CA1073813A

CA1073813A - Fluorescent immunoassay of aminoglycoside antibiotics

Info

Publication number: CA1073813A
Application number: CA276,094A
Authority: CA
Inventors: David S. Smith
Original assignee: Technicon Instruments Corp
Current assignee: Bayer Corp
Priority date: 1976-04-15
Filing date: 1977-04-13
Publication date: 1980-03-18
Also published as: JPS5338619A; SE7704244L; FR2348492B1; DE2716276C2; JPS6139623B2; GB1573212A; DE2716276A1; SE442679B; NL7704126A; US4150949A; FR2348492A1

Abstract

ABSTRACT
Aminoglycomide antibotics, especially gentamicin, in biological fluid samples are assayed fluori-metrically by mixing the sample with a fluorescent-labelled compound and with antibodies. The fluor-escence of the compound is reduced when the compound binds with the antibodies, and by measuring the fluorescence of the mixture, the amount of antibiotic in the sample can be determined.

Description

10'73~3~3 This invention is concerned with immunoassays and, more particularly, with a method of assayin~ gentamicin and similar aminoglycoside antibiotics in biological 1uid samples.
Gentamicin is one of the most important of the aminoglycoside class of antibiotics. It has the structure:

2 ~ ~ NH2 NHR' I I
R / ~ ~ o ~ o NH.CH3 Gentamicin Cl: R = R' = Me Gentamicin C2: R = Me, R' = 11 ~ Gentamicin Cla:R = R~ = H

; Gentamicin preparations normally consist of a mixture of these components.
Gentamicin is dangerously toxic, particularly towards the ears ~ototoxicity), when present in the circulation ; at as little as 2-3 times the optimum therapeutic level.
Furthermore, gentamicin clearance rates from the blood vary widely from patient to patient. It is essential, therefore, that clinical administration of gentamicin be followed in each individual case by monitoring of blood for gentamicin level.
- Currently the most common assay of gentamicin is a bioassay in which the ability of a blood sample to inhibit the gro~th 3Q of bacteria is estimated.

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1()73~313 This procedure is slow, imprecise and capable of only a very low throughput of samples. It is almost certain that these problems are at present restricting the - clinical use of gentamicin.
Recently, radioimmunoassays (RIA) for gentamicin have been developed and reported in the literature. These are faster and have greater precision than the bioassay procedure, and have the additional advantage of immuno-specificity. Dis-advantages of the RIA are, firstly, the usual radiation hazards, limited life of label, and necessity for radioactive counting facilities. Secondly, the necessity for a separation step to isolate the free or antibody-bound labelled fraction for quantitation. Thirdly, the problem of radio-labelling of gentamicin. Tritiated (3H-) gentamicin, obtainable to order commercially, is known to be impure, and use of the tritium label re~uires expensive liquid scintillation counting methods.
The advantages of radioiodine labelling ~e.g. simple gamma-counting quantitation) can only be realised either by first coupling gentamicin to a suitable carrier species, followed by radio-iodination of the carrier, or by coupling or reacting gentamicin with a previously iodinated carrier. These are complex procedures which often show poor reproducibility.
We have now devised an immunoassay procedure for gentamicin and other similar aminoglycoside antibiotics, which does not involve the use of radio-active materials and has a number of advantages over the bioassay procedure re-ferred to above. In particular, we have found that if these aminoglycoside antibiotics are labelled with a fluorescent group, such as fluorescein, the fluorescence of the label is reduced when the antibiotic is treated with specific ;

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1073~313 antibody. As a xesult~ it is possible reliably and effectively to assay biological fluid samples for these antibiotics by determining the fluorescence.
According to the invention, there is provided a method of assaying a biological fluid sample for gentamicin or a similar aminoglycoside antibiotic, which comprises form-ing a mixture of the sample, a fluorescent-labelled compound (as herein defined~ and antibody (e.g. antiserum or immuno-~globulins from antiserum) against the antibiotic under assay and the said compound, and measuring the fluorescence of the mixture so formed and thereby determining the amount of amino-glycoside antibiotic present in the sample.
By "fluorescent-labelled compound" we mean a com-pound which carries a fluorescent gxoup, the fluorescence of which is reduced when the compound binds with the antibody.
Thus, the fluorescence of the mixture will be less than the fluorescence of the labelled compound by an amount depending on the quantity of aminoglycoside antibiotic present in the original sample. By measuring the fluorescence of the ; 20 mixture and comparing it, for example, with a standaxd curve ~described in more detail below) the amount of aminoglycoside antibiotic can be determined.
The fluorescent-labelled compound has to be capable of complexing with the antibody used in the method, and the antibody must also be capable of complexing with the gentamicin (or other similar drug) under analysis. It follows, therefore, that the labelled compound must either be of identical structure (apart from the label) to the gentamicin or other drug under assay, or have a very closely similar structure, since otherwise it will not bind with the

