C, the cell membranes are covalently linked to the swabs through the cyanogen bromide. The swabs are then rinsed with distilled water, soaked in ethanolamine for two hours, rinsed again, and dried.
A conjugate of penicillanic acid and peroxidase was prepared as follows. An alkaline solution (pH=9.0) containing 28.1 mg 6 amino penicillanic acid is mixed with a phosphate buffered peroxidase solution containing 8.8 mg peroxidase and 8.2 mg car-bodiimide in water. The reaction mixture is maintained at 4C for 24 hours while stirring. As a result, the peroxidase ll~BS~6 1 and 6 amino penicillanic acid became covalently linked. The conjugate solution is next dialyzed against 0.05 M phosphate uffered saline (PBS); penicillin activity disappears from the PBS after the first change of solution. After four solution changes, the penicillin - peroxidase conjugate is ready for use.
The assay using the reagents prepared as set forth above is conducted by adding a cotton swab to respective test samples (or controls) together with 100 microliters of conjugate solution. The reaction mixture is incubated in a dry well incubator for three minutes at 50C. During this time, penicillin in the sample (if any) and the peroxidase-tagged penicillanic acid compete for sites of attachment on the cell membranes bonded to the swabs. The sample is then poured from the tube, the swab is washed with water to remove any unbound conjugate, and one ml of a substrate solution is added to the tubes. The substrate solution consists of 1.0 ml 0.3% H202, 25 mg 4 amino antipyrine, and 20 ml phenol dissolved in 50 ml of distilled water.
The swab plus substrate is then incubated at 25C. If less than 0.05 units of penicillin was present in the sample, sufficient conjugate becomes immobilized on the swab to produce, through the action of the peroxidase, a visually detectable pink to red colour in about 15 minutes. If after 15 minutes no colour develops, then the sample contained more than 0.05 units penicillin per milliliter.
For more precise results, the optical density of sub-strate may be read at 510 millimicrons on a spectrophotometer.
Results of exemplary tests are set forth below:
5~6 1 Penicillin Conc. O.D. (~a m~) 1) 0.05 units 0.75 0-05 units 1 7180 1.05 2) 0.05 units 0.70 0.77 0.00 units 1.05 1.00 àverage O.D. at 0.05 units penn./ml.- 0.75 average O.D. at 0.00 units penn./ml.- 1.05 EXAMPLE III
Another group of tests was run to demonstrate the speed and sensitivity with which streptomycin can be detected.
Six five-milliliter samples of milk were mixed with streptomycin to result in samples containing 0 ng/ml, 1 ng/ml, 10 ng/ml, 100 ng/ml, 1000 ng/ml, and 106 ng/ml. Each sample was mixed with tritium-labelled dihydrostreptomycin of 228,000 counts/
minute (commercially available for example from Amersham Searle Co.) and with _. stearothermophilus suspension prepared as described above for Example I and reconstituted from 0.2 ml freeze dried material diluted with 8 ml water. (One suitable concentration of the material prior to freeze drying is attained by resuspending the centrifugally-separated cells, as described in Example I, to a concentration such that a 1 to 500 dilution has an optical density (O.D.) of 0.60 at a wavelength of 600 millimicrons.) The mixtures were heated to facilitate the binding of the tritium-labelled dihydrostreptomycin at its binding site in the cells. In this example, accordingly, samples initially at 10-20 C were heated at 90 C in a dry block heater for three minutes so that the temperature of the samples reach 1 60-65C. The samples were then centrifuged for three minutes at 3,000 xg, the supernatants decanted, and each precipitate washed twice with distilled water. The precipitates (cells) were then transferred, with scintillation fluid, to scintillation vials, and the counts read for one minute. The results of these tests are in Table IV set forth below.
TABLE IV
Sample No. Streptomycin Count Count/
Concentration (cpm) Control Count (%) (ner ml) . . .
1 0 30,441 100 2 1 ng 25,849 84.9 3 10 ng 22,644 74.5 4 100 ng 18,123 59.6 1000 ng 14,819 48.7 6 106 ng 8,707 28.6 As can be appreciated from the foregoing table, a significant decrease in the count occurs as the amount of streptomycin in the sampl~ is increased from zero (sample 1) through one, ten 0 and 100 ng/ml, and greater. Accordingly, the presence of streptomycin in samples containing as little as 1 ng/ml can be detected by treating the sample in accordance with the foregoing procedure, and comparing the count with the data in the table.
Such determinations may be conducted in about eight minutes.
In view of the foregoing discussion, those skilled in the art will appreciate that it is possible to detect even smaller concentrations of antibiotics by, for example, incubating the sample with the antibiotic sensitive cells for sufficient time to enable all antibiotic present in the sample to attach to receptor sites. The tagged antibiotic would be added thereafter and would attach to any remaining receptor sites.
