The development of microbiological and immunological assays for antibiotics

29

trends in analytical chemistry, vol. 5, no. 2,1986

biotechnology focus

The development of microbiological and immunological assays for antibiotics L. 0. White Bristol, U.K. Antibiotics have traditionally been assayed mierobiologically. In the clinical laboratory there has been a trend over the last few years to assay aminoglycoside antibiotics in patients’ serum by non-isotopic immunoassay techniques since in the clinical situation their high speci@city and speed are advantageous. Quality control circulation results show that this trend towards immunoassays has resulted in more laboratories performing satisfactorily with regard to accuracy and precision.

There has always been a need to measure the concentration of drugs, especially for quality assurance in their manufacture and distribution, and traditionally most antibiotics have been measured by microbiological assay. The reasons are twofold: (i) the essential property of an antibiotic is its ability to stop the growth of a susceptible microorganism and, (ii) many early antibiotics were impure fermentation mixtures, the antimicrobial potency of which could not be precisely determined by chemical means. Microbiological assay A typical microbiological assay uses a square flatbottomed plate (25 x 25 cm) containing a thin (2-3 mm), uniform layer of solid nutrient medium (agar) seeded with an indicator microorganism. Samples containing antibiotic are applied to the agar on paper discs or in wells (up to 64 per plate). During incubation at a suitable temperature for up to 18 h the antibiotic diffuses into the agar and the indicator organism grows. Zones of inhibition of growth form around the discs or wells, the zone edge occurring when a minimum concentration of antibiotic capable of inhibiting the growth of the microorganism meets for the first time a population density too great for it to inhibit. The distance of the zone edge from the centre of the well or disc is therefore determined by original concentration of antibiotic, rate of diffusion, original population density, growth rate of the organism and other factors. Since all but the former can be kept constant, zone diameter can be related to antibiotic concentration. An assay, where a plot of log

concentration against zone diameter or square of zone diameter will be near linear over a chosen range of up to five doubling concentrations, can be devised by careful choice of sample size, indicator organism, population density, medium, temperature etc. By careful control of all possible variables, by the use of pseudo random (latin square) distribution of replicates on a single plate and by precise zone measurement with magnifying zone readers very high reproducibility can be obtained. Such high precision microbiological assays are still used extensively in the pharmaceutical industry to assay drug potency. Clinical antibiotic assays The early 1970s saw the introduction into clinical medicine of the wide spectrum parenteral aminoglycoside gentamicin. This antibiotic proved very effective for the treatment of life threatening infections caused by a large number of different bacterial pathogens but was in addition potentially ototoxic and nephrotoxic. Some medical microbiologists soon recognised the need to assay gentamicin concentrations in patients since efficacy and toxicity were related to serum concentrations and the margin between effective and potentially toxic concentrations was narrow. Typically, peak (post dose) concentrations of gentamicin of 5-8 mg/l were required for efficacy and concentrations over 12 mg/l were to be avoided. Also trough (pre-dose) concentrations of less than 2 mg/l were thought necessary to minimise the risk of ototoxicity’. This need to monitor serum concentrations also holds true for the more recently introduced aminoglycosides tobramycin, amikacin and netilmicin. Since aminoglycosides have a relatively short serum half life (approximately 2 h) they must be administered three or four times a day. Therefore to obtain pre- and post-dose serum concentrations upon which decisions about dosage individualisation could be made, hospital laboratories need to provide a rapid reliable assay service to clinicians. Clinical microbiological assays Many laboratories began to use modified @Elsevier

micro-

Science Publishers B.V.