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~` 1073813 antibody. Thus~ the labelled compound to b~ used in the assay of a drug A will either be A itseIf carrying a fluorescent label, or a compound which is very similar to A (and which carries a fluorescent label).
It is not essential in the method of the invention to use antibody which have been raised using either the particular drug under assay or (where applicable) the closely related compound which is to carry the fluorescent label.
~ The antibody can, instead, be raised using another material but this will necessarily be closely similar in structure to the drug under assay and to the label compound, since other-wise the antibody will not bind with these two materials.
Some o the aminoglycoside antibiotics are of very closely similax chemical structure, and we have found that, for example, rabbit anti-gentamicin antiserum will bind not only with gentamicin but also with sisomycin and Schering 1 20569. Cross-reactions of this type make it difficult to assay a sample contalning two or more such antibiotics but, in practice, such assays are rarely required. Normally, the biological fluid sample will contain only one aminoglycoside antibiotic and, in such cases, the possibility of cross- -reactions is advantagecus. Thus, in order for example ~o assay, by the method of the invention, for gentamicin sisomycin or Schering 20569, only one antiserum i5 needed, e.g. rabbit anti-gentamicin antiserum. Not only does this mean that fewer stocks are required in a clinical laboratory, but also it means that drugs can be assayed for which it is difficult to prepare specific antisera.
- In the method of the invention, it is preferred to add the labelled compound to the sample under test, and ., ' ' ' ' ~

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then to form the ~ utre with the antibody, AlternatiYely, the antibody can be mixed with'the'sample, and the labelled compound then a~aed.
The determination of aminoglycoside antibiotic in the sample from the fluorescence measurement, can con-veniently be effected using a standard curve. A standard curve for any particular system (i.e. antibiotic/fluorescent-labelled antibiotic or other compound/antiserum) may, for -example, be obtained as follows. Solutions of known con-centration of the antibiotic to be assayed are made up in pooled normal serum or a suitable buffer. To each solution is added a constant known amount of fluorescent-labelled antibiotic or other compound and sufficient antiserum to form a solution with a predetermined dilution of antiserum. !' The fluorescence intensities of the solutions are then measured and a standard curve of fluorescence intensity against the concentration of unlabelled antibiotic is plotted.
Such a curve may then be used in the method of the invention for example as follo~s. To a known volume of the biological fluid sample (containing the antibiotic to be assayed), which may comprise a buffer, is added the constant known amount of fluorescent-labelled antibiotic or other compound used for preparing the standard curve. An amount o antiserum is then added to provide the dilution thereof used in the standard curve determination. The' fluorescence intensity of the'resulting mixture is measured and from the , standard curve, the amount of antibiotic in the bioiogical fluid sample'can be determined.
The method of invention is particularly useful for assay of gentamicin, but it can also be used for other , .

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aminoglycoside antibiotics / such as 5~repton~cin~ tobra~ycin~neomycin, kanamycin~ amikacin, and the more recently dis-covered sisomycin and Schering 20569, for example. The chemical formulae of sisomycin ~nd Schering 20569 are:

~NH2 ~ ~N3 NH.CH3 SISOMYCIN

H2N yy NH-CH2 CH3 . NH2 1 ~ -~ ~ ~ ~ / \

OH ~CN3 H.CH3 .. . .. .. . ...

In the assay of gentamicin by the method of the - invention, the preferred fluorescent label is fluorescein.

This can be attached to gentamicin by, for example, reacting bm~J~

- ' . ' gentamicin with'fluorescein isothiocyanate to give fluorescein-thiocarbamyl gentamicin (hereafter "FTC-G"). Other fluore-scent groups can also be used, such as for example dansyl, rhodamine, fluorescamine, pyrene and 2-methoxy-2,4-diphenyl-3(2H)-furanone. The su'itability of any particular fluorescent group with any particular antibiotic/antiserum system can readlly be determined by routine trial and experiment. The fluorescent group should be one which is compatible with the system as a whole to show a reliable and reproducible fluore-scent quenching effect upon formatlon of the labelled antibiotlc/
antiserum complex.
In thi's connection, it should be noted that the '~ fluorescent quenching is dependent not only upon the particular fluorescent group used as label,~but also upon the nature of the antibiotic and antiserum. The latter must also be selected having regard to its suitability in the overall system. Again, routine trial and experiment will reveal the suitability or otherwise of a particular antiserum in a particular antibiotic/
fluorescent-labelled antibiotic system. We have found that with gentamicin labelled with fluorescein, rabbit antisera produced by injecting gentamicin coupled to bovine'serum albumin by the carbodiimide method are satisfactory.
In the'particular case of gentamicin/FTC-G~rabbit antiserum, we believe that fluorescent quenching occurs by inter-action between the fluorescont bm/,~