85~6 1 Aside from these modifications, the procedure would be sub-stantially identical to that set forth above and would result in a slightly slower but even more sensitive test.
The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be con-sidered in all respects as illustrative and not restrictive, the - scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
.
- Bacillus stearothermophilus was grown in accordance with the procedure set forth in Example I. The resulting cell suspension is then sonicated to lyse the cells, preferably while the sample is immersed in an ice bath. Unlysed cells are removed by centrifuging the sonicated suspension at 2500xg for five minutes. The cell membranes containing penicillin binding sites are isolated by centrifugation for 10 minutes at 5000 xg.
One hundred cotton swabs (cotton sections 6.5 g) were then added to 100 ml of a 30 g/l CNBr solution adjusted to pH=11.0 with 5 M NaOH. After 10 minutes, the swabs are removed, rinsed with Na~CO3 solution, and added to the cell membranes produced as set forth above contained in a suspension of NaHCO3 solution, pH=8.1. After stirring for 18 hr. at 4C, the cell membranes are covalently linked to the swabs through the cyanogen bromide. The swabs are then rinsed with distilled water, soaked in ethanolamine for two hours, rinsed again, and dried.
A conjugate of penicillanic acid and peroxidase was prepared as follows. An alkaline solution (pH=9.0) containing 28.1 mg 6 amino penicillanic acid is mixed with a phosphate buffered peroxidase solution containing 8.8 mg peroxidase and 8.2 mg car-bodiimide in water. The reaction mixture is maintained at 4C for 24 hours while stirring. As a result, the peroxidase ll~BS~6 1 and 6 amino penicillanic acid became covalently linked. The conjugate solution is next dialyzed against 0.05 M phosphate uffered saline (PBS); penicillin activity disappears from the PBS after the first change of solution. After four solution changes, the penicillin - peroxidase conjugate is ready for use.
The assay using the reagents prepared as set forth above is conducted by adding a cotton swab to respective test samples (or controls) together with 100 microliters of conjugate solution. The reaction mixture is incubated in a dry well incubator for three minutes at 50C. During this time, penicillin in the sample (if any) and the peroxidase-tagged penicillanic acid compete for sites of attachment on the cell membranes bonded to the swabs. The sample is then poured from the tube, the swab is washed with water to remove any unbound conjugate, and one ml of a substrate solution is added to the tubes. The substrate solution consists of 1.0 ml 0.3% H202, 25 mg 4 amino antipyrine, and 20 ml phenol dissolved in 50 ml of distilled water.
The swab plus substrate is then incubated at 25C. If less than 0.05 units of penicillin was present in the sample, sufficient conjugate becomes immobilized on the swab to produce, through the action of the peroxidase, a visually detectable pink to red colour in about 15 minutes. If after 15 minutes no colour develops, then the sample contained more than 0.05 units penicillin per milliliter.
For more precise results, the optical density of sub-strate may be read at 510 millimicrons on a spectrophotometer.
Results of exemplary tests are set forth below:
5~6 1 Penicillin Conc. O.D. (~a m~) 1) 0.05 units 0.75 0-05 units 1 7180 1.05 2) 0.05 units 0.70 0.77 0.00 units 1.05 1.00 àverage O.D. at 0.05 units penn./ml.- 0.75 average O.D. at 0.00 units penn./ml.- 1.05 EXAMPLE III
Another group of tests was run to demonstrate the speed and sensitivity with which streptomycin can be detected.
Six five-milliliter samples of milk were mixed with streptomycin to result in samples containing 0 ng/ml, 1 ng/ml, 10 ng/ml, 100 ng/ml, 1000 ng/ml, and 106 ng/ml. Each sample was mixed with tritium-labelled dihydrostreptomycin of 228,000 counts/
minute (commercially available for example from Amersham Searle Co.) and with _. stearothermophilus suspension prepared as described above for Example I and reconstituted from 0.2 ml freeze dried material diluted with 8 ml water. (One suitable concentration of the material prior to freeze drying is attained by resuspending the centrifugally-separated cells, as described in Example I, to a concentration such that a 1 to 500 dilution has an optical density (O.D.) of 0.60 at a wavelength of 600 millimicrons.) The mixtures were heated to facilitate the binding of the tritium-labelled dihydrostreptomycin at its binding site in the cells. In this example, accordingly, samples initially at 10-20 C were heated at 90 C in a dry block heater for three minutes so that the temperature of the samples reach 1 60-65C. The samples were then centrifuged for three minutes at 3,000 xg, the supernatants decanted, and each precipitate washed twice with distilled water. The precipitates (cells) were then transferred, with scintillation fluid, to scintillation vials, and the counts read for one minute. The results of these tests are in Table IV set forth below.
TABLE IV
Sample No. Streptomycin Count Count/
Concentration (cpm) Control Count (%) (ner ml) . . .