30

biological plate assays, but in the clinical setting microbiological assays suffer from two disadvantages, poor specificity and lack of speed, even when performed in such a way as to ensure suitable precision and accuracy. Poor specificity is a problem since patients are often treated with two or more antibiotics and thus it is necessary either to inactivate those antibiotics you do not wish to measure or to use an indicator organism insusceptible to them. The application of various inactivation procedures and the use of multiply resistant Gram negative indicator organisms such as Klebsiella edwardsii var. atlantae NCTC 10896 helped relieve this problem with aminoglycoside assays2. Unfortunately other antibiotics present in samples are often not disclosed by the requesting physicians; also over the last few years several new, highly active antibiotics to which many indicator organisms are sensitive have been introduced and are often co-prescribed with an aminoglycoside. In an attempt to speed up microbiological assays some laboratories either used elevated incubation temperatures or measured some biochemical parameter of bacterial growth such as urease activity3. It thus became possible to produce a result in less than one dosage interval (6-8 h) but the essential problem of poor specificity remained. lmmunoassays for aminoglycosides Immunoassay is a quantitative technique which uses specific antibody (Ab) as a reagent. It is very highly specific. The aminoglycosides (Ag) are ideal candidates for immunoassay because they are not significantly metabolised and are chemically suited to coupling with a protein carrier to produce an immunogenic conjugate (Ag.Con) and chemical labelling to produce a tracer reagent (Ag.Tr). Aminoglycoside immunoassays based on competitive binding (Fig. 1) soon appeared. A radioimmunoassay (RIA) using [3H]gentamicin was described in 1973 but 1251radioimmunoassays followed and were marketed as kits. Non-separation assays using fluoroscein-thiocarbamyl-gentamicin (FTC-G) as the tracer were described in 1976 and 1977. A polarisation fluoroimmunoassay (PFIA) exploited the fact that when excited by polarised light free FTC-G emitted non-polarised fluorescence whereas bound FTC-G emitted polarised fluorescence. A quenching fluoroimmunoassay (QFIA) utilised the fact that the fluorescence of FTC-G is quenched by antibody binding. Homogeneous enzyme immunoassay of the enzyme multiplied immunoassay technique (EMIT@) type appeared in 1978. The aminoglycoside was labelled with bacterial glucose-6-phosphate dehydrogenase. In the free state the enzyme was active but in the

trends in analytical chemistry, vol. 5,110.2, I986

Fig. 1. Competitive binding immunoassays. Ab is raised by immunising animals with Ag. Con. Tr. can be radioisotope, enzyme, fluorochrome etc. Measurement of Tr. in the bound fraction is achieved by separation or by using a Tr., the activity of which is altered when bound to antibody.

bound state activity was inhibited. Enzyme activity was estimated spectrophotometrically by measuring the rate of formation of NADH, from NAD. At about the same time a substrate-labelled fluoroimmunoassay (SLFIA) was described. Aminoglycoside was labelled with a fluorigenic enzyme substrate (umbelliferyl-@-D-galactoside). In the free fraction this was degraded by /?-galactosidase to a fluorescent product; in the bound fraction it was not. Thus with the advent of immunoassay for the first time highly specific aminoglycoside assays were available. These assays were also much more rapid than microbiological assays taking 1 h (RIA) or less (under 15 min for some homogeneous assays) and, in addition, required only very small volumes of serum. Thus two major drawbacks of microbiological assay were overcome. Performance and popularity In the U.K. in 1971 some laboratories exchanged gentamicin-containing sera and the results obtained showed such wide differences in the estimated concentration that a formal quality control circulation was set up. Its format has undergone slight modifications since it was formed but the basic method for estimating a laboratory’s performance remains the same. Participating laboratories assay six samples of known concentration and the modulus of the mean percentage error plus two standard deviations of the percentage error (M + 2 S.D.) is calculated for each laboratory. A (M + 2 S.D.) of O-30 is considered satisfactory for clinical purposes, 31-50 possibly satisfactory and over 51 unsatisfactory. In 1973 only 81 laboratories participated but the number of participants is now over 3004-6. A resume of the performance and popularity of microbiological and immunological assays is shown in Table I. Initially almost all assays were done microbiologically. Plate assays using gram-negative or gram-positive indicator organisms were the most popular; a third method which involved making doubling dilutions of the sample in a nutrient broth was used by about 20% of laboratories initially but soon disappeared because its performance was very poor, and has not been documented in the table. Over the years the gram-positive plate has lost popularity because of its poorer

trendrjn analyticalchemistry, vol. 5, no. 2,1986

TABLE

I. Popularity

and performance

31

of microbiological

vs. immunological

assays for gentamicin

in serum.