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label and gro~ps of the antibody molecule.
It will be appreciated that the method of the invention includes the so-called ~'competitive bindingl' technique, in which there is competition between the labelled and unlabelled antigen ~antibiotic) to bind with a limited amount of antibody (antiserwm). Competitive bind-ing immunoassays are very well known, and normally necessarily involve separation o~ the bound antibody : antigen complexes from free antigen. This separation step (which is, for example, necessary in RIA) is a practical inconvenience. The method of the present invention, however, does not 1nvolve any separation step, and this makes the method ideally suited to analyses of the continuous-flow type. Accordingly, the invention includes the method herein described effected in a continuous-flow manner, and also apparatus therefor. I
In continuous flow analyses according to the present invention, the mixture of sample, antibody and fluorescent labelled compound, is passed along a conduit and the fluore3cence is measured. In a preferred procedure, which is described in U.S. patent specification no. 2,797,1~9, individual segments of mixture are passed sequentially along the conduit, separated by an inert fluid segment (e.g. air) and~ if desired, a wash liquid segment. The mixture can be formed in the conduit itself, by supplying to the conduit, 2~ in phase with segments of components of the mixture already preæent therein, the one or more further components, mixing of the components occurring in the conduit as the mixture flows therethroughO
It is a highly advantageous feature of the present invention that analyses of the drugs in question can be carried ~ _ 9 _ (follo~ed by page 9a) 10'73813 `': ' out relatively simply in this continuous flow manner~ mainly as a result of the fact that no separation step iR required in the method of the invention. Thus, the mixture flowing in the conduit may be passed directly to (or through) a fluorescence cell for direot measurement. It is therefore possible by the method of the invention to assay these particular drugs on a continuou~ flow basis which has been hitherto impossible or ~ ' very difficult by prior known assay techniques.
Among the advantages of the present invention are the following:
1. FTC-G is readily prepared in excellent yield from easily ~vailable and cheap starting products.
2. ~TC-G has good shelf-life.
' 3. Neither radiation hazard nor the need for radioactive , counting facilities is involved.
4. No separation step is needed.

5. Measurement is by conventional fluorimetry.

- 6. Because of points 4 and 5, the procedure can be ;~ automated easily.
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7. The assay is very fast. Only a few minutes are necessary for attainment of immunological equilibrium between anti-body, FTC-G and gentamicint after which a single fluorescence measurement gives the result.

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8. The assay can be immunospecific for the particular antibiotic, subject to the comments made above con-cerning cross-reactions.
, 9. The serum sample size required is very small. 5 ~1 or less suffices for a discrete assay.
In the Figures:
EIG. 1 shows the results of an assay for gentamicin - content by radio-immunoassay and for fluorescein content by fluorimetry of the mixtures in accordance with Example l;
FIG. 2 shows the fluorescence intensity for the antiserum dilutions in accordance with Example 3(i);
FIG. 3 is the standard curve produced by subtract-ing the serum intrinsic fluorescence intensity from the total intensity of the assay mixtures in accordance with Example 3(ii);
FIG. 4 shows the correlation between gentamicin -~
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levels measured by fluorescence quenching immunoassay, and levels measured by an independent laboratory using an established bioassay;
~Q FIG 5. shows one form of flow system suitable for a continuous flow analysis; -FIG~ 6 shows the standard curve produced by subtracting the serum intrinsic fluorescence intensity from the net fluorescence intensi~y increase measured for each assay mixture in accordance with Example 6, and;
i ~ FIG. 7 shows the correlation between gentamicin levels measured by automated fluorescence quenching :
immunoassay, and levels measured by an independent laboratory using an esta~lished bioassay~
~0 I~ order that the invention may be more fully understood, the following Examples are given by way of illustration only.
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10'~3~13 EXAMPLES