1 0 30,441 100 2 1 ng 25,849 84.9 3 10 ng 22,644 74.5 4 100 ng 18,123 59.6 1000 ng 14,819 48.7 6 106 ng 8,707 28.6 As can be appreciated from the foregoing table, a significant decrease in the count occurs as the amount of streptomycin in the sampl~ is increased from zero (sample 1) through one, ten 0 and 100 ng/ml, and greater. Accordingly, the presence of streptomycin in samples containing as little as 1 ng/ml can be detected by treating the sample in accordance with the foregoing procedure, and comparing the count with the data in the table.
Such determinations may be conducted in about eight minutes.
In view of the foregoing discussion, those skilled in the art will appreciate that it is possible to detect even smaller concentrations of antibiotics by, for example, incubating the sample with the antibiotic sensitive cells for sufficient time to enable all antibiotic present in the sample to attach to receptor sites. The tagged antibiotic would be added thereafter and would attach to any remaining receptor sites.
85~6 1 Aside from these modifications, the procedure would be sub-stantially identical to that set forth above and would result in a slightly slower but even more sensitive test.
The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be con-sidered in all respects as illustrative and not restrictive, the - scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (21)
1. A process for detecting the presence of an antibiotic in a liquid sample, said process comprising the steps of:
A. incubating the sample with a microorganism or a part thereof which has receptor sites capable of binding with the antibiotic, the incubation being conducted under conditions to allow antibiotic molecules, if present in the sample, to bind with the receptor sites;
B. incubating the mixture of step A with a tagged sub-stance comprising an antibiotic, a derivative thereof, or a precursor thereof capable of binding with said receptor sites;
C. separating the solid phase from the liquid;
D. determining the amount of tagged substance associated either with the solid phase or with the liquid; and E. comparing the determination with a standard to obtain an indication of the presence of antibiotic in the sample.
A. incubating the sample with a microorganism or a part thereof which has receptor sites capable of binding with the antibiotic, the incubation being conducted under conditions to allow antibiotic molecules, if present in the sample, to bind with the receptor sites;
B. incubating the mixture of step A with a tagged sub-stance comprising an antibiotic, a derivative thereof, or a precursor thereof capable of binding with said receptor sites;
C. separating the solid phase from the liquid;
D. determining the amount of tagged substance associated either with the solid phase or with the liquid; and E. comparing the determination with a standard to obtain an indication of the presence of antibiotic in the sample.
2. The process as set forth in claim 1 wherein the amount of tagged substance associated with the solid phase is determined.
3. The process as set forth in claim 1 wherein steps A
and B are conducted simultaneously.
and B are conducted simultaneously.
4. The process as set forth in claim 1 wherein the tag on said tagged substance is a radioactive atom, an enzyme, or a coenzyme.
5. The process as set forth in claim 1 wherein the anti-biotic to be detected is a .beta. lactam antibiotic, the tagged sub-stance includes a .beta. lactam moiety, and the microorganism is a .beta. lactam antibiotic sensitive microorganism.
6. The process as set forth in claim 1 wherein the microorganism is Bacillus stearothermophilus.
7. The process as set forth in claim 1 wherein the anti-biotic sensitive microorganism is supersensitive to the anti-biotic to be detected.
8. The process as set forth in claim 1 wherein the antibiotic sensitive microorganism is a strain having a tem-perature of optimum growth above about 50°C.
9. The process as set forth in claim 1 wherein the separation step is effected by centrifugation.
10. The process as set forth in claim 1 wherein the microorganism or parts thereof are immobilized on a water insoluble support and said separation step is effected by re-moving the support from the liquid and washing.
11. The process as set forth in claim 1 wherein the liquid sample is selected from the group consisting of milk, body fluids, liquids extracted from meat, and fermentation broths.
12. The process as set forth in claim 1 wherein the antibiotic to be detected and the tagged antibiotic are the same.
13. The process as set forth in claim 1 wherein the anti-biotic to be detected is selected from the group consisting of benzylpenicillin, cephalosporin,ampicillin, oxacillin, methicillin, cloxacillin, cephalosporidine, and cephalothin,and the tagged antibiotic is a radioactive tagged substance selected from the group consisting of 6-amino penicillanic acid and benzylpenicillin.
14. The process as set forth in claim 1 wherein the antibiotic to be detected is streptomycin and the tagged substance is tritium-tagged tetrahydrostreptomycin.
15. The process as set forth in claim 1 wherein the standard used in step E is a standard curve of antibiotic concentration versus a quantitative indication of the amount of tagged substance associated with the solid phase.
16. The process as set forth in claim 1 wherein an enzyme tag is used, an indication of the quantity of enzyme present is obtained by incubation with an enzyme substrate solution capable of undergoing a colour change in response to the catalytic influence of the enzyme tag, and the standard used in step E comprises an optical density value made under standard conditions, said value corresponding to a selected antibiotic concentration.