Numbers in parentheses denote the percentage that is satisfactory. Year

73 74 79 79-80 80-81 81 81-82 82 83 83-84 84-85

Number labs

81 92 274 306 322 319 350 327 323 315 312

of

Percentage

of users (%)

Microbiological

assays

Gm- plate

Gm+ plate

30 53 83 78 71 68

38 32 9 9 6 4

(29) (18) (34) (27) (42) (56)

Urease

(23) (14) (8) (19) (26) (31)

6 4 2 1 1 1

(0) (25) (0) (25) (33) (33)

Immunoassay

techniques

RIA

EMIT

0 0 3 3 2 2

0 0 1 5 15 18

(38) (50) (50) (71)

(67) (57) (72) (78)

FIA

PFIA

0 0 1 1 2 2

0 0 0 0 0 0

(67) (50) (40) (40)

56 (38) 50 (36)

31 (67) (0)


2 (43) (50)

26 (55) 29 (52)

63 (40) (25)

29 (58) 23 (44)

1 (50) 1

(0)

1 (50)
1 (50) 1 (50)

41 (81) 44 (87)

11 (44) 12 (74)

3 (80) 5 (100)

19 (48)

1

(0)


1 (67)

45 (78)

13 (82)

9

performance and specificity. The gram-negative plate remains the most popular microbiological method and although performance has improved over the years it is rare for more than 50% of the laboratories to perform satisfactorily and its popularity has shown a steady decline. The decline in the popularity of microbiological assays is matched by an increase in the usage of immunoassays (except for RIA which never became very popular in the U.K.) especially EMIT which is currently the most popular method. Since 1982 several laboratories have changed to PFIA and this is solely attributable to the fully automated Abbott TDX assay. Performance of theimmunoassays is substantially better than microbiological assays; since 1983 the three most popular methods EMIT, FIA (almost all Ames SLFIA assays) and PFIA (Abbott TDX) have consistently had over 70% of users submitting satisfactory results. Concluding remarks In the U.K. clinical aminoglycoside assays were almost all performed by microbiological assay in 1973. Over the last twelve years there has been a steady decline in the popularity of microbiological assays in favour of non-isotopic commercially produced enzyme and fluoroimmunoassays. This is despite the fact that immunoassays are much more costly to perform both in terms of captial equipment and consumable items. Quality control circulation results show that this trend towards immunoassay has resulted in a significant improvement in performance. Acknowledgement Antibiotic Quality Control Circulation data quoted with permission of the Director DMRQC.

(0)

<;

(100)

(98)

References 1 D. S. Reeves

2

3

4 5

6

and L. 0. White, in A. Richens and V. Marks (Editors), Therupeutic Drug Monitoring, Churchill Livingstone, Edinburgh, 1980, pp. 445-456. D. S. Reeves and M. J. Bywater, in J. de Louvois (Editor), Selected Topics in Clinical Bacteriology, Balliere Tindal, London, 1976, pp. 21-78. J. Broughall, in D. S. Reeves, I. Phillips, J. D. Williams and R. Wise (Editors), Laboratory Methods in Antimicrobial Chemotherapy, Churchill Livingstone, Edinburgh, 1978, pp. 194-207. D. S. Reeves and M. J. Bywater, J. Antirnicrob. Chemother., l(l975) 103-116. M. J. Bywater, H. A. Holt and D. S. Reeves, in P. Periti and G. G. Grassi (Editors), Proceedings of the 12th International Congress of Chemotherapy, Florence, Italy, 19-24 July, 1981, American Society for Microbiology, Washington DC, 1982, pp. 795-797. M. J. Bywater, H. A. Holt and D. S. Reeves, presented at The 2nd European Congress of Clinical Microbiology, Brighton, l-5 September, 1985, unpublished results.

Les White obtained both his B.Sc. and Ph.D. at the University of Birmingham. After obtaining his Ph.D. he became an MRC Junior Research Fellow and worked with Professor H. Smith, FRS, on mechanisms of fungal pathogenic@. He is currently Principal Scientific Officer at Sothmead Hospital, Bristol, U.K.

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