EXAMPLE 1. Preparation of fluorescein-labelled gentamicin rFTC-G ) Gentamicin (lmM) and fluorescein isothiocyanate (FITC) (1.25mM) were allowed to react in O. 05M sodium carbonate/bicarbonate buffer, pH 9.0, for 2 hours at room temperature. 2ml of the reaction mixture was applied to a column (1 X 97cm) of G-15 grade Sephadex and eluted with carbonate/bicarbonate buffer as above, with a flow rate of 1.8ml/h. Column fractions ~1.8ml) were assayed for genta-micin content by radio-immunoassay and for fluorescein con-tent by fluorimetry. Figure 1 of the accompanying drawings shows the result. Unreacted gentamicin left the column first. The FTC-G product exited next, well separated from the gentamicin peak. Unreacted FITC was very strongly held on the G-15 column and was not eluted.
The FTC-G product was stored in solution, either frozen or at 4C. Electrophoretic characterisation showed the presence of one major band (presumed to be mono-FTC-~0 labelled gentamicin) and two minor bands (presumed to be poly-FTC-labelled gentamicin).

Co~centrations of FTC-G quoted below are based on the gentamicin content as assessed by radio-immunoassay.
EXAMPLE 2. Preparation of anti-gentimic n sera Rabbits were immunised with gentamicin coupled to bovine serum albumin by means of the carbodiimide method.

dc~i ~" 10'73813 EXAMPLE 3. Fluorescence quenching immunoassay for gentamicin (i) Antibody dilution curve In order to choose optimum assay conditions, 0.5 ml amounts of a 16.4 n~/ml solution of FTC-G in 0.1 M sodium phosphate buffer p~l 7.5 were added to doubling dilutions (1 ml) of an antiserum in the same buffer.
After allowing a few minutes for equilibration, fluorescence intensity was measured (see E`igure 2 of the accompanying draw-ings). From this curve, a final antihody dilution of 1/240 in 1.5 ml was chosen for construction of the standard curve.
(ii) Standard curve Gentamicin was added in known amounts to pooled normal human serum. Aliquats of these standard samples were diluted 1/5~ in 0.1 M sodium phosphate buffer pH
7 . 5 . To 0.5 ml of the diluted standard samples was added 0.5 ml of a 16.4 ng/ml solution of FTC-G in the same buffer, ~ollowed by 0.5 ml of antiserum diluted 1/8~ in the same buffer.
After allowing a few minutes for equilibration, the fluore-scence intensity of the assay mixtures was measured. The contribution of the intrinsic fluorescence of the pooled normal human serum to the total fluorescence intensity of the assay mixtures was estimated independently by adding 1 ml of the phosphate buffer to 0.5 ml of the diluted standard samples and measuring the fluorescence. 8y subtraction of the serum intrinsic fluorescence intensity from the total intensity of the assay mixtures, the standard curve shown in Figure 3 was produced.

EXAMPLE 4. Assay of gentamicin in patient samples Serum samples from patients receiving gentamicin therapy were assayed according to the procedure described in Example 3, sec~ion (ii). The contribution of the intrinsic fluorescence of each serum sample, measured independently, was subtracted bm/

10'73813 from the total fluorescence intensity of the corresponding assay mixture, and the result used to determine, from an appropriate-standard curve, the amount of gentamicin in-the serum sample.
Figure 4 shows the correlation between gentamicin levels measured by fluorescence quenching immunoassay, and levels measured by an independent laboratory using an established bioassay. The computed line of best fit is shown. Given the acknowledged inaccuracy of the bioassay technique, the agree-ment between the two methods is acceptable, and satisfactory for clinical purposes.

EXAMPLE 5 Continuous-flow system for automated assay of . . _ Figure 5 of the accompanying drawings shows one form of flow system, suitable for a continuous-flow analysis. The system comprises sample input line 1, FTC-G input line 2, air input line 3 and antiserum input line 4. Lines 2 and 3 meet at segmenter 6 which is connected to junction 7 where line 1 joins line 2. Downstream of jUnctiQn 7 line 2 is provided with a mixing coil 8 and then passes to junction 9 where line 4 joins.
Downstream of 9 is a mixing coil 10 and finally a fluorimeter 11 having a waste outlet 12 and an outlet-13 downstream of the fluorescence cell connected to line 5 and thence to waste. The ; fluorimeter 11 is operatively coupled to recorder 14.
In operation, a controlled amount of FTC-G enters line 2 and is segmented by air in segmenter 6. The sample to be ~ested (e.g. serum), diluted as necessary, is introduced into the segmented stream in junction 7, followed by mixing in coil 8, then a controlled amount of antiserum is introduced in junction 9, followed by mixing in coil 10 before passing to bm/J-~

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fluorimeter 11.