17. A test set for detecting the presence of antibiotic in a liquid sample, said set comprising, in combination A. concentration-stabilized cells or cell parts having receptor sites reactive with the antibiotic, B. a tagged antibiotic, antibiotic precursor, or derivative thereof capable of binding with the receptor sites when incubated with said cells or cell parts, and C. a standard to which the results of tests made with the cells and the tagged material may be compared.
18. The test set of claim 17 wherein reagent A comprises a water-insoluble support having cell parts of an antibiotic-supersensitive microorganism immobilized thereon and reagent B comprises an enzyme-tagged antibiotic, said set further com-prising an enzyme substrate solution capable of undergoing a colour change in response to the catalytic influence of the enzyme tag.
19. The test set of claim 17 wherein reagent A is made from Bacillus stearothermophilus.
20. The test set of claim 17 wherein reagent B is tagged with tritium, 125I, or 14C.
21. The test set of claim 17 wherein the standard includes at least one standard selected from the group consisting of a standard curve, at least one sample containing a known concentration of the antibiotic to be detected, and a tag readout value made under standard test conditions and correspond-ing to a selected antibiotic concentration.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US85354177A | 1977-11-21 | 1977-11-21 | |
| US853,541 | 1977-11-21 | ||
| US05/914,414 US4239852A (en) | 1978-06-12 | 1978-06-12 | Antibiotic detection method |
| US914,414 | 1978-06-12 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1108516A true CA1108516A (en) | 1981-09-08 |
Family
ID=27127165
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA316,133A Expired CA1108516A (en) | 1977-11-21 | 1978-11-10 | Antibiotic detection method |
Country Status (10)
| Country | Link |
|---|---|
| JP (1) | JPS587279B2 (en) |
| BR (1) | BR7807695A (en) |
| CA (1) | CA1108516A (en) |
| DE (1) | DE2850503C2 (en) |
| DK (1) | DK152383C (en) |
| FR (1) | FR2409517A1 (en) |
| GB (1) | GB2008248B (en) |
| IL (1) | IL55964A0 (en) |
| NL (1) | NL186930C (en) |
| NZ (1) | NZ188910A (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IL59723A (en) * | 1980-03-27 | 1983-05-15 | Teva Pharma | Determination of antibacterial agents |
| US4686182A (en) * | 1984-10-17 | 1987-08-11 | Minnesota Mining And Manufacturing Company | Method and kit for detecting antibiotics in a liquid sample |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| NL154600B (en) * | 1971-02-10 | 1977-09-15 | Organon Nv | METHOD FOR THE DETERMINATION AND DETERMINATION OF SPECIFIC BINDING PROTEINS AND THEIR CORRESPONDING BINDABLE SUBSTANCES. |
| NL171930C (en) * | 1972-05-11 | 1983-06-01 | Akzo Nv | METHOD FOR DETERMINING AND DETERMINING BITES AND TEST PACKAGING. |
-
1978
- 1978-11-10 CA CA316,133A patent/CA1108516A/en not_active Expired
- 1978-11-14 NZ NZ188910A patent/NZ188910A/en unknown
- 1978-11-16 GB GB7844810A patent/GB2008248B/en not_active Expired
- 1978-11-16 IL IL55964A patent/IL55964A0/en not_active IP Right Cessation
- 1978-11-20 DK DK516878A patent/DK152383C/en active
- 1978-11-20 FR FR7832671A patent/FR2409517A1/en active Granted
- 1978-11-21 DE DE2850503A patent/DE2850503C2/en not_active Expired
- 1978-11-21 BR BR7807695A patent/BR7807695A/en unknown
- 1978-11-21 NL NLAANVRAGE7811478,A patent/NL186930C/en not_active IP Right Cessation
- 1978-11-21 JP JP53142927A patent/JPS587279B2/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| DE2850503A1 (en) | 1979-05-23 |
| FR2409517B1 (en) | 1985-05-03 |
| DK152383B (en) | 1988-02-22 |
| IL55964A0 (en) | 1979-01-31 |
| GB2008248A (en) | 1979-05-31 |
| DE2850503C2 (en) | 1985-09-12 |
| JPS587279B2 (en) | 1983-02-09 |
| FR2409517A1 (en) | 1979-06-15 |
| BR7807695A (en) | 1979-07-31 |
| DK152383C (en) | 1988-07-25 |
| NL7811478A (en) | 1979-05-23 |
| NZ188910A (en) | 1981-01-23 |
| DK516878A (en) | 1979-05-22 |
| GB2008248B (en) | 1982-06-16 |
| NL186930C (en) | 1991-04-02 |
| JPS5496098A (en) | 1979-07-30 |
| NL186930B (en) | 1990-11-01 |
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