EXAMPLE 6. Automated assay of gentamicin: standard curve Gentamicin was added in known amounts to pooled normal human serum. Aliquots of these standard samples were diluted 1/100 in 0.1 M Tris/HCl buffer pH 7.5 containing 10 mM MgC1 ("Tris/MgC12 buffer"). Using the flow system of Figure 5, a standard curve was produced by means of the following procedure.
~he standard samples were pumped through input line 1 at 0.16 ml/min. Each sample was pumped for 1 min, then Tris/MgC12 buffer was pumped ~or a 1 min wash period before pumping of the next sample. FTC-G, 8.2 ng/ml in Tris/MgC12 buffer containing 0.1% V/V Triton X-lOO~detergent, was pumped through input line 2 at 0.16 ml/min. Antiserum, diluted 1/160 in Tris/MgC12 buffer containing 0.1% V/V Triton X-lOO~detergent, was pumped through input line 4 at 0.16 ml/min. The mixed streams were pulled through the fluorimeter flow cell at 0.42 ml/min (line 5). The i *otal fluorescence intensity of the mixed streams was recorded l on the chart recorder. The intensity recorded during the wash i~ 20 periods (corresponding to maximum binding of FTC-G to antiserum in absence of sample) was subtracted from the intensity re-corded during passage of each assay mixture through the fluorimeter flow cell. In this way, the net increase in fluorescence intensity due to each sample was measured. When the standard samples had passed through the system, the solution pumped through lines 2 and 4 was changed to Tris/MgC12 buffer containing 0.1~ V/V Triton X-lOO~detergent. The standard samples were then re-run through the system. The intrinsic ' fluorescence intensity of the standard serum sample~ was there-by measured. By subtraction of the serum intrinsic .~, .
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10~3813 fluorescence intensity from the net fluorescence intensity increase measured for each assay mixture, the standard curve shown in Figure 6 was produced.

EXAMPLE 7. Automated assay of gentamicin in patient samples . .
Serum samples from patients receiving gentamicin therapy were assayed according to the procedure described in Example 6.
The contribution of the intrinsic fluorescence of each serum sample, measured independently, was subtracted from the net fluorescence intensity increase measured for the corresponding assay mixture, and the result used to determine, from an appropriate standard curve, the amount of gentamicin in the serum sample.
Figure 7 shows the correiation between gentamicin leYels measured by automated fluorescence quenching immunoassay, the levels measured by an independent laboratory using an established bioassay. The computed line of best fit is shown.
Given the acknowledged inaccuracy of the bioassay technique, the agreement between the two methods is acceptable, and satisfactory for clinical purposes.
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Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY
OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A method of assaying a biological fluid sample for an antibiotic selected from gentamicin, sisomycin, Schering 20569, streptomycin, tobramycin, neomycin, kanamycin and amikacin, which comprises forming a mixture of:
(a) the sample;
(b) a fluorescent-labelled compound which is a compound which carries a fluorescent group, the fluorescence of which is reduced when the compound binds with antibody; and (c) antibody against the antibiotic and the fluorescent-labelled compound measuring the fluorescence of the mixture; comparing the said fluorescence with standard results and thereby determining the amount of said antibiotic present in the sample.

2. A method according to Claim 1 which is carried out in a continuous-flow manner.

3. A method of assaying a biological fluid sample for an aminoglycoside antibiotic comprising:
(a) forming a mixture of the sample, a fluorescent-labelled compound, the fluorescence of which is reduced when the compound binds with antibodies and antibody against the antibiotic under assay and the compound;
(b) measuring the fluorescence of the mixture to determine the amount of reduction of fluorescence of the compound to determine the amount of aminoglycoside antibiotic present in the sample,

4. A method according to Claim 3, wherein segments of the mixture are flowed along a conduit, separated by segments of an inert fluid, and the fluorescence of the mixture segments is measured without any step of separation of reaction product from the mixture.

5. A method according to Claim 3 which is carried out in a continuous-flow manner.

6. A method according to Claim 3 wherein the anti-biotic is gentamicin.

7. A method according to Claim 6 wherein the fluore-scent-labelled compound is fluorescein-labelled gentamicin.

8. A method according to Claim 7 wherein the fluore-scein-labelled gentamicin is fluoresceinthio-carbamyl gentamicin.

9. A method according to Claim 6 wherein the antibody is rabbit anti-gentamicin antiserum.

10. A method according to Claim 4 wherein the fluore-scence of the mixture segments is compared with standard results to determine the amount of aminoglycoside antibiotic under assay in the sample.

11. A method according to Claim 10 wherein segments of the said mixture are separated by segments of air.